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Recent Articles in The Journal of Experimental Biology

Hamilton JL, Edwards CR, Holt SR, Worden MK
Temperature dependent modulation of lobster neuromuscular properties by serotonin.
J Exp Biol. 2007 Mar;210(Pt 6):1025-35.
In cold-blooded species the efficacy of neuromuscular function depends both on the thermal environmental of the animal's habitat and on the concentrations of modulatory hormones circulating within the animal's body. The goal of this study is to examine how temperature variation within an ecologically relevant range affects neuromuscular function and its modulation by the neurohormone serotonin (5-HT) in Homarus americanus, a lobster species that inhabits a broad thermal range in the wild. The synaptic strength of the excitatory and inhibitory motoneurons innervating the lobster dactyl opener muscle depends on temperature, with the strongest neurally evoked muscle movements being elicited at cold (<5 degrees C) temperatures. However, whereas neurally evoked contractions can be elicited over the entire temperature range from 2 to >20 degrees C, neurally evoked relaxations of resting muscle tension are effective only at colder temperatures at which the inhibitory junction potentials are hyperpolarizing in polarity. 5-HT has two effects on inhibitory synaptic signals: it potentiates their amplitude and also shifts the temperature at which they reverse polarity by approximately +7 degrees C. Thus 5-HT both potentiates neurally evoked relaxations of the muscle and increases the temperature range over which neurally evoked muscle relaxations can be elicited. Neurally evoked contractions are maximally potentiated by 5-HT at warm (18 degrees C) temperatures; however, 5-HT enhances excitatory junction potentials in a temperature-independent manner. Finally, 5-HT strongly increases resting muscle tension at the coldest extent of the temperature range tested (2 degrees C) but is ineffective at 22 degrees C. These data demonstrate that 5-HT elicits several temperature-dependent physiological changes in the passive and active responses of muscle to neural input. The overall effect of 5-HT is to increase the temperature range over which neurally evoked motor movements can be elicited in this neuromuscular system. [Abstract/Link to Full Text]

Hiroi J, McCormick SD
Variation in salinity tolerance, gill Na+/K+-ATPase, Na+/K+/2Cl- cotransporter and mitochondria-rich cell distribution in three salmonids Salvelinus namaycush, Salvelinus fontinalis and Salmo salar.
J Exp Biol. 2007 Mar;210(Pt 6):1015-24.
We compared seawater tolerance, gill Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter (NKCC) abundance, and mitochondria-rich cell (MRC) morphology of three salmonids, lake trout Salvelinus namaycush, brook trout Salvelinus fontinalis and Atlantic salmon Salmo salar. They were transferred directly from 0 p.p.t. (parts per thousand; freshwater) to 30 p.p.t. seawater, or transferred gradually from 0 to 10, 20 and 30 p.p.t. at 1-week intervals and kept in 30 p.p.t. for 3 weeks. The survival rates of lake trout, brook trout and Atlantic salmon were 80%, 50% and 100% following direct transfer, and 80%, 100% and 100% during gradual transfer, respectively. Plasma Na(+), K(+) and Cl(-) concentrations in surviving lake trout increased rapidly and remained at high levels in 30 p.p.t. of both direct and gradual transfer, whereas those in brook trout showed a transient increase following direct transfer but did not change significantly during gradual transfer. Only minor changes in plasma ions were observed in Atlantic salmon smolts in both direct and gradual transfer. These results suggest that lake trout retains some degree of euryhalinity and that brook trout possesses intermediate euryhalinity between lake trout and Atlantic salmon smolts. Gill Na(+)/K(+)-ATPase activity of lake trout and brook trout increased in seawater, whereas that of Atlantic salmon smolts was already upregulated in freshwater and remained high after seawater exposure. NKCC abundance was upregulated in parallel with gill Na(+)/K(+)-ATPase activity in each species. Immunocytochemistry with anti-Na(+)/K(+)-ATPase alpha-subunit and anti-NKCC revealed that the two ion transporters were colocalized on the basolateral membrane of gill MRCs. Immunopositive MRCs were distributed on both primary filaments and secondary lamellae in all three species kept in freshwater; following transfer to seawater this pattern did not change in lake trout and brook trout but lamellar MRCs disappeared in Atlantic salmon. Previous studies on several teleost species have suggested that filament and lamellar MRCs would be involved in seawater and freshwater acclimation, respectively. However, our results in lake trout and brook trout suggest that lamellar MRCs could be also functional during seawater acclimation. [Abstract/Link to Full Text]

Behrens JW, Stahl HJ, Steffensen JF, Glud RN
Oxygen dynamics around buried lesser sandeels Ammodytes tobianus (Linnaeus 1785): mode of ventilation and oxygen requirements.
J Exp Biol. 2007 Mar;210(Pt 6):1006-14.
The oxygen environment around buried sandeels (Ammodytes tobianus) was monitored by planar optodes. The oxygen penetration depth at the sediment interface was only a few mm. Thus fish, typically buried at 1-4 cm depth, were generally in anoxic sediment. However, they induced an advective transport through the permeable interstice and formed an inverted cone of porewater with 93% air saturation in front of the mouth. From dye experiments the mean ventilatory flow rate was estimated at 0.26+/-0.02 ml min(-1) (86.9+/-7.3 ml min(-1) kg(-1)) (N=3). Expelled water from the gills induced a 1 cm circular plume with <15% air saturation around the gills. During this quasi-steady ventilation mode, fish extracted 86.2+/-4.8% (N=7) of the oxygen from the inspired water. However, 13% of the investigated fish (2 of 15) occasionally wriggled their bodies and thereby transported almost fully air-saturated water down along the body, referred to as ;plume ventilation'. Yet, within approximately 30 min the oxic plume was replenished by oxygen-depleted water from the gills. The potential for cutaneous respiration by the buried fish was thus of no quantitative importance. Calculations derived by three independent methods (each with N=3) revealed that the oxygen uptake of sandeel buried for 6-7 h was 40-50% of previous estimates on resting respirometry of non-buried fish, indicating lower O(2) requirements during burial on a diurnal timescale. Buried fish exposed to decreasing oxygen tensions gradually approached the sediment surface, but remained in the sediment until the inspired water reached 5-10% air saturation. [Abstract/Link to Full Text]

Brown SG, Boettner GH, Yack JE
Clicking caterpillars: acoustic aposematism in Antheraea polyphemus and other Bombycoidea.
J Exp Biol. 2007 Mar;210(Pt 6):993-1005.
Acoustic signals produced by caterpillars have been documented for over 100 years, but in the majority of cases their significance is unknown. This study is the first to experimentally examine the phenomenon of audible sound production in larval Lepidoptera, focusing on a common silkmoth caterpillar, Antheraea polyphemus (Saturniidae). Larvae produce airborne sounds, resembling ;clicks', with their mandibles. Larvae typically signal multiple times in quick succession, producing trains that last over 1 min and include 50-55 clicks. Individual clicks within a train are on average 24.7 ms in duration, often consisting of multiple components. Clicks are audible in a quiet room, measuring 58.1-78.8 dB peSPL at 10 cm. They exhibit a broadband frequency that extends into the ultrasound spectrum, with most energy between 8 and 18 kHz. Our hypothesis that clicks function as acoustic aposematic signals, was supported by several lines of evidence. Experiments with forceps and domestic chicks correlated sound production with attack, and an increase in attack rate was positively correlated with the number of signals produced. In addition, sound production typically preceded or accompanied defensive regurgitation. Bioassays with invertebrates (ants) and vertebrates (mice) revealed that the regurgitant is deterrent to would-be predators. Comparative evidence revealed that other Bombycoidea species, including Actias luna (Saturniidae) and Manduca sexta (Sphingidae), also produce airborne sounds upon attack, and that these sounds precede regurgitation. The prevalence and adaptive significance of warning sounds in caterpillars is discussed. [Abstract/Link to Full Text]

Donini A, Gaidhu MP, Strasberg DR, O'donnell MJ
Changing salinity induces alterations in hemolymph ion concentrations and Na+ and Cl- transport kinetics of the anal papillae in the larval mosquito, Aedes aegypti.
J Exp Biol. 2007 Mar;210(Pt 6):983-92.
Mosquito larvae are found in diverse aquatic habitats ranging from freshwater to hypersaline water and must often deal with rapid changes in habitat salinity. We transferred larvae of Aedes aegypti from freshwater to 30% seawater, or vice versa, and measured the time course of changes in their hemolymph ion concentrations, using ion-selective microelectrodes. We also reported the Michaelis-Menten kinetics of Na(+) and Cl(-) transport by the anal papillae for the first time using the scanning ion-selective electrode technique (SIET). Hemolymph concentrations of Na(+), Cl(-) and H(+) increased within 6 h, when larvae were transferred from freshwater to seawater and decreased within 6 h, when transferred from seawater to freshwater. Kinetic parameters for Na(+) and Cl(-) transport by the anal papillae were altered after only 5 h following transfer between freshwater (FW) and 30% seawater (30%SW). The J(max) (maximum transport rate) for both ions decreased when larvae were transferred to 30%SW, whereas the K(t) (a measure of transporter affinity) increased for Na(+) transport but was unaltered for Cl(-) transport, suggesting that Na(+) and Cl(-) uptake are independent. Data reveal significant changes in ion transport by the anal papillae of mosquito larvae when they are faced with changes in external salinity such that Na(+) and Cl(-) uptake decrease in higher salinity. The alterations in Na(+) and Cl(-) uptake may be a consequence of changes in hemolymph ion levels when larvae encounter altered salinity. The rapid changes in ion transport described here compliment the previously observed long term alterations in the morphology and ultrastructure of the anal papillae. [Abstract/Link to Full Text]

Chang YH, Kram R
Limitations to maximum running speed on flat curves.
J Exp Biol. 2007 Mar;210(Pt 6):971-82.
Why is maximal running speed reduced on curved paths? The leading explanation proposes that an increase in lateral ground reaction force necessitates a decrease in peak vertical ground reaction force, assuming that maximum leg extension force is the limiting factor. Yet, no studies have directly measured these forces or tested this critical assumption. We measured maximum sprint velocities and ground reaction forces for five male humans sprinting along a straight track and compared them to sprints along circular tracks of 1, 2, 3, 4 and 6 m radii. Circular track sprint trials were performed either with or without a tether that applied centripetal force to the center of mass. Sprinters generated significantly smaller peak resultant ground reaction forces during normal curve sprinting compared to straight sprinting. This provides direct evidence against the idea that maximum leg extension force is always achieved and is the limiting factor. Use of the tether increased sprint speed, but not to expected values. During curve sprinting, the inside leg consistently generated smaller peak forces compared to the outside leg. Several competing biomechanical constraints placed on the stance leg during curve sprinting likely make the inside leg particularly ineffective at generating the ground reaction forces necessary to attain maximum velocities comparable to straight path sprinting. The ability of quadrupeds to redistribute function across multiple stance legs and decouple these multiple constraints may provide a distinct advantage for turning performance. [Abstract/Link to Full Text]

Andersson J, Borg-Karlson AK, Vongvanich N, Wiklund C
Male sex pheromone release and female mate choice in a butterfly.
J Exp Biol. 2007 Mar;210(Pt 6):964-70.
In butterflies female mate choice is influenced by both visual and olfactory cues, the latter of which are important at close range. Males of the green-veined butterfly, Pieris napi, are known to release citral (mixture of geranial and neral, 1:1), but its role(s) and conditions of release are not known. Here, we show that male P. napi release citral when interacting with conspecific males, conspecific females, heterospecific males and also when alone. The amount of citral released correlated strongly with male flight activity, which explained more than 70% of the variation. This suggests that males do not exercise control over turning release on or off, but rather that citral is emitted as a passive physical process during flight. Electroantennogram experiments showed that female antennal response was ten times more sensitive to citral than male response. Females expressed acceptance behavior when exposed to models made with freshly excised male wings or those treated with citral following chemical extraction, but not to ones with extracted wings only. Hence, these behavioral and electrophysiological tests provide strong evidence that citral is a signal from the male directed to the female during courtship, and that it functions as a male sex pheromone. [Abstract/Link to Full Text]

Aguila JR, Suszko J, Gibbs AG, Hoshizaki DK
The role of larval fat cells in adult Drosophila melanogaster.
J Exp Biol. 2007 Mar;210(Pt 6):956-63.
In the life history of holometabolous insects, distinct developmental stages are tightly linked to feeding and non-feeding periods. The larval stage is characterized by extensive feeding, which supports the rapid growth of the animal and allows accumulation of energy stores, primarily in the larval fat body. In Drosophila melanogaster access to these stores during pupal development is possible because the larval fat body is preserved in the pupa as individual fat cells. These larval fat cells are refractive to autophagic cell death that removes most of the larval cells during metamorphosis. The larval fat cells are thought to persist into the adult stage and thus might also have a nutritional role in the young adult. We used cell markers to demonstrate that the fat cells in the young adult are in fact dissociated larval fat body cells, and we present evidence that these cells are eventually removed in the adult by a caspase cascade that leads to cell death. By genetically manipulating the lifespan of the larval fat cells, we demonstrate that these cells are nutritionally important during the early, non-feeding stage of adulthood. We experimentally blocked cell death of larval fat cells using the GAL4/UAS system and found that in newly eclosed adults starvation resistance increased from 58 h to 72 h. Starvation survival was highly correlated with the number of remaining larval fat cells. We discuss the implications of these results in terms of the overall nutritional status of the larva as an important factor in adult survival in environmental stresses such as starvation. [Abstract/Link to Full Text]

Suprenant KA, Bloom N, Fang J, Lushington G
The major vault protein is related to the toxic anion resistance protein (TelA) family.
J Exp Biol. 2007 Mar;210(Pt 6):946-55.
Vaults are barrel-shaped ribonucleoprotein particles that are abundant in certain tumors and multidrug resistant cancer cells. Prokaryotic relatives of the major vault protein, MVP, have not been identified. We used sequence analysis and molecular modeling to show that MVP and the toxic anion resistance protein, TelA of Rhodobacter sphaeroides strain 2.4.1, share a novel fold that consists of a three-stranded antiparallel beta-sheet. Because of this strong structural correspondence, we examined whether mammalian cell vaults respond to tellurite treatment. In the presence of the oxyanion tellurite, large vault aggregates, or vaultosomes, appear at the cell periphery in 15 min or less. Vaultosome formation is temperature-dependent, reversible, and occurs in normal human umbilical vein endothelial cells as well as transformed HeLa cervical cancer cells. Vaultosome formation is not restricted to tellurite and occurs in the presence of other toxic oxyanions (selenate, selinite, arsenate, arsenite, vanadate). In addition, vaultosomes form independently from other stress-induced ribonucleoprotein complexes, stress granules and aggresomes. Vaultosome formation is therefore a unique cellular response to an environmental toxin. [Abstract/Link to Full Text]

Tremblay Y, Roberts AJ, Costa DP
Fractal landscape method: an alternative approach to measuring area-restricted searching behavior.
J Exp Biol. 2007 Mar;210(Pt 6):935-45.
Quantifying spatial and temporal patterns of prey searching is of primary importance for understanding animals' critical habitat and foraging specialization. In patchy environments, animals forage by exhibiting movement patterns consisting of area-restricted searching (ARS) at various scales. Here, we present a new method, the fractal landscape method, which describes the peaks and valleys of fractal dimension along the animal path. We describe and test the method on simulated tracks, and quantify the effect of track inaccuracies. We show that the ARS zones correspond to the peaks from this fractal landscape and that the method is near error-free when analyzing high-resolution tracks, such as those obtained using the Global Positioning System (GPS). When we used tracks of lower resolution, such as those obtained with the Argos system, 9.6-16.3% of ARS were not identified, and 1-25% of the ARS were found erroneously. The later type of error can be partially flagged and corrected. In addition, track inaccuracies erroneously increased the measured ARS size by a factor of 1.2 to 2.2. Regardless, the majority of the times and locations were correctly flagged as being in or out of ARS (from 83.8 to 89.5% depending on track quality). The method provides a significant new tool for studies of animals' foraging behavior and habitat selection, because it provides a method to precisely quantify each ARS separately, which is not possible with existing methods. [Abstract/Link to Full Text]

James RS, Navas CA, Herrel A
How important are skeletal muscle mechanics in setting limits on jumping performance?
J Exp Biol. 2007 Mar;210(Pt 6):923-33.
Jumping is an important locomotor behaviour used by many animals. The power required to perform a jump is supplied by skeletal muscle. The mechanical properties of skeletal muscle, including the power it can produce, are determined by its composition, which in turn reflects trade-offs between the differing tasks performed by the muscle. Recent studies suggest that muscles used for jumping are relatively fast compared with other limb muscles. As animals get bigger absolute jump performance tends to increase, but recent evidence suggests that adult jump performance may be relatively independent of body size. As body size increases the relative shortening velocity of muscle decreases, whereas normalised power output remains relatively constant. However, the relative shortening velocity of the fastest muscle fibre types appears to remain relatively constant over a large body size range of species. It appears likely that in many species during jumping, other factors are compensating for, or allowing for, uncoupling of jumping performance from size-related changes in the mechanical properties of muscle. In some species smaller absolute body size is compensated for by rapid development of locomotor morphology to attain high locomotor performance early in life. Smaller animal species also appear to rely more heavily on elastic storage mechanisms to amplify the power output available from skeletal muscle. Adaptations involving increased relative hindlimb length and relative mass of jumping muscles, and beneficial alteration of the origin and/or insertion of jumping muscles, have all been found to improve animal jump performance. However, further integrative studies are needed to provide conclusive evidence of which morphological and physiological adaptations are the most important in enhancing jump performance. [Abstract/Link to Full Text]

Gibbs AG
Waterproof cockroaches: the early work of J. A. Ramsay.
J Exp Biol. 2007 Mar;210(Pt 6):921-2. [Abstract/Link to Full Text]

Kumar S, Kumar D, Paranjpe DA, R AC, Sharma VK
Selection on the timing of adult emergence results in altered circadian clocks in fruit flies Drosophila melanogaster.
J Exp Biol. 2007 Mar;210(Pt 5):906-18.
To investigate whether circadian clocks in fruit flies Drosophila melanogaster evolve as a consequence of selection on the timing of adult emergence, we raised four replicate populations each of early (early(1..4)) and late (late(1..4)) emerging flies by selecting for adults that emerged during the morning and the evening hours. We estimated the percentage of flies that emerged during the two selection windows to evaluate the direct response to selection, and the circadian phenotypes of adult emergence and locomotor activity rhythms under light/dark (LD) and constant darkness (DD) to assess the correlated response to selection. After 55 generations, the percentage of flies emerging during the morning window increased in the early populations, but decreased in the late populations. The percentage of flies emerging during the evening window increased in the late populations, but decreased in the early populations. The time course and waveform of emergence and locomotor activity rhythms of the selected populations diverged from each other as well as from the controls. Further, the circadian periodicity of the early populations was significantly shorter than the controls, while that of the late populations was significantly longer than the controls. The light-induced phase response curve of the selected populations differed significantly within groups as well as from the controls. Such modifications in the circadian phenotypes of the selected populations due to heritable changes in genetic architecture, in response to imposed selection pressure, suggest that the circadian clocks underlying emergence and locomotor activity rhythms in D. melanogaster evolve as a correlated response to selection on the timing of adult emergence. [Abstract/Link to Full Text]

Greene MJ, Gordon DM
Structural complexity of chemical recognition cues affects the perception of group membership in the ants Linephithema humile and Aphaenogaster cockerelli.
J Exp Biol. 2007 Mar;210(Pt 5):897-905.
Hydrocarbon profiles on the cuticle of social insects act as multi-component recognition cues used to identify membership in a species, a colony or, within colonies, cues about its reproductive status or task group. To examine the role of structural complexity in ant hydrocarbon recognition cues, we studied the species recognition response of two ant species, Linepithema humile and Aphaenogaster cockerelli, and the recognition of conspecifics by L. humile. The cuticular hydrocarbons of ants are composed of molecules of varying chain lengths from three structural classes, n-alkanes, methyl-branched alkanes and n-alkenes. We employed species recognition bioassays that measured the aggressive response of both species of ants to mixtures of hydrocarbon classes, single structural classes of hydrocarbons (n-alkanes, methyl-branched alkanes and n-alkenes), and controls. The results showed that a combination of at least two hydrocarbon structural classes was necessary to elicit an aggressive species recognition response. Moreover, no single class of hydrocarbons was more important than the others in eliciting a response. Similarly, in the recognition of conspecifics, Linepithema humile did not respond to a mixture of n-alkane cuticular hydrocarbons presented alone, but supplementation of nestmate hydrocarbon profiles with the n-alkanes did elicit high levels of aggression. Thus both L. humile and A. cockerelli required mixtures of hydrocarbons of different structural classes to recognize species and colony membership. It appears that information on species and colony membership is not in isolated components of the profile, but instead in the mixture of structural classes found in cuticular hydrocarbon profiles. [Abstract/Link to Full Text]

Ramamurti R, Sandberg WC
A computational investigation of the three-dimensional unsteady aerodynamics of Drosophila hovering and maneuvering.
J Exp Biol. 2007 Mar;210(Pt 5):881-96.
Three-dimensional unsteady computations of the flow past a fruit fly Drosophila under hovering and free flight conditions are computed. The kinematics of the wings and the body of the fruit fly are prescribed from experimental observations. The computed unsteady lift and thrust forces are validated with experimental results and are in excellent agreement. The unsteady aerodynamic origin of the time-varying yaw moment is identified. The differences in the kinematics between the right and the left wings show that subtle change in the stroke angle and deviation angle can result in the yaw moment for the turning maneuver. The computed yaw moment reaches a peak value at the beginning of the maneuver and remains positive throughout the remainder of the maneuver. The origin of the yaw moment is investigated by computing the center of pressures on each wing and the individual moment arms. This investigation leads to the conclusion that it is the forward force and a component of the lift force that combine to produce the turning moment while the side force alone produces the restoring torque during the maneuver. The vorticity shed from the wing's leading edge and the tips show a loop like structure that during stroke reversals pinches off into Lambda-like structures that have not been previously observed in the wakes of flapping fliers. [Abstract/Link to Full Text]

Oliva D, Medan V, Tomsic D
Escape behavior and neuronal responses to looming stimuli in the crab Chasmagnathus granulatus (Decapoda: Grapsidae).
J Exp Biol. 2007 Mar;210(Pt 5):865-80.
Behavioral responses to looming stimuli have been studied in many vertebrate and invertebrate species, but neurons sensitive to looming have been investigated in very few animals. In this paper we introduce a new experimental model using the crab Chasmagnathus granulatus, which allows investigation of the processes of looming detection and escape decision at both the behavioral and neuronal levels. By analyzing the escape response of the crab in a walking simulator device we show that: (i) a robust and reliable escape response can be elicited by computer-generated looming stimuli in all tested animals; (ii) parameters such as distance, speed, timing and directionality of the escape run, are easy to record and quantify precisely in the walking device; (iii) although the magnitude of escape varies between animals and stimulus presentations, the timing of the response is remarkably consistent and does not habituate at 3 min stimulus intervals. We then study the response of neurons from the brain of the crab by means of intracellular recordings in the intact animal and show that: (iv) two subclasses of previously identified movement detector neurons from the lobula (third optic neuropil) exhibit robust and reliable responses to the same looming stimuli that trigger the behavioral response; (v) the neurons respond to the object approach by increasing their rate of firing in a way that closely matches the dynamics of the image expansion. Finally, we compare the neuronal with the behavioral response showing that: (vi) differences in the neuronal responses to looming, receding or laterally moving stimuli closely reflect the behavioral differences to such stimuli; (vii) during looming, the crab starts to run soon after the looming-sensitive neurons begin to increase their firing rate. The increase in the running speed during stimulus approach faithfully follows the increment in the firing rate, until the moment of maximum stimulus expansion. Thereafter, the neurons abruptly stop firing and the animal immediately decelerates its run. The results are discussed in connection with studies of responses to looming stimuli in the locust. [Abstract/Link to Full Text]

