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Recent Articles in Journal of Pineal Research

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Furio AM, Brusco LI, Cardinali DP
Possible therapeutic value of melatonin in mild cognitive impairment: a retrospective study.
J Pineal Res. 2007 Nov;43(4):404-9.
Mild cognitive impairment (MCI) is an etiologically heterogeneous syndrome characterized by cognitive impairment preceding dementia. Approximately 12% of MCI patients convert to Alzheimer's disease (AD) or other dementia disorders every year. In the present report we retrospectively examined the initial and final neuropsychological assessment of 50 MCI outpatients, 25 of whom had received daily 3-9 mg of a fast-release melatonin preparation p.o. at bedtime for 9-18 months. Melatonin was given in addition to the standard medication prescribed by the attending psychiatrist. Patients treated with melatonin showed significantly better performance in Mini Mental State Examination and the cognitive subscale of the Alzheimer's Disease Assessment Scale. After application of a battery of neuropsychological tests including Mattis' test, Digit-symbol test, Trail A and B tasks and the Rey's verbal test, better performance was found in melatonin-treated patients, except for the Digit-symbol test score which remained unchanged. Abnormally high Beck Depression Inventory scores decreased in melatonin-treated patients, concomitantly with an improvement in wakefulness and sleep quality. The results suggest that melatonin can be a useful add-on drug for treating MCI in a clinical setting. [Abstract]

Jou MJ, Peng TI, Yu PZ, Jou SB, Reiter RJ, Chen JY, Wu HY, Chen CC, Hsu LF
Melatonin protects against common deletion of mitochondrial DNA-augmented mitochondrial oxidative stress and apoptosis.
J Pineal Res. 2007 Nov;43(4):389-403.
Defected mitochondrial respiratory chain (RC), in addition to causing a severe ATP deficiency, often augments reactive oxygen species (ROS) generation in mitochondria (mROS) which enhances pathological conditions and diseases. Previously, we demonstrated a potent endogenously RC defect-augmented mROS associated dose-dependently with a commonly seen large-scale deletion of 4977 base pairs of mitochondrial DNA (mtDNA), i.e. the common deletion (CD). As current treatments for CD-associated diseases are rather supplementary and ineffective, we investigated whether melatonin, a potential mitochondrial protector, provides beneficial protection for CD-augmented mitochondrial oxidative stress and apoptosis particularly upon the induction of a secondary oxidative stress. Detailed mechanistic investigations were performed by using laser scanning dual fluorescence imaging microscopy to provide precise spatial and temporal resolution of mitochondrial events at single cell level. We demonstrate, for the first time, that melatonin significantly prevents CD-augmented mROS formation under basal conditions as well as at early time-points upon secondary oxidative stress induced by H2O2 exposure. Thus, melatonin prevents mROS-mediated depolarization of mitochondrial membrane potential (DeltaPsim) and subsequent opening of the mitochondrial permeability transition pore (MPTP) and cytochrome c release. Moreover, melatonin prevents depletion of cardiolipin which appears to be crucial for postponing later MPTP opening, disruption of the mitochondrial membrane and apoptosis. Finally, the protection provided by melatonin is superior to those caused by the suppression of mitochondrial Ca2+ regulators including the mitochondrial Na+-Ca2) exchanger, the MPTP, and the mitochondrial Ca2+ uniporter and by antioxidants including vitamin E and mitochondria-targeted coenzyme Q, MitoQ. As RC defect-augmented endogenous mitochondrial oxidative stress is centrally involved in a variety of pathological conditions and diseases, melatonin thus may serve as a therapeutic drug to benefit many clinical conditions that involve malfunction of the mitochondria. [Abstract]

Hardeland R, Backhaus C, Fadavi A
Reactions of the NO redox forms NO+, *NO and HNO (protonated NO-) with the melatonin metabolite N1-acetyl-5-methoxykynuramine.
J Pineal Res. 2007 Nov;43(4):382-8.
The different NO redox forms, NO+, *NO and HNO (=protonated NO-), were compared for their capabilities of interacting with the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK), using NO+SbF6-, PAPA-NONOate and Angeli's salt as donors of the respective NO species. Particular attention was paid to stability and possible interconversions of the redox forms. *NO formation was followed by measuring the decolorization of 2-(trimethylammonio-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (TMA-PTIO), at different pH values, at which NO+ is, in aqueous solution, either highly unstable (pH 7.4) or relatively stable (pH 2.0). *NO donation by PAPA-NONOate, as indicated by TMA-PTIO decolorization, was similar at either pH and 3-acetamidomethyl-6-methoxycinnolinone (AMMC) was formed as the major product from AMK, at pH 7.4 more efficiently than at pH 2.0. At pH 2.0, TMA-PTIO decolorization by NO+SbF6- was much weaker than by PAPA-NONOate, but AMMC was produced at substantial rates, whereas neither TMA-PTIO decolorization nor AMMC formation was observed with the NO+ donor at pH 7.4. As NO+ is also stable in organic, especially aprotic solvents, NO+SbF6- was reacted with AMK in acetonitrile, ethanol, butanol, and ethyl acetate. In all these cases, AMMC was the only or major product. In ethyl acetate, N1-acetyl-5-methoxy-3-nitrokynuramine (AMNK) was also formed, presumably as a consequence of organic peroxides emerging in that solvent. Presence of tert-butylhydroperoxide in an ethanolic solution of NO+SbF6- and AMK also resulted in AMNK formation, in addition to AMMC and two red-fluoresecent, to date unknown products. However, hydrogen peroxide enhanced *NO-dependent AMMC production from AMK and also from N1-acetyl-N2-formyl-5-methoxykynuramine. HNO donation by Angeli's salt (Na2N2O3) also caused AMMC formation from AMK at pH 7.4, with a somewhat lower efficiency than PAPA-NONOate, but no AMNK nor any other product was detected. Therefore, all three NO congeners are, in principle, capable of nitrosating AMK and forming AMMC, but in biological material the reaction with NO+ is strongly limited by the extremely short life-time of this redox form. [Abstract]

Guha M, Maity P, Choubey V, Mitra K, Reiter RJ, Bandyopadhyay U
Melatonin inhibits free radical-mediated mitochondrial-dependent hepatocyte apoptosis and liver damage induced during malarial infection.
J Pineal Res. 2007 Nov;43(4):372-81.
We showed earlier that malarial infection significantly induces liver apoptosis mediated by oxidative stress mechanisms. Thus, a nontoxic antioxidant-antiapoptotic molecule may be beneficial for hepatoprotection. Melatonin remarkably prevents hepatocyte apoptosis in mice induced during malaria as indicated by caspase 3 and TUNEL assays as well as transmission electron microscopy (TEM) of the liver tissue. The mitochondrial apoptotic pathway, which plays a critical role in liver cell death during malarial infection, was almost completely suppressed by melatonin as it corrects both the overexpression of Bax and down-regulation of bcl-2 as revealed by semiquantitative RT-PCR. Fluorometric studies using JC-1 documented that melatonin also restores mitochondrial transmembrane potential (DeltaPsim) in malaria-infected mice liver. The antiapoptotic effect of melatonin is associated with its antioxidant role because melatonin protects liver from oxidative stress induced during malaria by scavenging the hydroxyl radicals, preventing the depletion of reduced glutathione, inhibiting lipid peroxidation and protein carbonyl formation. The effective antioxidant dose of melatonin to protect liver from oxidative stress during malaria is 20 times lower than that of known antioxidants, vitamin C and vitamin E. Apoptosis of hepatocytes during malarial infection is well correlated with dysfunction of the liver while melatonin offers hepatoprotective effects as indicated by different liver function tests. Thus, melatonin may well be effective in combating oxidative stress-induced apoptosis and liver damage during malaria infection. [Abstract]

Pontes GN, Cardoso EC, Carneiro-Sampaio MM, Markus RP
Pineal melatonin and the innate immune response: the TNF-alpha increase after cesarean section suppresses nocturnal melatonin production.
J Pineal Res. 2007 Nov;43(4):365-71.
The nocturnal surge of melatonin is the endocrine expression of the circadian system and is essential for organizing the timing of various endogenous processes. Previous works suggest that, in the beginning of a defense response, the increase in circulating tumor necrosis factor-alpha (TNF-alpha) leads to a transient block of nocturnal melatonin production and promotes a disruption of internal time organization. In the present paper, the concentration of melatonin and cytokines [TNF-alpha, interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12] in the colostrum (postdelivery day 3) and in the milk (postdelivery days 10, 15, 20 and 30) obtained at midday and midnight from mothers who gave birth by vaginal or cesarean section were compared. The nocturnal melatonin surge observed 3 days after vaginal delivery was absent after cesarean section. IL-12 presented no daily variation in either case, while daily variations in IFN-gamma, IL-10, IL-4 and IL-5 were observed after vaginal delivery and cesarean section. On the other hand, the increase in TNF-alpha after cesarean section resulted in suppression of the nocturnal melatonin surge. Daily variation of IL-2 was only observed after recovery of the nocturnal melatonin surge, 30 days after cesarean section. The present paper supports the hypothesis of a cross-talk between the pineal gland and the immune system, which could represent a putative immune-pineal axis. [Abstract]

Beraldo FH, Mikoshiba K, Garcia CR
Human malarial parasite, Plasmodium falciparum, displays capacitative calcium entry: 2-aminoethyl diphenylborinate blocks the signal transduction pathway of melatonin action on the P. falciparum cell cycle.
J Pineal Res. 2007 Nov;43(4):360-4.
The malarial parasite senses the environment to modulate its own cycle. Knowledge of the mechanisms for regulation signaling processes at the invasion, maturation, as well as division of Plasmodium falciparum before reinvasion would represent a major breakthrough and, therefore, might open new avenues for therapy. We have previously reported that melatonin modulates the circadian rhythm of malarial parasites through the activation of phospholipase C (PLC), production of InsP3, and induction of calcium release from intracellular stores. To further investigate the molecular mechanism of melatonin's action, we have used the InsP3 modulator 2-aminoethyl diphenylborinate (2-APB) given in a culture of P. falciparum parasites. Here we show that the melatonin acts on Plasmodium cell cycle through InsP3 signaling as 2-APB blocks melatonin's effect on calcium release. The function of the InsP3 signaling can be regarded as an important event for parasite invasion and maturation process, since addition of the PLC inhibitor, U73122 into Plasmodium-infected red blood cells impairs parasite invasion in vitro. By using 8BrcAMP, we also report here that Plasmodia displays a 'capacitative calcium entry' mechanism for amplification of calcium signals throughout the cytoplasm. [Abstract]

Carr R, Wasdell MB, Hamilton D, Weiss MD, Freeman RD, Tai J, Rietveld WJ, Jan JE
Long-term effectiveness outcome of melatonin therapy in children with treatment-resistant circadian rhythm sleep disorders.
J Pineal Res. 2007 Nov;43(4):351-9.
To date, there have been no prospective long-term studies of melatonin therapy in children. We report here data from a prospective follow-up study of 44 children with neurodevelopmental disabilities and treatment-resistant circadian rhythm sleep disorders (CRSD) who had participated in a placebo controlled, double blind cross-over trial of sustained-release melatonin. The follow-up study involved a structured telephone interview of caregivers every 3 months for upto 3.8 yr. The caregivers provided ratings of satisfaction, adverse effects, benefits, persistence with treatment and additional medications. Changes in melatonin dose were recorded. Open ended questions were included to capture caregivers' impressions and comments concerning melatonin therapy. Adverse reaction to melatonin therapy and development of tolerance were not evident. Better sleep was associated with reported improvement in health, behavior and learning. At the end of the study, the parental comments regarding the effectiveness of long-term melatonin therapy were highly positive. Parents whose children had sleep maintenance difficulties expressed a wish to have a commercially available controlled-release melatonin product which would promote sleep for 8-10 hr. Hypnotics for children with CRSD should be considered a second line of treatment for those who fail to respond to sleep hygiene and/or melatonin. [Abstract]

Kopczak A, Korth HG, de Groot H, Kirsch M
N-nitroso-melatonin releases nitric oxide in the presence of serotonin and its derivatives.
J Pineal Res. 2007 Nov;43(4):343-50.
A novel reaction was observed between 5-hydroxytryptophan derivatives like serotonin and N-nitroso-melatonin (NOMela). This reaction decreased the concentration of serotonin by about 50% and generated initially as detectable products nitric oxide and melatonin with stoichiometrical yields. The other expected product, a serotonin-derived radical, could not be detected by electron spin resonance (ESR) spectrometry, probably because the self-decay of phenoxyl type radicals proceed at the diffusion-controlled limit. From the facts that the decay rate of NOMela corresponded very well with the nitric oxide releasing rate and that nitrite was the only thermodynamically stable nitrogen oxide-containing product, it is concluded that the NOMela-serotonin reaction proceeded quantitatively. The observed reaction might be a possibility to counteract a pharmacologically abnormal high serotonin concentration in various diseases. [Abstract]

Gorfine T, Yeshurun Y, Zisapel N
Nap and melatonin-induced changes in hippocampal activation and their role in verbal memory consolidation.
J Pineal Res. 2007 Nov;43(4):336-42.
Overnight sleep contributes to memory consolidation; even a short nap improves memory performance. Such improvement has been linked to hippocampal activity during sleep. Melatonin has been shown to affect the human hippocampus and to induce 'sleep like' changes in brain activation. We therefore conducted and compared two functional magnetic resonance imaging studies: the first study assessed the effect of a 2-hr mid-day nap versus an equal amount of wakefulness on a verbal memory task (unrelated word pair association); the second assessed the effect of melatonin versus placebo (both conditions without nap) on a similar task. We report that following a nap relative to wakefulness, successful retrieval-related activation in the parahippocampus is decreased. A smaller decrease is seen in wakefulness with melatonin but not placebo. In parallel, an improvement in verbal memory recall was found after a nap compared with wakefulness but not with melatonin during wakefulness compared with placebo. Our findings demonstrate effects of melatonin that resemble those of sleep on verbal memory processing in the hippocampus thus suggesting that melatonin, like sleep, can initiate offline plastic changes underlying memory consolidation; they also suggest that concomitant rest without interferences is necessary for enhanced performance. [Abstract]

Migaud H, Davie A, Martinez Chavez CC, Al-Khamees S
Evidence for differential photic regulation of pineal melatonin synthesis in teleosts.
J Pineal Res. 2007 Nov;43(4):327-35.
The aim of this study was to compare the circadian control of melatonin production in teleosts. To do so, the effects of ophthalmectomy on circulating melatonin rhythms were studied along with ex vivo pineal culture in six different teleosts. Results strongly suggested that the circadian control of melatonin production could have dramatically changed with at least three different systems being present in teleosts when one considers the photic regulation of pineal melatonin production. First, salmonids presented a decentralized system in which the pineal gland responds directly to light independently of the eyes. Then, in seabass and cod both the eyes and the pineal gland are required to sustain full night-time melatonin production. Finally, a third type of circadian control of melatonin production is proposed in tilapia and catfish in which the pineal gland would not be light sensitive (or only slightly) and required the eyes to perceive light and inhibit melatonin synthesis. Further studies (anatomical, ultrastructural, retinal projections) are needed to confirm these results. Ex vivo experiments indirectly confirmed these results, as while the pineal gland responded normally to day-night rhythms in salmonids, seabass and cod, only very low levels were obtained at night in tilapia and no melatonin could be measured from isolated pineal glands in catfish. Together, these findings suggest that mechanisms involved in the perception of light and the transduction of this signal through the circadian axis has changed in teleosts possibly as a reflection of the photic environment in which they have evolved in. [Abstract]

Papis K, Poleszczuk O, Wenta-Muchalska E, Modlinski JA
Melatonin effect on bovine embryo development in vitro in relation to oxygen concentration.
J Pineal Res. 2007 Nov;43(4):321-6.
Melatonin promotes mouse embryo development in vitro. An effect of melatonin on bovine embryo development is described here. Slaughterhouse derived oocytes were subjected to standard in vitro maturation and fertilization procedures. Presumptive zygotes were cultured for 2 days in CR1aaLA medium supplemented with melatonin (10(-4) m) or without melatonin (control). Culture was performed under two different gas atmospheres containing physiological (7%) or atmospheric (20%) oxygen concentrations (2x2 factorial analysis). After day 2, embryos from each treatment group developed to at least four-cell stage, were cultured without melatonin until day 10 at optimum 7% O2 atmosphere. Blastocyst formation rates of presumptive zygotes and of four-cell embryos were calculated for each group. Significant interactions between oxygen tension and the melatonin treatment were found. Out of four-cell embryos put into in vitro culture after initial incubation in medium containing melatonin, decreased blastocyst rate was observed in melatonin group (47.7%) compared with control (67.7%; P=0.0327) when lower oxygen concentration was applied. A beneficial effect of melatonin was observed in 20% O2: out of 61 embryos, 42 (68.9%) developed to the blastocyst stage after treatment in melatonin versus 32 of 63 (50.8%; P=0.0458) blastocysts that developed in control group. In conclusion, beneficial or harmful effects of melatonin on bovine embryo development in vitro were observed, depending on the oxygen tension during the treatment. [Abstract]

Tan DX, Manchester LC, Terron MP, Flores LJ, Tamura H, Reiter RJ
Melatonin as a naturally occurring co-substrate of quinone reductase-2, the putative MT3 melatonin membrane receptor: hypothesis and significance.
J Pineal Res. 2007 Nov;43(4):317-20.
The nature of the MT3 melatonin receptor/binding site has been a long pondered mystery for scientists. Even though it is a presumptive membrane receptor, neither its transduction cascade nor its biological consequences, after its stimulation, have been uncovered. Moreover, solid data support the idea that the MT3 melatonin binding site is an enzyme, quinone reductase 2 (QR2), rather than a membrane melatonin receptor. Based on the data available and our preliminary studies, we hypothesize that melatonin is a co-substrate of QR2. We surmise that melatonin binds to a co-substrate binding site (MT3 binding site) donating an electron to the enzyme co-factor, flavin adenine dinucleotide (FAD). FAD can be reduced to either FADH or FADH2 while melatonin is converted to N1-acetyl-N2-formyl-5-methoxykynuramine and/or cyclic 3-hydroxymelatonin. QR2 is considered to be a detoxifying and antioxidant enzyme and its behavior changes depending on available co-substrates. As a naturally occurring substance, melatonin's levels fluctuate with the light/dark cycle, with aging and with health/disease state. As a result, these alterations in melatonin production under physiological or pathological conditions would probably influence the activity of QR2. [Abstract]

Morera AL, Abreu P
Daytime/night-time and summer/winter melatonin and malondialdehyde rhythms: an inverse relationship.
J Pineal Res. 2007 Oct;43(3):313-4. [Abstract]

Subramanian P, Mirunalini S, Dakshayani KB, Pandi-Perumal SR, Trakht I, Cardinali DP
Prevention by melatonin of hepatocarcinogenesis in rats injected with N-nitrosodiethylamine.
J Pineal Res. 2007 Oct;43(3):305-12.
N-nitrosodiethylamine (NDEA) is a potent carcinogenic agent that induces liver cancer. To evaluate the chemopreventive function of melatonin in this experimental model, Wistar male rats received a single i.p. injection of NDEA or vehicle followed by weekly s.c. injections of carbon tetrachloride or vehicle for 6 weeks. Melatonin (5 mg/kg body weight) or its vehicle (0.5 mL saline) was given i.p. on a daily basis 2 hr before lights off for 20 wk. At the end of this period the rats were killed and liver and blood samples were taken for histological and biochemical studies. As markers for liver function, the activity of aspartate transaminase (AST) and alanine transaminase (ALT) and the levels of alpha-fetoprotein were measured in serum. To assess lipid peroxidation and the antioxidant status in liver and blood, the levels of thiobarbituric acid reactive substances (TBARS) and of reduced glutathione (GSH) were measured. The activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) was assessed in liver and erythrocyte fraction of NDEA-treated rats. NDEA administration inhibited body weight, macro- and microscopically detectable liver tumors and increased levels of plasma AST, ALT and alpha-fetoprotein. NDEA treatment decreased liver TBARS levels and CAT and SOD activities and increased liver GSH levels and GST and GPx activities. Plasma TBARS were augmented, while plasma GSH levels and the activities of erythrocyte CAT, SOD, GST and GPx decreased, in NDEA-treated rats. Melatonin administration significantly curtailed tumor development and counteracted all the biochemical effects. [Abstract]

St Hilaire MA, Gronfier C, Zeitzer JM, Klerman EB
A physiologically based mathematical model of melatonin including ocular light suppression and interactions with the circadian pacemaker.
J Pineal Res. 2007 Oct;43(3):294-304.
The rhythm of plasma melatonin concentration is currently the most accurate marker of the endogenous human circadian pacemaker. A number of methods exist to estimate circadian phase and amplitude from the observed melatonin rhythm. However, almost all these methods are limited because they depend on the shape and amplitude of the melatonin pulse, which vary among individuals and can be affected by environmental influences, especially light. Furthermore, these methods are not based on the underlying known physiology of melatonin secretion and clearance, and therefore cannot accurately quantify changes in secretion and clearance observed under different experimental conditions. A published physiologically-based mathematical model of plasma melatonin can estimate synthesis onset and offset of melatonin under dim light conditions. We amended this model to include the known effect of melatonin suppression by ocular light exposure and to include a new compartment to model salivary melatonin concentration, which is widely used in clinical settings to determine circadian phase. This updated model has been incorporated into an existing mathematical model of the human circadian pacemaker and can be used to simulate experimental protocols under a number of conditions. [Abstract]

Stokkan KA, van Oort BE, Tyler NJ, Loudon AS
Adaptations for life in the Arctic: evidence that melatonin rhythms in reindeer are not driven by a circadian oscillator but remain acutely sensitive to environmental photoperiod.
J Pineal Res. 2007 Oct;43(3):289-93.
In reindeer Rangifer tarandus, a high latitude species, the rhythmic production of melatonin periodically dissipates under natural photoperiods when, in mid-winter, there is near permanent darkness and again, in summer, when there is permanent light. In spring and autumn, as expected, melatonin production reflects the ambient light:dark (LD) cycle. We investigated the expression of circadian mechanisms on blood levels of melatonin in reindeer. Two experiments were conducted in which animals were transferred from natural photic conditions into continuous darkness for 3 days: (i) in February, when they had been exposed to an LD cycle (11L:13D) and (ii) in July, when they had been exposed to permanent light. In July, plasma levels of melatonin rose abruptly on exposure to darkness but then declined over 24 hr before displaying a second rise and decline over the following 36 hr. In contrast, in February, levels of melatonin rose abruptly but then remained elevated for more than 60 hr in darkness. Melatonin secretion upon exposure to darkness did not conform to a circadian pattern and did not, therefore, support the hypothesis that pineal activity in reindeer is tightly regulated by circadian mechanisms. Instead the secretion of melatonin appeared to be acutely and directly sensitive to ambient lighting. The results are consistent with a model in which Arctic resident animals have adapted to extreme photic conditions by disconnecting the generation of the pineal melatonin signal from their circadian machinery and relying, instead, on its being driven by the LD cycle for just a few weeks annually in spring and autumn. [Abstract]

Rodriguez-Osorio N, Kim IJ, Wang H, Kaya A, Memili E
Melatonin increases cleavage rate of porcine preimplantation embryos in vitro.
J Pineal Res. 2007 Oct;43(3):283-8.
Melatonin has been used to promote in vitro embryo development in different species. This study determined the effects of melatonin on in vitro porcine embryo development; in particular, cleavage rate, blastocyst rate, and blastocyst cell number. Starting 5 hr after insemination, porcine zygotes were cultured in porcine zygote medium 3 (PZM-3) culture medium supplemented with melatonin at increasing concentrations (10(-12) M, 10(-9) M, 10(-6) M, 10(-3) M). Melatonin at a concentration of 10(-9) M had a positive effect on cleavage rates, while the highest concentration of melatonin (10(-3) M) significantly decreased cleavage rates. Although blastocyst rates were not increased by 10(-9) M melatonin, blastocyst cell numbers were significantly higher for embryos subjected to 10(-9) M melatonin. The expression levels of the pro-apoptotic gene BAX and anti-apoptotic gene BCL2L1 in blastocysts were not affected by the presence of melatonin in the culture medium. To further study the protective properties of 10(-9) M melatonin against stressful conditions, hydrogen peroxide (0.01 mm) and heat (40 degrees C) were used during embryo culture. The addition of melatonin to embryos subjected to 40 degrees C for 3 hr increased cleavage rates, but had no protective effect for embryos subjected to 0.01 mm H(2)O(2), probably because the physiological levels of melatonin could not counteract the pharmacological levels of H(2)O(2). Our data indicate that 10(-9) M melatonin has a positive effect on porcine embryo cleavage rates and blastocyst total cell numbers and it might have a protective effect against heat stress. [Abstract]

de Lima VR, Caro MS, Tavares MI, Creczynski-Pasa TB
Melatonin location in egg phosphatidylcholine liposomes: possible relation to its antioxidant mechanisms.
J Pineal Res. 2007 Oct;43(3):276-82.
Although it is known that the antioxidant properties of melatonin can be modulated by its effect on membrane fluidity, there are few studies on this subject reported in the literature and they are controversial. In this study, viscosimetry and nuclear magnetic resonance (NMR) techniques were used to determine melatonin's effect and location on egg phosphatidylcholine bilayers mobility. Melatonin decreases the dynamic viscosity of the lipid dispersion. (31)P-NMR line width analysis indicated that melatonin induces a slight but uniform restriction of the lipid motional freedom in the polar head. However, melatonin changes in choline (13)C dynamics was only observed through chemical shift analysis. On the other hand, melatonin can induce an increase in the lipid nonpolar chain mobility, as observed by (13)C and (1)H relaxation time analysis. These results suggest the interfacial location of melatonin in the membrane. Additionally, the results of the analysis of the lipid (1)H-fitted exponential relaxation times suggest that melatonin promotes a molecular rearrangement of the bilayers. The melatonin effect and location in the lipid membrane may explain its antioxidant properties against lipid peroxidation induced by reactive species. [Abstract]

Ruiz-Rabelo JF, Vázquez R, Perea MD, Cruz A, González R, Romero A, Muñoz-Villanueva MC, Túnez I, Montilla P, Muntané J, Padillo FJ
Beneficial properties of melatonin in an experimental model of pancreatic cancer.
J Pineal Res. 2007 Oct;43(3):270-5.
Pancreatic cancer is a major health problem because of the aggressiveness of the disease and the lack of effective systemic therapies. Melatonin has antioxidant activity and prevents experimental genotoxicity. However, the effect of melatonin in pancreatic cancer has not been tested. Pancreatic carcinogenesis was induced by N-nitrosobis (2-oxopropyl)amine (BOP) in Syrian hamsters. Melatonin was administered during the BOP-induction phase (12 wk) and/or following the postinduction phase (12 wk). Different parameters of oxidative stress including lipid peroxides (LPO) and antioxidants (superoxide dismutase, catalase, reduced glutathione and glutathione peroxidase) were determined in pancreatic tissue. Also, the presence of atypical hyperplasia (AH), well and moderately differentiated adenomacarcinoma (ADC-WD and ADC-MD, respectively) were studied. The administration of BOP induced an intense oxidative stress and ADC induction in the pancreas. The administration of melatonin during the induction or postinduction phase reduced LPO and improved the antioxidant status, as well as drastically reducing the presence of ADC but some AH remained. In conclusion, treatment with melatonin reduced oxidative damage and cancer nodules induced by BOP in the pancreas. [Abstract]

Peña C, Rincon J, Pedreanez A, Viera N, Mosquera J
Chemotactic effect of melatonin on leukocytes.
J Pineal Res. 2007 Oct;43(3):263-9.
Melatonin seems to be an important stimulatory factor of the immune system. This indolamine is capable of inducing activation of leukocytes. Tissue leukocyte infiltration is a key feature of inflammatory and immune responses; however, there is no information about the effect of melatonin on leukocyte chemotaxis. Therefore, the aim of this study was to examine the in vitro and in vivo effects of melatonin on leukocyte chemotaxis, on modulation of leukocyte chemotaxis to other chemoattractants and on the in vivo induction of leukocyte chemokines. Neutrophils and mononuclear leukocytes (PBMC) were isolated by a discontinuous gradient on Hystopaque. Chemotaxis was performed in blind well Boyden's chambers. In vivo chemotaxis was determined after intraperitoneal injection of melatonin into rats. Leukocyte chemotactic response and leukocyte chemokine expression were determined in human volunteers treated with 20 mg daily of melatonin. Increased neutrophils and PBMC chemotaxis in response to 1.2 nm melatonin was observed in vitro. Peritoneal leukocytes were found increased after melatonin injection. Humans treated with melatonin showed an increased neutrophil chemotactic response to a physiological chemoattractant and increased expression of intracellular chemokines; however, decreased chemotactic response and no chemokine expression were observed in PBMC. These data suggest that melatonin could have a relevant role during the tissue leukocyte infiltration in inflammatory and immune responses. [Abstract]

Kokkola T, Vaittinen M, Laitinen JT
Inverse agonist exposure enhances ligand binding and G protein activation of the human MT1 melatonin receptor, but leads to receptor down-regulation.
J Pineal Res. 2007 Oct;43(3):255-62.
Melatonin binds and activates G protein-coupled melatonin receptors. The density and affinity of the endogenous melatonin receptors change throughout the 24-hr day, and the exposure of recombinant melatonin receptors to melatonin often results in desensitization of the receptors. Receptor density, G protein activation and expression level were analyzed in CHO cell lines stably expressing the human MT1 receptors after 1 or 72 hr of exposure to melatonin (agonist, 10 nm) and luzindole (antagonist/inverse agonist, 10 microm). The 72-hr exposure to luzindole significantly increased the apparent receptor density in cell lines with both high and low MT1 receptor expression levels (MT1(high) and MT1(low) cells, respectively). In the constitutively active MT1(high) cells, luzindole pretreatment also stimulated the functional response to melatonin in [(35)S]GTPgammaS binding assays, whereas melatonin pretreatment attenuated the functional response at both time points. Receptor ELISA was used to analyze the cell membrane and total expression level of the MT1 receptor in intact and permeabilized cells, respectively. Luzindole pretreatment decreased the total cellular level of MT1 receptor in the MT1(high) cells at both time points but increased the cell surface expression of MT1 receptor at 72 hr. Melatonin significantly decreased MT1 receptor cell surface expression only in MT1(high) cells after a 1-hr treatment. These results indicate that melatonin treatment desensitizes MT1 receptors, whereas luzindole increases ligand binding and G-protein activation. Luzindole also stimulates downregulation of the MT1 receptor protein, interfering with the synthesis and/or degradation of the receptor. [Abstract]

Sharma R, McMillan CR, Niles LP
Neural stem cell transplantation and melatonin treatment in a 6-hydroxydopamine model of Parkinson's disease.
J Pineal Res. 2007 Oct;43(3):245-54.
Melatonin has multiple roles including neuroprotection. Melatonin signaling involves diverse targets including two G-protein-coupled receptors, MT(1) and MT(2), which have both been localized to the nigrostriatal pathway. Previous studies in our laboratory demonstrated preservation of tyrosine hydroxylase immunoreactivity, following chronic treatment with a physiological dose of melatonin, in the 6-hydroxydopamine rat model of Parkinson's disease. Additionally, we reported the presence of the melatonin MT(1) receptor subtype in cultured C17.2 neural stem cells (NSCs). In the present study, we examined the effects of C17.2 NSC transplantation on dopaminergic denervation following 6-hydroxydopamine lesioning in the rat striatum. Moreover, based on our detection of the MT(1) in these cells, we examined the effects of combined C17.2 NSC transplantation and melatonin treatment, following striatal lesioning. Behavioral studies indicated a marked inhibition of apomorphine-induced rotations in lesioned animals that received C17.2 NSC transplantation, melatonin, or the combined regimen. In addition, these treatments resulted in a significant protection of tyrosine hydroxylase immunoreactivity in the striatum and substantia nigra of lesioned animals, when compared with untreated controls. Lesioned animals treated with C17.2 NSCs, melatonin or a combination of both agents exhibited no significant differences in the number of tyrosine hydroxylase-positive cells in the substantia nigra or ventral tegmental area ipsilateral or contralateral to the lesioned striatum. These findings suggest that stem cell therapy and concomitant use of neuroprotective agents such as melatonin could be a viable approach in Parkinson's disease. [Abstract]

Martín V, Herrera F, García-Santos G, Antolín I, Rodriguez-Blanco J, Medina M, Rodriguez C
Involvement of protein kinase C in melatonin's oncostatic effect in C6 glioma cells.
J Pineal Res. 2007 Oct;43(3):239-44.
Classical anticancer therapies often are ineffective in patients with malignant glioma who have a survival of <1 year. Our previous studies showed a potent inhibitory effect of melatonin on glioma cell proliferation. This effect seems to be mediated by the well-known antioxidant properties of this molecule and the negative regulation of some intracellular effectors, such as the kinase Akt or the transcription factor nuclear factor (NF)-kappaB. Finally, protein kinase C (PKC) also seems to be implicated in this effect although the intracellular pathways involved have not been elucidated. In this study, we analyzed the role of PKC in the regulation by melatonin of intracellular effectors leading to inhibition of cell proliferation. Activation of PKC by incubation with triphorbol ester acetate (TPA) blocks the inhibitory effect of melatonin on Akt and NF-kappaB activity. Moreover, incubation with melatonin induces a decrease in p21 expression in these cells that is partially blocked by co-incubation with TPA. Taken together, these results suggest that melatonin's oncostatic effect on glioma cells is mediated, at least in part, by the inhibition of PKC activity which, in turn, results in Akt and NF-kappaB activity inhibition and modulation of cell cycle-related gene expression. [Abstract]

Liu YJ, Zhuang J, Zhu HY, Shen YX, Tan ZL, Zhou JN
Cultured rat cortical astrocytes synthesize melatonin: absence of a diurnal rhythm.
J Pineal Res. 2007 Oct;43(3):232-8.
Melatonin not only plays a major role in the regulation of circadian rhythms, but is also involved in antioxidative defense and immunomodulation. Circulating melatonin levels are derived primarily from the pineal gland while other sources of melatonin have also been reported. Here, we show for the first time that astrocytes from the rat cortex and glioma C6 cell line synthesize melatonin in vitro. In addition, we show the presence of serotonin, the precursor of melatonin and the two key enzymes in the pathway of melatonin synthesis, i.e. N-acetyltransferase and hydroxyndole-O-methyltransferase in the cultured rat cortical astrocytes. Release of melatonin into the culture medium showed no diurnal changes. These point to astrocytes as a local source of melatonin in the rat brain. Its exact physiological function remains a topic for future studies. [Abstract]

Baydas G, Koz ST, Tuzcu M, Etem E, Nedzvetsky VS
Melatonin inhibits oxidative stress and apoptosis in fetal brains of hyperhomocysteinemic rat dams.
J Pineal Res. 2007 Oct;43(3):225-31.
Moderate hyperhomocysteinemia is a risk factor for neurodegenerative diseases and complications during pregnancy. Increased homocysteine levels during pregnancy may elevate developmental risk on fetal brain structure and function. However, little is known about the mechanism of action of homocysteine on the degeneration of the fetal brain. Hence in this study, we examined the effects of maternal hyperhomocysteinemia on oxidative stress and apoptosis in brain tissues and investigated whether administration of melatonin to the mother would prevent homocysteine-induced oxidative cerebral damage in pups. Hyperhomocysteinemia was induced in female rats by administration of methionine at a dose of 1 g/kg body weight dissolved in drinking water during pregnancy. Some animals received methionine plus 10 mg/kg/day melatonin subcutaneously throughout pregnancy. After delivery, the level of lipid peroxidation (malondialdehyde + 4-hydroxyalkenals) was determined in different subfractions of pup brains. Furthermore, DNA fragmentation, levels of Bcl-2 protein and p53 mRNA expression were determined to evaluate apoptosis. Significant elevation was found in the levels of lipid peroxidation in subcellular fractions of the brain of pups of hyperhomocysteinemic dams. Increased DNA fragmentation and p53 mRNA expression was observed in the brain of pups of homocysteine-treated rats, while a significant reduction was seen in the levels of anti-apoptotic Bcl-2 levels. Melatonin administration prevented markers of oxidative stress and biochemical signs of apoptosis. In conclusion, therapeutic administration of melatonin protects against the induction of oxidative stress and neural tissue injury and might prevent congenital malformations of fetal brain caused by maternal hyperhomocysteinemia. [Abstract]

Navara KJ, Nelson RJ
The dark side of light at night: physiological, epidemiological, and ecological consequences.
J Pineal Res. 2007 Oct;43(3):215-24.
Organisms must adapt to the temporal characteristics of their surroundings to successfully survive and reproduce. Variation in the daily light cycle, for example, acts through endocrine and neurobiological mechanisms to control several downstream physiological and behavioral processes. Interruptions in normal circadian light cycles and the resulting disruption of normal melatonin rhythms cause widespread disruptive effects involving multiple body systems, the results of which can have serious medical consequences for individuals, as well as large-scale ecological implications for populations. With the invention of electrical lights about a century ago, the temporal organization of the environment has been drastically altered for many species, including humans. In addition to the incidental exposure to light at night through light pollution, humans also engage in increasing amounts of shift-work, resulting in repeated and often long-term circadian disruption. The increasing prevalence of exposure to light at night has significant social, ecological, behavioral, and health consequences that are only now becoming apparent. This review addresses the complicated web of potential behavioral and physiological consequences resulting from exposure to light at night, as well as the large-scale medical and ecological implications that may result. [Abstract]

Todisco M
Melatonin makes splenectomy unnecessary in two patients with idiopathic thrombocytopenic purpura refractory to corticosteroids.
J Pineal Res. 2007 Sep;43(2):214. [Abstract]

Toma CD, Svoboda M, Arrich F, Ekmekcioglu C, Assadian O, Thalhammer T
Expression of the melatonin receptor (MT) 1 in benign and malignant human bone tumors.
J Pineal Res. 2007 Sep;43(2):206-13.
The beneficial effects of melatonin on bone homeostasis have been shown in various diseases. As this indoleamine causes dose-dependent modulation of bone-forming osteoblast and bone-resorbing osteoclast activities by receptor-independent and -dependent pathways, we investigated the expression of G-protein-coupled melatonin receptors (MTs) in malignant and non-malignant human bone lesions. By TaqMan polymerase chain reaction (PCR), we analyzed 30 specimens from osteosarcoma and 11 from benign bone tumors for MT1-mRNA expression. Furthermore, we determined mRNA expression levels of the osteoclast activity-stimulating receptor activator of nuclear factor-kappa B ligand (RANKL) and its counterpart osteoprotegerin (OPG). Although mean MT1-mRNA levels were similar (P = 0.596) in malignant (4.39 +/- 4.98-fold) and benign samples (4.64 +/- 6.81-fold), the highest MT1-mRNA levels (up to 27-fold) were observed in individual osteosarcomas, particularly, in two specimens of patients with local recurrence of the tumor. Moreover, mean RANKL- and OPG-mRNA levels were similar in malignant and benign specimens (RANKL: 7.38 +/- 9.61-fold versus 3.57 +/- 3.11-fold, P = 0.207; OPG: 23.45 +/- 32.76 versus 8.07 +/- 7.23-fold, P = 0.133). Again, highest RANKL- and OPG-mRNA levels (up to 41- and 160-fold, respectively) were observed in individual osteosarcomas. Expression of MT1-mRNA was confirmed in two human osteosarcoma cell lines (HOS, MG63). High expression levels of MT1-mRNA together with low OPG-mRNA were found in both osteosarcoma cell lines, while in normal human osteoblasts and bone marrow stromal cells, high OPG-mRNA levels were associated with low MT1-mRNA levels. These data on the abundant expression of MT1-mRNA in human bone tumors and osteosarcoma cells lines suggest an important role for MT1 in bone pathology. [Abstract]

García-Navarro A, González-Puga C, Escames G, López LC, López A, López-Cantarero M, Camacho E, Espinosa A, Gallo MA, Acuña-Castroviejo D
Cellular mechanisms involved in the melatonin inhibition of HT-29 human colon cancer cell proliferation in culture.
J Pineal Res. 2007 Sep;43(2):195-205.
The antiproliferative and proapoptotic properties of melatonin in human colon cancer cells in culture were recently reported. To address the mechanisms involved in these actions, HT-29 human colon cancer cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum at 37 degrees C. Cell proliferation was assessed by the incorporation of [(3)H]-thymidine into DNA. Cyclic nucleotide levels, nitrite concentration, glutathione peroxidase and reductase activities, and glutathione levels were assessed after the incubation of these cells with the following drugs: melatonin membrane receptor agonists 2-iodo-melatonin, 2-iodo-N-butanoyl-5-methoxytryptamine, 5-methoxycarbonylamino-N-acetyltryptamine (GR-135,531), and the antagonists luzindole, 4-phenyl-2-propionamidotetralin, and prazosin; the melatonin nuclear receptor agonist CGP 52608, and four synthetic kynurenines analogs to melatonin 2-acetamide-4-(3-methoxyphenyl)-4-oxobutyric acid, 2-acetamide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid, 2-butyramide-4-(3-methoxyphenyl)-4-oxobutyric acid and 2-butyramide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid. The results show that the membrane receptors are not necessary for the antiproliferative effect of melatonin and the participation of the nuclear receptor in this effect is suggested. Moreover, the antioxidative and anti-inflammatory actions of melatonin, counteracting the oxidative status and reducing the production of nitric oxide by cultured HT-29 cells seem to be directly involved in the oncostatic properties of melatonin. Some of the synthetic kynurenines exert higher antiproliferative effects than melatonin. The results reinforce the clinical interest of melatonin due to the different mechanisms involved in its oncostatic role, and suggest a new synthetic pathway to obtain melatonin agonists with clinical applications to oncology. [Abstract]

Ackermann K, Dehghani F, Bux R, Kauert G, Stehle JH
Day-night expression patterns of clock genes in the human pineal gland.
J Pineal Res. 2007 Sep;43(2):185-94.
Rhythm generation within the mammalian circadian system is achieved by clock genes and their protein products. As an integral part of this system, the pineal gland serves the need to tune the body to the temporal environment by the rhythmic synthesis and release of melatonin. A number of human disorders and syndromes are associated with alterations in circadian rhythms of clock genes and their protein products and/or a dysfunction in melatonin synthesis. In the human, little is known about the molecular signature of time management. Pineal tissue from regular autopsies was allocated to asserted time-of-death groups (dawn, day, dusk, night), and analyzed by RT-PCR, immunoblotting, immunohistochemistry, and confocal laser scanning microscopy for expression of clock genes. Despite the observed diurnal rhythms in activity of the arylalkylamine N-acetyltransferase and in melatonin content, mRNA levels for the clock genes Period1, Cryptochrome1, Clock, and Bmal1, and also amounts of corresponding clock gene proteins showed no differences between time- of-death groups. In contrast, a time-of-day-dependent nucleocytoplasmic shuttling of clock gene proteins was detected. These data confirm the minor importance of a transcriptional regulation for dynamics in the human pineal gland, and offer a novel twist in the molecular competence of clock gene proteins. [Abstract]

Gögenur I, Middleton B, Burgdorf S, Rasmussen LS, Skene DJ, Rosenberg J
Impact of sleep and circadian disturbances in urinary 6-sulphatoxymelatonin levels, on cognitive function after major surgery.
J Pineal Res. 2007 Sep;43(2):179-84.
Sleep and circadian disturbances may underlie cognitive dysfunction after major surgery. The aim of this study was to examine the association between sleep and circadian disturbances (as assessed by changes in the melatonin rhythm) and postoperative cognitive dysfunction (POCD). We measured subjective and objective sleep quality, excretion of the major metabolite of melatonin, 6-sulphatoxymelatonin (aMT6s) in urine and cognitive function before and 4 days after major abdominal surgery in 36 patients. Subjective sleep quality was measured by visual analogue scale, objective sleep quality was measured by actigraphy, and cognitive function was assessed by neuropsychological testing. Eighteen patients (50%) had POCD on day 4 after surgery. At that time, the excretion of aMT6s was disturbed with significantly higher daytime excretion and a reduced night/day ratio compared with the preoperative measure (P = 0.05). Patients with POCD had significantly worse sleep quality and more night awakenings (P < 0.05) but we found no significant differences in day time (06:00-22:00 hr), night-time (22:00-06:00 hr) or total aMT6s excretion (mug/24 hr). A significant correlation was found between the total excretion of aMT6s and actigraphically measured sleep efficiency (r(s) = 0.45, P = 0.03) and wakefulness after sleep onset (r(s) = -0.44, P = 0.04). In conclusion, POCD was associated with worse subjective sleep quality and more awakenings. Circadian rhythmicity as assessed by aMT6s excretion was disturbed after surgery but we were unable to show an association with POCD. Strategies to improve postoperative sleep quality should be investigated in the future. [Abstract]

Kurcer Z, Oguz E, Ozbilge H, Baba F, Aksoy N, Celik H, Cakir H, Gezen MR
Melatonin protects from ischemia/reperfusion-induced renal injury in rats: this effect is not mediated by proinflammatory cytokines.
J Pineal Res. 2007 Sep;43(2):172-8.
The pathophysiologic mechanisms leading to acute ischemic renal failure are not completely understood. Melatonin, a compound with well-known antioxidant properties, reduces IR-induced renal injury. The purpose of the present study was to investigate the changes in levels of tumor necrosis factor (TNF)-alpha, IL-beta, and IL-6 in postischemic reperfused renal tissue, and to determine whether the protective effect of melatonin is related the modulation of the production of these inflammatory molecules. Male Wistar albino rats were unilaterally nephrectomized and subjected to 1 hr of renal pedicle occlusion followed by 2 hr or 24 hr of reperfusion. Melatonin (10 mg/kg, i.p.) or vehicle was administrated at 10 min prior to ischemia. After 24 hr of the reperfusion, following decapitation, kidney samples were taken both for histologic examination and for the determination of malondialdehyde (MDA), myeloperoxidase (MPO) activity, total antioxidant capacity (TAC), total oxidative stress (TOS), creatinine, and blood urea nitrogen (BUN). These were measured in serum samples. TNF-alpha, IL-beta, and IL-6 were measured in kidney samples after 2 hr of reperfusion. IR caused a significant increase in renal MDA, MPO, TOS, creatinine, and BUN while decrease TAC without any change in TNF-alpha, IL-beta, and IL-6 levels. Melatonin treatment reduced the biochemical indices without any change in the cytokine levels and ameliorated histopathologic alterations induced by IR. The protective effect of melatonin on IR-induced renal injury is related to its antioxidant properties but not to proinflammatory cytokines. [Abstract]

Lin AM, Fang SF, Chao PL, Yang CH
Melatonin attenuates arsenite-induced apoptosis in rat brain: involvement of mitochondrial and endoplasmic reticulum pathways and aggregation of alpha-synuclein.
J Pineal Res. 2007 Sep;43(2):163-71.
In the present study, the protective effect of melatonin on sodium arsenite (arsenite)-induced apoptosis was investigated. Local infusion of arsenite elevated lipid peroxidation and depleted glutathione content in the infused substantia nigra (SN), as well as reduced striatal dopamine content. Systemic administration of melatonin diminished arsenite-induced oxidative injury. Furthermore, melatonin attenuated arsenite-induced increases in heat shock protein 70 and heme oxygenase-1 as well as phosphorylation of p38 mitogen-activated protein kinase and elevations in cyclooxygenase II and inducible nitric oxide synthase expression. Inhibition by melatonin of arsenite-induced apoptosis was determined by its attenuation of DNA fragmentation and terminal deoxytransferase-mediated dUTP-nick end labeling's positive cells in the infused SN of melatonin-treated rats. Melatonin reduced arsenite-induced apoptosis through mitochondrial and endoplasmic reticulum (ER) pathways. In the mitochondrial pathway, systemic melatonin inhibited arsenite-induced elevations in Bcl-2 and cytosolic cytochrome c as well as arsenite-induced reductions in procaspase-3 levels and elevations in active caspase-3 levels in the infused SN. Regarding the ER pathway, melatonin attenuated arsenite-induced elevations in activating transcription factor-4, CCAAT/enhancer binding protein (C/EBP) homologues protein, X-bon binding protein (XBP-1) and cytosolic immunoglobulin binding protein (BIP) as well as reductions in procaspase 12 levels. Moreover, aggregation of alpha-synuclein was reduced in the arsenite-infused SN of melatonin-treated rats. Our in vitro data showed that melatonin ameliorated arsenite-induced lipid peroxidation. Taken together, our data suggest that melatonin is neuroprotective against arsenite-induced oxidative injury in the nigrostriatal dopaminergic system of rat brain. Furthermore, the neuroprotective effects by melatonin on arsenite-induced apoptosis were mediated via inhibiting both mitochondrial and ER pathways. Accordingly, melatonin may be therapeutically useful for the treatment of arsenite-induced apoptosis in central nervous system. [Abstract]

Radogna F, Paternoster L, Albertini MC, Cerella C, Accorsi A, Bucchini A, Spadoni G, Diamantini G, Tarzia G, De Nicola M, D'Alessio M, Ghibelli L
Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells.
J Pineal Res. 2007 Sep;43(2):154-62.
Among the non-neurological functions of melatonin, much attention is being directed to the ability of melatonin to modulate the immune system, whose cells possess melatonin-specific receptors and biosynthetic enzymes. Melatonin controls cell behaviour by eliciting specific signal transduction actions after its interaction with plasma membrane receptors (MT(1), MT(2)); additionally, melatonin potently neutralizes free radicals. Melatonin regulates immune cell loss by antagonizing apoptosis. A major unsolved question is whether this is due to receptor involvement, or to radical scavenging considering that apoptosis is often dependent on oxidative alterations. Here, we provide evidence that on U937 monocytic cells, apoptosis is antagonized by melatonin by receptor interaction rather than by radical scavenging. First, melatonin and a set of synthetic analogues prevented apoptosis in a manner that is proportional to their affinity for plasma membrane receptors but not to their antioxidant ability. Secondly, melatonin's antiapoptotic effect required key signal transduction events including G protein, phospholipase C and Ca(2+) influx and, more important, it is sensitive to the specific melatonin receptor antagonist luzindole. [Abstract]

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Recent Articles in Developmental Dynamics: An Official Publication of the American Association of Anatomists

