In vivo oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment

The effects of frequency of follicular aspiration and treatment of donor cattle with FSH on in vivo oocyte recovery and in vitro embryo production were studied. Simmental heifers (n = 24) formed 8 replicates of 3 treatments in which oocyte donors were aspirated 1) once a week, 2) twice a week, or 3) once a week following treatment with FSH for 3 d prior to aspiration. Oocytes were graded, washed, matured for 20 to 24 h and then inseminated with frozen/thawed semen from a single sire, followed by co-culture on granulosa cell layers. Embryo development was observed until Day 7 after insemination. Significantly fewer follicles per heifer per week were counted (14.7+/-2.3 vs. 27.4+/-3.1 vs. 23.1+/-2.8) and aspirated (12.0+/-2.0 vs. 21.8+/-2.7 vs. 20.1+/-2.6) in heifers on the once-weekly than twice-weekly aspiration treatment (P<0.01) or on the once-weekly aspiration after FSH treatment (P<0.05). There were no significant differences between treatments in the total number of oocytes recovered per week (5.6+/-1.2 vs. 8.9+/-1.5 vs. 6.1+/-1.2), but significantly more oocytes per heifer per week recovered from animals treated with FSH were graded Category 1 (2.8+/-0.4), i.e., >4 layers good cumulus with a clear, even cytoplasm, than from animals aspirated once (0.9+/-0.2; P<0.01) or twice a week (1.5+/-0.3; P<0.05). The number of transferable morulae plus blastocysts produced per heifer per week was higher from animals aspirated twice a week (2.4+/-0.4; P<0.05) or once a week following FSH treatment (2.1+/-0.4; P<0.05) than from animals aspirated once a week without FSH treatment (1.0+/-0.3). In conclusion, FSH treatment of bovine oocyte donors aspirated once a week enabled a similar number of transferable embryos to be produced per donor week as aspiration twice a week without FSH treatment. These 2 treatments produced twice as many transferable embryos per donor week as aspiration once a week without FSH treatment.     

Biomechanical modeling and sensitivity analysis of bipedal running ability. I. Extant taxa

I used a simple mathematical model of the inverse dynamics of locomotion to estimate the minimum muscle masses required to maintain quasi-static equilibrium about the four main limb joints at mid-stance of fast running. Models of 10 extant taxa (a human, a kangaroo, two lizards, an alligator, and five birds) were analyzed in various bipedal poses to examine how anatomy, size, limb orientation, and other model parameters influence running ability. I examined how the muscle masses required for fast running compare to the muscle masses that are actually able to exert moments about the hip, knee, ankle, and toe joints, to see how support ability varies across the limb. I discuss the assumptions and limitations of the models, using sensitivity analysis to see how widely the results differed with feasible parameter input values. Even with a wide range of input values, the models validated the analysis procedure. Animals that are known to run bipedally were calculated as able to preserve quasi-static equilibrium about their hindlimb joints at mid-stance, whereas non-bipedal runners (iguanas and alligators) were recognized as having too little muscle mass to run quickly in bipedal poses. Thus, this modeling approach should be reliable for reconstructing running ability in extinct bipeds such as nonavian dinosaurs. The models also elucidated how key features are important for bipedal running capacity, such as limb orientation, muscle moment arms, muscle fascicle lengths, and body size. None of the animals modeled had extensor muscle masses acting about any one joint that were 7% or more of their body mass, which provides a reasonable limit for how much muscle mass is normally apportioned within a limb to act about a particular joint. The models consistently showed that a key biomechanical limit on running ability is the capacity of ankle extensors to generate sufficiently large joint moments. Additionally, the analysis reveals how large ratite birds remain excellent runners despite their larger size; they have apomorphically large extensor muscles with relatively high effective mechanical advantage. Finally, I reconstructed the evolution of running ability in the clade Reptilia, showing that the ancestors of extant birds likely were quite capable runners, even though they had already reduced key hip extensors such as M. caudofemoralis longus.     

Chemistry and biology of maculalactone A from the marine cyanobacterium <Emphasis Type="Italic">Kyrtuthrix maculans</Emphasis>

Maculalactone A is the most abundant secondary metabolite in Kyrtuthrix maculans, a marine cyanobacterium found in the mid-high shore of moderately exposed to sheltered rocky shores in Hong Kong and South East Asia. This species appears to survive as pure colonies forming distinct black zones on the rock. Maculalactone A may provide K. maculans with a chemical defense against several marine organisms, including the common grazer, Chlorostoma argyrostoma and settlement by larvae of the barnacles, Tetraclita japonica, Balanus amphitrite and Ibla cumingii. The natural concentration of maculalactone A varied with season and also with tidal height on the shore and although a strong positive linear correlation was observed between maculalactone A concentration and herbivore grazing pressure, manipulative experiments demonstrated that grazing pressure was not directly responsible for inducing the biosynthesis of this metabolite. The potential of maculalactone A as a natural marine anti-fouling agent (i.e. as an alternative to environmentally-damaging copper- and tin-based anti-fouling paints) was investigated after achieving a gram-scale synthesis of this compound. Preliminary field trials with anti-fouling paints which contained synthetic maculalactone A as the active principle have confirmed that this compound seems to have a specific activity against molluscan settlers.     