Telang A, Frame L, Brown MR
Larval feeding duration affects ecdysteroid levels and nutritional reserves regulating pupal commitment in the yellow fever mosquito Aedes aegypti (Diptera: Culicidae).
J Exp Biol. 2007 Mar;210(Pt 5):854-64.
What little is known about the endocrine regulation of mosquito development suggests that models based on Lepidoptera and Drosophila may not apply. We report on basic parameters of larval development and the commitment to metamorphosis in the yellow fever mosquito Aedes aegypti that are affected by varying the length of feeding time for last instar larvae. A critical mass for pupal commitment was achieved after 24 h of feeding by last instars, also the age at which tissue production and hemolymph titers of ecdysteroids are increasing. A greater proportion of last instars successfully pupated and eclosed as adults as the length of their feeding time increased. Less than 24 h of feeding time resulted in last instars that were developmentally arrested; these larvae tolerated starvation conditions for up to 2 weeks and retained the capacity to pupate if re-fed. Starvation tolerance may be a common trait among container-inhabiting species, and this period is an important factor to be considered for vectorial capacity and control measures. To distinguish cues for metamorphosis related to a larva's nutritional status versus its age, newly molted last instars were fed for different periods of time but sampled at the same age; ecdysteroid levels, body mass and nutrient reserves were then measured for each group. Our data suggest that metamorphic capacity is dependent on a larva's nutritional condition and not just the age at which ecdysteroid titers increase. Last instars that have fed for a particular length of time may initiate their metamorphic molt when both threshold levels of nutrient reserves and ecdysteroid titer have been met. Future studies will lead to a conceptual model specific for the nutritional and hormonal regulation of mosquito post-embryonic development. This model should facilitate the exploitation of current and novel insect growth regulators that are among favored strategies for vector population suppression. [Abstract/Link to Full Text]

Dacke M, Srinivasan MV
Honeybee navigation: distance estimation in the third dimension.
J Exp Biol. 2007 Mar;210(Pt 5):845-53.
Honeybees determine distance flown by gauging the extent to which the image of the environment moves in the eye as they fly towards their goal. Here we investigate how this visual odometer operates when a bee flies along paths that include a vertical component. By training bees to fly to a feeder along tunnels of various three-dimensional configurations, we find that the odometric signal depends only upon the total distance travelled along the path and is independent of its three-dimensional configuration. Hence, unlike walking desert ants, which measure the distance travelled in the horizontal plane whilst traversing undulating terrain, flying bees simply integrate the image motion that is experienced on the way to the goal, irrespective of the direction in which the image moves across the eyes. These findings raise important questions about how honeybee recruits navigate reliably to find the food sources that are advertised by scouts. [Abstract/Link to Full Text]

Hayward SA, Rinehart JP, Sandro LH, Lee RE, Denlinger DL
Slow dehydration promotes desiccation and freeze tolerance in the Antarctic midge Belgica antarctica.
J Exp Biol. 2007 Mar;210(Pt 5):836-44.
Adaptations to low moisture availability are arguably as important as cold resistance for polar terrestrial invertebrates, especially because water, in the form of ice, is biologically inaccessible for much of the year. Desiccation responses under ecologically realistic soil humidity conditions--those close to the wilting points of plants [98.9% relative humidity (RH)]--have not previously been examined in polar insect species. In the current study we show that, when desiccated at 98.2% RH, larvae of the Antarctic midge Belgica antarctica are more tolerant of dehydration than larvae desiccated at lower humidities (75% RH), and develop an increased tolerance to freezing. The slow rate of desiccation at this high RH enabled more than 50% of larvae to survive the loss of >75% of their osmotically active water (OAW). Survival rates were further increased when rehydration was performed at 100% RH, rather than by direct contact with water. Two days at 98.2% RH resulted in a approximately 30% loss of OAW, and dramatically increased the freeze tolerance of larvae to -10 and -15 degrees C. The supercooling point of animals was not significantly altered by this desiccation treatment, and all larvae were frozen at -10 degrees C. This is the first evidence of desiccation increasing the freeze tolerance of a polar terrestrial arthropod. Maximum water loss and body fluid osmolality were recorded after 5 days at 98.2% RH, but osmolality values returned to predesiccated levels following just 1 h of rehydration in water, well before all the water lost through desiccation had been replenished. This suggests active removal of osmolytes from the extracellular fluids during the desiccation process, presumably to intracellular compartments. Heat-shock proteins appear not to contribute to the desiccation tolerance we observed in B. antarctica. Instead, we suggest that metabolite synthesis and membrane phospholipid adaptation are likely to be the underpinning physiological mechanisms enhancing desiccation and cold tolerance in this species. [Abstract/Link to Full Text]

Symonds BL, James RS, Franklin CE
Getting the jump on skeletal muscle disuse atrophy: preservation of contractile performance in aestivating Cyclorana alboguttata (Gunther 1867).
J Exp Biol. 2007 Mar;210(Pt 5):825-35.
Prolonged immobilisation or unloading of skeletal muscle causes muscle disuse atrophy, which is characterised by a reduction in muscle cross-sectional area and compromised locomotory function. Animals that enter seasonal dormancy, such as hibernators and aestivators, provide an interesting model for investigating atrophy associated with disuse. Previous research on the amphibian aestivator Cyclorana alboguttata (G�nther 1867) demonstrated an absence of muscle disuse atrophy after 3 months of aestivation, as measured by gastrocnemius muscle contractile properties and locomotor performance. In this study, we aimed to investigate the effect of aestivation on iliofibularis and sartorius muscle morphology and contractile function of C. alboguttata over a longer, more ecologically relevant time-frame of 9 months. We found that whole muscle mass, muscle cross-sectional area, fibre number and proportions of fibre types remained unchanged after prolonged disuse. There was a significant reduction in iliofibularis fibre cross-sectional area (declined by 36% for oxidative fibre area and 39% for glycolytic fibre area) and sartorius fibre density (declined by 44%). Prolonged aestivation had little effect on the isometric properties of the skeletal muscle of C. alboguttata. There was a significant reduction in the isometric contraction times of the relatively slow-twitch iliofibularis muscle, suggesting that the muscle was becoming slower after 9 months of aestivation (time to peak twitch increased by 25%, time from peak twitch to half relaxation increased by 34% and time from last stimulus to half tetanus relation increased by 20%). However, the results of the work-loop analysis clearly demonstrate that, despite changes to muscle morphology and isometric kinetics, the overall contractile performance and power output levels of muscles from 9-month aestivating C. alboguttata are maintained at control levels. [Abstract/Link to Full Text]

Wujcik JM, Wang G, Eastman JT, Sidell BD
Morphometry of retinal vasculature in Antarctic fishes is dependent upon the level of hemoglobin in circulation.
J Exp Biol. 2007 Mar;210(Pt 5):815-24.
We quantitatively assessed ocular vascular patterns of six Antarctic notothenioid fishes that vary in their expression of the circulating oxygen-binding protein, hemoglobin (Hb). Digital image analyses revealed marked differences in vessel morphometries among notothenioid species. Hemoglobinless (-Hb) icefishes display mean vessel length densities that are greater (Chaenocephalus aceratus, 5.51+/-0.32 mm mm(-2); Champsocephalus gunnari, 5.15+/-0.50 mm mm(-2)) than those observed in red-blooded (+Hb) species (Gymnodraco acuticeps, 5.20+/-0.46 mm mm(-2); Parachaenichthyes charcoti, 4.40+/-0.30 mm mm(-2); Trematomus hansoni, 3.94+/-0.08 mm mm(-2); Notothenia coriiceps, 2.48+/-0.21 mm mm(-2)). -Hb fishes also have mean vessel diameters that are approximately 1.5 times greater than vessel diameters of +Hb species (-Hb, 0.193+/-0.006 mm; +Hb, 0.125+/-0.005 mm). Vascular density index (VDI), a stereological index that is affected by both vessel number and length, is greatest in -Hb C. aceratus (3.51+/-0.20) and lowest in +Hb N. coriiceps (1.58+/-0.14). Among four +Hb species, there is a direct relationship between red blood cell content and retinal vasculature. Hematocrit (Hct) is inversely correlated to vascular density (r(2)=0.934) and positively correlated to intervessel distance (r(2)= 0.898) over a >2.3-fold range of Hct. These results indicate that anatomical capacity to supply blood to the retina increases to compensate for decreases in oxygen-carrying capacity of the blood. [Abstract/Link to Full Text]

Pereira AC, Rodr�guez-Cattaneo A, Castell� ME, Caputi AA
Post-natal development of the electromotor system in a pulse gymnotid electric fish.
J Exp Biol. 2007 Mar;210(Pt 5):800-14.
Some fish emit electric fields generated by the coordinated activation of electric organs. Such discharges are used for exploring the environment and for communication. This article deals with the development of the electric organ and its discharge in Gymnotus, a pulse genus in which brief discharges are separated by regular silent intervals. It is focused on the anatomo-functional study of fish sized between 10 and 300 mm from the species of Gymnotus, in which electrogenic mechanisms are best known. It was shown that: (1) electroreception and electromotor control is present from early larval stages; (2) there is a single electric organ from larval to adult stages; (3) pacemaker rhythmicity becomes similar to that of the adult when the body length becomes greater than 45 mm and (4) there is a consistent developmental profile of the electric organ discharge in which waveform components are added according to a programmed sequence. The analysis of these data allowed us to identify three main periods in post-natal development of electrogenesis: (1) before fish reach 55 mm in length, when maturation of neural structures is the main factor determining a characteristic sequence of changes observed in the discharge timing and waveform; (2) between 55 and 100 mm in length, when peripheral maturation of the effector cells and changes in post-effector mechanisms due to the fish's growth determine minor changes in waveform and the increase in amplitude of the discharge and (3) beyond 100 mm in length, when homothetic growth of the fish body explains the continuous increase in electric power of the discharge. [Abstract/Link to Full Text]

Douglas JM, Cronin TW, Chiou TH, Dominy NJ
Light habitats and the role of polarized iridescence in the sensory ecology of neotropical nymphalid butterflies (Lepidoptera: Nymphalidae).
J Exp Biol. 2007 Mar;210(Pt 5):788-99.
The exploitation of polarized light may increase perceived visual contrast independent of spectrum and intensity and thus have adaptive value in forest habitats, where illumination varies greatly in brightness and spectral properties. Here we investigate the extent to which Costa Rican butterflies of the family Nymphalidae exhibit polarized wing reflectance and evaluate the types of habitats in which the trait is commonly found. We also examine the degree of polarized reflectance of wing patterns in representative species belonging to the nymphalid subfamilies Charaxinae, Heliconiinae, Morphinae and Nymphalinae. Polarized reflectance was evaluated using museum specimens illuminated with a light source that simulated the spectrum of ambient sunlight and viewed through a polarized filter. Of the 144 species examined, 75 species exhibited polarized reflectance patterns. These species were significantly more likely to occupy forest habitats than open habitats. A concentrated changes test performed on a phylogeny of the Nymphalidae, with the Papilionidae as an outgroup, provides further support for the correlated evolution of polarized iridescence and life in a forest light environment. These results are consistent with the hypothesis that the production and detection of polarized light may have adaptive communicative value in those species inhabiting forest habitats with complex light conditions. The potential utility of polarized iridescence and iridescent wing coloration within differing ambient spectral environments is discussed to provide a basis for future investigation of the polarized light ecology of butterflies. [Abstract/Link to Full Text]

Schulte-Pelkum N, Wieskotten S, Hanke W, Dehnhardt G, Mauck B
Tracking of biogenic hydrodynamic trails in harbour seals (Phoca vitulina).
J Exp Biol. 2007 Mar;210(Pt 5):781-7.
For seals hunting in dark and murky waters one source of sensory information for locating prey consists of fish-generated water movements, which they can detect using their highly sensitive mystacial vibrissae. As water movements in the wake of fishes can persist for several minutes, hydrodynamic trails of considerable length are generated. It has been demonstrated that seals can use their vibrissae to detect and track hydrodynamic trails generated artificially by miniature submarines. In the present study, we trained a harbour seal to swim predefined courses, thus generating biogenic hydrodynamic trails. The structure of these trails was measured using Particle Image Velocimetry. A second seal was trained to search for and track the trail after the trail-generating seal had left the water. Our trail-following seal was able to detect and accurately track the hydrodynamic trail, showing search patterns either mostly congruent with the trail or crossing the trail repeatedly in an undulatory way. The undulatory trail-following search pattern might allow a seal to relocate a lost trail or successfully track a fleeing, zigzagging prey fish. [Abstract/Link to Full Text]

Marasco PD, Catania KC
Response properties of primary afferents supplying Eimer's organ.
J Exp Biol. 2007 Mar;210(Pt 5):765-80.
The mole's nose is covered with mechanosensory structures called Eimer's organs. Each organ contains Merkel cell-neurite complexes, Paciniform corpuscles and intraepidermal free nerve endings. The function of Eimer's organ has been the subject of speculation since the 1800s, but responses from the afferents have never been investigated. Our goal was to explore the function of Eimer's organ by recording primary afferent responses to a range of mechanosensory stimuli. Unit activity from the trigeminal ganglion was recorded from coast (Scapanus orarius) and star-nosed (Condylura cristata) moles, while stimulating the nose with a Chubbuck mechanosensory stimulator, a piezo-electric sweeping stimulator, and hand-held probes. Stimuli included static indentations, sinusoidal displacements, different indentation velocities, displacement amplitudes, and directional stimuli across the skin. Receptive fields were small, sometimes restricted to single Eimer's organs. Responses were consistent with a slowly adapting Merkel cell-neurite complex-like receptor class and a dynamically sensitive Pacinian-like rapidly adapting class. A second rapidly adapting class was hypothesized to represent activity of prominent free nerve endings within a central cell column. Some receptors were most sensitive to stimuli applied in particular directions across the skin. Most receptors relayed mechanosensory input with high temporal fidelity. In addition some receptors were tuned to respond best when stimulated at a velocity matching the velocity of the nose during foraging. These results support the hypothesis that Eimer's organ functions to detect small surface features and textures by encoding and integrating deflection information for multiple Eimer's organs during brief touches. [Abstract/Link to Full Text]

Rank NE, Bruce DA, McMillan DM, Barclay C, Dahlhoff EP
Phosphoglucose isomerase genotype affects running speed and heat shock protein expression after exposure to extreme temperatures in a montane willow beetle.
J Exp Biol. 2007 Mar;210(Pt 5):750-64.
Eastern Sierra Nevada populations of the willow beetle Chrysomela aeneicollis commonly experience stressfully high and low environmental temperatures that may influence survival and reproduction. Allele frequencies at the enzyme locus phosphoglucose isomerase (PGI) vary across a climatic latitudinal gradient in these populations, with PGI allele 1 being most common in cooler regions and PGI allele 4 in warmer ones. PGI genotypes differ in heat and cold tolerance and in expression of a 70 kDa heat shock protein. Here we examine genetic, behavioral and environmental factors affecting a performance character, running speed, for willow beetles, and assess effects of consecutive cold and heat exposure on running speed and expression of Hsp70 in the laboratory. In nature, running speed depends on air temperature and is higher for males than females. Mating beetles ran faster than single beetles, and differences among PGI genotypes in male running speed depended on the presence of females. In the laboratory, exposure to cold reduced subsequent running speed, but the amount of this reduction depended on PGI genotype and previous thermal history. Effects of exposure to heat also depended on life history stage and PGI genotype. Adults possessing allele 1 ran fastest after a single exposure to stressful temperature, whereas those possessing allele 4 ran faster after repeated exposure. Larvae possessing allele 4 ran fastest after a single stressful exposure, but running speed generally declined after a second exposure to stressful temperature. The ranking of PGI genotypes after the second exposure depended on whether a larva had been exposed to cold or heat. Effects of temperature on Hsp70 expression also varied among PGI genotypes and depended on type of exposure, especially for adults (single heat exposure, two cold exposures: PGI 1-1>1-4>4-4; other multiple extreme exposures: 4-4>1-4>1-1). There was no consistent association between alleles at other polymorphic enzyme loci and running speed or Hsp70 expression. These data suggest that variation at PGI is associated with considerable plasticity in running speed. Differences in Hsp70 expression among PGI genotypes suggest that the heat-shock response may buffer differences in thermal tolerance and performance among genotypes and help maintain the PGI polymorphism in a thermally variable environment. [Abstract/Link to Full Text]

Hoffman TC, Walsberg GE, DeNardo DF
Cloacal evaporation: an important and previously undescribed mechanism for avian thermoregulation.
J Exp Biol. 2007 Mar;210(Pt 5):741-9.
We present the first experimental evidence that a bird is capable of evaporating enough water from the cloaca to be important for thermoregulation. We measured rates of evaporation occurring from the mouth, the skin, and the cloaca of Inca doves Columbina inca Lesson and Eurasian quail Coturnix coturnix Linnaeus. Inca doves showed no significant increase in cutaneous evaporation in response to curtailment of buccopharyngeal evaporation. Cloacal evaporation in doves was negligible at ambient temperatures of 30 degrees , 35 degrees and 40 degrees C. However, at 42 degrees C, the apportionment of total evaporation in doves was 53.4% cutaneous, 25.4% buccopharyngeal and 21.2% cloacal, with cloacal evaporation shedding, on average, 150 mW of heat. In contrast, the evaporative apportionment in quail at 32 degrees C (the highest ambient temperature tolerated by this species) was 58.2% cutaneous, 35.4% buccopharyngeal and 6.4% cloacal. These results suggest that, for some birds, cloacal evaporation can be controlled and could serve as an important emergency tactic for thermoregulation at high ambient temperatures. [Abstract/Link to Full Text]

Kato H, Jochim RC, Lawyer PG, Valenzuela JG
Identification and characterization of a salivary adenosine deaminase from the sand fly Phlebotomus duboscqi, the vector of Leishmania major in sub-Saharan Africa.
J Exp Biol. 2007 Mar;210(Pt 5):733-40.
Two transcripts coding for an adenosine deaminase (ADA) were identified by sequencing a Phlebotomus duboscqi salivary gland cDNA library. Adenosine deaminase was previously reported in the saliva of the sand fly Lutzomyia longipalpis but it was not present in the saliva of the sand flies Phlebotomus papatasi, P. argentipes, P. perniciosus and P. ariasi, suggesting that this enzyme is only present in the saliva of sand flies from the genus Lutzomyia. In the present work, we tested the hypothesis that the salivary gland transcript coding for ADA in Phlebotomus duboscqi, a sister species of Phlebotomus papatasi, produces an active salivary ADA. Salivary gland homogenates of P. duboscqi converted adenosine to inosine, suggesting the presence of ADA activity in the saliva of this species of sand fly; furthermore, this enzymatic activity was significantly reduced when using either salivary glands of recently blood-fed sand flies or punctured salivary glands, suggesting that this enzyme is secreted in the saliva of this insect. This enzymatic activity was absent from the saliva of P. papatasi. In contrast to other Phlebotomus sand flies, we did not find AMP or adenosine in P. duboscqi salivary glands as measured by HPLC-photodiode array. To confirm that the transcript coding for ADA was responsible for the activity observed in the saliva of this sand fly, we cloned this transcript into a prokaryotic expression vector and produced a soluble and active recombinant protein of approximately 60 kDa that was able to convert adenosine to inosine. Extracts of bacteria transformed with control plasmids did not show this activity. These results suggest that P. duboscqi transcripts coding for ADA are responsible for the activity detected in the salivary glands of this sand fly and that P. duboscqi acquired this activity independently from other Phlebotomus sand flies. This is another example of a gene recruitment event in salivary genes of blood-feeding arthropods that may be relevant for blood feeding and, because of the role of ADA in immunity, it may also play a role in parasite transmission. [Abstract/Link to Full Text]

Moran AL, Woods HA
Oxygen in egg masses: interactive effects of temperature, age, and egg-mass morphology on oxygen supply to embryos.
J Exp Biol. 2007 Feb;210(Pt 4):722-31.
Embryos of many marine invertebrates are encased in gelatinous masses for part or all of development. Because gel and intervening embryos retard oxygen flux, such a life-history mode profoundly affects partial pressures of metabolic gases surrounding embryos. However, little is known about relationships between egg-mass structure and the opportunities and constraints imposed on structure by metabolic gas transport. We examined the effects of four factors (temperature, embryo age, embryo density and egg-mass size) on the metabolism of egg masses using both natural egg masses of a nudibranch and artificial egg masses made from sand dollar embryos and low-melting point agarose. Both temperature and embryo age strongly affected metabolic rates of nudibranch embryos. For embryos of a given age (stage), rates of oxygen consumption roughly doubled between 12 and 21 degrees C; from early cleavage to the veliger stage, consumption rose two- to fourfold, depending on temperature. Oxygen profiles in egg masses showed that advanced embryonic age, and to a lesser extent high temperature, both led to steeper oxygen gradients into egg masses. Egg masses containing advanced embryos at 21 degrees C had very low central oxygen levels. Small-diameter artificial masses (2 mm diameter) had virtually no internal oxygen gradients regardless of embryo density or temperature, while medium (4 mm) and large diameter (10 mm) artificial masses had oxygen profiles that depended strongly and interactively on embryo density and temperature. Together, our data on natural and artificial egg masses suggest that (i) multiple factors have strong effects on metabolic rate; (ii) rates of oxygen transport are relatively invariant with temperature in simple, artificial systems but may vary more strongly with temperature in natural egg masses; and (iii) the four factors--temperature, embryo age, embryo density and egg-mass size--interact in important ways bearing on egg mass design. A simple mathematical model is developed to provide a quantitative means of estimating primary and interactive effects of the different factors. We also show that in T. diomedea the gel itself is the main barrier to oxygen transport into egg masses, and that the metabolic activity of embryos increases substantially when embryos are artificially released from the capsules that contain them within the gel mass. [Abstract/Link to Full Text]