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Warkman AS, Yatskievych TA, Hardy KM, Krieg PA, Antin PB
Myocardin expression during avian embryonic heart development requires the endoderm but is independent of BMP signaling.
Dev Dyn. 2007 Dec 10;
Myocardin, a serum response factor cofactor, plays an important role in regulating heart and smooth muscle development. To investigate myocardin function during early stages of heart development, we isolated the chicken orthologue of myocardin and characterized its expression between Hamburger and Hamilton stages 3 and 15. At stage 4, myocardin transcripts are detected in the lateral and extraembryonic mesoderm, become progressively localized to the precardiac mesoderm and the differentiated myocardium and are also seen in smooth muscle cells of the developing vascular plexus. Surprisingly, myocardin expression within the developing chicken embryo precedes that of the homeodomain transcription factor Nkx2.5. Embryonic dissection studies demonstrate that signals from the endoderm are required for myocardin expression within the precardiac mesoderm. However, unlike Nkx2.5, myocardin expression is not regulated by bone morphogenetic protein (BMP) signaling. These results suggest that initial expression of myocardin in the precardiac mesoderm is regulated by a signaling pathway that is parallel to, and independent of, Nkx2.5 expression. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Singh S, Robinson M, Ismail I, Saha M, Auer H, Kornacker K, Robinson ML, Bates CM, McHugh KM
Transcriptional profiling of the megabladder mouse: A unique model of bladder dysmorphogenesis.
Dev Dyn. 2007 Dec 10;
Recent studies in our lab identified a mutant mouse model of obstructive nephropathy designated mgb for megabladder. Homozygotic mgb mice (mgb-/-) develop lower urinary tract obstruction in utero due to a lack of bladder smooth muscle differentiation. This defect is the result of a random transgene insertion/translocation into chromosomes 11 and 16. Transcriptional profiling identified a significantly over-expressed cluster of gene products located on the translocated fragment of chromosome 16 including urotensin II-related peptide (Urp), which was shown to be preferentially over-expressed in developing mgb-/- bladders. Pathway analysis of mgb microarray data indicated dysregulation of at least 60 gene products associated with smooth muscle development. In conclusion, the results of this study indicate that the molecular pathways controlling normal smooth muscle development are severely altered in mgb-/- bladders, and provide the first evidence that Urp may play a critical role in bladder smooth muscle development. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Cann GM, Guignabert C, Ying L, Deshpande N, Bekker JM, Wang L, Zhou B, Rabinovitch M
Developmental expression of LC3alpha and beta: Absence of fibronectin or autophagy phenotype in LC3beta knockout mice.
Dev Dyn. 2007 Dec 10;
Murine light chain 3 (LC3) exists as two isoforms, LC3alpha and beta: LC3beta is an RNA-binding protein that enhances fibronectin (FN) mRNA translation, and is also a marker of autophagy. We report embryonic expression patterns for LC3alpha and LC3beta, with some overlap but notable differences in the brain, and in tissues of non-neuronal origin. LC3beta knockout (-/-) mice develop normally without a compensatory increase in LC3alpha. LC3beta-/- embryonic fibroblasts (MEFs) exhibit reduced FN synthesis but maintain wild type (WT) levels of FN protein. No significant changes in proteins associated with FN turnover, i.e., caveolin-1, LRP-1, or matrix metalloproteinases were identified. Autophagosomes form in amino acid-starved LC3beta-/-MEFs, and Caesarean-delivered pups survive as long as WT pups without an increase in LC3-related proteins linked to autophagy. These results suggest novel compensatory mechanisms for loss of LC3beta, ensuring proper FN accumulation and autophagy during fetal and neonatal life. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Hakmé A, Huber A, Dollé P, Schreiber V
The macroPARP genes parp-9 and parp-14 are developmentally and differentially regulated in mouse tissues.
Dev Dyn. 2007 Dec 10;
The macroPARPs Parp-9 and Parp-14 are macro domain containing poly(ADP-ribose) polymerases involved in transcriptional regulation in response to immunoregulatory cytokines. Their genes reside in the same locus (16B3), and the Parp-9 gene lies head-to-head and shares its promoter with the gene encoding its partner, Bbap. Here, we provide a detailed analysis of Parp-9, Parp-14, and Bbap expression during mouse development and adulthood. Parp-9 is developmentally regulated, and prominently expressed in the thymus and specific regions of the brain and gut. In adults, highest expression is maintained in the thymus and intestine. Parp-14 is more weakly expressed, mainly in the thymus during development and in adulthood. In addition, we show that Bbap is essentially coexpressed with Parp-9 during development and in adult mouse. However, the different levels of their transcripts detected in the developing brain and gut suggest that Bbap and Parp-9 display both common and independent tissue-specific regulations. Developmental Dynamics 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Wagstaff LJ, Bellett G, Mogensen MM, Münsterberg A
Multicellular rosette formation during cell ingression in the avian primitive streak.
Dev Dyn. 2007 Dec 10;
Cell movements are a fundamental feature during the development of multi-cellular organisms. In amniote gastrulation, cells ingress through the primitive streak, which identifies the anterior-posterior axis of the embryo. We investigated the cytoskeletal architecture during these morphogenetic processes and characterized microtubule organisation in whole chick embryos. This revealed the distribution of cells with polarized and radial microtubule (MT) arrays across different regions of the embryo. Cells in the epiblast usually displayed radial MT-arrays, while the majority of cells in the primitive streak had polarized MT-arrays. Within the primitive streak, many cells organized into groups and were arranged in rosette-like structures with a distinct centre characterized by an accumulation of actin. Extended confocal microscopy and three-dimensional image reconstruction identified tips of polarized cells that were protruding from the plane of rosettes, usually from the centre. We propose that organization into higher order structures facilitates cell ingression during gastrulation. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Itman C, Loveland KL
SMAD expression in the testis: An insight into BMP regulation of spermatogenesis.
Dev Dyn. 2007 Dec 10;
Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta superfamily, extensively influence events that establish male fertility, affecting germ cells and somatic cells throughout fetal and postnatal life. BMP signals are relayed by SMAD proteins, transcription factors that translocate to the nucleus upon ligand stimulation. We show that BMP signaling in the testis may be regulated by selective expression of BMP-responsive and inhibitory SMADs, with expression differing between the first wave and adult spermatogenesis. Smad1, Smad5, Smad8, Smad4, Smad6, and Smad7 expression is ubiquitous during testis development but becomes cell-specific in the adult. Furthermore, regulated SMAD6 protein expression at the onset of spermatogenesis suggests differential responsiveness of spermatogonial subpopulations to ligands. In vitro, immature Sertoli cells and spermatogonia transduce BMP2 and BMP4 signals by means of SMAD1, SMAD5, and SMAD8. Based on these findings, we extrapolate these data to interpret BMP mutant testis phenotypes in terms of SMAD availability for signal transduction. Developmental Dynamics 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Bracken CM, Mizeracka K, McLaughlin KA
Patterning the embryonic kidney: BMP signaling mediates the differentiation of the pronephric tubules and duct in Xenopus laevis.
Dev Dyn. 2007 Dec 10;
The Bone morphogenetic proteins (BMPs) mediate a wide range of diverse cellular behaviors throughout development. Previous studies implicated an important role for BMP signaling during the differentiation of the definitive mammalian kidney, the metanephros. In order to examine whether BMP signaling also plays an important role during the patterning of earlier renal systems, we examined the development of the earliest nephric system, the pronephros. Using the amphibian model system Xenopus laevis, in combination with reagents designed to inhibit BMP signaling during specific stages of nephric development, we revealed an evolutionarily conserved role for this signaling pathway during renal morphogenesis. Our results demonstrate that conditional BMP inhibition after specification of the pronephric anlagen is completed, but prior to the onset of morphogenesis and differentiation of renal tissues, results in the severe malformation of both the pronephric duct and tubules. Importantly, the effects of BMP signaling on the developing nephron during this developmental window are specific, only affecting the developing duct and tubules, but not the glomus. These data, combined with previous studies examining metanephric development in mice, provide further support that BMP functions to mediate morphogenesis of the specified renal field during vertebrate embryogenesis. Specifically, BMP signaling is required for the differentiation of two types of nephric structures, the pronephric tubules and duct. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

O'Farrell F, Esfahani SS, Engström Y, Kylsten P
Regulation of the Drosophila lin-41 homologue dappled by let-7 reveals conservation of a regulatory mechanism within the LIN-41 subclade.
Dev Dyn. 2007 Dec 10;
Drosophila Dappled (DPLD) is a member of the RBCC/TRIM superfamily, a protein family involved in numerous diverse processes such as developmental timing and asymmetric cell divisions. DPLD belongs to the LIN-41 subclade, several members of which are micro RNA (miRNA) regulated. We re-examined the LIN-41 subclade members and their relation to other RBCC/TRIMs and dpld paralogs, and identified a new Drosophila muscle specific RBCC/TRIM: Another B-Box Affiliate, ABBA. In silico predictions of candidate miRNA regulators of dpld identified let-7 as the strongest candidate. Overexpression of dpld led to abnormal eye development, indicating that strict regulation of dpld mRNA levels is crucial for normal eye development. This phenotype was sensitive to let-7 dosage, suggesting let-7 regulation of dpld in the eye disc. A cell-based assay verified let-7 miRNA down-regulation of dpld expression by means of its 3'-untranslated region. Thus, dpld seems also to be miRNA regulated, suggesting that miRNAs represent an ancient mechanism of LIN-41 regulation. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Minami S, Iwamoto R, Mekada E
HB-EGF decelerates cell proliferation synergistically with TGFalpha in perinatal distal lung development.
Dev Dyn. 2007 Dec 10;
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors that is suggested to be involved in distal lung development. In HB-EGF null (HB(del/del)) newborns, a histopathologic analysis revealed abnormally thick saccular walls occurring from embryonic day 18.5 that reduced the terminal saccular space area. HB-EGF gene deletion resulted in a significant increase in cell proliferation, indicating that HB-EGF suppresses distal lung cell proliferation. Furthermore, an analysis of saccular morphology and proliferation in HB-EGF and transforming growth factor-alpha (TGFalpha) double-mutant newborns revealed that HB-EGF and TGFalpha function synergistically in this suppression. Finally, crosses between HB(del/del) mice and waved 2 mice, a hypomorphic EGF receptor (EGFR) mutant strain, suggest that HB-EGF and EGFR cooperate in this process. Thus, HB-EGF has a novel suppressive function that contributes to decelerating distal lung cell proliferation synergistically with TGFalpha through EGFR in perinatal distal lung development. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Rodgers LS, Lalani S, Runyan RB, Camenisch TD
Differential growth and multicellular villi direct proepicardial translocation to the developing mouse heart.
Dev Dyn. 2007 Dec 3;
In the mammalian system the proepicardium (PE) arises from mesothelium of the septum transversum before translocation to the heart where it forms the epicardium and progenitor cells of the coronary vessels. Despite its importance, the process in which PE cells translocate to the myocardium in mammals is not well defined. The current paradigm states that cellular cysts of PE float across the pericardial space and contact the outer surface of the myocardium. This mechanism does not provide a satisfactory explanation for the directionality or localization of PE migration. To better define PE migration, we performed a detailed study of mouse PE development. We provide thorough documentation that redefines the size of the PE migratory field and the mechanism of migration. Our new model incorporates differential growth and direct contact between multicellular PE villi and the myocardium as mechanisms in formation of the epicardium. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Urness LD, Li C, Wang X, Mansour SL
Expression of ERK signaling inhibitors Dusp6, Dusp7, and Dusp9 during mouse ear development.
Dev Dyn. 2007 Dec 3;
The levels of fibroblast growth factor (FGF) signaling play important roles in coordinating development of the mouse inner, middle, and outer ears. Extracellular signal-regulated kinases (ERKs) are among the effectors that transduce the FGF signal to the nucleus and other cellular compartments. Attenuation of ERK activity by dephosphorylation is necessary to modulate the magnitude and duration of the FGF signal. Recently, we showed that inactivation of the ERK phosphatase, dual specificity phosphatase 6 (DUSP6), causes partially penetrant postnatal lethality, hearing loss and skeletal malformations. To determine whether other Dusps may function redundantly with Dusp6 during otic development, we surveyed the expression domains of the three ERK-specific DUSP transcripts, Dusp6, Dusp7, and Dusp9, in the embryonic mouse ear. We show that each is expressed in partially overlapping patterns that correspond to regions of active FGF signaling, suggesting combinatorial roles in negative regulation of this pathway during ear development. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Porras D, Brown CB
Temporal-spatial ablation of neural crest in the mouse results in cardiovascular defects.
Dev Dyn. 2007 Dec 3;
Neural crest cells are thought to play a critical role in human conotruncal morphogenesis and dysmorphogenesis. Much of our understanding of the contribution of neural crest to cardiovascular patterning comes from ablation and transplantation experiments in avian species. Although fate mapping experiments in mice suggests a conservation of function, the functional requirement for neural crest in cardiovascular development in mammals has not been formally tested. We used a novel two component genetic system for the temporal-spatial ablation of neural crest in the mouse. Affected embryos displayed a spectrum of cardiovascular outflow tract defects and aortic arch patterning abnormalities. We show that the severity of the cardiovascular phenotype is directly related to the level and extent of neural crest ablation. This is the first report of cardiac neural crest ablation in mammals, and it provides important insight into the role of the mammalian neural crest during cardiovascular development. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Liedtke D, Winkler C
Midkine-b regulates cell specification at the neural plate border in zebrafish.
Dev Dyn. 2007 Dec 3;
Midkines compose a family of secreted, heparin-binding growth factors with neurotrophic activity in vitro, but largely unknown functions in mammals in vivo. Here we show that one member of this family, Midkine-b (Mdkb), is responsible for establishment of the neural plate border in zebrafish. We propose that MdkB acts downstream of several signaling factors, most notably retinoic acid, implicated in neural crest cell (ncc) induction and refines a zone of competence for ncc and Rohon-Beard (RB) sensory neuron formation. Overexpression of Mdkb expands the cell numbers of various ncc precursor subtypes and results in a significant increase in the number of RB neurons at the neural plate border. On the other hand, Morpholino-mediated knockdown of Mdkb leads to a dramatic reduction of ncc and a loss of sensory neurons. Our results imply that Mdkb is required for the earliest steps of cell specification at the neural plate border in zebrafish. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Boughner JC, Hallgrímsson B
Biological spacetime and the temporal integration of functional modules: A case study of dento-gnathic developmental timing.
Dev Dyn. 2007 Dec 3;
For the individual, coordination between tooth and jaw development is important to proper food acquisition and ingestion later in life. Among and within species, variation in dental and gnathic size, shape, and, in the case of teeth, number, must be mutually accommodating and functionally compatible. For these reasons, the development and evolution of these two systems should be closely integrated. Furthermore, the timing of dental development correlates tightly with life history events such as weaning. This correlation hints at a central regulation of the developmental timing of multiple systems that have tandem effects on physiology and behaviour. Important work on embryonic oral development continues to tease apart the molecular mechanisms that pattern jaw identity and establish tooth morphology and position in the alveolar bone. Still very poorly understood is what underlies rates and periods of gene activity such that pre- and postnatal tooth and jaw development are coordinated. Recent literature suggests at least some level of autonomy between permanent tooth and mandibular ontogenetic timing. However, whether the timing of these various signaling pathways is directly regulated or is an outcome of the pathways themselves is untested. Here, we review what is currently known about the embryonic signaling pathways that regulate tooth and jaw development in the context of time rather than space, as has been traditional. We hypothesize that the timing of mandibular and dental development is not directly mediated by a common factor but is an indirect outcome of strong selection for coordinated molecular pathways and growth trajectories. The mandible and lower jaw dentition is a powerful model with which to investigate the mechanisms that facilitate morphological change-in this case, the development and evolution-of organs that are closely integrated in terms of function, space and time. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Bit-Avragim N, Rohr S, Rudolph F, Van Der Ven P, Fürst D, Eichhorst J, Wiesner B, Abdelilah-Seyfried S
Nuclear localization of the zebrafish tight junction protein nagie oko.
Dev Dyn. 2007 Dec 3;
The tight junctions-associated MAGUK protein nagie oko is closely related to Drosophila Stardust, mouse protein associated with lin-seven 1 (Pals1), and human MAGUK p55 subfamily member 5 (Mpp5). As a component of the evolutionarily conserved Crumbs protein complex, nagie oko is essential for the maintenance of epithelial cell polarity. Here, we show that nagie oko contains a predicted nuclear export and two conserved nuclear localization signals. We find that loss of the predicted nuclear export signal results in nuclear protein accumulation. We show that nagie oko nuclear import is redundantly controlled by the two nuclear localization signals and the evolutionarily conserved region 1 (ECR1), which links nagie oko with Par6-aPKC. Finally, deletion forms of nagie oko that lack nuclear import and export signals complement several nagie oko mutant defects in cell polarity and epithelial integrity. This finding provides an entry point to potentially novel and unknown roles of this important cell polarity regulator. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Itoh N, Ornitz DM
Functional evolutionary history of the mouse Fgf gene family.
Dev Dyn. 2007 Dec 3;
Fibroblast Growth Factors (FGFs) are polypeptides with diverse activities in development and physiology. The mammalian Fgf family can be divided into the intracellular Fgf11/12/13/14 subfamily (iFGFs), the hormone-like Fgf15/21/23 subfamily (hFGFs), and the canonical Fgf subfamilies, including Fgf1/2/5, Fgf3/4/6, Fgf7/10/22, Fgf8/17/18, and Fgf9/16/20. However, all Fgfs are evolutionarily related. We propose that an Fgf13-like gene is the ancestor of the iFgf subfamily and the most likely evolutionary ancestor of the entire Fgf family. Potential ancestors of the canonical and hFgf subfamilies, Fgf4-, Fgf5-, Fgf8-, Fgf9-, Fgf10-, and Fgf15-like, appear to have derived from an Fgf13-like ancestral gene. Canonical FGFs function in a paracrine manner, while hFGFs function in an endocrine manner. We conclude that the ancestral Fgfs for these subfamilies acquired this functional diversity before the evolution of vertebrates. During the evolution of early vertebrates, the Fgf subfamilies further expanded to contain three or four members in each subfamily. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Tanjore H, Zeisberg EM, Gerami-Naini B, Kalluri R
beta1 integrin expression on endothelial cells is required for angiogenesis but not for vasculogenesis.
Dev Dyn. 2007 Dec 3;
Integrins are a family of cell adhesion receptors that are involved in cell-matrix and cell-cell communications. They facilitate cell proliferation, migration, and survival. Using the Cre-Lox system, we deleted beta1 integrin on Tie2-positive (Tie2-cre beta1 Int (fl/fl)) vascular endothelial cells. Deletion of beta1 integrin on vascular endothelial cells results in embryonic lethality. Blood vessel defects are encountered in the Tie2-Cre beta1 Int (fl/fl) embryos at embryonic age (E9.5), and embryos die before reaching E10.5. The embryos exhibit growth retardation and both histological evaluation and PECAM-1 staining of E9.5 embryos revealed defects in angiogenic sprouting and vascular branching morphogenesis. Large and medium-size vessel formation is not affected in these embryos. Angiogenic defects were observed in several regions of the embryo and yolk sacs. These results indicate that beta1 integrin expression on vascular endothelial cells is crucial for embryonic angiogenesis but dispensable for vasculogenesis. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Wang Q, Elghazi L, Martin S, Martins I, Srinivasan RS, Geng X, Sleeman M, Collombat P, Houghton J, Sosa-Pineda B
ghrelin is a novel target of Pax4 in endocrine progenitors of the pancreas and duodenum.
Dev Dyn. 2007 Dec 3;
Pax4-deficient mice have a severe gastrointestinal endocrine deficiency: they lack most pancreatic cells that produce insulin or somatostatin and various duodenal endocrine cell types. Remarkably, Pax4-deficient mice also have an overabundance of ghrelin-expressing cells in the pancreas and duodenum. Detailed analysis of the Pax4 nullizygous pancreas determined that the mutant islets are largely composed of a distinctive endocrine cell type that expresses ghrelin, glucagon, islet amyloid polypeptide (IAPP), and low levels of Pdx1. Lineage-tracing analysis revealed that most of these unique endocrine cells directly arose from Pax4-deficient progenitors. Previous in vitro work reported that Pax4 is a transcriptional repressor of islet amyloid polypeptide (IAPP) and glucagon. In this study, we expanded those results by showing that Pax4 is also a repressor of gherlin. Together, our data further support the notion that Pax4 activity is necessary to establish appropriate patterns of gene expression in endocrine progenitors of the digestive tract. Developmental Dynamics, 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]


DD ArtPix.
Dev Dyn. 2007 Dec;236(12):fvii. [Abstract]

Brachmann I, Jakubick VC, Shakèd M, Unsicker K, Tucker KL
A simple slice culture system for the imaging of nerve development in embryonic mouse.
Dev Dyn. 2007 Dec;236(12):3514-23.
Newborn neurons elaborate an axon that undertakes a complicated journey to find its ultimate target in the brain or periphery. Although major progress in the study of this process has been made by analysis of dissociated neurons in vitro, one would like to observe and manipulate axonal outgrowth and pathfinding as it occurs in situ, as fasciculated nerves growing within the tissue itself. Here, we present a simple technique to do this, through cultivation of embryonic mouse slices expressing enhanced green fluorescent protein (EGFP) specifically in newborn neurons. This system allows for imaging of outgrowth of peripheral nerves into structures such as the developing limb. We demonstrate a reproduction of normal innervation patterns by spinal nerves derived from spinal cord motor neurons and sensory neurons of the dorsal root ganglia. The slices can be manipulated pharmacologically as well as genetically, by crossing the EGFP-expressing line with lines containing targeted mutations in genes of interest. Developmental Dynamics 236:3514-3523, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Kiefer J
Highlights in DD.
Dev Dyn. 2007 Dec;236(12):fvi. [Abstract]

Demontis F, Dahmann C
Apical and lateral cell protrusions interconnect epithelial cells in live Drosophila wing imaginal discs.
Dev Dyn. 2007 Dec;236(12):spc1.
3D-rendering of a living Drosophila melanogaster wing imaginal disc expressing CD8-GFP in the dorsal compartment and stained with the lipophilic dye FM4-64. See Demontis and Dahmann, Developmental Dynamics 236:3408-3418. (c) 2007 Wiley-Liss, Inc. [Abstract]

Clark PA, Treisman DM, Ebben J, Kuo JS
Developmental signaling pathways in brain tumor-derived stem-like cells.
Dev Dyn. 2007 Dec;236(12):3297-308.
Recently, a subpopulation of cells highly efficient in tumor initiation and growth has been isolated from brain tumors. Of interest, these brain tumor initiating cells exhibit many stem-like properties, including self-renewal, extended proliferation, and multipotency, and are both phenotypically and genetically similar to normal neural stem cells (NSCs). Aberrant expression of developmental pathways, such as WNT, Hedgehog, Notch, and transforming growth factor-beta/bone morphogenetic protein, have been demonstrated in brain tumors, and extrinsic regulation of these pathways may be used to target brain tumor stem-like cells (BTSCs) and form the basis of novel biological therapies. Because of regulatory redundancy during normal development, future therapeutic strategies to inhibit BTSC-mediated tumor growth and minimize NSC-related deleterious effects may require detailed understanding and regulation of multiple cellular mechanisms. This review analyzes the role developmental pathways play in brain tumors, focusing on the potential effects of pathway regulation on BTSC-driven tumorigenesis. Developmental Dynamics 236:3297-3308, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Guo YL, Ye J, Huang F
p38alpha MAP kinase-deficient mouse embryonic stem cells can differentiate to endothelial cells, smooth muscle cells, and neurons.
Dev Dyn. 2007 Dec;236(12):3383-92.
p38 MAP kinase alpha (p38alpha) regulates various cellular processes in adult cells, but little is known about its function in stem cells. We investigated the potential of wild type and p38alpha deficient mouse embryonic stem cells (ESCs) to differentiate into endothelial cells (ECs), smooth muscle cells (SMCs), and neurons. Our differentiation methods allowed simultaneous development of all these cell types. ECs formed monolayers similar to mature ECs and could assemble into vessel-like structures. SMCs had well-organized actin filaments with morphology similar to adult SMCs. Neurons exhibited well-developed cell bodies and elongated axons. Deletion of the p38alpha gene did not significantly compromise ESC differentiation since p38alpha-/- cells could express cell-specific markers and displayed similar overall morphology to the cells differentiated from p38alpha+/+ ESCs. Although p38alpha regulates various cellular activities of adult SMCs, ECs, and neurons, our data demonstrate that p38alpha is not essential for ESC differentiation to these cell types. Developmental Dynamics 236:3383-3392, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Yoshida-Kashikawa M, Shibata N, Takechi K, Agata K
DjCBC-1, a conserved DEAD box RNA helicase of the RCK/p54/Me31B family, is a component of RNA-protein complexes in planarian stem cells and neurons.
Dev Dyn. 2007 Dec;236(12):3436-50.
The stem cells of planarians, known as neoblasts, can give rise to all cell types in planarians. Neoblasts can be identified by electron microscopy as cells with electron-dense chromatoid bodies, which are large RNP (ribonucleoprotein) complexes, in their cytoplasm. However, the components and function of chromatoid bodies are still relatively unknown. Here we identified a DEAD box RNA helicase gene of the RCK/p54/Me31B family from a planarian EST database and showed the localization of its product in chromatoid bodies by immunoelectron microscopy. We named this gene Djcbc-1 (Dugesia japonica chromatoid body component 1). Djcbc-1 was also strongly expressed in the brain and in the germline stem cells of sexualized planarians. We observed chromatoid body-like electron-dense bodies in brain neurons, where DjCBC-1 was also expressed. These observations suggest that common molecular components of RNP complexes may be involved in the regulation of somatic and germline stem cells, and neurons in planarians. Departmental Dynamics 236:3436-3450, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Chaw RC, Vance E, Black SD
Gastrulation in the spider Zygiella x-notata involves three distinct phases of cell internalization.
Dev Dyn. 2007 Dec;236(12):3484-95.
The cell movements of gastrulation were analyzed in embryos of the spider Zygiella x-notata, using time-lapse video, cell tracing, and improved histology. Cells are internalized near the center of the germ disc in three distinct phases. First, cumulus mesenchyme cells ingress and migrate as a group beneath the superficial layer. Second, mass internalization through a blastopore yields a diffusely organized deep layer. Third, superficial cells accumulate at the center of the germ disc to form the caudal bud. The floor is internalized, and the caudal bud moves over the nascent dorsal field to form the caudal lobe. This pattern of gastrulation differs from the canonical pattern described in the historical literature: (1) the cumulus of Z. x-notata is completely formed before any other cells internalize; and (2) the caudal lobe is formed by means of the caudal bud, which is a locus of cell internalization. Developmental Dynamics 236:3484-3495, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Fisher SA
The developing embryonic cardiac outflow tract is highly sensitive to oxidant stress.
Dev Dyn. 2007 Dec;236(12):3496-502.
This study tested the hypothesis that the remodeling of the cardiac outflow tract (OFT) may represent a developmental window of vulnerability to reactive oxygen species (ROS). Chick embryos were exposed in ovo or ex ovo to increasing concentrations of the stable oxidant hydrogen peroxide (H2O2). As assessed by trypan blue staining, H2O2 induced cell injury in the stage 25-30 OFT at concentrations as low as 1 nM. Higher concentrations were required to induce cell injury in the ventricular and atrial myocardium. Using DCFDA as an indicator of oxidant stress, H2O2 also induced a greater fluorescent signal in the OFT myocardium. H2O2 at these low concentrations also induced Caspase activity, indicative of activation of the pathway of PCD. Interestingly, the induction of Caspase-3 activity was predominately in the OFT cushion mesenchymal cells. Thus, the developing OFT is particularly sensitive to ROS-mediated injury, suggesting that ROS could play a role in the development of congenital defects of the cardiac OFT. Developmental Dynamics 236:3496-3502, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Gribble SL, Nikolaus OB, Dorsky RI
Regulation and function of Dbx genes in the zebrafish spinal cord.
Dev Dyn. 2007 Dec;236(12):3472-83.
Dbx homeodomain proteins are important for spinal cord dorsal/ventral patterning and the production of multiple spinal cord cell types. We have examined the regulation and function of Dbx genes in the zebrafish. We report that Hedgehog signaling is not required for the induction or maintenance of these genes; in the absence of Hedgehog signaling, dbx1a/1b/2 are expanded ventrally with concomitant increases in postmitotic neurons that differentiate from this domain. Also, we find that retinoic acid signaling is not required for the induction of Dbx expression. Furthermore, we are the first to report that knockdown of Dbx1 function causes a dorsal expansion of nkx6.2, which is thought to be the cross-repressive partner of Dbx1 in mouse. Our data confirm that the dbx1a/1b/2 domain in zebrafish spinal cord development behaves similarly to amniotes, while extending knowledge of Dbx1 function in spinal cord patterning. Developmental Dynamics 236:3472-3483, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Berger C, Renner S, Lüer K, Technau GM
The commonly used marker ELAV is transiently expressed in neuroblasts and glial cells in the Drosophila embryonic CNS.
Dev Dyn. 2007 Dec;236(12):3562-8.
Glial cells in the Drosophila embryonic nervous system can be monitored with the marker Reversed-polarity (Repo), whereas neurons lack Repo and express the RNA-binding protein ELAV (Embryonic Lethal, Abnormal Vision). Since the first description of the ELAV protein distribution in 1991 (Robinow and White), it is believed that ELAV is an exclusive neuronal and postmitotic marker. Looking at ELAV expression, we unexpectedly observed that, in addition to neurons, ELAV is transiently expressed in embryonic glial cells. Furthermore, it is transiently present in the proliferating longitudinal glioblast, and it is transcribed in embryonic neuroblasts. Likewise, elav-Gal4 lines, which are generally used as postmitotic neuronal driver lines, show expression in neural progenitor cells and nearly all embryonic glial cells. Thus, in the embryo, elav can no longer be considered an exclusive marker or driver for postmitotic neurons. elav loss-of-function mutants show no obvious effects on the number and pattern of embryonic glia. Developmental Dynamics 236:3562-3568, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Hara A, Kadoya Y, Kojima I, Yamashina S
Rat pancreatic islet is formed by unification of multiple endocrine cell clusters.
Dev Dyn. 2007 Dec;236(12):3451-8.
The organogenesis of islets in rat pancreas was studied by three-dimensional reconstructions from serial section micrographs. On embryonic day (E) 12, an endocrine cluster consisting mainly of glucagon-expressing cells maintained connection with the pancreatic endoderm at several regions. On E15-E17, the cluster enlarged by fusion of newly formed buds. Although the proportion of insulin-expressing cells increased, they were located in the periphery of the cluster. On the day of birth, insulin-expressing cell clusters enlarged and fused to form several cores within the islet. The glucagon-expressing cell mass expanded to form a thin mantle covering the cores. During islet organogenesis, proliferation activity was high in the exocrine duct system. Moreover, the endocrine cell clusters maintained contact with the duct epithelium throughout. We conclude that the pancreatic islet is generated by the unification of multiple endocrine clusters originated from separate regions of the duct system. The mechanism of mantle-core formation is discussed. Developmental Dynamics 236:3451-3458, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Cooper CA, Walsh LA, Damjanovski S
Peroxisome biogenesis occurs in late dorsal-anterior structures in the development of Xenopus laevis.
Dev Dyn. 2007 Dec;236(12):3554-61.
Metabolism and development are two important processes not often examined in the same context. The focus of the present study is the expression of specific peroxisomal genes, the subsequent biogenesis of peroxisomes, and their potential role in the metabolism associated with the development of Xenopus laevis embryos. The temporal and expression patterns of six peroxisomal genes (PEX5, ACO, PEX19, PMP70, PEX16, and catalase) were elucidated using RT-PCR. Functionally related peroxisomal genes exhibited similar expression patterns with their RNA levels elevated relatively late during embryogenesis. Using immunohistochemistry PMP70 and catalase protein was localized largely to dorsal-anterior structures. Peroxisomal function was assayed with peroxisomal targeted-GFP, which when microinjected, revealed peroxisomes in dorsal-anterior structures at stage 45. A requirement for peroxisomal function appears to be present only late in development as organogenesis is finishing, yolk stores are depleted, and ingestion commences. Developmental Dynamics 236:3554-3561, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Spence JR, Wells JM
Translational embryology: Using embryonic principles to generate pancreatic endocrine cells from embryonic stem cells.
Dev Dyn. 2007 Dec;236(12):3218-27.
Diseases that affect endodermally derived organs such as the lungs, liver, and pancreas include cystic fibrosis, chronic hepatitis, and diabetes, respectively. Despite the prevalence of these diseases, cures remain elusive. While several promising transplantation-based therapies exist for some diseases such as Type 1 diabetes, they are currently limited by the availability of donor-derived tissues. Embryonic stem cells are a promising and renewable source of tissue for transplantation; however, directing their differentiation into specific, adult cell lineages remains a significant challenge. In this review, we will focus on one endodermally derived organ, the pancreas, and discuss how studies of embryonic pancreas development have been used as the basis for the directed, step-wise differentiation of mouse and human embryonic stem cells into pancreatic endocrine cells that are capable of rescuing Type 1 diabetes in animal models. Developmental Dynamics 236:3218-3227, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Park HC, Shin J, Roberts RK, Appel B
An olig2 reporter gene marks oligodendrocyte precursors in the postembryonic spinal cord of zebrafish.
Dev Dyn. 2007 Dec;236(12):3402-7.
Continuous production of new neurons and glia in adult mammals occurs within specialized proliferation zones of the forebrain. Neural cell proliferation and neurogenesis is more widespread in adult amphibians, reptiles, and fish but the identity of neural stem cell populations in these organisms has not been fully described. We investigated expression of a reporter gene driven by olig2 regulatory DNA at postembryonic stages in zebrafish. We show that olig2 expression marks a discrete population of spinal cord radial glia in larvae and adults that divide continuously. olig2(+) radial glia have hallmarks of stem cells and their divisions appear to be asymmetric, producing new oligodendrocytes but not neurons or astrocytes. Developmental Dynamics 236:3402-3407, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Kwakowsky A, Schwirtlich M, Zhang Q, Eisenstat DD, Erdélyi F, Baranyi M, Katarova ZD, Szabó G
GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: Correlation with Dlx2 and Dlx5.
Dev Dyn. 2007 Dec;236(12):3532-44.
Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter of the adult nervous system and its biosynthetic enzyme glutamic acid decarboxylase (GAD) are abundantly expressed in the embryonic nervous system and are involved in the modulation of cell proliferation, migration, and differentiation. Here we describe for the first time the expression of GABA and embryonic and adult GAD isoforms in the developing mouse lens. We show that the GAD isoforms are sequentially induced with specific spatiotemporal profiles: GAD65 and embryonic GAD isoforms prevail in primary fibers, while GAD67 is the predominant GAD expressed in the postnatal secondary fibers. This pattern correlates well with the expression of Dlx2 and Dlx5, known as upstream regulators of GAD. GABA and GAD are most abundant at the tips of elongating fibers and are absent from organelle-free cells, suggesting their involvement is primarily in shaping of the cytoskeleton during fiber elongation stages. Developmental Dynamics 236:3532-3544, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Dooley J, Erickson M, Larochelle WJ, Gillard GO, Farr AG
FGFR2IIIb signaling regulates thymic epithelial differentiation.
Dev Dyn. 2007 Dec;236(12):3459-71.
Heterogeneous epithelial populations comprising the thymic environment influence early and late stages of T-cell development. The processes that regulate the differentiation of thymic epithelium and that are responsible for this heterogeneity are not well understood, although mesenchymal/epithelial interactions are clearly involved. Here, we show that targeted expression by thymocytes of an fibroblast growth factor receptor-2IIIb (FGFR2IIIb) ligand, FGF10, profoundly alters the differentiation and function of thymic epithelium (TE). Reconstitution of irradiated lckFGF10 mice with normal bone marrow restores normal thymic organization and function, while wild-type mice reconstituted with lckFGF10 bone marrow recapitulates some of the thymic alterations seen in lckFGF10 mice. We also demonstrate that interference with FGFR2IIIb signaling in the thymus with a soluble FGFR2IIIb dominant-negative fusion protein leads to precocious reductions in thymic size and cellularity that resemble age-related thymic involution. These findings indicate that TE compartments are dynamically maintained and that FGF signals are involved in this process. Developmental Dynamics 236:3459-3471, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

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Recent Articles in Microscopy Research and Technique

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Rocha T, Leonardo MB, De Souza BM, Palma MS, Da Cruz-Höfling MA
Mastoparan effects in skeletal muscle damage: An ultrastructural view until now concealed.
Microsc Res Tech. 2007 Dec 10; .
Animal venoms have been valuable sources for development of new drugs and important tools to understand cellular functioning in health and disease. The venom of Polybia paulista, a neotropical social wasp belonging to the subfamily Polistinae, has been sampled by headspace solid phase microextraction and analyzed by gas chromatography-mass spectrometry. Recent study has shown that mastoparan, a major basic peptide isolated from the venom, reproduces the myotoxic effect of the whole venom. In this study, Polybia-MPII mastoparan was synthesized and studies using transmission electron microscopy were carried out in mice tibial anterior muscle to identify the subcellular targets of its myotoxic action. The effects were followed at 3 and 24 h, 3, 7, and 21 days after mastoparan (0.25 mug/muL) intramuscular injection. The peptide caused disruption of the sarcolemma and collapse of myofibril arrangement in myofibers. As a consequence, fibers presented heteromorphic amorphous masses of agglutinated myofilaments very often intermingled with denuded sarcoplasmic areas sometimes only surrounded by a persistent basal lamina. To a lesser extent, a number of fibers apparently did not present sarcolemma rupture but instead appeared with multiple small vacuoles. The results showed that sarcolemma, sarcoplasmic reticulum (SR), and mitochondria were the main targets for mastoparan. In addition, a number of fibers showed apoptotic-like nuclei suggesting that the peptide causes death both by necrosis and apoptosis. This study presents a hitherto unexplored view of the effects of mastoparan in skeletal muscle and contributes to discuss how the known pharmacology of the peptide is reflected in the sarcolemma, SR, mitochondria, and nucleus of muscle fibers, apparently its subcellular targets. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Chi H, Feng M, Xiao Z, Lu Z
Preservation and fluorescence of the microfossils from Neoproterozoic Doushantuo formation.
Microsc Res Tech. 2007 Dec 10;
The phosphatized microfossils from Doushantuo Formation, Southeast China show us the biodiversity about 600 million years ago, which is a unique window for the evolution of the early life on earth. However, the process of phosphatic fossilization in detail still remains unknown. Here we report our study on the preservation state of the fossils by using confocal laser scanning microscopy. We found that fluorescent signal of the fossil could reflect the preservation state when compared with the transmission light microscopy. First, we found the fluorescent signal of the decayed cells of the fossil was weaker than that of the nondecayed part. Second, we found that the three-dimensional reconstruction of the fluorescent signals could help to judge the degree of mineralization of the fossil cells, compared with the observation by transmission light microscope. Third, we found that almost all of the fossil specimens we observed could fluoresce more or less when excited by laser light. Therefore, the fluorescent microscopy provides a useful method for the study of the preservation state of the phosphatic fossil cells. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Rieppo J, Hallikainen J, Jurvelin JS, Kiviranta I, Helminen HJ, Hyttinen MM
Practical considerations in the use of polarized light microscopy in the analysis of the collagen network in articular cartilage.
Microsc Res Tech. 2007 Dec 10;
Polarized light microscopy is a traditional method for visualizing the collagen network architecture of articular cartilage. Articular cartilage repair and tissue engineering studies have raised new demands for techniques capable of quantitative characterization of the scar and repair tissues, including properties of the collagen network. Modern polarized light microscopy can be used to measure collagen fibril orientation, parallelism, and birefringence. New commercial instruments are computer controlled and the measurements are easy to perform. However, often the interpretation of results causes difficulties, even errors, because the theoretical aspects of the technique are demanding. The aim of this study was to describe the instrumentation and properties of a modern polarized light microscope, to point out some sources of error in the interpretation of the results, and to recall the theoretical background of the polarized light microscopy. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Kinoshita Y, Johnson EM, Gordon RE, Negri-Bell H, Evans MT, Coolbaugh J, Rosario-Peralta Y, Samet J, Slusser E, Birkenbach MP, Daniel DC
Colocalization of MCM8 and MCM7 with proteins involved in distinct aspects of DNA replication.
Microsc Res Tech. 2007 Dec 10;
Minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. A subcomplex of the MCM2-7 family members, initially characterized in yeast, is thought to serve as a eukaryotic DNA replicative helicase. MCM8 is a new family member, not present in yeast, which may function alone or with other family members in aspects of DNA metabolism, including replication initiation and elongation. Through the use of chromatin immunoprecipitation, we find that MCM8, like MCM7, colocalizes on a specific DNA segment of the c-MYC replication initiation zone (c-MYC replicator) with Cdc6, a protein potentially involved in loading MCM proteins onto DNA. The association between MCM8 and MCM7 peaks in mid G1, at the time of assembly of the prereplication complex. The association of both MCM proteins with Cdc6, however, continues even after DNA replication is complete. We also find that MCM8 colocalizes at the c-MYC replicator with chromatin-bound Cdk2. Our data indicate that any role MCM8 may play in elongation is likely to be discontinuous, in its association with DNA, from a potential role in initiation. Using immunogold electron microscopy we show that MCM8 and MCM7 differ in spatial relation to RPA70 during S phase. Our data strongly suggest that MCM8 functions with other known replication proteins in processes which accompany DNA replication, especially initiation, and which are specifically adapted to suit higher eukaryotes. Microsc. Res. Tech, 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Tsai YS, Chung IF, Simpson JC, Lee MI, Hsiung CC, Chiu TY, Kao LS, Chiu TC, Lin CT, Lin WC, Liang SF, Lin CC
Automated recognition system to classify subcellular protein localizations in images of different cell lines acquired by different imaging systems.
Microsc Res Tech. 2007 Dec 10;
Systemic analysis of subcellular protein localization (location proteomics) provides clues for understanding gene functions and physiological condition of the cells. However, recognition of cell images of subcellular structures highly depends on experience and becomes the rate-limiting step when classifying subcellular protein localization. Several research groups have extracted specific numerical features for the recognition of subcellular protein localization, but these recognition systems are restricted to images of single particular cell line acquired by one specific imaging system and not applied to recognize a range of cell image sources. In this study, we establish a single system for automated subcellular structure recognition to identify cell images from various sources. Two different sources of cell images, 317 Vero (http://gfp-cdna.embl.de) and 875 CHO cell images of subcellular structures, were used to train and test the system. When the system was trained by a single source of images, the recognition rate is high and specific to the trained source. The system trained by the CHO cell images gave high average recognition accuracy for CHO cells of 96%, but this was reduced to 46% with Vero images. When we trained the system using a mixture of CHO and Vero cell images, an average accuracy of recognition reached 86.6% for both CHO and Vero cell images. The system can reject images with low confidence and identify the cell images correctly recognized to avoid manual reconfirmation. In summary, we have established a single system that can recognize subcellular protein localizations from two different sources for location-proteomic studies. studies. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Alessandrini A, Gavazzo P, Picco C, Facci P
Voltage-induced morphological modifications in oocyte membranes containing exogenous K(+) channels studied by electrochemical scanning force microscopy.
Microsc Res Tech. 2007 Nov 30;
We report on a novel use of electrochemical scanning force microscopy (SFM) for the investigation of morphological modifications occurring in plasma membranes containing voltage-gated ion channels, on membrane potential variation. Membrane patches of Xenopus laevis oocytes microinjected with exogenous KAT1 cRNA, deposited by a stripping method at the surface of a derivatized gold film in inside-out configuration, have been imaged by SFM in an electrochemical cell. A potentiostat was used to maintain a desired potential drop across the membrane. Performing imaging at potential values corresponding to open (-120 mV) and closed (+20 mV) states for KAT1, morphological differences in localized sample zones were observed. Particularly, cross-shaped features involving a significant membrane portion appear around putative channel locations. The reported approach constitutes the first demonstration of an SPM-based experimental technique suitable to investigate the rearrangements occurring to the plasma membrane containing voltage-gated channels on transmembrane potential variation. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Rosa RG, Tarsitano CA, Hyslop S, Yamada AT, Toledo OM, Joazeiro PP
Relaxation of the mouse pubic symphysis during late pregnancy is not accompanied by the influx of granulocytes.
Microsc Res Tech. 2007 Nov 28;
In some animals, such as mice and guinea pigs, a hormonally controlled mechanism increases the flexibility of the pubic symphysis and enhances the cervical remodeling necessary for safe delivery. Cervical ripening during pregnancy is associated with a paradoxical influx of leukocytes. However, the changes in cell metabolism during relaxation of the mouse pubic symphysis for delivery have not been extensively studied. In this work, we used light microscopy and transmission and scanning electron microcopy, as well as immunohistochemistry and Western blotting for MMP-8, to investigate the involvement of granulocytes or resident stromal cells in the relaxation of the virgin pubic symphysis during late pregnancy (days 18 and 19, before delivery) in vivo and in explanted joints. MMP-8 was studied because this collagenase is a hallmark for cervical ripening associated with the influx of granulocytes during late pregnancy. Extensive dissolution and disorganization of the extracellular matrix was seen around fibroblastic-like cells in late pregnancy. In contrast to the cervix (positive control), morphological and immunohistochemical analyses revealed that there was no characteristic cellular inflammatory response in the interpubic tissue. Staining for MMP-8 was observed in chondroid and fibroblastic-like cells of virgin and relaxed interpubic ligament, respectively. However, no granulocytes were seen during the extensive remodeling of the pubic joint in late pregnancy. These results indicate that constitutive stromal cells may have an important role in tissue relaxation during remodeling of the pubic symphysis in pregnancy. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Laksameethanasan D, Brandt SS, Engelhardt P, Renaud O, Shorte SL
A Bayesian reconstruction method for micro-rotation imaging in light microscopy.
Microsc Res Tech. 2007 Nov 28;
The authors present a three-dimensional (3D) reconstruction algorithm and reconstruction-based deblurring method for light microscopy using a micro-rotation device. In contrast to conventional 3D optical imaging where the focal plane is shifted along the optical axis, micro-rotation imaging employs dielectric fields to rotate the object inside a fixed optical set-up. To address this entirely new 3D-imaging modality, the authors present a reconstruction algorithm based on Bayesian inversion theory and use the total variation function as a structure prior. The spectral properties of the reconstruction by simulations that illustrate the strengths and the weaknesses of the micro-rotation approach, compared with conventional 3D optical imaging, were studied. The reconstruction from real data sets shows that this method is promising for 3D reconstruction and offers itself as a deblurring method using a reconstruction-based procedure for removing out-of-focus light from the micro-rotation image series. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Germanà A, Marino F, Guerrera MC, Campo S, de Girolamo P, Montalbano G, Germanà GP, Ochoa-Erena FJ, Ciriaco E, Vega JA
Expression and distribution of S100 protein in the nervous system of the adult zebrafish (Danio rerio).
Microsc Res Tech. 2007 Nov 27;
S100 proteins are EF-hand calcium-binding protein highly preserved during evolution present in both neuronal and non-neuronal tissues of the higher vertebrates. Data about the expression of S100 protein in fishes are scarce, and no data are available on zebrafish, a common model used in biology to study development but also human diseases. In this study, we have investigated the expression of S100 protein in the central nervous system of adult zebrafish using PCR, Western blot, and immunohistochemistry. The central nervous system of the adult zebrafish express S100 protein mRNA, and contain a protein of approximately 10 kDa identified as S100 protein. S100 protein immunoreactivity was detected widespread distributed in the central nervous system, labeling the cytoplasm of both neuronal and non-neuronal cells. In fact, S100 protein immunoreactivity was primarily found in glial and ependymal cells, whereas the only neurons displaying S100 immunoreactivity were the Purkinje's neurons of the cerebellar cortex and those forming the deep cerebellar nuclei. Outside the central nervous system, S100 protein immunoreactivity was observed in a subpopulation of sensory and sympathetic neurons, and it was absent from the enteric nervous system. The functional role of S100 protein in both neurons and non-neuronal cells of the zebrafish central nervous system remains to be elucidated, but present results might serve as baseline for future experimental studies using this teleost as a model. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Iliescu M, Hoemann CD, Shive MS, Chenite A, Buschmann MD
Ultrastructure of hybrid chitosan-glycerol phosphate blood clots by environmental scanning electron microscopy.
Microsc Res Tech. 2007 Nov 27;
Chitosan-based polymers have been extensively studied for biomedical applications. Recently, liquid solutions of chitosan in a glycerol phosphate buffer (chitosan-GP) with physiological pH and osmolality were mixed with autologous blood to form hybrid chitosan-GP/blood implants that improved the repair of articular cartilage lesions in a large animal model. The mixture of chitosan-GP and blood forms a viscous liquid, which solidifies in minutes via normal blood coagulation as well as chitosan-mediated mechanisms. Here we have examined the ultrastructure of these chitosan-GP/blood clots as well as regular blood clots and chitosan-GP gels, the latter produced by heating. Both unfixed and fixed samples of chitosan-GP/blood clots, regular blood clots, and chitosan-GP gels were investigated by environmental scanning electron microscopy (ESEM) in conjunction with energy dispersive X-ray spectrometry (EDS), the former permitting direct observation of the ultrastructure in hydrated conditions simulating the natural state. By examination of unfixed specimens using ESEM we found that chitosan formed a network structure in both chitosan-GP gels and chitosan-GP/blood clots; however this structure was altered by aldehyde fixation to produce artifactual aggregates of chitosan microparticles. We were also able to identify chitosan in chitosan-GP/blood clots by washing samples in low concentration NaCl solutions followed by local EDS analyses to identify excess chloride versus sodium, and thus presence of cationic chitosan in analyzed features. Additional results indicated that the majority of glycerol phosphate diffuses freely from chitosan-GP gels (by EDS of phosphorus) and that hyperosmotic paraformaldehyde-based fixatives (i.e. 4% w/v) significantly disturb erythrocyte morphology in fixed whole blood clots. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Richter B, Fragner K, Weissenböck H
Simultaneous detection of protozoa in the tissues of snakes by double in situ hybridization.
Microsc Res Tech. 2007 Nov 27;
Different methods have been established for the simultaneous detection of different pathogens in tissue samples, each with certain advantages and disadvantages. Chromogenic in situ hybridization combines specific molecular pathogen detection with microscopic evaluation of pathogen quantity, morphology and distribution, as well as associated tissue damage. Furthermore, only a minimum of usually costly technical equipment is needed. The aim of our study was to detect two different protozoa simultaneously in tissue samples using exclusively digoxigenin (DIG)-labeled probes and alkaline phosphatase-coupled anti-DIG-antibodies and the chromogens Vector Red and NBT/BCIP with standard protocols. Gastrointestinal tissue samples from 15 snakes infected with either one or two protozoan species were investigated. All expected protozoa stained clearly dark purple or bright red, respectively, depending on the chromogen used. This technique can be used in pathogenicity studies of various pathogens in any kind of tissue. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Corrêa CL, da Silva PG, Pereira MJ, Allodi S, Martinez AM
Electron microscopy and morphometric analyses of microtubules in two differently sized types of axons in the protocerebral tract of a crustacean.
Microsc Res Tech. 2007 Nov 19;
Despite several reports on the morphology and functions associated with the morphometry of the vertebrate axoplasm cytoskeleton, the subject has not been thoroughly explored in invertebrates. In vertebrates, among many other functions, microtubules (MTs) serve as scaffolding for axon assembly, and neurofilaments (NFs) as the elements that determine the axon caliber. Intermediate filaments have never been described by electron microscopy in arthropods, although NF proteins have been revealed in the MT side-arms of the axoplasm of certain species, such as the crab Ucides cordatus. Thus, it is not known which elements of the cytoskeleton of invertebrates are responsible for determination of the axon caliber. We studied, by electron microscopy and morphometric analyses, the MT and axon area variability in differently sized axons of the protocerebral tract of the crab Ucides cordatus. Our results revealed differences in the distance between MTs, in MT density and number, and in the areas of differently sized axons. The number of MTs increases with the axon area, but this relationship is not directly proportional. Therefore, MT density is greater in smaller axons than in medium axons, similar to the morphometry of the vertebrate axon MT. The distance between MTs is, however, directly related to the axonal area. On the basis of the results shown here, and on previous reports by us and others, we suggest that MTs may be involved in the determination of the axon caliber, possibly due to the presence of NF proteins found in the side-arms. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Colyer RA, Lee C, Gratton E
A novel fluorescence lifetime imaging system that optimizes photon efficiency.
Microsc Res Tech. 2007 Nov 15;
Fluorescence lifetime imaging (FLIM) is a powerful microscopy technique for providing contrast of biological and other systems by differences in molecular species or their environments. However, the cost of equipment and the complexity of data analysis have limited the application of FLIM. We present a mathematical model and physical implementation for a low cost digital frequency domain FLIM (DFD-FLIM) system, which can provide lifetime resolution with quality comparable to time-correlated single photon counting methods. Our implementation provides data natively in the form of phasors. On the basis of the mathematical model, we present an error analysis that shows the precise parameters for maximizing the quality of lifetime acquisition, as well as data to support this conclusion. The hardware and software of the proposed DFD-FLIM method simplifies the process of data acquisition for FLIM, presents a new interface for data display and interpretation, and optimizes the accuracy of lifetime determination. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Rocha GM, Miranda K, Weissmüller G, Bisch PM, de Souza W
Ultrastructure of Trypanosoma cruzi revisited by atomic force microscopy.
Microsc Res Tech. 2007 Nov 8;
Most advances in atomic force microscopy (AFM) have been accomplished in recent years. Previous attempts to use AFM to analyze the organization of pathogenic protozoa did not significantly contribute with new structural information. In this work, we introduce a new perspective to the study of the ultrastructure of the epimastigote form of Trypanosoma cruzi by AFM. Images were compared with those obtained using field emission scanning electron microscopy of critical point dried cells and transmission electron microscopy of negative stained detergent-extracted and air-dried cells. AFM images of epimastigote forms showed a flagellum furrow separating the axoneme from the paraflagellar rod (PFR) present from the emergence of the flagellar pocket to the tip of the flagellum. At high magnification, a row of periodically organized structures, which probably correspond to the link between the axoneme, the PFR and the flagellar membrane were seen along the furrow. In the origin of the flagellum, two basal bodies were identified. Beyond the basal bodies, small periodically arranged protrusions, positioned at 400 nm from the flagellar basis were seen. This structure was formed by nine substructures distributed around the flagellar circumference and may correspond to the flagellar necklace. Altogether, our results demonstrate the importance of the application of AFM in the structural characterization of the surface components and cytoskeleton on protozoan parasites. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Guan YQ, Cai YY, Zhang X, Lee YT, Opas M
Adaptive correction technique for 3D reconstruction of fluorescence microscopy images.
Microsc Res Tech. 2007 Nov 8;
Recent advances in high-resolution imaging have provided valuable novel insights into structural relationships within cells and tissues both in vitro and in vivo. An analysis of this kind is regularly done by optical sectioning using either confocal or deconvolution microscopy. However, the reconstruction of 3D images suffers from light scattering and absorption with increasing depth by finite transparency of the used media. Photobleaching of fluorochromes has been especially troublesome and often the only remedy for loss of signal during optical sectioning is to reduce the number of sections. This causes disparities in the x-y and z dimensions of voxels, which lead to vertical distortion of the original stack of images and necessitates interpolation. Interpolation is necessary to fill up the gaps between consecutive sections in the original image stack to obtain cubic voxels. The present manuscript describes a novel method for adaptive compensation of attenuation of light intensity in stacks of fluorescence microscopy images that is based on a physical model of light attenuation. First, we use a fast interpolation technique to generate a cubic voxel-based volume stack with the aid of a contribution look up table. With the contribution look up table, multiple calculations are avoided, which substantially reduces the computational time without compromising the accuracy of the restoration procedure. Second, each section within the resulting volume is processed to rectify its intensity values that have been altered due to photobleaching and scattering and absorption. The method allows to define the last good section in the stack and the correction is then done automatically. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Reale L, Kaiser J, Reale A, Lai A, Flora F, Balerna A, Cinque G, Fanelli M, Ruggieri F, Faenov A, Pikuz T, Tucci A, Poma A, Zuppella P, Li?ka M, Malina R
Mapping the intake of different elements in vegetal tissues by dual-energy X-ray imaging at DaPhine synchrotron light source.
Microsc Res Tech. 2007 Nov 8;
This article reports on the first utilization of the soft X-ray beamline at the DaPhine synchrotron light source for mapping the intake of different elements in plant tissues. As a test, the method of dual-energy X-ray microradiography was applied to the investigation of the natural sulfur content in dried leaf and root samples. Our ultimate goal was to monitor the pollutant lead and its intake, which was added in controlled doses to the hydroponic medium of laboratory-controlled samples of vegetal species. The results obtained by the nondestructive X-ray radiographic analysis are compared to the values of concentrations determined by a standard chemical analysis utilizing atomic absorption spectroscopy. From this comparison the validity of the X-ray detection of heavy metals in biological samples has been confirmed. The superposition of the dual energy results on the simple planar radiography shows the representation of the pollutant intake directly on the sample structures. It should be pointed out that this method, developed here for plant root and leaves could be applied to any biological sample of interest, but the preparation and observation conditions necessitate different strategies according to the type of sample under analysis. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Azevedo DD, Matiello-Guss CP, Rönnau M, Zanuncio JC, Serrão JE
Post-embryonic development of the antennal sensilla in Melipona quadrifasciata anthidioides (Hymenoptera: Meliponini).
Microsc Res Tech. 2007 Nov 8;
The sensilla are sensory organs formed by cuticular and cellular structures specialized in reception of chemical and physical stimuli from the environment and transmission to the insect's central nervous system. In function of the great concentration of sensilla, the antennae are the main organs for interaction between bees and with the environment. This work studied the presence of antennal sensilla in the different phases of pupal development of the stingless bee Melipona quadrifasciata anthidioides by means of scanning electron microscopy and light microscopy. The results showed that antennal sensilla begin their development in the transition of the prepupae to the white-eyed pupae and finish it in the pigmented-body pupae phase. The antennal sensilla were exposed to the environment in the black-eyed pupae when the old cuticle is completely digested, suggesting that only in the final pupal phases can these bees perceive the environmental stimuli. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Dezfuli BS, Lui A, Giari L, Boldrini P, Giovinazzo G
Ultrastructural study on the body surface of the acanthocephalan parasite Dentitruncus truttae in brown trout.
Microsc Res Tech. 2007 Nov 8;
Scanning electron microscope (SEM) investigations on the holdfast elements, proboscis hooks, and trunk spines of Dentitruncus truttae (Acanthocephala, Palaeacanthocephala), an endoparasite of Salmo trutta (brown trout), provide more data about the surface of these taxonomic relevant structures. In both acanthocephalan sexes, the fully everted cylindrical proboscis possessed 18 longitudinal rows of hooks with 18 hooks per row (rarely 19-20). Hook length varied according to position on the proboscis; apical hooks were 40-52 mum long, middle hooks were 31.7-36.6 mum, and basal hooks were 38.1-40 mum. Starting from the anterior end of the metasoma, numerous cuticular spines (26.7-30 mum in length) were visible and their number progressively decreased posteriorly. SEM observations of D. truttae hooks and spines revealed the presence of many surface striations on each proboscis hook. These surface striations were absent from trunk spines. From the base of the hook, the striations ran parallel toward the point of convergence. Additionally, survey of longitudinal and transversal sections of the hook using transmission electron microscope confirmed that the hook surface was not smooth. SEM comparison with the hooks of several palaeacanthocephalan species, as well as with the hooks of species belonging to Eoacanthocephala and Polyacanthocephala, indicated that the striations are currently exclusive to D. truttae proboscis hooks. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Zhang D, Zhang H, Liu C, Jiang J
Microscopic observation and laser-controlled microoptothermal drive mechanism.
Microsc Res Tech. 2007 Oct 24;
The mechanism of novel optothermal microactuators (OTA) was analyzed with a microscope system, a charge-coupled device-combined light microscope, and a computer system. One OTA, two optothermal microswitches (OTS) with different length and shape were machined by an excimer laser micromatching system using single layer material. They all had two thin expansion arms with different widths. The mechanism of the OTA/OTS is that the different optothermal expansion controlled by asymmetric topology and shape of the arms causes a magnified lateral deflection or vibration. A red laser diode (650 nm) with maximum power output of 30 mW was employed as the external power source to drive the OTA/OTS. Experiments were carried out with the microscope system, and serial images and videos were acquired. The results indicate that the structure of the OTA/OTS is simple and easy to be manufactured. They can practically generate an obvious lateral deflection or vibration. When the location of the laser spot on the OTA changed, the direction of its deflection is also changed. When the value of laser power that irradiated on the OTS increased, the lateral deflection of the OTS enlarged, the maximum could be larger than 30 mum. This kind of novel microactuator has the advantages of remote wireless controlling, large displacement, simple structure, easy to be machined, and therefore will be quite useful for the practical applications in the fields of micro/nanotechnology. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Teschke O, De Souza EF, Silva-Stenico ME, Fiore MD, Etchegaray A
Quorum sensing detected by atomic force microscopy imaging of corrals surrounding multicellular arrangement of bacteria.
Microsc Res Tech. 2007 Oct 17;
Connectivity of the glycocalyx covering of small communities of Acidithiobacillus ferrooxidans bacteria deposited on hydrophilic mica plates was imaged by atomic force microscopy. When part of the coverage was removed by water rinsing, an insoluble structure formed by corrals surrounding each individual bacterium was observed. A collective ring structure with clustered bacteria (>/=3) was observed, which indicates that the bacteria perceived the neighborhood in order to grow a protective structure that results in smaller production of exopolysaccharides material. The most surprising aspect of these collective corral structures was that they occur at a low bacterial cell density. The deposited layers were also analyzed by confocal Raman microscopy and shown to contain polysaccharides, protein, and glucoronic acid. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Nakadate M, Amizuka N, Li M, Freitas PH, Oda K, Nomura S, Uoshima K, Maeda T
Histological evaluation on bone regeneration of dental implant placement sites grafted with a self-setting alpha-tricalcium phosphate cement.
Microsc Res Tech. 2007 Oct 17;
This study aimed to evaluate the histological characteristics of the new bone formed at dental implant placement sites concomitantly grafted with a self-setting tricalcium phosphate cement (BIOPEX-R(R)). Standardized defects were created adjacent to the implants in maxillae of 4-week-old male Wistar rats, and were concomitantly filled with BIOPEX-R(R). Osteogenesis was examined in two sites of extreme clinical relevance: (1) the BIOPEX-R(R)-grafted surface corresponding to the previous alveolar ridge (alveolar ridge area), and (2) the interface between the grafting material and implants (interface area). At the alveolar ridge area, many tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts had accumulated on the BIOPEX-R(R) surface and were shown to migrate toward the implant. After that, alkaline phosphatase (ALPase)-positive osteoblasts deposited new bone matrix, demonstrating their coupling with osteoclasts. On the other hand, the interface area showed several osteoclasts initially invading the narrow gap between the implant and graft material. Again, ALPase-positive osteoblasts were shown to couple with osteoclasts, having deposited new bone matrix after bone resorption. Transmission electron microscopic observations revealed direct contact between the implant and the new bone at the interface area, although few thin cells could still be identified. At both the alveolar ridge and the interface areas, newly formed bone resembled compact bone histologically. Also, concentrations of Ca, P, and Mg were much alike with those of the preexistent cortical bone. In summary, when dental implant placement and grafting with BIOPEX-R(R) are done concomitantly, the result is a new bone that resembles compact bone, an ideal achievement in reconstructive procedures for dental implantology. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Sun Y, Tan HY, Lin SJ, Lee HS, Lin TY, Jee SH, Young TH, Lo W, Chen WL, Dong CY
Imaging tissue engineering scaffolds using multiphoton microscopy.
Microsc Res Tech. 2007 Oct 17;
In this study, we combined two-photon autofluorescence and second harmonic generation imaging to investigate the three-dimensional microstructure and nonlinear optical properties of tissue engineering scaffolds. We focused on five different types of scaffold materials commonly used in tissue engineering, including: open-cell polylactic acid, polyglycolic acid, collagen composite scaffold, collagraft bone graft matrix strip, and nylon. By the use of multiphoton microscopy and a motorized stage, we obtained high resolution, spectrally resolved structural information of the scaffolds over large areas or in three-dimensions. Our results show that the nonlinear optical properties of the scaffolds will enable us to spectrally and morphologically distinguish the different types of scaffold materials investigated. We envision multiphoton microscopy to be a useful technique in tissue engineering applications in understanding the interplay between cultured cells and the scaffold materials. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Dalal RB, Digman MA, Horwitz AF, Vetri V, Gratton E
Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode.
Microsc Res Tech. 2007 Oct 15;
We describe a method to obtain the brightness and number of molecules at each pixel of an image stack obtained with a laser scanning microscope. The method is based on intensity fluctuations due to the diffusion of molecules in a pixel. For a detector operating in the analog mode, the variance must be proportional to the intensity. Once this constant has been calibrated, we use the ratio between the variance and the intensity to derive the particle brightness. Then, from the ratio of the intensity to the brightness we obtain the average number of particles in the pixel. We show that the method works with molecules in solution and that the results are comparable to those obtained with fluctuation correlation spectroscopy. We compare the results obtained with the detector operating in the analog and photon counting mode. Although the dynamic range of the detector operating in the photon counting mode is superior, the performance of the analog detector is acceptable under common experimental conditions. Since most commercial laser scanning microscopes operate in the analog mode, the calculation of brightness and number of particles can be applied to data obtained with these instruments, provided that the variance is proportional to the intensity. We demonstrate that the recovered brightness of mEGFP, independent of concentration, is similar whether measured in solution or in two different cell types. Furthermore, we distinguish between mobile and immobile components, and introduce a method to correct for slow variations in intensity. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Jovanovi? I, Jordovi? B, Petkovi? M, Ignjatovi? N, Uskokovi? D
Preparation of smallest microparticles of poly-d,l-lactide by modified precipitation method: Influence of the process parameters.
Microsc Res Tech. 2007 Oct 15;
Biodegradable microspheres such as those made of poly-D,L-lactide (PDLLA) are widely investigated delivery systems for drugs or antigens. The aim of this study was to examine experimental conditions in order to produce PDLLA microspheres with the best properties for controlled and sustained drug delivery by the modified precipitation method. For this purpose, the following parameters were varied: co-solvent (methanol or ethanol), the concentration of stabilizer polyvinyl alcohol (PVA), chloroform-to-water ratio and the speed and time of homogenization. Scanning electron microscopy (SEM) and stereological analysis were used to characterize the particles. The average size and morphology of the microspheres varied substantially with preparation conditions from 8.44-1.25 mum. Results showed that the smallest particles were obtained with methanol, 1% PVA and with 10 min of homogenization at 21,000 rpm. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Cornillie P, Van Den Broeck W, Simoens P
Three-dimensional reconstruction of the remodeling of the systemic vasculature in early pig embryos.
Microsc Res Tech. 2007 Oct 15;
Current research on angiogenesis and vascular regression is mainly focused on pathological conditions such as tumor growth and diabetic retinopathy, while a suitable physiological model to study the controlling factors in these processes is still lacking. The remodeling pattern of the embryonic vasculature into the adult configuration, such as the branchial arch arterial system developing into the aorta or the early embryonic veins building the caudal vena cava can potentially serve as a model. However, practical applications of the embryonic vascular patterning are impeded by the current controversy over the exact development of the caudal vena cava in mammals. To elucidate these ambiguities, specific developmental stages of vascular development in pig embryos were mapped by means of computer-assisted 3D reconstructions starting from histological serial sections of Bouin's fixed embryos. Special attention was given to venous segments in the lumbar region, as their origin and fate are equivocally described in literature. Here we demonstrate that these venous segments originate from the caudal cardinal veins which are forced to migrate during development into a more dorsal position due to the expansion of the developing metanephroi and the more dorsal relocation of the umbilical arteries. These findings are in contrast with the generally accepted theory that the venous segments in the lumbar region arise from newly formed veins that are located dorsal to the early caudal cardinal system. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Neethirajan S, Thomson DJ, Jayas DS, White ND
Characterization of the surface morphology of durum wheat starch granules using atomic force microscopy.
Microsc Res Tech. 2007 Oct 15;
Knowledge of the structure and properties of microscopic surfaces of durum wheat starch granules is essential for understanding the functional and physico-chemical properties. The nanoscale surface undulations on the starch granules inside durum wheat macroscopically influence the milling properties. The objective of this study was to visualize the surface morphology and the size of starch grains of vitreous and nonvitreous durum wheat kernels using atomic force microscopy. The distribution of starch granules in the vitreous and nonvitreous durum wheat starch samples were examined and compared. The results of our study confirm the 'blocklet' model of the ultrastructure of the starch granule surface. Image contrast enhancement using UV/ozone treatment of microtomed starch samples improved the imaging of growth rings on the starch samples. The observation of growth rings in the nonvitreous starch granule surfaces indicates that amylopectin is more common than amylose in nonvitreous starch when compared with vitreous starch. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Chen WL, Sun Y, Lo W, Tan HY, Dong CY
Combination of multiphoton and reflective confocal imaging of cornea.
Microsc Res Tech. 2007 Sep 27;
We combine reflective confocal microscopy with multiphoton microscopy to form a minimally invasive technique to observe the cornea. The two imaging modalities allow detection of complementary information from the cornea. The autofluorescence signal shows the cytoplasm of epithelial cells, and the second harmonic generation signal is used to detect collagen, found mostly in the stroma of the cornea. The reflective confocal imaging allows detection of epithelial cells and keratocytes in the stroma. The system is first tested on bovine cornea. Assessment of the result on the bovine eye will be used to evaluate the potential of the system as a technique for in vivo clinical application. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Li J, Yi T, Lai HS, Xue D, Jiang H, Peng HC, Zhang H
Application of microscopy in authentication of traditional Tibetan medicinal plant Halenia elliptica.
Microsc Res Tech. 2007 Sep 27;
Halenia elliptica D. Don, a popularly used ethnodrug from Qinghai-Tibetan plateau, was studied to reveal the indispensable morphoanatomic details. The fixed, sectioned, and stained plant materials as well as the epidermis, powder, and maceration materials were studied using light microscope according to the usual microscopic techniques. The results of the microscopic features were systematic described and illustrated. In the root, an endodermal cell was divided into 8-16-22 and 38-50-62 daughter cells in transverse section and in face view, respectively, and 9-11-13 phloem strands were present in primary structure; in the stem, stone cells were observed in the cortex, pericycle, and external phloem while 17-19-21 internal phloem strands were present in an incontinuous ring; in the pedicel, 8-10-12 internal phloem strands were observed to form an incontinuous ring; anisocytic and anomocytic stomata were present in leaf and sepal epidermis; pollen grain was with three germinal apertures and furrows; a few tracheids, a large number of spiral vessels, and various fibers were observed. Also, semiquantitative and quantitative micrographic parameter tables were simultaneously presented. Further, the key authentication parameters were concluded. The study indicated that light microscopy and related techniques could be unambiguously applied to the authentication of Halenia elliptica. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Fiolka R, Belyaev Y, Ewers H, Stemmer A
Even illumination in total internal reflection fluorescence microscopy using laser light.
Microsc Res Tech. 2007 Sep 20;
In modern fluorescence microscopy, lasers are a widely used source of light, both for imaging in total internal reflection and epi-illumination modes. In wide-field imaging, scattering of highly coherent laser light due to imperfections in the light path typically leads to nonuniform illumination of the specimen, compromising image analysis. We report the design and construction of an objective-launch total internal reflection fluorescence microscopy system with excellent evenness of specimen illumination achieved by azimuthal rotation of the incoming illuminating laser beam. The system allows quick and precise changes of the incidence angle of the laser beam and thus can also be used in an epifluorescence mode. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Lee JD, Chang YF, Kao FJ, Kao LS, Lin CC, Lu AC, Shyu BC, Chiou SH, Yang DM
Detection of the interaction between SNAP25 and rabphilin in neuroendocrine PC12 cells using the FLIM/FRET technique.
Microsc Res Tech. 2007 Sep 20;
Exocytosis has been proposed to contain four sequential steps, namely docking, priming, fusion, and recycling, and to be regulated by various proteins-protein interactions. Synaptosomal-associated protein of 25 kDa (SNAP25) has recently been found to bind rabphilin, the Rab3A specific binding protein, in vitro. However, it is still unclear whether SNAP25 and rabphilin interact during exocytosis within cells in vivo. This problem was addressed by the integration of fluorescence resonance energy transfer (FRET) with high sensitivity fluorescence lifetime imaging microscopy (FLIM) to observe this protein-protein interaction. Enhanced green fluorescence protein-labeled SNAP25 (donor) and red fluorescence protein-labeled rabphilin (acceptor) were expressed in neuroendocrine PC12 cells as a FRET pair and ATP stimulation was carried out for various durations. With 10 s stimulation, a 0.17-ns left shift of the lifetime peak was found when compared with donor only. Analysis of the lifetime image further suggested that the lifetime recovered to a similar level as the donor only in a time dependent manner. Four-dimensional (4D) images by FLIM provided useful information indicating that the interaction of SNAP25 and rabphilin occurred particularly within optical sections near cell membrane. Together the results suggest that SNAP25 bound rabphilin loosely at docking step before exocytosis and the binding became tighter at the very start of exocytosis. Finally, these two proteins dissociated after stimulation. To our knowledge, this is the first report to demonstrate the interaction of SNAP25 and rabphilin in situ using the FLIM-FRET technique within neuroendocrine cells. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Dragomir NM, Goh XM, Roberts A
Three-dimensional refractive index reconstruction with quantitative phase tomography.
Microsc Res Tech. 2007 Sep 20;
Optical tomography based on quantitative phase microscopy is used to determine nondestructively and with high spatial resolution the three-dimensional (3D) refractive index distributions within optical fiber devices. After obtaining a series of phase images of the fiber as it is rotated around its longitudinal axis at regularly-spaced angular positions, filtered backprojection is used to reconstruct a 3D map of the refractive index. The 3D refractive index distribution of the join region between two fusion spliced optical fibers is reconstructed with accuracy better than 10(-3). Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Wang L, Ou-Yang L, Yau SL
Adlayer structure of octa-alkoxy-substituted copper(II) phthalocyanine on Au(111) by electrochemical scanning tunneling microscopy.
Microsc Res Tech. 2007 Sep 20;
Electrochemical scanning tunneling microscopy (ECSTM) has been used to examine the adlayer of octa-alkoxy-substituted copper(II) phthalocyanines (CuPc(OC(8)H(17))(8)) on Au(111) in 0.1 M HClO(4), where the molecular adlayer was prepared by spontaneous adsorption from a benzene solution containing this molecule. Topography STM scans revealed long-range ordered, interweaved arrays of CuPc(OC(8)H(17))(8) with coexistent rectangular and hexagonal symmetries. High-quality STM molecular resolution yielded the internal molecular structure and the orientation of CuPc(OC(8)H(17))(8) admolecules. These STM results could shed insight into the method of generating ordered molecular assemblies of phthalocyanine molecules with long-chained substitutes on metal surface. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Wai MS, Lorke DE, Zhang A, Kung HF, Yew DT
Study of the spinal cords of the sturgeon Acipenser schrenckii, gar Lepisosteus oculatus, and goldfish Carassius auratus by morphological, immunohistochemical, and biochemical approaches.
Microsc Res Tech. 2007 Dec;70(12):
Little is known about the spinal cords of phylogenetically ancient actinopterygeans. The spinal cords of the chondrostean Acipenser schrenckii (Amur sturgeon), holostean Lepisosteus oculatus (spotted gar), and teleost Carassius auratus (goldfish) were, therefore, analyzed by immunohistochemistry, electron microscopy and two-dimensional gel electrophoresis. Morphology showed numerous similarities between sturgeons and gars. In both, a dorsal column between the two dorsal horns was lacking, giving the grey matter an inverted Y-shape. In goldfish, a small dorsal column was seen, the grey matter occupied a larger area, neuronal density was much higher, and a ventral commissure was apparent, which was absent in sturgeons and gars. In the white matter of sturgeons and gars, small caliber axons predominated, whereas larger axons were frequent in goldfish. Choline acetyltransferase immunoreactive neurons were prevalent in the ventral horns of all three fish, mainly in motoneurons, but stained fibers were only found in sturgeons and gars. gamma-aminobutyric acid positive cells were seen in both the ventral and the dorsal horns of all three fish. Distribution of serotonin (5-HT) and tyrosine hydroxylase (TH) immunoreaction was similar in sturgeons and gars, being located in both the ventral and the dorsal horns. In goldfish, 5-HT label was confined to the ventral horn and TH label was mainly observed in a cell group located ventromedially. Two-dimensional gel electrophoresis showed a gradual increase in protein number from sturgeons to gars to goldfish. In conclusion, the spinal cords of sturgeons and gars share many morphological and chemical features, distinguishing them from the goldfish spinal cord. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Hashimoto H, Kusakabe M, Ishikawa H
A novel method for three-dimensional observation of the vascular networks in the whole mouse brain.
Microsc Res Tech. 2007 Sep 14;
A novel method for acquiring serial images suitable for three-dimensional reconstruction of vascular networks in the whole brain of mouse was developed. The brain infused with a White India ink-gelatin solution was fixed and embedded in paraffin containing Sudan Black B through xylene also containing Sudan Black B. Each sliced surface of the paraffin block was coated with liquid paraffin and its image was serially acquired. Coating with liquid paraffin extremely improved the quality of the image. The series of serial images was free of distortion and a three-dimensional image was reconstructed without the problem of the alignment and registration of adjacent images. The volume-rendered image indicated three-dimensional distribution of blood vessels in a whole brain. No ghost or shadow was observed on a volume-rendered image of the White India ink-gelatin infused brain. The z-axial resolution examined on the orthogonal sections reconstituted from serial images obtained at an interval of 5 mum showed no cross talk, indicating that the z-axial resolution was no larger than 5 mum. A proper understanding of the vascular system in a whole brain is indispensable to reveal the development of the vascular system in the brain of normal and genetically manipulated mouse and vascular alterations in pathological situation, such as stroke and neurodegenerative disease. Although simple and inexpensive, this method will provide fundamental information on the vascular system in a whole brain. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Ward TS, Rosen GD, von Bartheld CS
Optical disector counting in cryosections and vibratome sections underestimates particle numbers: Effects of tissue quality.
Microsc Res Tech. 2007 Sep 14;
Optical disector counting is currently applied most often to cryosections, followed in frequency by resin-embedded tissues, paraffin, and vibratome sections. The preservation quality of these embedding options differs considerably; yet, the effect of tissue morphology on numerical estimates is unknown. We tested whether different embedding media significantly influence numerical estimates in optical disector counting, using the previously calibrated trochlear motor nucleus of hatchling chickens. Animals were perfusion-fixed with paraformaldehyde (PFA) only or in addition with glutaraldehyde (GA), or by Methacarn immersion fixation. Brains were prepared for paraffin, cryo-, vibratome- or celloidin sectioning. Complete penetration of the thionin stain was verified by z-axis analysis. Neuronal nuclei were counted using an unbiased counting rule, numbers were averaged for each group and compared by ANOVA. In paraffin sections, 906 +/- 12 (SEM) neurons were counted, similar to previous calibrated data series, and results obtained from fixation with Methacarn or PFA were statistically indistinguishable. In celloidin sections, 912 +/- 28 neurons were counted-not statistically different from paraffin. In cryosections, 812 +/- 12 neurons were counted (underestimate of 10.4%) when fixed with PFA only, but 867 +/- 17 neurons were counted when fixed with PFA and GA. Vibratome sections had the most serious aberration with 729 +/- 31 neurons-a deficit of 20%. Thus, our analysis shows that PFA-fixed cryosections and vibratome sections result in a substantial numerical deficit. The addition of GA to the PFA fixative significantly improved counts in cryosections. These results may explain, in part, the significant numerical differences reported from different labs and should help investigators select optimal conditions for quantitative morphological studies. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

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Recent Articles in Journal of Morphology

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Shearman RM
Chondrogenesis and ossification of the lissamphibian pectoral girdle.
J Morphol. 2007 Nov 12; .
Knowledge of amphibian shoulder development is requisite for further understanding of gnathostome pectoral girdle evolution. Fish and amniotes share few pectoral girdle elements, but modern amphibians exhibit a unique combination of traits that bridge the morphological gap between these two groups. I analyzed patterns of chondrogenesis, ossification, and bone histology of the pectoral girdles of two anuran species (Xenopus laevis and Bombina orientalis) and two urodele species (Ambystoma mexicanum and Desmognathus aeneus) to provide new insight into the evolution of the tetrapod pectoral girdle. Comparisons reveal the following: 1) variation in the pattern of chondrogenesis among the anuran species analyzed correlates to variation in adult pectoral girdle morphology; 2) morphologically similar pectoral skeletons do not necessarily have similar patterns of bone histology; and 3) the urodele and anuran pectoral girdles included herein share a common morphology despite differences in patterns of chondrogenesis. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Sever DM, Siegel DS, Bagwill A, Eckstut ME, Alexander L, Camus A, Morgan C
Renal sexual segment of the Cottonmouth snake, Agkistrodon piscivorous (Reptilia, Squamata, Viperidae).
J Morphol. 2007 Nov 12;
The seasonal variation of the renal sexual segment (RSS) of males of the Cottonmouth snake, Agkistrodon piscivorous, is described using light and electron microscopy. This study is the first to describe the ultrastructure of the RSS of a viper (Viperidae) and only the fourth on a snake. Renal sexual segments from males collected February to May and from August to November are similar in appearance. The cells are eosinophilic and react with periodic acid/Schiff procedure (PAS) for neutral carbohydrates and bromphenol blue (BB) for proteins. At the ultrastructure level, the cells contain large (2 mum diameter), electron-dense secretory granules and smaller vesicles with a diffuse material, and these structures abut against the luminal border and upon clear vacuoles continuous with intercellular canaliculi. Evidence was found for both apocrine and merocrine processes of product release. In June and July, the RSS are significantly smaller in diameter, largely basophilic, and have only scattered granules that are PAS+ and BB+. Cytologically, the RSS from June to July lack electron-dense secretory granules and the smaller vesicles with diffuse material. Numerous condensing vacuoles and abundant rough endoplasmic reticulum, however, indicate that active product synthesis is occurring. This is the first report of significant seasonal variation in the histology and ultrastructure of the RSS of a snake, although such reports exist for lizards. The seasons when the RSS is most highly hypertrophied correspond to the fall and spring mating seasons of A. piscivorous, as determined by other studies. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Drea CM, Weil A
External genital morphology of the ring-tailed lemur (Lemur catta): Females are naturally "masculinized"
J Morphol. 2007 Oct 30;
The extravagance and diversity of external genitalia have been well characterized in male primates; however, much less is known about sex differences or variation in female form. Our study represents a departure from traditional investigations of primate reproductive anatomy because we 1) focus on external rather than internal genitalia, 2) measure both male and female structures, and 3) examine a strepsirrhine rather than an anthropoid primate. The subjects for morphological study were 21 reproductively intact, adult ring-tailed lemurs (Lemur catta), including 10 females and 11 males, two of which (one per sex) subsequently died of natural causes and also served as specimens for gross anatomical dissection. Male external genitalia presented a typical masculine configuration, with a complex distal penile morphology. In contrast, females were unusual among mammals, presenting an enlarged, pendulous external clitoris, tunneled by the urethra. Females had a shorter anogenital distance and a larger urethral meatus than did males, but organ diameter and circumference showed no sex differences. Dissection confirmed these characterizations. Noteworthy in the male were the presence of a "levator penis" muscle and discontinuity in the corpus spongiosum along the penile shaft; noteworthy in the female were an elongated clitoral shaft and glans clitoridis. The female urethra, while incorporated within the clitoral body, was not surrounded by erectile tissue, as we detected no corpus spongiosum. The os clitoridis was 43% the length and 24% the height of the os penis. On the basis of these first detailed descriptions of strepsirrhine external genitalia (for either sex), we characterize those of the female ring-tailed lemur as moderately "masculinized." Our results highlight certain morphological similarities and differences between ring-tailed lemurs and the most male-like of female mammals, the spotted hyena (Crocuta crocuta), and call attention to a potential hormonal mechanism of "masculinization" in female lemur development. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Pulcini D, Costa C, Aguzzi J, Cataudella S
Light and shape: A contribution to demonstrate morphological differences in diurnal and nocturnal teleosts.
J Morphol. 2007 Oct 30;
Light intensity is an important environmental factor affecting the structure of fish assemblages during the day-night cycle. Light influences how organisms perceive their environment, modulating their intraspecific and interspecific relationships. The relationship between light intensity variations and biological cycles should be observed at the level of organismal morphology. In this study the relationship between activity rhythms, thus light intensity experienced by fish in the period of major activity and external morphology, have been investigated. The morphological traits of 97 selected fish species were compared in order to determine the existence of a common morphological plan in agreement with their diurnal or nocturnal activity rhythm. Species sorting was performed by maximizing the diversity of activity rhythm, habitat choice, ecology, and trophic habits within the same family, to assess the importance of the day-night cycle on species morphology in relation to other environmental features. The morphological characters selected for the geometric morphometric analysis were body profile and the position of mouth, eye, pelvic, pectoral, dorsal, and caudal fin. The present analysis allowed different consensus forms for nocturnal and for diurnal species to be identified. Two-block Partial Least Squares analysis was then performed for the purpose of modeling the covariation between the form and two important external variables (ecology and activity). J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Egger GF, Witter K, Weissengruber G, Forstenpointner G
Articular cartilage in the knee joint of the African elephant, Loxodonta africana, Blumenbach 1797.
J Morphol. 2007 Oct 30;269(1):
Knee joints of one adult and three juvenile African elephants were dissected. The specific features of the articular cartilage with particular reference to matrix components were studied by light and electron microscopy and immunohistochemistry. The elephant knee joint cartilage contains an unusually low concentration of proteoglycans resulting in rather eosinophilic staining properties of the matrix. The very thick collagen fibers of the cartilage possibly represent collagen I. Except for the different thickness of cartilage at the weight-bearing surfaces of femur ( approximately 6.7 mm) and tibia ( approximately 11.2 mm) in juvenile elephants, light and electron microscopy did not reveal distinct topographical differences in cartilage structure, perhaps because of the high congruency of the articulating surfaces and resulting uniform load distribution in the knee. The number of cell profiles per section area of both femoral ( approximately 950 cell profiles/mm(2)) and tibial cartilage ( approximately 898 cell profiles/mm(2)) was low, indicating excessive matrix production by the chondrocytes during cartilage development. These unique properties could be a result of the enormous compressive load resting on the elephant knee. Maintenance of the equilibrium between biological function and resistance to compression seems to be crucial in the elephant knee joint cartilage. Any disturbance that interferes with this equilibrium appears to lead to arthrotic alterations, as particularly seen in captive elephants. J. Morphol., 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Fastnacht M
Tooth replacement pattern of Coloborhynchus robustus (pterosauria) from the Lower Cretaceous of Brazil.
J Morphol. 2007 Oct 25;
The well preserved anterior upper and lower jaw fragment of an adult specimen of Coloborhynchus robustus (Pterosauria: Ornithocheiridae), SMNK 2302 PAL, allowed investigations of the replacement pattern of the dentition macroscopically and by using CT scans. The quantification of the dentition by Zahnreihen, Z-Spacing, and replacement waves indicates a complex pattern of different replacement stages in which large gaps within the dentition were avoided. The specialized prey-catching apparatus of Coloborhynchus thus could retain its function even following tooth replacement. The replacement process in the specimen took about 2/3 of the total life-time of a tooth, and damaged teeth in the anterior jaw region may have been replaced more rapidly than posterior teeth. The distolingual replacement of the functional teeth delayed the time of their shedding in comparison with the circular resorption present in crocodiles. In contrast to these, the distolingual position of the replacement tooth did not decrease the biomechanical stability of the functional tooth, which can also be observed as a convergence in other thecodont dentitions, e.g., recent carnivore mammals. Teeth were shed when their replacement had reached about 60% of the full-grown height. A comparison of the observed pattern is constricted by the preservation and preparation of other specimens. Unfortunately, no known specimen in public collections reaches the quality of Coloborhynchus robustus, SMNK 2302 PAL, so that comparable patterns in other specimens are not likely to be detected. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Bairati A, Gioria M
An ultrastructural study of cell junctions and the cytoskeleton in epithelial cells of the molluscan integument.
J Morphol. 2007 Oct 25;
Cell junctions and the cytoskeleton of integumental epidermal cells from six bivalves, four gastropods, and two cephalopods were studied by transmission electron microscopy. In all species examined, the junctions in supporting cells presented the following similar pattern: an apical-lateral adhesion belt (occluding junctions were not observed); (b) a lateral complex of septate junctions and smooth septate junctions, with interdigitations between adjacent cells while the gap junctions were not constantly present, and a basal complex with hemidesmosomes, focal contacts, and sometimes basolateral adherent junctions. Desmosomes were never observed. Microfilamentous and microgranular material were present throughout the cells, as bundles of microfilaments within microvilli and the terminal web, within interdigitations, and as cytoplasmic plaques forming part of the adherent junctions, hemidesmosomes, and focal contacts. Bundles of intermediate filaments that originated from basal hemidesmosomes were located close to and oriented parallel with the lateral plasma membrane and terminated within the terminal web. In cells of Aplysia depilans, intermediate filaments converged apically to terminate in hemidesmosome-like structures at the bases of the microvilli. In the cephalopods, hemidesmosomes were never observed and intermediate filaments made direct contact with the basal cell membrane. Some functional interpretations and hypotheses were also discussed. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Vickaryous MK, Hall BK
Development of the dermal skeleton in Alligator mississippiensis (Archosauria, Crocodylia) with comments on the homology of osteoderms.
J Morphol. 2007 Oct 25;
The dermal skeleton (= exoskeleton) has long been recognized as a major determinant of vertebrate morphology. Until recently however, details of tissue development and diversity, particularly among amniotes, have been lacking. This investigation explores the development of the dermatocranium, gastralia, and osteoderms in the American alligator, Alligator mississippiensis. With the exception of osteoderms, elements of the dermal skeleton develop early during skeletogenesis, with most initiating ossification prior to mineralization of the endoskeleton. Characteristically, circumoral elements of the dermatocranium, including the pterygoid and dentigerous elements, are among the first to form. Unlike other axially arranged bones, gastralia develop in a caudolateral to craniomedial sequence. Osteoderms demonstrate a delayed onset of development compared with the rest of the skeleton, not appearing until well after hatching. Osteoderm development is asynchronous across the body, first forming dorsally adjacent to the cervical vertebrae; the majority of successive elements appear in caudal and lateral positions. Exclusive of osteoderms, the dermal skeleton initiates osteogenesis via intramembranous ossification. Following the establishment of skeletal condensations, some preossified spicules become engorged with many closely packed clusters of chondrocyte-like cells in a bone-like matrix. This combination of features is characteristic of chondroid bone, a tissue otherwise unreported among nonavian reptiles. No secondary cartilage was identified in any of the specimens examined. With continued growth, dermal bone (including chondroid bone) and osteoid are resorbed by multinucleated osteoclasts. However, there is no evidence that these cells contribute to the rugose pattern of bony ornamentation characteristic of the crocodylian dermatocranium. Instead, ornamentation develops as a result of localized concentrations of bone deposited by osteoblasts. Osteoderms develop in the absence of osteoblastic cells, osteoid, and periosteum; bone develops via the direct transformation of the preexisting dense irregular connective tissue. This mode of bone formation is identified as metaplasia. Importantly, it is also demonstrated that osteoderms are not histologically uniform but involve a range of tissues including calcified and uncalcified dense irregular connective tissue. Between taxa, not all osteoderms develop by homologous processes. However, it is concluded that all osteoderms may share a deep homology, connected by the structural and skeletogenic properties of the dermis. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Sustaita D
Musculoskeletal underpinnings to differences in killing behavior between North American accipiters (Falconiformes: Accipitridae) and falcons (Falconidae).
J Morphol. 2007 Oct 25;
Accipiters (Accipiter spp.) and falcons (Falco spp.) both use their feet to seize prey, but falcons kill primarily with their beaks, whereas accipiters kill with their feet. This study examines the mechanistic basis to differences in their modes of dispatching prey, by focusing on the myology and biomechanics of the jaws, digits, and distal hindlimb. Bite, grip, and distal hindlimb flexion forces were estimated from measurements of physiological cross-sectional area (PCSA) and indices of mechanical advantage (MA) for the major jaw adductors, and digit and tarsometatarsal flexors. Estimated bite force, total jaw adductor PCSA, and jaw MA (averaged over adductors) tended to be relatively and absolutely greater in falcons, reflecting their emphasis on biting for dispatching their prey. Differences between genera in estimated grip force, total digit flexor PCSA, and digit MA (averaged over inter-phalangeal joints and digits) were not as clear-cut; each of these parameters scaled positively allometric in accipiters, which may reflect the scaling of both prey size, and the proportion of mammalian prey consumed by this lineage with increasing body size. Estimated tarsometatarsal force was greater in falcons than in accipiters, due to their greater MA, which may reflect selection for incurring greater forces during prey strikes. Conversely, the comparatively lower tarsometatarsal MA in accipiters reflects their capacity for greater foot speed potentially necessary for grasping elusive prey. Thus, this study elucidates how differences in jaw and hindlimb musculoskeletal morphology of accipiters and falcons are reflected in differences in their killing modes, and through differences in their force-generating capacities. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Spieß R, Schoofs A, Heinzel HG
Anatomy of the stomatogastric nervous system associated with the foregut in Drosophila melanogaster and Calliphora vicina third instar larvae.
J Morphol. 2007 Oct 25;
The stomatogastric nervous system (SNS) associated with the foregut was studied in 3rd instar larvae of Drosophila melanogaster and Calliphora vicina (blowfly). In both species, the foregut comprises pharynx, esophagus, and proventriculus. Only in Calliphora does the esophagus form a crop. The position of nerves and neurons was investigated with neuronal tracers in both species and GFP expression in Drosophila. The SNS is nearly identical in both species. Neurons are located in the proventricular and the hypocerebral ganglion (HCG), which are connected to each other by the proventricular nerve. Motor neurons for pharyngeal muscles are located in the brain not, as in other insect groups, in the frontal ganglion. The position of the frontal ganglion is taken by a nerve junction devoid of neurons. The junction is composed of four nerves: the frontal connectives that fuse with the antennal nerves (ANs), the frontal nerve innervating the cibarial dilator muscles and the recurrent nerve that innervates the esophagus and projects to the HCG. Differences in the SNS are restricted to a crop nerve only present in Calliphora and an esophageal ganglion that only exists in Drosophila. The ganglia of the dorsal organs give rise to the ANs, which project to the brain. The extensive conformity of the SNS of both species suggests functional parallels. Future electrophysiological studies of the motor circuits in the SNS of Drosophila will profit from parallel studies of the homologous but more accessible structures in Calliphora. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Santagata S
The morphology and evolutionary significance of the ciliary fields and musculature among marine bryozoan larvae.
J Morphol. 2007 Oct 25;
Despite the embryological and anatomical disparities present among lophotrochozoan phyla, there are morphological similarities in the cellular arrangements of ciliated cells used for propulsion among the nonfeeding larval forms of kamptozoans, nemerteans, annelids, mollusks, and bryozoans. Evaluating whether these similarities are the result of convergent selective pressures or a shared (deep) evolutionary history is hindered by the paucity of detailed cellular information from multiple systematic groups from lesser-known, and perhaps, basal evolutionary phyla such as the Bryozoa. Here, I compare the ciliary fields and musculature among the major morphological grades of marine bryozoan larvae using light microscopy, SEM, and confocal imaging techniques. Sampling effort focused on six species from systematic groups with few published accounts, but an additional four well-known species were also reevaluated. Review of the main larval types among species of bryozoans and these new data show that, within select systematic groups of marine bryozoans, there is some conservation of the cellular arrangement of ciliary fields and larval musculature. However, there is much more morphological diversity in these structures than previously documented, especially among nonfeeding ctenostome larval types. This structural and functional diversification reflects species differences in the orientation of the apical disc during swimming and crawling behaviors, modification of the presumptive juvenile tissues, elongation of larval forms in the aboral-oral axis, maximizing the surface area of cell types with propulsive cilia, and the simplification of ciliary fields and musculature within particular lineages due to evolutionary loss. Considering the embryological origins and functional plasticity of ciliated cells within bryozoan larvae, it is probable that the morphological similarities shared between the coronal cells of bryozoan larvae and the prototrochal cells of trochozoans are the result of convergent functional solutions to swimming in the plankton. However, this does not rule out cell specification pathways shared by more closely related spiralian phyla. Overall, among the morphological grades of larval bryozoans, the structural variation and arrangement of the main cell groups responsible for ciliary propulsion have been evolutionarily decoupled from the more divergent modifications of larval musculature. The structure of larval ciliary fields reflects the functional demands of swimming and substrate exploration behaviors before metamorphosis, but this is in contrast to the morphology of larval musculature and presumptive juvenile tissues that are linked to macroevolutionary differences in morphogenetic movements during metamorphosis. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Crockett CJ, Peters SE
Hindlimb muscle fiber types in two frogs (Rana catesbeiana and Litoria caerulea) with different locomotor behaviors: Histochemical and enzymatic comparison.
J Morphol. 2007 Oct 25;
To test how differences in locomotor behaviors may be reflected in muscle fiber-type diversity within anurans, a comparison of hindlimb muscles between the powerful terrestrial hopper, Rana catesbeiana, and the tree frog, Litoria caerulea, was done. One postural muscle (tibialis posticus, TP) and one primary hopping muscle (plantaris longus, PL), were characterized to identify muscle fiber types using standard histochemical methods. In addition, spectophotometric analysis of activity levels of the oxidative enzyme citrate synthase (CS) and the glycolytic enzyme lactate dehydrogenase (LDH) were done in each muscle. In spite of presumed differences in behavior between the species, we found no significant differences in the proportions of the identified fiber types when the muscles were compared across species. In addition, there were no significant differences in the proportions of the different fiber types between the postural versus phasic muscles within species. Within Rana, the postural muscle (TP) had greater oxidative capacity (as measured by CS activity) than did the phasic muscle (PL). Both muscles had equivalent LDH activities. Within Litoria, PL and TP did not differ in either LDH or CS activities. Both PL and TP of Litoria had less LDH activity and greater CS activity than their homologs in Rana. Thus, in spite of the uniform populations of fiber types between muscles and species, the metabolic diversity based on enzyme activity is consistent with behavioral differences between the species. These results suggest that the range of functional diversity within fiber types may be very broad in anurans, and histochemical fiber typing alone is not a clear indicator of their metabolic or functional properties. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Rougerie R, Estradel Y
Morphology of the preimaginal stages of the African emperor moth Bunaeopsis licharbas (Maassen and Weyding): Phylogenetically informative characters within the Saturniinae (Lepidoptera: Saturniidae).
J Morphol. 2007 Oct 23;
The current classification of African representatives of the family Saturniidae is largely unsatisfactory and needs to be revised following a proper and rigorous phylogenetic analysis. In this regard, the use of characters of preimaginal stages in phylogeny reconstruction was already emphasized in the literature; however, few details are known about the morphology of the eggs, larvae, and pupae of these moths. To fill this gap and to provide a preliminary basis for further phylogenetic studies, the early stages of Bunaeopsis licharbas, a member of the tribe Bunaeini within the Saturniinae, are described. Egg chorionic ultrastructure, chaetotaxy of the larva in every instar, and morphology of the pupa are described and illustrated. A total of 22 previously overlooked characters was found and their potential phylogenetic significance is discussed in the light of a comparative study which includes a large set of representatives of all five tribes in the subfamily. Until further phylogenetic analyses, some characters are considered to be potential synapomorphies supporting, in particular, the monophyly of the tribe Bunaeini, the close relationship between this tribe and part of the Urotini, as well as the existence of an Afrotropical lineage within the Saturniinae uniting the tribes Bunaeini, Urotini, and Micragonini. In a group of moths whose preimaginal instars are among the best known within the Lepidoptera, the unexpected, and relatively high number of potentially informative characters found by the present study stresses the value of minute and exhaustive descriptive studies to make use of morphological characters available for phylogenetic studies. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Hofmann RR, Streich WJ, Fickel J, Hummel J, Clauss M
Convergent evolution in feeding types: Salivary gland mass differences in wild ruminant species.
J Morphol. 2007 Oct 23;
In the ongoing debate about divergent evolutionary morphophysiological adaptations of grazing and browsing ruminants, the size of the salivary glands has received special attention. Here, we report the most comprehensive dataset on ruminant salivary glands so far, with data on the Glandula parotis (n = 62 species), Gl. mandibularis (n = 61), Gl. buccalis ventralis (n = 44), and Gl. sublingualis (n = 30). All four salivary gland complexes showed allometric scaling with body mass (BM); in all cases, the 95% confidence interval for the allometric exponent included 0.75 but did not include 1.0 (linearity); therefore, like other parameters linked to the process of food intake, salivary gland mass appears to be correlated to metabolic body weight (BM(0.75)), and comparisons of relative salivary gland mass between species should rather be made on the basis of BM(0.75) than as a percentage of BM. In the subsequent analyses, the percentage of grass (%grass) in the natural diet was used to characterize the feeding type; the phylogenetic tree used for a controlled statistical evaluation was entirely based on mitochondrial DNA information. Regardless of phylogenetic control in the statistical treatment, there was, for all four gland complexes, a significant positive correlation of BM and gland mass, and a significant negative correlation between %grass in the natural diet and gland mass. If the Gl. parotis was analyzed either for cervid or for bovid species only, the negative correlation of gland mass and %grass was still significant in either case; an inspection of certain ruminant subfamilies, however, suggested that a convergent evolutionary adaptation can only be demonstrated if a sufficient variety of ruminant subfamilies are included in a dataset. The results support the concept that ruminant species that ingest more grass have smaller salivary glands, possibly indicating a reduced requirement for the production of salivary tannin-binding proteins. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Plochocki JH, Rivera JP, Zhang C, Ebba SA
Bone modeling response to voluntary exercise in the hindlimb of mice.
J Morphol. 2007 Oct 23;
The functional adaptation of juvenile mammalian limb bone to mechanical loading is necessary to maintain bone strength. Diaphyseal size and shape are modified during growth through the process of bone modeling. Although bone modeling is a well-documented response to increased mechanical stress on growing diaphyseal bone, the effect of proximodistal location on bone modeling remains unclear. Distal limb elements in cursorial mammals are longer and thinner, most likely to conserve energy during locomotion because they require less energy to move. Therefore, distal elements are hypothesized to experience greater mechanical loading during locomotion and may be expected to exhibit a greater modeling response to exercise. In this study, histomorphometric comparisons are made between femora and tibiae of mice treated with voluntary exercise and a control group (N = 20). We find that femora of exercised mice exhibit both greater bone growth rates and growth areas than do controls (P < 0.05). The femora of exercised mice also have significantly greater cortical area, bending rigidity, and torsional rigidity (P < 0.05), although bending and torsional rigidity are comparable when standardized by bone length. Histomorphometric and cross-section geometric properties of the tibial midshaft of exercised and control mice did not differ significantly, although tibial length was significantly greater in exercised mice (P < 0.05). Femora of exercised mice were able to adapt to increased mechanical loading through increases in compressive, bending, and torsional rigidity. No such adaptations were found in the tibia. It is unclear if this is a biomechanical adaptation to greater stress in proximal elements or if distal elements are ontogenetically constrained in a tradeoff of bone strength of distal elements for bioenergetic efficiency during locomotion. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Ricci C, Caprioli M, Fontaneto D, Melone G
Volume and morphology changes of a bdelloid rotifer species (Macrotrachela quadricornifera) during anhydrobiosis.
J Morphol. 2007 Oct 23;
Following a study on the changes occurring in a bdelloid species (Macrotrachela quadricornifera, Rotifera, Bdelloidea) when entering anhydrobiosis, we investigated the changes in morphology, including weight and volume during the transition from the active hydrated to the dormant anhydrobiotic state by scanning electron microscopy, confocal microscopy and light microscopy. We compared sizes and morphologies of hydrated extended, hydrated contracted and anhydrobiotic specimens. Bdelloid musculature is defined: longitudinal muscles are contracted in the hydrated contracted animal (head and foot are retracted inside the trunk), but appear loose in the anhydrobiotic animal. When anhydrobiotic, M. quadricornifera appears much smaller in size, with a volume reduction of about 60% of the hydrated volume, and its internal organization undergoes remarkable modifications. Internal body cavities, clearly distinguishable in the hydrated extended and contracted specimens, are no longer visible in the anhydrobiotic specimen. Concomitantly, M. quadricornifera loses more than 95% of its weight when anhydrobiotic; this is more than expected from the volume reduction data and could indicate the presence of space-filling molecular species in the dehydrated animal. We estimate that the majority of body mass loss and volume reduction can be ascribed to the water loss from the body cavity during desiccation. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Moore BC, Uribe-Aranzábal MC, Boggs AS, Guillette LJ
Developmental morphology of the neonatal alligator (Alligator mississippiensis) ovary.
J Morphol. 2007 Oct 23;
American alligator (Alligator mississippiensis) ovary development is incomplete at hatching. During the months following hatching, the cortical processes of oogenesis started in ovo continues and folliculogenesis is initiated. Additionally, the medullary region of the gonad undergoes dramatic restructuring. We describe alligator ovarian histology at hatching, 1 week, 1 month, and 3 months of age in order to characterize the timing of morphological development and compare these findings to chicken ovary development. At hatching, the ovarian cortex presents a germinal epithelium containing oogonia and a few primary oocytes irregularly scattered between somatic epithelial cells. The hatchling medulla shows fragmentation indicative of the formation of lacunae. By 1 week of age, oocytes form growing nests and show increased interactions with somatic cells, indicative of the initiation of folliculogenesis. Medullary lacunae increase in diameter and contain secretory material in their lumen. At 1 month, nest sizes and lacunar diameters continue to enlarge. Pachytene oocytes surrounded by somatic cells are more frequent. Trabeculae composed of dense irregular connective tissue divide cortical nests. Three months after hatching oocytes in meiotic stages of prophase I up to diplotene are present. The ovary displays many enlarged follicles with oocytes in diplotene arrest, thecal layers, lampbrush chromosomes, and complete layers of follicular cells. The medulla is an elaborated complex of vascularized lacunae underlying the cortex and often containing discrete lymphoid aggregates. While the general morphology of the alligator ovary is similar to that of the chicken ovary, the progression of oogenesis and folliculogenesis around hatching is notably slower in alligators. Diplotene oocytes are observed at hatching in chickens, but not until 3 months in alligators. Folliculogenesis is completed at 3 weeks in chickens whereas it is still progressing at 3 months in alligators. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Castanet J, Harrison FW
Eighth international congress of vertebrate morphology paris, france, july 16-21, 2007.
J Morphol. 2007 Dec;268(12): [Abstract]