Navas CA, James RS
Sexual dimorphism of extensor carpi radialis muscle size, isometric force, relaxation rate and stamina during the breeding season of the frog Rana temporaria Linnaeus 1758.
J Exp Biol. 2007 Feb;210(Pt 4):715-21.
Mating success of individual male frogs within explosive breeding species can depend on their ability to compete for a mate and to hold onto that mate during amplexus. Such importance of amplexus has resulted in the evolution of sexual dimorphism in the morphology and contractile characteristics of the anuran forelimb muscles used during amplexus. The aims of our study were to use an explosive breeding frog (Rana temporaria) during the breeding season to compare extensor carpi radialis (ECR) muscle length, mass, isometric activation times, relaxation times, absolute force, relative force (stress) and fatigue between male and female frogs. We found that ECR muscle mass and length were greater (tenfold and 1.4-fold, respectively), absolute tetanic muscle force and relative tetanic force (stress) were greater (16-fold and 2.2-fold, respectively) and relaxation times were slower in males than in females. Male ECR muscles incompletely relaxed during fatigue tests and showed less fatigue than female muscles. These sex differences are likely to be beneficial to the male frogs in allowing them to produce relatively high absolute muscle forces for prolonged periods of time to hold onto their mate during amplexus. [Abstract/Link to Full Text]

Recent Articles in Biological Procedures Online

Kato M
Evaluation of intra- and interspecific divergence of satellite DNA sequences by nucleotide frequency calculation and pairwise sequence comparison.
Biol Proced Online. 2003;563-68.
Satellite DNA sequences are known to be highly variable and to have been subjected to concerted evolution that homogenizes member sequences within species. We have analyzed the mode of evolution of satellite DNA sequences in four fishes from the genus Diplodus by calculating the nucleotide frequency of the sequence array and the phylogenetic distances between member sequences. Calculation of nucleotide frequency and pairwise sequence comparison enabled us to characterize the divergence among member sequences in this satellite DNA family. The results suggest that the evolutionary rate of satellite DNA in D. bellottii is about two-fold greater than the average of the other three fishes, and that the sequence homogenization event occurred in D. puntazzo more recently than in the others. The procedures described here are effective to characterize mode of evolution of satellite DNA. [Abstract/Link to Full Text]

Bechard ME, Chhatwal S, Garcia RE, Rasche ME
Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.
Biol Proced Online. 2003;569-77.
Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase. [Abstract/Link to Full Text]

Mukherjee S, Sousa R
Use of Site-Specifically Tethered Chemical Nucleases to Study Macromolecular Reactions.
Biol Proced Online. 2003;578-89.
During a complex macromolecular reaction multiple changes in molecular conformation and interactions with ligands may occur. X-ray crystallography may provide only a limited set of snapshots of these changes. Solution methods can augment such structural information to provide a more complete picture of a macromolecular reaction. We analyzed the changes in protein conformation and protein:nucleic acid interactions which occur during transcription initiation by using a chemical nuclease tethered to cysteines introduced site-specifically into the RNA polymerase of bacteriophage T7 (T7 RNAP). Changes in cleavage patterns as the polymerase steps through transcription reveal a series of structural transitions which mediate transcription initiation. Cleavage by tethered chemical nucleases is seen to be a powerful method for revealing the conformational dynamics of macromolecular reactions, and has certain advantages over cross-linking or energy transfer approaches. [Abstract/Link to Full Text]

Artlett CM, Dito CG, Christner PJ
Methodology for Detecting Trace Amounts of Microchimeric DNA from Peripheral Murine White Blood Cells by Real-Time PCR.
Biol Proced Online. 2003;5103-107.
Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report methodology using primers for a portion of the H2-k(b) murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 microM primer, a 20-fold increase in the median manufacturer's recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 microg of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection limit half as sensitive as that found when no carrier DNA was present. [Abstract/Link to Full Text]

Mahajan SD, Schwartz SA, Nair MP
Immunological assays for chemokine detection in in-vitro culture of CNS cells.
Biol Proced Online. 2003;590-102.
Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene array analysis, northern blot analysis, Ribonuclease Protection assay, Flow cytometry, ELISPOT, western blot analysis, and ELISA. No single method of analysis meets the criteria for a comprehensive, biologically relevant assessment of the chemokines, therefore more than one assay might be necessary for correct data interpretation, a choice that is based on development of a scientific rationale for the method with emphasis on the reliability and relevance of the method. [Abstract/Link to Full Text]

Reimer M, Sas D, Ali I
Biological Procedures: An electronic techniques journal serving biochemists and biologists.
Biol Proced Online. 1998 May 14;1I. [Abstract/Link to Full Text]

Recent Articles in BMC Developmental Biology

Lizotte PP, Hanford LE, Enghild JJ, Nozik-Grayck E, Giles BL, Oury TD
Developmental expression of the receptor for advanced glycation end-products (RAGE) and its response to hyperoxia in the neonatal rat lung.
BMC Dev Biol. 2007;715.
BACKGROUND: The receptor for advanced glycation end products (mRAGE) is associated with pathology in most tissues, while its soluble form (sRAGE) acts as a decoy receptor. The adult lung is unique in that it expresses high amounts of RAGE under normal conditions while other tissues express low amounts normally and up-regulate RAGE during pathologic processes. We sought to determine the regulation of the soluble and membrane isoforms of RAGE in the developing lung, and its expression under hyperoxic conditions in the neonatal lung. RESULTS: Fetal (E19), term, 4 day, 8 day and adult rat lung protein and mRNA were analyzed, as well as lungs from neonatal (0-24 hrs) 2 day and 8 day hyperoxic (95% O2) exposed animals. mRAGE transcripts in the adult rat lung were 23% greater than in neonatal (0-24 hrs) lungs. On the protein level, rat adult mRAGE expression was 2.2-fold higher relative to neonatal mRAGE expression, and adult sRAGE protein expression was 2-fold higher compared to neonatal sRAGE. Fetal, term, 4 day and 8 day old rats had a steady increase in both membrane and sRAGE protein expression evaluated by Western Blot and immunohistochemistry. Newborn rats exposed to chronic hyperoxia showed significantly decreased total RAGE expression compared to room air controls. CONCLUSION: Taken together, these data show that rat pulmonary RAGE expression increases with age beginning from birth, and interestingly, this increase is counteracted under hyperoxic conditions. These results support the emerging concept that RAGE plays a novel and homeostatic role in lung physiology. [Abstract/Link to Full Text]

Mamo S, Gal AB, Bodo S, Dinnyes A
Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro.
BMC Dev Biol. 2007;714.
BACKGROUND: Real-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data. RESULTS: Quantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a) in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly. CONCLUSION: Compared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb, the classical housekeeping gene, showed the least stability. We recommend the use of the geometric averages of those three genes for normalization in mouse preimplantation-stage gene expression studies. [Abstract/Link to Full Text]

Escudero LM, Freeman M
Mechanism of G1 arrest in the Drosophila eye imaginal disc.
BMC Dev Biol. 2007;713.
BACKGROUND: Most differentiating cells are arrested in G1-phase of the cell cycle and this proliferative quiescence appears important to allow differentiation programmes to be executed. An example occurs in the Drosophila eye imaginal disc, where all cells are synchronized and arrested in G1 phase prior to making a fate choice either to initiate the first round of photoreceptor differentiation or to re-enter one terminal mitosis. RESULTS: We have analysed the mechanism of this temporally regulated G1-phase in order to develop an integrated model of this proliferative regulation. We find that an overlapping set of cell cycle inhibitors combine to form an efficient barrier to cell cycle progression. This barrier depends on both the primary secreted signals that drive retinal development, Dpp and Hh. Each of these has distinct, as well as partially overlapping functions, in ensuring that Cyclin E and dE2F1 are kept in check. Additionally, inhibition of Cyclin A by Roughex is essential, and this regulation is independent of Dpp and Hh. CONCLUSION: One implication of these results is to further support the idea that Cyclin A has important functions in S-phase entry as well as in mitosis. The unexpectedly complex network of regulation may reflect the importance of cells being uniformly ready to respond to the inductive signals that coordinate retinal differentiation. [Abstract/Link to Full Text]

Laslett AL, Grimmond S, Gardiner B, Stamp L, Lin A, Hawes SM, Wormald S, Nikolic-Paterson D, Haylock D, Pera MF
Transcriptional analysis of early lineage commitment in human embryonic stem cells.
BMC Dev Biol. 2007;712.
BACKGROUND: The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells. RESULTS: We used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling) to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation. CONCLUSION: These findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo. [Abstract/Link to Full Text]

Alviano F, Fossati V, Marchionni C, Arpinati M, Bonsi L, Franchina M, Lanzoni G, Cantoni S, Cavallini C, Bianchi F, Tazzari PL, Pasquinelli G, Foroni L, Ventura C, Grossi A, Bagnara GP
Term Amniotic membrane is a high throughput source for multipotent Mesenchymal Stem Cells with the ability to differentiate into endothelial cells in vitro.
BMC Dev Biol. 2007;711.
BACKGROUND: Term Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC). RESULTS: The recovery of hMSCs and their in vitro expansion potential were greater in amniotic membrane than in bone marrow stroma. At flow cytometry analysis AM-hMSCs showed an immunophenotypical profile, i.e., positive for CD105, CD73, CD29, CD44, CD166 and negative for CD14, CD34, CD45, consistent with that reported for bone marrow-derived MSCs. In addition, amniotic membrane-isolated cells underwent in vitro osteogenic (von Kossa stain), adipogenic (Oil Red-O stain), chondrogenic (collagen type II immunohistochemichal detection) and myogenic (RT-PCR MyoD and Myogenin expression as well as desmin immunohistochemical detection) differentiation. In angiogenic experiments, a spontaneous differentiation into endothelial cells was detected by in vitro matrigel assay and this behaviour has been enhanced through Vascular Endothelial Growth Factor (VEGF) induction. According to these findings, VEGF receptor 1 and 2 (FLT-1 and KDR) were basally expressed in AM-hMSCs and the expression of endothelial-specific markers like FLT-1 KDR, ICAM-1 increased after exposure to VEGF together with the occurrence of CD34 and von Willebrand Factor positive cells. CONCLUSION: The current study suggests that AM-hMSCs may emerge as a remarkable tool for the cell therapy of multiple diseased tissues. AM-hMSCs may potentially assist both bone and cartilage repair, nevertheless, due to their angiogenic potential, they may also pave the way for novel approaches in the development of tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required. [Abstract/Link to Full Text]

Straub JA, Sholler GL, Nishi R
Embryonic sympathoblasts transiently express TrkB in vivo and proliferate in response to brain-derived neurotrophic factor in vitro.
BMC Dev Biol. 2007;710.
BACKGROUND: Nerve growth factor and neurotrophin-3 are involved in the development of sympathetic neurons; however, whether brain derived neurotrophic factor also plays a role is not known. The purpose of this study was to determine whether BDNF and its receptor, TrkB, are expressed during the development of paravertebral sympathetic ganglia in vivo and to determine the effect of BDNF in vitro. RESULTS: As neural crest cells coalesce to form sympathetic ganglia, TrkB-positive cells are seen in both chicken and mouse embryos. In chicken embryos, TrkB-expressing cells first appear at Hamburger-Hamilton Stage (St) 27 and they co-express HNK-1, confirming that they are migrating neural crest cells. The TrkB-positive cells lack neural markers at this stage; however, they migrate with other neurally differentiating cells that are TrkA and TrkC-positive. By St. 29/30, TrkB-positive cells begin to express the neural specific markers Hu C/D and Islet-1; eventually, all TrkB positive cells commence neural differentiation. By St. 34, TrkB and TrkC staining are lost. BDNF transcript expression parallels that of TrkB. In the mouse, TrkB-positive cells surround newly formed sympathetic ganglia and a small number of TrkB positive cells that co-express tyrosine hydroxylase are seen within ganglia between E13.5-15. In cell culture, many cells from St. 29-30 chicken lumbar sympathetic ganglia express neural markers and are dividing, indicating that they are sympathoblasts. Sympathoblasts and neurons require both nerve growth factor and neurotrophin-3 for survival. BDNF increases the number of cells expressing neural markers in culture by increasing number of cells that incorporate bromodeoxyuridine. In contrast, most TrkB-positive sympathetic cells in vivo are not actively proliferating between E6-E8. CONCLUSION: Developing paravertebral sympathetic ganglia in avian and murine embryos contain a subpopulation of sympathoblasts that transiently express TrkB and ultimately commence neuronal differentiation. These TrkB expressing sympathoblasts are not actively dividing in vivo; yet, when placed in vitro, will divide in response to BDNF. This suggests that the availability of BDNF in vivo fails to reach a threshold necessary to induce proliferation. We suggest that excess TrkB stimulation of sympathoblasts in vivo may lead to the genesis of neuroblastoma. [Abstract/Link to Full Text]

Slavik MA, Allen-Hoffmann BL, Liu BY, Alexander CM
Wnt signaling induces differentiation of progenitor cells in organotypic keratinocyte cultures.
BMC Dev Biol. 2007;79.
BACKGROUND: Interfollicular skin develops normally only when the activity of the progenitor cells in the basal layer is counterbalanced by the exit of cells into the suprabasal layers, where they differentiate and cornify to establish barrier function. Distinct stem and progenitor compartments have been demonstrated in hair follicles and sebaceous glands, but there are few data to describe the control of interfollicular progenitor cell activity. Wnt signaling has been shown to be an important growth-inducer of stem cell compartments in skin and many other tissues. RESULTS: Here, we test the effect of ectopic Wnt1 expression on the behavior of interfollicular progenitor cells in an organotypic culture model, and find that Wnt1 signaling inhibits their growth and promotes terminal differentiation. CONCLUSION: These results are consistent with the phenotypes reported for transgenic mice engineered to have gain or loss of function of Wnt signaling in skin, which would recommend our culture model as an accurate one for molecular analysis. Since it is known that canonical ligands are expressed in skin, it is likely that this pathway normally regulates the balance of growth and differentiation, and suggests it could be important to pathogenesis. [Abstract/Link to Full Text]

Frankenberg S, Smith L, Greenfield A, Zernicka-Goetz M
Novel gene expression patterns along the proximo-distal axis of the mouse embryo before gastrulation.
BMC Dev Biol. 2007;78.
BACKGROUND: To date, the earliest stage at which the orientation of the anterior-posterior axis in the mouse embryo is distinguishable by asymmetric gene expression is shortly after E5.5. At E5.5, prospective anterior markers are expressed at the distal tip of the embryo, whereas prospective posterior markers are expressed more proximally, close to the boundary with the extraembryonic region. RESULTS: To contribute to elucidating the mechanisms underlying the events involved in early patterning of the mouse embryo, we have carried out a microarray screen to identify novel genes that are differentially expressed between the distal and proximal parts of the E5.5 embryo. Secondary screening of resulting candidates by in situ hybridisation at E5.5 and E6.5 revealed novel expression patterns for known and previously uncharacterised genes, including Peg10, Ctsz1, Cubilin, Jarid1b, Ndrg1, Sfmbt2, Gjb5, Talia and Plet1. The previously undescribed gene Talia and recently identified Plet1 are expressed specifically in the distal-most part of the extraembryonic ectoderm, adjacent to the epiblast, and are therefore potential candidates for regulating early patterning events. Talia and the previously described gene XE7 define a gene family highly conserved among metazoans and with a predicted protein structure suggestive of a post-transcriptional regulative function, whilst Plet1 appears to be mammal-specific and of unknown function. CONCLUSION: Our approach has allowed us to compare expression between dissected parts of the egg cylinder and has identified multiple genes with novel expression patterns at this developmental stage. These genes are potential candidates for regulating tissue interactions following implantation. [Abstract/Link to Full Text]

Montesano R, Carrozzino F, Souli� P
Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells.
BMC Dev Biol. 2007;77.
BACKGROUND: Formation of branching tubes is a fundamental step in the development of glandular organs. To identify extracellular cues that orchestrate epithelial tubulogenesis, we employed an in vitro assay in which EpH4-J3B1A mammary epithelial cells form spheroidal cysts when grown in collagen gels under serum-free conditions, but form branching tubules in the presence of fetal calf serum (FCS). RESULTS: Initial experiments showed that the tubulogenesis-inducing activity of FCS was markedly increased by heating (70 degrees C) or transient acidification to pH3. We therefore hypothesized that the tubulogenic agent was transforming growth factor-beta (TGF-beta), a cytokine that is present in serum in latent form and can be activated by heat or acid treatment. We found indeed that the tubulogenic activity of acidified FCS is abrogated by addition of either SB-431542, a selective inhibitor of the TGF-beta type I receptor, or a neutralizing antibody to TGF-beta-1. On the other hand, addition of low concentrations (20-100 pg/ml) of exogenous TGF-beta-1 recapitulated the effect of acidified FCS in inducing morphogenesis of hollow tubes. In contrast, higher concentrations of TGF-beta-1 induced the formation of thin cellular cords devoid of a detectable lumen. To gain insight into the mechanisms underlying TGF-beta-1-induced tube formation, we assessed the potential role of matrix metalloproteinases (MMPs). By western blot and gelatin zymography, we observed a dose-dependent increase in MMP-9 upon TGF-beta-1 treatment. Tube formation was suppressed by a synthetic broad-spectrum metalloproteinase inhibitor, by recombinant tissue inhibitor of metalloproteinases-2 (TIMP-2) and by a selective inhibitor of MMP-9, indicating that this morphogenetic process requires the activity of MMP-9. CONCLUSION: Altogether, our results provide evidence that, at low concentrations, TGF-beta-1 promotes MMP-dependent branching tubulogenesis by mammary epithelial cells in vitro, and suggest that it plays a similar role during mammary gland development in vivo. [Abstract/Link to Full Text]

Yang HS, Hinds PW
pRb-mediated control of epithelial cell proliferation and Indian hedgehog expression in mouse intestinal development.
BMC Dev Biol. 2007;76.
BACKGROUND: Self-renewal of the epithelium of the small intestine is a highly regulated process involving cell proliferation and differentiation of stem cells or progenitor cells located at the bottom of the crypt, ending ultimately with extrusion of the terminally differentiated cells at the tip of villus. RESULTS: Here, we utilized the Cre/loxP system to investigate the function of the retinoblastoma protein, pRb in intestinal epithelium. pRb null mice displayed a profoundly altered development of the intestine with increased proliferation and abnormal expression of differentiation markers. Loss of pRb induces cell hyperproliferation in the proliferative region (crypt) as well as in the differentiated zone (villi). The absence of pRb further results in an increase in the population of enterocytes, goblet, enteroendocrine and Paneth cells. In addition, differentiated enteroendocrine cells failed to exit the cell cycle in the absence of pRb. These proliferative changes were accompanied by increased expression of Indian hedgehog and activation of hedgehog signals, a known pathway for intestinal epithelial cell proliferation. CONCLUSION: Our studies have revealed a unique function of pRb in intestine development which is critical for controlling not only the proliferation of a stem cell or progenitor cell population but that of terminally differentiated cells as well. [Abstract/Link to Full Text]

Hans S, Christison J, Liu D, Westerfield M
Fgf-dependent otic induction requires competence provided by Foxi1 and Dlx3b.
BMC Dev Biol. 2007;75.
BACKGROUND: The inner ear arises from a specialized set of cells, the otic placode, that forms at the lateral edge of the neural plate adjacent to the hindbrain. Previous studies indicated that fibroblast growth factors (Fgfs) are required for otic induction; in zebrafish, loss of both Fgf3 and Fgf8 results in total ablation of otic tissue. Furthermore, gain-of-function studies suggested that Fgf signaling is not only necessary but also sufficient for otic induction, although the amount of induced ectopic otic tissue reported after misexpression of fgf3 or fgf8 varies among different studies. We previously suggested that Foxi1 and Dlx3b may provide competence to form the ear because loss of both foxi1 and dlx3b results in ablation of all otic tissue even in the presence of a fully functional Fgf signaling pathway. RESULTS: Using a transgenic line that allows us to misexpress fgf8 under the control of the zebrafish temperature-inducible hsp70 promoter, we readdressed the role of Fgf signaling and otic competence during placode induction. We find that misexpression of fgf8 fails to induce formation of ectopic otic vesicles outside of the endogenous ear field and has different consequences depending upon the developmental stage. Overexpression of fgf8 from 1-cell to midgastrula stages leads to formation of no or small otic vesicles, respectively. Overexpression of fgf8 at these stages never leads to ectopic expression of foxi1 or dlx3b, contrary to previous studies that indicated that foxi1 is activated by Fgf signaling. Consistent with our results we find that pharmacological inhibition of Fgf signaling has no effect on foxi1 or dlx3b expression, but instead, Bmp signaling activates foxi1, directly and dlx3b, indirectly. In contrast to early activation of fgf8, fgf8 overexpression at the end of gastrulation, when otic induction begins, leads to much larger otic vesicles. We further show that application of a low dose of retinoic acid that does not perturb patterning of the anterior neural plate leads to expansion of foxi1 and to a massive Fgf-dependent otic induction. CONCLUSION: These results provide further support for the hypothesis that Foxi1 and Dlx3b provide competence for cells to respond to Fgf and form an otic placode. [Abstract/Link to Full Text]

Wells JM, Esni F, Boivin GP, Aronow BJ, Stuart W, Combs C, Sklenka A, Leach SD, Lowy AM
Wnt/beta-catenin signaling is required for development of the exocrine pancreas.
BMC Dev Biol. 2007;74.
BACKGROUND: Beta-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of beta-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that beta-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of beta-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of beta-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of beta-catenin disrupts pancreatic development. A Pdx1-cre mouse line was used to delete a floxed beta-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis. RESULTS: Pdx1-cre floxed beta-catenin animals were viable but demonstrated small body size and shortened median survival. The pancreata from knockout mice were hypoplastic and histologically demonstrated a striking paucity of exocrine pancreas, acinar to duct metaplasia, but generally intact pancreatic islets containing all lineages of endocrine cells. In animals with extensive acinar hypoplasia, putative hepatocyte transdifferention was occasionally observed. Obvious and uniform pancreatic hypoplasia was observed by embryonic day E16.5. Transcriptional profiling of Pdx1-cre floxed beta-catenin embryonic pancreata at E14.5, before there was a morphological phenotype, revealed significant decreases in the beta-catenin target gene N-myc, and the basic HLH transcription factor PTF1, and an increase of several pancreatic zymogens compared to control animals. By E16.5, there was a dramatic loss of exocrine markers and an increase in Hoxb4, which is normally expressed anterior to the pancreas. CONCLUSION: We conclude that beta-catenin expression is required for development of the exocrine pancreas, but is not required for development of the endocrine compartment. In contrast, beta-catenin/Wnt signaling appears to be critical for proliferation of PTF1+ nascent acinar cells and may also function, in part, to maintain an undifferentiated state in exocrine/acinar cell precursors. Finally, beta-catenin may be required to maintain positional identity of the pancreatic endoderm along the anterior-posterior axis. This data is consistent with the findings of frequent beta-catenin mutations in carcinomas of acinar cell lineage seen in humans. [Abstract/Link to Full Text]

Herpin A, Rohr S, Riedel D, Kluever N, Raz E, Schartl M
Specification of primordial germ cells in medaka (Oryzias latipes).
BMC Dev Biol. 2007;73.
BACKGROUND: Primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types. PGCs specification in zebrafish appears to depend on inheritance of germ plasm in which several RNA molecules such as vasa and nanos reside. Whether the specification mode of PGCs found in zebrafish is general for other fish species was brought into question upon analysis of olvas expression--the vasa homologue in another teleost, medaka (Oryzias latipes). Here, in contrast to the findings in zebrafish, the PGCs are found in a predictable position relative to a somatic structure, the embryonic shield. This finding, coupled with the fact that vasa mRNA, which is localized to the germ plasm of zebrafish but does not label a similar structure in medaka opened the possibility of fundamentally different mechanisms governing PGC specification in these two fish species. RESULTS: In this study we addressed the question concerning the mode of PGC specification in medaka using embryological experiments, analysis of RNA stability in the PGCs and electron microscopy observations. Dramatic alterations in the somatic environment, i.e. induction of a secondary axis or mesoderm formation alteration, did not affect the PGC number. Furthermore, the PGCs of medaka are capable of protecting specific RNA molecules from degradation and could therefore exhibit a specific mRNA expression pattern controlled by posttrancriptional mechanisms. Subsequent analysis of 4-cell stage medaka embryos using electron microscopy revealed germ plasm-like structures located at a region corresponding to that of zebrafish germ plasm. CONCLUSION: Taken together, these results are consistent with the idea that in medaka the inheritance of maternally provided asymmetrically-localized cytoplasmic determinants directs cells to assume the germ line fate similar to zebrafish PGCs. [Abstract/Link to Full Text]

Fong B, Watson PH, Watson AJ
Mouse preimplantation embryo responses to culture medium osmolarity include increased expression of CCM2 and p38 MAPK activation.
BMC Dev Biol. 2007;72.
BACKGROUND: Mechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK) occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM) was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2). The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation embryos to hyperosmotic environments. RESULTS: Our results indicate that Ccm2 along with upstream p38 MAPK pathway constituents (Map3k3, Map2k3, Map2k6, and Map2k4) are expressed throughout mouse preimplantation development. CCM2, MAP3K3 and the phosphorylated forms of MAP2K3/MAP2K6 and MAP2K4 were also detected throughout preimplantation development. Embryo culture in hyperosmotic media increased p38 MAPK activity in conjunction with elevated CCM2 levels. CONCLUSION: These results define the expression of upstream activators of p38 MAPK during preimplantation development and indicate that embryo responses to hyperosmotic environments include elevation of CCM2 and activation of p38 MAPK. [Abstract/Link to Full Text]

Chen YH, Wang YH, Chang MY, Lin CY, Weng CW, Westerfield M, Tsai HJ
Multiple upstream modules regulate zebrafish myf5 expression.
BMC Dev Biol. 2007;71.
BACKGROUND: Myf5 is one member of the basic helix-loop-helix family of transcription factors, and it functions as a myogenic factor that is important for the specification and differentiation of muscle cells. The expression of myf5 is somite- and stage-dependent during embryogenesis through a delicate regulation. However, this complex regulatory mechanism of myf5 is not clearly understood. RESULTS: We isolated a 156-kb bacterial artificial chromosome clone that includes an upstream 80-kb region and a downstream 70-kb region of zebrafish myf5 and generated a transgenic line carrying this 156-kb segment fused to a green fluorescent protein (GFP) reporter gene. We find strong GFP expression in the most rostral somite and in the presomitic mesoderm during segmentation stages, similar to endogenous myf5 expression. Later, the GFP signals persist in caudal somites near the tail bud but are down-regulated in the older, rostral somites. During the pharyngula period, we detect GFP signals in pectoral fin buds, dorsal rostral myotomes, hypaxial myotomes, and inferior oblique and superior oblique muscles, a pattern that also corresponds well with endogenous myf5 transcripts. To characterize the specific upstream cis-elements that regulate this complex and dynamic expression pattern, we also generated several transgenic lines that harbor various lengths within the upstream 80-kb segment. We find that (1) the -80 kb/-9977 segment contains a fin and cranial muscle element and a notochord repressor; (2) the -9977/-6213 segment contains a strong repressive element that does not include the notochord-specific repressor; (3) the -6212/-2938 segment contains tissue-specific elements for bone and spinal cord; (4) the -2937/-291 segment contains an eye enhancer, and the -2937/-2457 segment is required for notochord and myocyte expression; and (5) the -290/-1 segment is responsible for basal transcription in somites and the presomitic mesoderm. CONCLUSION: We suggest that the cell lineage-specific expression of myf5 is delicately orchestrated by multiple modules within the distal upstream region. This study provides an insight to understand the molecular control of myf5 and myogenesis in the zebrafish. [Abstract/Link to Full Text]

Cheong SM, Choi SC, Han JK
Xenopus Dab2 is required for embryonic angiogenesis.
BMC Dev Biol. 2006;663.
BACKGROUND: The molecular mechanisms governing the formation of the embryonic vascular system remain poorly understood. Here, we show that Disabled-2 (Dab2), a cytosolic adaptor protein, has a pivotal role in the blood vessel formation in Xenopus early embryogenesis. RESULTS: Xenopus Disabled-2 (XDab2) is spatially localized to the blood vessels including the intersomitic veins (ISV) in early embryos. Both antisense morpholino oligonucleotide (MO)-mediated knockdown and overexpression of XDab2 inhibit the formation of ISV, which arise from angiogenesis. In addition, we found that activin-like signaling is essential for this angiogenic event. Functional assays in Xenopus animal caps reveal that activin-like signals induce VEGF expression and this induction can be inhibited by XDab2 depletion. However, XDab2 MO has no effects on the induction of other target genes by activin-like signals. Furthermore, we show that the disruption of the sprouting ISV in XDab2-depleted embryos can be rescued by coexpression of VEGF. CONCLUSION: Taking together, we suggest that XDab2 regulates the embryonic angiogenesis by mediating the VEGF induction by activin-like signaling in Xenopus early development. [Abstract/Link to Full Text]

Behesti H, Holt JK, Sowden JC
The level of BMP4 signaling is critical for the regulation of distinct T-box gene expression domains and growth along the dorso-ventral axis of the optic cup.
BMC Dev Biol. 2006;662.
BACKGROUND: Polarised gene expression is thought to lead to the graded distribution of signaling molecules providing a patterning mechanism across the embryonic eye. Bone morphogenetic protein 4 (Bmp4) is expressed in the dorsal optic vesicle as it transforms into the optic cup. Bmp4 deletions in human and mouse result in failure of eye development, but little attempt has been made to investigate mammalian targets of BMP4 signaling. In chick, retroviral gene overexpression studies indicate that Bmp4 activates the dorsally expressed Tbx5 gene, which represses ventrally expressed cVax. It is not known whether the Tbx5 related genes, Tbx2 and Tbx3, are BMP4 targets in the mammalian retina and whether BMP4 acts at a distance from its site of expression. Although it is established that Drosophila Dpp (homologue of vertebrate Bmp4) acts as a morphogen, there is little evidence that BMP4 gradients are interpreted to create domains of BMP4 target gene expression in the mouse. RESULTS: Our data show that the level of BMP4 signaling is critical for the regulation of distinct Tbx2, Tbx3, Tbx5 and Vax2 gene expression domains along the dorso-ventral axis of the mouse optic cup. BMP4 signaling gradients were manipulated in whole mouse embryo cultures during optic cup development, by implantation of beads soaked in BMP4, or the BMP antagonist Noggin, to provide a local signaling source. Tbx2, Tbx3 and Tbx5, showed a differential response to alterations in the level of BMP4 along the entire dorso-ventral axis of the optic cup, suggesting that BMP4 acts across a distance. Increased levels of BMP4 caused expansion of Tbx2 and Tbx3, but not Tbx5, into the ventral retina and repression of the ventral marker Vax2. Conversely, Noggin abolished Tbx5 expression but only shifted Tbx2 expression dorsally. Increased levels of BMP4 signaling caused decreased proliferation, reduced retinal volume and altered the shape of the optic cup. CONCLUSION: Our findings suggest the existence of a dorsal-high, ventral-low BMP4 signaling gradient across which distinct domains of Tbx2, Tbx3, Tbx5 and Vax2 transcription factor gene expression are set up. Furthermore we show that the correct level of BMP4 signaling is critical for normal growth of the mammalian embryonic eye. [Abstract/Link to Full Text]

Sharma N, Liu S, Tang L, Irwin J, Meng G, Rancourt DE
Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation.
BMC Dev Biol. 2006;661.
BACKGROUND: We have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1) and uterus (ISP1 and ISP2). These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers. RESULTS: Here, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo. CONCLUSION: On the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation. [Abstract/Link to Full Text]

LaLonde M, Janssens H, Yun S, Crosby J, Redina O, Olive V, Altshuller YM, Choi SY, Du G, Gergen JP, Frohman MA
A role for Phospholipase D in Drosophila embryonic cellularization.
BMC Dev Biol. 2006;660.
BACKGROUND: Cellularization of the Drosophila embryo is an unusually synchronous form of cytokinesis in which polarized membrane extension proceeds in part through incorporation of new membrane via fusion of apically-translocated Golgi-derived vesicles. RESULTS: We describe here involvement of the signaling enzyme Phospholipase D (Pld) in regulation of this developmental step. Functional analysis using gene targeting revealed that cellularization is hindered by the loss of Pld, resulting frequently in early embryonic developmental arrest. Mechanistically, chronic Pld deficiency causes abnormal Golgi structure and secretory vesicle trafficking. CONCLUSION: Our results suggest that Pld functions to promote trafficking of Golgi-derived fusion-competent vesicles during cellularization. [Abstract/Link to Full Text]

Patrat C, Auer J, Fauque P, Leandri RL, Jouannet P, Serres C
Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm.
BMC Dev Biol. 2006;659.
BACKGROUND: The functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilized oocytes. RESULTS: The hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilized oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilized oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilized oocytes (61.6 +/- 6.2% vs 60.7 +/- 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilized oocytes (19% vs 57% respectively, p < 0.01). We investigated the status of ZP2 in the oocytes used in the functional tests, and demonstrated that sperm binding and acrosome reaction induction, but not ZP penetration, occurred whether or not ZP2 was cleaved. CONCLUSION: The change in ZP function induced by fertilisation could be different in human and mouse species. Our results suggest a zona blocking to polyspermy based at the sperm penetration level in humans. [Abstract/Link to Full Text]

Ribeiro LA, Turba ME, Zannoni A, Bacci ML, Forni M
Gelatinases, endonuclease and Vascular Endothelial Growth Factor during development and regression of swine luteal tissue.
BMC Dev Biol. 2006;658.
BACKGROUND: The development and regression of corpus luteum (CL) is characterized by an intense angiogenesis and angioregression accompanied by luteal tissue and extracellular matrix (ECM) remodelling. Vascular Endothelial Growth Factor (VEGF) is the main regulator of angiogenesis, promoting endothelial cell mitosis and differentiation. After the formation of neovascular tubes, the remodelling of ECM is essential for the correct development of CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). During luteal regression, characterized by an apoptotic process and successively by an intense ECM and luteal degradation, the activation of Ca++/Mg++-dependent endonucleases and MMPs activity are required. The levels of expression and activity of VEGF, MMP-2 and -9, and Ca++/Mg++-dependent endonucleases throughout the oestrous cycle and at pregnancy were analyzed. RESULTS: Different patterns of VEGF, MMPs and Ca++/Mg++-dependent endonuclease were observed in swine CL during different luteal phases and at pregnancy. Immediately after ovulation, the highest levels of VEGF mRNA/protein and MMP-9 activity were detected. On days 5-14 after ovulation, VEGF expression and MMP-2 and -9 activities are at basal levels, while Ca++/Mg++-dependent endonuclease levels increased significantly in relation to day 1. Only at luteolysis (day 17), Ca++/Mg++-dependent endonuclease and MMP-2 spontaneous activity increased significantly. At pregnancy, high levels of MMP-9 and VEGF were observed. CONCLUSION: Our findings, obtained from a precisely controlled in vivo model of CL development and regression, allow us to determine relationships among VEGF, MMPs and endonucleases during angiogenesis and angioregression. Thus, CL provides a very interesting model for studying factors involved in vascular remodelling. [Abstract/Link to Full Text]

Kumar S, Vaze KM, Kumar D, Sharma VK
Selection for early and late adult emergence alters the rate of pre-adult development in Drosophila melanogaster.
BMC Dev Biol. 2006;657.
BACKGROUND: Circadian clocks have been implicated in the regulation of pre-adult development of fruit flies Drosophila melanogaster. It is believed that faster clocks speed up development and slower clocks slow it down. We established three sets of D. melanogaster populations (early, control and late). The early and late populations were raised by selecting for flies that emerged either in the morning or in the evening under 12:12 hr light/dark (LD) cycles. After 75 generations of selection, the time course and waveform of the adult emergence and activity rhythms of the early and the late populations diverged from each other as well as from the controls. In this paper, we report the consequence of this selection on the rate of pre-adult development. RESULTS: We assayed the pre-adult development time of the selected and control populations under 12:12 hr LD cycles and constant darkness (DD). Under LD cycles, the early populations develop faster than the controls, while the late populations develop slower than the controls. Although flies take longer to develop under DD than in LD, the relative differences between the mean development times of the selected and control populations remain unaltered in DD. In a separate experiment designed to investigate the effect of time of egg collection and experimental conditions on the duration of pre-adult stage, we assayed the development time of the selected and control populations by collecting eggs at different times of the day (morning and evening) and by assaying their pre-adult development time under constant light (LL), LD, and DD conditions. Irrespective of the time of egg collection and assay light regime, the late flies continue to develop slower than the early flies. CONCLUSION: The results of our study clearly indicate that selection on the timing of adult emergence alters the rate of pre-adult development in D. melanogaster. The timing of egg collection as well as assay light regime does not have any measurable effect on the relative differences between the developmental rates of the early and the late flies. Taken together these results appear to suggest that pleiotropic effects of clock genes mediate correlated changes in the timing of adult emergence and the rate of pre-adult development in D. melanogaster. [Abstract/Link to Full Text]

Andrieu D, Meziane H, Marly F, Angelats C, Fernandez PA, Muscatelli F
Sensory defects in Necdin deficient mice result from a loss of sensory neurons correlated within an increase of developmental programmed cell death.
BMC Dev Biol. 2006;656.
BACKGROUND: The human NECDIN gene is involved in a neurodevelopmental disorder, Prader-Willi syndrome (PWS). Previously we reported a mouse Necdin knock-out model with similar defects to PWS patients. Despite the putative roles attributed to Necdin, mainly from in vitro studies, its in vivo function remains unclear. In this study, we investigate sensory-motor behaviour in Necdin deficient mice. We reveal cellular defects and analyse their cause. RESULTS: We report sensory differences in Necdin deficient mice compared to wild type animals. These differences led us to investigate sensory neuron development in Necdin deficient mouse embryos. First, we describe the expression pattern of Necdin in developing DRGs and report a reduction of one-third in specified sensory neurons in dorsal roots ganglia and show that this neuronal loss is achieved by E13.5, when DRGs sensory neurons are specified. In parallel, we observed an increase of 41% in neuronal apoptosis during the wave of naturally occurring cell death at E12.5. Since it is assumed that Necdin is a P75NTR interactor, we looked at the P75NTR-expressing cell population in Necdin knock-out embryos. Unexpectedly, Necdin loss of function has no effect on p75NTR expressing neurons suggesting no direct genetic interaction between Necdin and P75NTR in this context.Although we exclude a role of Necdin in axonal outgrowth from spinal sensory neurons in early developmental stages; such a role could occur later in neuronal differentiation. Finally we also exclude an anti-proliferative role of Necdin in developing sensory neurons. CONCLUSION: Overall, our data show clearly that, in early development of the nervous system, Necdin is an anti-apoptotic or survival factor. [Abstract/Link to Full Text]

Ramos DM, Kamal F, Wimmer EA, Cartwright AN, Monteiro A
Temporal and spatial control of transgene expression using laser induction of the hsp70 promoter.
BMC Dev Biol. 2006;655.
BACKGROUND: Precise temporal and spatial regulation of transgene expression is a critical tool to investigate gene function in developing organisms. The most commonly used technique to achieve tight control of transgene expression, however, requires the use of specific DNA enhancers that are difficult to characterize in non-model organisms. Here, we sought to eliminate the need for this type of sequence-based gene regulation and to open the field of functional genetics to a broader range of organisms. RESULTS: We have developed a new laser mediated method to heat shock groups of cells that provides precise spatio-temporal control of gene expression without requiring knowledge of specific enhancer sequences. We tested our laser-system in a transgenic line of Bicyclus anynana butterflies containing the EGFP reporter gene attached to the heat sensitive hsp70 promoter of Drosophila melanogaster. Whole organismal heat shocks demonstrated that this Drosophila promoter can drive gene expression in butterflies, and the subsequent laser heat shocks showed that it was possible to activate cell-specific gene expression in very precise patterns on developing pupal wings. CONCLUSION: This laser-mediated gene expression system will enable functional genetic investigations, i.e., the ectopic expression of genes and their knock-down in targeted groups of cells in model and non-model organisms with little or no available regulatory data, as long as a compatible heat-shock promoter is used and the target tissue is accessible to a laser beam. This technique will also be useful in evolutionary developmental biology as it will enable the study of the evolution of gene function across a variety of organisms. [Abstract/Link to Full Text]

Lozyk MD, Papp S, Zhang X, Nakamura K, Michalak M, Opas M
Ultrastructural analysis of development of myocardium in calreticulin-deficient mice.
BMC Dev Biol. 2006;654.
BACKGROUND: Calreticulin is a Ca2+ binding chaperone of the endoplasmic reticulum which influences gene expression and cell adhesion. The levels of both vinculin and N-cadherin are induced by calreticulin expression, which play important roles in cell adhesiveness. Cardiac development is strictly dependent upon the ability of cells to adhere to their substratum and to communicate with their neighbours. RESULTS: We show here that the levels of N-cadherin are downregulated in calreticulin-deficient mouse embryonic hearts, which may lead to the disarray and wavy appearance of myofibrils in these mice, which we detected at all investigated stages of cardiac development. Calreticulin wild type mice exhibited straight, thick and abundant myofibrils, which were in stark contrast to the thin, less numerous, disorganized myofibrils of the calreticulin-deficient hearts. Interestingly, these major differences were only detected in the developing ventricles while the atria of both calreticulin phenotypes were similar in appearance at all developmental stages. Glycogen also accumulated in the ventricles of calreticulin-deficient mice, indicating an abnormality in cardiomyocyte metabolism. CONCLUSION: Calreticulin is temporarily expressed during heart development where it is required for proper myofibrillogenesis. We postulate that calreticulin be considered as a novel cardiac fetal gene. [Abstract/Link to Full Text]

Holohan EE, zur Lage PI, Jarman AP
Multiple enhancers contribute to spatial but not temporal complexity in the expression of the proneural gene, amos.
BMC Dev Biol. 2006;653.
BACKGROUND: The regulation of proneural gene expression is an important aspect of neurogenesis. In the study of the Drosophila proneural genes, scute and atonal, several themes have emerged that contribute to our understanding of the mechanism of neurogenesis. First, spatial complexity in proneural expression results from regulation by arrays of enhancer elements. Secondly, regulation of proneural gene expression occurs in distinct temporal phases, which tend to be under the control of separate enhancers. Thirdly, the later phase of proneural expression often relies on positive autoregulation. The control of these phases and the transition between them appear to be central to the mechanism of neurogenesis. We present the first investigation of the regulation of the proneural gene, amos. RESULTS: Amos protein expression has a complex pattern and shows temporally distinct phases, in common with previously characterised proneural genes. GFP reporter gene constructs were used to demonstrate that amos has an array of enhancer elements up- and downstream of the gene, which are required for different locations of amos expression. However, unlike other proneural genes, there is no evidence for separable enhancers for the different temporal phases of amos expression. Using mutant analysis and site-directed mutagenesis of potential Amos binding sites, we find no evidence for positive autoregulation as an important part of amos control during neurogenesis. CONCLUSION: For amos, as for other proneural genes, a complex expression pattern results from the sum of a number of simpler sub-patterns driven by specific enhancers. There is, however, no apparent separation of enhancers for distinct temporal phases of expression, and this correlates with a lack of positive autoregulation. For scute and atonal, both these features are thought to be important in the mechanism of neurogenesis. Despite similarities in function and expression between the Drosophila proneural genes, amos is regulated in a fundamentally different way from scute and atonal. [Abstract/Link to Full Text]

Monteiro A, Glaser G, Stockslager S, Glansdorp N, Ramos D
Comparative insights into questions of lepidopteran wing pattern homology.
BMC Dev Biol. 2006;652.
BACKGROUND: Butterfly and moth eyespots can share a similar appearance, involving multiple concentric rings of colored scales, but usually occurring in non-homologous positions on the wing. Within the butterflies, on the other hand, spots that share the same homologous position may not share the concentric ring structure; and, in butterfly species that have eyespots with concentric rings, ectopic eyespots with a similar ring structure can be induced by means of a simple epidermal wound. The extent to which all these eyespots, natural or induced, share similar genes and developmental mechanisms is investigated here by means of protein in-situ localizations in selected butterfly and moth species. In addition to looking at some of the transcription factors previously identified as being involved in eyespot formation, we also tested the involvement of candidate genes from the Wingless and TGF-beta signaling pathways as putative morphogens for eyespot development. RESULTS: Saturniid moth and nymphalid butterfly eyespots with concentric rings of color express at least two transcription factors, Distal-less and Engrailed, in the center of the future pattern. Nymphalid eyespots centers also express the ligand Wingless and an activated signal transducer, a phosphorylated Smad protein, but neither these proteins nor the previous two proteins are found in pierid spot centers, which consist of a single patch of color. Both butterfly wing patterns, however, express a third transcription factor, Spalt, a portion of whose expression domain maps to the black scales on the adult wing. Wounding a nymphalid wing, on the other hand, leads to upregulation of Distal-less, engrailed and spalt in subsets of cells around the wounding site, mimicking concentric eyespot development. CONCLUSION: Wingless and TGF-beta ligands are both candidate morphogens involved in nymphalid butterfly eyespot formation. These eyespots, as well as saturniid moth eyespots with concentric circles, share two genes that are associated with the differentiation of the signaling cells in nymphalid eyespots. This commonality suggests that they may be produced via the same developmental mechanism despite their non-homologous location. By contrast, pierid butterfly spots of a single color share some of the same genes but appear to be produced by a different mechanism. Eyespots with concentric rings may have co-opted a wound healing genetic network during their evolution. [Abstract/Link to Full Text]

Wang J, Nagy A, Larsson J, Dudas M, Sucov HM, Kaartinen V
Defective ALK5 signaling in the neural crest leads to increased postmigratory neural crest cell apoptosis and severe outflow tract defects.
BMC Dev Biol. 2006;651.
BACKGROUND: Congenital cardiovascular diseases are the most common form of birth defects in humans. A substantial portion of these defects has been associated with inappropriate induction, migration, differentiation and patterning of pluripotent cardiac neural crest stem cells. While TGF-beta-superfamily signaling has been strongly implicated in neural crest cell development, the detailed molecular signaling mechanisms in vivo are still poorly understood. RESULTS: We deleted the TGF-beta type I receptor Alk5 specifically in the mouse neural crest cell lineage. Failure in signaling via ALK5 leads to severe cardiovascular and pharyngeal defects, including inappropriate remodeling of pharyngeal arch arteries, abnormal aortic sac development, failure in pharyngeal organ migration and persistent truncus arteriosus. While ALK5 is not required for neural crest cell migration, our results demonstrate that it plays an important role in the survival of post-migratory cardiac neural crest cells. CONCLUSION: Our results demonstrate that ALK5-mediated signaling in neural crest cells plays an essential cell-autonomous role in the pharyngeal and cardiac outflow tract development. [Abstract/Link to Full Text]