Riner K, Boos A, Hässig M, Liesegang A
Vitamin D receptor distribution in intestines of domesticated sheep Ovis ammon f. aries.
J Morphol. 2007 Oct 12;
The biologically active form of vitamin D, i.e., 1,25-dihydroxycholecalciferol or calcitriol, plays an important role in bone metabolism and calcium homeostasis, which is often disturbed at the onset of lactation in high milk-yielding domestic ruminants. Gene transcription is modulated via vitamin D receptors, but nongenomic effects of vitamin D via membrane receptors have also been described. In the intestines, vitamin D promotes calcium absorption via vitamin D receptors. Vitamin D receptors are of clinical relevance, but have not been systematically assessed within all segments of the intestine in any species. Thus, we present for the first time an immunohistochemical study of the distribution patterns of the vitamin D receptor protein in sheep, which may be the basis for present and future investigations on mineral homeostasis in domestic ruminants. Tissue probes of the intestines were collected from five lambs and five nonlactating and nonpregnant dams, fixed in formalin, embedded in paraffin, and used for the assessment of vitamin D receptor protein. Nuclear vitamin D receptor immunoreaction was scored semiquantitatively and exhibited a segment-specific distribution pattern. Goblet cells always were devoid of any vitamin D receptor immunoreaction. Surface epithelial cells and enterocytes of the crypt openings generally demonstrated only a weak immunoreaction. Basally and/or intermediately located crypt epithelial cells exhibited stronger immunoreactions in duodenum, jejunum, and colon descendens. This basal/intermediate to superficial gradient was most pronounced in the duodenum and less evident in jejunum and colon descendens and not observed in ileum and cecum. There were no age-dependent variations in vitamin D receptor protein expression. Results demonstrate that intestinal vitamin D receptor distribution patterns are segment-specific and strongest immunoreactions correlate with highest intestinal calcium absorptive activities, as reported in literature. Strong expression of vitamin D receptors within the lower half of crypts also suggests a role for calcitriol in epithelial differentiation and cellular homeostasis. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Koch M, Edgecombe GD
The peristomatic structures of Lithobiomorpha (Myriapoda, Chilopoda): comparative morphology and phylogenetic significance.
J Morphol. 2007 Oct 12;
A comparative survey of the epipharynx and hypopharynx of lithobiomorph centipedes by light and scanning electron microscopy examines 18 species that sample the major groups of both families, the Lithobiidae and Henicopidae. Cladistic analysis of 11 characters of the peristomatic structures together with 29 additional morphological characters serves as a basis for interpreting the evolution of the lithobiomorph peristomatic structures. Scutigeromorpha is used for outgroup comparison in the framework of a homology scheme for the basic components of the epi- and hypopharynx. Compared to other chilopods, the monophyly of Lithobiomorpha is supported by a row of distinctive bottle-shaped gland openings at the border between the labral and clypeal parts of the epipharynx, as well as by a distinctive shape of the hypopharynx. Paired rows of elongate spines on the clypeal part of the epipharynx are an apomorphic character of Lithobiidae. The transformation of these spine rows into a few groups of branching spines is characteristic for the Monotarsobius group sensu Verhoeff. Similar groups of branching clypeal spines characterize the Anopsobiinae within Henicopidae, whereas Henicopinae possess a dense cluster of short, simple spines instead. The recently described genus Dzhungaria is resolved closer to Henicopinae than to Anopsobiinae, a hypothesis supported by a field of grooves on the medial labral part of the epipharynx. Monophyly of Henicopidae does not receive unique support from the peristomatic structures although two homoplastic characters contribute to this node; among these, the reduction of a median spine field between clypeal and labral parts of the epipharynx to a narrow transverse band also supports a close relationship between the Ezembius group and Hessebius within Lithobiidae. An Ezembius + Hessebius clade is additionally supported by the absence of a transverse bulge between the clypeal and labral parts of the epipharynx, a character otherwise present in all lithobiomorph species studied so far. Lithobius is resolved as polyphyletic, with different species being most closely related to such genera as Australobius, Hessebius and Pleurolithobius. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Siegel DS, Sever DM
Sperm aggregations in female Agkistrodon piscivorus (Reptilia:Squamata): A histological and ultrastructural investigation.
J Morphol. 2007 Oct 12;
Upon copulation in female Agkistrodon piscivorus, sperm migrate up the oviduct to sperm storage tubules (SSTs) in the posterior infundibulum. The epithelium of the SSTs is composed of ciliated and secretory cells and differs ultrastructurally from that of the epithelium lining the lumen of the posterior infundibulum. Sperm pass through an area composed primarily of ciliated cells at the opening of each gland before aligning themselves in parallel arrays with their nuclei facing an area composed primarily of secretory cells at the base of the tubules. Sperm are also found embedded inter- and intracellularly in the SSTs. The secretory vacuoles in the SSTs become highly electron dense after the start of the fall mating season along with the synthesis of lipid droplets. Histochemical analysis reveals that the alteration in secretory material density is caused by the production of neutral carbohydrates. Some sperm remain in aggregates in the nonglandular section of the posterior uterus until the time of ovulation. However, ultrastructural evidence indicates these sperm degrade before ovulation. Therefore, sperm in posterior aggregates have no role in fertilization of ovulated ova. The data presented here support the hypothesis that infundibular sperm storage is the mode that snakes utilize to sequester viable sperm until ovulation. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Frédérich B, Pilet A, Parmentier E, Vandewalle P
Comparative trophic morphology in eight species of damselfishes (Pomacentridae).
J Morphol. 2007 Oct 12;
Damselfishes show significant biodiversity in the coral reefs. To better understand such diversity, an ecomorphological approach was investigated in the trophic morphology of eight species of Pomacentridae (Chromis acares, C. margaritifer, Dascyllus aruanus, D. flavicaudus, Pomacentrus pavo, Plectroglyphidodon johnstonianus, Pl. lacrymatus and Stegastes nigricans) belonging to different trophic guilds (zooplankton, algal, coral polyp feeders and omnivores). Geometric morphometrics were used to quantify size and shape variations in four skeletal units: (1) neurocranium, (2) suspensorium and opercle, (3) mandible and (4) premaxilla. This method allowed us to reveal shape and size differences correlated to functional diversity both within and between trophic guilds. Among zooplanktivores, C. margaritifer, D. aruanus and D. flavicaudus have a high and long supraoccipital crest, short mandibles forming a small mouth and high suspensoria and opercles. These three species can be considered to be suction feeders. In the same guild, C. acares shows opposite characteristics (long and thin mandibles, lengthened neurocranium and suspensorium) and can be considered as a ram feeder. Among herbivores and corallivores, the two species of Plectroglyphidodon and S. nigricans can be considered as grazers. Differences in skeletal shape are mainly related to improving the robustness of some skeletal parts (broad hyomandibular, short and high mandibles). The shapes of P. pavo, which feeds largely on algae, strongly differ from that of the other three grazers exhibiting similar morphological characteristics to C. acares (e.g., long and shallow suspensorium, lengthened neurocranium). This highlights likely differences concerning cutting or scraping method. Finally, no strong correlations exist between size and shapes in the eight studied species. Size difference among species having a very similar shape could be viewed as a factor optimizing resource partitioning. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Rasch EM, Wyngaard GA, Connelly BA
Heterochromatin endoreduplication prior to gametogenesis and chromatin diminution during early embryogenesis in Mesocyclops edax (Copepoda: Crustacea).
J Morphol. 2007 Oct 11;
The segregation of progenitor somatic cells from those of the primordial germ cells that sequester and retain elevated levels of DNA during subsequent developmental events, poses an interesting, alternative pathway of chromosome behavior during the reproductive cycle of certain species of cyclopoid copepods and several other organisms. Separation of maternal and paternal chromosome sets during very early cleavages (gonomery) is often a feature following marked elevations of DNA levels in germ cells for some of these species. Here, we report on the accumulation of large amounts of DNA in germ line nuclei of both female and male juveniles and adults of a freshwater copepod, Mesocyclops edax (Forbes, 1890). We also report the robust uptake of (3)H-thymidine by germ cells prior to gametogenesis in this species. By using cytophotometric analysis of the DNA levels in both germ line cells and somatic cells from the same specimens we demonstrate that germ cell nuclei accumulate high levels of DNA prior to the onset of gametogenesis. These elevated amounts coincide with the levels of heterochromatic DNA discarded during chromatin diminution. A new model is proposed of major cytological events accompanying the process of chromatin diminution in M. edax. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Kieselbach D, Hausen H
Chaetal arrangement provides no support for a close relationship of Sabellidae and Sabellariidae (Annelida).
J Morphol. 2007 Oct 9;269(1):
Sabellid and sabellariid polychaetes are regarded as sister groups in a number of recent phylogenetic analyses. This is based mainly on a shared specific arrangement of chaetae referred to as chaetal inversion. Remarkably, the uncini have a notopodial position in the abdomen, whereas capillary chaetae occur in the neuropodia in both taxa in contrast to the situation in putative relatives. However, in sabellids uncini and capillary chaetae change their position completely at the border between thorax and abdomen, whereas uncini are missing in the parathorax of Sabellariidae. Due to this difference the significance of the chaetal inversion for systematics has been subject to discussion for years. Serial semithin sections of parapodia of the Sabellidae Sabella pavonina, Branchiomma bombyx, Fabricia stellaris, and of the Sabellariidae Sabellaria alveolata were studied in order to obtain detailed information on their chaetal arrangement and sites of chaetal origin. SEM investigations and computer-aided 3D-reconstructions provide deep insight into the spatial organization of the rami. Though differing externally, the principal chaetal arrangement and the location of the formative sites turned out to be almost identical within the species of Sabellidae. Most chaetae are aligned in straight transverse rows with a dorsal site of origin within neuropodia and a ventral one in notopodia as is common in sedentary polychaetes. Semicircular and spiral arrangements are revealed to be modified transverse rows. Only in thoracic notopodia does an additional dorsocaudal formative site form distinct rows. The chaetal inversion in Sabellidae is additionally characterized by an abrupt change of capillary chaetae and uncini along with a sudden change of the parapodial morphology at the border between thorax and abdomen. All chaetae of S. alveolata are aligned in transverse rows with the same location of the formative sites as in sabellids and other sedentary polychaetes. However, in contrast to sabellids the chaetae are not inverted across a parathoracic abdominal border. Moreover, there is no inversion of the parapodial structure from parathorax to abdomen and the neuropodial chaetal composition changes gradually from parathorax to abdomen. The chaetal arrangement in Sabellariidae thus cannot be described as inverted along the body-axis as in Sabellidae. Evolutionary steps implied by the assumption of an inverted chaetal pattern in a supposed common ancestor are discussed. It is concluded that the specific chaetal arrangement of Sabellidae and Sabellariidae arose independently and therefore provides no support for a sistergroup relationship of sabellids and sabellariids. J. Morphol., 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Huja SS, Rummel AM, Beck FM
Changes in mechanical properties of bone within the mandibular condyle with age.
J Morphol. 2007 Oct 9;
The purpose of the study was to compare indentation modulus (IM) and hardness of condylar bone in young and adult dogs. In addition we desired to examine histologic sections for bone formation activity in the two groups. Mandibular condyles were obtained from adult (1- to 2-year-old) and young ( approximately 5-m old) dogs. Two sections/condyle were obtained and one was processed for histomorphometry and the other for mechanical analyses. Indents were made on moist condylar trabecular bone to a depth of 500 nm at a loading rate of 10 nm/s using a custom-made hydration system to obtain IM and hardness. Histomorphometric analyses measured the bone volume/total volume (BV/TV%) and ratio of labeled to unlabeled bone within the condyle. Data were analyzed using a repeated-measures factorial analysis of variance and Tukey-Kramer method. Overall, the IM of the adult condyles (10.0 +/- 3.4 GPa, Mean +/- SD) were significantly (P < 0.0001) higher than in young dogs (5.6 +/- 2.6 GPa). There was a greater bone mass in the young (60.2%) versus the adult condyles (42%). Also, significantly more labeled bone in the young (66.1%) condylar bone suggested higher bone forming activity than in adult condyles (27.5%). With age there is a change in mass and material properties in the trabecular bone of the mandibular condyle in dogs. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Schnell NK, Bernstein P, Maier W
The "pseudo-craniovertebral articulation" in the deep-sea fish Stomias boa (Teleostei: Stomiidae).
J Morphol. 2007 Oct 9;
Many predatory deep-sea fishes show highly specialized modifications of their feeding apparatus, e.g., elongate jaws studded with long daggerlike teeth, often combined with a very distensible stomach, to be capable of swallowing relatively large prey. These striking features can be observed in members of the marine teleost family Stomiidae. The present study gives a detailed morphological description of the mesopelagic predatory fish, Stomias boa, based on a combined approach of clearing and double staining, serial sections and dissection. In this genus, large pads made of dense connective tissue extend from the first enlarged neural arch to the ventral side of the chordal sheath, embracing the prominent exoccipitals and thus constituting a kind of double ball- and socket joint for the head. The notochordal occipito-vertebral gap is enlarged, probably not by loss of vertebral centra as is proposed for other genera of the stomiid family, e.g., in Astronesthes or Photostomias. We conclude that this "pseudo-craniovertebral articulation" serves as a functional substitute for the absent vertebrae and strengthens the flexible, anterior part of the vertebral column during extreme dorsal expansion of the gape during prey capture and swallowing. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

Quagliata S, Pacini S, Punzi T, Malentacchi C, Ruggiero M, Delfino G
Bombesin promotes vasculogenesis and angiogenesis in chick chorio-allantoic membrane: A morphometric, structural, and ultrastructural study.
J Morphol. 2007 Sep 27;269(1):
Experiments were performed on the chorio-allantoic membrane (CAM) of the chick to evaluate the effects of bombesin (BN) on vascular neoformation. In morphometrical assays, 10(-13)-10(-4) M BN promoted dose-dependent vascular development. Newly formed vessels converged toward the BN release site in a spoked wheel arrangement, suggesting a diffusion gradient mechanism. Structural and ultrastructural analysis of CAM specimens collected near the BN release site showed that both vasculogenetic and angiogenetic processes cooperated in vascular neoformation that involved committed cells from the mesenchyme (angioblasts) as well as endothelial cells. No pattern of vascular development was detected away from the BN release site. Findings from the present study emphasize the role of BN in vascular net development of respiratory organs. J. Morphol., 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Eastman JT, Lannoo MJ
Brain and sense organ anatomy and histology of the Falkland Islands mullet, Eleginops maclovinus (Eleginopidae), the sister group of the Antarctic notothenioid fishes (Perciformes: Notothenioidei).
J Morphol. 2007 Sep 27;269(1):
The perciform notothenioid fish Eleginops maclovinus, representing the monotypic family Eleginopidae, has a non-Antarctic distribution in the Falkland Islands and southern South America. It is the sister group of the five families and 103 species of Antarctic notothenioids that dominate the cold shelf waters of Antarctica. Eleginops is the ideal subject for documenting the ancestral morphology of nervous and sensory systems that have not had historical exposure to the unusual Antarctic thermal and light regimes, and for comparing these systems with those of the phyletically derived Antarctic species. We present a detailed description of the brain and cranial nerves of Eleginops and ask how does the neural and sensory morphology of this non-Antarctic notothenioid differ from that seen in the phyletically derived Antarctic notothenioids? The brain of Eleginops is similar to those of visually oriented temperate and tropical perciforms. The tectum is smaller but it has well-developed olfactory and mechanoreceptive lateral line areas and a large, caudally projecting corpus cerebellum. Eye diameter is about twofold smaller in Eleginops than in many Antarctic species. Eleginops has a duplex (rod and cone) retina with single and occasional twin cones conspicuous centrally. Ocular vascular structures include a large choroid rete mirabile and a small lentiform body; a falciform process and hyaloid arteries are absent. The olfactory rosette is oval with 50-55 lamellae, a large number for notothenioids. The inconspicuous bony canals of the cephalic lateral line system are simple with membranous secondary branches that lack neuromasts. In Antarctic species, the corpus cerebellum is the most variable brain region, ranging in size from large and caudally projecting to small and round. "Stalked" brains showing reduction in the size of the telencephalon, tectum, and corpus cerebellum are present in the deep-living artedidraconid Dolloidraco longedorsalis and in most of the deep-living members of the Bathydraconini. Eye diameter is generally larger in Antarctic species but there is a phylogenetic loss of cellularity in the retina, including cone photoreceptors. Some deep-living Antarctic species have lost most of their cones. Mechanosensation is expanded in some species, most notably the nototheniid Pleuragramma antarcticum, the artedidraconid genera Dolloidraco and Pogonophryne, and the deep living members of the bathydraconid tribe Bathydraconini. Reduction in retinal cellularity, expansion of mechanoreception, and stalking are the most noteworthy departures from the morphology seen in Eleginops. These features reflect a modest depth or deep-sea effect, and they are not uniquely "Antarctic" attributes. Thus, at the level of organ system morphology, perciform brain and sensory systems are suitable for conditions on the Antarctic shelf, with only minor alterations in structure in directions exhibited by other fish groups inhabiting deep water. Notothenioids retain a relative balance among their array of senses that reflects their heritage as inshore perciforms. J. Morphol., 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Danos N, Fisch N, Gemballa S
The musculotendinous system of an anguilliform swimmer: Muscles, myosepta, dermis, and their interconnections in Anguilla rostrata.
J Morphol. 2007 Sep 21;269(1):
Eel locomotion is considered typical of the anguilliform swimming mode of elongate fishes and has received substantial attention from various perspectives such as swimming kinematics, hydrodynamics, muscle physiology, and computational modeling. In contrast to the extensive knowledge of swimming mechanics, there is limited knowledge of the internal body morphology, including the body components that contribute to this function. In this study, we conduct a morphological analysis of the collagenous connective tissue system, i.e., the myosepta and skin, and of the red muscle fibers that sustain steady swimming, focusing on the interconnections between these systems, such as the muscle-tendon and myosepta-skin connections. Our aim is twofold: (1) to identify the morphological features that distinguish this anguilliform swimmer from subcarangiform and carangiform swimmers, and (2) to reveal possible pathways of muscular force transmission by the connective tissue in eels. To detect gradual morphological changes along the trunk we investigated anterior (0.4L), midbody (0.6L), and posterior body positions (0.75L) using microdissections, histology, and three-dimensional reconstructions. We find that eel myosepta have a mediolaterally oriented tendon in each the epaxial and hypaxial regions (epineural or epipleural tendon) and two longitudinally oriented tendons (myorhabdoid and lateral). The latter two are relatively short (4.5-5% of body length) and remain uniform along a rostrocaudal gradient. The skin and its connections were additionally analyzed using scanning electron microscopy (SEM). The stratum compactum of the dermis consists of approximately 30 layers of highly ordered collagen fibers of alternating caudodorsal and caudoventral direction, with fiber angles of 60.51 +/- 7.05 degrees (n = 30) and 57.58 +/- 6.92 degrees (n = 30), respectively. Myosepta insert into the collagenous dermis via fiber bundles that pass through the loose connective tissue of the stratum spongiosum of the dermis and either weave into the layers of the stratum compactum (weaving fiber bundles) or traverse the stratum compactum (transverse fiber bundles). These fiber bundles are evenly distributed along the insertion line of the myoseptum. Red muscles insert into lateral and myorhabdoid myoseptal tendons but not into the horizontal septum or dermis. Thus, red muscle forces might be distributed along these tendons but will only be delivered indirectly into the dermis and horizontal septum. The myosepta-dermis connections, however, appear to be too slack for efficient force transmission and collagenous connections between the myosepta and the horizontal septum are at obtuse angles, a morphology that appears inadequate for efficient force transmission. Though the main modes of undulatory locomotion (anguilliform, subcarangiform, and carangiform) have recently been shown to be very similar with respect to their midline kinematics, we are able to distinguish two morphological classes with respect to the shape and tendon architecture of myosepta. Eels are similar to subcarangiform swimmers (e.g., trout) but are substantially different from carangiform swimmers (e.g., mackerel). This information, in addition to data from kinematic and hydrodynamic studies of swimming, shows that features other than midline kinematics (e.g., wake patterns, muscle activation patterns, and morphology) might be better for describing the different swimming modes of fishes. J. Morphol., 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Sobotník J, Alberti G, Weyda F, Hubert J
Ultrastructure of the digestive tract in Acarus siro (Acari: Acaridida).
J Morphol. 2007 Sep 21;269(1):
The gut of the mite Acarus siro is characterized on the ultrastructural level. It consists of the foregut (pharynx, esophagus), midgut (ventriculus, caeca, colon, intercolon, postcolonic diverticula, postcolon), and hindgut (anal atrium). The gut wall is formed by a single-layered epithelium; only regenerative cells are located basally and these have no contact with the lumen. Eight cell types form the whole gut: (i) simple epithelial cells forming fore- and hindgut; (ii) cells that probably produce the peritrophic membrane; (iii) regenerative cells occurring in the ventriculus, caeca, colon, and intercolon; (iv) spherite cells and (v) digestive cells forming the ventriculus and caeca; (vi) colonic cells and (vii) intercolonic cells; and (viii) cells forming the walls of postcolonic diverticula and postcolon. Spherite and digestive cells change in structure during secretory cycles, which are described and discussed. The cycle of spherite, colonic, and intercolonic cells is terminated by apoptosis. Ingested food is packed into a food bolus surrounded by a single homogeneous peritrophic membrane formed by addition of lamellae that subsequently fuse together. The postcolonic diverticula serve as a shelter for filamentous bacteria, which also are abundant in the intercolon. J. Morphol., 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Spa?ek-Wo?czy?ska A, Klag J, Bielecki A, Swi?tek P
Oogenesis in four species of Piscicola (Hirudinea, Rhynchobdellida).
J Morphol. 2007 Sep 21;269(1):
Piscicola has a pair of elongated sac-shaped ovaries. Inside the ovaries are numerous small somatic cells and regularly spherical egg follicles. Each follicle is composed of three types of cells: many (average 30) germ cells (cystocytes) interconnected by intercellular bridges in clones (cysts), one intermediate cell, and three to five outer follicle cells (envelope cells). Each germ cell in a clone has one intercellular bridge connecting it to the central anucleate cytoplasmic mass, the cytophore. Each cluster of germ cells is completely embedded inside a single huge somatic follicle cell, the intermediate (interstitial) cell. The most spectacular feature of the intermediate cell is its development of a system of intracytoplasmic canals apparently formed of invaginations of its cell membrane. Initially the complex of germ cell cluster + intermediate cell is enclosed within an envelope composed of squamous cells. As oogenesis progresses the envelope cells gradually degenerate. All the germ cells that have terminated their mitotic divisions are of similar size and enter meiotic prophase, but one of the cystocytes promptly starts to grow faster and differentiates into the oocyte, whereas the remaining cystocytes withdraw from meiosis and become nurse cells (trophocytes). Numerous mitochondria, ER, and a vast amount of ribosomes are transferred from the trophocytes via the cytophore toward the oocyte. Eventually the oocyte ingests all the content of the cytophore, and the trophocytes degenerate. Little vitellogenesis takes place; the oocyte gathers nutrients in the form of small lipid droplets. At the end of oogenesis, an electron-dense fibrous vitelline envelope appears around the oocyte, among short microvilli. At the same time, electron-dense cortical granules occur in the oocyte cortical cytoplasm; at the end of oogenesis they are numerous, but after fertilization they disappear from the ooplasm. In the present article we point out many differences in the course of oogenesis in two related families of rhynchobdellids: piscicolids and glossiphoniids. J. Morphol., 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Sjölin E, Gustavsson LM
An ultrastructural study of the cuticle in the marine annelid Heterodrilus (Tubificidae, Clitellata).
J Morphol. 2007 Sep 21;269(1):
The ultrastructure of the cuticle in four species of the marine Heterodrilus (H. paucifascis, H. pentcheffi, H. flexuosus, H. minisetosus) is investigated with transmission electron microscopy. The noncellular cuticle consists of several parts; closest to the epidermis is a thick zone of collagen fibers embedded in a matrix. The matrix continues outside the fiber zone, forming a layered epicuticle. The external surface of the epicuticle is covered by evenly distributed, membrane-bound bodies, termed epicuticular projections. The epicuticular projections have their longitudinal axis perpendicular to the surface of the cuticle and are attached to the surface by either the surrounding membrane itself or by short pedestals. Microvilli, extensions from the epidermal cells, penetrate and sometimes pass completely through the cuticle. There is interspecific variation in the morphology of the cuticle. The four studied species differ in the arrangement of the collagen fibers, from irregularly distributed fibril bundles to orthogonally arranged fiber layers, as well as in the number and density of layers in the epicuticle. One of the studied species, H. paucifascis, shows intraspecific variation, which is associated with sample locality. The Bahamian specimens of H. paucifascis have four layers in the epicuticle, club-shaped epicuticular projections, and collagen fibers forming a less defined orthogonal grid, while the Belizean specimens have three layers in the epicuticle, epicuticular projections with a bulging part at midlevel, and a distinct orthogonal grid. Based on these findings the variation in the morphology of the cuticle appears to be dependent on both phylogenetic constraints, and functional and environmental factors. J. Morphol., 2008. (c) 2007 Wiley-Liss, Inc. [Abstract]

Tsuihiji T
Homologies of the longissimus, iliocostalis, and hypaxial muscles in the anterior presacral region of extant diapsida.
J Morphol. 2007 Nov;268(11):
Homologies of muscles of the m. longissimus and m. iliocostalis groups in the dorsal and cervical regions, as well as those of the subvertebral muscles and mm. intercostales externi that continue from the dorsal into the cervical regions, in extant Diapsida are proposed based on detailed dissections and published accounts of lepidosaurs, crocodylians, and birds. The morphology of tendons and innervation patterns suggest that the avian "m. iliocostalis" in the dorsal region include the homologs of both m. longissimus and m. iliocostalis in non-avian diapsids. The conserved nature of the morphology of tendons in palaeognath birds also revealed that the avian mm. intertransversarii in the cervical region consist of muscles of the both m. longissimus and m. iliocostalis groups despite having been treated as a single series of muscles, and thus are not homologous with muscles of the same name in Lepidosauria or Crocodylia. The avian mm. inclusi that lie medial to mm. intertransversarii are homologous with mm. intercostales externi in Lepidosauria and mm. intercostales externi and m. scalenus combined in Crocodylia. Innervation patterns suggest that a muscle ("m. iliocostalis capitis") connecting the atlas rib and occiput in Crocodylia includes contributions from the subvertebral layer and m. cucullaris complex, and possibly m. iliocostalis as well. The present findings may serve as a basis for revising the currently used avian nomenclature so that it will reflect homologies of muscles with their non-avian counterparts. [Abstract]

Claeson KM, Bemis WE, Hagadorn JW
New interpretations of the skull of a primitive bony fish Erpetoichthys calabaricus (Actinopterygii: Cladistia).
J Morphol. 2007 Nov;268(11):
Polypterid fishes are considered the basalmost group of extant actinopterygians and may be a direct link to understanding the systematics and evolution of the first bony fishes. Several investigations have been conducted on one member genus, Polypterus; however, since the first specimens of its sister taxon Erpetoichthys calabaricus were described, remarkably little work has been done on the species. We review terminology critical to understanding cranial morphology in polypterids and present a new description of the skull of E. calabaricus as observed through classical methods of skeletal preparation, X-radiographic microfocus computed tomography, and 3D-digital reconstruction. Differences among E. calabaricus and at least three species of Polypterus (P. bichir, P. senegalus, and P. endlicheri), besides the gross variation in size, include an overall elongation of the skull roof observable in most elements of E. calabaricus with a shortening of most associated processes. In addition, several elements present in species of Polypterus are absent in E. calabaricus. As a result, Polypterus should not be used as a proxy for the family Polypteridae to the exclusion of E. calabaricus in phylogenetic studies, which examine early actinopterygians. Each should be treated separately, to resolve inter- and intrarelationships of Polypteridae. [Abstract]

Rorandelli R, Paoli F, Cannicci S, Mercati D, Giusti F
Characteristics and fate of the spermatozoa of Inachus phalangium (Decapoda, Majidae): Description of novel sperm structures and evidence for an additional mechanism of sperm competition in brachyura.
J Morphol. 2007 Sep 5;
Various aspects of the reproductive anatomy of the spider crab Inachus phalangium are investigated utilizing light and electron microscopy. Spermatozoal ultrastructure reveals the presence of a glycocalyx in the peripheral region of the periopercular rim, never recorded before in crustacean sperm cells. Sperm cell morphological traits such as semi-lunar acrosome shape, centrally perforate and flat operculum, and absence of a thickened ring, are shared only with Macropodia longirostris, confirming a close phylogenetic relationship of these species and their separation from the other members of the family Majidae. Spermatozoa are transferred to females inside spermatophores of different sizes, but during ejaculate transfer, larger spermatophores might be ruptured by tooth-like structures present on the ejaculatory canal of the male first gonopod, releasing free sperm cells. Such a mechanism could represent the first evidence of a second form of sperm competition in conflict with sperm displacement, the only mechanism of sperm competition known among Brachyura, enabling paternity for both dominant and smaller, non-dominant, males. In addition, we propose several hypotheses concerning the remote and proximal causes of the existence of large seminal receptacles in females of I. phalangium. Among these, genetically diverse progeny, reduction of sexual harassment and phylogenetic retention seem the most plausible, while acquisition of nutrients from seminal fluids, demonstrated in other arthropods, and suggested by previous studies, could be discarded on the basis of the presented data. J. Morphol., 2007. (c) 2007 Wiley-Liss, Inc. [Abstract]

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Recent Articles in Anatomy and Embryology

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Bennis M, Repérant J, Ward R, Rio JP, M'hamed SB, Jay B
The postnatal development of the optic nerve of a reptile (Vipera aspis): A quantitative ultrastructural study.
Anat Embryol (Berl). 2006 Nov;211(6):691-705.
The number of axons in the optic nerve of the ovoviviparous reptile Vipera aspis was estimated from electron micrographs taken during the first 5 weeks of postnatal life. One to two days after birth, the optic nerve contains about 170,000 fibres, of which about 9% are myelinated. At the end of the fifth postnatal week, the number of optic fibres has fallen to about 100,000, of which about 42% are myelinated. This fibre loss continues after the fifth postnatal week, since in the adult viper the nerve contains about 60,000 fibres, of which 85% are myelinated; overall, about 65% of the optic nerve fibres present at birth disappear before the number of axons stabilises at the adult level. This study shows, for the first time, that the mode of development of the visual axons of reptiles is not that of anamniote vertebrates but similar to that of birds and mammals. [Abstract]

Kritzenberger M, Wrobel KH
Immunophenotyping and spatio-temporal distribution of aortic cell clusters in the bovine embryo.
Anat Embryol (Berl). 2006 Nov;211(6):739-55.
In the present study the temporal and spatial appearance of aortic cell clusters in bovine embryos is described. Aorta-associated c-kit-positive cell clusters can be observed first in 23 days post inseminationem (dpi) bovine embryos and disappear after 34 dpi. For the first time, it was shown that the immunophenotype of these aortic cluster cells changes during embryonic development. Aortic cell clusters are c-kit+/CD45-/STA-, when they are first detected in the 23 dpi embryo, and acquire a c-kit+/CD45+/STA- phenotype in 27-29 embryos and a c-kit+/CD45+/STA+ immunophenotype in 32-34-day-old specimens. Cell clusters are most prominent in the vicinity of lateral and ventral aortic branches, but rare in omphalomesenteric arteries and absent in Aa. umbilicales. Free c-kit-positive cells in an intravasal position are common, suggesting separation from the clusters in order to colonize subsequent hematopoietic organs, i.e., the liver and the mesonephros. Transmission electron microscopic analysis reveals the existence of primitive desmosomes between the clusters cells and adjacent endothelial cells as well as a fine basal lamina as a demarcation between the cluster cells and underlying mesenchymal cells. Material resembling extracellular matrix is found in large vacuoles in cluster cells of 23 dpi embryos. Immunocytochemistry reveals an intense accumulation of heparan sulfate proteoglycan and collagen IV in the aortic wall at the sites where cell clusters are attached. These observations suggest that the hematopoietic cell clusters induce the formation of a specific microenvironment within the aortic wall. [Abstract]

Usunoff KG, Itzev DE, Rolfs A, Schmitt O, Wree A
Nitric oxide synthase-containing neurons in the amygdaloid nuclear complex of the rat.
Anat Embryol (Berl). 2006 Nov;211(6):721-37.
The nitric oxide-producing neurons in the rat amygdala (Am) were studied, using reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. Almost all nuclei of the Am contained NADPHd-positive neurons and fibers, but the somatodendritic morphology and the intensity of staining of different subpopulations varied. The strongly stained neurons displayed labeling of the perikaryon and the dendritic tree with Golgi impregnation-like quality, whilst the dendrites of the lightly stained neurons were less successfully followed. Many strongly positive neurons were located in the external capsule and within the intraamygdaloid fiber bundles. A large number of small, strongly stained cells was present in the amygdalostriatal transition area. In the Am proper, a condensation of deeply stained cells occurred in the lateral amygdaloid nucleus. In the basolateral nucleus, the strongly NADPHd-positive neurons were few, and were located mainly along the lateral border of the nucleus. These cells clearly differed from the large, pyramidal, and efferent cells. The basomedial nucleus contained numerous positive cells but most of them were only lightly labeled. A moderate number of strongly stained neurons appeared in the medial division of the central nucleus, and a larger accumulation of strongly positive cells was present in the lateral and the capsular divisions. The medial amygdaloid nucleus contained numerous moderately stained neurons and displayed the strongest diffuse neuropil staining in Am. In the nucleus of the lateral olfactory tract, the first layer contained only NADPHd-stained axons, in the second layer, there were numerous moderately stained cells, and in the third layer, a few but deeply stained neurons. From the cortical nuclei, the most appreciable number of stained neurons was seen in the anterior cortical nucleus. The anterior amygdaloid area contained numerous NADPHd-positive neurons; in its dorsal part the majority of cells were only moderately stained, whereas in the ventral part the neurons were very strongly stained. The intercalated amygdaloid nucleus lacked NADPHd-positive neurons but an appreciable plexus of fine, tortuous axons was present. In the intra-amygdaloid part of the bed nucleus of the stria terminalis (st) some lightly stained cells were seen but along the entire course of st strongly stained neurons were observed. Some Am nuclei, and especially the central lateral nucleus and the intercalated nucleus, display considerable species differences when compared with the primate Am. The age-related changes of the nitrergic Am neurons, as well as their involvement in neurodegenerative diseases is discussed. [Abstract]

Fukunishi K, Sawada K, Kashima M, Sakata-Haga H, Fukuzaki K, Fukui Y
Development of cerebral sulci and gyri in fetuses of cynomolgus monkeys (Macaca fascicularis).
Anat Embryol (Berl). 2006 Nov;211(6):757-64.
This study aimed to clarify the development of sulci and gyri on the external surface of the cerebrum of cynomolgus monkeys. Sulcus formation began with the appearance of the lateral fissure on embryonic day (ED) 70, followed by delineations of four cerebral lobes by the emergence of the parietooccipital sulcus, central sulcus, and preoccipital notch on EDs 80-90. The following primary sulci were then visible until ED 120: the superior temporal sulcus on ED 90; the intraparietal sulcus, lunate sulcus, inferior occipital sulcus, and arcuate sulcus on ED 100; and the principle sulcus on ED 110; the occipitotemporal sulcus, anterior middle temporal sulcus, and superior postcentral dimple on ED 120. These sulci demarcated the superior temporal gyrus on ED 90, the precentral gyrus, supramarginal gyrus, and angular gyrus on ED 100, and the inferior and middle temporal gyri, postocentral gyrus, superior parietal lobule, superior, middle and inferior frontal gyri, and inferior occipital gyrus on ED 120. Except for the intermediate and lateral orbitofrontal sulci, the sulci that appeared on ED 130 and thereafter were not related to the gyrus demarcations. Intriguingly, the brain markedly gained weight on EDs 100 and 120, corresponding to the embryonic ages when almost all gyri were visible. The results suggest that a rapid growth of the cerebrum involves convolutions of the gyri by a regular sequence of the sulcus formation in cynomolgus monkeys. This study further provides a standard of reference for normal development in the cerebral cortical morphology of cynomolgus monkeys. [Abstract]

Wilting J, Becker J
Two endothelial cell lines derived from the somite.
Anat Embryol (Berl). 2006 Dec;211 Suppl 157-63.
Somites are sequentially formed, metameric units of the paraxial mesoderm of vertebrate embryos. They are the most obvious correlative of the segmental patterning along the cranio-caudal axis and transfer segmentation to other tissues such as the spinal nerves and dorsal aortic branches. Furthermore, somites are the source of numerous mesodermal cell types such as smooth and striated muscle, cartilage and tendon cells, and soft connective tissue. They also give rise to endothelial cells. Here we focus on the finding that two lineages of endothelial cells, blood vascular endothelial cells and lymphatic endothelial cells are derived from the somite. Their precursors, angioblasts, and lymphangioblasts, respectively, are born in the somite at different time points. Angioblasts are characterized by the expression of vascular endothelial growth factor receptor-2, whereas lymphangioblasts express the homeobox transcription factor Prox1. There seem to be two types of lymphangioblasts. Type 1 is derived from venous endothelium, while type 2 originates from mesenchymal precursor cells. The molecular networks of angioblast and lymphangioblast development and the relation between the two cell types and hematopoietic cells are discussed. [Abstract]

Rupik W, Stawierej A, Stolarczyk I, Wid?ak W
Promoter of the heat shock testis-specific Hsp70.2/Hst70 gene is active in nervous system during embryonic development of mice.
Anat Embryol (Berl). 2006 Nov;211(6):631-8.
The Hsp70.2/Hst70 gene is a unique member of the 70 kDa heat shock proteins multigene family whose activity is regulated developmentally; in adult mice and rats its expression is restricted mostly to meiotic and postmeiotic male germ cells. In aim to analyze activity of the Hsp70.2/Hst70 promoter in developing embryos we have constructed transgenic mice expressing EGFP reporter gene under control of the rat Hst70 promoter. The appearance of EGFP fluorescence coincides with series of major developmental events, such as extra-embryonic membranes formation, axial rotation, formation of neural tube and the primordium of central nervous system, formation of differentiated somites, extensive remodeling of the heart, development of fingers and toes, and sensory organs formation. Activity of the Hst70 promoter localizes mostly inside nervous system indicating the role of Hsp70.2/Hst70 gene in development of this system. [Abstract]

Bombardi C, Grandis A, Chiocchetti R, Lucchi ML
Distribution of calbindin-D28k, neuronal nitric oxide synthase, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the lateral nucleus of the sheep amygdaloid complex.
Anat Embryol (Berl). 2006 Nov;211(6):707-20.
This study describes calbindin-D28k (CB), neuronal nitric oxide synthase (nNOS), and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) expression in the lateral nucleus of the sheep amygdaloid complex. Double immunofluorescence protocol was used in order to determine whether there is colocalization of CB and nNOS. The CB-immunoreactive (IR) neuronal population was composed especially of non-pyramidal neurons, but a few pyramidal cells were also present. The non-pyramidal neurons showed a multipolar and, occasionally, a fusiform morphology. The comparison between single-labeled CB-IR non-pyramidal neurons and cells belonging to CB-IR neuronal population showed they were identical for morphology, mean size, and distribution. The single-labeled CB-IR non-pyramidal neurons were only the 17.8% of the total non-pyramidal neurons counted. The nNOS-IR neuronal population was represented by non-pyramidal multipolar and fusiform neurons. Single-labeled nNOS-IR non-pyramidal neurons had the same morphology, mean area, and distribution as cells belonging to nNOS-IR neuronal population. Single-labeled nNOS-IR non-pyramidal neurons were more numerous than single-labeled CB-IR, and represented the 73.7% of total non-pyramidal neurons counted. NADPH-d-positive cells had the same morphology and distribution as the nNOS-IR neurons. Double immunolabeling (CB/nNOS) was found mostly in non-pyramidal multipolar neurons and only in a few non-pyramidal fusiform cells. These neurons had a mean perikaryal area significantly higher and significantly smaller than that of single-labeled nNOS and single-labeled CB-IR non-pyramidal neurons, respectively. CB and nNOS coexist only in a minority of non-pyramidal neurons (8.5%). The 32.4% of all CB-IR non-pyramidal neurons were nNOS-positive; only 10.4% of nNOS-IR non-pyramidal neurons were CB-positive. These results indicate that CB and nNOS are expressed by selective neurons and that the majority of nNOS-IR non-pyramidal neurons are lacking in CB. [Abstract]

Evans DJ, Valasek P, Schmidt C, Patel K
Skeletal muscle translocation in vertebrates.
Anat Embryol (Berl). 2006 Dec;211 Suppl 143-50.
It is now over 30 years since Bodo Christ first demonstrated that the musculature of the limb originated from the somites and overturned the then prevailing view that limb muscle develops from a local source. Subsequently, using electron microscopy and histological procedures, Bodo Christ identified that cells of the somites undergo an epithelial to mesenchymal transition which enabled them to move from their paraxial point of origin to distal locations. These studies defined this translocation as one of the major mechanisms allowing myogenic cells to translocate around the body. The other means used to translocate muscle involves the movement of cells as a sheet. The deployment of one of these two mechanisms has been postulated to be involved in the formation of all the hypaxial musculature of the vertebrate body. In this paper we describe the formation of muscles both in the head and in the body, which use a translocatory mechanism during their development. We highlight recent data showing that muscle translocation is a far more complex process than first thought but which in itself can be used as a valuable tool to address questions regarding tissue patterning and development. [Abstract]

Buckingham M, Bajard L, Daubas P, Esner M, Lagha M, Relaix F, Rocancourt D
Myogenic progenitor cells in the mouse embryo are marked by the expression of Pax3/7 genes that regulate their survival and myogenic potential.
Anat Embryol (Berl). 2006 Dec;211 Suppl 151-6.
The transcription factors Pax3 and Pax7 are important regulators of myogenic cell fate, as demonstrated by genetic manipulations in the mouse embryo. Pax3 lies genetically upstream of MyoD and has also been shown recently to directly control Myf5 transcription in derivatives of the hypaxial somite, where it also plays an important role in ensuring cell survival. Both Pax3 and Pax7 are expressed in myogenic progenitor cells derived from the central dermomyotome that make a major contribution to skeletal muscle growth. In Pax3/Pax7 double mutants, the myogenic determination genes, Myf5 and MyoD, are not activated in these cells which become incorporated into other tissues or die. This again demonstrates the dual function of Pax factors in regulating the entry of progenitor cells into the myogenic programme and in ensuring their survival. Pax3 expression marks cells in the dermomyotome that either become myogenic or downregulate Pax3 and assume another cell fate. The latter include the smooth muscle cells of the dorsal aorta that share a common clonal origin with the skeletal muscle of the myotome, thus illustrating the initial multipotency of Pax3 expressing cells. [Abstract]

Patel K, Scaal M
Special issue dedicated to Bodo Christ.
Anat Embryol (Berl). 2006 Dec;211 Suppl 11-2. [Abstract]

Yusuf F, Brand-Saberi B
The eventful somite: patterning, fate determination and cell division in the somite.
Anat Embryol (Berl). 2006 Dec;211 Suppl 121-30.
The segmental somites not only determine the vertebrate body plan, but also represent turntables of cell fates. The somite is initially naive in terms of its fate restriction as shown by grafting and rotation experiments whereby ectopically grafted or rotated tissue of newly formed somites yielded the same pattern of normal derivatives. Somitic derivatives are determined by local signalling between adjacent embryonic tissues, in particular the neural tube, notochord, surface ectoderm and the somitic compartments themselves. The correct spatio-temporal specification of the deriving tissues, skeletal muscle, cartilage, endothelia and connective tissue is achieved by a sequence of morphogenetic changes of the paraxial mesoderm, eventually leading to the three transitory somitic compartments: dermomyotome, myotome and sclerotome. These structures are specified along a double gradient from dorsal to ventral and from medial to lateral. The establishment and controlled disruption of the epithelial state of the somitic compartments are crucial for development. In this article, we give a synopsis of some of the most important signalling events involved in somite patterning and cell fate decisions. Particular emphasis has been laid on the issue of epithelio-mesenchymal transition and different types of cell division in the somite. [Abstract]

Phillips RJ, Rhodes BS, Powley TL
Effects of age on sympathetic innervation of the myenteric plexus and gastrointestinal smooth muscle of Fischer 344 rats.
Anat Embryol (Berl). 2006 Nov;211(6):673-83.
Loss of myenteric neurons with age is well documented, however little is known about age-related changes of the sympathetic innervation of the myenteric plexus and gastrointestinal smooth muscle. The goal of the present study, therefore, was to evaluate the influence of age on the sympathetic innervation of the myenteric plexus throughout the gastrointestinal tract. Ad libitum fed virgin male Fischer 344 rats at 3, 15-16, 24, and 27-28 months of age were sampled. Whole mounts of the stomach, small intestine, and large intestine were processed with an antibody to tyrosine hydroxylase (TH). Additionally, some specimens labeled for TH were stained for NADPH-diaphorase to selectively label the nitrergic subpopulation of neurons in the myenteric plexus. Age-related changes in the TH-positive axons occurred as early as 15-16 months and became more pronounced by 27-28 months. Changes included markedly swollen axons and terminals and a decrease in the intensity of TH staining in some of the surviving processes. Similarly, swollen NADPH-diaphorase-positive axons were found in the myenteric ganglia and secondary plexus between ganglia in the whole mounts of rats 15-28 months of age, but swollen nitrergic axons and dystrophic TH-positive axons were never present in the same ganglion or connective. Therefore, in the aged rat, deterioration of the sympathetic innervation of the myenteric plexus could be one possible mechanism for the age-related decline in gastrointestinal motor function evidenced in the elderly. [Abstract]

Aulehla A, Pourquié O
On periodicity and directionality of somitogenesis.
Anat Embryol (Berl). 2006 Dec;211 Suppl 13-8.
It is currently thought that the mechanism underlying somitogenesis is linked to a molecular oscillator, the segmentation clock, and to gradients of signaling molecules within the paraxial mesoderm. Here, we review the current picture of this segmentation clock and gradients, and use this knowledge to critically ask: What is the basis for periodicity and directionality of somitogenesis? [Abstract]

Ettl AK, Holzschuh J, Driever W
The zebrafish mutation m865 affects formation of dopaminergic neurons and neuronal survival, and maps to a genetic interval containing the sepiapterin reductase locus.
Anat Embryol (Berl). 2006 Dec;211 Suppl 173-86.
The zebrafish mutation m865 was isolated during a large-scale mutagenesis screen aimed at identifying genes involved in the development and maintenance of subgroups of neurons in the zebrafish central nervous system. The phenotype of m865 mutant embryos shows defects in the development of dopaminergic neurons in the pretectum and of retinal amacrine cells, as well as abnormal caudal dopaminergic cluster in the diencephalon. The effects of the mutation appear not to be restricted to dopaminergic neurons, as development of other neurotransmitter systems (serotonergic and cholinergic) is impaired as well. Furthermore, increased apoptosis is localized to the m865 mutant retina and in the optic tectum starting at 24hpf, and may lead to the observed reduced size of the mutant head and eye. Early patterning is not affected in m865 mutant embryos, and expression of genes known to play a role in dopaminergic cell differentiation is normal except for reduced expression of nurr1 in the mutant retina. Thus the m865 mutation does not specifically affect dopaminergic neuron development. m865 was genetically mapped to linkage group 5, and the critical genomic interval could be narrowed down to a region of 110 kb, containing four candidate genes. For one of these candidate genes, sepiapterin reductase (spr), a requirement for neuronal survival has previously been implicated, including dopaminergic neurons. Identification of the mutated gene should lead to a more detailed understanding of the defects observed in m865 mutant embryos, and potentially could enhance the understanding of the development and maintenance of specific dopaminergic neuronal populations. [Abstract]

Geetha-Loganathan P, Nimmagadda S, Huang R, Scaal M, Christ B
Expression pattern of BMPs during chick limb development.
Anat Embryol (Berl). 2006 Dec;211 Suppl 187-93.
In vertebrates, BMPs (bone morphogenic proteins) play critical roles in establishing the basic embryonic body plan and are involved in the development of a large variety of organs and tissues. Here, we analyzed the expression pattern of various BMPs (2, 4, 5 and 7) by whole mount in situ hybridization during chick limb development. In limb, expression of BMPs suggests evolutionary conserved mechanisms of BMP-dependent differentiation between lower and higher vertebrates. During the early developmental stages, BMP-2 and BMP-7 are expressed in the posterior distal mesenchyme leaving a less prominent expression anteriorly. BMP-4 is initially expressed in the anterior mesenchyme and spreads later to the whole mesenchyme leaving a stronger expression at the anterior side. From HH-stage 25, expression of BMP-4 is observed in the anterior-posterior margins of the limb bud. The BMPs 2, 4 and 7 are expressed strongly in the AER, whereas BMP-5 is expressed as a weak signal in the distal mesoderm during the early stages of limb development. Later from HH-stage 25 onwards, BMP-5 is expressed in the dorsal and ventral muscular mass of the developing limb. As digits become identifiable, expression of BMPs are observed in the interdigital mesenchyme and can also be detected along the contours of the developing phalanges and at the distal tips of the digits. All these BMPs are found to be expressed in the developing feather buds from day 8 onwards. [Abstract]

Steffl M, Schweiger M, Wessler I, Kunz L, Mayerhofer A, Amselgruber WM
Non-neuronal acetylcholine and choline acetyltransferase in oviductal epithelial cells of cyclic and pregnant pigs.
Anat Embryol (Berl). 2006 Nov;211(6):685-90.
Certain female reproductive tissues are known to express the non-neuronal cholinergic system. Using different experimental approaches, we tested the hypothesis that acetylcholine (ACh) in the porcine oviduct may also be derived from non-neuronal structures. Immunohistochemistry was performed to detect acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) in different segments of the oviduct of cyclic and pregnant sows. Immunohistochemical experiments revealed strong immunoexpression of ChAT in the entire oviductal epithelium at metoestrus. Thereby, a particular pronounced staining was found in the supranuclear region of almost all epithelial cells. Immunostaining of ChAT decreased markedly during dioestrus and prooestrus stages, respectively. At prooestrus, ChAT immunoreactivity was confined to ciliated cells. Furthermore, we found elevated level of staining intensity of ChAT in the pregnant oviduct at day 13. Using the same ChAT antibody for Western blot analyses, we detected immunoreactive bands of MW 69,000 and 46,000 mainly in ampulla, while MW 58,000 and 30,000 forms were present mainly in infundibulum and isthmus. Furthermore ACh was detected by HPLC and fluorimetric methods in oviductal epithelium. In conclusion, we show expression of ChAT in oviductal epithelial cells at different stages of the oestrus cycle and pregnancy, indicating that these cells can synthesize ACh in a cycle-dependent manner. These results suggest as yet unexplored roles of epithelial ACh in the oviduct. [Abstract]

Zhang X, Dai F, Weise C, Yusuf F, Bonafede A, Morosan-Puopolo G, Rehimi R, Wang J, Brand-Saberi B
Expression of the avian gene cNOC2 encoding nucleolar complex associated protein 2 during embryonic development.
Anat Embryol (Berl). 2006 Nov;211(6):649-57.
Genetic information that directs a cell during different phases of embryogenesis is locked up in the genome. Therein is contained the road map for growth, proliferation, differentiation and morphogenesis. The cellular transportation machinery plays a major role to ensure that all the components for transcription and translation are available at the right place at the right time. Nucleolar complex associated protein2 (NOC2) has a highly conserved UPF0120 domain, and is an element involved in ribosome transportation from the nucleoplasm to the cytoplasm. However, its gene expression pattern is still unknown. We chose the developing chick embryo to investigate the possible involvement of avian NOC2 (cNOC2) in developmental processes, particularly neurogenesis and myogenesis. For this purpose, we constructed a fragment of chicken cNOC2, which contains the UPF0120 domain coding sequence, into pDrive vector, and performed in situ hybridization on chicken embryos of different stages with this gene probe. A dynamic expression pattern of cNOC2 transcripts can be seen beginning as early as from stage HH7 until stage HH32. Using in situ hybridization we could detect that cNOC2 transcripts were expressed ubiquitously, but prominent expression could be found in the neural tissue, the somites and in the developing limbs. Comparison of cNOC2 gene expression with the proliferation marker gene cPCNA, muscle specific marker genes cMyf5 and cMyoD in single or double in situ hybridisation show that cNOC2 is expressed in the myotome, similar to cMyf5 and cMyoD, but not like cPCNA, which is hardly detectable in the myotome. Our results suggest that cNOC2 is involved in the development of neural tissue, somites and limbs. [Abstract]

Ribeiro AA
Size and number of binucleate and mononucleate superior cervical ganglion neurons in young capybaras.
Anat Embryol (Berl). 2006 Nov;211(6):607-17.
The total number of neurons in the superior cervical ganglion (SCG) of adult capybaras is known from a previous study, where a marked occurrence of binucleate neurons (13%) was also noted. Here, distribution, number and fate of binucleate neurons were examined in younger, developing capybaras, aged 3 months. The mean neuronal cross-sectional area was 575.2 microm2 for mononucleate neurons and 806.8 microm2 in binucleate neurons. Frequency of binucleate neurons was about 36%. The mean ganglion volume was about 190 mm3 in young capybaras and the mean neuronal density was about 9,517 neurons/mm3. The total number of neurons per ganglion was about 1.81 mill. Neuronal cell bodies constituted 22.5% of the ganglion volume and the average neuronal volume was 23,600 microm3. By comparing the present data with those previously published the conclusion is drawn that the maturation period was characterized by the following points: a 26% remarkable decrease in neuronal density which was significant (P < 0.05) and a significant 16% (P < 0.05) decrease in the total number of SCG neurons accompanied by a 23% decrease in the total number of SCG binucleate neurons. [Abstract]

Huang R, Christ B, Patel K
Regulation of scapula development.
Anat Embryol (Berl). 2006 Dec;211 Suppl 165-71.
The scapula is a component of the shoulder girdle. Its structure has changed greatly during evolution. For example, in humans it is a large quite flat triangular bone whereas in chicks it is a long blade like structure. In this review we describe the mechanisms that control the formation of the scapula. To assimilate our understanding regarding the development of the scapula blade we start by addressing the issue concerning the origin of the scapula. Experiments using somite extirpation, chick-quail cell marking system and genetic cell labelling techniques in a variety of species have suggested that the scapula had its origin in the somites. For example we have shown in the chick that the scapula blade originates from the somite, while the cranial part, which articulates with the upper limb, is derived from the somatopleure of the forelimb field. In the second and third part of the review we discuss the compartmental origin of this bone and the signalling molecules that control the scapula development. It is very interesting that the scapula blade originates from the dorsal compartment, dermomyotome, which has been previously been associated as a source of muscle and dermis, but not of cartilage. Thus, the development of the scapula blade can be considered a case of dermomyotomal chondrogenesis. Our results show that the dermomyotomal chondrogenesis differ from the sclerotomal chondrogenesis. Firstly, the scapula precursors are located in the hypaxial domain of the dermomyotome, from which the hypaxial muscles are derived. The fate of the scapula precursors, like the hypaxial muscle, is controlled by ectoderm-derived signals and BMPs from the lateral plate mesoderm. Ectoderm ablation and inhibition of BMP activity interfers the scapula-specific Pax1 expression and scapula blade formation. However, only somite cells in the cervicothoracic transition region appear to be committed to form scapula. This indicates that the intrinsic segment specific information determines the scapula forming competence of the somite cells. Taken together, we conclude that the scapula forming cells located within the hypaxial somitic domain require BMP signals derived from the somatopleure and as yet unidentified signals from ectoderm for activation of their coded intrinsic segment specific chondrogenic programme. In the last part we discuss the new data that provides evidence that neural crest contributes for the development of the scapula. [Abstract]

Scaal M, Wiegreffe C
Somite compartments in anamniotes.
Anat Embryol (Berl). 2006 Dec;211 Suppl 19-19.
Somites are a common feature of the phylotypic stage of embryos of all higher chordates. In amniote species like mouse and chick, somite development has been the subject of intense research over many decades, giving insight into the morphological and molecular processes leading to somite compartmentalization and subsequent differentiation. In anamniotes, somite development is much less understood. Except for recent data from zebrafish, and morphological studies in Xenopus, very little is known about the formation of somite compartments and the differentiation of somite derivatives in anamniotes. Here, we give a brief overview on the development of myotome, sclerotome and dermomyotome in various anamniote organisms, and point out the different mechanisms of somite development between anamniotes and the established amniote model systems. [Abstract]