Del Giacco L, Sordino P, Pistocchi A, Andreakis N, Tarallo R, Di Benedetto B, Cotelli F
Differential regulation of the zebrafish orthopedia 1 gene during fate determination of diencephalic neurons.
BMC Dev Biol. 2006;650.
BACKGROUND: The homeodomain transcription factor Orthopedia (Otp) is essential in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates. However, there is little information on the intercellular factors that regulate Otp expression during development. RESULTS: Here, we identified two otp orthologues in zebrafish (otp1 and otp2) and explored otp1 in the context of the morphogenetic pathways that specify neuroectodermal regions. During forebrain development, otp1 is expressed in anterior groups of diencephalic cells, positioned in the preoptic area (PO) (anterior alar plate) and the posterior tuberculum (PT) (posterior basal plate). The latter structure is characterized by Tyrosine Hydroxylase (TH)-positive cells, suggesting a role for otp1 in the lineage restriction of catecholaminergic (CA) neurons. Disruptions of Hedgehog (HH) and Fibroblast Growth Factor (FGF) pathways point to the ability of SHH protein to trigger otp1 expression in PO presumptive neuroblasts, with the attenuating effect of Dzip1 and FGF8. In addition, our data disclose otp1 as a determinant of CA neurons in the PT, where otp1 activity is strictly dependent on Nodal signaling and it is not responsive to SHH and FGF. CONCLUSION: In this study, we pinpoint the evolutionary importance of otp1 transcription factor in cell states of the diencephalon anlage and early neuronal progenitors. Furthermore, our data indicate that morphogenetic mechanisms differentially regulate otp1 expression in alar and basal plates. [Abstract/Link to Full Text]

Gui ZZ, Lee KS, Kim BY, Choi YS, Wei YD, Choo YM, Kang PD, Yoon HJ, Kim I, Je YH, Seo SJ, Lee SM, Guo X, Sohn HD, Jin BR
Functional role of aspartic proteinase cathepsin D in insect metamorphosis.
BMC Dev Biol. 2006;649.
BACKGROUND: Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. RESULTS: Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage. CONCLUSION: Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis. [Abstract/Link to Full Text]

Recent Articles in BMC Evolutionary Biology

Fjerdingstad EJ, Schtickzelle N, Manhes P, Gutierrez A, Clobert J
Evolution of dispersal and life history strategies--Tetrahymena ciliates.
BMC Evol Biol. 2007;7133.
BACKGROUND: Considerable attention has focused on how selection on dispersal and other core life-history strategies (reproductive effort, survival ability, colonization capacity) may lead to so-called dispersal syndromes. Studies on genetic variation in these syndromes within species could importantly increase our understanding of their evolution, by revealing whether traits co-vary across genetic lineages in the manner predicted by theoretical models, and by stimulating further hypotheses for experimental testing. Yet such studies remain scarce. Here we studied the ciliated protist Tetrahymena thermophila, a particularly interesting organism due to cells being able to transform into morphs differing dramatically in swim-speed. We investigated dispersal, morphological responses, reproductive performance, and survival in ten different clonal strains. Then, we examined whether life history traits co-varied in the manner classically predicted for ruderal species, examined the investment of different strains into short- and putative long-distance dispersal, while considering also the likely impact of semi-sociality (cell aggregation, secretion of 'growth factors') on dispersal strategies. RESULTS: Very significant among-strain differences were found with regard to dispersal rate, morphological commitment and plasticity, and almost all core life-history traits (e.g. survival, growth performance and strategy), with most of these traits being significantly intercorrelated. Some strains showed high short-distance dispersal rates, high colonization capacity, bigger cell size, elevated growth performance, and good survival abilities. These well performing strains, however, produced fewer fast-swimming dispersal morphs when subjected to environmental degradation than did philopatric strains performing poorly under normal conditions. CONCLUSION: Strong evidence was found for a genetic covariation between dispersal strategies and core life history traits in T. thermophila, with a fair fit of observed trait associations with classic colonizer models. However, the well performing strains with high colonization success and short-distance dispersal likely suffered under a long-distance dispersal disadvantage, due to producing fewer fast-swimming dispersal morphs than did philopatric strains. The smaller cell size at carrying capacity of the latter strains and their poor capacity to colonize as individual cells suggest that they may be adapted to greater levels of dependency on clone-mates (stronger sociality). In summary, differential exposure to selection on competitive and cooperative abilities, in conjunction with selective factors targeting specifically dispersal distance, likely contributed importantly to shaping T. thermophila dispersal and life history evolution. [Abstract/Link to Full Text]

Castric V, Vekemans X
Evolution under strong balancing selection: how many codons determine specificity at the female self-incompatibility gene SRK in Brassicaceae?
BMC Evol Biol. 2007;7132.
BACKGROUND: Molecular lock-and-key systems are common among reproductive proteins, yet their evolution remains a major puzzle in evolutionary biology. In the Brassicaceae, the genes encoding self-incompatibility have been identified, but technical challenges currently prevent detailed analyses of the molecular interaction between the male and female components. In the present study, we investigate sequence polymorphism in the female specificity determinant SRK of Arabidopsis halleri from throughout Europe. Using a comparative approach based on published SRK sequences in A. lyrata and Brassica, we track the signature of frequency-dependent selection acting on these genes at the codon level. Using simulations, we evaluate power and accuracy of our approach and estimate the proportion of codon sites involved in the molecular interaction. RESULTS: We identified several members of the S-gene family, together with 22 putative S-haplotypes. Linkage to the S-locus and the presence of a kinase domain were formally demonstrated for four and six of these haplotypes, respectively, and sequence polymorphism was extremely high. Twenty-five codons showed signs of positive selection in at least one species, and clustered significantly (but not exclusively) within hypervariable regions. We checked that this clustering was not an artifact due to variation in evolution rate at synonymous sites. Simulations revealed that the analysis was highly accurate, thus providing a reliable set of candidates for future functional analyses, but with an overall power not higher than 60 %. Assuming similar power, we infer from our results that about 23% of all codons in the S-domain may actually be involved in recognition. Interestingly, while simulations demonstrated that this comparison remained reliable even at very high levels of divergence, codons identified in Brassica had higher posterior rates of non-synonymous to synonymous substitutions than codons identified in A. halleri or A. lyrata, possibly suggesting more intense selection in Brassica. CONCLUSION: The signature of balancing selection can be identified reliably at the codon level even in cases of very high sequence divergence, provided that a sufficiently large set of sequences are analyzed. Altogether, our results indicate that a large proportion of codons may be involved in recognition and confirm the particular importance of hypervariable regions. The more intense signature of positive selection detected in Brassica suggests that allelic diversification in this genus was very recent, possibly following a recent demographic bottleneck. [Abstract/Link to Full Text]

Harlin-Cognato AD, Markowitz T, W�rsig B, Honeycutt RL
Multi-locus phylogeography of the dusky dolphin (Lagenorhynchus obscurus): passive dispersal via the west-wind drift or response to prey species and climate change?
BMC Evol Biol. 2007;7131.
BACKGROUND: The dusky dolphin (Lagenorhynchus obscurus) is distributed along temperate, coastal regions of New Zealand, South Africa, Argentina, and Peru where it feeds on schooling anchovy, sardines, and other small fishes and squid tightly associated with temperate ocean sea surface temperatures. Previous studies have suggested that the dusky dolphin dispersed in the Southern Hemisphere eastward from Peru via a linear, temperate dispersal corridor provided by the circumpolar west-wind drift. With new mitochondrial and nuclear DNA sequence data, we propose an alternative phylogeographic history for the dusky dolphin that was structured by paleoceanographic conditions that repeatedly altered the distribution of its temperate prey species during the Plio-Pleistocene. RESULTS: In contrast to the west-wind drift hypothesis, phylogenetic analyses support a Pacific/Indian Ocean origin, with a relatively early and continued isolation of Peru from other regions. Dispersal of the dusky dolphin into the Atlantic is correlated with the history of anchovy populations, including multiple migrations from New Zealand to South Africa. Additionally, the cooling of the Eastern Equatorial Pacific led to the divergence of anchovy populations, which in turn explains the north-south equatorial transgression of L. obliquidens and the subsequent divergence of L. obscurus in the Southern Hemisphere. CONCLUSION: Overall, our study fails to support the west-wind drift hypothesis. Instead, our data indicate that changes in primary productivity and related abundance of prey played a key role in shaping the phylogeography of the dusky dolphin, with periods of ocean change coincident with important events in the history of this temperate dolphin species. Moderate, short-term changes in sea surface temperatures and current systems have a powerful effect on anchovy populations; thus, it is not infeasible that repeated fluctuations in anchovy populations continue to play an important role in the history of coastal dolphin populations. [Abstract/Link to Full Text]

Rensing SA, Ick J, Fawcett JA, Lang D, Zimmer A, Van de Peer Y, Reski R
An ancient genome duplication contributed to the abundance of metabolic genes in the moss Physcomitrella patens.
BMC Evol Biol. 2007;7130.
BACKGROUND: Analyses of complete genomes and large collections of gene transcripts have shown that most, if not all seed plants have undergone one or more genome duplications in their evolutionary past. RESULTS: In this study, based on a large collection of EST sequences, we provide evidence that the haploid moss Physcomitrella patens is a paleopolyploid as well. Based on the construction of linearized phylogenetic trees we infer the genome duplication to have occurred between 30 and 60 million years ago. Gene Ontology and pathway association of the duplicated genes in P. patens reveal different biases of gene retention compared with seed plants. CONCLUSION: Metabolic genes seem to have been retained in excess following the genome duplication in P. patens. This might, at least partly, explain the versatility of metabolism, as described for P. patens and other mosses, in comparison to other land plants. [Abstract/Link to Full Text]

te Velthuis AJ, Admiraal JF, Bagowski CP
Molecular evolution of the MAGUK family in metazoan genomes.
BMC Evol Biol. 2007;7129.
BACKGROUND: Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla. RESULTS: Our results indicate that at least four of the seven MAGUK subfamilies were present in early metazoan lineages, such as Porifera. We employed domain sequence and structure based methods to infer a model for the evolutionary history of the MAGUKs. Notably, the phylogenetic trees for the guanylate kinase (GK)-, the PDZ- and the SH3-domains all suggested a matching evolutionary model which was further supported by molecular modeling of the 3D structures of different GK domains. We found no MAGUK in plants, fungi or other unicellular organisms, which suggests that the MAGUK core structure originated early in metazoan history. CONCLUSION: In summary, we have characterized here the molecular and structural evolution of the large MAGUK family. Using the MAGUKs as an example, our results show that it is possible to derive a highly supported evolutionary model for important multidomain families by analyzing encoded protein domains. It further suggests that larger superfamilies encoded in the different genomes can be analyzed in a similar manner. [Abstract/Link to Full Text]

Kochiwa H, Tomita M, Kanai A
Evolution of ribonuclease H genes in prokaryotes to avoid inheritance of redundant genes.
BMC Evol Biol. 2007;7128.
BACKGROUND: A theoretical model of genetic redundancy has proposed that the fates of redundant genes depend on the degree of functional redundancy, and that functionally redundant genes will not be inherited together. However, no example of actual gene evolution has been reported that can be used to test this model. Here, we analyzed the molecular evolution of the ribonuclease H (RNase H) family in prokaryotes and used the results to examine the implications of functional redundancy for gene evolution. RESULTS: In prokaryotes, RNase H has been classified into RNase HI, HII, and HIII on the basis of amino acid sequences. Using 353 prokaryotic genomes, we identified the genes encoding the RNase H group and examined combinations of these genes in individual genomes. We found that the RNase H group may have evolved in such a way that the RNase HI and HIII genes will not coexist within a single genome--in other words, these genes are inherited in a mutually exclusive manner. Avoiding the simultaneous inheritance of the RNase HI and HIII genes is remarkable when RNase HI contains an additional non-RNase H domain, double-stranded RNA, and an RNA-DNA hybrid-binding domain, which is often observed in eukaryotic RNase H1. This evolutionary process may have resulted from functional redundancy of these genes, because the substrate preferences of RNase HI and RNase HIII are similar. CONCLUSION: We provide two possible evolutionary models for RNase H genes in which functional redundancy contributes to the exclusion of redundant genes from the genome of a species. This is the first empirical study to show the effect of functional redundancy on changes in gene constitution during the course of evolution. [Abstract/Link to Full Text]

Kon T, Nohara M, Yamanoue Y, Fujiwara Y, Nishida M, Nishikawa T
Phylogenetic position of a whale-fall lancelet (Cephalochordata) inferred from whole mitochondrial genome sequences.
BMC Evol Biol. 2007;7127.
BACKGROUND: The lancelet Asymmetron inferum (subphylum Cephalochordata) was recently discovered on the ocean floor off the southwest coast of Japan at a depth of 229 m, in an anaerobic and sulfide-rich environment caused by decomposing bodies of the sperm whale Physeter macrocephalus. This deep sulfide-rich habitat of A. inferum is unique among the lancelets. The distinguishing adaptation of this species to such an extraordinary habitat can be considered in a phylogenetic framework. As the first step of reconstruction of the evolutionary processes in this species, we investigated its phylogenetic position based on 11 whole mitochondrial genome sequences including the newly determined ones of the whale-fall lancelet A. inferum and two coral-reef congeners. RESULTS: Our phylogenetic analyses showed that extant lancelets are clustered into two major clades, the Asymmetron clade and the Epigonichthys + Branchiostoma clade. A. inferum was in the former and placed in the sister group to A. lucayanum complex. The divergence time between A. inferum and A. lucayanum complex was estimated to be 115 Mya using the penalized likelihood (PL) method or 97 Mya using the nonparametric rate smoothing (NPRS) method (the middle Cretaceous). These are far older than the first appearance of large whales (the middle Eocene, 40 Mya). We also discovered that A. inferum mitogenome (mitochondrial genome) has been subjected to large-scale gene rearrangements, one feature of rearrangements being unique among the lancelets and two features shared with A. lucayanum complex. CONCLUSION: Our study supports the monophyly of genus Asymmetron assumed on the basis of the morphological characters. Furthermore, the features of the A. inferum mitogenome expand our knowledge of variation within cephalochordate mitogenomes, adding a new case of transposition and inversion of the trnQ gene. Our divergence time estimation suggests that A. inferum remained a member of the Mesozoic and the early Cenozoic large vertebrate-fall communities before shifting to become a whale-fall specialist. [Abstract/Link to Full Text]

Ackermann M, Schauerte A, Stearns SC, Jenal U
Experimental evolution of aging in a bacterium.
BMC Evol Biol. 2007 Jul 28;7(1):126.
ABSTRACT: BACKGROUND: Aging refers to a decline in reproduction and survival with increasing age. According to evolutionary theory, aging evolves because selection late in life is weak and mutations exist whose deleterious effects manifest only late in life. Whether the assumptions behind this theory are fulfilled in all organisms, and whether all organisms age, has not been clear. We tested the generality of this theory by experimental evolution with Caulobacter crescentus, a bacterium whose asymmetric division allows mother and daughter to be distinguished. RESULTS: We evolved three populations for 2000 generations in the laboratory under conditions where selection was strong early in life, but very weak later in life. All populations evolved faster growth rates, mostly by decreasing the age at first division. Evolutionary changes in aging were inconsistent. The predominant response was the unexpected evolution of slower aging, revealing the limits of theoretical predictions if mutations have unanticipated phenotypic effects. However, we also observed the spread of a mutation causing earlier aging of mothers whose negative effect was reset in the daughters. CONCLUSIONS: Our results confirm that late-acting deleterious mutations do occur in bacteria and that they can invade populations when selection late in life is weak. They suggest that very few organisms - perhaps none- can avoid the accumulation of such mutations over evolutionary time, and thus that aging is probably a fundamental property of all cellular organisms. [Abstract/Link to Full Text]

Enikeeva FN, Kotelnikova EA, Gelfand MS, Makeev VJ
A model of evolution with constant selective pressure for regulatory DNA sites.
BMC Evol Biol. 2007;7125.
BACKGROUND: Molecular evolution is usually described assuming a neutral or weakly non-neutral substitution model. Recently, new data have become available on evolution of sequence regions under a selective pressure, e.g. transcription factor binding sites. To reconstruct the evolutionary history of such sequences, one needs evolutionary models that take into account a substantial constant selective pressure. RESULTS: We present a simple evolutionary model with a single preferred (consensus) nucleotide and the neutral substitution model adopted for all other nucleotides. This evolutionary model has a rate matrix in which all substitutions that do not involve the consensus nucleotide occur with the same rate. The model has two time scales for achieving a stationary distribution; in the general case only one of the two rate parameters can be evaluated from the stationary distribution. In the middle-time zone, a counterintuitive behavior was observed for some parameter values, with a probability of conservation for a non-consensus nucleotide greater than that for the consensus nucleotide. Such an effect can be observed only in the case of weak preference for the consensus nucleotide, when the probability to observe the consensus nucleotide in the stationary distribution is less than 1/2. If the substitution rate is represented as a product of mutation and fixation, only the fixation can be calculated from the stationary distribution. The exhibited conservation of non-consensus nucleotides does not take place if the elements of mutation matrix are identical, and can be related to the reduced mutation rate between the non-consensus nucleotides. This bias can have no effect on the stationary distribution of nucleotide frequencies calculated over the ensemble of multiple alignments, e.g. transcription factor binding sites upstream of different sets of co-regulated orthologous genes. CONCLUSION: The derived model can be used as a null model when analyzing the evolution of orthologous transcription factor binding sites. In particular, our findings show that a nucleotide preferred at some position of a multiple alignment of binding sites for some transcription factor in the same genome is not necessarily the most conserved nucleotide in an alignment of orthologous sites from different species. However, this effect can take place only in the case of a mutation matrix whose elements are not identical. [Abstract/Link to Full Text]

Rosa A, Ornelas C, Jobling MA, Brehm A, Villems R
Y-chromosomal diversity in the population of Guinea-Bissau: a multiethnic perspective.
BMC Evol Biol. 2007;7124.
BACKGROUND: The geographic and ethnolinguistic differentiation of many African Y-chromosomal lineages provides an opportunity to evaluate human migration episodes and admixture processes, in a pan-continental context. The analysis of the paternal genetic structure of Equatorial West Africans carried out to date leaves their origins and relationships unclear, and raises questions about the existence of major demographic phenomena analogous to the large-scale Bantu expansions. To address this, we have analysed the variation of 31 binary and 11 microsatellite markers on the non-recombining portion of the Y chromosome in Guinea-Bissau samples of diverse ethnic affiliations, some not studied before. RESULTS: The Guinea-Bissau Y chromosome pool is characterized by low haplogroup diversity (D = 0.470, sd 0.033), with the predominant haplogroup E3a*-M2 shared among the ethnic clusters and reaching a maximum of 82.2% in the Mandenka people. The Felupe-Djola and Papel groups exhibit the highest diversity of lineages and harbor the deep-rooting haplogroups A-M91, E2-M75 and E3*-PN2, typical of Sahel's more central and eastern areas. Their genetic distinction from other groups is statistically significant (P = 0.01) though not attributable to linguistic, geographic or religious criteria. Non sub-Saharan influences were associated with the presence of haplogroup R1b-P25 and particular lineages of E3b1-M78. CONCLUSION: The predominance and high diversity of haplogroup E3a*-M2 suggests a demographic expansion in the equatorial western fringe, possibly supported by a local agricultural center. The paternal pool of the Mandenka and Balanta displays evidence of a particularly marked population growth among the Guineans, possibly reflecting the demographic effects of the agriculturalist lifestyle and their putative relationship to the people that introduced early cultivation practices into West Africa. The paternal background of the Felupe-Djola and Papel ethnic groups suggests a better conserved ancestral pool deriving from East Africa, from where they have supposedly migrated in recent times. Despite the overall homogeneity in a multiethnic sample, which contrasts with their social structure, minor clusters suggest the imprints of multiple peoples at different timescales: traces of ancestral inhabitants in haplogroups A-M91 and B-M60, today typical of hunter-gatherers; North African influence in E3b1-M78 Y chromosomes, probably due to trans-Saharan contacts; and R1b-P25 lineages reflecting European admixture via the North Atlantic slave trade. [Abstract/Link to Full Text]

Jiang ZJ, Castoe TA, Austin CC, Burbrink FT, Herron MD, McGuire JA, Parkinson CL, Pollock DD
Comparative mitochondrial genomics of snakes: extraordinary substitution rate dynamics and functionality of the duplicate control region.
BMC Evol Biol. 2007;7123.
BACKGROUND: The mitochondrial genomes of snakes are characterized by an overall evolutionary rate that appears to be one of the most accelerated among vertebrates. They also possess other unusual features, including short tRNAs and other genes, and a duplicated control region that has been stably maintained since it originated more than 70 million years ago. Here, we provide a detailed analysis of evolutionary dynamics in snake mitochondrial genomes to better understand the basis of these extreme characteristics, and to explore the relationship between mitochondrial genome molecular evolution, genome architecture, and molecular function. We sequenced complete mitochondrial genomes from Slowinski's corn snake (Pantherophis slowinskii) and two cottonmouths (Agkistrodon piscivorus) to complement previously existing mitochondrial genomes, and to provide an improved comparative view of how genome architecture affects molecular evolution at contrasting levels of divergence. RESULTS: We present a Bayesian genetic approach that suggests that the duplicated control region can function as an additional origin of heavy strand replication. The two control regions also appear to have different intra-specific versus inter-specific evolutionary dynamics that may be associated with complex modes of concerted evolution. We find that different genomic regions have experienced substantial accelerated evolution along early branches in snakes, with different genes having experienced dramatic accelerations along specific branches. Some of these accelerations appear to coincide with, or subsequent to, the shortening of various mitochondrial genes and the duplication of the control region and flanking tRNAs. CONCLUSION: Fluctuations in the strength and pattern of selection during snake evolution have had widely varying gene-specific effects on substitution rates, and these rate accelerations may have been functionally related to unusual changes in genomic architecture. The among-lineage and among-gene variation in rate dynamics observed in snakes is the most extreme thus far observed in animal genomes, and provides an important study system for further evaluating the biochemical and physiological basis of evolutionary pressures in vertebrate mitochondria. [Abstract/Link to Full Text]