Heise CE, Mitrofanis J
Fos immunoreactivity in some locomotor neural centres of 6OHDA-lesioned rats.
Anat Embryol (Berl). 2006 Nov;211(6):659-71.
In this study, we explore Fos expression (a measure of cell activity) in three nuclei associated with locomotion, namely the zona incerta, pedunculopontine tegmental nucleus and cuneiform nucleus (the latter two form the mesencephalic locomotor region) in hemiparkinsonian rats. Sprague-Dawley rats had small volumes of either saline (control) or 6 hydroxydopamine (6OHDA) injected into the medial forebrain bundle, the major tract carrying dopaminergic nigrostriatal axons. After various post-lesion survival periods, ranging from 2 h to 28 days, rats were perfused with formaldehyde and their brains processed for routine tyrosine hydroxylase and Fos immunocytochemistry. Our results showed a significant increase (P < 0.05) in the number of strongly labelled Fos+ cells in the cuneiform nucleus in the 6OHDA-lesioned cases compared to the controls after 7 and 28 days survival periods. By contrast, there were no significant differences (P > 0.05) in the number of strong-labelled Fos+ cells in the zona incerta and pedunculopontine nucleus of 6OHDA-lesioned rats compared to controls at any survival period. Many of the Fos+ cells within the pedunculopontine and cuneiform nuclei were glutamatergic (35-60%), while none or very few were nitric oxide synthase+. In conclusion, we reveal an increase in the number of strongly labelled Fos+ cells within the cuneiform nucleus of the so-called defensive locomotive system in 6OHDA-lesioned rats. In relation to Parkinson disease, we suggest that this increase is associated with the akinesia or lack of movement seen in patients. [Abstract]

Weise C, Dai F, Pröls F, Ketelsen UP, Dohrmann U, Kirsch M, Brand-Saberi B
Myogenin (Myf4) upregulation in trans-differentiating fibroblasts from a congenital myopathy with arrest of myogenesis and defects of myotube formation.
Anat Embryol (Berl). 2006 Nov;211(6):639-48.
Congenital myopathies often have an unclear aetiology. Here, we studied a novel case of a severe congenital myopathy with a failure of myotube formation. Polymerase chain reaction-based analysis was performed to characterize the expression patterns of the Desmin, p21, p57, and muscle regulatory factors (MRFs) MyoD, Myf4, Myf5 and Myf6 in differentiating skeletal muscle cells (SkMCs), normal human fibroblasts and patient-derived fibroblasts during trans-differentiation. The temporal and spatial pattern of MRFs was further characterized by immunocyto- and immunohistochemical stainings. In differentiating SkMCs, each MRF showed a characteristic expression pattern. Normal trans-differentiating fibroblasts formed myotubes and expressed all of the MRFs, which were detected. Interestingly, the patient's fibroblasts also showed some fusion events during trans-differentiation with a comparable expression profile for the MRFs, particularly, with increased expression of Myf4 and p21. Immunohistochemical analysis of normal and patient-derived skeletal musculature revealed that Myf4, which is downregulated during normal fetal development, was still present in patient-derived skeletal head muscle, which was also positive for Desmin and sarcomeric actin. The abnormal upregulation of Myf4 and p21 in the patient who suffered from a severe congenital myopathy suggests that the regulation of Myf4 and p21 gene expression during myogenesis might be of interest for further studies. [Abstract]

Vasyutina E, Birchmeier C
The development of migrating muscle precursor cells.
Anat Embryol (Berl). 2006 Dec;211 Suppl 137-41.
A major subclass of hypaxial muscle groups is derived from long-range migrating precursor cells that delaminate from the dermomyotome. Migrating precursors are generated on particular axial levels only, i.e. occipitally, cervically, and on the levels of the fore and hind limbs. They express the homeobox gene Lbx1, which provides a useful marker for their visualization. In the mouse, migrating precursor cells give rise to muscles of the extremities, the hypoglossal chord, and the diaphragm. We discuss here the development of this migrating lineage, which critically depends on the correct specification of the precursors in the dermomyotome, their delamination and correct migration. Finally, proliferation at the targets is essential to ensure a correct size of the precursor pool and of the muscle that derives thereof. [Abstract]

Kalcheim C, Kahane N, Cinnamon Y, Ben-Yair R
Mechanisms of lineage segregation in the avian dermomyotome.
Anat Embryol (Berl). 2006 Dec;211 Suppl 131-6.
The somite and its intermediate derivatives, sclerotome and dermomyotome (DM), are composed of distinct subdomains based on lineage analysis and gene expression patterns. This sets the grounds for elucidating the mechanisms underlying differential cell specification and morphogenesis. By examining the in vivo roles of N-cadherin on discrete domains of the somitic epithelium at various times, our recent studies highlight the existence of a regional and temporal heterogeneity in cellular responsiveness. As examples of this assortment, we document a coupling between asymmetric cell division and fate segregation in the DM sheet, sequential effects of N-cadherin-mediated adhesion on early myogenic specification compared to later myofiber patterning, and a differential behavior of pioneer myoblasts compared to later myogenic waves. [Abstract]

Pérez FA, Roma SM, Cabada MO, Marini PE
Sperm binding glycoprotein is differentially present surrounding the lumen of isthmus and ampulla of the pig's oviduct.
Anat Embryol (Berl). 2006 Nov;211(6):619-24.
In several mammals a sperm reservoir is formed at the isthmus of the Fallopian tube, providing viable, potentially fertile sperm for an extensive period. In pig (Sus scrofa) the spermadhesin AQN-1 seems to be involved in the establishment of the sperm reservoir. The pig oviductal protein, sperm binding glycoprotein (SBG), binds to sperm and exposes carbohydrate groups that can be recognized by AQN-1. In this study we obtain anti-SBG polyclonal antibodies and use them to localize SBG in the oviduct. Immunohistochemical analysis shows that SBG is present at the apical surface of isthmic and ampullar epithelial cells. The presence of SBG is limited to the upper two-thirds of the crypts of the isthmus and to cells located near the oviductal lumen in the ampulla. The ratio of the amount of SBG detected by western blot is 1:3 (ampulla:isthmus). Sperm entering the Fallopian tube probably contact the epithelial cells at the lumen before they reach the cells at the bottom of the folds. In vitro sperm can bind to isthmus and, at less extent, to ampulla. Thus, the localization and the relative amount of SBG in the isthmus and ampulla of pig's oviduct are compatible with its possible function in sperm binding to oviductal epithelial cells. [Abstract]

Tsujimura T, Ikeda R, Aiyama S
Changes in the number and distribution of myoepithelial cells in the rat parotid gland during postnatal development.
Anat Embryol (Berl). 2006 Oct;211(5):567-74.
The mature rat parotid gland shows hardly any cell bodies of myoepithelial cells around the acini, only a few cell processes being visible. However, in the early postnatal period, the rat parotid gland shows many myoepithelial cell bodies around the acini, including the intercalated ducts. In order to clarify the reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development, changes in the number and distribution of myoepithelial cells in the rat parotid gland were examined histochemically and chronologically, with particular reference to cell proliferation and cell death. From day 7 to day 14, many myoepithelial cells showing a positive reaction with anti-actin antiserum were found around the acini and intercalated ducts, but thereafter the number of such cells decreased gradually, particularly around the acini, and had almost disappeared after day 35. BrdU/PCNA-positive myoepithelial cells surrounding the acini were easily detected on day 14, but disappeared by day 21, whereas BrdU/PCNA-positive acinar cells remained numerous even after day 21. TUNEL/ISEL staining showed no positive myoepithelial cells throughout the observation period. Transmission electron microscopy also demonstrated no myoepithelial cells with chromatin condensation characteristic of apoptosis through the observation period. These findings suggest that the main reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development is the large difference between the number of myoepithelial cells and that of acinar cells, because the acinar cells retain their proliferative activity even after myoepithelial cells have become quiescent. [Abstract]

Zhang GH, Deng SP, Li LL, Li HT
Developmental change of alpha-gustducin expression in the mouse fungiform papilla.
Anat Embryol (Berl). 2006 Nov;211(6):625-30.
Developmental changes in the distribution pattern of taste buds in newborn mouse have not been previously elucidated, and little work has been done to examine the postnatal alteration of the expression of alpha-gustducin in the mouse taste buds. In the present paper, we delineated the development and frequency distribution of the taste buds as well as the immunohistochemical expression of alpha-gustducin, a G protein closely related to the transduction of taste stimuli in the fungiform papilla from the birthday till the age of week 9. At birth, more than 45 taste buds (with or without pores) were observed on the fungiform papilla, then the number of mature taste buds increased rapidly, and resulted in 66% (80.2 +/- 0.6 of 122.2 +/- 1.3) of fungiform papilla taste buds containing taste pores at week 3. By age the total counts of pored taste buds continuously increased and their morphological features became quite discernible. They became ellipse in shape, characterized by distinct pores. Quantitative analysis of alpha-gustducin expression at different postnatal ages revealed a significant increase in the number of immunolabeled taste buds and alpha-gustducin-positive cells in single taste buds from week 1 to 7, by week 7, the number reached the value found in adults (99.3 +/- 0.9 and 8.3 +/- 0.3, respectively). These results indicated that taste buds within fungiform papilla play an important role in the detection of nutrients in the postnatal mouse. [Abstract]

Lametschwandtner A, Lametschwandtner U, Radner Ch, Minnich B
Spatial growth and pattern formation in the small intestine microvascular bed from larval to adult Xenopus laevis: a scanning electron microscope study of microvascular corrosion casts.
Anat Embryol (Berl). 2006 Oct;211(5):535-47.
The microvascular anatomy of the small intestine of metamorphosing tadpoles of the South African Clawed Toad, Xenopus laevis (Daudin) is studied from developmental stages 55 to 65 and in adults by scanning electron microscopy (SEM) of vascular corrosion casts (VCCs) and light microscopy. Up to stage 62, VCCs reveal a dense two-dimensional vascular network ensheating the intestinal tube, whose proximal portion forms a clockwise spiralling outer and its distal portion an anti-clockwise spiralling inner coil. Vessels of the intestinal network impose flat and run circularly to slightly obliquely. Locally, dense capillary plexus with small "holes" indicating ongoing intussusceptive microvascular growth (IMG) and vessel maturation, are present. The typhlosole, an invagination along the proximal portion of the small intestine, reveals a dense capillary bed with locally ongoing IMG. VCCs of stages 62/63 for the first time reveal a three-dimensional vascular bed with longitudinal intestinal folds of varying size and heights greatly enlarging the luminal exchange area of the intestinal tube. From stage 65 onwards, longitudinal intestinal folds undulate and, though smaller in size and less mature as indicated in VCCs by the presence of wider, sinus-like vessels with small "holes" interposed between, closely resemble the intestinal folds present in the small intestine of adult Xenopus. Our data suggest that maturation of the vascular pattern in the small intestine of X. laevis tadpoles takes place successively after stages 62-63, and growth during this period is preferentially by intussusception. [Abstract]

Liersch J, Räder C, Görcs T, Scholten A, Kremmer E, Plüm J, Pöggel S, Zilles K
Immunohistochemical localization of Ih channel HCN3 in the rat brain.
Anat Embryol (Berl). 2006 Aug 8; [Abstract]

Saburkina I, Pauza DH
Location and variability of epicardiac ganglia in human fetuses.
Anat Embryol (Berl). 2006 Nov;211(6):585-94.
The aim of the study was to determine the morphology of epicardiac ganglia in human fetuses at different stages of their development as these ganglia are considered to be of a pivotal clinical importance. Twenty-one fetal hearts were investigated applying a technique of histochemistry for acetylcholinesterase to visualize the epicardiac neural ganglionated plexus with its subsequent stereoscopic examination on total organs. In all of the examined fetuses, epicardiac neural plexus with numerous ganglia was well recognizable and could be clearly differentiated into seven ganglionated subplexuses, topography and structural organization of which were typical for hearts of adult human. The largest ganglion number comprising 77% of all counted ganglia was identified on the dorsal atrial surface. Fetal epicardiac plexus in gestation period of 15-40 weeks contained 929 +/- 62 ganglia, but ganglion amount did vary substantially from heart to heart. In conclusion, this study implies that the human fetal epicardiac ganglia occupy their definitive location already at gestation period from 15 weeks and their number as well as distribution on heart surface presumably is not age dependent. [Abstract]

Berger UV, Hediger MA
Distribution of the glutamate transporters GLT-1 (SLC1A2) and GLAST (SLC1A3) in peripheral organs.
Anat Embryol (Berl). 2006 Nov;211(6):595-606.
The glutamate transporters GLT-1 and GLAST are widely expressed in astrocytes in the brain where they fulfill important functions during glutamatergic neurotransmission. The present study examines their distribution in peripheral organs using in situ hybridization (ISH) and immunocytochemistry. GLAST was found to be more widely distributed than GLT-1. GLAST was expressed primarily in epithelial cells, cells of the macrophage-lineage, lymphocytes, fat cells, interstitial cells, and salivary gland acini. GLT-1 was primarily expressed in glandular tissue, including mammary gland, lacrimal gland, and ducts and acini in salivary glands, but also by perivenous hepatocytes and follicular dendritic cells in spleen and lymph nodes. The findings demonstrate that, although expressed by the same cells in the brain, these two glutamate transporters have different distribution patterns in peripheral tissues and that they fulfill glutamate transport functions apart from glutamatergic neurotransmission in these areas. [Abstract]

Bjaalie JG, Zilles K
New article category in anatomy and embryology: Methodological standards.
Anat Embryol (Berl). 2006 Aug;211(4):255. [Abstract]

Holmseth S, Lehre KP, Danbolt NC
Specificity controls for immunocytochemistry.
Anat Embryol (Berl). 2006 Aug;211(4):257-66.
Antibodies have been in widespread use for more than three decades as invaluable tools for the specific detection of proteins or other molecules in biological samples. In spite of such a long experience, the field of immunocytochemistry is still troubled by spurious results due to insufficient specificity of antibodies or procedures used. The importance of keeping a high standard is increasing because massive sequencing of entire genomes leads to the identification of numerous new proteins. All the identified proteins and their variants will have to be localized precisely and quantitatively at high resolution throughout the development of all species. Consequently, antibody generation and immunocytochemical investigations will be done on a large scale. It will be economically important to secure an optimal balance between the risk of publishing erroneous data (which are expensive to correct) and the costs of specificity testing. Because proofs of specificity are never absolute, but rather represent failures to detect crossreactivity, there is no limit to the number of control experiments that can be performed. The aims of the present paper are to increase the awareness of the difficulties in proving the specificity of immunocytochemical labeling and to initiate a discussion on optimized standards. The main points are: (1) antibodies should be described properly, (2) the labeling obtained with an antibody to a single epitope needs additional verification and (3) the investigators should be required to outline in detail how they arrive at the conclusion that the immunocytochemical labeling is specific. [Abstract]

Ozegbe PC, Aire TA, Soley JT
The morphology of the efferent ducts of the testis of the ostrich, a primitive bird.
Anat Embryol (Berl). 2006 Oct;211(5):559-65.
The efferent duct of the ostrich consists of two segments, the proximal efferent duct (PED) and the distal efferent duct (DED) that are continuous, as in some other birds. Both segments of the duct possess an epithelium comprising non-ciliated and ciliated cells in varying proportions between the two segments. The non-ciliated cell (type I) of the PED contains a well-developed, subapical endocytic apparatus of apical tubules and endocytic vacuoles, a solitary, large, heterogeneous lipid droplet, and numerous, oval, dense bodies in the supranuclear region of the cell. Mitochondria tend to concentrate in the basal part of the cell. Intercellular spaces between the non-ciliated cells are enlarged, especially in the basal half of the epithelium. Together, these morphological features confer on the PED an efficient fluid absorption capability. The DED epithelium displays the type II non-ciliated cell whose poorly developed subapical endocytic apparatus as well as the absence of dilated basal intercellular spaces indicate its limited fluid absorptive capacity. [Abstract]

Martinelli C, Sartori P, De Palo S, Ledda M, Pannese E
The perineuronal glial tissue of spinal ganglia. Quantitative changes in the rabbit from youth to extremely advanced age.
Anat Embryol (Berl). 2006 Oct;211(5):455-63.
The volumes of the nerve cell bodies and those of the enveloping satellite cell sheaths from spinal ganglia were determined by morphometric methods applied to electron micrographs in young, adult, old and very old rabbits. The mean volume of the nerve cell bodies increased progressively with age; this is probably related to the increase with age of the body size of the rabbits studied. The mean volume of the satellite cell sheaths did not differ significantly in young, adult and old animals, but was significantly smaller in very old animals. It is extremely unlikely that this marked reduction in the volume of the satellite cell sheath is the result of a pathological process. The mean value of the volume ratio between the satellite cell sheaths and the related nerve cell bodies did not differ significantly in young and adult animals, but was significantly smaller in old and very old animals. This ratio was particularly low in very old animals. Our analysis showed that in each age group the volume of the satellite cell sheath is linearly related to the volume of the related nerve cell body. This result suggests that in rabbit spinal ganglia the quantitative relations between glial and nervous tissue are tightly controlled throughout life. It is suggested that ganglionic neurons release signals to influence and control the volume of their associated glial tissue. Since satellite cells have important support roles for the neurons they surround, it is likely that the marked reduction in the volume of perineuronal sheaths in the extremely advanced age is accompanied by a reduction of those roles, with negative consequences for neuronal activity. [Abstract]

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Recent Articles in Journal of Anatomy

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Butler CM, Shaw G, Clark J, Renfree MB
The functional development of Leydig cells in a marsupial.
J Anat. 2007 Dec 5;
Leydig cells are the major source of androgen in the male mammal. We describe here for the first time the development of the Leydig cell in a macropodid marsupial, the tammar wallaby, Macropus eugenii. Leydig cells are first recognized morphologically 2 days after birth with the appearance of lipid droplets in the cytoplasm of certain interstitial cells. Lipid content closely matches the steroid content of the developing testis and marks the maturation of the steroid synthesis pathway in the tammar testis. Morphologically mature Leydig cells, marked by distinct mitochondria with tubular cristae and an extensive anastomosing network of smooth endoplasmic reticulum, are developed by day 10 after birth - the time of peak testosterone content in perinatal tammar testes. The volume percentage of each cell type in the testis does not change over time so the growth of each cellular component keeps pace with growth of the whole testis. There was no morphological or quantitative evidence of a change from one population of Leydig cells to another in the tammar testis as has been reported in several other species including the rat, mouse and human. Maturation of the testis is also marked by the development of tight junctions between the cell membranes of adjacent Sertoli cells. These appear around day 30 after birth and coincide with the onset of mitotic arrest in male germ cells. Overall, the development of the Leydig cell in the tammar wallaby follows a similar pattern to that seen in other mammals, although the start of Leydig cell differentiation is, like many other organ systems in marsupials, post natal, not fetal and there appears to be only a single population of Leydig cells. [Abstract]

Porzionato A, Macchi V, Guidolin D, Sarasin G, Parenti A, De Caro R
Anatomic distribution of apoptosis in medulla oblongata of infants and adults.
J Anat. 2007 Dec 7;
The aim of the study was to evaluate the distribution of apoptosis in the medullary nuclei of infants and adults who died of hypoxic-ischaemic injury. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) in brainstems from 22 adults (7 subjects who died of opiate intoxication, 15 who died of other hypoxic-ischaemic injury) and 10 infants. The nuclei examined included the hypoglossal, dorsal motor nucleus of the vagus, nucleus tractus solitarii, nucleus of the spinal trigeminal tract, cuneate, vestibular and inferior olivary nuclei. A morphometric analysis with the optical disector method was performed to calculate the mean percentages (+/- standard deviation) of TUNEL-positive neuronal and glial cells for the sample populations. Opiate deaths did not have higher apoptotic indices than other adult hypoxic-ischaemic deaths. Statistically significant differences between adults and infants were found in the neuronal apoptotic indices of the cuneate (28.2 +/- 16.3% vs. 6.9 +/- 8.7%), vestibular (24.7 +/- 15.0% vs. 11.3 +/- 11.4%), nucleus tractus solitarii (11.2 +/- 11.2% vs. 2.3 +/- 2.4%), dorsal motor nucleus of the vagus (6.8 +/- 8.5% vs. 0.1 +/- 0.2%) and hypoglossal (6.6 +/- 5.7% vs. 0.1 +/- 0.2%), indicating higher resistance of the neuronal populations of these infant medullary nuclei to terminal hypoxic-ischaemic injury or post-mortem changes. Differences in neuronal apoptotic index were also statistically significant among nuclei, suggesting differential characteristics of survival. Nuclei with higher neuronal apoptotic indices were the cuneate, vestibular and nucleus of the spinal trigeminal tract, which are located in the lateral medullary tegmentum and share the same vascular supply from the posterior inferior cerebellar artery. [Abstract]

Giachini FR, Carriel V, Capelo LP, Tostes RC, Carvalho MH, Fortes ZB, Zorn TM, San Martin S
Maternal diabetes affects specific extracellular matrix components during placentation.
J Anat. 2007 Dec 6;
During embryo implantation, invasive trophoblast cells mediate embryo invasion into the decidualized stroma, forming a rich network of lacunae that connect the embryonic tissues to the maternal blood vessels. Placentation is probably guided by the composition and organization of the endometrial extracellular matrix. Certain pathological conditions that occur during pregnancy, including diabetes, have been linked to abnormal placental morphology and consequent fetal morbidity. We used immunoperoxidase techniques to identify members of the collagen, proteoglycan and glycoprotein families in the various compartments of the rat placenta and to determine whether experimentally induced diabetes affects placental morphology and alters the distribution of these molecules during pregnancy. Single injections of alloxan (40 mg kg(-1) i.v.) were used to induce diabetes on day 2 of pregnancy in Wistar rats. Placentas were collected on days 14, 17, and 20. Type I and III collagen, as well as the proteoglycans decorin and biglycan, were found to be distributed throughout the placentas of control and diabetic rats. In both groups, laminin expression decreased at the end of pregnancy. In contrast, fibronectin was detected in the labyrinth region of diabetic rats at all gestational stages studied, whereas it was detected only at term pregnancy in the placentas of control rats. These results show for the first time that some extracellular matrix molecules are modulated during placental development. However, as diabetic rats presented increased fibronectin deposition exclusively in the labyrinth region, we speculate that diabetes alters the microenvironment at the maternal-fetal interface, leading to developmental abnormalities in the offspring. [Abstract]

Fernández B, Durán AC, Fernández MC, Fernández-Gallego T, Icardo JM, Sans-Coma V
The coronary arteries of the C57BL/6 mouse strains: implications for comparison with mutant models.
J Anat. 2007 Dec 6;
There are few detailed descriptions of the coronary arterial patterns in the mouse. Some recent reports on coronary anomalies in mutant mouse models have uncovered the importance of several genes (i.e. iv and connexin43) in coronary morphogenesis. These mutations spontaneously appeared (iv) or were generated (connexin43) in a C57BL/6 background, which is widely used for the development of mutant mice. We have studied the origin and course of the main coronary arteries of two C57BL/6 mouse strains. Unusual anatomical coronary arterial patterns were found, including: solitary ostium in aorta, accessory ostium, high take-off, aortic intramural course, slit-like ostium, sinus-like ostium and origin of a septal artery from the left coronary artery. In humans, some of these conditions are clinically relevant. Most of these patterns, which differ from those observed in wild mice and Swiss albino mice, coincide with those previously found in iv/iv and connexin43 knockout mice. The results indicate that there is variability in the coronary arterial arrangement of the laboratory mouse. Care should be taken when analysing coronary phenotypes of mutant mouse models. [Abstract]

Li X, Liu J, Davey M, Duce S, Jaberi N, Liu G, Davidson G, Tenent S, Mahood R, Brown P, Cunningham C, Bain A, Beattie K, McDonald L, Schmidt K, Towers M, Tickle C, Chudek S
Micro-magnetic resonance imaging of avian embryos.
J Anat. 2007 Dec;211(6):798-809.
Chick embryos are useful models for probing developmental mechanisms including those involved in organogenesis. In addition to classic embryological manipulations, it is possible to test the function of molecules and genes while the embryo remains within the egg. Here we define conditions for imaging chick embryo anatomy and for visualising living quail embryos. We focus on the developing limb and describe how different tissues can be imaged using micro-magnetic resonance imaging and this information then synthesised, using a three-dimensional visualisation package, into detailed anatomy. We illustrate the potential for micro-magnetic resonance imaging to analyse phenotypic changes following chick limb manipulation. The work with the living quail embryos lays the foundations for using micro-magnetic resonance imaging as an experimental tool to follow the consequences of such manipulations over time. [Abstract]

Pugener LA, Maglia AM
Skeletal morphology and development of the olfactory region of Spea (Anura: Scaphiopodidae).
J Anat. 2007 Dec;211(6):754-68.
The nasal capsules of anurans are formed by an intricate set of sac-like cavities that house the olfactory organ and constitute the beginning of the respiratory system. In tadpoles, nasal capsules do not have a respiratory function, but each is composed of a single soft tissue cavity lined with olfactory epithelium. Our study has revealed that in Spea the nasal cartilages and septomaxillae are de novo adult structures that form dorsal to the larval skeleton of the ethmoid region. The only element of the adult nasal capsule that is partially derived from the larval skeleton is the solum nasi. Development of the nasal skeleton begins at about Gosner Stage 31, with chondrification of the septum nasi and lamina orbitonasalis. The alary cartilage and superior prenasal cartilage are the first of the anterior nasal cartilages to chondrify at Gosner Stage 37. By Gosner Stages 40/41, the ethmoid region is composed of the larval structures ventrally and the adult structures dorsally. By Stage 44, the larval structures have eroded. The adult nasal capsule is characterized by: (1) a septum nasi that projects ventrally beyond the plane of the nasal floor; (2) a paranasal commissure that forms the ventral margin of the fenestra nasolateralis; and (3) a large skeletal support for the eminentia olfactoria formed by the nasal floor and vomer. The timing of chondrification of the anterior nasal cartilages and the development of the postnasal wall, inferior prenasal cartilage, fenestra nasolateralis, and paranasal commissure are discussed and compared with those of other anuran species. This study also includes a discussion of the morphology of the skeletal support for the eminentia olfactoria, a structure best developed in distinctly ground-dwelling frogs such as spadefoot toads. Finally, we propose a more precise restriction of the terminology that is used to designate the posterior structures of the olfactory region of anurans. [Abstract]

Haas SJ, Petrov S, Kronenberg G, Schmitt O, Wree A
Orthotopic transplantation of immortalized mesencephalic progenitors (CSM14.1 cells) into the substantia nigra of hemiparkinsonian rats induces neuronal differentiation and motoric improvement.
J Anat. 2007 Nov 23;
Neural progenitor cell grafting is a promising therapeutic option in the treatment of Parkinson's disease. In previous experiments we grafted temperature-sensitive immortalized CSM14.1 cells, derived from the ventral mesencephalon of E14-rats, bilaterally in the caudate putamen of adult hemiparkinsonian rats. In these studies we were not able to demonstrate either a therapeutic improvement or neuronal differentiation of transplanted cells. Here we examined whether CSM14.1 cells grafted bilaterally orthotopically in the substantia nigra of hemiparkinsonian rats have the potential to differentiate into dopaminergic neurons. Adult male rats received 6-hydroxydopamine into the right medial forebrain bundle, and successful lesions were evaluated with apomorphine-induced rotations 12 days after surgery. Two weeks after a successful lesion the animals received bilateral intranigral grafts consisting of either about 50 000 PKH26-labelled undifferentiated CSM14.1 cells (n = 16) or a sham-graft (n = 9). Rotations were evaluated 3, 6, 9 and 12 weeks post-grafting. Animals were finally perfused with 4% paraformaldehyde. Cryoprotected brain slices were prepared for immunohistochemistry using the freeze-thaw technique to preserve PKH26-labelling. Slices were immunostained against neuronal epitopes (NeuN, tyrosine hydroxylase) or glial fibrillary acidic protein. The CSM14.1-cell grafts significantly reduced the apomorphine-induced rotations 12 weeks post-grafting compared to the sham-grafts (P < 0.05). There was an extensive mediolateral migration (400-700 microm) of the PKH26-labelled cells within the host substantia nigra. Colocalization with NeuN or glial fibrillary acidic protein in transplanted cells was confirmed with confocal microscopy. No tyrosine hydroxylase-immunoreactive grafted cells were detectable. The therapeutic effect of the CSM14.1 cells could be explained either by their glial cell-derived neurotrophic factor-expression or their neural differentiation with positive effects on the basal ganglia neuronal networks. [Abstract]

Smith CD, Masouros SD, Hill AM, Wallace AL, Amis AA, Bull AM
Tensile properties of the human glenoid labrum.
J Anat. 2007 Nov 21;
Human fresh-frozen cadaveric glenoid labrae from 16 donors were harvested and ten of these had no gross degeneration. These ten were divided into eight equal circumferential sections. Each section was cut to produce test-samples from the core layer with a cross-section of 1 x 1 mm. Tensile testing was performed within a controlled environment unit at 37 +/- 1 degrees C and 100% relative humidity. Each test-sample was precycled to a quasi-static state to alleviate the effects of deep-freezing, prior to final testing. The tangent modulus was calculated for each test-sample before and after a 5-min period of stress relaxation and at yield. The mean elastic modulus and yield stress of the glenoid labrum were 22.8 +/- 11.4 and 2.5 +/- 2.1 MPa, respectively. The anterosuperior portion had a lower elastic modulus and lower yield stress than the inferior portion (both P < 0.02). The pre-stress relaxation tangent modulus was significantly lower than the post-stress relaxation tangent modulus for all portions of the labrum. The glenoid labrum has similar tensile material properties to articular cartilage. Its elastic modulus varies around its circumference. This suggests that the labrum may encounter different forces at different positions. [Abstract]

Hildreth V, Webb S, Bradshaw L, Brown NA, Anderson RH, Henderson DJ
Cells migrating from the neural crest contribute to the innervation of the venous pole of the heart.
J Anat. 2007 Nov 21;
Cells migrating from the neural crest are known to septate the outflow tract of the developing heart, and to contribute to the formation of the arterial valves, their supporting sinuses, the coronary arteries and cardiac neural ganglia. Neural crest cells have also been suggested to contribute to development of the venous pole of the heart, but the extent and fate of such cells remains unclear. In this study, in the mouse, it is shown that cells from the neural crest contribute to the parasympathetic and, to a lesser extent, the sympathetic innervation of the venous pole of the heart. Nerves within the venous pole of the heart are shown to be of mixed origin, with some being derived from the neural crest, while others have an alternative origin, presumably placodal. The neurons innervating the nodal tissue, which can exert chronotropic effects on cardiac conduction, are shown not to be derived from the neural crest. In particular, no evidence was found to support previous suggestions that cells from the neural crest make a direct contribution to the myocardial atrioventricular conduction axis, although a small subset of these cells do co-localize with the developing left bundle branch. We have therefore confirmed that cells from the neural crest migrate to the venous pole of the heart, and that their major role is in the development of the parasympathetic innervation. In addition, in some embryos, a population of cells derived from the neural crest persist in the leaflets of the atrioventricular valves, but their role in subsequent development remains unknown. [Abstract]

Sperber GH, Sperber SM
Embryology. Board Review Series.
J Anat. 2007 Nov 21; [Abstract]

Sorg H, Krueger C, Vollmar B
Intravital insights in skin wound healing using the mouse dorsal skin fold chamber.
J Anat. 2007 Dec;211(6):810-8.
The skin fold chamber is one of the most accepted animal models for studying the microcirculation both in health and disease. Here we describe for the first time the alternative use of the skin fold chamber in mice for intravital microscopic investigation of skin regeneration after creating a full dermal thickness wound. The dorsal skin fold chamber was implanted in hairless SKH1-hr mice and a full dermal thickness wound (area ~4 mm(2)) was created. By means of intravital fluorescence microscopy, the kinetics of wound healing were analyzed for 12 days post wounding with assessment of epithelialization and nutritive perfusion. The morphology of the regenerating skin was characterized by hematoxylin-eosin histology and immunohistochemistry for proliferation and microvessel density. The model allows the continuous visualization of wound closure with complete epithelialization at day 12. Furthermore, a sola cutis se reficientis could be described by an inner circular ring of vessels at the wound margin surrounded by outer radial passing vessels. Inner circular vessels presented initially with large diameters and matured towards diameters of less than 15 microm for conversion into radial spreading outer vessels. Furthermore, wound healing showed all diverse core issues of skin repair. In summary, we were able to establish a model for the analysis of microcirculation in the healing skin of the mouse. This versatile model allows distinct analysis of new vessel formation and maturation in regenerating skin as well as evaluation of skin healing under different pathologic conditions. [Abstract]

Robertson J, Zhang W, Liu JJ, Muir KR, Maciewicz RA, Doherty M
Radiographic assessment of the index to ring finger ratio (2D:4D) in adults.
J Anat. 2007 Nov 13;
The smaller index to ring finger (2D:4D) ratio has been considered as a 'male finger pattern' and is associated with sporting ability and a number of conditions. However, the ratio may vary according to what is measured, the hand selected and the method used. This study aimed to determine: (1) which bones (phalanges, metacarpals or both) account for variation in the 2D:4D ratio; (2) whether the ratio shows right-left symmetry or relates to hand dominance; and (3) the correlation between visual classification and measured determinations of the ratio based on radiographs. Hand radiographs obtained as part of a large osteoarthritis genetic study were examined. Each hand was classified visually into three types according to the relative length of the index and ring finger: Type 1 (index longer than ring), Type 2 (index = ring) and Type 3 (index shorter than ring). For both index and ring fingers we measured (1) from base of proximal to tip of distal phalanx and (2) metacarpal length. Reproducibility of the classification and measurements were examined using kappa and intraclass correlation coefficient; symmetry between left and right hands was examined using Bland and Altman's agreement analysis; and correlation between visual classification and 2D:4D ratio data was analysed using the anova linearity test. Data were obtained from 3172 radiographs (1636 men, 1536 women; mean age 67 +/- 7.9 years, range 45-86 years). Prevalence of Type 3 hand was 61% in men and 37% in women (P < 0.001). Men had smaller 2D:4D ratios than women for phalanges (0.908 versus 0.922, P < 0.01), metacarpals (1.152 versus 1.157, P < 0.01) and the sum of phalanges plus metacarpals (1.005 versus 1.015, P < 0.01). The mean difference between right and left was -0.001 (95% limit of agreement -0.035, 0.032) for the phalangeal ratio and 0.003 (95% limit of agreement -0.051 to 0.057) for the metacarpal ratio. The 2D:4D ratio did not associate with handedness or age. There was a linear trend between the visual classification of hand type and the 2D:4D ratio data (P < 0.001). More technical difficulties (due to positioning, finger trauma, osteoarthritis) were encountered with the phalangeal ratio and visual categorization than with the metacarpal ratio: the latter could be measured in 98.7% of the study population. We concluded that measured 2D:4D ratios and visual categorization can be derived from hand radiographs. The phalanges and metacarpals both contribute to the variation in 2D:4D ratio with smaller ratios observed in men than in women. The ratio is symmetrical with only very small differences between right and left hands. Visual classification may be a useful simple tool for future epidemiological studies but is more prone to bias from positioning than direct measurement. If radiographs are used for this purpose, we recommend the metacarpal ratio with measurement of a single index hand or an average of both as it is least affected by bias from malpositioning, trauma or common joint disease. [Abstract]

Sperber GH
Core Anatomy - Illustrated - By I. Parkin, B. M. Logan and M. McCarthy.
J Anat. 2007 Dec;211(6):832. [Abstract]

Laberge F, Roth G
Is there a structure equivalent to the mammalian basolateral amygdaloid complex in amphibians?
J Anat. 2007 Dec;211(6):830. [Abstract]

Moreno N, González A
Reply.
J Anat. 2007 Dec;211(6):830-1. [Abstract]

Batista Lobo S, Denyer M, Britland S, Javid FA
Development of an intestinal cell culture model to obtain smooth muscle cells and myenteric neurones.
J Anat. 2007 Dec;211(6):819-29.
This paper reports on the development of an entirely new intestinal smooth muscle cell (ISMC) culture model using rat neonates for use in pharmacological research applications. Segments of the duodenum, jejunum and ileum were obtained from Sprague-Dawley rat neonates. The cell extraction technique consisted of ligating both ends of the intestine and incubating (37 masculineC) in 0.25% trypsin for periods of 30-90 min. Isolated cells were suspended in DMEM-HEPES, plated and allowed to proliferate for 7 days. Cell culture quality was assessed via a series of viability tests using the dye exclusion assay. In separate experiments, tissues were exposed to trypsin for varying durations and subsequently histological procedures were applied. Cell purification techniques included differential adhesion technique for minimizing fibroblasts. Selective treatments with neurotoxin scorpion venom (30 microg mL(-1)) and anti-mitotic cytosine arabinoside (6 microm) were also applied to purify respectively ISMC and myenteric neurones selectively. The different cell populations were identified in regard to morphology and growth characteristics via immunocytochemistry using antibodies to smooth muscle alpha-actin, alpha-actinin and serotonin-5HT(3) receptors. Based on both viability and cell confluence experiments, results demonstrated that intestinal cells were best obtained from segments of the ileum dissociated in trypsin for 30 min. This provided the optimum parameters to yield highly viable cells and confluent cultures. The finding was further supported by histological studies demonstrating that an optimum incubation time of 30 min is required to isolate viable cells from the muscularis externae layer. When cell cultures were treated with cytosine arabinoside, the non-neuronal cells were abolished, resulting in the proliferation of cell bodies and extended neurites. Conversely, cultures treated with scorpion venom resulted in complete abolition of neurones and proliferation of increasing numbers of ISMC, which were spindle-shaped and uniform throughout the culture. When characterized by immunocytochemistry, neurones were stained with antibody to 5HT(3) receptors but not with antibodies to alpha-smooth muscle actin and alpha-actinin. Conversely, ISMC were stained with antibodies to alpha-smooth muscle actin and alpha-actinin but not with antibody to 5HT(3) receptors. The present study provides evidence that our method of dissociation and selectively purifying different cell populations will allow for pharmacological investigation of each cell type on different or defined mixtures of different cell types. [Abstract]

Yamamoto Y, Sato Y, Taniguchi K
Distribution of TRPV1- and TRPV2-immunoreactive afferent nerve endings in rat trachea.
J Anat. 2007 Dec;211(6):775-83.
Nociception in the trachea is important for respiratory modulation. We investigated the distribution, neurochemical characteristics, and origin of nerve endings with immunoreactivity for candidate sensor channels, TRPV1 and TRPV2, in rat trachea. In the epithelial layer, the intraepithelial nerve endings and dense subepithelial network of nerve fibers were immunoreactive for TRPV1. In contrast, TRPV2 immunoreactivity was observed mainly in nerve fibers of the tracheal submucosal layer and in several intrinsic ganglion cells in the peritracheal plexus. Double immunostaining revealed that some TRPV1-immunoreactive nerve fibers were also immunoreactive for substance P or calcitonin gene-related peptide, but neither neuropeptide colocalized with TRPV2. Injection of the retrograde tracer, fast blue, into the tracheal wall near the thoracic inlet demonstrated labeled neurons in the jugular, nodose, and dorsal root ganglia at segmental levels of C2-C8. In the jugular and nodose ganglia, 59.3% (70/118) and 10.7% (17/159), respectively, of fast blue-labeled neurons were immunoreactive for TRPV1, compared to 8.8% (8/91) and 2.6% (5/191) for TRPV2-immunoreactive. Our results indicate that TRPV1-immunoreactive nerve endings are important for tracheal nociception, and the different expression patterns of TRPV1 and TRPV2 with neuropeptides may reflect different subpopulations of sensory neurons. [Abstract]

Gordon Z, Elad D, Almog R, Hazan Y, Jaffa AJ, Eytan O
Anthropometry of fetal vasculature in the chorionic plate.
J Anat. 2007 Dec;211(6):698-706.
Normal fetal development is dependent on adequate placental blood perfusion. The functional role of the placenta takes place mainly in the capillary system; however, ultrasound imaging of fetal blood flow is commonly performed on the umbilical artery, or on its first branches over the chorionic plate. The objective of this study was to evaluate the structural organization of the feto-placental vasculature of the chorionic plate. Casting of the placental vasculature was performed on 15 full-term placentas using a dental polymer mixed with colored ink. Observations of the cast models revealed that the branching architecture of the chorionic vessel is a combination of dichotomous and monopodial patterns, where the first two to three generations are always of a dichotomous nature. Analysis of the daughter-to-mother diameter ratios in the chorionic vessels provided a maximum in the range of 0.6-0.8 for the dichotomous branches, whereas in monopodial branches it was in the range of 0.1-0.3. Similar to previous studies, this study reveals that the vasculature architecture is mostly monopodial for the marginal cord insertion and mostly dichotomous for the central insertion. The more marginal the umbilical cord insertion is on the chorionic plate, the more monopodial branching patterns are created to compensate the dichotomous pattern deficiency to perfuse peripheral placental territories. [Abstract]

Makanya AN, Mortola JP
The structural design of the bat wing web and its possible role in gas exchange.
J Anat. 2007 Dec;211(6):687-97.
The structure of the skin in the epauletted fruit bat (Epomophorus wahlbergi) wing and body trunk was studied with a view to understanding possible adaptations for gas metabolism and thermoregulation. In addition, gas exchange measurements were performed using a respirometer designed for the purpose. The body skin had an epidermis, a dermis with hair follicles and sweat glands and a fat-laden hypodermis. In contrast, the wing web skin was made up of a thin bilayered epidermis separated by a connective tissue core with collagen and elastic fibres and was devoid of hair follicles and sweat glands. The wings spanned 18-24 cm each, with about 753 cm(2) of surface exposed to air. The body skin epidermis was thick (61 +/- 3 microm, SEM), the stratum corneum alone taking a third of it (21 +/- 3 microm). In contrast, the wing web skin epidermis was thinner at 9.8 +/- 0.7 microm, with a stratum corneum measuring 4.1 +/- 0.3 microm (41%). The wing capillaries in the wing web skin ran in the middle of the connective tissue core, with a resultant surface-capillary diffusion distance of 26.8 +/- 3.2 microm. The rate of oxygen consumption (VO(2)) of the wings alone and of the whole animal measured under light anaesthesia at ambient temperatures of 24 masculineC and 33 masculineC, averaged 6% and 10% of the total, respectively. Rate of carbon dioxide production had similar values. The membrane diffusing capacity for the wing web was estimated to be 0.019 ml O(2) min(-1) mmHg(-1). We conclude that in Epomophorus wahlbergi, the wing web has structural modifications that permit a substantial contribution to the total gas exchange. [Abstract]

Frey R, Volodin I, Volodina E
A nose that roars: anatomical specializations and behavioural features of rutting male saiga.
J Anat. 2007 Dec;211(6):717-36.
The involvement of the unique saiga nose in vocal production has been neglected so far. Rutting male saigas produce loud nasal roars. Prior to roaring, they tense and extend their noses in a highly stereotypic manner. This change of nose configuration includes dorsal folding and convex curving of the nasal vestibulum and is maintained until the roar ends. Red and fallow deer males that orally roar achieve a temporary increase of vocal tract length (vtl) by larynx retraction. Saiga males attain a similar effect by pulling their flexible nasal vestibulum rostrally, allowing for a temporary elongation of the nasal vocal tract by about 20%. Decrease of formant frequencies and formant dispersion, as acoustic effects of an increase of vtl, are assumed to convey important information on the quality of a dominant male to conspecifics, e.g. on body size and fighting ability. Nasal roaring in saiga may equally serve to deter rival males and to attract females. Anatomical constraints might have set a limit to the rostral pulling of the nasal vestibulum. It seems likely that the sexual dimorphism of the saiga nose was induced by sexual selection. Adult males of many mammalian species, after sniffing or licking female urine or genital secretions, raise their head and strongly retract their upper lip and small nasal vestibulum while inhalating orally. This flehmen behaviour is assumed to promote transport of non-volatile substances via the incisive ducts into the vomeronasal organs for pheromone detection. The flehmen aspect in saiga involves the extensive flexible walls of the greatly enlarged nasal vestibulum and is characterized by a distinctly concave configuration of the nose region, the reverse of that observed in nasal roaring. A step-by-step model for the gradual evolution of the saiga nose is presented here. [Abstract]

van der Werf M, Lezuo P, Maissen O, van Donkelaar CC, Ito K
Inhibition of vertebral endplate perfusion results in decreased intervertebral disc intranuclear diffusive transport.
J Anat. 2007 Dec;211(6):769-74.
Impaired nutrition of the intervertebral disc has been hypothesized to be one of the causes of disc degeneration. However, no causal relationship between decreased endplate perfusion and limited nutrient transport has been demonstrated to support this pathogenic mechanism. To determine the importance of endplate perfusion on solute diffusion into the nucleus pulposus and to show causality of endplate perfusion on intranuclear diffusion in large animal lumbar intervertebral discs, diffusive transport into ovine lumbar intervertebral discs was evaluated after inhibiting adjacent vertebral endplate perfusion. Partial perfusion blocks were created in vertebrae close and parallel to both endplates of lumbar discs of anaesthetized sheep. To assess diffusivity of small molecules through the endplate, N(2)O was introduced into the inhalation gas mixture and concentrations of intranuclear N(2)O were measured for 35 min thereafter. Post mortem, procion red was infused through the spinal vasculature and perfusion through the endplate was assessed by quantifying the density of dye-perfused endplate vascular buds in histology sections. Perfusion of the endplates overlying the nucleus pulposus was inhibited by almost 50% in the partially blocked discs relative to the control discs. There was also a nine-fold decreased transport rate of intranuclear N(2)O in partially blocked discs compared with control discs. The density of perfused endplate vascular buds correlated significantly to the amount of transported intranuclear N(2)O (r(2) = 0.52, P = 0.008). The vertebral endplate was demonstrated to be the main route of intravascular solute transport into the nucleus pulposus of intervertebral discs, and inhibition of endplate perfusion can cause inhibited solute transport into the disc intranuclear tissue. [Abstract]

Mohammad MG, Chilmonczyk S, Birch D, Aladaileh S, Raftos D, Joss J
Anatomy and cytology of the thymus in juvenile Australian lungfish, Neoceratodus forsteri.
J Anat. 2007 Dec;211(6):784-97.
The anatomy, histology and ultrastructure of the thymus of a dipnoan, the Australian lungfish, Neoceratodus forsteri, was studied by light and transmission electron microscopy. The thymic tissue showed clear demarcation into a cortex and medulla with ample vascularization. Large cells including foamy and giant multinucleated cells with periodic acid Schiff/Alcian blue positive staining properties were localized mainly in the medulla. The major cellular components were epithelial cells and lymphoid cells. The epithelial cells were classified by location and ultrastructure into six sub-populations: capsular cells, cortical and medullary reticular cells, perivascular endothelial cells, intermediate cells, nurse-like cells and Hassall-like corpuscles. Myoid cells were found mainly in the cortico-medullary boundary and medulla. Macrophages and secretory-like cells were also present. These findings will provide a base of knowledge about the cellular immune system of lungfish. [Abstract]

Scheyer TM
Skeletal histology of the dermal armor of Placodontia: the occurrence of 'postcranial fibro-cartilaginous bone' and its developmental implications.
J Anat. 2007 Dec;211(6):737-53.
Placodontia (Reptilia: Sauropterygia) is a group of enigmatic armored marine reptiles restricted to the Triassic time period. Only a single row of osteoderms dorsal to the spine is present in the basal placodontoid Placodus gigas, whereas derived cyamodontoids superficially resemble turtles in enclosing their body in an armor shell. Despite the extensive occurrence of the dermal armor in the derived cyamodontoid group, little research has focused on its bone histology and development. Here, I present an overview of the bone microstructures that reveals the unique presence of cartilaginous tissue in the postcranial armor plates. Placodont armor plates stand in contrast to osteoderms of other tetrapods that develop intramembraneously or through metaplastic ossification without cartilaginous preformation. The different developmental pathways leading to this 'postcranial fibro-cartilaginous bone' tissue found in placodont plates compared to the dermal bone tissues of most other tetrapod osteoderms indicate the non-homology of these structures. A resulting morphogenetic model of histogenesis is given to exemplify how the derived armor morphologies (i.e. spiked, flat polygonal and hexagonal, and rhomboidal shapes) together with the peculiar bone histologies could have developed through differential growth. In accordance with the pachyostotic limb bones of placodonts, the presence of the compact 'postcranial fibro-cartilaginous bone' is interpreted as an osteosclerotic trend in the armor plates which aids in buoyancy control and affects maneuverability and swimming speed. [Abstract]

Hamilton RD, Foss AJ, Leach L
Establishment of a human in vitro model of the outer blood-retinal barrier.
J Anat. 2007 Dec;211(6):707-16.
The outer blood-retinal barrier is composed of a monolayer of retinal pigment epithelium, Bruch's membrane and the choriocapillaris which is fenestrated. Endothelial proliferation and breaching of Bruch's membrane leads to the neovascular form of age-related macula degeneration (ARMD). The aim of this study was to generate an in vitro model that mimics more faithfully the phenotype of the choriocapillaris and the trilayer architecture in vitro. A trilayer culture model was generated with retinal pigment epithelium (ARPE-19) cell cultures on the epithelial surface of amniotic membrane and with human umbilical vein-derived endothelial cells on the other surface. A control model for the effect of retinal pigment epithelium on endothelial changes was generated with corneal epithelial cells replacing the ARPE-19. Both human umbilical vein-derived endothelial and ARPE-19 cells formed confluent monolayers on respective surfaces of the amnion. The human umbilical vein-derived endothelial cells in the trilayer became fenestrated when co-cultured with the ARPE-19 cells, but not with corneal epithelial cells, or when grown as monolayers on the amnion, showing a loss of fidelity of origin in the presence of ARPE-19 cells. These cells also revealed VE-cadherin and ZO-1 at cell-cell contacts from 24 h in the trilayer. The tight junctional molecules, occludin and ZO-1, were localized to cell-cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. Permeability of the trilayer was tested by using fluorescein and fluorescein-conjugated tracers under flow. At 72 h the trilayer severely restricted transfer of sodium fluorescein (NaF) (ten-fold reduction) whilst transfer of a 4 kDa FITC-conjugated dextran was virtually occluded, confirming a restrictive barrier. Ultrastructural studies showed the retinal pigment epithelium monolayer was polarized with microvilli present on the apical surface. Paracellular clefts showed numerous tight junctional-like appositions, similar to that seen on amnion alone. This study demonstrates that ARPE-19 and human umbilical vein-derived endothelial cells can be co-cultured on the amniotic membrane and that the resultant cross-talk leads to formation of a fenestrated endothelium, whilst maintaining a polarized restrictive epithelial layer. The fenestrated endothelial phenotype achieved in this human in vitro trilayer model is a first and offers an outer-retinal barrier which approaches the in vivo state and has potential for studies into induced junctional disruption, endothelial proliferation and migration: features of ARMD. [Abstract]

Montaudon M, Desbarats P, Berger P, de Dietrich G, Marthan R, Laurent F
Assessment of bronchial wall thickness and lumen diameter in human adults using multi-detector computed tomography: comparison with theoretical models.
J Anat. 2007 Nov;211(5):579-88.
A thickened bronchial wall is the morphological substratum of most diseases of the airway. Theoretical and clinical models of bronchial morphometry have so far focused on bronchial lumen diameter, and bronchial length and angles, mainly assessed from bronchial casts. However, these models do not provide information on bronchial wall thickness. This paper reports in vivo values of cross-sectional wall area, lumen area, wall thickness and lumen diameter in ten healthy subjects as assessed by multi-detector computed tomography. A validated dedicated software package was used to measure these morphometric parameters up to the 14th bronchial generation, with respect to Weibel's model of bronchial morphometry, and up to the 12th according to Boyden's classification. Measured lumen diameters and homothety ratios were compared with theoretical values obtained from previously published studies, and no difference was found when considering dichotomic division of the bronchial tree. Mean wall area, lumen area, wall thickness and lumen diameter were then provided according to bronchial generation order, and mean homothety ratios were computed for wall area, lumen area and wall thickness as well as equations giving the mean value of each parameter for a given bronchial generation with respect to its value in generation 0 (trachea). Multi-detector computed tomography measurements of bronchial morphometric parameters may help to improve our knowledge of bronchial anatomy in vivo, our understanding of the pathophysiology of bronchial diseases and the evaluation of pharmacological effects on the bronchial wall. [Abstract]

Kayacan R
The effect of staining on the monotonic tensile mechanical properties of human cortical bone.
J Anat. 2007 Nov;211(5):654-61.
Microdamage in the form of microcracks has been observed in cortical bone following in vivo and in vitro fatigue loading. It has been suggested that bone has an inherent ability to repair microdamage at physiological activity levels. If the biological remodelling and repair process cannot keep up with the rate of damage accumulation, as in ageing bone and in individuals such as athletes and military recruits, microdamage may accumulate even at physiological activity levels. Such microdamage accumulation is thought to contribute to stress and fragility fractures. It is therefore important to obtain quantitative data on the rate of damage accumulation so as to understand the etiology of skeletal fractures. Sequential labelling of microdamage using fluorochrome stains at different stages of mechanical loading is becoming standard for assessing damage evolution. Although verification of this staining technique is provided in the literature, it has not yet been reported if the stains change the mechanical properties of cortical bone. In this study, monotonic tensile tests were performed to investigate the effect of the staining on the monotonic tensile mechanical properties of cortical bone. Forty-eight specimens were machined from human femora obtained from three male subjects, aged 52-55 years, and all 48 specimens were systematically divided into one control and three treatment groups. Specimens in the first (n = 12) and second treatment groups (n = 12) were stained with alizarin complexone and calcein (0.0005 M), respectively, for 16 h under 50 mmHg vacuum. Specimens in the third treatment group (n = 12) were kept in calcium-supplemented saline solution under the same conditions of the first and second treatment groups. Specimens in the control group (n = 12) were removed from the freezer prior to testing and allowed to thaw at room temperature in saline solution. Differences among the mean values of the mechanical properties for four testing groups were determined by the Mann-Whitney test at a significance level of P < 0.05. The statistical results indicated that the chelating stains and the staining conditions have no significant effect on the mechanical properties of the cortical bone under monotonic tensile loading. This study demonstrated that microcrack labelling with the chelating stains under aforementioned conditions (stain concentration, staining time, etc.) is a reliable method in that staining cortical bone with alizarin complexone and calcein prior to testing does not affect tensile properties. [Abstract]