S�nchez JA, Aguilar C, Dorado D, Manrique N
Phenotypic plasticity and morphological integration in a marine modular invertebrate.
BMC Evol Biol. 2007;7122.
BACKGROUND: Colonial invertebrates such as corals exhibit nested levels of modularity, imposing a challenge to the depiction of their morphological evolution. Comparisons among diverse Caribbean gorgonian corals suggest decoupling of evolution at the polyp vs. branch/internode levels. Thus, evolutionary change in polyp form or size (the colonial module sensu stricto) does not imply a change in colony form (constructed of modular branches and other emergent features). This study examined the patterns of morphological integration at the intraspecific level. Pseudopterogorgia bipinnata (Verrill) (Octocorallia: Gorgoniidae) is a Caribbean shallow water gorgonian that can colonize most reef habitats (shallow/exposed vs. deep/protected; 1-45 m) and shows great morphological variation. RESULTS: To characterize the genotype/environment relationship and phenotypic plasticity in P. bipinnata, two microsatellite loci, mitochondrial (MSH1) and nuclear (ITS) DNA sequences, and (ITS2) DGGE banding patterns were initially compared among the populations present in the coral reefs of Belize (Carrie Bow Cay), Panama (Bocas del Toro), Colombia (Cartagena) and the Bahamas (San Salvador). Despite the large and discrete differentiation of morphotypes, there was no concordant genetic variation (DGGE banding patterns) in the ITS2 genotypes from Belize, Panama and Colombia. ITS1-5.8S-ITS2 phylogenetic analysis afforded evidence for considering the species P. kallos (Bielschowsky) as the shallow-most morphotype of P. bipinnata from exposed environments. The population from Carrie Bow Cay, Belize (1-45 m) was examined to determine the phenotypic integration of modular features such as branch thickness, polyp aperture, inter-polyp distance, internode length and branch length. Third-order partial correlation coefficients suggested significant integration between polypar and colonial traits. Some features did not change at all despite 10-fold differences in other integrated features. More importantly, some colonial features showed dependence on modular features. CONCLUSION: Consequently, module integration in gorgonian corals can be shifted, switched or canalized along lineages. Modular marine organisms such as corals are variations on a single theme: their modules can couple or decouple, allowing them to adapt to all marine benthic environments. [Abstract/Link to Full Text]

Pfenninger M, Schwenk K
Cryptic animal species are homogeneously distributed among taxa and biogeographical regions.
BMC Evol Biol. 2007;7121.
BACKGROUND: Cryptic species are two or more distinct but morphologically similar species that were classified as a single species. During the past two decades we observed an exponential growth of publications on cryptic species. Recently published reviews have demonstrated cryptic species have profound consequences on many biological disciplines. It has been proposed that their distribution is non-random across taxa and biomes. RESULTS: We analysed a literature database for the taxonomic and biogeographical distribution of cryptic animal species reports. Results from regression analysis indicate that cryptic species are almost evenly distributed among major metazoan taxa and biogeographical regions when corrected for species richness and study intensity. CONCLUSION: This indicates that morphological stasis represents an evolutionary constant and that cryptic metazoan diversity does predictably affect estimates of earth's animal diversity. Our findings have direct theoretical and practical consequences for a number of prevailing biological questions with regard to global biodiversity estimates, conservation efforts and global taxonomic initiatives. [Abstract/Link to Full Text]

Liongue C, Ward AC
Evolution of Class I cytokine receptors.
BMC Evol Biol. 2007;7120.
BACKGROUND: The Class I cytokine receptors have a wide range of actions, including a major role in the development and function of immune and blood cells. However, the evolution of the genes encoding them remains poorly understood. To address this we have used bioinformatics to analyze the Class I receptor repertoire in sea squirt (Ciona intestinalis) and zebrafish (Danio rerio). RESULTS: Only two Class I receptors were identified in sea squirt, one with homology to the archetypal GP130 receptor, and the other with high conservation with the divergent orphan receptor CLF-3. In contrast, 36 Class I cytokine receptors were present in zebrafish, including representative members for each of the five structural groups found in mammals. This allowed the identification of 27 core receptors belonging to the last common ancestor of teleosts and mammals. CONCLUSION: This study suggests that the majority of diversification of this receptor family occurred after the divergence of urochordates and vertebrates approximately 794 million years ago (MYA), but before the divergence of ray-finned from lobe-finned fishes around 476 MYA. Since then, only relatively limited lineage-specific diversification within the different Class I receptor structural groups has occurred. [Abstract/Link to Full Text]

Neafsey DE, Galagan JE
Positive selection for unpreferred codon usage in eukaryotic genomes.
BMC Evol Biol. 2007;7119.
BACKGROUND: Natural selection has traditionally been understood as a force responsible for pushing genes to states of higher translational efficiency, whereas lower translational efficiency has been explained by neutral mutation and genetic drift. We looked for evidence of directional selection resulting in increased unpreferred codon usage (and presumably reduced translational efficiency) in three divergent clusters of eukaryotic genomes using a simple optimal-codon-based metric (Kp/Ku). RESULTS: Here we show that for some genes natural selection is indeed responsible for causing accelerated unpreferred codon substitution, and document the scope of this selection. In Cryptococcus and to a lesser extent Drosophila, we find many genes showing a statistically significant signal of selection for unpreferred codon usage in one or more lineages. We did not find evidence for this type of selection in Saccharomyces. The signal of positive selection observed from unpreferred synonymous codon substitutions is coincident in Cryptococcus and Drosophila with the distribution of upstream open reading frames (uORFs), another genic feature known to reduce translational efficiency. Functional enrichment analysis of genes exhibiting low Kp/Ku ratios reveals that genes in regulatory roles are particularly subject to this type of selection. CONCLUSION: Through genome-wide scans, we find recent selection for unpreferred codon usage at approximately 1% of genetic loci in a Cryptococcus and several genes in Drosophila. Unpreferred codons can impede translation efficiency, and we find that genes with translation-impeding uORFs are enriched for this selection signal. We find that regulatory genes are particularly likely to be subject to selection for unpreferred codon usage. Given that expression noise can propagate through regulatory cascades, and that low translational efficiency can reduce expression noise, this finding supports the hypothesis that translational efficiency may be suppressed in some cases to reduce stochastic noise in gene expression. [Abstract/Link to Full Text]

Mayer G, Harzsch S
Immunolocalization of serotonin in Onychophora argues against segmental ganglia being an ancestral feature of arthropods.
BMC Evol Biol. 2007;7118.
BACKGROUND: Onychophora (velvet worms) represent the most basal arthropod group and play a pivotal role in the current discussion on the evolution of nervous systems and segmentation in arthropods. Although there is a wealth of information on the immunolocalization of serotonin (5-hydroxytryptamine, 5-HT) in various euarthropods, as yet no comparable localization data are available for Onychophora. In order to understand how the onychophoran nervous system compares to that of other arthropods, we studied the distribution of serotonin-like immunoreactive neurons and histological characteristics of ventral nerve cords in Metaperipatus blainvillei (Onychophora, Peripatopsidae) and Epiperipatus biolleyi (Onychophora, Peripatidae). RESULTS: We demonstrate that paired leg nerves are the only segmental structures associated with the onychophoran nerve cord. Although the median commissures and peripheral nerves show a repeated pattern, their arrangement is independent from body segments characterized by the position of legs and associated structures. Moreover, the somata of serotonin-like immunoreactive neurons do not show any ordered arrangement in both species studied but are instead scattered throughout the entire length of each nerve cord. We observed neither a serially iterated nor a bilaterally symmetric pattern, which is in contrast to the strictly segmental arrangement of serotonergic neurons in other arthropods. CONCLUSION: Our histological findings and immunolocalization experiments highlight the medullary organization of the onychophoran nerve cord and argue against segmental ganglia of the typical euarthropodan type being an ancestral feature of Onychophora. These results contradict a priori assumptions of segmental ganglia being an ancestral feature of arthropods and, thus, weaken the traditional Articulata hypothesis, which proposes a sistergroup relationship of Annelida and Arthropoda. [Abstract/Link to Full Text]

Behere GT, Tay WT, Russell DA, Heckel DG, Appleton BR, Kranthi KR, Batterham P
Mitochondrial DNA analysis of field populations of Helicoverpa armigera (Lepidoptera: Noctuidae) and of its relationship to H. zea.
BMC Evol Biol. 2007;7117.
BACKGROUND: Helicoverpa armigera and H. zea are amongst the most significant polyphagous pest lepidopteran species in the Old and New Worlds respectively. Separation of H. armigera and H. zea is difficult and is usually only achieved through morphological differences in the genitalia. They are capable of interbreeding to produce fertile offspring. The single species status of H. armigera has been doubted, due to its wide distribution and plant host range across the Old World. This study explores the global genetic diversity of H. armigera and its evolutionary relationship to H zea. RESULTS: We obtained partial (511 bp) mitochondrial DNA (mtDNA) Cytochrome Oxidase-I (COI) sequences for 249 individuals of H. armigera sampled from Australia, Burkina Faso, Uganda, China, India and Pakistan which were associated with various host plants. Single nucleotide polymorphisms (SNPs) within the partial COI gene differentiated H. armigera populations into 33 mtDNA haplotypes. Shared haplotypes between continents, low F-statistic values and low nucleotide diversity between countries (0.0017-0.0038) suggests high mobility in this pest. Phylogenetic analysis of four major Helicoverpa pest species indicates that H. punctigera is basal to H. assulta, which is in turn basal to H. armigera and H. zea. Samples from North and South America suggest that H. zea is also a single species across its distribution. Our data reveal short genetic distances between H. armigera and H. zea which seem to have been established via a founder event from H. armigera stock at around 1.5 million years ago. CONCLUSION: Our mitochondrial DNA sequence data supports the single species status of H. armigera across Africa, Asia and Australia. The evidence for inter-continental gene flow observed in this study is consistent with published evidence of the capacity of this species to migrate over long distances. The finding of high genetic similarity between Old World H. armigera and New World H. zea emphasises the need to consider work on both pests when building pest management strategies for either. [Abstract/Link to Full Text]

Gordo I, Campos PR
Patterns of genetic variation in populations of infectious agents.
BMC Evol Biol. 2007;7116.
BACKGROUND: The analysis of genetic variation in populations of infectious agents may help us understand their epidemiology and evolution. Here we study a model for assessing the levels and patterns of genetic diversity in populations of infectious agents. The population is structured into many small subpopulations, which correspond to their hosts, that are connected according to a specific type of contact network. We considered different types of networks, including fully connected networks and scale free networks, which have been considered as a model that captures some properties of real contact networks. Infectious agents transmit between hosts, through migration, where they grow and mutate until elimination by the host immune system. RESULTS: We show how our model is closely related to the classical SIS model in epidemiology and find that: depending on the relation between the rate at which infectious agents are eliminated by the immune system and the within host effective population size, genetic diversity increases with R0 or peaks at intermediate R0 levels; patterns of genetic diversity in this model are in general similar to those expected under the standard neutral model, but in a scale free network and for low values of R0 a distortion in the neutral mutation frequency spectrum can be observed; highly connected hosts (hubs in the network) show patterns of diversity different from poorly connected individuals, namely higher levels of genetic variation, lower levels of genetic differentiation and larger values of Tajima's D. CONCLUSION: We have found that levels of genetic variability in the population of infectious agents can be predicted by simple analytical approximations, and exhibit two distinct scenarios which are met according to the relation between the rate of drift and the rate at which infectious agents are eliminated. In one scenario the diversity is an increasing function of the level of transmission and in a second scenario it is peaked around intermediate levels of transmission. This is independent of the type of host contact structure. Furthermore for low values of R0, very heterogeneous host contact structures lead to lower levels of diversity. [Abstract/Link to Full Text]

Aanen DK, Ros VI, de Fine Licht HH, Mitchell J, de Beer ZW, Slippers B, Rouland-Lef�vre C, Boomsma JJ
Patterns of interaction specificity of fungus-growing termites and Termitomyces symbionts in South Africa.
BMC Evol Biol. 2007;7115.
BACKGROUND: Termites of the subfamily Macrotermitinae live in a mutualistic symbiosis with basidiomycete fungi of the genus Termitomyces. Here, we explored interaction specificity in fungus-growing termites using samples from 101 colonies in South-Africa and Senegal, belonging to eight species divided over three genera. Knowledge of interaction specificity is important to test the hypothesis that inhabitants (symbionts) are taxonomically less diverse than 'exhabitants' (hosts) and to test the hypothesis that transmission mode is an important determinant for interaction specificity. RESULTS: Analysis of Molecular Variance among symbiont ITS sequences across termite hosts at three hierarchical levels showed that 47 % of the variation occurred between genera, 18 % between species, and the remaining 35 % between colonies within species. Different patterns of specificity were evident. High mutual specificity was found for the single Macrotermes species studied, as M. natalensis was associated with a single unique fungal haplotype. The three species of the genus Odontotermes showed low symbiont specificity: they were all associated with a genetically diverse set of fungal symbionts, but their fungal symbionts showed some host specificity, as none of the fungal haplotypes were shared between the studied Odontotermes species. Finally, bilaterally low specificity was found for the four tentatively recognized species of the genus Microtermes, which shared and apparently freely exchanged a common pool of divergent fungal symbionts. CONCLUSION: Interaction specificity was high at the genus level and generally much lower at the species level. A comparison of the observed diversity among fungal symbionts with the diversity among termite hosts, indicated that the fungal symbiont does not follow the general pattern of an endosymbiont, as we found either similar diversity at both sides or higher diversity in the symbiont. Our results further challenge the hypothesis that transmission-mode is a general key-determinant of interaction specificity in fungus-growing termites. [Abstract/Link to Full Text]

Schrempf A, Darrouzet E, Heinze J
Mating success and potential male-worker conflict in a male-dimorphic ant.
BMC Evol Biol. 2007;7114.
BACKGROUND: Males of many species adjust their reproductive tactics with regard to their condition and status. For example, large males may develop weapons and fight for access to females, whereas small or undernourished males do not express costly weapons or ornaments and sneak copulations. Different condition-dependent reproductive tactics may be associated with unequal average fitness, but the tactic chosen by a given male under given circumstances is thought to result in the highest possible fitness return.The ant species Cardiocondyla obscurior exhibits an environment-controlled polymorphism of docile, winged males and aggressive "ergatoid" males. Ergatoid males, which can replenish their sperm supply throughout their lives, engage in lethal fighting, and attempt to monopolize all female sexuals available in their nests, were previously assumed to gain higher lifetime reproductive success than the peaceful, winged males, which disperse to mate away from the nest and whose spermatogenesis is limited to the first days of adult life. However, precise data on male mating success have as yet not been available.Here, we compare the average mating success of the two male morphs, taking the high mortality rate of immature ergatoid males into account. Because individuals in insect societies may have opposing interests about their own development, we also investigate whether the interests of male larvae coincide with those of the workers and the rest of the society. RESULTS: When the survival probability of males is taken into account, winged males are more likely to mate multiply and in consequence have a higher estimated average mating success than ergatoid males. Therefore, male larvae are expected to prefer developing into winged instead of ergatoid adults. CONCLUSION: Though male larvae can expect a higher average mating success when developing into winged males, most colonies produce only ergatoid males under standard conditions. This might point at a novel type of potential kin conflict within the social insect colony. Because workers in insect societies usually control male larval development, ergatoid male production under normal conditions probably reflects the optimal allocation strategy of workers to maximise their inclusive fitness. [Abstract/Link to Full Text]

Chopra SS, Watanabe H, Zhong TP, Roden DM
Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates.
BMC Evol Biol. 2007;7113.
BACKGROUND: Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish). RESULTS: We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3) and duplicate genes for beta4 (zbeta4.1, zbeta4.2). Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. CONCLUSION: The identification of conserved orthologs to all 4 voltage-gated sodium channel beta subunit genes in zebrafish and the lack of evidence for beta subunit genes in invertebrate chordates together indicate that this gene family emerged early in vertebrate evolution, prior to the divergence of teleosts and tetrapods. The evolutionary history of sodium channel beta subunits suggests that these genes may have played a key role in the diversification and specialization of electrical signaling in early vertebrates. [Abstract/Link to Full Text]

Biedler JK, Tu Z
The Juan non-LTR retrotransposon in mosquitoes: genomic impact, vertical transmission and indications of recent and widespread activity.
BMC Evol Biol. 2007;7112.
BACKGROUND: In contrast to DNA-mediated transposable elements (TEs), retrotransposons, particularly non-long terminal repeat retrotransposons (non-LTRs), are generally considered to have a much lower propensity towards horizontal transfer. Detailed studies on site-specific non-LTR families have demonstrated strict vertical transmission. More studies are needed with non-site-specific non-LTR families to determine whether strict vertical transmission is a phenomenon related to site specificity or a more general characteristic of all non-LTRs. Juan is a Jockey clade non-LTR retrotransposon first discovered in mosquitoes that is widely distributed in the mosquito family Culicidae. Being a non-site specific non-LTR, Juan offers an opportunity to further investigate the hypothesis that non-LTRs are genomic elements that are primarily vertically transmitted. RESULTS: Systematic analysis of the ~1.3 Gbp Aedes aegypti (Ae. aegypti) genome sequence suggests that Juan-A is the only Juan-type non-LTR in Aedes aegypti. Juan-A is highly reiterated and comprises approximately 3% of the genome. Using minimum cutoffs of 90% length and 70% nucleotide (nt) identity, 663 copies were found by BLAST using the published Juan-A sequence as the query. All 663 copies are at least 95% identical to Juan-A, while 378 of these copies are 99% identical to Juan-A, indicating that the Juan-A family has been transposing recently in evolutionary history. Using the 0.34 Kb 5' UTR as the query, over 2000 copies were identified that may contain internal promoters, leading to questions on the genomic impact of Juan-A. Juan sequences were obtained by PCR, library screening, and database searches for 18 mosquito species of six genera including Aedes, Ochlerotatus, Psorophora, Culex, Deinocerites, and Wyeomyia. Comparison of host and Juan phylogenies shows overall congruence with few exceptions. CONCLUSION: Juan-A is a major genomic component in Ae. aegypti and it has been retrotransposing recently in evolutionary history. There are also indications that Juan has been recently active in a wide range of mosquito species. Furthermore, our research demonstrates that a Jockey clade non-LTR without target site-specificity has been sustained by vertical transmission in the mosquito family. These results strengthen the argument that non-LTRs tend to be genomic elements capable of persistence by vertical descent over a long evolutionary time. [Abstract/Link to Full Text]

Carbone I, Ramirez-Prado JH, Jakobek JL, Horn BW
Gene duplication, modularity and adaptation in the evolution of the aflatoxin gene cluster.
BMC Evol Biol. 2007;7111.
BACKGROUND: The biosynthesis of aflatoxin (AF) involves over 20 enzymatic reactions in a complex polyketide pathway that converts acetate and malonate to the intermediates sterigmatocystin (ST) and O-methylsterigmatocystin (OMST), the respective penultimate and ultimate precursors of AF. Although these precursors are chemically and structurally very similar, their accumulation differs at the species level for Aspergilli. Notable examples are A. nidulans that synthesizes only ST, A. flavus that makes predominantly AF, and A. parasiticus that generally produces either AF or OMST. Whether these differences are important in the evolutionary/ecological processes of species adaptation and diversification is unknown. Equally unknown are the specific genomic mechanisms responsible for ordering and clustering of genes in the AF pathway of Aspergillus. RESULTS: To elucidate the mechanisms that have driven formation of these clusters, we performed systematic searches of aflatoxin cluster homologs across five Aspergillus genomes. We found a high level of gene duplication and identified seven modules consisting of highly correlated gene pairs (aflA/aflB, aflR/aflS, aflX/aflY, aflF/aflE, aflT/aflQ, aflC/aflW, and aflG/aflL). With the exception of A. nomius, contrasts of mean Ka/Ks values across all cluster genes showed significant differences in selective pressure between section Flavi and non-section Flavi species. A. nomius mean Ka/Ks values were more similar to partial clusters in A. fumigatus and A. terreus. Overall, mean Ka/Ks values were significantly higher for section Flavi than for non-section Flavi species. CONCLUSION: Our results implicate several genomic mechanisms in the evolution of ST, OMST and AF cluster genes. Gene modules may arise from duplications of a single gene, whereby the function of the pre-duplication gene is retained in the copy (aflF/aflE) or the copies may partition the ancestral function (aflA/aflB). In some gene modules, the duplicated copy may simply augment/supplement a specific pathway function (aflR/aflS and aflX/aflY) or the duplicated copy may evolve a completely new function (aflT/aflQ and aflC/aflW). Gene modules that are contiguous in one species and noncontiguous in others point to possible rearrangements of cluster genes in the evolution of these species. Significantly higher mean Ka/Ks values in section Flavi compared to non-section Flavi species indicate increased positive selection acting in the evolution of genes in OMST and AF gene clusters. [Abstract/Link to Full Text]

Stich M, Briones C, Manrubia SC
Collective properties of evolving molecular quasispecies.
BMC Evol Biol. 2007;7110.
BACKGROUND: RNA molecules, through their dual appearance as sequence and structure, represent a suitable model to study evolutionary properties of quasispecies. The essential ingredient in this model is the differentiation between genotype (molecular sequences which are affected by mutation) and phenotype (molecular structure, affected by selection). This framework allows a quantitative analysis of organizational properties of quasispecies as they adapt to different environments, such as their robustness, the effect of the degeneration of the sequence space, or the adaptation under different mutation rates and the error threshold associated. RESULTS: We describe and analyze the structural properties of molecular quasispecies adapting to different environments both during the transient time before adaptation takes place and in the asymptotic state, once optimization has occurred. We observe a minimum in the adaptation time at values of the mutation rate relatively far from the phenotypic error threshold. Through the definition of a consensus structure, it is shown that the quasispecies retains relevant structural information in a distributed fashion even above the error threshold. This structural robustness depends on the precise shape of the secondary structure used as target of selection. Experimental results available for natural RNA populations are in qualitative agreement with our observations. CONCLUSION: Adaptation time of molecular quasispecies to a given environment is optimized at values of the mutation rate well below the phenotypic error threshold. The optimal value results from a trade-off between diversity generation and fixation of advantageous mutants. The critical value of the mutation rate is a function not only of the sequence length, but also of the specific properties of the environment, in this case the selection pressure and the shape of the secondary structure used as target phenotype. Certain functional motifs of RNA secondary structure that withstand high mutation rates (as the ubiquitous hairpin motif) might appear early in evolution and be actually frozen evolutionary accidents. [Abstract/Link to Full Text]

Kawahara BT, Quinn MT, Lambeth JD
Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes.
BMC Evol Biol. 2007;7109.
BACKGROUND: NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes. RESULTS: We assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to canonical motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity, as verified by mutational analysis. The presence of these non-canonical, but highly conserved regions suggests that all Nox/Duox may possess a common biological function remained in a long history of Nox/Duox evolution. CONCLUSION: This report provides the first comprehensive analysis of the evolution and conserved functions of Nox and Duox family members, including identification of conserved amino acid residues. These results provide a guide for future structure-function studies and for understanding the evolution of biological functions of these enzymes. [Abstract/Link to Full Text]