Kress A, Morson G
Changes in the oviducal epithelium during the estrous cycle in the marsupial Monodelphis domestica.
J Anat. 2007 Oct;211(4):503-17.
The Monodelphis oviduct can be divided into four anatomical segments: preampulla (comprising fimbriae and infundibulum), ampulla, isthmus with crypts and uterotubal junction. Ovaries are enclosed in a periovarial sac, the bursa, and in some specimens tubules of an epoophoron could be identified. In both structures non-ciliated cells develop small translucent vesicles, which accumulate in the cell apices and presumably produce fluid as often seen in the bursa and in the tubules of the epooophoron. These vesicles do not stain with Alcian blue or PAS. The same applies also to the non-ciliated cells of the fimbriae. The oviducal epithelium of ampulla and the surface epithelium of the isthmus consisting of ciliated and non-ciliated, secretory cells undergo considerable changes during the estrous cycle. Proestrus shows low numbers of ciliated cells, some are in the process of neo-ciliogenesis, non-ciliated cells carry solitary cilia and few remnant secretory granules from the previous cycle may be found. At estrus the amount of ciliated cells in ampulla and isthmus has increased, most non-cililated cells lost the solitary cilia, developed longer microvilli and formed numerous secretory granules in their cell apices. At postestrus secretory products, often surrounded by membranes, are extruded into the oviducal lumen and contribute towards egg coat formation. First signs of deciliation processes are apparent. Solitary cilia reappear. At metestrus only few secretory cells are left with some secretory material. The lumen is often filled with shed cilia and cell apices. Proliferation of basal bodies within non-secretory cells indicate the formation of new ciliated cells. The non-ciliated epithelial cells of the isthmic crypts form no secretory granules but accumulate a great number of translucent vesicles, which in contrast to the secretory granules do not stain with Alcian blue or PAS. [Abstract]

Bentley VA, Sample SJ, Livesey MA, Scollay MC, Radtke CL, Frank JD, Kalscheur VL, Muir P
Morphologic changes associated with functional adaptation of the navicular bone of horses.
J Anat. 2007 Nov;211(5):662-72.
Failure of functional adaptation to protect the skeleton from damage is common and is often associated with targeted remodeling of bone microdamage. Horses provide a suitable model for studying loading-related skeletal disease because horses are physically active, their exercise is usually regulated, and adaptive failure of various skeletal sites is common. We performed a histologic study of the navicular bone of three groups of horses: (1) young racing Thoroughbreds (n = 10); (2) young unshod ponies (n = 10); and (3) older horses with navicular syndrome (n = 6). Navicular syndrome is a painful condition that is a common cause of lameness and is associated with extensive remodeling of the navicular bone; a sesamoid bone located within the hoof which articulates with the second and third phalanges dorsally. The following variables were quantified: volumetric bone mineral density; cortical thickness (Ct.Th); bone volume fraction, microcrack surface density; density of osteocytes and empty lacunae; and resorption space density. Birefringence of bone collagen was also determined using circularly polarized light microscopy and disruption of the lacunocanalicular network was examined using confocal microscopy. Remodeling of the navicular bone resulted in formation of transverse secondary osteons orientated in a lateral to medial direction; bone collagen was similarly orientated. In horses with navicular syndrome, remodeling often led to the formation of intracortical cysts and development of multiple tidemarks at the articular surface. These changes were associated with high microcrack surface density, low bone volume fraction, low density of osteocytes, and poor osteocyte connectivity. Empty lacunae were increased in Thoroughbreds. Resorption space density was not increased in horses with navicular syndrome. Taken together, these data suggest that the navicular bone may experience habitual bending across the sagittal plane. Consequences of cumulative cyclic loading in horses with navicular syndrome include arthritic degeneration of adjacent joints and adaptive failure of the navicular bone, with accumulation of microdamage and associated low bone mass, poor osteocyte connectivity, and low osteocyte density, but not formation of greater numbers of resorption spaces. [Abstract]

Butti C, Corain L, Cozzi B, Podestà M, Pirone A, Affronte M, Zotti A
Age estimation in the Mediterranean bottlenose dolphin Tursiops truncatus (Montagu 1821) by bone density of the thoracic limb.
J Anat. 2007 Nov;211(5):639-46.
The determination of age is an important step in defining the life history traits of individuals and populations. Age determination of odontocetes is mainly based on counting annual growth layer groups in the teeth. However, this useful method is always invasive, requiring the cutting of at least one tooth, and sometimes the results are difficult to interpret. Based on the concept that bone matrix is constantly deposited throughout life, we analysed the bone mineral density of the arm and forearm of a series of bottlenose dolphins (Tursiops truncatus, Montagu 1821) stranded along the Italian coast of the Adriatic Sea or maintained in confined waters. The bone mineral density values we obtained were evaluated as possible age predictors of the Mediterranean population of this species, considering age as determined by counting growth layer groups in sections of the teeth and the total body length of the animal as references. Comparisons between left and right flipper showed no difference. Our results show that bone mineral density values of the thoracic limb are indeed reliable age predictors in Tursiops truncatus. Further investigations in additional odontocete species are necessary to provide strong evidence of the reliability of bone mineral density as an indicator of growth and chronological wear and tear in toothed-whales. [Abstract]

Fraher JP, Dockery P, O'Donoghue O, Riedewald B, O'Leary D
Initial motor axon outgrowth from the developing central nervous system.
J Anat. 2007 Nov;211(5):600-11.
Rat and chick studies show that the earliest motor rootlet axon bundles emerge from all levels of the neural tube between radial glial end feet which comprise the presumptive glia limitans. The loose arrangement of the end feet at the time of emergence facilitates this passage. The points of emergence are regularly spaced in relation to the long axis of the neural tube and are not defined by any cell contact with its surface. Each rootlet carries a covering of basal lamina from the neural tube surface, which forms a sleeve around it. It is only after bundles of ventral rootlet axons have emerged that cells associate with them, forming clusters on the rootlet surface at a distance peripheral to the CNS surface of both species. A tight collar of glial end feet develops around the axon bundle at the neural tube surface shortly after initial emergence. These arrangements are in sharp contrast to those seen in the sensory rootlets, where clusters of boundary cap cells prefigure the sensory entry zones at the attachments of the prospective dorsal spinal and cranial sensory rootlets. Boundary cap cells resemble cluster cells and a neural crest origin seems the most likely for them. The study clearly demonstrates that no features resembling boundary caps are found in relation to the developing motor exit points. [Abstract]

Jenkins D, Winyard PJ, Woolf AS
Immunohistochemical analysis of Sonic hedgehog signalling in normal human urinary tract development.
J Anat. 2007 Nov;211(5):620-9.
Studies of mouse mutants have demonstrated that Sonic hedgehog (SHH) signalling has a functional role in morphogenesis and differentiation at multiple sites within the forming urinary tract, and urinary tract malformations have been reported in humans with mutations that disrupt SHH signalling. However, there is only strikingly sparse and fragmentary information about the expression of SHH and associated signalling genes in normal human urinary tract development. We used immunohistochemistry to demonstrate that SHH protein was localised in distinct urinary tract epithelia in developing normal humans, in the urothelium of the nascent bladder and in kidney medullary collecting ducts. The expression patterns of the SHH-transducing proteins Patched (PTCH) and Smoothened (SMO) were consistent with long-range paracrine signalling associated with detrusor smooth muscle differentiation in the urogenital sinus. In the developing kidney, SHH and PTCH were expressed in epithelia of the collecting system between 16-26 weeks--surprisingly, SMO was not detected. Analysis of cell proliferation and Cyclin B1 immunohistochemistry at 26 weeks, as compared with a 28 week sample in which SHH expression was down-regulated, was consistent with the idea that SHH and PTCH might influence medullary collecting duct growth by regulating the subcellular localisation of Cyclin B1 independently of SMO. Collectively, these descriptive results generate new hypotheses regarding SHH signal transduction in human urinary tract development and help to explain the varied urinary tract malformation phenotypes noted in individuals with mutations in the SHH pathway. [Abstract]

Fernandes T, Costa C
Klippel-Feil syndrome with other associated anomalies in a medieval Portuguese skeleton (13th-15th century).
J Anat. 2007 Nov;211(5):681-5.
Klippel-Feil syndrome, or synostosis of the cervical spine, is the result of an abnormal division of somites during embryonic development. This report analyses an adult male (exhumed from a Portuguese graveyard dating from the 13th to the 15th century) with malformations in the cranium and vertebral column. Besides the lesions that are typical of Klippel-Feil syndrome type II, other defects usually linked to this pathology are described (occipito-atlantal fusion, hemivertebrae, butterfly vertebrae, cervical rib, changes in normal number of vertebral segments and a possible Sprengel deformity). [Abstract]

O'Rahilly R, Müller F
The development of the neural crest in the human.
J Anat. 2007 Sep;211(3):335-51.
The first systematic account of the neural crest in the human has been prepared after an investigation of 185 serially sectioned staged embryos, aided by graphic reconstructions. As many as fourteen named topographical subdivisions of the crest were identified and eight of them give origin to ganglia (Table 2). Significant findings in the human include the following. (1) An indication of mesencephalic neural crest is discernible already at stage 9, and trigeminal, facial, and postotic components can be detected at stage 10. (2) Crest was not observed at the level of diencephalon 2. Although pre-otic crest from the neural folds is at first continuous (stage 10), crest-free zones are soon observable (stage 11) in Rh.1, 3, and 5. (3) Emigration of cranial neural crest from the neural folds at the neurosomatic junction begins before closure of the rostral neuropore, and later crest cells do not accumulate above the neural tube. (4) The trigeminal, facial, glossopharyngeal and vagal ganglia, which develop from crest that emigrates before the neural folds have fused, continue to receive contributions from the roof plate of the neural tube after fusion of the folds. (5) The nasal crest and the terminalis-vomeronasal complex are the last components of the cranial crest to appear (at stage 13) and they persist longer. (6) The optic, mesencephalic, isthmic, accessory, and hypoglossal crest do not form ganglia. Cervical ganglion 1 is separated early from the neural crest and is not a Froriep ganglion. (7) The cranial ganglia derived from neural crest show a specific relationship to individual neuromeres, and rhombomeres are better landmarks than the otic primordium, which descends during stages 9-14. (8) Epipharyngeal placodes of the pharyngeal arches contribute to cranial ganglia, although that of arch 1 is not typical. (9) The neural crest from rhombomeres 6 and 7 that migrates to pharyngeal arch 3 and from there rostrad to the truncus arteriosus at stage 12 is identified here, for the first time in the human, as the cardiac crest. (10) The hypoglossal crest provides cells that accompany those of myotomes 1-4 and form the hypoglossal cell cord at stages 13 and 14. (11) The occipital crest, which is related to somites 1-4 in the human, differs from the spinal mainly in that it does not develop ganglia. (12) The occipital and spinal portions of the crest migrate dorsoventrad and appear to traverse the sclerotomes before the differentiation into loose and dense zones in the latter. (13) Embryonic examples of synophthalmia and anencephaly are cited to emphasize the role of the neural crest in the development of cranial ganglia and the skull. [Abstract]

Stanley RL, Fleck RA, Becker DL, Goodship AE, Ralphs JR, Patterson-Kane JC
Gap junction protein expression and cellularity: comparison of immature and adult equine digital tendons.
J Anat. 2007 Sep;211(3):325-34.
Injury to the energy-storing superficial digital flexor tendon is common in equine athletes and is age-related. Tenocytes in the superficial digital flexor tendon of adult horses appear to have limited ability to respond adaptively to exercise or prevent the accumulation of strain-induced microdamage. It has been suggested that conditioning exercise should be introduced during the growth period, when tenocytes may be more responsive to increased quantities or intensities of mechanical strain. Tenocytes are linked into networks by gap junctions that allow coordination of synthetic activity and facilitate strain-induced collagen synthesis. We hypothesised that there are reductions in cellular expression of the gap junction proteins connexin (Cx) 43 and 32 during maturation and ageing of the superficial digital flexor tendon that do not occur in the non-injury-prone common digital extensor tendon. Cryosections from the superficial digital flexor tendon and common digital extensor tendon of 5 fetuses, 5 foals (1-6 months), 5 young adults (2-7 years) and 5 old horses (18-33 years) were immunofluorescently labelled and quantitative confocal laser microscopy was performed. Expression of Cx43 and Cx32 protein per tenocyte was significantly higher in the fetal group compared with all other age groups in both tendons. The density of tenocytes was found to be highest in immature tissue. Higher levels of cellularity and connexin protein expression in immature tendons are likely to relate to requirements for tissue remodelling and growth. However, if further studies demonstrate that this correlates with greater gap junctional communication efficiency and synthetic responsiveness to mechanical strain in immature compared with adult tendons, it could support the concept of early introduction of controlled exercise as a means of increasing resistance to later injury. [Abstract]

Pazzaglia UE, Bonaspetti G, Rodella LF, Ranchetti F, Azzola F
Design, morphometry and development of the secondary osteonal system in the femoral shaft of the rabbit.
J Anat. 2007 Sep;211(3):303-12.
The architecture of the diaphyseal bone is closely correlated with the cortical vessel network, whose pattern develops in the course of growth. Various methods have been applied to clarify the three-dimensional anatomy of the cortical canal system, but there is still disagreement about the geometry, blood supply, flux dynamics and factors controlling canal geometry during bone growth and remodeling. A modification of the currently employed dye-injection method was applied to study the vessel network of the whole hemi-shaft of the rabbit femur in mature bones (8-month-old rabbits) and growing bones (1.5-month-old rabbits). The cortical vascular tree of the hemi-shaft of the femur was injected with black China ink and observed in full-thickness specimens of the cortex. The same specimens were then processed for histology. A comparative study of the middle diaphysis (mid-shaft) with the distal extremity (distal shaft) was performed in both young and old rabbit femurs. The longitudinally oriented pattern of the vessel network was seen to develop in the diaphysis of mature femurs, while at the extremity of the shaft of the same specimen the network showed a reticular organization without a dominant polarization. The vessels were significantly higher in the mid-shaft than in the distal shaft of the old femurs (P < 0.0001), as was their diameter (P < 0.05). In the group of young rabbits at mid-shaft level the longitudinally oriented pattern of the vessel network was not yet completely developed, without their being significant differences in length and diameter between the mid-shaft and distal shaft. The differentiation of the mid-shaft from the distal shaft was confirmed histologically by the presence, in the latter, of longitudinal calcified cartilage septa between osteons. This pattern of structural organization and development of the intracortical vascular network has not been previously reported. The cells primarily involved in polarization of the remodeling process were the osteoclasts at the top of the cutting cones advancing from the proximal and distal metaphyses toward the mid-shaft. This suggests, first, a relationship with the longitudinally oriented structures already present in the cortex near the metaphysis (the calcified cartilage septa) and then with the columns of interosteonic breccia, which were formed as a secondary effect of the longitudinal polarization of the remodeling process. Our observations did not enable us to substantiate the model of two different systems, one of longitudinal vessels (Havers) and the other of connecting transversal vessels (Volkmann), but suggested instead that there is a network whose loops lengthen in the direction of the major bone axis in the course of growth and secondary modeling. The associated morphology supported the view that the type of structural organization of the tubular bone cortex is primarily determined by an inherited constitutional factor rather than by mechanical strains. [Abstract]

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Recent Articles in The Anatomical Record

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Langevin HM, Yandow JA
Relationship of acupuncture points and meridians to connective tissue planes.
Anat Rec. 2002 Dec 15;269(6):257-65.
Acupuncture meridians traditionally are believed to constitute channels connecting the surface of the body to internal organs. We hypothesize that the network of acupuncture points and meridians can be viewed as a representation of the network formed by interstitial connective tissue. This hypothesis is supported by ultrasound images showing connective tissue cleavage planes at acupuncture points in normal human subjects. To test this hypothesis, we mapped acupuncture points in serial gross anatomical sections through the human arm. We found an 80% correspondence between the sites of acupuncture points and the location of intermuscular or intramuscular connective tissue planes in postmortem tissue sections. We propose that the anatomical relationship of acupuncture points and meridians to connective tissue planes is relevant to acupuncture's mechanism of action and suggests a potentially important integrative role for interstitial connective tissue. [Abstract]

Mizer LA, Farnum CE, Schenck PD
The Modular Resource Center: integrated units for the study of the anatomical sciences in a problem-based curriculum.
Anat Rec. 2002 Dec 15;269(6):249-56.
The Modular Resource Center (MRC) at the College of Veterinary Medicine at Cornell University was created in 1993 as a way to provide visual resources in support of a newly implemented problem-based curriculum in which the anatomical sciences are taught primarily in the first tutorial-based course, The Animal Body. Over two dozen modules have been created specifically in support of this course, whereas additional modules have been created in support of other basic science courses. The basic unit of organization of the MRC is a module presented in a carrel that provides students a way to study, either alone or in groups, a given topic. The topic is presented through a script and an integrated set of anatomical materials including plastinated dissected specimens, vascular casts, skeletal preparations, models, radiographs, histological slides, and photo- and electron micrographs. The key feature of this resource center is that it is not a museum; rather it is more analogous to an interactive library, that can be used for reference, study, and review, not only by veterinary students but also by faculty, interns, residents, and undergraduates. A unique aspect is that all materials have been made by veterinary students working with faculty during the summer. Although started as a resource in support of a tutorial-based curriculum, the MRC has evolved over a decade into an anatomy resource that would be highly valued in any curricular format. [Abstract]

Rizzolo LJ
Human dissection: an approach to interweaving the traditional and humanistic goals of medical education.
Anat Rec. 2002 Dec 15;269(6):242-8.
Anatomy remains one of the core courses of medical school, but the time devoted to it is decreasing. To accommodate the explosion of medical knowledge, educators search to streamline the curriculum. Because it is time-consuming, dissection comes under increased scrutiny. Even in the face of these pressures to reduce course hours, I would like to propose broadening, not reducing, the responsibilities of the anatomy instructor. Anatomy instructors can play a crucial role in helping medical schools meet the critical need to cultivate humanistic values, especially in the arena of end-of-life care. Anatomy can--and should--play an important role in a curriculum-wide effort to address this issue. Just as dissection remains an essential technique to teach three-dimensional concepts, the cadaver dissection lab is an ideal place to introduce concepts of humanistic care. The lab evokes the students' memories, speculations, and fears about serious illness in themselves, their families, and loved ones. Some programs address these reactions with supplemental activities, such as journaling, essay writing, and small group discussion. Valuable as these activities may be, anatomy instructors can achieve more by recognizing their role as a mentor, who can integrate humanistic values into traditional course objectives in a way that adds little time to the curriculum. The attitude of the instructor in ministering to the students' needs as they undertake the emotionally charged task of dissection can provide a model for how the students will respond, in turn, to the hopes and fears of their patients-and to their own reactions to dying. This approach will allow students to implement and practice humanistic values immediately, laying a foundation for their clinical training. [Abstract]

Tattersall I, Schwartz JH
Is paleoanthropology science? Naming new fossils and control of access to them.
Anat Rec. 2002 Dec 15;269(6):239-41.
Progress in paleoanthropology is impeded when new fossil materials are published but unavailable for comparative study, as is too often the case. In this commentary, we review the stages of description and analysis that new fossils must undergo and conclude that it is disingenuous to argue that fossils have not been properly "published" when descriptions and new names formulated in accordance with the International Code of Zoological Nomenclature have appeared in leading scientific journals. Once such names and descriptions have been published, it is imperative that the original fossils concerned be available to the scientific community for comparative analysis, for by the very nature of science, no statement about such fossils, however carefully prepared by the original describers (or anyone else), can be regarded as definitive. Science is a system of provisional knowledge that constantly requires re-examination and testing. It cannot function as a system in which assertions have to be left unchallenged for want of free access to the primary data. [Abstract]

Ridgway SH, Marino L, Lipscomb TP
Description of a poorly differentiated carcinoma within the brainstem of a white whale (Delphinapterus leucas) from magnetic resonance images and histological analysis.
Anat Rec. 2002 Dec 1;268(4):441-9.
In this study we used magnetic resonance imaging (MRI) to investigate neuroanatomical structure in the brain of a white whale (Delphinapterus leucas) that died from a large tumor within the brainstem. This specimen was also compared with a normal white whale brain using MRI. MRI scans of the white whale specimen show how the tumor deformed surrounding brain structure. Histopathological analysis indicated a poorly differentiated carcinoma of uncertain origin. These analyses demonstrate the usefulness of supplementing histological analyses of pathology with studies of gross morphology facilitated by MRI. [Abstract]

Ritty TM, Ditsios K, Starcher BC
Distribution of the elastic fiber and associated proteins in flexor tendon reflects function.
Anat Rec. 2002 Dec 1;268(4):430-40.
The elastic fiber is known to be an important component of skin, lung, and vasculature. Much less is known about the distribution of elastin and elastic fiber-related proteins in connective tissues, yet genetic defects of elastic fiber constituents can lead to deficiencies in these tissues. For the first time, we determine the distribution of elastin, fibrillins 1 and 2, and microfibril-associated glycoproteins (MAGPs) 1 and 2 in the flexor digitorum profundus (FDP) tendon. Three functionally distinct regions of the FDP tendon, the fibrocartilagenous (FC) region, avascular/tensional (AV/T) region, and insertion region, were evaluated by immunohistochemical methods for these five proteins. Biochemical analysis of desmosine content, an elastin-specific cross-link, demonstrated the presence of elastin in each region, and this was verified histochemically. The fibrillins were found with elastin and also pericellularly with internal fibroblasts where elastin was not detected. Although there was overlapping distribution, fibrillin 2 was more prominent in the interior of the tendon while fibrillin 1 was prominent in outer cell layers that contained elastic fibers. Both MAGP-1 and -2 were found throughout the tendon, although the greatest abundance was near the tendon insertion to bone. Surprisingly, MAGP-1 demonstrated a filamentous appearance within the fibrocartilage that did not correspond to the fibrillin 1 or 2 or MAGP-2 staining pattern. Lastly, we have shown that a vincular membrane located along the dorsal surface of the tendon near the insertion has a very high elastin content and a unique interface with the tendon that consists of an elastic anchor within the tendon body. [Abstract]

Marino L, Sudheimer KD, Pabst DA, McLellan WA, Filsoof D, Johnson JI
Neuroanatomy of the common dolphin (Delphinus delphis) as revealed by magnetic resonance imaging (MRI).
Anat Rec. 2002 Dec 1;268(4):411-29.
In this study, magnetic resonance (MR) images of the brain of an adult common dolphin (Delphinus delphis) were acquired in the coronal plane at 66 antero-posterior levels. From these scans a computer-generated set of resectioned virtual images in orthogonal planes was constructed using the programs VoxelView and VoxelMath (Vital Images, Inc., Michigan State Univ.). Sections in all three planes reveal major neuroanatomical structures. These structures in the adult common dolphin brain are compared with those from a fetal common dolphin brain from a previously published study as well as with MR images of adult brains of other odontocetes. This study, like previous ones, demonstrates the utility of MR imaging (MRI) for comparative neuroanatomical investigations of dolphin brains. [Abstract]

Jo Mauch T, Albertine KH
Urorectal septum malformation sequence: Insights into pathogenesis.
Anat Rec. 2002 Dec 1;268(4):405-10.
We characterize the urorectal septum malformation sequence (URSMS) in discordant fetal lambs and relate it to the human syndromes with which URSMS is associated. We found abnormal external genitalia, imperforate anus, and fistulous connections between the rectum, bladder, and vagina. Discordance among the dizygous twins eliminated teratogens as a likely etiologic factor. We summarize the relevant literature and propose a molecular model for the pathogenesis of the URSMS, in which alterations in sonic hedgehog and homeobox genes lead to caudal mesodermal deficiency during blastogenesis. [Abstract]

Fazan VP, Ma X, Chapleau MW, Barreira AA
Qualitative and quantitative morphology of renal nerves in C57BL/6J mice.
Anat Rec. 2002 Dec 1;268(4):399-404.
The detailed morphology of the renal nerves in mice has not been reported previously. The aims of this study were to describe the general morphology of the extrinsic renal nerve in C57BL/6 mice, and determine its morphometric parameters. The major renal nerve innervating the left kidney was isolated in five mice. Thin sections of the nerve segments were then examined by transmission electron microscopy. The renal nerve averaged 35.4 +/- 3.6 (S.E.M.) microm in diameter and 741 +/- 104 microm in area. The renal nerve contained an average of 830 +/- 169 unmyelinated fibers and only 4.6 +/- 1.7 myelinated fibers. The axon diameter of myelinated and unmyelinated fibers averaged 2.2 +/- 0.3 microm and 0.76 +/- 0.02 microm, respectively. The diameter of the unmyelinated fibers ranged from 0.3 to 2.0 microm, and the distribution histogram was unimodal. The majority of fibers (85%) had diameters of 0.6-1.0 microm. These results are similar to those obtained for renal nerves of rats with respect to the predominance of unmyelinated fibers. However, the diameter of unmyelinated fibers is larger in rats and the distribution histogram of rat unmyelinated fibers is bimodal, in contrast to the unimodal distribution in mice. The morphological description of the renal nerves in mice provides baseline data for further investigations of the structural basis of altered autonomic reflexes. The results will be useful in analyses of genes that influence the development and structure of sympathetic and sensory innervation of the kidney in genetically manipulated mice. [Abstract]

Icardo JM, Colvee E, Cerra MC, Tota B
The structure of the conus arteriosus of the sturgeon (Acipenser naccarii) heart: II. The myocardium, the subepicardium, and the conus-aorta transition.
Anat Rec. 2002 Dec 1;268(4):388-98.
Sturgeons constitute a family of living "fossil" fish whose heart is related to that of other ancient fish and the elasmobranches. We have undertaken a systematic study of the structure of the sturgeon heart aimed at unraveling the relationship between the heart structure and the adaptive evolutionary changes. In a related paper, data were presented on the conus valves and the subendocardium. Here, the structure of the conus myocardium, the subepicardial tissue, and the conus-aorta transition were studied by conventional light, transmission, and scanning electron microscopy. In addition, actin localization by fluorescent phalloidin was used. The conus myocardium is organized into bundles whose spatial organization changes along the conus length. The variable orientation of the myocardial cell bundles may be effective in emptying the conus lumen during contraction and in preventing reflux of blood. Myocardial cell bundles are separated by loose connective tissue that contains collagen and elastin fibers, vessels, and extremely flat cells separating the cell bundles and enclosing blood vessels and collagen fibers. The ultrastructure of the myocardial cells was found to be very similar to that of other fish groups, suggesting that it is largely conservative. The subepicardium is characterized by the presence of nodular structures that contain lympho-hemopoietic (thymus-like) tissue in the young sturgeons and a large number of lymphocytes after the sturgeons reach sexual maturity. This tissue is likely implicated in the establishment and maintenance of the immune responses. The intrapericardial ventral aorta shows a middle layer of circumferentially oriented cells and internal and external layers with cells oriented longitudinally. Elastin fibers completely surround each smooth muscle cell, and the spaces between the different layers are occupied by randomly arranged collagen bundles. The intrapericardial segment of the ventral aorta is a true transitional segment whose structural characteristics are different from those of both the conus subendocardium and the rest of the ventral aorta. [Abstract]

Komatsu K, Mosekilde L, Viidik A, Chiba M
Polarized light microscopic analyses of collagen fibers in the rat incisor periodontal ligament in relation to areas, regions, and ages.
Anat Rec. 2002 Dec 1;268(4):381-7.
We prepared decalcified sagittal sections (20 microm thick) from the incisal, middle, and basal regions of the mandibular incisor of male Wistar rats aged 2, 6, 12, and 24 months, and examined the sections using polarized light microscopy. Most of the birefringent fibers appeared to run obliquely across the periodontal ligament. Birefringent fibers running parallel to the long axis of the incisor were also found in the intermediate area of the ligament. Similar fiber architecture was observed in all four age groups. Quantitative analysis showed that the retardation values of collagen were higher in the bone- and tooth-related areas and lower in the intermediate area of the ligament. The values for the bone- and tooth-related areas increased from the basal toward the incisal regions in all four age groups. Age-related changes in the retardation values were found only in the incisal region of the incisor. In the incisal region, the values for the bone- and tooth-related areas increased markedly from 2-24 months of age, whereas those for the intermediate area increased slightly but significantly with age. Our findings indicate that the degrees of molecular organization and alignment of collagen fibers in the bone- and tooth-related areas of the ligament are higher than those in the intermediate area and increase near the incisal region and with age. It is also suggested that the collagen fibers in the intermediate area remain immature along the long axis of the incisor throughout the life span of the animal. [Abstract]

Morita M, Kudo H, Doi Y, Hirano T, Ikemura K, Fujimoto S
Enhanced immunocytochemical expression of antioxidant enzymes in rat submandibular gland after normobaric oxygenation.
Anat Rec. 2002 Dec 1;268(4):371-80.
In order to clarify the role of antioxidant enzymes in the male rat submandibular gland against short-term normobaric oxygenation, we performed immunocytochemical staining of manganese-containing superoxide dismutase (Mn-SOD), copper- and zinc-containing SOD (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase, and glutathione S-transferases (GST alpha, GST mu, and GST pi) between days 1 and 7 after normobaric oxygenation. Ultrastructural alterations and immunoreactivities for malondialdehyde (MDA), a lipid peroxidation-related molecule, of the acinar and ductal cells after the oxygenation were also investigated. Immunoreactivity for MDA was exhibited in the acinar cells throughout the experiment. On the other hand, immunoreactivity for the SODs, CAT, and GSTs was not altered, when compared to that of controls, but was significantly elevated in the granular, striated, and excretory ductal cells. Since an increase of lipid peroxidation as indicated by enhanced immunoreactivity for MDA was detected in the acinar and intercalated ductal cells, the results indicate that the enhanced antioxidant enzymes in the granular, striated, and excretory ductal cells play a crucial role in the self-defense system of the male rat submandibular gland against normobaric oxygenation. [Abstract]

Shea JE, Hallows RK, Ricks S, Bloebaum RD
Microvascularization of the hypermineralized calcified fibrocartilage and cortical bone in the sheep proximal femur.
Anat Rec. 2002 Dec 1;268(4):365-70.
It is well known that the incidence of hip fractures is increasing as the population ages, and that vascularity is one of the most important characteristics for any tissue (the proximal femur being no exception). Additionally, calcified fibrocartilage from tendon and ligament insertions comprises a significant portion of the fractional area of the proximal femur's cortical shell. The goal of the present investigation was to quantify and compare the microvascularity of the cortical bone and calcified fibrocartilage of the proximal femur in a sheep model. There were no regional differences in the vascular density of the cortical bone. However, the calcified fibrocartilage from tendon and capsular insertions were determined to be avascular, and regions of the proximal femur with insertions lacked a vascularized periosteum. If a vessel was present in the calcified fibrocartilage, it was located within an isolated region of bone tissue or osteoid. Since blood vessels appear to be a significant contributor to the health and remodeling of mineralized tissue, it is hypothesized that the large areas of avascular calcified fibrocartilage present on the elderly femoral neck may predispose these regions to damage accumulation. Therefore future research should examine the role of the vascularity to the proximal femur in the mechanisms of numerous pathological conditions, such as avascular necrosis, osteopenia, and hip fractures. [Abstract]

Zavala WD, De Simone DS, Sacerdote FL, Cavicchia JC
Variation in Langerhans cell number and morphology between the upper and lower regions of the human esophageal epithelium.
Anat Rec. 2002 Dec 1;268(4):360-4.
Langerhans cells (LCs) are dendritic components of stratified epithelia, presenting antigens to other cells of the immune system that play a crucial role in local defense. The paucity of information about their significance in the esophageal mucosa was addressed by studying their distribution and morphology in this particular location. LCs were identified by immunohistochemical detection of CD1a, a cell-specific marker, using a monoclonal antibody, as well as by electron microscopic identification of characteristic Birbeck granules, among other typical morphological features. Cell counts carried out at 25 and 35 cm distal to the dental arch demonstrated significant differences in number and size between the two locations. The upper region contained 10.4 +/- 0.8 cells (mean +/- SEM) vs. 18.4 +/- 1.4 cells in the lower region. Also, cells in the lower region were larger and appeared to have longer dendritic processes. To our knowledge this is the first report of regional differences in number and morphology of LCs in human esophageal mucosa. [Abstract]

Crivellato E, Ribatti D, Mallardi F, Beltrami CA
Granule changes of human and murine endocrine cells in the gastrointestinal epithelia are characteristic of piecemeal degranulation.
Anat Rec. 2002 Dec 1;268(4):353-9.
Piecemeal degranulation is a unique pattern of cell secretion that consists of a slow release of granule contents from cytoplasmic secretory granules, which leaves empty chambers that do not fuse with each other or with the plasma membrane. To our knowledge, no cell types other than mast cells, basophils, and eosinophils have been reported in the literature to show morphological features of piecemeal degranulation. In the present study we provide evidence for ultrastructural morphologies characteristic of piecemeal degranulation in entero-endocrine cells of the human and murine gastrointestinal epithelia. Human biopsy samples were taken from the mucosa of the distal duodenum, proximal jejunum, and colon in 10 patients undergoing endoscopic examination for malabsorption, diarrhea, and/or abdominal pain. Murine gastrointestinal samples were obtained from 10 adult C57 mice. All specimens were prepared for transmission electron microscopy (TEM) according to standard protocols. Results showed that different types of gastrointestinal entero-endocrine cells, in both humans and mice, were recognizable with ultrastructural features diagnostic for piecemeal degranulation, including specific granule and cytoplasmic changes. In the granules, the content was found to be loosely packed or diminished. Notably, altered granules did not fuse with each other or with the plasma membrane, and were characteristically intermingled with normal, resting granules. At times, the release events transformed the granules into enlarged, empty containers. Numerous entero-endocrine cells presented a rich supply of membrane-bound vesicles (50-200 nm in diameter) that were free in the cytoplasm or attached to granules. This finding of piecemeal degranulation in gastrointestinal entero-endocrine cells suggests that such a secretory model might be a general degranulation pattern in cells involved in paracrine-endocrine secretion. [Abstract]

Pérez-Pomares JM, Muñoz-Chápuli R
Epithelial-mesenchymal transitions: a mesodermal cell strategy for evolutive innovation in Metazoans.
Anat Rec. 2002 Nov 1;268(3):343-51.
Epithelial-mesenchymal transitions (EMTs) are well known processes in which new mesenchyme is locally generated from epithelia. During the development of the vertebrate embryo, EMTs are a source of mesenchyme in diverse places and stages through embryonic morphogenesis, especially in mesodermal domains. In the present work we consider the embryo as a two-state system in which epithelium and mesenchyme represent the stable and unstable states, respectively. We think that a pattern of recurrent oscillations between the plasticity and exploratory behaviour of the mesenchyme and the stability of the epithelia can be recognized in the embryogenesis of vertebrates and, probably, in most tripoblastic Metazoans. Mesoderm, in particular, might be regarded as a cell layer able to oscillate between epithelial and mesenchymal states. The cellular and molecular mechanisms that enable these recurrent oscillations between stable (epithelial) and unstable (mesenchymal) states during embryogenesis provide the mesoderm with a large plasticity, an extended potential for innovation, and a better control of the three-dimensional (3D) body organization. In this scenario, it is conceivable that the origin of the mesoderm itself might be related to ancestral mechanisms regulating cell adhesion and detachment. We conclude that EMTs played a key role in the evolution of Metazoans, and are involved in the pathological and reparative processes of adult organisms. [Abstract]

Furusawa C, Kaneko K
Origin of multicellular organisms as an inevitable consequence of dynamical systems.
Anat Rec. 2002 Nov 1;268(3):327-42.
The origin of multicellular organisms is studied by considering a cell system that satisfies minimal conditions, that is, a system of interacting cells with intracellular biochemical dynamics, and potentiality in reproduction. Three basic features in multicellular organisms-cellular diversification, robust developmental process, and emergence of germ-line cells-are found to be general properties of such a system. Irrespective of the details of the model, such features appear when there are complex oscillatory dynamics of intracellular chemical concentrations. Cells differentiate from totipotent stem cells into other cell types due to instability in the intracellular dynamics with cell-cell interactions, as explained by our isologous diversification theory (Furusawa and Kaneko, 1998a; Kaneko and Yomo, 1997). This developmental process is shown to be stable with respect to perturbations, such as molecular fluctuations and removal of some cells. By further imposing an adequate cell-type-dependent adhesion force, some cells are released, from which the next generation cell colony is formed, and a multicellular organism life-cycle emerges without any finely tuned mechanisms. This recursive production of multicellular units is stabilized if released cells are few in number, implying the separation of germ cell lines. Furthermore, such an organism with a variety of cellular states and robust development is found to maintain a larger growth speed as an ensemble by achieving a cooperative use of resources, compared to simple cells without differentiation. Our results suggest that the emergence of multicellular organisms is not a "difficult problem" in evolution, but rather is a natural consequence of a cell colony that can grow continuously. [Abstract]

DeAngelis PL
Evolution of glycosaminoglycans and their glycosyltransferases: Implications for the extracellular matrices of animals and the capsules of pathogenic bacteria.
Anat Rec. 2002 Nov 1;268(3):317-26.
Glycosaminoglycans (linear polysaccharides with a repeating disaccharide backbone containing an amino sugar) are essential components of extracellular matrices of animals. These complex molecules play important structural, adhesion, and signaling roles in mammals. Direct detection of glycosaminoglycans has been reported in a variety of organisms, but perhaps more definitive tests for the glycosyltransferase genes should be utilized to clarify the distribution of glycosaminoglycans in metazoans. Recently, glycosyltransferases that form the hyaluronan, heparin/heparan, or chondroitin backbone were identified at the molecular level. The three types of glycosyltransferases appear to have evolved independently based on sequence comparisons and other characteristics. All metazoans appear to possess heparin/heparan. Chondroitin is found in some worms, arthropods, and higher animals. Hyaluronan is found only in two of the three main branches of chordates. The presence of several types of glycosaminoglycans in the body allows multiple communication channels and adhesion systems to operate simultaneously. Certain pathogenic bacteria produce extracellular coatings, called capsules, which are composed of glycosaminoglycans that increase their virulence during infection. The capsule helps shield the microbe from the host defenses and/or modulates host physiology. The bacterial and animal polysaccharides are chemically identical or at least very similar. Therefore, no immune response is generated, in contrast to the vast majority of capsular polymers from other bacteria. In microbial systems, it appears that in most cases functional convergent evolution of glycosaminoglycan glycosyltransferases occurred, rather than direct horizontal gene transfer from their vertebrate hosts. [Abstract]

Exposito JY, Cluzel C, Garrone R, Lethias C
Evolution of collagens.
Anat Rec. 2002 Nov 1;268(3):302-16.
The extracellular matrix is often defined as the substance that gives multicellular organisms (from plants to vertebrates) their structural integrity, and is intimately involved in their development. Although the general functions of extracellular matrices are comparable, their compositions are quite distinct. One of the specific components of metazoan extracellular matrices is collagen, which is present in organisms ranging from sponges to humans. By comparing data obtained in diploblastic, protostomic, and deuterostomic animals, we have attempted to trace the evolution of collagens and collagen-like proteins. Moreover, the collagen story is closely involved with the emergence and evolution of metazoa. The collagen triple helix is one of numerous modules that arose during the metazoan radiation which permit the formation of large multimodular proteins. One of the advantages of this module is its involvement in oligomerization, in which it acts as a structural organizer that is not only relatively resistant to proteases but also permits the creation of multivalent supramolecular networks. [Abstract]

Dolan MF, Melnitsky H, Margulis L, Kolnicki R
Motility proteins and the origin of the nucleus.
Anat Rec. 2002 Nov 1;268(3):290-301.
Hypotheses on the origin of eukaryotic cells must account for the origin of the microtubular cytoskeletal structures (including the mitotic spindle, undulipodium/cilium (so-called flagellum) and other structures underlain by the 9(2)+2 microtubular axoneme) in addition to the membrane-bounded nucleus. Whereas bacteria with membrane-bounded nucleoids have been described, no precedent for mitotic, cytoskeletal, or axonemal microtubular structures are known in prokaryotes. Molecular phylogenetic analyses indicate that the cells of the earliest-branching lineages of eukaryotes contain the karyomastigont cytoskeletal system. These protist cells divide via an extranuclear spindle and a persistent nuclear membrane. We suggest that this association between the centriole/kinetosome axoneme (undulipodium) and the nucleus existed from the earliest stage of eukaryotic cell evolution. We interpret the karyomastigont to be a legacy of the symbiosis between thermoacidophilic archaebacteria and motile eubacteria from which the first eukaryote evolved. Mutually inconsistent hypotheses for the origin of the nucleus are reviewed and sequenced proteins of cell motility are discussed because of their potential value in resolving this problem. A correlation of fossil evidence with modern cell and microbiological studies leads us to the karyomastigont theory of the origin of the nucleus. [Abstract]

Thompson RF, Langford GM
Myosin superfamily evolutionary history.
Anat Rec. 2002 Nov 1;268(3):276-89.
The superfamily of myosin proteins found in eukaryotic cells is known to contain at least 18 different classes. Members are classified based on the phylogenetic analysis of the head domains located at the amino terminus of the polypeptide. While phylogenetic relationships provide insights into the functional relatedness of myosins within and between families, the evolutionary history of the myosin superfamily is not revealed by such studies. In order to establish the evolutionary history of the superfamily, we analyzed the representation of myosin gene families in a range of organisms covering the taxonomic spectrum. The amino acid sequences of 232 myosin heavy chains, as well as 65 organisms representing the protist, plant, and animal kingdoms, were included in this study. A phylogenetic tree of organisms was constructed based on several complementary taxonomic classification schemes. The results of the analysis support an evolutionary hypothesis in which myosins II and I evolved the earliest of all the myosin groups. Myosins V and XI evolved from a common myosin II-like ancestor, but the two families diverged to either the plant (XI) or animal (V) lineage. Class VII myosin appeared fourth among the families, and classes VI and IX appeared later during the early period of metazoan radiation. Myosins III, XV, and XVIII appeared after this group, and X appeared during the formative phases of vertebrate evolution. The remaining members of the myosin superfamily (IV, VI, XII, XIII, XIV, XVI, and XVII) are limited in distribution to one or more groups of organisms. The evolutionary data permits one to predict the likelihood that myosin genes absent from a given species are either missing (not found yet because of insufficient data) or lost due to a mutation that removed the gene from an organism's lineage. In conclusion, an analysis of the evolutionary history of the myosin superfamily suggests that early-appearing myosin families function as generalists, carrying out a number of functions in a variety of cell types, while more recently evolved myosin families function as specialists and are limited to a few organisms or a few cell types within organisms. [Abstract]

Davis GE, Bayless KJ, Mavila A
Molecular basis of endothelial cell morphogenesis in three-dimensional extracellular matrices.
Anat Rec. 2002 Nov 1;268(3):252-75.
Although many studies have focused on blood vessel development and new blood vessel formation associated with disease processes, the question of how endothelial cells (ECs) assemble into tubes in three dimensions (i.e., EC morphogenesis) remains unanswered. EC morphogenesis is particularly dependent on a signaling axis involving the extracellular matrix (ECM), integrins, and the cytoskeleton, which regulates EC shape changes and signals the pathways necessary for tube formation. Recent studies reveal that genes regulating this matrix-integrin-cytoskeletal (MIC) signaling axis are differentially expressed during EC morphogenesis. The Rho GTPases represent an important class of molecules involved in these events. Cdc42 and Rac1 are required for the process of EC intracellular vacuole formation and coalescence that regulates EC lumen formation in three-dimensional (3D) extracellular matrices, while RhoA appears to stabilize capillary tube networks. Once EC tube networks are established, supporting cells, such as pericytes, are recruited to further stabilize these networks, perhaps by regulating EC basement membrane matrix assembly. Furthermore, we consider recent work showing that EC morphogenesis is balanced by a tendency for newly formed tubes to regress. This morphogenesis-regression balance is controlled by differential gene expression of such molecules as VEGF, angiopoietin-2, and PAI-1, as well as a plasmin- and matrix metalloproteinase-dependent mechanism that induces tube regression through degradation of ECM scaffolds that support EC-lined tubes. It is our hope that this review will stimulate increased interest and effort focused on the basic mechanisms regulating capillary tube formation and regression in 3D extracellular matrices. [Abstract]

Morris CE
How did cells get their size?
Anat Rec. 2002 Nov 1;268(3):239-51.
Cells exercise size homeostasis, and the origins of their ability to do so is the topic of this essay. Before there were cells, there were protocells. The most basic questions about protocells as objects are: What were they made of, and how big were they? Asking how big they were implies that the answer to the first part includes a boundary. The best candidate for that boundary is a self-assembling lipid bilayer. Therefore, protocells are defined here as Darwinian liposomes-bilayer vesicles with mutable on-board replicases linked to phenotypes. Because liposomes undergo spontaneous fission and fusion, and are subject to osmotic forces, size regulation in the earliest protocells would essentially have been liposome physics. For successful protocells, averting osmotic lysis would have been the first order of business. However, from the outset size mattered too, because of sex and reproduction (i.e., genome mixing and genome copying in entities with phenotypes). Protocell fission and fusion would have blended seamlessly into protocell sex and reproduction, making any gene product that furnished control over protocell size changes doubly adaptive. A recurrent theme is the feedback role of bilayer tension in protocell size control. Ways in which primitive peptides and their aggregates (e.g., channels) might have allowed liposomes to gain improved volume and surface area homeostasis are suggested. Domain-swapped proteins that polymerize as filaments are discussed as the origin of cytoskeleton structures that diversify and stabilize liposome shapes and sizes. Throughout, attention is paid to the question of set points for cell size. [Abstract]

Beznoussenko GV, Mironov AA
Models of intracellular transport and evolution of the Golgi complex.
Anat Rec. 2002 Nov 1;268(3):226-38.
We have performed a systematic analysis of models explaining the mechanisms of the intracellular biosecretory transport. The models assessed include not only those based on one mechanism (the dissociation model (and its individual case, the vesicular model), the progression model (and its individual cases, the cisterna maturation/progression and the carrier maturation models), and the lateral diffusion model (and its individual case, the bolus model), but also combined models of transport (the percolating-vesicles model and the synthetic model), including several transport mechanisms. Most of these models are not able to explain recent data on the evolution of genes involved in intracellular transport and Golgi evolution. The carrier maturation model proposing that fusion of the large cargo domain with the distal (closer to the plasmalemma) compartment precedes fission of the domain from the proximal compartment exhibits the best performance in correlation with the available information on evolution of the biosecretory pathway. [Abstract]

Svetina S, Zeks B
Shape behavior of lipid vesicles as the basis of some cellular processes.
Anat Rec. 2002 Nov 1;268(3):215-25.
The basic principles that govern the shape behavior of phospholipid vesicle shapes are discussed. The important membrane parameters of the system are defined by presenting the expressions for the relevant contributions to the system's mechanical energy. In the description of the rather unique shape behavior of lipid vesicles, the emphasis is on providing a qualitative understanding of the dependence of vesicle shape on the parameters of the system. The vesicle shape behavior is then related to biologically important phenomena. Some examples are given of how the results of the shape behavior of lipid vesicles can be applied to the analysis of cellular systems. Red blood cell shape and shape transformations, vesicle fission and fusion processes, and the phenomenon of cellular polarity are considered. It is reasoned that the current biological processes that involve changes of membrane conformation may have their origin in the general shape behavior of closed lamellar membranes. [Abstract]

Luisi PL
Toward the engineering of minimal living cells.
Anat Rec. 2002 Nov 1;268(3):208-14.
The article focuses on the notion of a synthetic or semi-synthetic minimal cell, defined as a system that has the minimal and sufficient structural conditions for cellular life. It is emphasized that two complementary approaches are in principle possible, defined as "bottom-up" and "top-down" approaches. The first one aims at the construction of a minimal cell starting from scratch, and it is argued that a very serious bottle-neck to this pathway lies in the origination of specific macro-molecular sequences, as in nature those were constructed most likely by a particular contingent set of conditions. The top-down approaches utilize extant genes and enzymes, and the work in this case is based on the incorporation of the minimal and sufficient amount of such macromolecules into liposomes, as models for the shell of biological cells. The first phase of this ambitious project foresees the study of conditions under which complex molecular biology reactions takes place in the compartments of liposomes. Examples of these reactions are provided, for example, the production of RNA throughout Q-beta replicase in a self-reproducing vesicle system; or PC Reaction in phospholipid vesicles; or even the incorporation of ribosomes in liposomes, with the production of polypeptide chains. The use of giant vesicles is also illustrated. These systems, due to their large size, offer the advantage that by way of special micro-injection techniques, all sort of biochemical agents can be directly introduced in the compartment; and that the reaction can be followed by optical microscopy. In the final part of the article, the outlook of increasing the complexity of these liposome systems so as to arrive at first semi-synthetic cells is discussed. [Abstract]

Monnard PA, Deamer DW
Membrane self-assembly processes: steps toward the first cellular life.
Anat Rec. 2002 Nov 1;268(3):196-207.
This review addresses the question of the origin of life, with emphasis on plausible boundary structures that may have initially provided cellular compartmentation. Some form of compartmentation is a necessary prerequisite for maintaining the integrity of interdependent molecular systems that are associated with metabolism, and for permitting variations required for speciation. The fact that lipid-bilayer membranes define boundaries of all contemporary living cells suggests that protocellular compartments were likely to have required similar, self-assembled boundaries. Amphiphiles such as short-chain fatty acids, which were presumably available on the early Earth, can self-assemble into stable vesicles that encapsulate hydrophilic solutes with catalytic activity. Their suspensions in aqueous media have therefore been used to investigate nutrient uptake across simple membranes and encapsulated catalyzed reactions, both of which would be essential processes in protocellular life forms. [Abstract]

Simoneit BR
Molecular indicators (biomarkers) of past life.
Anat Rec. 2002 Nov 1;268(3):186-95.
Biomarkers in geological samples on Earth are products derived from biochemical precursors (i.e., natural products) by reductive and oxidative alteration processes (e.g., cholestanes from cholesterol). Generally, lipids, pigments, and some biomembranes are preserved best over longer geological times, and labile compounds such as amino acids, sugars, etc. are useful biomarkers for recent times. Thus, the detailed characterization of biomarker composition permits the assessment of the major contributing species of extinct and/or extant life. Nonbiomarkers and abiogenic organic compounds are also discussed. In the case of the early Earth, work has progressed to elucidate biomarker structures and carbon isotopic signals preserved in ancient sedimentary rocks. In addition, the combination of bacterial biochemistry with the organic geochemistry of contemporary and ancient hydrothermal ecosystems permits the modeling of the nature, behavior, and preservation potential of primitive microbial communities. This approach entails combined molecular and isotopic analyses to characterize lipids and biopolymers produced by cultured bacteria (representative of ancient strains) and to test a variety of culture conditions that affect their biosynthesis processes. In regards to Mars, the biomarkers from lipids and biopolymers would be expected to be preserved best if life flourished there during its early history (3.5-4 x 10(9) years ago). Both oxidized and reduced products would be expected. This is based on the inference that hydrothermal activity occurred during that time, with the concomitant preservation of biochemically-derived carbonaceous matter. Known biomarkers (i.e., as elucidated for early terrestrial samples and for primitive terrestrial microbiota) as well as novel, potentially unknown compounds, should be characterized. [Abstract]

Fox A
Chemical markers for bacteria in extraterrestrial samples.
Anat Rec. 2002 Nov 1;268(3):180-5.
Interplanetary missions to collect pristine Martian surface samples for analysis of organic molecules, and to search for evidence of life, are in the planning phases. The only extraterrestrial samples currently on Earth are lunar dust and rocks, brought back by the Apollo (U.S.) and Luna (Soviet Union) missions to the moon, and meteorites. Meteorites are contaminated when they pass through the Earth's atmosphere, and during environmental exposure on Earth. Lunar fines have been stored on Earth for over 30 years under conditions designed to avoid chemical but not microbiological contamination. It has been extremely difficult to draw firm conclusions about the origin of chemicals (including amino acids) in extraterrestrial samples. Of particular concern has been the possibility of bacterial contamination. Recent work using state-of-the-art gas chromatography tandem mass spectrometry (GC-MS/MS) has dramatically lowered the chemical background, allowing a clear demonstration that lunar fines are remarkably different from terrestrial dust in that they generally lack certain chemical markers (muramic acid and 3-hydroxy fatty acids) characteristic of Earth's bacteria. Thus, lunar dust might be used as a negative control, in conjunction with GC-MS/MS analyses, in future analytical studies of lunar dust and meteorites. Such analyses may also be important in studies designed to search for the presence of life on Mars. [Abstract]