Asher RJ
A web-database of mammalian morphology and a reanalysis of placental phylogeny.
BMC Evol Biol. 2007;7108.
BACKGROUND: Recent publications concerning the interordinal phylogeny of placental mammals have converged on a common signal, consisting of four major radiations with some ambiguity regarding the placental root. The DNA data with which these relationships have been reconstructed are easily accessible from public databases; access to morphological characters is much more difficult. Here, I present a graphical web-database of morphological characters focusing on placental mammals, in tandem with a combined-data phylogenetic analysis of placental mammal phylogeny. RESULTS: The results reinforce the growing consensus regarding the extant placental mammal clades of Afrotheria, Xenarthra, Euarchontoglires, and Laurasiatheria. Unweighted parsimony applied to all DNA sequences and insertion-deletion (indel) characters of extant taxa alone support a placental root at murid rodents; combined with morphology this shifts to Afrotheria. Bayesian analyses of morphology, indels, and DNA support both a basal position for Afrotheria and the position of Cretaceous eutherians outside of crown Placentalia. Depending on treatment of third codon positions, the affinity of several fossils (Leptictis,Paleoparadoxia, Plesiorycteropus and Zalambdalestes) vary, highlighting the potential effect of sequence data on fossils for which such data are missing. CONCLUSION: The combined dataset supports the location of the placental mammal root at Afrotheria or Xenarthra, not at Erinaceus or rodents. Even a small morphological dataset can have a marked influence on the location of the root in a combined-data analysis. Additional morphological data are desirable to better reconstruct the position of several fossil taxa; and the graphic-rich, web-based morphology data matrix presented here will make it easier to incorporate more taxa into a larger data matrix. [Abstract/Link to Full Text]

Manchado M, Infante C, Asensio E, Ca�avate JP, Douglas SE
Comparative sequence analysis of the complete set of 40S ribosomal proteins in the Senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.) (Teleostei: Pleuronectiformes): phylogeny and tissue- and development-specific expression.
BMC Evol Biol. 2007;7107.
BACKGROUND: Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. Their levels can vary as a function of organism growth and development; however, some RPs have been associated with other cellular processes or extraribosomal functions. Their high representation in cDNA libraries has resulted in the increase of RP sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis. RESULTS: The development of large-scale genomics of Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species, has made possible the identification and systematic analysis of the complete set of RP sequences for the small (40S) ribosome subunit. Amino acid sequence comparisons showed a high similarity both between these two flatfish species and with respect to other fish and human. EST analysis revealed the existence of two and four RPS27 genes in Senegalese sole and Atlantic halibut, respectively. Phylogenetic analysis clustered RPS27 in two separate clades with their fish and mammalian counterparts. Steady-state transcript levels for eight RPs (RPS2, RPS3a, RPS15, RPS27-1, RPS27-2, RPS27a, RPS28, and RPS29) in sole were quantitated during larval development and in tissues, using a real-time PCR approach. All eight RPs exhibited different expression patterns in tissues with the lowest levels in brain. On the contrary, RP transcripts increased co-ordinately after first larval feeding reducing progressively during the metamorphic process. CONCLUSION: The genomic resources and knowledge developed in this survey will provide new insights into the evolution of Pleuronectiformes. Expression data will contribute to a better understanding of RP functions in fish, especially the mechanisms that govern growth and development in larvae, with implications in aquaculture. [Abstract/Link to Full Text]

Desmond E, Brochier-Armanet C, Gribaldo S
Phylogenomics of the archaeal flagellum: rare horizontal gene transfer in a unique motility structure.
BMC Evol Biol. 2007;7106.
BACKGROUND: As bacteria, motile archaeal species swim by means of rotating flagellum structures driven by a proton gradient force. Interestingly, experimental data have shown that the archaeal flagellum is non-homologous to the bacterial flagellum either in terms of overall structure, components and assembly. The growing number of complete archaeal genomes now permits to investigate the evolution of this unique motility system. RESULTS: We report here an exhaustive phylogenomic analysis of the components of the archaeal flagellum. In all complete archaeal genomes, the genes coding for flagellum components are co-localized in one or two well-conserved genomic clusters showing two different types of organizations. Despite their small size, these genes harbor a good phylogenetic signal that allows reconstruction of their evolutionary histories. These support a history of mainly vertical inheritance for the components of this unique motility system, and an interesting possible ancient horizontal gene transfer event (HGT) of a whole flagellum-coding gene cluster between Euryarchaeota and Crenarchaeota. CONCLUSION: Our study is one of the few exhaustive phylogenomics analyses of a non-informational cell machinery from the third domain of life. We propose an evolutionary scenario for the evolution of the components of the archaeal flagellum. Moreover, we show that the components of the archaeal flagellar system have not been frequently transferred among archaeal species, indicating that gene fixation following HGT can also be rare for genes encoding components of large macromolecular complexes with a structural role. [Abstract/Link to Full Text]

Lindqvist C, Laakkonen L, Albert VA
Polyglutamine variation in a flowering time protein correlates with island age in a Hawaiian plant radiation.
BMC Evol Biol. 2007;7105.
BACKGROUND: A controversial topic in evolutionary developmental biology is whether morphological diversification in natural populations can be driven by expansions and contractions of amino acid repeats in proteins. To promote adaptation, selection on protein length variation must overcome deleterious effects of multiple correlated traits (pleiotropy). Thus far, systems that demonstrate this capacity include only ancient or artificial morphological diversifications. The Hawaiian Islands, with their linear geological sequence, present a unique environment to study recent, natural radiations. We have focused our research on the Hawaiian endemic mints (Lamiaceae), a large and diverse lineage with paradoxically low genetic variation, in order to test whether a direct relationship between coding-sequence repeat diversity and morphological change can be observed in an actively evolving system. RESULTS: Here we show that in the Hawaiian mints, extensive polyglutamine (CAG codon repeat) polymorphism within a homolog of the pleiotropic flowering time protein and abscisic acid receptor FCA tracks the natural environmental cline of the island chain, consequent with island age, across a period of 5 million years. CAG expansions, perhaps following their natural tendency to elongate, are more frequent in colonists of recently-formed, nutrient-rich islands than in their forebears on older, nutrient-poor islands. Values for several quantitative morphological variables related to reproductive investment, known from Arabidopsis fca mutant studies, weakly though positively correlate with increasing glutamine tract length. Together with protein modeling of FCA, which indicates that longer polyglutamine tracts could induce suboptimally mobile functional domains, we suggest that CAG expansions may form slightly deleterious alleles (with respect to protein function) that become fixed in founder populations. CONCLUSION: In the Hawaiian mint FCA system, we infer that contraction of slightly deleterious CAG repeats occurred because of competition for resources along the natural environmental cline of the island chain. The observed geographical structure of FCA variation and its correlation with morphologies expected from Arabidopsis mutant studies may indicate that developmental pleiotropy played a role in the diversification of the mints. This discovery is important in that it concurs with other suggestions that repetitive amino acid motifs might provide a mechanism for driving morphological evolution, and that variation at such motifs might permit rapid tuning to environmental change. [Abstract/Link to Full Text]

Mayer WE, Herrmann M, Sommer RJ
Phylogeny of the nematode genus Pristionchus and implications for biodiversity, biogeography and the evolution of hermaphroditism.
BMC Evol Biol. 2007;7104.
BACKGROUND: The nematode Pristionchus pacificus has originally been developed as a satellite organism for comparison to Caenorhabditis elegans. A 10X coverage of the whole genome of P. pacificus is available, making P. pacificus the first non-Caenorhabditis nematode with a fully sequenced genome. The macroevolutionary comparison between P. pacificus and C. elegans has been complemented by microevolutionary studies of closely related strains and species within the genus Pristionchus. In addition, new understanding of the biology of Pristionchus from field studies, demonstrating a close association with various scarab beetles and the Colorado potato beetle, supports consideration of this nematode in studies of ecosystems. In the course of field studies on four continents more than 1,200 isolates were established from 15,000 beetle specimens representing 18 Pristionchus species. Two remarkable features of the Pristionchus-beetle association are the high species specificity of the interaction and the interception of the beetle's sex communication system for host recognition by the nematodes, as suggested by chemotaxis studies. Evolutionary interpretations of differences in developmental, behavioral and ecological patterns require a phylogenetic framework of the genus Pristionchus. RESULTS: Here, we provide a robust phylogeny of all 18 available Pristionchus species based on a set of 27 ribosomal protein genes encompassing a total of 10,971 bp. The phylogenetic tree provides evidence for North American and European clades, which are embedded in a deeper clade that includes Asian species. It also indicates putative invasion events. Of the 18 Pristionchus species, 13 are gonochoristic and five are hermaphroditic. The phylogeny indicates that all hermaphroditic species have arisen independently within the genus Pristionchus. CONCLUSION: Combined ribosomal protein cDNA data can provide the basis for reconstruction of a robust phylogenetic framework for microevolutionary and biogeographic analyses. An additional major implication of our studies is the use of Pristionchus for nematode biodiversity assessments. While some species are represented by more than 100 isolates, others were found less than four times. Such patterns were observed on all continents and in all phylogenetic clades indicating that species asymmetry is a widespread phenomenon, which can now be further investigated by molecular tools. [Abstract/Link to Full Text]

Recent Articles in BMC Structural Biology

Kovalenko OV, Metcalf DG, DeGrado WF, Hemler ME
Structural organization and interactions of transmembrane domains in tetraspanin proteins.
BMC Struct Biol. 2005;511.
BACKGROUND: Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed. RESULTS: Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)n. In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80. CONCLUSION: Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that defines TM1-TM2 interaction in tetraspanins is the specific packing of bulky residues against small residues. [Abstract/Link to Full Text]

Rigden DJ
An inactivated nuclease-like domain in RecC with novel function: implications for evolution.
BMC Struct Biol. 2005;59.
BACKGROUND: The PD-(D/E)xK superfamily, containing a wide variety of other exo- and endonucleases, is a notable example of general function conservation in the face of extreme sequence and structural variation. Almost all members employ a small number of shared conserved residues to bind catalytically essential metal ions and thereby effect DNA cleavage. The crystal structure of the RecBCD prokaryotic DNA repair machinery shows that RecB contains such a nuclease domain at its C-terminus. The RecC C-terminal region was reported as having a novel fold. RESULTS: The RecC C-terminal region can be divided into an alpha/beta domain and a smaller alpha-helical bundle domain. Here we show that the alpha/beta domain is homologous to the RecB nuclease domain but lacks the features necessary for catalysis. Instead, the domain has a novel function within the nuclease superfamily--providing a hoop through which single-stranded DNA passes. Comparison with other structures of nuclease domains bound to DNA reveals strikingly different modes of ligand binding. The alpha-helical bundle domain contributes the pin which splits the DNA duplex. CONCLUSION: The demonstrated homology of RecB and RecC shows how evolution acted to produce the present RecBCD complex through aggregation of new domains as well as functional divergence and structural redeployment of existing domains. Distantly homologous nuclease(-like) domains bind DNA in highly diverse manners. [Abstract/Link to Full Text]

Pahlke D, Freund C, Leitner D, Labudde D
Statistically significant dependence of the Xaa-Pro peptide bond conformation on secondary structure and amino acid sequence.
BMC Struct Biol. 2005;58.
BACKGROUND: A reliable prediction of the Xaa-Pro peptide bond conformation would be a useful tool for many protein structure calculation methods. We have analyzed the Protein Data Bank and show that the combined use of sequential and structural information has a predictive value for the assessment of the cis versus trans peptide bond conformation of Xaa-Pro within proteins. For the analysis of the data sets different statistical methods such as the calculation of the Chou-Fasman parameters and occurrence matrices were used. Furthermore we analyzed the relationship between the relative solvent accessibility and the relative occurrence of prolines in the cis and in the trans conformation. RESULTS: One of the main results of the statistical investigations is the ranking of the secondary structure and sequence information with respect to the prediction of the Xaa-Pro peptide bond conformation. We observed a significant impact of secondary structure information on the occurrence of the Xaa-Pro peptide bond conformation, while the sequence information of amino acids neighboring proline is of little predictive value for the conformation of this bond. CONCLUSION: In this work, we present an extensive analysis of the occurrence of the cis and trans proline conformation in proteins. Based on the data set, we derived patterns and rules for a possible prediction of the proline conformation. Upon adoption of the Chou-Fasman parameters, we are able to derive statistically relevant correlations between the secondary structure of amino acid fragments and the Xaa-Pro peptide bond conformation. [Abstract/Link to Full Text]

Rossbach M, Daumke O, Klinger C, Wittinghofer A, Kaufmann M
Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold.
BMC Struct Biol. 2005;57.
BACKGROUND: aaTHEP1, the gene product of aq_1292 from Aquifex aeolicus, shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro. Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown. RESULTS: Here we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 A resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (alpha/beta/alpha)-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the beta-sheet consisting of four additional beta-strands. CONCLUSION: We conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family. [Abstract/Link to Full Text]

Cheek S, Ginalski K, Zhang H, Grishin NV
A comprehensive update of the sequence and structure classification of kinases.
BMC Struct Biol. 2005;56.
BACKGROUND: A comprehensive update of the classification of all available kinases was carried out. This survey presents a complete global picture of this large functional class of proteins and confirms the soundness of our initial kinase classification scheme. RESULTS: The new survey found the total number of kinase sequences in the protein database has increased more than three-fold (from 17,310 to 59,402), and the number of determined kinase structures increased two-fold (from 359 to 702) in the past three years. However, the framework of the original two-tier classification scheme (in families and fold groups) remains sufficient to describe all available kinases. Overall, the kinase sequences were classified into 25 families of homologous proteins, wherein 22 families (approximately 98.8% of all sequences) for which three-dimensional structures are known fall into 10 fold groups. These fold groups not only include some of the most widely spread proteins folds, such as the Rossmann-like fold, ferredoxin-like fold, TIM-barrel fold, and antiparallel beta-barrel fold, but also all major classes (all alpha, all beta, alpha+beta, alpha/beta) of protein structures. Fold predictions are made for remaining kinase families without a close homolog with solved structure. We also highlight two novel kinase structural folds, riboflavin kinase and dihydroxyacetone kinase, which have recently been characterized. Two protein families previously annotated as kinases are removed from the classification based on new experimental data. CONCLUSION: Structural annotations of all kinase families are now revealed, including fold descriptions for all globular kinases, making this the first large functional class of proteins with a comprehensive structural annotation. Potential uses for this classification include deduction of protein function, structural fold, or enzymatic mechanism of poorly studied or newly discovered kinases based on proteins in the same family. [Abstract/Link to Full Text]

Saad AS
Orientation determination by wavelets matching for 3D reconstruction of very noisy electron microscopic virus images.
BMC Struct Biol. 2005;55.
BACKGROUND: In order to perform a 3D reconstruction of electron microscopic images of viruses, it is necessary to determine the orientation (Euler angels) of the 2D projections of the virus. The projections containing high resolution information are usually very noisy. This paper proposes a new method, based on weighted-projection matching in wavelet space for virus orientation determination. In order to speed the retrieval of the best match between projections from a model and real virus particle, a hierarchical correlation matching method is also proposed. RESULTS: A data set of 600 HSV-1 capsid particle images in different orientations was used to test the proposed method. An initial model of about 40 A resolutions was used to generate projections of an HSV-1 capsid. Results show that a significant improvement, in terms of accuracy and speed, is obtained for the initial orientation estimates of noisy herpes virus images. For the bacteriophage (P22), the proposed method gave the correct reconstruction compared to the model, while the classical method failed to resolve the correct orientations of the smooth spherical P22 viruses. CONCLUSION: This paper introduces a new method for orientation determination of low contrast images and highly noisy virus particles. This method is based on weighted projection matching in wavelet space, which increases the accuracy of the orientations. A hierarchical implementation of this method increases the speed of orientation determination. The estimated number of particles needed for a higher resolution reconstruction increased exponentially. For a 6 A resolution reconstruction of the HSV virus, 50,000 particles are necessary. The results show that the proposed method reduces the amount of data needed in a reconstruction by at least 50 %. This may result in savings 2 to 3 man-years invested in acquiring images from the microscope and data processing. Furthermore, the proposed method is able to determine orientations for some difficult particles like P22 with accuracy and consistency. Recently a low PH sindbis capsid was determined with the proposed method, where other methods based on the common line fail. [Abstract/Link to Full Text]

Bella J, Humphries MJ
Calpha-H...O = C hydrogen bonds contribute to the specificity of RGD cell-adhesion interactions.
BMC Struct Biol. 2005;54.
BACKGROUND: The Arg-Gly-Asp (RGD) cell adhesion sequence occurs in several extracellular matrix molecules known to interact with integrin cell-surface receptors. Recently published crystal structures of the extracellular regions of two integrins in complex with peptides containing or mimicking the RGD sequence have identified the Arg and Asp residues as key specificity determinants for integrin recognition, through hydrogen bonding and metal coordination interactions. The central Gly residue also appears to be in close contact with the integrin surface in these structures. RESULTS: When hydrogen atoms are modelled on the central Gly residue with standard stereochemistry, the interaction between this residue and a carbonyl group in the integrin surface shows all the hallmarks of Calpha-H...O = C hydrogen bonding, as seen in the collagen triple helix and in many crystal structures of small organic molecules. Moreover, molecular dynamic simulations of the docking of RGD-containing fragments on integrin surfaces support the occurrence of these interactions. There appears to be an array of four weak and conventional hydrogen bonds lining up the RGD residues with main chain carbonyl groups in the integrin surface. CONCLUSIONS: The occurrence of weak Calpha-H...O = C hydrogen bonds in the RGD-integrin interaction highlights the importance of the conserved Gly residue in the RGD motif and its contribution to integrin-ligand binding specificity. Our analysis shows how weak hydrogen bonds may also play important biological roles by contributing to the specificity of macromolecular recognition. [Abstract/Link to Full Text]

Armengaud J, Dedieu A, Solques O, Pellequer JL, Quemeneur E
Deciphering structure and topology of conserved COG2042 orphan proteins.
BMC Struct Biol. 2005;53.
BACKGROUND: The cluster of orthologous group COG2042 has members in all sequenced Eukaryota as well as in many Archaea. The cellular function of these proteins of ancient origin remains unknown. PSI-BLAST analysis does not indicate a possible link with even remotely-related proteins that have been functionally or structurally characterized. As a prototype among COG2042 orthologs, SSO0551 protein from the hyperthermophilic archaeon Sulfolobus solfataricus was purified to homogeneity for biophysical characterization. RESULTS: The untagged protein is thermostable and behaves as a monomeric protein in gel filtration experiment. Several mass spectrometry-based strategies were combined to obtain a set of low resolution structural information. Kinetic data from limited proteolysis with various endoproteases are concordant in pointing out that region Glu73-Arg78 is hyper-sensitive, and thus accessible and flexible. Lysine labeling with NHS-biotin and cross-linking with DTSSP revealed that the 35 amino acid RLI motif at the N terminus is solvent exposed. Cross-links between Lys10-Lys14 and Lys23-Lys25 indicate that these residues are spatially close and in adequate conformation to be cross-linked. These experimental data have been used to rank multiple three-dimensional models generated by a de novo procedure. CONCLUSION: Our data indicate that COG2042 proteins may share a novel fold. Combining biophysical, mass-spectrometry data and molecular model is a useful strategy to obtain structural information and to help in prioritizing targets in structural genomics programs. [Abstract/Link to Full Text]

Chmiel AA, Bujnicki JM, Skowronek KJ
A homology model of restriction endonuclease SfiI in complex with DNA.
BMC Struct Biol. 2005;52.
BACKGROUND: Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified. RESULTS: SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN--NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN--NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis. CONCLUSIONS: The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the alpha-class (EcoRI-like), and the beta-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses. [Abstract/Link to Full Text]

da Silveira NJ, Arcuri HA, Bonalumi CE, de Souza FP, Mello IM, Rahal P, Pinho JR, de Azevedo WF
Molecular models of NS3 protease variants of the Hepatitis C virus.
BMC Struct Biol. 2005;51.
BACKGROUND: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. RESULTS: The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. CONCLUSIONS: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis. [Abstract/Link to Full Text]

Wernimont AK, Weissenhorn W
Crystal structure of subunit VPS25 of the endosomal trafficking complex ESCRT-II.
BMC Struct Biol. 2004 Dec 4;4(1):10.
BACKGROUND: Down-regulation of plasma membrane receptors via the endocytic pathway involves their monoubiquitylation, transport to endosomal membranes and eventual sorting into multi vesicular bodies (MVB) destined for lysosomal degradation. Successive assemblies of Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II and III) largely mediate sorting of plasma membrane receptors at endosomal membranes, the formation of multivesicular bodies and their release into the endosomal lumen. In addition, the human ESCRT-II has been shown to form a complex with RNA polymerase II elongation factor ELL in order to exert transcriptional control activity. RESULTS: Here we report the crystal structure of Vps25 at 3.1 A resolution. Vps25 crystallizes in a dimeric form and each monomer is composed of two winged helix domains arranged in tandem. Structural comparisons detect no conformational changes between unliganded Vps25 and Vps25 within the ESCRT-II complex composed of two Vps25 copies and one copy each of Vps22 and Vps36 12. CONCLUSIONS: Our structural analyses present a framework for studying Vps25 interactions with ESCRT-I and ESCRT-III partners. Winged helix domain containing proteins have been implicated in nucleic acid binding and it remains to be determined whether Vps25 has a similar activity which might play a role in the proposed transcriptional control exerted by Vps25 and/or the whole ESCRT-II complex. [Abstract/Link to Full Text]

Osborne MJ, Siddiqui N, Iannuzzi P, Gehring K
The solution structure of ChaB, a putative membrane ion antiporter regulator from Escherichia coli.
BMC Struct Biol. 2004 Aug 11;49.
BACKGROUND: ChaB is a putative regulator of ChaA, a Na+/H+ antiporter that also has Ca+/H+ activity in E. coli. ChaB contains a conserved 60-residue region of unknown function found in other bacteria, archaeabacteria and a series of baculoviral proteins. As part of a structural genomics project, the structure of ChaB was elucidated by NMR spectroscopy. RESULTS: The structure of ChaB is composed of 3 alpha-helices and a small sheet that pack tightly to form a fold that is found in the cyclin-box family of proteins. CONCLUSION: ChaB is distinguished from its putative DNA binding sequence homologues by a highly charged flexible loop region that has weak affinity to Mg2+ and Ca2+ divalent metal ions. [Abstract/Link to Full Text]

Wang K, Fain B, Levitt M, Samudrala R
Improved protein structure selection using decoy-dependent discriminatory functions.
BMC Struct Biol. 2004 Jun 18;48.
BACKGROUND: A key component in protein structure prediction is a scoring or discriminatory function that can distinguish near-native conformations from misfolded ones. Various types of scoring functions have been developed to accomplish this goal, but their performance is not adequate to solve the structure selection problem. In addition, there is poor correlation between the scores and the accuracy of the generated conformations. RESULTS: We present a simple and nonparametric formula to estimate the accuracy of predicted conformations (or decoys). This scoring function, called the density score function, evaluates decoy conformations by performing an all-against-all Calpha RMSD (Root Mean Square Deviation) calculation in a given decoy set. We tested the density score function on 83 decoy sets grouped by their generation methods (4state_reduced, fisa, fisa_casp3, lmds, lattice_ssfit, semfold and Rosetta). The density scores have correlations as high as 0.9 with the Calpha RMSDs of the decoy conformations, measured relative to the experimental conformation for each decoy.We previously developed a residue-specific all-atom probability discriminatory function (RAPDF), which compiles statistics from a database of experimentally determined conformations, to aid in structure selection. Here, we present a decoy-dependent discriminatory function called self-RAPDF, where we compiled the atom-atom contact probabilities from all the conformations in a decoy set instead of using an ensemble of native conformations, with a weighting scheme based on the density scores. The self-RAPDF has a higher correlation with Calpha RMSD than RAPDF for 76/83 decoy sets, and selects better near-native conformations for 62/83 decoy sets. Self-RAPDF may be useful not only for selecting near-native conformations from decoy sets, but also for fold simulations and protein structure refinement. CONCLUSIONS: Both the density score and the self-RAPDF functions are decoy-dependent scoring functions for improved protein structure selection. Their success indicates that information from the ensemble of decoy conformations can be used to derive statistical probabilities and facilitate the identification of near-native structures. [Abstract/Link to Full Text]