Schwabe C
Genomic potential hypothesis of evolution: a concept of biogenesis in habitable spaces of the universe.
Anat Rec. 2002 Nov 1;268(3):171-9.
The new hypothesis of evolution establishes a contiguity of life sciences with cosmology, physics, and chemistry, and provides a basis for the search for life on other planets. Chemistry is the sole driving force of the assembly of life, under the subtle guidance exerted by bonding orbital geometry. That phenomenon leads to multiple origins that function on the same principles but are different to the extent that their nucleic acid core varies. Thus, thoughts about the origins of life and the development of complexity have been transferred from the chance orientation of the past to the realm of atomic structures, which are subject to the laws of thermodynamics and kinetics. Evolution is a legitimate subject of basic science, and the complexity of life will submit to the laws of chemistry and physics as the problem is viewed from a new perspective. The paradigm connects life to the big events that formed every sphere of our living space and that keeps conditions fine-tuned for life to persist, perhaps a billion years or more. The "genomic potential" hypothesis leads to the prediction that life like ours is likely to exist in galaxies that are as distant from the origin of the universe as the Milky Way, and that the habitable zone of our galaxy harbors other living planets as well. [Abstract]

Blumberg BS
Astrobiology: an introduction.
Anat Rec. 2002 Nov 1;268(3):169-70. [Abstract]

Trelease RB
Anatomical informatics: Millennial perspectives on a newer frontier.
Anat Rec. 2002 Oct 15;269(5):224-35.
One of the most ancient of sciences, anatomy has evolved over many centuries. Its methods have progressively encompassed dissection instruments, manual illustration, stains, microscopes, cameras and photography, and digital imaging systems. Like many other more modern scientific disciplines in the late 20th century, anatomy has also benefited from the revolutionary development of digital computers and their automated information management and analytical capabilities. By using newer methods of computer and information sciences, anatomists have made outstanding contributions to science, medicine, and education. In that regard, there is a strong rationale for recognizing anatomical informatics as a proper subdiscipline of anatomy. A high-level survey of the field reveals important anatomical applications of computer sciences methods in imaging, image processing and visualization, virtual reality, modeling and simulation, structural database processing, networking, and artificial intelligence. Within this framework, computational anatomy is a developing field focusing on data-driven mathematical models of bodily structures. Mastering such computer sciences and informatics methods is crucial for new anatomists, who will shape the future in research, clinical knowledge, and teaching. [Abstract]

Yamamoto Y, Shinohara K
Application of X-ray microscopy in analysis of living hydrated cells.
Anat Rec. 2002 Oct 15;269(5):217-23.
Because there is a limit for analysis of fine hydrophilic cell structures of living cells in medium by ordinary techniques, including electron microscopy, the development of a new technology to overcome such limitation is highly desirable. In this regard, soft X-ray microscopy (high-resolution X-ray imaging of structures), which does not require any special procedures for sample preparation, has been developed and applied to analyze structures of biological specimens. In this article, application of two types of X-ray microscopes, which use laser-produced plasma X-rays or synchrotron radiation to image the structure of macrophage cells, is introduced as an example of a novel approach to analysis of biological specimens. Both types of X-ray microscopy show the network of fine fibrillar surface structures on macrophages in medium. Ordinary transmission and scanning electron microscopy and light microscopy also show the presence of such structures, but electron microscopy showed alterations due to sample processing and light microscopy did not show a clear image due to low resolution. Thus, X-ray microscopy has the potential capability to analyze structures of live cells in a hydrated condition and may reveal a function-related structural alignment of cells in their natural form. [Abstract]

Geuna S, Giacobini-Robecchi MG
The use of brainstorming for teaching human anatomy.
Anat Rec. 2002 Oct 15;269(5):214-6.
Interactive teaching techniques have been used mainly in clinical teaching, with little attention given to their use in basic science teaching. With the aim of partially filling this gap, this study outlines an interactive approach to teaching anatomy based on the use of "brainstorming." The results of the students' critique of the teaching techniques are also included. Seventy-five students from the first-year nursing curriculum were tested by a structured questionnaire after three brainstorming sessions. The overall response to these sessions was very positive, indicating that students perceived this interactive technique as both interesting and useful. Furthermore, this approach may provide a useful strategy when learning the clinical courses of the upcoming academic years. [Abstract]

Haines DE
The A.J. Ladman AAA/Wiley Exemplary Service Award.
Anat Rec. 2002 Oct 15;269(5):212-3. [Abstract]

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Recent Articles in Applied Immunohistochemistry and Molecular Morphology: AIMM / Official Publication of the Society for Applied Immunohistochemistry

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Buccoliero AM, Gheri CF, Castiglione F, Ammannati F, Gallina P, Taddei A, Garbini F, Degl'Innocenti DR, Arganini L, Di Lorenzo N, Mennonna P, Taddei GL
Merlin expression in secretory meningiomas: evidence of an NF2-independent pathogenesis? Immunohistochemical study.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):353-7.
One of the most common chromosomal regions implicated in the meningiomas tumorigenesis is 22q12 where the neurofibromatosis 2 (NF2) gene resides. The NF2 tumor-suppressor gene encodes for the merlin/schwannomin protein, which is responsible for the inherited disease neurofibromatosis 2. NF2 gene mutations predominantly occur in transitional and fibroblastic meningiomas, whereas the meningothelial variant is less affected. Secretory meningioma is an infrequent meningioma subtype. Its most typical morphologic feature is the presence of intracytoplasmic or extracytoplasmic round hyaline, eosinophilic, and periodic acid Shiff-positive bodies in a lesion frequently otherwise classifiable as meningothelial meningioma. This study reviews the immunohistochemical merlin expression in 14 consecutive secretory meningiomas. Our purpose was to investigate if secretory meningiomas, analogous to meningothelial meningiomas, follow a molecular route of pathogenesis independent of the neurorofibromatosis 2 gene-associated pathway. All meningiomas showed positive immunocoloration involving the majority of the hyaline inclusions and secretory cells; in 12 (86%) meningiomas, a positive immunoreaction was also documented in nonsecretory tumoral cells. Our results may indicate a molecular, besides morphologic, similarity between secretory and meningothelial meningiomas: the almost constant merlin immunohistochemical expression in our series gives evidence for a possible NF2 gene-independent pathogenesis in secretory meningiomas. [Abstract]

Padilla-Rodríguez AL, Bembassat M, Lazaro M, Ortiz-Hidalgo C
Intra-abdominal follicular dendritic cell sarcoma with marked pleomorphic features and aberrant expression of neuroendocrine markers: report of a case with immunohistochemical analysis.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):346-52.
Follicular dendritic cell sarcoma (FDCS) is a very rare malignant tumor arising most frequently in lymph nodes with only few reports of extranodal locations. We report the case of a 35-year-old man with a large retroperitoneal mass. Histologically the tumor was composed of highly pleomorphic cells exhibiting some uncommon features such as an epithelioid appearance, cystic spaces, and multinucleated cells with morphologic features of emperipolesis. Immunohistochemically the neoplastic cells were immunoreactive for CD21, CD23 and CD35. A previously unreported expression of neuroendocrine markers (Synaptophisyn and Neuron-Specific-Enolase) was present. Ultrastructurally no neuroendocrine secretory granules were detected. FDCS can mimic a wide variety of other malignant tumors, and a correct diagnosis requires exclusion of other neoplasms and immunohistochemical confirmation. [Abstract]

Vogel UF, Bode J, Bueltmann B
Increasing the efficiency of paraffin tissue microarrays by packing the paraffin tissue core biopsies in a honeycomb pattern.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):343-5.
Paraffin tissue microarrays (PTMAs) are blocks of paraffin holding up to 1000 paraffin tissue core biopsies (PTCBs) for high throughput molecular analysis. The number of PTCBs in a PTMA depends on the surface area of the PTMA, the diameter of and the distance between the PTCBs and on their arrangement inside the assembled PTMA. The PTCBs are usually arranged in a rectangular x-y pattern of rows and columns. This design facilitates the construction of a PTMA because the operator simply turns the wheels of an x-y-table for a set, unchanging distance. The evaluation of the stained sections is also relatively easy. However, this rectangular arrangement means wasted space in the PTMA. To reclaim this space, the PTCBs could be arranged in a honeycomblike pattern. For every 8 rows in the conventional rectangular arrangement, 1 additional row of PTCBs can be packed. However, the researcher has to become accustomed to this uncommon arrangement when filling and evaluating the PTMA. Automatic slide readers and specially designed computer programs for the digital evaluation of the PTMAs can be helpful. In summary, the arrangement of PTCBs in a honeycomblike pattern increases the density and number of specimens stored in a PTMA, thereby enhancing its efficiency. [Abstract]

Castiglione F, Degl'Innocenti DR, Taddei A, Garbini F, Buccoliero AM, Raspollini MR, Pepi M, Paglierani M, Asirelli G, Freschi G, Bechi P, Taddei GL
Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):338-42.
In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation. [Abstract]

Dirsch O, Ji Y, Bohr J, Shen K, Levison D, Dahmen U
Probe production for in situ hybridization by PCR and subsequent covalent labeling with fluorescent dyes.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):332-7.
A simple procedure for fluorescent labeling of probes just before in situ hybridization is provided. Aminoallyl-dUTP is introduced during probe production by polymerase chain reaction (PCR). The aminoallyl-dUTP functions as a reactive site for subsequent labeling of the probe. Activated fluorescent dyes such as fluorescein are covalently attached to the probe through the formation of a stable amide bond. Labeled probes are purified by size-exclusion gel chromatography to remove unincorporated dye. Target genes used to demonstrate the efficacy of this technique with in situ hybridization are rat Y-chromosome and rat granulocyte colony-stimulating factor receptor. PCR amplicons containing aminoallyl-dUTP were produced in high yield. Probes obtained after labeling with activated fluorophores demonstrated high intrinsic activity within in situ hybridizations. The introduction of aminoallyl-dUTP into the PCR reaction enables the production of "unlabeled" probes by PCR having a shelf life, which is not limited by the storage and stability challenges of fluorophore-labeled probes. Subsequent labeling of the probes with activated fluorescent dyes just before use allows one step in situ hybridization with high activity and minimal background staining. [Abstract]

Phillips T, Murray G, Wakamiya K, Askaa J, Huang D, Welcher R, Pii K, Allred DC
Development of standard estrogen and progesterone receptor immunohistochemical assays for selection of patients for antihormonal therapy.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):325-31.
Estrogen receptor (ER) and progesterone receptor (PR) status in breast carcinomas are considered validated predictive factors for selecting patients for antihormonal therapy. Published surveys have shown a significant rate of disagreement and lack of reproducibility of immunohistochemistry (IHC) results from laboratories around the world. To address these limitations IHC assays for ER and PR were developed using characterized reagents, after careful calibration of the sensitivity and specificity to match established assays previously validated in large clinical studies. The ER assay uses a cocktail of 2 mouse monoclonal antibodies (1D5 and ER-2-123) and the PR assay uses 1 mouse monoclonal antibody (PgR 1294); both are followed by a polymer-peroxidase-based detection system. All antibodies were tested for specificity by epitope mapping. The sensitivity of the new assays was calibrated to be equivalent to previously validated IHC assays followed by a comparison with the validated assays in a concordance study involving over 200 specimens. All slides were scored with the "Allred Score," also used for scoring of the original validated assays. The overall concordance between the new and the established IHC assays was nearly perfect (99%). The concordance study demonstrated greater than 98% positive agreement and 100% negative agreement of the new IHC assays with the previously validated IHC assays. This equivalence establishes the clinical validation of the assays and, as they are based on newer generation reagents and are produced and tested under stringent quality control conditions to ensure their consistency, they add additional advantages to the user and patients. [Abstract]

Ni R, Mulligan AM, Have C, O'Malley FP
PGDS, a novel technique combining chromogenic in situ hybridization and immunohistochemistry for the assessment of ErbB2 (HER2/neu) status in breast cancer.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):316-24.
Given the important prognostic and predictive utility of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ErbB2) [human epidermal growth factor receptor-2 (HER2/neu)] in breast cancer, it is recommended that ErbB2 testing be performed on all invasive breast cancers at the time of diagnosis. A consensus, however, has not yet been reached as to the optimal method of evaluating ErbB2 status. Immunohistochemistry to detect protein overexpression and fluorescence in situ hybridization (FISH) to detect ErbB2 gene amplification are the most frequently used methods. As no one detection method fulfills all necessary requirements of reliability, reproducibility, and ease of use, we developed a novel approach in the form of a simple assay we refer to as protein and gene double staining (PGDS) which simultaneously evaluates protein overexpression and gene amplification by combining immunohistochemistry with chromogenic in situ hybridization (CISH). A total of 134 invasive breast carcinomas, including 81 cases with a full-face section and 53 cases included in a tissue microarray (TMA), were assessed by PGDS, and the results were correlated with ErbB2 gene amplification status as determined by FISH. ErbB2 gene copy number determined by CISH analysis in the PGDS assay showed excellent concordance with that of FISH (correlation coefficient 0.82; P<0.001 with full-face section cases, and 0.98; P<0.001 with cases in a TMA). The overall concordance rate for gene amplification status between PGDS and FISH was 90.12% in cases with a full-face section and 92.45% with TMA cases. Perfect correlation was seen between the PGDS assay and FISH in cases that were considered either nonamplified or highly amplified by the dual assay. Of the 17 cases that showed low amplification by PGDS, 5 were classified as nonamplified by FISH. Correction for chromosome 17 copy number in the FISH assessment contributed to the discordance between CISH and FISH results. This newly developed PGDS method represents a novel approach to ErbB2 status determination that combines the assessment of both protein overexpression and gene amplification in one simple assay. It is likely that this assay will aid in immunohistochemical calibration and will also increase the sensitivity and specificity of ErbB2 testing. [Abstract]

Bakshi N, Kunju LP, Giordano T, Shah RB
Expression of renal cell carcinoma antigen (RCC) in renal epithelial and nonrenal tumors: diagnostic Implications.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):310-5.
Antibody to renal cell carcinoma (RCC) antigen, a normal human proximal brush border antigen, has recently become commercially available and reported to be highly specific and a relatively sensitive marker for RCC. Of the nonrenal tumors occasional carcinomas have been reported to express RCC, notably breast carcinoma. Using tissue microarrays, we investigated the use of RCC on a large number of renal epithelial neoplasms (RENs) and nonrenal tumors, especially those potentially confused with REN. Three tissue microarrays containing 241 REN samples, 192 samples of a wide variety of neoplasms and 170 adrenal tumor samples, respectively, were stained with RCC monoclonal antibody. RCC expression was scored for staining intensity and percentage expression. Out of 241 REN, 173 were positive for RCC (sensitivity 72%): clear cell 72%, papillary 95%, chromophobe 91%, unclassified 85%, oncocytoma 75%, sarcomatoid 20%, and metastatic RCC 40%. The overall immunostaining intensity was consistently much higher in papillary and clear cell RCC than in other tumors. Seventy-six out of 362 nonrenal tumor samples demonstrated either focal or diffuse expression for RCC (specificity 79%). These included: adrenocortical neoplasms 37/170 (22%), colonic 11/29 (37.5%), breast 9/27 (33%), prostate 5/18 (27.7%), ovary 2/17 (11.7%), melanoma 3/18 (16.6%), lung 3/21 (14.2%), and parathyroid 3/3 (100%). RCC expression was seen equally among adrenal adenoma and carcinoma group. Eight out of 28 (28.5%) normal adrenal cores also stained for RCC. RCC is a relatively useful marker in the differential diagnosis of REN only if used in a panel with other positive and negative markers. RCC does not reliably differentiate REN, especially classic clear cell type, from adrenocortical neoplasms, which are frequently confused due to close anatomic proximity and similar morphology. RCC also does not reliably differentiate subtypes of renal epithelial neoplasms. [Abstract]

Buckley AF, Kakar S
Comparison of the Dako EGFR pharmDx kit and Zymed EGFR antibody for assessment of EGFR status in colorectal adenocarcinoma.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):305-9.
Immunohistochemistry is widely used to assess epidermal growth factor receptor (EGFR) expression on colorectal carcinomas to select patients for treatment with cetuximab, an anti-EGFR antibody. The data comparing different commercial EGFR antibodies is limited, and no cost comparisons have been made. We analyzed 65 advanced colorectal cancers from 36 patients using the EGFR pharmDx kit (DakoCytomation) and Clone 31G7 (Zymed Laboratories, Inc). EGFR expression was seen in 35 (53%) tumors (21 primary, 14 metastatic) with the Dako pharmDx kit. The Zymed antibody showed positive results in 41 (63%) tumors (25 primary, 16 metastatic). The cost per test was $40.00 with the pharmDx kit and $3.52 with the Zymed antibody. The Zymed antibody detects 10% more cases of colorectal cancer as EGFR positive, and is 10 times cheaper than the Dako pharmDx kit. There is little justification for the use of expensive kits for testing EGFR expression, when other available antibodies without the kit can give comparable or superior results. [Abstract]

Euscher ED, Marsh WL, Lucas JG, Frankel WL
Histologic and immunohistochemical changes in the stented common bile duct.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):299-304.
Many patients with pancreatic carcinoma have stent placement for biliary obstruction before resection. Stent-associated atypia, found in common bile duct (CBD) margins at the time of resection, may be confused with malignancy. We evaluated histologic and immunohistochemical changes in CBD margins from resection specimens for pancreatic carcinoma. Histologic findings in CBDs, including ulcer and inflammation; epithelial metaplasia, atypia, and gland complexity; and increased wall thickness, nerve entrapment, and smooth muscle content, were compared in 30 stented and 31 nonstented CBD margins from pancreaticoduodenectomies for carcinoma and 13 normal CBDs from autopsy material. The proliferation index was calculated for stented and nonstented CBDs after Ki-67 immunohistochemical staining. Immunostaining for Ki-67, p53, and c-erbB-2 was performed in stented CBDs and corresponding carcinomas. All the histologic changes occurred more frequently in stented and nonstented CBD margins from carcinoma patients than in normal CBDs. Stented CBDs had significantly increased epithelial changes and Ki-67 proliferation rate as compared with nonstented CBDs. The stented CBDs had significantly less p53 and c-erbB-2 expression as compared with corresponding pancreatic carcinomas.Caution should be applied when interpreting atypia in CBD margins from patients with a history of CBD stenting. Changes found in stented CBDs are characteristic, and in most cases can be distinguished from malignancy. In difficult cases, immunohistochemistry may be useful. [Abstract]

Jang KT, Lee KT, Lee JG, Choi SH, Heo JS, Choi DW, Ahn G
Immunohistochemical expression of Sonic hedgehog in intraductal papillary mucinous tumor of the pancreas.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):294-8.
Aberrant expression of Sonic hedgehog (Shh) has been reported in many human cancers including ductal carcinoma of the pancreas. The intraductal papillary mucinous tumor (IPMT) has been considered as one of the precursor lesions of invasive ductal carcinoma of the pancreas. Shh expression in pancreatic IPMT has not been reported. We investigated an immunohistochemical (IHC) expression of Shh in 55 cases of pancreatic IPMT. We analyzed the IHC expression of Shh in the following histologic grades of tumor: adenoma (AD), moderate dysplasia (MD), noninvasive carcinoma (NIC), and invasive carcinoma (IC), and with the following histologic subtype classification: intestinal, pancreatobiliary, null, and unclassifiable type. IHC Shh expression was noted in 6 (46.2%) of 13 AD, 5 (35.7%) of 14 MD, 12 (80%) of 15 NIC, and 11 (84.6%) of 13 IC. Shh expression was significantly increased in malignant IPMT (NIC+IC) compared with nonmalignant IPMT (AD+MD) (82.1% vs. 40.7%, P=0.0005). IHC Shh expression was found in 11 (68.8%) of 16 intestinal types, 13 (92.8%) of 14 pancreatobiliary types, 8 (38.1%) of 21 null types, and 2 (50%) of 4 unclassifiable types. Intestinal and pancreatobiliary subtypes showed a high expression of Shh compared with the null and unclassifiable type of IPMT. All 3 cases of node metastasis showed IHC Shh expression in tumor cells of metastatic lymph nodes. Therefore, Shh expression may have a critical role in the late stage of carcinogenesis of IPMT, and may impact metastatic progression to the lymph nodes in malignant IPMT. [Abstract]

Leslie KK, Walter SA, Torkko K, Stephens JK, Thompson C, Singh M
Effect of tamoxifen on endometrial histology, hormone receptors, and cervical cytology: a prospective study with follow-up.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):284-93.
OBJECTIVES: Our major hypothesis for these studies was that tamoxifen's varied effects on the endometrium might be due in part to differences in effect on estrogen and progesterone receptors [ER, progesterone receptor isoform A (PRA), and progesterone receptor isoform B (PRB)]. We aimed to evaluate the changes in histology in serial endometrial biopsies (Em bx), Papanicolaou smears (Pap smears), and endometrial ultrasounds as well as changes in the expression of ER, PRA, and PRB in response to tamoxifen. We propose that understanding and correlating the dynamics of receptor expression with histologic and cytologic changes will help us better understand the effect of tamoxifen on the endometrium and its role in the development of endometrial carcinoma in some patients. METHODS: Forty-two patients to be started on tamoxifen underwent a pretreatment Em bx and Pap smear. Follow-up serial Em bxs and Pap smears were obtained at sixth month and then at yearly intervals for up to 6 biopsies per case. Maturation indices (MIs) were determined on the Pap smears, and ER, PRA, and PRB immunostains were performed on the biopsies. Follow-up data is for a maximum of 10 years. Trends in changes in endometrial histology were analyzed and when atrophic or inactive endometrium changed to proliferative endometrium on treatment it was considered to be an increase in estrogen effect and the vice versa changes as a decrease in estrogen effect. RESULTS: None of the subjects developed hyperplasia or malignancy. Two patients' Em bx demonstrated atypical cells associated with eosinophilic metaplasia, but subsequent biopsies had no atypia. Of the 42 patients, 37 had serial Em bxs in which evaluation for trends could be performed. Twelve of 37 (32.4%) had an overall decrease in estrogen effect on endometrial histology with another 12/37 (32.4%) showing no estrogenic effect on endometrial histology. Six of 37 patients (16.2%) showed an increased estrogen effect on endometrial histology. Seven of 37 (18.9%) had variable endometrial histology with no definable pattern. There was a statistically significant increase in PRA expression compared with baseline as time progressed (P<0.05). The PRB showed a contrasting significant decrease in expression at 2.5 and 3.5 years (P<0.05). There was no significant change in ER expression over the course of the study (P>0.05). Seven of 12 (58.3%) with a decreased estrogenic effect on endometrial histology had a concordant decrease in PRB expression. Seven of 12 (58.3%) with no change in endometrial histology also had a concordant decrease in PRB expression. Comparing the MI of Pap smears with histologic activity of the endometrium revealed minimal correlation between the two. However, in the patients with an increased estrogen effect on endometrial histologic activity, there was no correlation with the MI. Additionally, 57% of patients showed no correlation between endometrial histologic activity and ultrasound findings. CONCLUSIONS: Tamoxifen had an antiestrogenic or neutral effect on endometrial histology and Pap smears of most subjects, but estrogenic, or variable effects were also observed in a minority of patients. Tamoxifen treatment was accompanied by an uncoupling of the regulation of PRA and PRB expression without effect on ER expression. Overall, expression of PRB decreased whereas that of PRA increased. [Abstract]

Tringler B, Grimm C, Dudek G, Zeillinger R, Tempfer C, Speiser P, Joura E, Reinthaller A, Hefler LA
p16INK4a expression in invasive vulvar squamous cell carcinoma.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):279-83.
p16, a member of the INK4a family of cyclin-dependent kinase inhibitors, is known as a negative regulator of cell cycle progression and differentiation. Although p16 has been shown to be a promising biomarker for the detection of cervical intraepithelial neoplasia, few data have been published on vulvar cancer. Using immunohistochemistry, we evaluated the expression of p16 in 80 cases of invasive vulvar squamous cell carcinoma. Results were correlated with clinicopathologic parameters and survival data to determine the prognostic significance of p16 in vulvar cancer. p16 expression was detected in 34 of 80 (43%) cases of invasive vulvar squamous cell carcinoma. The expression was localized to the cytoplasm and the nuclei of the tumor cells. Correlations between p16 expression status and any clinicopathologic variables failed to be of statistical significance. In a univariate analysis, groin lymph node status, tumor stage, and tumor grade were associated with disease-free and overall survival, respectively. Patients positive for p16 expression showed a significantly longer disease-free and overall survival by univariate analysis. p16 expression was not associated with survival in a multivariate Cox-regression model. Our data add on those published in the literature and suggest that p16 may be of prognostic significance in invasive vulvar squamous cell carcinoma. [Abstract]

Genelhu MC, Gobbi H, Arantes DC, Cardoso SV, Cassali GD
Immunolocalization of beta-catenin in pleomorphic adenomas and carcinomas ex-pleomorphic adenomas of salivary glands.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):273-8.
Beta-catenin plays a central role in cadherin/catenin cell-cell adhesion complex and is involved in cell signaling pathway. Change in beta-catenin distribution has been associated with several human cancers including salivary gland tumors. We studied the immunolocalization of beta-catenin in a series of pleomorphic adenomas (PA) and carcinomas ex-pleomorphic adenomas (Ca ex-PA). Ten samples of PA and ten of Ca ex-PA were evaluated by immunohistochemistry using streptavidin-biotin-peroxidase technique and a monoclonal antibody against beta-catenin (E-5). Cell membrane/cytoplasmic staining of beta-catenin was observed in normal gland parenchyma, PA, and in well-differentiated Ca ex-PA. Cytoplasmic/nuclear beta-catenin staining was observed in poorly differentiated carcinomas and, interestingly, in one case of PA. Our data showed decreased cell membrane beta-catenin expression in higher-grade tumors suggesting that beta-catenin may play an important role in histologic differentiation and transition to malignant phenotype of Ca ex-PA. [Abstract]

Takahashi H, Murai Y, Tsuneyama K, Nomoto K, Okada E, Fujita H, Takano Y
High labeling indices of cdc25B is linked to progression of gastric cancers and associated with a poor prognosis.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):267-72.
To clarify the significance of cdc25B, which plays an important physiologic role in regulation of the G2/M check point, in progression of gastric cancer, 125 samples of paraffin-embedded gastric cancers were investigated by immunohistochemistry. In addition, 3 human gastric cancer cell lines were studied to determine the cellular localization of cdc25B by immunohistochemistry and cell fractionation followed by Western blotting. In the cell lines cdc25B was found to be present in both nuclei and cytoplasm, but predominantly in nuclei. High labeling indices of cdc25B in invasion front of gastric cancer was observed in 31 out of 125 cases (24.8%), linked to an advanced depth of cancer invasion (P=0.02), high rates of lymphatic invasion (P=0.03), and lymph node metastasis (P<0.01). Furthermore, the Kaplan-Meier method demonstrated a poor prognosis for cdc25B high labeling indices cases (P=0.02), although multivariate analysis revealed it not to be an independent factor. In conclusion, it seems likely that cdc25B is located predominantly in nuclei when overexpressed and this has some linkage with progression of gastric cancer. [Abstract]

Dabbs DJ, Kaplai M, Chivukula M, Kanbour A, Kanbour-Shakir A, Carter GJ
The spectrum of morphomolecular abnormalities of the E-cadherin/catenin complex in pleomorphic lobular carcinoma of the breast.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):260-6.
Pleomorphic lobular carcinoma of the breast is a high nuclear grade variant of lobular carcinoma. E-cadherin, a tumor-invasion suppressor gene, codes for a transmembrane protein that functions in intercellular adhesion. The E-cadherin protein internal domain binds with alpha, beta, gamma, and p120 catenins to anchor the E-cadherin complex to the actin cytoskeleton of the cell. The E-cadherin gene is routinely mutated in lobular neoplasia. This study examines the morphomolecular spectrum of the components of the E-cadherin-catenin complex in lobular neoplasia. Fifteen cases of pleomorphic lobular neoplasia, 8 cases of classic lobular neoplasia and 4 ductal carcinomas were studied. Normal breast epithelium and invasive ductal carcinomas all showed intense linear cell membrane immunostaining with antibodies to E-cadherin, alpha, beta, gamma, and P120 catenins. Membrane immunostaining of the catenin antibodies in lobular neoplasia was negative, except for rare cases that displayed beaded or dotlike patterns. Cytoplasmic immunostaining patterns for all lobular lesions included coarse paranuclear granules of beta catenin or diffuse intense cytoplasmic staining for P120 catenin. These immunostaining patterns demonstrate that catenins alpha, beta, gamma, and p120 are routinely dislocated from the cell membrane into the cytoplasm in lobular neoplasia and that the disrupted catenin patterns parallel absence of membrane E-cadherin in all cases. The diffuse cytoplasmic immunostaining of p120 in lobular neoplasia may be useful diagnostically as a positive marker for lobular neoplasia. [Abstract]

Nassar A, Radhakrishnan A, Cabrero IA, Cotsonis G, Cohen C
COX-2 expression in invasive breast cancer: correlation with prognostic parameters and outcome.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):255-9.
Lipoxygenases (LOX) and cyclooxygenases (COX) are key mediators of arachidonic acid metabolism. Recently, studies have reported that human breast carcinomas aberrantly express LOX and cyclooxygenase-2 (COX-2), and that decreased levels of 15-lipoxygenase (15-LOX) and raised levels of COX-2 and 12-LOX have prognostic value in patients with breast cancer. 15-LOX was significantly reduced with increasing stage, and in patients who developed metastatic disease, local recurrence, and/or died. With high COX-2, patients developed local recurrence, died from breast cancer and had reduced disease-free and disease-related overall survival in estrogen receptor (ER)-negative but not ER-positive disease. COX-2 expression is also associated with increased angiogenesis, lymph node metastasis, and Her2-neu overexpression. The purpose of this study is to evaluate COX-2 expression in breast cancer and to determine its correlation with prognostic parameters and outcome. Five tissue microarrays were constructed from 43 breast carcinomas and 5 normal breast tissues, represented by 1 mm cores in triplicate from each of 3 foci. Tissue microarray cores were immunostained with monoclonal COX-2. Expression was assessed as intensity and scored as percentage of cells positive. Prognostic parameters and follow-up information were obtained from the hospital records of Mexican Oncology Hospital, Mexico, where the carcinomas were diagnosed. Ninety-five percent (41/43) of the breast carcinomas showed cytoplasmic COX-2 expression. COX-2 intensity and percentage of cells positive correlated significantly with size of carcinoma (P=0.0271; P=0.0539, respectively). COX-2 intensity correlated significantly with histologic grade (P=0.0182). COX-2 did not correlate with outcome (disease-free and overall survival). There was no significant correlation between COX-2 and ER. In conclusion, COX-2 correlates with poor prognostic markers in breast cancer (large tumor size and high tumor grade), but not with outcome. The therapeutic value of COX-2 inhibitors in COX-2 positive breast cancer patients requires further investigation. [Abstract]

Dunphy CH, Nies MK, Gabriel DA
Correlation of plasma cell percentages by CD138 immunohistochemistry, cyclin D1 status, and CD56 expression with clinical parameters and overall survival in plasma cell myeloma.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):248-54.
CONTEXT: Plasma cell myelomas (PCMs) are traditionally diagnosed by the percentage (%) of plasma cells (PCs) in the bone marrow aspirate differential combined with clinical parameters and radiographic findings. PCs are most reliably quantitated in bone marrow (BM) tissues by CD138 immunohistochemistry (IHC). However, there are no correlations of % CD138+ cells with clinical parameters or overall survival (OS). The presence of cyclin D1 has correlated with worst prognosis, but cyclin D1 has not been correlated with routine cytogenetics. CD56+, although not significantly reported in reactive plasmacytoses, monoclonal gammopathy of undetermined significance (MGUS), nor in lymphoplasmacytic lymphomas (LPLs), has not been evaluated in borderline diagnostic (borderline) cases. OBJECTIVES: It includes: (1) correlating the percentages of PCs by CD138 IHC, cyclin D1 status, and CD56 expression with clinical parameters and OS in PCMs, (2) correlating cyclin D1 status with routine cytogenetics in PCMs, borderline cases, and MGUSs, and (3) analyzing CD56 expression in PCMs, borderline cases, MGUSs, and LPLs. DESIGN: Bone marrow aspirates, BM touch preparations, and BM clot and/or biopsy sections with CD138/kappa/lambda IHC (44-PCMs, 9-MGUSs, 17-borderline cases, 3-LPLs, and 3-reactive plasmacytoses) were reviewed and stained with CD56 and cyclin D1. RESULTS/CONCLUSIONS: Increased CD138+ cells did not correlate significantly with clinical parameters or OS. Cyclin D1+ did not correlate with the presence of a t(11;14) by routine cytogenetics [although detected in all t(11;14)+ cases], clinical parameters, nor OS. CD56 expression was identified in PCMs, MGUSs, and LPL but not in reactive plasmacytoses. CD56+ did not distinguish PCMs, MGUS, and LPLs, and did not correlate with clinical parameters or OS. CD56 and cyclin D1 IHC were better evaluated in BM clot than biopsy sections. [Abstract]

Peterson MR, Piao Z, Bazhenova LA, Weidner N, Yi ES
Terminal respiratory unit type lung adenocarcinoma is associated with distinctive EGFR immunoreactivity and EGFR mutations.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):242-7.
Approximately 10% to 20% of nonsmall cell lung cancer patients respond to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, such as gefitinib. Responders are mostly nonsmokers and women with tumors displaying bronchioloalveolar features. Mutations of the tyrosine kinase domain of the EGFR gene have been associated with a clinical response to gefitinib. A recent study reported that the terminal respiratory unit (TRU)-type adenocarcinoma shares the clinical profile and EGFR mutations of gefitinib responders. EGFR immunoreactivity in this context has not been reported in the literature. We performed a detailed immunohistochemical analysis of EGFR expression on 124 consecutive lung resection specimens for malignancy, to survey the EGFR immunoreactivity in lung cancers in general and to correlate EGFR immunoreactivity with EGFR mutations and TRU-type histology. EGFR positivity was seen most frequently in squamous cell carcinomas (77%), followed by TRU-type adenocarcinomas (63%), large cell carcinomas (23%), and non-TRU-type adenocarcinomas (12%). A distinctive basally oriented cytoplasmic positivity was observed exclusively in TRU-type adenocarcinomas. EGFR mutation was identified in 6 of 54 cases studied and all 6 cases were TRU-type adenocarcinomas. Five of six cases with EGFR mutation were positive for EGFR immunostain with the basal cytoplasmic localization. In conclusion, EGFR immunoreactivity with basal cytoplasmic pattern was exclusively seen in TRU-type adenocarcinoma and a subset of these cases was seen with EGFR mutations in the responders to EGFR inhibitor therapy. [Abstract]

Yaziji H, Taylor CR
Begin at the beginning, with the tissue! The key message underlying the ASCO/CAP Task-force Guideline Recommendations for HER2 testing.
Appl Immunohistochem Mol Morphol. 2007 Sep;15(3):239-41. [Abstract]

Lin AY, Ai Z, Lee SC, Bajcsy P, Pe'er J, Leach L, Maniotis AJ, Folberg R
Comparing vasculogenic mimicry with endothelial cell-lined vessels: techniques for 3D reconstruction and quantitative analysis of tissue components from archival paraffin blocks.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):113-9.
We previously described techniques to generate 3-dimensional reconstructions of the tumor microcirculation using immunofluorescence histochemistry and laser scanning confocal microscopy on serial sections from archival formalin-fixed, paraffin-embedded tissues. By aligning sequential z-stacks in an immersive visualization environment (ImmersaDesk), the need to insert fiduciary markers into tissue was eliminated. In this study, we developed methods to stitch overlapping confocal z-series together to extend the surface area of interest well beyond that captured by the confocal microscope objective and developed methods to quantify the distribution of markers of interest in 3 dimensions. These techniques were applied to the problem of comparing the surface area of nonendothelial cell-lined, laminin-rich looping vasculogenic mimicry (VM) patterns that are known to transmit fluid, with the surface area of endothelial cell-lined vessels in metastatic uveal melanoma to the liver in 3 dimensions. After labeling sections with antibodies to CD34 and laminin, the surface area of VM patterns to vessels was calculated by segmenting out structures that labeled with laminin but not with CD34 from those structures labeling with CD34, or CD34 and laminin. In metastatic uveal melanoma tissues featuring colocalization of high microvascular density [66.4 microvessels adjusted for 0.313 mm2 area (range 56.7 to 72.7)] and VM patterning, the surface area of VM patterns was 11.6-fold greater (range 10.8 to 14.1) than the surface provided by CD34-positive vessels. These methods may be extended to visualize and quantify molecular markers in 3 dimensions in a variety of pathologic entities from archival paraffin-embedded tissues. [Abstract]

Kong X, Zhao Y, Ksionsk M, Zhou M, Walden P, Bosland M, Pei Z, Lee P, Melamed J
Radiographic determination of tissue thickness in paraffin blocks: application to the construction of tissue microarrays.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):108-12.
The determination of tissue thickness in paraffin blocks in the histology laboratory has been largely based on visual estimates. More accurate methods are required for the construction of tissue microarrays (TMAs) to assure a greater yield of cores in sections through the TMA block. We describe an accurate radiographic method to determine tissue thickness in donor paraffin blocks and have validated its application to TMA construction. Individual radiographic analysis was performed on paraffin donor blocks used for the construction of TMAs for determination of donor block tissue thickness. Consecutive numbered slide sections through the TMA block were then examined for the presence or loss of cores in the 150th TMA slide (from the final third of the TMA block) and correlated with the thickness of the individual donor blocks determined radiographically. At the 150th TMA slide, 202 of 1340 cores (15.1%) were depleted. Radiographic measurement showed a greater thickness of the donor paraffin block tissue (2.02 mm) corresponding to the retained cores as compared with the donor tissue (1.54 mm) of the depleted cores (P < 0.001). With progressive slide sections through a TMA block, the retention of tissue cores shows a significant correlation with donor block tissue thickness. Radiographic determination of tissue thickness in donor paraffin blocks can be used in TMA construction. Prior knowledge of tissue thickness in TMA construction can prompt compensatory steps that can enhance the yield of valuable samples and assure sufficient numbers of adequate cores for statistical analysis in biomarker evaluations. [Abstract]

Vosse BA, Seelentag W, Bachmann A, Bosman FT, Yan P
Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):103-7.
The aim of this study was to evaluate specific immunostaining and background staining in formalin-fixed, paraffin-embedded human tissues with the 2 most frequently used immunohistochemical detection systems, Avidin-Biotin-Peroxidase (ABC) and EnVision+. A series of fixed tissues, including breast, colon, kidney, larynx, liver, lung, ovary, pancreas, prostate, stomach, and tonsil, was used in the study. Three monoclonal antibodies, 1 against a nuclear antigen (Ki-67), 1 against a cytoplasmic antigen (cytokeratin), and 1 against a cytoplasmic and membrane-associated antigen and a polyclonal antibody against a nuclear and cytoplasmic antigen (S-100) were selected for these studies. When the ABC system was applied, immunostaining was performed with and without blocking of endogenous avidin-binding activity. The intensity of specific immunostaining and the percentage of stained cells were comparable for the 2 detection systems. The use of ABC caused widespread cytoplasmic and rare nuclear background staining in a variety of normal and tumor cells. A very strong background staining was observed in colon, gastric mucosa, liver, and kidney. Blocking avidin-binding capacity reduced background staining, but complete blocking was difficult to attain. With the EnVision+ system no background staining occurred. Given the efficiency of the detection, equal for both systems or higher with EnVision+, and the significant background problem with ABC, we advocate the routine use of the EnVision+ system. [Abstract]

Powell WC, Hicks DG, Prescott N, Tarr SM, Laniauskas S, Williams T, Short S, Pettay J, Nagle RB, Dabbs DJ, Scott KM, Brown RW, Grogan T, Roche PC, Tubbs RR
A new rabbit monoclonal antibody (4B5) for the immunohistochemical (IHC) determination of the HER2 status in breast cancer: comparison with CB11, fluorescence in situ hybridization (FISH), and interlaboratory reproducibility.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):94-102.
The 2 methodologies in current clinical use to assess HER2 status in breast cancer are: fluorescence in situ hybridization (FISH) (gene amplification) and immunohistochemistry (protein over-expression). A consistent finding has been that 3% to 15% of breast cancers over-express HER2 protein without evidence for gene amplification. Accurate determination of the HER2 status has implications for selecting patients most likely to respond to trastuzumab. We report here our preliminary experience with a new anti-HER2 rabbit monoclonal antibody, 4B5. The evaluation of HER2 status in 2 different cohorts of breast cancer cases (Single Institution (SI) and Multinational (MN)) with a total of 322 breast cancer cases was performed on an automated staining system (Ventana Medical Systems, Inc, Tucson, AZ) and scored by 3 pathologists (0-3+), for comparison with CB11 staining results (PATHWAY) and FISH (PathVysion). Interlaboratory reproducibility of automated staining results and interpretation was determined on a subset of the SI cohort at 3 separate laboratories. Rabbit monoclonal 4B5 demonstrated sharper membrane staining with less cytoplasmic and stromal background staining than CB11. In the SI cohort, the staining results for 4B5 were highly comparable with those obtained for CB11 with an overall concordance of 93.3%. In the multinational cohort, the overall concordance with CB11 was 84.7%. This lower level of concordance was associated with a much higher overall agreement of 4B5 with FISH (89.5%), compared with agreement of CB11 with FISH (81.2%). The difference in the performance of CB11 in the MN cohort versus the SI cohort may be due to differences in tissue fixation and processing in a centralized, high volume laboratory in an academic medical center versus multiple sites in the international community with potentially nonstandardized techniques. The staining results with 4B5 indicate that it has a more robust performance than CB11 because the correlation of 4B5 with FISH was nearly equivalent (88.2% MN; 89.3% SI) in both cohorts. Interlaboratory reproducibility was also excellent (kappa 1.0). RMoAb 4B5 provides excellent sensitivity, specificity, and interlaboratory reproducibility for the detection of HER2 status in breast cancer. [Abstract]

Leong AS, Haffajee Z, Clarke M
Microwave enhancement of CISH for HER2 oncogene.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):88-93.
We describe a modification to the prescribed procedure for the Zymed Spot-Light HER2 chromogenic in situ hybridization kit (84-0146, Zymed Laboratories, San Francisco, CA) by substituting the heat pretreatment step with MW irradiation in citrate buffer 10 mmol/L at pH 6.0 at 98 degrees C for 10 minutes and repeating the procedure afterenzyme digestion with time and temperature controlled in the Mega T/ T oven (Milestone s.r.l., Sorisole, Italy). The subsequent procedure leading up to hybridized was as per manufacturer's instructions. Invasive breast carcinoma previously scored by immunohistochemistry for HER2, comprising 18 cases of 3+, 18 cases of 2+, and 12 cases of 1+, were examined by chromogenic in situ hybridization using this modified procedure, with a parallel set of cases examined by the prescribed Zymed method. The introduction of the "MW retrieval" steps resulted in consistently a greater number of hybridization signals in amplified tumor cells with benign epithelial cells and lymphocytes displaying 2 clear dots compared with the weaker and less consistent signals obtained with the standard procedure. MW exposed sections showed larger numbers of large and small clusters that often allowed identification of amplified tumors without having to count single dots with crisp staining and absence of background precipitation. [Abstract]

Willmore-Payne C, Metzger K, Layfield LJ
Effects of fixative and fixation protocols on assessment of Her-2/neu oncogene amplification status by fluorescence in situ hybridization.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):84-7.
Fluorescence in situ hybridization (FISH) is used to determine amplification status of the Her-2/neu gene in specimens of newly diagnosed breast carcinoma. The Vysis kit for FISH analysis stipulates that the tissue be formalin-fixed and paraffin-embedded. Concerns regarding carcinogenicity of formalin and environmental effects of formalin waste have led to the development of formalin replacement products. An increasing number of breast biopsy specimens are being fixed in these substitutes. We tested 6 non-formalin-based fixatives to determine their impact on FISH testing for Her-2/neu gene amplification status by comparison with formalin-fixed control specimens from the same neoplasm. Specimens fixed in Pen-Fix, Prefer, Histochoice, UniFix, and GTF were associated with absent or technically compromised staining in at least one of the 3 neoplasms tested for each fixative when compared to the formalin-fixed control. O-Fix did not seem to compromise staining quality in 3 paired specimens tested. [Abstract]

Jäger D, Filonenko V, Gout I, Frosina D, Eastlake-Wade S, Castelli S, Varga Z, Moch H, Chen YT, Busam KJ, Seil I, Old LJ, Nissan A, Frei C, Gure AO, Knuth A, Jungbluth AA
NY-BR-1 is a differentiation antigen of the mammary gland.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):77-83.
NY-BR-1 was recently identified by autologous serological typing of the recombinant expression library in a breast cancer patient. Extensive reverse transcriptase-polymerase chain reaction analysis revealed the presence of NY-BR-1 in normal breast tissue and tumors derived thereof. Except normal testis, no other normal tissue or tumors showed NY-BR-1 expression. However, nothing is known about the expression of its actual antigen. In the present study, we describe the generation of 2 new monoclonal antibodies, NY-BR-1#2 and NY-BR-1#3, to NY-BR-1 for the analysis of its expression on a protein level employing recombinant NY-BR-1 protein for the immunization of BALB/c mice. In normal tissues, immunohistochemical testing demonstrates NY-BR-1 in a mostly focal fashion in the epithelia of ducts and acini of the mammary gland. No other tissue was immunopositive including testis. In tumors, homogenous staining can be seen in almost all ductal carcinomas in situ and/or the intraductal component of invasive carcinomas. Invasive carcinomas show a lower number of NY-BR-1-positive tumors. Initial higher numbers of NY-BR-1 mRNA-positive invasive carcinomas are most likely based on sample error owing to the contamination of tumor tissue with remnants of normal breast epithelium. Sweat gland carcinomas, which are related to breast cancer, are also positive in about one-third of the cases. These data indicate that NY-BR-1 is a differentiation antigen of the mammary gland that could be useful for diagnosis and/or immunotherapy of breast carcinomas. [Abstract]

Masi L, Recenti R, Silvestri S, Pinzani P, Pepi M, Paglierani M, Brandi ML, Franchi A
Expression of cyclooxygenase-2 in osteosarcoma of bone.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):70-6.
Several studies indicate that cyclooxygenase-2 (COX-2) is overexpressed in human malignancies, where it produces high levels of prostaglandins and contributes to tumor growth. In this study we have analyzed the expression of COX-2 in a series of 48 skeletal osteosarcomas of different subtypes by immunohistochemistry. In addition, we examined the effects of the specific COX-2 inhibitor Celecoxib on the growth of the human osteosarcoma cell line SaOS-2. Immunoreactivity for COX-2 was observed in 39 out of 48 tumors (81.2%), 30 (76.9%) of which showed a moderate or diffuse immunostaining. Considering the group of 42 primary osteosarcomas, COX-2 immunoreactivity was significantly higher in high grade osteosarcomas, where moderate or diffuse expression was detected in 23 out of 32 cases (71.8%), than in low grade osteosarcomas, where moderate or diffuse expression was detected in 2 out of 10 cases (20%) (P = 0.008, Fisher exact test). In addition, low COX-2 expression was always associated with a good response to chemotherapy (5 out of 5 cases), whereas moderate or diffuse COX-2 expression was associated with a good response in 11 out of 20 cases (55%) (P = 0.12, Fisher exact test). In SaOS-2 osteosarcoma cells, which express COX-2, treatment with Celecoxib determined inhibition of cell proliferation and induction of apoptosis. These results indicate that COX-2 is expressed at high levels in high grade osteosarcomas and support the use of COX-2 inhibitors to improve both the tumor response to chemotherapy and the outcome of osteosarcoma patients. [Abstract]

Sabah M, Cummins R, Leader M, Kay E
Immunoreactivity of p53, Mdm2, p21(WAF1/CIP1) Bcl-2, and Bax in soft tissue sarcomas: correlation with histologic grade.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):64-9.
Tumor growth depends on 2 distinctive pathways: cell proliferation and apoptosis. The p53 pathway is an important regulator of the cell cycle as it triggers growth arrest or leads to apoptosis in response to cellular stress and therefore is commonly targeted during tumorigenesis. Apoptosis is also controlled by the Bcl-2 family, which includes proapoptotic and antiapoptotic proteins. The aim of this study was to investigate the expression of proteins that are involved in the p53 pathway and apoptosis in different types of soft tissue sarcomas and to correlate the expression of these proteins with the histologic grade of sarcoma cases. One hundred fifty-two cases of different types of soft tissue sarcomas were analyzed. The cases consisted of 54 low-grade, 40 intermediate-grade, and 58 high-grade sarcomas. Immunohistochemical stains for p21(WAF1/CIP1), p53, Mdm2, Bcl-2, and Bax proteins were carried out on tissue microarrays. Nuclear reactivity for p53 was detected in 49 cases (32.2%). Overexpression of Mdm2 was found in 18 cases (11.8%) and p21(WAF1/CIP1) immunostaining was seen in 28 tumors (18.4%). p53 and p21(WAF1/CIP1) expression correlated with the tumor grade (low grade, 5.6% and 3.7%; intermediate grade, 22.5% and 20%; high grade, 63.8% and 31%, respectively). Expression of Bax protein was a common finding in soft tissue sarcoma cases. It was detected in 141 cases (92.8%). Bcl-2 was identified in 59 tumors (38.8%) and was more prevalent in high-grade sarcomas (low grade, 25.9%; intermediate grade, 32.5%; high grade, 55.2%). It was concluded that alterations in the p53 pathway and genes that regulate apoptosis are common events in soft tissue sarcomas. The expression of p53, p21(WAF1/CIP1), and Bcl-2 is closely associated with the histologic grade of the tumor, and therefore these proteins may be used as prognostic markers. [Abstract]

Nash JW, Bhardwaj A, Wen P, Frankel WL
Maspin is useful in the distinction of pancreatic adenocarcinoma from chronic pancreatitis: a tissue microarray based study.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):59-63.
Maspin, a member of the serpin family of serine protease inhibitors, has been shown to limit invasion and metastases in breast and prostate carcinomas. Maspin gene expression is up-regulated in pancreatic cancer, but not in normal pancreatic tissue. Maspin expression has been documented using immunohistochemical studies in pancreatic adenocarcinoma and high-grade intraductal dysplasia. We studied pancreatic ductal adenocarcinomas and chronic pancreatitis utilizing tissue microarray technology to determine the utility of maspin in differentiating these lesions. Immunohistochemistry was performed on tissue microarrays made from 72 cases of pancreatic ductal adenocarcinoma and 24 cases of chronic pancreatitis. Carcinomas were graded as well, moderately, or poorly differentiated using the WHO criteria. The primary antibody used was monoclonal antimaspin antibody (clone G167-70, 1:800, PharMingen, San Diego, CA). Nuclear and/or cytoplasmic staining for maspin was qualitatively scored from 1 + to 3 + based on intensity. Cases were considered positive if one or more cores demonstrated staining. Cases of chronic pancreatitis showed focal, weak (1 + to 2 +) staining within occasional benign ductal epithelial cells in 29% of cases (7/24). Diffuse and intense (3 +) staining was present in ducts with squamous metaplasia (3 cases). The majority of ducts showed no staining. Ductal adenocarcinomas showed diffuse staining in 91% (66/72) of cases with generally more intense staining than cases of chronic pancreatitis. Maspin may be helpful in differentiating ductal adenocarcinoma from chronic pancreatitis, once squamous metaplasia is ruled out. [Abstract]

Haynik DM, Roma AA, Prayson RA
HER-2/neu expression in glioblastoma multiforme.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):56-8.
BACKGROUND: The HER-2/neu oncogene encodes for a transmembrane glycoprotein with intracellular tyrosine kinase activity. The HER-2/neu receptor belongs to the family of epidermal growth factor receptors that are crucial in the activation of subcellular signal transduction pathways controlling epithelial cell growth and differentiation. Overexpression of HER-2/neu is observed in 20% to 40% of breast cancers and other solid tumors. Although information is limited, one study suggested that 15% of glioblastoma multiforme (GBM) express HER-2/neu by immunohistochemistry (IHC); gene amplification by fluorescence in situ hybridization (FISH) was not investigated. Studies in this area are potentially significant owing to the role of recombinant monoclonal anti-HER-2/neu antibody traztuzumab (Herceptin) in the treatment of tumors. DESIGN: A retrospective clinicopathologic review of 49 patients with GBM with HER-2/neu IHC staining and HER-2/neu gene amplification by FISH was performed. RESULTS: The study included 44 patients (17 women, 27 men; age range 20 to 79 y, mean 57.9 y). Initial surgery involved tumor debulking or subtotal resection in 34 patients. Thirty-six patients received adjuvant radiation therapy and 19 patients received adjuvant chemotherapy. At follow-up (range 1.0 to 49.5 mo, mean 10.5 mo), 40 patients died with tumor and 4 patients were lost to follow-up. All tumors were negative for HER-2/neu protein by IHC and for HER-2/neu gene amplification by FISH. CONCLUSIONS: No GBM demonstrates HER-2/neu protein by IHC or amplification of the HER-2/neu gene by FISH. The HER-2/neu oncogene does not seem to play a role in the pathogenesis of GBM. [Abstract]

Mai KT, Burns BF, Stinson WA, Morash C
The 3-dimensional structure of isolated and small foci of prostatic adenocarcinoma: the morphologic relationship between prostatic adenocarcinoma and prostatic intraepithelial neoplasia.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):50-5.
BACKGROUND: Transitional histopathologic changes from high-grade prostatic intraepithelial neoplasia (HGPIN) into early prostatic adenocarcinoma (PAC) have not been well studied to date. To investigate the histogenesis of PAC, we examined isolated and small foci of PAC (ISPAC) found in prostatectomy specimens and the 3-dimensional structure of these foci. DESIGN: Twelve consecutive radical prostatectomy specimens having ISPAC, performed for peripheral zone PAC (10 cases) and for transitional zone PAC (2 cases), of Gleason score were studied. One to 2 tissue blocks with representative sections were used. RESULTS: Eight ISPAC, with Gleason score 3 + 3 had complete serial sections of the entire lesion. PAC consisted of continuous, tortuous and branching tubules and acini arising from benign ducts displaying: (a) HGPIN in 5 ISPAC and (b) no HGPIN in 3 ISAPC. At the junctions between benign epithelia with or without HGPIN and malignant epithelia, there were transitional lesions with HGPIN involving small ducts and acini. CONCLUSIONS: PAC develops as a result of multiple outpouchings of the epithelium with formation of small ducts and acini showing cytologic atypia and gradual or abrupt loss of basal cells. Grade 3 ISPAC consists of a system of continuous duct pushing into the stroma. There is also evidence suggestive of HGPIN as being both a precursor lesion and an accompanying lesion of PAC. [Abstract]

Quraishi I, Rishi M, Feldman M, Wargovich MJ, Weber B
Clinical validation of breast cancer biomarkers using tissue microarray technology.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):45-9.
The results of previous studies done in our laboratory on breast cancer gene expression profile, using DNA microarrays, led to the discovery of several genes associated with breast cancer progression. Further evaluation of these genes and their involvement at various stages of cancer progression required performance of immunohistochemistry on thousands of different tissue blocks. Tissue microarray (TMA) technology facilitates rapid translation of DNA microarrays results to clinical specimens by using immunohistochemical analysis of protein expression. DNA microarray analysis done in our laboratory showed a significantly higher expression of prostatic-specific antigen (PSA) in invasive ductal carcinomas as compared to ductal carcinoma in situ, a finding contrary to the previously published data for PSA immunoreactivity in breast carcinomas. To find out whether TMA strategy could be used to explore the expression of the candidate genes involved in the breast cancer progression, we constructed a breast cancer progression TMA. It consisted of 2 normal ductal epithelium, 8 ductal carcinoma in situ, 19 invasive ductal carcinomas, and 3 metastatic ductal carcinomas of breast in triplets. Two prostatic adenocarcinomas and 2 normal colons were used as positive and negative controls, respectively. We first used well-documented and well-tested markers, such as antibodies to estrogen receptor, progesterone receptor, and p53. Results of these 3 antibodies were according to the previously published data. To validate our result, we then used antibody to PSA and looked for the expression of this protein on breast cancer progression TMA. Except for the 2 positive controls all 98 cores were found to be negative for PSA expression highlighting the importance of validation studies for DNA microarray results. [Abstract]

Barrionuevo C, Zaharia M, Martinez MT, Taxa L, Misad O, Moscol A, Sarria G, Guerrero I, Casanova L, Flores C, Zevallos-Giampietri EA
Extranodal NK/T-cell lymphoma, nasal type: study of clinicopathologic and prognosis factors in a series of 78 cases from Peru.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):38-44.
It is well known that extranodal NK/T-cell lymphoma (NK/TCL) nasal type clusters in Asian countries. A large series of 78 cases of nasal NK/TCL from Peru is analyzed in the present study. Two histologic groups 1 (monomorphic) and 2 (polymorphic), were segregated according to the proportion of large cells (above and below 30%, respectively). Catalyzed signal amplification technique was performed for enhancement of immunohistochemistry reactivities. Epstein-Barr virus (EBV) sequences and types were investigated using polymerase chain reaction. Clinical characteristics, stage, outcome, and response to treatment were evaluated in both groups. Fourteen cases (18%) and 64 cases (82%) corresponded to groups 1 and 2, respectively. Except for nasal obstruction, more common in group 2, all other symptoms were similar in both groups. Local extension and staging were also comparable. Both groups showed CD3c+ CD2+ CD56+ CD3s- CD20- immunophenotype. All cases were positive for EBV. In this series type-2 EBV was found more frequent than type-1 EBV, contrarily to that observed in Asian series. However, about one-third of cases simultaneously harbored both viral types. Both groups received an average of 50-Gy dose of radiation therapy (RT), with or without chemotherapy. Complete therapeutic response was achieved in 89% of group 1 and in 74% of group 2, but this difference was not statistically significant. There were no significant differences between the groups regarding disease-free survival, failure-free survival, relapse, and overall survival. The overall survival, in both groups, was longer for patients treated with RT alone compared with those treated with combined RT therapy and chemotherapy. The present study has shown that dividing nasal NK/TCL in monomorphic and polymorphic variants, according to frequency of large cells, does not correlate with clinical and prognostic factors. [Abstract]

Ulukus EC, Kargi HA, Sis B, Lebe B, Oztop I, Akkoclu A, Onen A, Sanli A
Survivin expression in non-small-cell lung carcinomas: correlation with apoptosis and other apoptosis-related proteins, clinicopathologic prognostic factors and prognosis.
Appl Immunohistochem Mol Morphol. 2007 Mar;15(1):31-7.
The role of survivin that regulates the biological behavior of non-small-cell lung carcinoma (NSCLC) is still controversial. We aimed to investigate survivin expression in NSCLC and to define any correlation with expressions of p53, bcl-2, bax, apoptotic index (AI), tumor cell proliferation, clinicopathologic variables, and overall survival. Tumors of 63 patients with NSCLC were examined for expressions of survivin, p53, bcl-2, bax, and Ki-67 by immunohistochemistry. AI was also evaluated. Results for each antibody were correlated with each other, and with clinicopathologic variables including age, sex, histologic subtype, TNM (T: primary tumor, N: regional lymph node metastasis, M: distant metastasis) stage, lymph node status, smoking history, and prognosis. Nuclear survivin expression was inversely correlated with p53 expression (P = 0.04, r = - 0.367), and tumor stage (P = 0.03, r = - 0.273), and positively correlated with tumor cell proliferation (P = 0.009, r = 0.329). Cytoplasmic survivin expression positively correlated with smoking history (P = 0.02, r = 0.282). Survivin/bax ratio was inversely correlated with AI (r: - 0.004). By Kaplan-Meier analysis, TNM stage (P < or = 0.001), lymph node metastasis (P = 0.04), and Ki-67 index (P < or = 0.001) were associated with survival, whereas survivin was not. In multivariate analysis, only TNM stage was an independent predictor. Although survivin and other apoptosis-related protein expressions fail to predict the clinical outcome, the present findings suggest that survivin is involved in tumor cell apoptosis and proliferation and may play a role in critical steps of cancer progression in NSCLC. [Abstract]

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Recent Articles in Cells, Tissues, Organs

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Arráez-Aybar LA, Turrero-Nogués A, Marantos-Gamarra DG
Embryonic Cardiac Morphometry in Carnegie Stages 15-23, from the Complutense University of Madrid Institute of Embryology Human Embryo Collection.
Cells Tissues Organs. 2007 Dec 5;
Aims: We performed a morphometric study of cardiac development on human embryos to complement the scarce data on human embryonic cardiac morphometry and to attempt to establish, from these, algorithms describing cardiac growth during the second month of gestation. Methods: Thirty human embryos from Carnegie stages 15-23 were included in the study. Shrinkage and compression effects from fixation and inclusion in paraffin were considered in our calculations. Results: Growth of the cardiac (whole heart) volume and volume of ventricular myocardium through the Carnegie stages were analysed by ANOVA. Linear correlation was used to describe the relationship between the ventricular myocardium and cardiac volumes. Comparisons of models were carried out through the R(2) statistic. The relationship volume of ventricular myocardium versus cardiac volume is expressed by the equation: cardiac volume = 0.6266 + 2.4778 volume of ventricular myocardium. The relationship cardiac volume versus crown-rump length is expressed by the equation: cardiac volume = 1.3 e(0.126 CR length), where e is the base of natural logarithms. Conclusion: At a clinical level, these results can contribute towards the establishment of a normogram for cardiac development, useful for the design of strategies for early diagnosis of congenital heart disease. They can also help in the study of embryogenesis, for example in the discussion of ventricular trabeculation. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Nolte SV, Xu W, Rennekampff HO, Rodemann HP
Diversity of Fibroblasts - A Review on Implications for Skin Tissue Engineering.
Cells Tissues Organs. 2007 Nov 28;
Enormous advances in the development of skin substitutes have occurred in the past 3 decades. Major obstacles yet to be overcome in the quest for an optimal skin substitute include controlling scar formation, contraction and the loss of adnexal structures. Mesenchyme-derived signals are essential for epithelial proliferation, skin morphogenesis, homeostasis and differentiation. Having previously shown that fibroblasts differentiate along a lineage from highly proliferative progenitor fibroblasts with characteristic spindle-shaped appearance to differentiated postmitotic polygonal fibrocytes, we have now established that the different subsets of fibroblasts exert significantly different patterns of cytokine release and that the highest levels of keratinocyte growth factor and transforming growth factor-beta(1) expression result from differentiated fibroblasts. Coculture studies with keratinocytes reveal that postmitotic fibroblasts stimulate keratinocyte proliferation to a greater extent than progenitor fibroblasts. Acellular and fibroblast-seeded dermal substitutes have been shown to improve scarring and contraction in animal studies, the latter substitutes yielding the most favorable results. Fibroblasts from different body sites display different functional properties which may affect their suitability for dermal substitutes. Future in vivo human studies in tissue-engineered dermal substitutes will likely focus on fibroblast-seeded lattices and the impact of fibroblast subpopulations and bone marrow-derived mesenchymal stem cells on dermal regeneration. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Gérard C, Blouin K, Tchernof A, Doillon CJ
Adipogenesis in Nonadherent and Adherent Bone Marrow Stem Cells Grown in Fibrin Gel and in the Presence of Adult Plasma.
Cells Tissues Organs. 2007 Nov 28;
Bone marrow-derived mesenchymal stem cells (i.e., adherent cells) are known to differentiate into fat tissue in the presence of adipogenic supplements in cultures. Induction of adipogenesis has not been investigated within the nonadherent cell fraction that includes predominantly hematopoietic cells. In the present study, murine nonadherent bone marrow-derived stem cells (96% CD45+ cells) were seeded and then grown in fibrin gel to form cell clusters in which most cells were positive to DiI-acetylated low-density lipoprotein uptake. Amongst different culture media supplemented either in fetal bovine serum, horse serum, murine plasma, human plasma or adipogenic supplements, a subpopulation of nonadherent stem cells within clusters differentiated into adipocytes, specifically in the presence of adult syngeneic plasma. This was confirmed by the observation and quantification of oil red O-positive cells, the measurement of glycerol-3-phosphate dehydrogenase activity and peroxisome proliferator-activated receptor-gamma mRNA expression. Similarly, adipogenesis was also observed in the presence of murine plasma with adherent mesenchymal stem cells and 3T3-L1 preadipocytes which were grown either in monolayer plastic cultures or in fibrin gel. Thus, it is possible that nonadherent cells, once in a 3-dimensional environment, can further differentiate towards adipogenesis. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Burdan F, Rozylo-Kalinowska I, Szumilo J, Dudka J, Klepacz R
Cyclooxygenase Inhibitors Affect Bone Mineralization in Rat Fetuses.
Cells Tissues Organs. 2007 Nov 9;
Intrauterine growth retardation, increased incidence of developmental variations, lack of cartilage and joint developmental side effects were previously reported for nonselective (ibuprofen, piroxicam, tolmetin) and selective (DFU) cyclooxygenase (COX)-2 inhibitors, also known as nonsteroidal anti-inflammatory drugs. The aim of the present study was to evaluate the lumbar vertebra mineralization in fetuses prenatally exposed to COX inhibitors. All the tested compounds were administered intragastrically to pregnant rats from gestational days 8 to 21. Fetuses were delivered on gestational day 21, and after digital radiological examination were double-stained with alcian blue and alizarin. Decrease of alizarin staining, as a qualitative sign of mineralization, was significantly greater in groups exposed to the highest doses of the nonselective COX inhibitors. Decrease of vertebra mineralization in drug-exposed groups was also revealed using quantitative radiological analysis. However, significant differences were noted only for the fifth and sixth lumbar vertebrae in the group exposed to the highest dose of tolmetin. Strong influence of the total protein level in maternal sera on the fetal bone optic density was found. It should be stressed that unlike DFU, the examined nonselective COX inhibitors decreased fetal bone mineralization when administered in high maternal toxic doses. Moreover, maternal health status determined fetal bone mineralization. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Inukai Y, Ikeda R, Aiyama S
Effect of Glucocorticoid on the Differentiation and Development of Terminal Tubules in the Fetal Rat Submandibular Gland.
Cells Tissues Organs. 2007 Nov 5;
Glucocorticoids (CORT) are known to promote branching of the epithelial cords during the development of the rat submandibular gland. The aim of this study was to examine the effect of CORT (triamcinolone) on the differentiation of cells forming the terminal tubules in the developing fetal rat submandibular gland and the properties of the secretory granules. Light and electron microscopy showed that the terminal tubules of the glands in the experimental group contained more type III cells, which have been identified as proacinar cells, than those in the control group, whereas the relative number of type I cells, which have been identified as terminal tubule cells, was reduced. Immunoelectron microscopy using an antibody against neonatal submandibular gland secretory protein B (SMGB) revealed the presence of more gold particles over type III cell granules in the experimental group than in the control group. Lectin histochemistry demonstrated more wheat germ agglutinin (WGA)-labeled gold particles over type III cell granules in the experimental group than in the control group. These findings suggest that CORT promote the differentiation of type III cells, and moreover stimulate the production of secretory granules reactive for SMGB and WGA by acting on the terminal tubules of the developing rat submandibular gland. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Mizuno H, Itoi Y, Kawahara S, Ogawa R, Akaishi S, Hyakusoku H
In vivo Adipose Tissue Regeneration by Adipose-Derived Stromal Cells Isolated from GFP Transgenic Mice.
Cells Tissues Organs. 2007 Nov 2;
We have previously demonstrated that pluripotent stem cells can be obtained from green fluorescence protein (GFP) transgenic mouse adipose tissue. In this study, we sought to determine whether adipose tissue regeneration can be induced in vivo using adipose-derived stromal cells (ASCs) from GFP mice. ASCs were isolated from inguinal fat pads of GFP mice, as described in our previous publication. After incubation in two passages in the control medium, the cells were incubated in the induction medium to induce adipogenesis. Induced ASCs were merged with fibrin glue, and the mixture was injected subcutaneously into the dorsum of athymic mice. Finally, specimens were harvested and analyzed morphologically and histologically. The regenerated tissue was macroscopically semitransparent and soft with slight angiogenesis. Fluorescence microscopy revealed that the specimens strongly emitted green fluorescence, suggesting that the transplanted ASCs had contributed to adipogenesis. Both hematoxylin and eosin and oil red O staining revealed that cells containing small lipid droplets had been regenerated histologically. These findings suggest that ASCs could contribute to adipose tissue regeneration in vivo. ASCs may be an ideal source for adipose tissue regeneration, which may in turn play an important role in augmentation surgery in surgically treated cancer or trauma patients. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Morjane A, Dahmane R, Ravnik D, Hribernik M
Anatomy and Surgical Relevance of the Hepatocaval Ligament. A Study on Cadaveric Livers.
Cells Tissues Organs. 2007 Oct 23;
Background: There are nearly no data on the hepatocaval ligament (HCL) in the anatomical literature, though it is of high importance during surgery of the right hemiliver. Aim: The aim of this study was to determine the frequency of the HCL, its description and its relations to the inferior vena cava (IVC) and the right hepatic vein (RHV) as well as the evaluation of the surgical relevance of the data obtained. Materials and Methods: The dissection of the livers of 43 cadavers of both sexes was performed and the presence of the HCL was established. The ligament was measured and dissected to expose the IVC and the extrahepatic part of the RHV from its inflow to the liver parenchyma. Results: The ligament was present in 77% of the cases. It was 12-35 mm long and 3-18 mm wide. The extrahepatic part of the RHV was 2-12 mm long. Conclusion: Dissection of the HCL revealed the terminal extrahepatic part of the RHV in all cases. Anatomically, resection of the right hemiliver with elective vascular control would be possible in 85% of the cases in which the length of the extrahepatic part of the RHV was >/=3 mm. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Lavulo LT, Uaesoontrachoon K, Mirams M, White JD, Cockett NE, Mackie EJ, Pagel CN
Myoblasts Isolated from Hypertrophy-Responsive Callipyge Muscles Show Altered Growth Rates and Increased Resistance to Serum Deprivation-Induced Apoptosis.
Cells Tissues Organs. 2007 Oct 23;
Back and hind limb muscles of sheep paternally heterozygous for the callipyge single nucleotide polymorphism undergo extensive hypertrophy shortly after birth. We have established cell cultures from foetal semitendinosus and longissimus dorsi muscles of normal and callipyge animals. Cultures were assessed for rates of proliferation, cell death, myogenicity and DLK1 expression. Myoblasts from callipyge semitendinosus, but not longissimus dorsi muscles, proliferated faster than myoblasts isolated from normal semitendinosus muscle, and cells isolated from either callipyge muscle were more resistant to serum deprivation-induced apoptosis than equivalent cells isolated from normal individuals. Theseobservations indicate that there are intrinsic differences in the behaviour of isolated myoblasts, which are associated with their muscle and genotype of origin. As myoblasts are the cells responsible for hypertrophy of muscle fibres, the observed differences in cell growth may play a role in the hypertrophy of certain muscles in callipyge animals. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Wu L, Zhu F, Wu Y, Lin Y, Nie X, Jing W, Qiao J, Liu L, Tang W, Zheng X, Tian W
Dentin Sialophosphoprotein-Promoted Mineralization and Expression of Odontogenic Genes in Adipose-Derived Stromal Cells.
Cells Tissues Organs. 2007 Oct 23;
Dentin sialophosphoprotein (DSPP) is an extracellular matrix, typically dentin- and bone-specific gene, which plays an important role in dentin mineralization and tooth development. Adipose-derived stromal cells (ADSCs) are considered to contain a group of pluripotent mesenchymal stem cells which are capable of mineralization either in vitro or in vivo. In the present study, we hypothesized that overexpression of DSPP would promote mineralization in ADSCs. Our results showed that infection of DSPP-expressing adenovirus (Ad-DSPP) enhanced expression of genes related to mineralization, such as Cbfa1,Osx,BSP, OCN and DMP1 in ADSCs. Alkaline phosphatase activity was also confirmed in Ad-DSPP-infected ADSCs by cytochemistry and alkaline phosphatase activity assay. Mineralization assay indicated that Ad-DSPP-infected ADSCs were able to form mineralized nodules. Another finding in this study is that early odontogenic marker genes such as Msx1, Msx2, Lhx7 and Pax9 were expressed in DSPP-overexpressed ADSCs. Thus, our results suggested that overexpression of DSPP promoted mineralization of ADSCs, and together with the expression of early odontogenic marker genes, implied that these cells may differentiate into functional odontoblast-like cells. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Xiao S, Zhu S, Ma B, Xia ZF, Yang J, Wang G
A New System for Cultivation of Human Keratinocytes on Acellular Dermal Matrix Substitute with the Use of Human Fibroblast Feeder Layer.
Cells Tissues Organs. 2007 Oct 16;
To improve the proliferative potential of human keratinocytes (HK) cultured on acellular dermal matrix (ADM), HK and mitomycin C-treated human fibroblasts (MMC-HF) were seeded onto ADM to form four types of composite skin: type I, HK were seeded onto the epidermal side of ADM; type II, both HK and MMC-HF were seeded onto the epidermal side; type III, MMC-HF were preseeded onto the dermal side of ADM, and then HK were seeded onto the epidermal side, and type IV, where MMC-HF were preseeded onto both sides, and then HK were seeded onto the epidermal side. Compared with type I and III, the proliferative potential of HK of type II and IV was significantly higher on day 3, 5, 7 and 9 in vitro. In type I and III, HK grew into one layer on day 7-9, while in type II and IV keratinocytes grew into a confluent monolayer by day 4-6. The adherence to ADM of HK in types II and IV was stronger than that in type I and III. The take rate of type II and IV composite skin was also significantly higher. In conclusion, when MMC-HF and HK were cocultured on the epidermal side of ADM, MMC-HF could serve as excellent feeder cells. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Hao W, Hu YY, Wei YY, Pang L, Lv R, Bai JP, Xiong Z, Jiang M
Collagen I Gel Can Facilitate Homogenous Bone Formation of Adipose-Derived Stem Cells in PLGA-beta-TCP Scaffold.
Cells Tissues Organs. 2007 Oct 15;
Cell-based tissue engineering is thought to be a new therapy for treatment of bone defects and nonunions after trauma and tumor resection. In this study, we explore the in vitro and in vivo osteogenesis of a novel biomimetic construct fabricated by using collagen I gel to suspend rabbit adipose-derived stem cells (rASCs) into a porous poly(lactic-co-glycolic)acid-beta-tricalcium phosphate (PLGA-beta-TCP) scaffold (rASCs-COL/PLGA-beta-TCP). In vitro and in vivo studies of the rASCs-COL/PLGA-beta-TCP composite (group A) were carried out compared with the single combination of rASCs and PLGA-beta-TCP (rASCs/PLGA-beta-TCP; group B), the combination of acellular collagen I gel and PLGA-beta-TCP (COL/PLGA-beta-TCP; group C), and the PLGA-beta-TCP scaffold (group D). Composites of different groups were cultured in vitro for 2 weeks in osteogenic medium and then implanted into the autologous muscular intervals for 8 weeks. After 2 weeks of in vitro culture, alkaline phosphatase activity and extracellular matrix mineralization in group A were significantly higher than in group B (p < 0.01, n = 4). In vivo osteogenesis was evaluated by radiographic and histological analyses. The calcification level was radiographically evident in group A, whereas no apparent calcification was observed in groups B, C and D (n = 4). In group A, woven bone with a trabecular structure was formed, while in group B, only osteoid tissue was observed. Meanwhile, the bone-forming area in group A was significantly higher than in group B (p < 0.01, n = 4). No bone formation was observed in groups C or D (n = 4). In conclusion, by using collagen I gel to suspend rASCs into porous PLGA-beta-TCP scaffold, osteogenic differentiation of rASCs can be improved and homogeneous bone tissue can be successfully formed in vivo. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Muraoka T, Omuro K, Wakahara T, Muramatsu T, Kanehisa H, Fukunaga T, Kanosue K
Effects of Muscle Cooling on the Stiffness of the Human Gastrocnemius Muscle in vivo.
Cells Tissues Organs. 2007 Oct 15;
Background/Aims: The effects of muscle cooling on the stiffness of the human gastrocnemius muscle (GAS) were examined in vivo. Methods: The knee joint was passively extended from 90 to 0 degrees (0 degrees = full knee extended position) with a constant ankle angle of 10 degrees dorsiflexed position (0 degrees = the sole of the foot is approximately perpendicular to the anterior margin of the shaft of the tibia) in a control condition (room temperature of 18-23 degrees C) and a cooling condition (muscle temperature decreased by 5.8 +/- 1.7 degrees C after cooling using a cold water bath at a temperature of 5-8 degrees C for 60 min). The change in passive Achilles tendon force, muscle fascicle length of GAS and muscle temperature were measured (n = 6) during the motion. Results and Conclusion: GAS stiffness was significantly greater in the cooling condition (20 +/- 8 N/mm) than the control condition (18 +/- 8 N/mm). There was no cooling effect on the muscle slack length, beyond which passive muscle force arises. The maximum passive Achilles tendon force significantly increased by 19 +/- 20% after cooling. These results suggested that cooling increased the passive muscle force due to the increase in the muscle stiffness rather than the shift of the muscle slack length. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Chinnathambi S, Tomanek-Chalkley A, Bickenbach JR
HSP70 and EndoG Modulate Cell Death by Heat in Human Skin Keratinocytes in vitro.
Cells Tissues Organs. 2007 Oct 15;
We examined how young and old keratinocytes died from heat stress in vitro. We found that keratinocyte cell death was not due to oxidative stress as neither Mn-SOD nor Cu-Zn-SOD was produced in either young or old heated keratinocytes. Instead, analysis of the anti-apoptotic factors, Bcl2 and HSP70, and the pro-apoptotic factors, caspase 3, caspase 8, Apaf-1, cytochrome c, AIF, and EndoG, indicated that keratinocyte cell death occurred via the caspase-independent EndoG apoptotic pathway. We found that both young and old keratinocytes died via the same pathway, and that we could specifically reduce both young and old keratinocyte death by addition of the EndoG inhibitor NEM. Further analysis suggested that the difference between young and old keratinocyte death was due to the synthesis of HSP70 protein, with the increase in response to heat more pronounced in young keratinocytes than in old keratinocytes. When we inhibited HSP70 by adding quercetin, death was increased in both young and old keratinocytes, but more so in old keratinocytes. These data suggest that old keratinocytes may die more readily than young keratinocytes when heated because they synthesize HSP70 at a lower efficiency. Such findings suggest that HSP70 production may be age-dependent. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Blevins TL, Peterson SB, Lee EL, Bailey AM, Frederick JD, Huynh TN, Gupta V, Grande-Allen KJ
Mitral Valvular Interstitial Cells Demonstrate Regional, Adhesional, and Synthetic Heterogeneity.
Cells Tissues Organs. 2007 Sep 12;
Background/Aims: Because various regions of the mitral valve contain distinctive extracellular matrix enabling the tissues to withstand diverse mechanical environments, we investigated phenotype and matrix production of porcine valvular interstitial cells (VICs) from different regions. Methods: VICswere isolated from the chordae (MCh), the center of the anterior leaflet (AlCtr), and the posterior leaflet free edge (PlFree), then assayed for metabolic, growth, and adhesion rates; collagen and glycosaminoglycan (GAG) production, and phenotype using biochemical assays, flow cytometry, and immunocytochemistry. Results: The AlCtr VICs exhibited the fastest metabolism but slowest growth. PlFree cells grew the fastest, but demonstrated the least smooth muscle alpha-actin, vimentin, and internal complexity. AlCtr VICs secreted less collagen into the culture medium but more 4-sulfated GAGs than other cells. Adhesion-based separation resulted in altered secretion of sulfated GAGs by MCh and AlCtr cells but not by the PlFree cells. Conclusions: VICs isolated from various regions of the mitral valve demonstrate phenotypic differences in culture, corresponding to the ability of the mitral valve to accommodate the physical stresses or altered hemodynamics that occur with injury or disease. Further understanding of VIC and valve mechanobiology could lead to novel medical or tissue engineering approaches to treat valve diseases. Copyright (c) 2007 S. Karger AG, Basel. [Abstract]

Soulintzi N, Zagris N
Spatial and temporal expression of perlecan in the early chick embryo.
Cells Tissues Organs. 2007;186(4):243-56.
Perlecan is a major heparan sulfate proteoglycan that binds growth factors and interacts with various extracellular matrix proteins and cell surface molecules. The expression and spatiotemporal distribution of perlecan was studied by RT-PCR, immunoprecipitation and immunofluorescence in the chick embryo from stages X (morula) to HH17 (29 somites). Combined RT-PCR and immunohistochemistry demonstrated the expression of perlecan as early as stage X and its presence may be fundamental to the first basement membrane assembly on the epiblast ventral surface at stage XIII (blastula). Perlecan fluorescence was intense in the cells ingressing through the primitive streak and was strong lining the epiblast ventral surface lateral to the streak at stage HH3-4 (gastrula). At stage HH5-6 (neurula), perlecan fluorescence was low in the neuroepithelium and stronger in the apical surface of the neural plate. At stage HH10-11 (12 somites), perlecan fluorescence was intense in the neuroepithelium and was then essentially nondetectable in the neuroepithelium, and the intensity had shifted to the basement membranes of encephalic vesicles by stage HH17. Perlecan immunofluorescence was intense in neural crest cells, strong in pharyngeal arches, intense in thymus and lung rudiments, intense in aortic arches and in dorsal aorta, strong in lens and retina and intense in intraretinal space and in optic stalk, strong in the dorsal mesocardium, myocardium and endocardium, strong in dermomyotome, low in sclerotome in somites, intense in mesonephric duct and tubule rudiments, intense in the lining of the gut luminal surface. Inhibition of the function of perlecan by blocking antibodies showed that perlecan is crucial for maintaining basement membrane integrity which mediates the epithelialization, adhesive separation and maintenance of neuroepithelium in brain, somite epithelialization, and tissue architecture during morphogenesis of the heart tube, dorsal aorta and gut. An intriguing possibility is that perlecan, as a signaling molecule that modulates the activity of growth factors and cytokines, participates in the signaling pathways that guide gastrulation movements and neural crest cell migration, proliferation and survival, cardiac cell proliferation and paraxial mesoderm (somitic) cell proliferation and segmentation. [Abstract]

Zeiler M, Leiser R, Johnson GA, Tinneberg HR, Pfarrer C
Development of an in vitro model for bovine placentation: a comparison of the in vivo and in vitro expression of integrins and components of extracellular matrix in bovine placental cells.
Cells Tissues Organs. 2007;186(4):229-42.
BACKGROUND/AIMS: Interaction of trophoblastic integrins with the extracellular matrix plays a role in embryo implantation and trophoblast invasion. The phenomenon of restricted trophoblast invasion, observed in the bovine epitheliochorial placenta offers intriguing conditions to study invasive processes. The migration of bovine trophoblast giant cells is accompanied by the expression of specific integrins and corresponding extracellular matrix ligands. METHODS: Primary cultures of different cell populations from cow placentomes were established and characterized, and in vitro phenotypes were compared with in vivo conditions by immunofluorescence. RESULTS: Propagated epithelial cells were positive for cytokeratin and vimentin, while fibroblasts contained alpha-smooth muscle actin, desmin and vimentin. Epithelial cells coexpressed integrin subunits alpha(6) and beta(1) with laminin, and fibroblast cells were positive for alpha(v), beta(3), fibronectin and laminin. In contrast to cells in vivo, cultured epithelial cells secreted fibronectin, while collagen IV was not detected. The occurrence of integrin subunits was confirmed at mRNA level by RT-PCR. CONCLUSION: We have established cell cultures isolated from maternal and fetal components of bovine placentomes expressing typical cytoskeletal filaments and integrin receptors also present in their in vivo counterparts. These bovine placentomal cells provide a suitable in vitro model for the study of cell-cell interactions. [Abstract]

Li F, Liu Y, Chen D, Lin X, Li J, Wang J, Peng Y, Wang S, Wang Y
Leukemia inhibitory factor-expressing human embryonic lung fibroblasts as feeder cells for human embryonic germ cells.
Cells Tissues Organs. 2007;186(4):221-8.
A robust culture system is critical for maintaining both proliferation and the developmental potential of human embryonic germ (hEG) cells. Here, we use human embryonic lung fibroblasts (hELF) overexpressing leukemia inhibitory factor (LIF) as feeder cells to support the self-renewal of hEG cells. We examine the morphology, gene expression, and developmental potential of hEG cells grown on a feeder layer of LIF-expressing hELF (hELF/lif) cells. hEG cells were positive for alkaline phosphatase (AP), stage-specific embryonic antigen (SSEA)-1, SSEA-4, tumor rejection antigen (TRA)-1-60, and TRA-1-81. In addition, hEG cells maintained on hELF/lif expressed higher levels of pluripotency genes such as Oct4 and Nanog. In addition, hEG cells maintained on hELF/lif cells gave rise to differentiated tissues when grown as embryoid bodies, consistent with the broad developmental potential of the starting population. Our results suggest that a hELF/lif feeder layer can support the proliferation of hEG cells, and that LIF signaling plays an essential role in this process. This human-derived culture system provides an attractive alternative to more commonly used mouse-derived feeder layers for use in clinical applications. [Abstract]

Wanschitz F, Stein E, Sutter W, Kneidinger D, Smolik K, Watzinger F, Turhani D
Expression patterns of Ets2 protein correlate with bone-specific proteins in cell-seeded three-dimensional bone constructs.
Cells Tissues Organs. 2007;186(4):213-20.
The transcription factor Ets2 and its transcriptional targets osteopontin (OPN) and osteocalcin (OC) are expressed in tissue-engineered bone constructs in vitro. Up to now little is known about the role of Ets2 in tissue-engineering applications. This study was intended to investigate the hypothesis that protein expression of Ets2 is correlated with the expression of bone-specific proteinsin tissue-engineeredbone constructs. Cell-seeded three-dimensional bone constructs manufactured with osteoblastic cells and poly(lactic-co-glycolic acid) polymer fleeces over a period of 21 days were analyzed by SDS-PAGE and Western blotting. The protein expression of OPN, OC, osteonectin and collagen type I was analyzed. Cellularity, alkaline phosphatase-specific activity and histology confirmed the osteoblastic phenotype of the constructs. Correlations between Ets2 expression and OPN and Ets2 and collagen type I expression could be detected during the phase of late osteoblastic differentiation between days 9 and 21. The correlation between OC and collagen type I was significant in this late stage of osteoblastic differentiation. These results suggest that there is a strong interplay of Ets2 with bone-specific proteins in cell-seeded three-dimensional bone constructs. This study is a crucial step to elucidate the complex interplay of bone-related proteins in the application of bone tissue engineering. [Abstract]

Sorrell JM, Baber MA, Caplan AI
A self-assembled fibroblast-endothelial cell co-culture system that supports in vitro vasculogenesis by both human umbilical vein endothelial cells and human dermal microvascular endothelial cells.
Cells Tissues Organs. 2007;186(3):157-68.
The construction of vascularized connective tissues is an important goal in tissue engineering in that the presence of a patent bio-engineered vasculature should facilitate vascularization of an implant. Fibroblasts play an essential role in the angiogenic process through their production of extracellular matrix molecules and through their release of essential growth factors. Therefore, the aim of this study is to develop a thin 3-dimensional model in which fibroblasts support endothelial cells in the formation of tube-like structures. Macro- and microvascular endothelial cells were seeded onto confluent lawns of human fibroblasts and were cultured in the presence of high levels of ascorbate 2-phosphate to create a tissue-like structure in which endothelial cell organized into tube-like structures. The process was visualized in the culture dish through labeling of cells with a long-lasting fluorescent vital dye. Intact sheet-like structures were created in which endothelial cell tube-like structures were encased by fibroblasts and were surrounded by a basement membrane. These structures appeared to contain a lumen and remained stable for up to 5 weeks in culture. This culture system provides an in vitro method to study fibroblast-endothelial cell interactions and to study the effects of pro- and anti-angiogenic factors on endothelial cell differentiation. This system also provides an experimental basis for developing vascularized tissue-engineered connective tissue. [Abstract]

Laurson J, Selden C, Clements M, Mavri-Damelin D, Coward S, Lowdell M, Hodgson HJ
Putative human liver progenitor cells in explanted liver.
Cells Tissues Organs. 2007;186(3):180-91.
BACKGROUND/AIMS: Hepatocyte progenitors have frequently been cultured from rodents but reports from human liver are rare. METHODS: Non-parenchymal cell fraction isolated from 19 explant livers (removed at orthotopic liver transplantation for acute or chronic liver disease) and histologically normal human liver was cultured. RESULTS: Proliferating epithelioid colonies were identifiable after 2-3 weeks culture as a very rare event (<1 per million cells plated) expressing mRNAs and protein antigens of mixed hepatocytic/biliary phenotype. Colony survival could be prolonged by transduction of the catalytic sub-unit of telomerase. Hepatocyte growth factor, epidermal growth factor and oncostatin M did not further enhance hepatocytic differentiation. The expression of markers associated with hepatocyte precursor status was investigated by flow cytometry. Cells expressing the stem cell-associated markers CD133 and CD117 were identified at low frequency. The proportion of cells expressing the integrin CD49f was higher in diseased liver than in normal liver, but the proportion expressing the hepatocyte growth factor receptor c-met was lower. Successful enrichment of plated populations for progenitors was not achieved. CONCLUSION: Although there is clear histological evidence of hepatocyte precursors in human explant livers, predictable culture of such cells with differentiation toward mature hepatocyte phenotype remains elusive. [Abstract]


Proceedings of the 2nd International EMT (epithelial-mesenchymal transitions) Meeting, October 1-3, 2005, Vancouver, Canada.
Cells Tissues Organs. 2007;185(1-3):5-241. [Abstract]

Nakamura Y, Yi SQ, Terayama H, Naito M, Li J, Moriyama H, Tsuchida A, Itoh M
Sequential histopathology of pancreatic tissues in aly/aly mice.
Cells Tissues Organs. 2007;186(3):204-9.
C57BL/6J strain mice carrying the homozygous autosomal recessive mutation alymphoplasia (aly) lack peripheral lymph nodes and Peyer's patches and exhibit chronic infiltration of lymphocytes into various organs. Pancreatitis, one of the inflammatory lesions, is considered to be of autoimmune origin; however, the target autoantigens have not yet been determined. In this study, pancreatic tissues of male aly/aly mice and wild-type mice at 1-65 weeks of age were light- and electron-microscopically examined to investigate when and how pancreatitis develops. The results showed that macrophages had first appeared and remained in the lymphatic lumen at 3 weeks of age and then a lot of eosinophilic granulocytes infiltrated into the interlobular connective tissues at 5 weeks of age. After the subsidence of eosinophilic inflammation, macrophages and B220+ cells appeared at the perivascular tissues at 9 weeks of age. Thereafter, both CD4+ and CD8+ cells finally participated in the interstitial inflammation from 11 weeks of age. It was noted that these leukocytes had infiltrated into the perivascular interstitium rather than the parenchymal tissues during the course of pancreatitis, although a large parenchymal area was finally degenerated and replaced by adipose tissue. [Abstract]

Li D, Wang GY, Dong BH, Zhang YC, Wang YX, Sun BC
Biological characteristics of human placental mesenchymal stem cells and their proliferative response to various cytokines.
Cells Tissues Organs. 2007;186(3):169-79.
The placenta is an attractive new source of mesenchymal stem cells (MSCs), but the biological characteristics of placenta-derived MSCs (P-MSCs) have not yet been characterized. We successfully isolated, cultured and expanded P-MSCs using routine methods. Under appropriate induction conditions, these cells can differentiate into bone, cartilage, fat and hepatocyte-like cells. In addition, the proliferative response of P-MSCs to different cytokines was monitored using the MTT assay. The results show that low concentrations of proinflammatory cytokines, e.g. RANTES, interleukin (IL)-1, IL-6 and IL-8 can stimulate the proliferation of P-MSCs in a dose-dependent manner, peaking at concentrations of 40 ng/ml of RANTES, 10 ng/ml of IL-1 and IL-6, and 150 ng/ml of IL-8 (p < 0.01). The level of proliferation decreased when the concentration of these four cytokines increased beyond these values. On the other hand, anti-inflammatory cytokines hepatocyte growth factor and IL-4 had an inhibitory effect on P-MSCs. In conclusion, the placenta contains MSCs that are consistent with the characteristics of bone marrow MSCs. Low concentrations of proinflammatory chemokines stimulated the proliferation of P-MSCs while anti-inflammatory cytokines inhibited the growth of P-MSCs in a dose-dependent manner. [Abstract]

Wang Y, Zhang M, Middleton FA, Horton JA, Pritchard M, Spadaro JA, Farnum CE, Damron TA
Connective tissue growth factor and insulin-like growth factor 2 show upregulation in early growth plate radiorecovery response following irradiation.
Cells Tissues Organs. 2007;186(3):192-203.
INTRODUCTION: The growth plate response following radiotherapy is poorly understood. In particular, little is known about the changes in growth plate growth factors and cytokines following irradiation. The hypothesis was that a limited number of growth factors and cytokines play a role in growth plate proliferative and hypertrophic chondrocyte radio-recovery. METHODS: The right limbs of 6 rats were irradiated (17.5 Gy), leaving the left limbs as controls. Limbs were harvested 1 (n = 3) and 2 (n = 3) weeks later. Microarrays were constructed from chondrocytes obtained by laser microdissection from the proliferative zone (PZ) and the hypertrophic zone (HZ) of normal and irradiated tibia growth plates. Real-time PCR was used to confirm the expression of parathyroid hormone receptor 1 (Pthr1), connective tissue growth factor (CTGF), insulin-like growth factor I receptor (IGF1R), insulin-like growth factor II (IGF2), interleukin 17beta (IL17b) and chemokine ligand 12 (CXCL12). RESULTS AND CONCLUSIONS: IGF2 is upregulated in the PZ and CTGF is upregulated in both the PZ and HZ 1 week after irradiation, prior to the histomorphometric appearance of growth plate recovery in this immature animal radiation model, supporting their role in stimulating early return of the growth plate. By 2 weeks after irradiation, a number of growth factors and cytokines, including CTGF and Pthr1 in both zones, CXCL12 and its receptor in the PZ, and IL17b and bone morphogenetic protein 2 in the HZ, show upregulation, suggesting a possible later role in radiorecovery. The effects of irradiation on Pthr1, CTGF, IGF2 and CXCL12 in PZ and Pthr1, CTGF, IL17b and IGF1R in the HZ determined by microarray and real-time RT-PCR was highly correlated (r = 0.797, p < 0.05 in the PZ and r = 0.875, p < 0.01 in the HZ, respectively). [Abstract]

Wright T
The molecular control of and clinical variations in root formation.
Cells Tissues Organs. 2007;186(1):86-93.
Roots of teeth perform critical functions to anchor the teeth in the jaws and transmit the masticatory forces in such a way as to minimize fracture and wear of the dentition. Tooth root development involves a variety of cell types, epithelial-mesenchymal interactions, the enumeration of specialized extracellular matrices, processing of these matrices and strict control over the microenvironment to allow the cementum and dentin to mineralize. While many of the specific molecular mechanisms involved in root formation remain poorly understood, our knowledge of these events and pathways has advanced markedly over the past decade. The molecular bases of many hereditary conditions having associated dental root anomalies are now known. Therapeutic approaches based on the molecular biology of root formation have and will continue to emerge and be translated into improved clinical care. The purpose of this study was to review our knowledge regarding developmental defects of root formation, the molecular mechanisms involved, and the impact of root variants on clinical dentistry. [Abstract]

Hu JC, Chun YH, Al Hazzazzi T, Simmer JP
Enamel formation and amelogenesis imperfecta.
Cells Tissues Organs. 2007;186(1):78-85.
Dental enamel is the epithelial-derived hard tissue covering the crowns of teeth. It is the most highly mineralized and hardest tissue in the body. Dental enamel is acellular and has no physiological means of repair outside of the protective and remineralization potential provided by saliva. Enamel is comprised of highly organized hydroxyapatite crystals that form in a defined extracellular space, the contents of which are supplied and regulated by ameloblasts. The entire process is under genetic instruction. The genetic control of amelogenesis is poorly understood, but requires the activities of multiple components that are uniquely important for dental enamel formation. Amelogenesis imperfecta (AI) is a collective designation for the variety of inherited conditions displaying isolated enamel malformations, but the designation is also used to indicate the presence of an enamel phenotype in syndromes. Recently, genetic studies have demonstrated the importance of genes encoding enamel matrix proteins in the etiology of isolated AI. Here we review the essential elements of dental enamel formation and the results of genetic analyses that have identified disease-causing mutations in genes encoding enamel matrix proteins. In addition, we provide a fresh perspective on the roles matrix proteins play in catalyzing the biomineralization of dental enamel. [Abstract]

Hart PS, Hart TC
Disorders of human dentin.
Cells Tissues Organs. 2007;186(1):70-7.
Dentin, the most abundant tissue in teeth, is produced by odontoblasts, which differentiate from mesenchymal cells of the dental papilla. Dentinogenesis is a highly controlled process that results in the conversion of unmineralized predentin to mineralized dentin. By weight, 70% of the dentin matrix is mineralized, while the organic phase accounts for 20% and water constitutes the remaining 10%. Type I collagen is the primary component (>85%) of the organic portion of dentin. The non-collagenous part of the organic matrix is composed of various proteins, with dentin phosphoprotein predominating, accounting for about 50% of the non-collagenous part. Dentin defects are broadly classified into two major types: dentinogenesis imperfectas (DIs, types I-III) and dentin dysplasias (DDs, types I and II). To date, mutations in DSPP have been found to underlie the dentin disorders DI types II and III and DD type II. With the elucidation of the underlying genetic mechanisms has come the realization that the clinical characteristics associated with DSPP mutations appear to represent a continuum of phenotypes. Thus, these disorders should likely be called DSPP-associated dentin defects, with DD type II representing the mild end of the phenotypic spectrum and DI type III representing the severe end. [Abstract]

D'Souza RN, Klein OD
Unraveling the molecular mechanisms that lead to supernumerary teeth in mice and men: current concepts and novel approaches.
Cells Tissues Organs. 2007;186(1):60-9.
Supernumerary teeth are defined as those that are present in excess of the normal complement of human dentition and represent a unique developmental anomaly of patterning and morphogenesis. Despite the wealth of information generated from studies on normal tooth development, the genetic etiology and molecular mechanisms that lead to congenital deviations in tooth number are poorly understood. For developmental biologists, the phenomenon of supernumerary teeth raises interesting questions about the development and fate of the dental lamina. For cell and molecular biologists, the anomaly of supernumerary teeth inspires several questions about the actions and interactions of transcription factors and growth factors that coordinate morphogenesis, cell survival and programmed cell death. For human geneticists, the condition as it presents itself in either syndromic or non-syndromic forms offers an opportunity to discover mutations in known or novel genes. For clinicians faced with treating the dental complications that arise from the presence of supernumerary teeth, knowledge about the basic mechanisms involved is essential. The purpose of this manuscript is to review current knowledge about how supernumerary teeth form, the molecular insights gained through studies on mice that are deficient in certain tooth signaling molecules and the questions that require further research in the field. [Abstract]

Iwase M, Kaneko S, Kim H, Satta Y, Takahata N
Evolutionary history of sex-linked mammalian amelogenin genes.
Cells Tissues Organs. 2007;186(1):49-59.
Amelogenin (AMEL) arose prior to the emergence of tetrapods and transposed into an intron of the Rho GTPase-activating protein 6 gene. In the mammalian lineage leading to eutherians, a pair of homologous autosomes with this nested gene structure fused with the then already differentiating sex chromosomes by suppressing homologous recombination. As sex-chromosomal differentiation extended to the fused region, a pair of homologous AMEL genes too differentiated from each other in two steps; first in the 5' region (the promoter region to transposon MER5 in intron 2) and second in the remaining 3' region. This resulted in gametologous AMELX and AMELY in the eutherian sex chromosomes. Although the early differentiation of the 5' region between AMELX and AMELY is consistent with the lowered expression level of AMELY, there is no indication for deterioration of AMELY at the amino acid level. Rather, both AMELX and AMELY in particular lineages might undergo positive selection, followed by negative selection to preserve established function. Based on patterns and levels of AMELX and AMELY polymorphisms in the human population, it is also argued that a recombination cold spot near AMELX might be related to the cause of the ancient pseudoautosomal boundary. [Abstract]

Sire JY, Davit-Béal T, Delgado S, Gu X
The origin and evolution of enamel mineralization genes.
Cells Tissues Organs. 2007;186(1):25-48.
BACKGROUND/AIMS: Enamel and enameloid were identified in early jawless vertebrates, about 500 million years ago (MYA). This suggests that enamel matrix proteins (EMPs) have at least the same age. We review the current data on the origin, evolution and relationships of enamel mineralization genes. METHODS AND RESULTS: Three EMPs are secreted by ameloblasts during enamel formation: amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). Recently, two new genes, amelotin (AMTN) and odontogenic ameloblast associated (ODAM), were found to be expressed by ameloblasts during maturation, increasing the group of ameloblast-secreted proteins to five members. The evolutionary analysis of these five genes indicates that they are related: AMEL is derived from AMBN, AMTN and ODAM are sister genes, and all are derived from ENAM. Using molecular dating, we showed that AMBN/AMEL duplication occurred >600 MYA. The large sequence dataset available for mammals and reptiles was used to study AMEL evolution. In the N- and C-terminal regions, numerous residues were unchanged during >200 million years, suggesting that they are important for the proper function of the protein. CONCLUSION: The evolutionary analysis of AMEL led to propose a dataset that will be useful to validate AMEL mutations leading to X- linked AI. [Abstract]

Kawasaki K, Buchanan AV, Weiss KM
Gene duplication and the evolution of vertebrate skeletal mineralization.
Cells Tissues Organs. 2007;186(1):7-24.
The mineralized skeleton is a critical innovation that evolved early in vertebrate history. The tissues found in dermal skeletons of ancient vertebrates are similar to the dental tissues of modern vertebrates; both consist of a highly mineralized surface hard tissue, enamel or enameloid, more resilient body dentin, and basal bone. Many proteins regulating mineralization of these tissues are evolutionarily related and form the secretory calcium-binding phosphoprotein (SCPP) family. We hypothesize here the duplication histories of SCPP genes and their common ancestors, SPARC and SPARCL1. At around the same time that Paleozoic jawless vertebrates first evolved mineralized skeleton, SPARCL1 arose from SPARC by whole genome duplication. Then both before and after the split of ray-finned fish and lobe-finned fish, tandem gene duplication created two types of SCPP genes, each residing on the opposite side of SPARCL1. One type was subsequently used in surface tissue and the other in body tissue. In tetrapods, these two types of SCPP genes were separated by intrachromosomal rearrangement. While new SCPP genes arose by duplication, some old genes were eliminated from the genome. As a consequence, phenogenetic drift occurred: while mineralized skeleton is maintained by natural selection, the underlying genetic basis has changed. [Abstract]

Simmer JP
Evolution and genetics of teeth.
Cells Tissues Organs. 2007;186(1):4-6. [Abstract]

Takezawa T, Nitani A, Shimo-Oka T, Takayama Y
A protein-permeable scaffold of a collagen vitrigel membrane useful for reconstructing crosstalk models between two different cell types.
Cells Tissues Organs. 2007;185(1-3):237-41.
Soft and turbid collagen gel disks were previously converted into strong and transparent gel membranes utilizing a concept for the vitrification of heat-denatured of proteins. This novel stable and transparent gel has been termed 'vitrigel'. By encompassing the collagen vitrigel membrane in a nylon frame, it can be easily handled with tweezers, and functions as an excellent scaffold for three-dimensional cell culture models, as cells can be cultured on both sides. Here, we investigated the molecular permeability of the collagen vitrigel membrane in a time course-dependent manner using glucose and serum proteins. Glucose penetrated through the collagen vitrigel membrane to the opposite side, and concentrations on each side were found to be equilibrated within 24 h. Serum proteins up to a molecular weight >100 kDa also gradually passed through the collagen vitrigel membrane. In addition, human microvascular endothelial cells (HMVECs) were cultured on one surface of the collagen vitrigel membrane with a nylon frame, and human dermal fibroblasts (HDFs) or HT-29 (a human colon carcinoma cell line) cells were cocultured on the opposite surface. Histomorphological observations revealed the formation of three-dimensional crosstalk models composed of HMVECs and HDFs or HMVECs and HT-29 cells. Resulting data suggest that the protein-permeable scaffold composed of the collagen vitrigel membrane is useful for the reconstruction and/or modeling of 'crosstalk' between two different cells types. Hereafter, such crosstalk models in vitro could be applied to research not only of paracrine factors, but also to epithelial- or endothelial-mesenchymal transitions. [Abstract]

Bessette DC, Wong PC, Pallen CJ
PRL-3: a metastasis-associated phosphatase in search of a function.
Cells Tissues Organs. 2007;185(1-3):232-6.
The molecular and cellular events involved in cancer progression and metastasis remain much less well-defined than those involved in oncogenesis, despite the fact that cell metastasis is the major factor in cancer mortality. Thus, the discovery that the expression of a protein tyrosine phosphatase, protein of regenerating liver-3 (PRL-3), is upregulated in colon cancer metastases provided an exciting indication that the altered regulation of specific protein tyrosine phosphorylation events and signaling pathways might characterize these metastatic cells and/or be key in promoting the tumor-to-metastasis transition in this, and perhaps other, cancers of epithelial origin. However, the cellular substrate(s) of PRL-3 has not been identified, and little is known of PRL-3-mediated cellular signaling pathways. This review illustrates the significance of PRL-3 in promoting metastasis and the importance of determining the endogenous role of PRL-3. [Abstract]

Burns WC, Kantharidis P, Thomas MC
The role of tubular epithelial-mesenchymal transition in progressive kidney disease.
Cells Tissues Organs. 2007;185(1-3):222-31.
The accumulation of interstitial matrix represents the final common pathway of most forms of kidney disease. Much of this matrix is synthesized by interstitial myofibroblasts, recruited from resident fibroblasts and circulating precursors. In addition, a significant proportion is derived from epithelial-mesenchymal transition (EMT) of tubuloepithelial cells. The importance of EMT has been demonstrated in experimental models, where blockade of EMT attenuates renal fibrosis. Although a number of factors may initiate EMT in the kidney, the most potent is transforming growth factor-beta1 (TGF-beta1). Moreover, many other prosclerotic factors have effects on EMT indirectly, via induction of TGF-beta1. Signaling events in this pathway include activation of Smad/integrin-linked kinase (ILK) and connective tissue growth factor (CTGF). Basement membrane integrity is also a key regulator of EMT. In particular, overexpression of matrix metalloproteinase-2 has a key role in the initiation of EMT, membrane dissolution, and the interstitial transit of transformed mesenchymal cells. Endogenous inhibitors of EMT also play an important counterregulatory role both to prevent EMT and stimulate uncommitted cells to regain their tubular phenotype (mesenchymal-epithelial transition). Such inhibitors represent a potential therapeutic approach, offering a mechanism to slow or even redress established renal fibrosis. [Abstract]