Mira H, Vilar M, Esteve V, Martinell M, Kogan MJ, Giralt E, Salom D, Mingarro I, Pe�arrubia L, P�rez-Pay� E
Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.
BMC Struct Biol. 2004 Jun 4;47.
BACKGROUND: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH. RESULTS: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in beta-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide. CONCLUSION: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future. [Abstract/Link to Full Text]

Degtyarenko K, Contrino S
COMe: the ontology of bioinorganic proteins.
BMC Struct Biol. 2004 Feb 27;43.
BACKGROUND: Many characterised proteins contain metal ions, small organic molecules or modified residues. In contrast, the huge amount of data generated by genome projects consists exclusively of sequences with almost no annotation. One of the goals of the structural genomics initiative is to provide representative three-dimensional (3-D) structures for as many protein/domain folds as possible to allow successful homology modelling. However, important functional features such as metal co-ordination or a type of prosthetic group are not always conserved in homologous proteins. So far, the problem of correct annotation of bioinorganic proteins has been largely ignored by the bioinformatics community and information on bioinorganic centres obtained by methods other than crystallography or NMR is only available in literature databases. RESULTS: COMe (Co-Ordination of Metals) represents the ontology for bioinorganic and other small molecule centres in complex proteins. COMe consists of three types of entities: 'bioinorganic motif' (BIM), 'molecule' (MOL), and 'complex proteins' (PRX), with each entity being assigned a unique identifier. A BIM consists of at least one centre (metal atom, inorganic cluster, organic molecule) and two or more endogenous and/or exogenous ligands. BIMs are represented as one-dimensional (1-D) strings and 2-D diagrams. A MOL entity represents a 'small molecule' which, when in complex with one or more polypeptides, forms a functional protein. The PRX entities refer to the functional proteins as well as to separate protein domains and subunits. The complex proteins in COMe are subdivided into three categories: (i) metalloproteins, (ii) organic prosthetic group proteins and (iii) modified amino acid proteins. The data are currently stored in both XML format and a relational database and are available at http://www.ebi.ac.uk/come/. CONCLUSION: COMe provides the classification of proteins according to their 'bioinorganic' features and thus is orthogonal to other classification schemes, such as those based on sequence similarity, 3-D fold, enzyme activity, or biological process. The hierarchical organisation of the controlled vocabulary allows both for annotation and querying at different levels of granularity. [Abstract/Link to Full Text]

Scheich C, Leitner D, Sievert V, Leidert M, Schlegel B, Simon B, Letunic I, B�ssow K, Diehl A
Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis.
BMC Struct Biol. 2004 Mar 8;44.
BACKGROUND: High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination. RESULTS: 88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC) was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains) were quickly identified as being well folded and suitable for structural analysis. CONCLUSION: The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics. [Abstract/Link to Full Text]

Bracher A, Weissenhorn W
Crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei reveals conformational plasticity at its C-terminus.
BMC Struct Biol. 2004 Mar 15;46.
BACKGROUND: Intracellular membrane fusion processes are mediated by the spatial and temporal control of SNARE complex assembly that results in the formation of a four-helical bundle, composed of one vesicle SNARE and three target membrane SNARE polypeptide chains. Syntaxins are essential t-SNAREs and are characterized by an N-terminal Habc domain, a flexible linker region, a coiled-coil or SNARE motif and a membrane anchor. The N-terminal Habc domain fulfills important regulatory functions while the coiled-coil motif, present in all SNAREs, is sufficient for SNARE complex formation, which is thought to drive membrane fusion. RESULTS: Here we report the crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei, s-syntaxin. Squid Habc crystallizes as a dimer and the monomer structure consists of a three-helical bundle. One molecule is strikingly similar to mammalian syntaxin 1A while the second one shows a structural deviation from the common fold in that the C-terminal part of helix C unwinds and adopts an extended conformation. CONCLUSION: Conservation of surface residues indicates that the cytosolic part of s-syntaxin can adopt an auto-inhibitory closed conformation that may bind squid neuronal Sec1, s-Sec1, in the same manner as observed in structure of the rat nSec1/syntaxin 1A complex. Furthermore, despite the overall structural similarity, the observed changes at the C-terminus of one molecule indicate structural plasticity in neuronal syntaxin. Implications of the structural conservation and the changes are discussed with respect to potential Habc domain binding partners such as Munc13, which facilitates the transition from the closed to the open conformation. [Abstract/Link to Full Text]

Teplyakov A, Pullalarevu S, Obmolova G, Doseeva V, Galkin A, Herzberg O, Dauter M, Dauter Z, Gilliland GL
Crystal structure of the YffB protein from Pseudomonas aeruginosa suggests a glutathione-dependent thiol reductase function.
BMC Struct Biol. 2004 Mar 8;45.
BACKGROUND: The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. RESULTS: The structure was determined at 1.0 A resolution by single-wavelength anomalous diffraction. The fold is very similar to that of arsenate reductase, which is an extension of the thioredoxin fold. CONCLUSION: Given the conservation of the functionally important residues and the ability to bind glutathione, YffB is likely to function as a GSH-dependent thiol reductase. [Abstract/Link to Full Text]

Rudenskaya GN, Kislitsin YA, Rebrikov DV
Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes.
BMC Struct Biol. 2004 Jan 20;42.
BACKGROUND: In this paper, we describe cDNA cloning of a new anionic trypsin and a collagenolytic serine protease from king crab Paralithodes camtschaticus and the elucidation of their primary structures. Constructing the phylogenetic tree of these enzymes was undertaken in order to prove the evolutionary relationship between them. RESULTS: The mature trypsin PC and collagenolytic protease PC contain 237 (Mcalc 24.8 kDa) and 226 amino acid residues (Mcalc 23.5 kDa), respectively. Alignments of their amino acid sequences revealed a high degree of the trypsin PC identity to the trypsin from Penaeus vannamei (approximately 70%) and of the collagenolytic protease PC identity to the collagenase from fiddler crab Uca pugilator (76%). The phylogenetic tree of these enzymes was constructed. CONCLUSIONS: Primary structures of the two mature enzymes from P. camtschaticus were obtained and compared with those of other proteolytic proteins, including some enzymes from brachyurans. A phylogenetic analysis was also carried out. These comparisons revealed that brachyurins are closely related to their vertebrate and bacterial congeners, occupy an intermediate position between them, and their study significantly contributes to the understanding of the evolution and function of serine proteases. [Abstract/Link to Full Text]

Taylor WR, Stoye JP
Consensus structural models for the amino terminal domain of the retrovirus restriction gene Fv1 and the murine leukaemia virus capsid proteins.
BMC Struct Biol. 2004 Jan 12;41.
BACKGROUND: The mouse Fv1 (friend virus) susceptibility gene inhibits the development of the murine leukaemia virus (MLV) by interacting with its capsid (CA) protein. As no structures are available for these proteins we have constructed molecular models based on distant sequence similarity to other retroviral capsid proteins. RESULTS: Molecular models were constructed for the amino terminal domains of the probable capsid-like structure for the mouse Fv1 gene product and the capsid protein of the MLV. The models were based on sequence alignments with a variety of other retrovirus capsid proteins. As the sequence similarity of these proteins with MLV and especially Fv1 is very distant, a threading method was employed that incorporates predicted secondary structure and multiple sequence information. The resulting models were compared with equivalent models constructed using the sequences of the capsid proteins of known structure. CONCLUSIONS: These comparisons suggested that the MLV model should be accurate in the core but with significant uncertainty in the loop regions. The Fv1 model may have some additional errors in the core packing of its helices but the resulting model gave some support to the hypothesis that it adopts a capsid-like structure. [Abstract/Link to Full Text]

Saad AS
Wavelets filtering for classification of very noisy electron microscopic single particles images--application on structure determination of VP5-VP19C recombinant.
BMC Struct Biol. 2003 Dec 11;39.
BACKGROUND: Images of frozen hydrated [vitrified] virus particles were taken close-to-focus in an electron microscope containing structural signals at high spatial frequencies. These images had very low contrast due to the high levels of noise present in the image. The low contrast made particle selection, classification and orientation determination very difficult. The final purpose of the classification is to improve the signal-to-noise ratio of the particle representing the class, which is usually the average. In this paper, the proposed method is based on wavelet filtering and multi-resolution processing for the classification and reconstruction of this very noisy data. A multivariate statistical analysis (MSA) is used for this classification. RESULTS: The MSA classification method is noise dependent. A set of 2600 projections from a 3D map of a herpes simplex virus--to which noise was added--was classified by MSA. The classification shows the power of wavelet filtering in enhancing the quality of class averages (used in 3D reconstruction) compared to Fourier band pass filtering. A 3D reconstruction of a recombinant virus (VP5-VP19C) is presented as an application of multi-resolution processing for classification and reconstruction. CONCLUSION: The wavelet filtering and multi-resolution processing method proposed in this paper offers a new way for processing very noisy images obtained from electron cryo-microscopes. The multi-resolution and filtering improves the speed and accuracy of classification, which is vital for the 3D reconstruction of biological objects. The VP5-VP19C recombinant virus reconstruction presented here is an example, which demonstrates the power of this method. Without this processing, it is not possible to get the correct 3D map of this virus. [Abstract/Link to Full Text]

Wlodawer A, Durell SR, Li M, Oyama H, Oda K, Dunn BM
A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases.
BMC Struct Biol. 2003 Nov 11;38.
BACKGROUND: Tripeptidyl-peptidase I, also known as CLN2, is a member of the family of sedolisins (serine-carboxyl peptidases). In humans, defects in expression of this enzyme lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. Similar enzymes have been found in the genomic sequences of several species, but neither systematic analyses of their distribution nor modeling of their structures have been previously attempted. RESULTS: We have analyzed the presence of orthologs of human CLN2 in the genomic sequences of a number of eukaryotic species. Enzymes with sequences sharing over 80% identity have been found in the genomes of macaque, mouse, rat, dog, and cow. Closely related, although clearly distinct, enzymes are present in fish (fugu and zebra), as well as in frogs (Xenopus tropicalis). A three-dimensional model of human CLN2 was built based mainly on the homology with Pseudomonas sp. 101 sedolisin. CONCLUSION: CLN2 is very highly conserved and widely distributed among higher organisms and may play an important role in their life cycles. The model presented here indicates a very open and accessible active site that is almost completely conserved among all known CLN2 enzymes. This result is somehow surprising for a tripeptidase where the presence of a more constrained binding pocket was anticipated. This structural model should be useful in the search for the physiological substrates of these enzymes and in the design of more specific inhibitors of CLN2. [Abstract/Link to Full Text]

Ladner JE, Obmolova G, Teplyakov A, Howard AJ, Khil PP, Camerini-Otero RD, Gilliland GL
Crystal structure of Escherichia coli protein ybgI, a toroidal structure with a dinuclear metal site.
BMC Struct Biol. 2003 Sep 30;37.
BACKGROUND: The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function. RESULTS: The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid. CONCLUSION: The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme. [Abstract/Link to Full Text]

Zunszain PA, Ghuman J, Komatsu T, Tsuchida E, Curry S
Crystal structural analysis of human serum albumin complexed with hemin and fatty acid.
BMC Struct Biol. 2003 Jul 7;36.
BACKGROUND: Human serum albumin (HSA) is an abundant plasma protein that binds a wide variety of hydrophobic ligands including fatty acids, bilirubin, thyroxine and hemin. Although HSA-heme complexes do not bind oxygen reversibly, it may be possible to develop modified HSA proteins or heme groups that will confer this ability on the complex. RESULTS: We present here the crystal structure of a ternary HSA-hemin-myristate complex, formed at a 1:1:4 molar ratio, that contains a single hemin group bound to subdomain IB and myristate bound at six sites. The complex displays a conformation that is intermediate between defatted HSA and HSA-fatty acid complexes; this is likely to be due to low myristate occupancy in the fatty acid binding sites that drive the conformational change. The hemin group is bound within a narrow D-shaped hydrophobic cavity which usually accommodates fatty acid; the hemin propionate groups are coordinated by a triad of basic residues at the pocket entrance. The iron atom in the centre of the hemin is coordinated by Tyr161. CONCLUSION: The structure of the HSA-hemin-myristate complex (PDB ID 1o9x) reveals the key polar and hydrophobic interactions that determine the hemin-binding specificity of HSA. The details of the hemin-binding environment of HSA provide a structural foundation for efforts to modify the protein and/or the heme molecule in order to engineer complexes that have favourable oxygen-binding properties. [Abstract/Link to Full Text]

Hughes SJ, Tanner JA, Hindley AD, Miller AD, Gould IR
Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment.
BMC Struct Biol. 2003 Jun 4;35.
BACKGROUND: Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. RESULTS: Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations.Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. CONCLUSIONS: The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-tRNA synthetases, and may play an important functional role. We suggest a manner in which catalytic efficiency could be improved by LysU operating in an alternating sites mechanism. [Abstract/Link to Full Text]

Rekha N, Srinivasan N
Structural basis of regulation and substrate specificity of protein kinase CK2 deduced from the modeling of protein-protein interactions.
BMC Struct Biol. 2003 May 9;34.
BACKGROUND: Protein Kinase Casein Kinase 2 (PKCK2) is an ubiquitous Ser/Thr kinase expressed in all eukaryotes. It phosphorylates a number of proteins involved in various cellular processes. PKCK2 holoenzyme is catalytically active tetramer, composed of two homologous or identical and constitutively active catalytic (alpha) and two identical regulatory (beta) subunits. The tetramer cannot phosphorylate some substrates that can be phosphorylated by PKCK2alpha in isolation. The present work explores the structural basis of this feature using computational analysis and modeling. RESULTS: We have initially built a model of PKCK2alpha bound to a substrate peptide with a conformation identical to that of the substrates in the available crystal structures of other kinases complexed with the substrates/ pseudosubstrates. In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2alpha and is highly solvent exposed. Interaction of the acidic residue is observed if the substrate peptide adopts conformations as seen in beta turn, alpha helix, or 3(10) helices. This type of conformation is observed and accommodated well by PKCK2alpha in calmodulin where the phosphorylation site is at the central helix. PP2A carries sequence patterns for PKCK2alpha phosphorylation. While the possibility of PP2A being phosphorylated by PKCK2 has been raised in the literature we use the model of PP2A to generate a model of PP2A-PKCK2alpha complex. PKCK2beta undergoes phosphorylation by holoenzyme at the N-terminal region, and is accommodated very well in the limited space available at the substrate-binding site of the holoenzyme while the space is insufficient to accommodate the binding of PP2A or calmodulin in the holoenzyme. CONCLUSION: Charge and shape complimentarity seems to play a role in substrate recognition and binding to PKCK2alpha, along with the consensus pattern. The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported. The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme. [Abstract/Link to Full Text]

Briese L, Willbold D
Structure determination of human Lck unique and SH3 domains by nuclear magnetic resonance spectroscopy.
BMC Struct Biol. 2003 May 7;33.
BACKGROUND: Protein tyrosine kinases are involved in signal transduction pathways that regulate cell growth, differentiation, activation and transformation. Human lymphocyte specific kinase (Lck) is a 56 kDa protein involved in T-cell- and IL2-receptor signaling. Three-dimensional structures are known for SH3, SH2 and kinase domains of Lck as well as for other tyrosine kinases. No structure is known for the unique domain of any Src-type tyrosine kinase. RESULTS: Lck(1-120) comprising unique and SH3 domains was structurally investigated by nuclear magnetic resonance spectroscopy. We found the unique domain, in contrast to the SH3 part, to have basically no defined structural elements. The solution structure of the SH3 part could be determined with very high precision. It does not show significant differences to Lck SH3 in the absence of the unique domain. Minor differences were observed to the X-ray structure of Lck SH3. CONCLUSION: The unique domain of Lck does not contain any defined structure elements in the absence of ligands and membranes. Presence of the unique domain is not relevant to the three-dimensional structure of the Lck SH3 domain. [Abstract/Link to Full Text]

Jenwitheesuk E, Samudrala R
Improved prediction of HIV-1 protease-inhibitor binding energies by molecular dynamics simulations.
BMC Struct Biol. 2003 Apr 1;32.
BACKGROUND: The accurate prediction of enzyme-substrate interaction energies is one of the major challenges in computational biology. This study describes the improvement of protein-ligand binding energy prediction by incorporating protein flexibility through the use of molecular dynamics (MD) simulations. RESULTS: Docking experiments were undertaken using the program AutoDock for twenty-five HIV-1 protease-inhibitor complexes determined by x-ray crystallography. Protein-rigid docking without any dynamics produced a low correlation of 0.38 between the experimental and calculated binding energies. Correlations improved significantly for all time scales of MD simulations of the receptor-ligand complex. The highest correlation coefficient of 0.87 between the experimental and calculated energies was obtained after 0.1 picoseconds of dynamics simulation. CONCLUSION: Our results indicate that relaxation of protein complexes by MD simulation is useful and necessary to obtain binding energies that are representative of the experimentally determined values. [Abstract/Link to Full Text]

Iyer LM, Koonin EV, Aravind L
Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases.
BMC Struct Biol. 2003 Jan 28;31.
BACKGROUND: The eukaryotic RNA-dependent RNA polymerase (RDRP) is involved in the amplification of regulatory microRNAs during post-transcriptional gene silencing. This enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria. No evolutionary relationship between RDRP and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery. RESULTS: Using extensive sequence profile searches, we identified bacteriophage homologs of the eukaryotic RDRP. The comparison of the eukaryotic RDRP and their homologs from bacteriophages led to the delineation of the conserved portion of these enzymes, which is predicted to harbor the catalytic site. Further, detailed sequence comparison, aided by examination of the crystal structure of the DNA-dependent RNA polymerase (DDRP), showed that the RDRP and the beta' subunit of DDRP (and its orthologs in archaea and eukaryotes) contain a conserved double-psi beta-barrel (DPBB) domain. This DPBB domain contains the signature motif DbDGD (b is a bulky residue), which is conserved in all RDRPs and DDRPs and contributes to catalysis via a coordinated divalent cation. Apart from the DPBB domain, no similarity was detected between RDRP and DDRP, which leaves open two scenarios for the origin of RDRP: i) RDRP evolved at the onset of the evolution of eukaryotes via a duplication of the DDRP beta' subunit followed by dramatic divergence that obliterated the sequence similarity outside the core catalytic domain and ii) the primordial RDRP, which consisted primarily of the DPBB domain, evolved from a common ancestor with the DDRP at a very early stage of evolution, during the RNA world era. The latter hypothesis implies that RDRP had been subsequently eliminated from cellular life forms and might have been reintroduced into the eukaryotic genomes through a bacteriophage. Sequence and structure analysis of the DDRP led to further insights into the evolution of RNA polymerases. In addition to the beta' subunit, beta subunit of DDRP also contains a DPBB domain, which is, however, distorted by large inserts and does not harbor a counterpart of the DbDGD motif. The DPBB domains of the two DDRP subunits together form the catalytic cleft, with the domain from the beta' subunit supplying the metal-coordinating DbDGD motif and the one from the beta subunit providing two lysine residues involved in catalysis. Given that the two DPBB domains of DDRP contribute completely different sets of active residues to the catalytic center, it is hypothesized that the ultimate ancestor of RNA polymerases functioned as a homodimer of a generic, RNA-binding DPBB domain. This ancestral protein probably did not have catalytic activity and served as a cofactor for a ribozyme RNA polymerase. Subsequent evolution of DDRP and RDRP involved accretion of distinct sets of additional domains. In the DDRPs, these included a RNA-binding Zn-ribbon, an AT-hook-like module and a sandwich-barrel hybrid motif (SBHM) domain. Further, lineage-specific accretion of SBHM domains and other, DDRP-specific domains is observed in bacterial DDRPs. In contrast, the orthologs of the beta' subunit in archaea and eukaryotes contains a four-stranded alpha + beta domain that is shared with the alpha-subunit of bacterial DDRP, eukaryotic DDRP subunit RBP11, translation factor eIF1 and type II topoisomerases. The additional domains of the RDRPs remain to be characterized. CONCLUSIONS: Eukaryotic RNA-dependent RNA polymerases share the catalytic double-psi beta-barrel domain, containing a signature metal-coordinating motif, with the universally conserved beta' subunit of DNA-dependent RNA polymerases. Beyond this core catalytic domain, the two classes of RNA polymerases do not have common domains, suggesting early divergence from a common ancestor, with subsequent independent domain accretion. The beta-subunit of DDRP contains another, highly diverged DPBB domain. The presence of two distinct DPBB domains in two subunits of DDRP is compatible with the hypothesis that the ith the hypothesis that the ultimate ancestor of RNA polymerases was a RNA-binding DPBB domain that had no catalytic activity but rather functioned as a homodimeric cofactor for a ribozyme polymerase. [Abstract/Link to Full Text]

Anishetty S, Pennathur G, Anishetty R
Tripeptide analysis of protein structures.
BMC Struct Biol. 2002 Dec 21;29.
BACKGROUND: An efficient building block for protein structure prediction can be tripeptides. 8000 different tripeptides from a dataset of 1220 high resolution (<or= 2.0 degrees A) structures from the Protein Data Bank (PDB) have been looked at, to determine which are structurally rigid and non-rigid. This data has been statistically analyzed, discussed and summarized. The entire data can be utilized for the building of protein structures. RESULTS: Tripeptides have been classified into three categories: rigid, non-rigid and intermediate, based on the relative structural rigidity between Calpha and Cbeta atoms in a tripeptide. We found that 18% of the tripeptides in the dataset can be classified as rigid, 4% as non-rigid and 78% as intermediate. Many rigid tripeptides are made of hydrophobic residues, however, there are tripeptides with polar side chains forming rigid structures. The bulk of the tripeptides fall in the intermediate class while very small numbers actually fall in the non-rigid class. Structurally all rigid tripeptides essentially form two structural classes while the intermediate and non-rigid tripeptides fall into one structural class. This notion of rigidity and non-rigidity is designed to capture side chain interactions but not secondary structures. CONCLUSIONS: Rigid tripeptides have no correlation with the secondary structures in proteins and hence this work is complementary to such studies. Tripeptide data may be used to predict plausible structures for oligopeptides and for denovo protein design. [Abstract/Link to Full Text]