Screening protein refolding using surface plasmon resonance

Surface plasmon resonance (SPR) measurements were used to screen refolding conditions to identify a physicochemical environment which gives an acceptable refolding yield for samples of glutathione-S-transferase (GST) denatured in 6 M guanidine hydrochloride and 32 mM dithiothreitol. The SPR measurements were performed on carboxymethylcellulose coated chips that could accommodate two separate flow paths. One side of the chip was derivatized with immobilized glutathione and the other with goat anti-GST antibody. This created a dual-derivatized chip capable of showing both the presence of GST and providing a measure of enzyme activity. The dual-derivatized chip could be regenerated using a two-step washing procedure and reused to analyze multiple samples from a screening study of protein refolding conditions. SPR measurements have been shown to be suitable for screening protein refolding conditions due to the high sensitivity, ease of chip regeneration and the ability to incorporate a control in the experimental design. The combination of such advantages with the high-throughput automated SPR systems currently available may be a valuable approach to determine conditions suitable for protein refolding following insoluble expression in a bacterial host.     

Candidate protein selection for diagnostic markers of African trypanosomiasis

The ability to accurately diagnose the presence of an infective micro-organism is not only important for individual human and animal health and wellbeing, but is also central to surveillance programmes. Effective and sustainable control of many diseases in the developing world depends on the availability of field applicable diagnostics that are cheap, reliable, simple in design and application, and which provide immediate results. This review examines how the genome sequences can be used in the selection of potential candidate proteins for developing new serodiagnostics for African trypanosomiasis.     

Molecular phylogeography and systematics of the arid-zone members of the Egernia whitii (Lacertilia: Scincidae) species group

We assembled a molecular phylogeny for the arid-zone members of the Egernia whitii species group to test Pianka's [Zoogeography and speciation of Australian desert lizards: an ecological perspective, Copeia (1972) 127-145] hypothesis that habitat specificity to the three major arid-zone vegetation communities is the primary cause of lizard speciation within the arid interior of Australia. This hypothesis predicts that species should exhibit phylogeographic structuring concordant with the major arid-zone vegetation types. Sequence data were obtained from four of the five arid-zone members of the E. whitii species group, and from across the ranges of the ecologically generalized E. inornata and E. multiscutata and the more specialized E. striata. We targeted a fragment (696 base pair (bp)) of the mitochondrial genome comprising the 3' half of the ND4 gene. We analysed the data using parsimony, maximum likelihood and Bayesian methods. Our phylogeny confirms the monophyly of the arid-zone members of the species group, although the phylogenetic relationships among species were not fully resolved. Although our topology does not support the recognition of the existing subspecies within E. multiscutata, there is a substantial phylogeographic break between South Australian/Victorian (Clade 1) and Western Australian (Clade 2) populations. We found considerable phylogeographic structure within E. inornata, with six major clades identified. However, these clades were not concordant with the distribution of habitat types in the arid-zone. Phylogeographic structure was also observed in the more specialized E. striata, although our analysis revealed close phylogenetic affinities between the sympatric species E. striata and E. kintorei. Shimodaira-Hasegawa topology tests were equivocal in regard to whether the phylogeographic structure within E. striata was in accordance with Pianka's predictions. Although our data failed to provide strong support for the suggestion that ecological and habitat factors are responsible for the diversification of arid-zone lizards, most E. inornata and E. striata populations had similar habitats, indicating that adaptation to particular habitats may have some role in the speciation of lizards in the Australian arid-zone.     

A model of structure and catalysis for ketoreductase domains in modular polyketide synthases

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.     

Effects of chair restraint on the strength of the tibia in rhesus monkeys

To determine the effects of the relative inactivity and unloading on the strength of the tibias of monkeys, Macaca mulatta, we used a non-invasive test to measure bending stiffness, or EI (Nm2), a mechanical property. The technique was validated by comparisons of in vivo measurements with standard measures of EI in the same bones post-mortem (r2 = 0.95, P < 0.0001). Inter-test precision was 4.28+/-1.4%. Normative data in 24 monkeys, 3.0+/-0.7 years and 3.6+/-0.6 kg, revealed EI to be 16% higher in the right than left tibia (4.4+/-1.6 vs. 3.7+/-1.6 Nm2, P < 0.05). Five monkeys, restrained in chairs for 14 days, showed decreases in EI. There were no changes in EI in two chaired monkeys that lost weight during a 2-week space flight. The factors that account for both the decreases in bone mechanical properties after chair restraint at 1 g and lack of change after microgravity remain to be identified. Metabolic factors associated with body weight changes are suggested by our results.     

A C-type lectin from the tunicate, Styela plicata, that modulates cellular activity

Previous studies have identified proteins from tunicates (invertebrate members of the Phylum Chordata) that have physicochemical and functional properties similar to those of the inflammatory cytokine, interleukin 1 (IL-1). Here we characterize one of those proteins from the tunicate, Styela plicata, that can stimulate tunicate and mammalian cell proliferation, activate phagocytosis, increase interleukin 2 (IL-2) secretion by mammalian peripheral blood mononuclear cells and enhance IL-2 receptor (IL-2R) expression by mammalian EL-4.IL-2 cells. Partial amino acid sequence data showed that the S. plicata protein resembles three C-type lectins (TC14, TC14-1 and TC14-2) from a closely related tunicate species, Polyandrocarpa misakiensis. Its similarity to carbohydrate recognition domains (CRDs) from P. misakiensis lectins suggests that the S. plicata protein modulates the activities of mammalian immunocompetent cells by interacting with carbohydrate moieties of glycosylated cell surface receptors.     

Pelvic and hindlimb musculature of Tyrannosaurus rex (Dinosauria: Theropoda)

In this article, we develop a new reconstruction of the pelvic and hindlimb muscles of the large theropod dinosaur Tyrannosaurus rex. Our new reconstruction relies primarily on direct examination of both extant and fossil turtles, lepidosaurs, and archosaurs. These observations are placed into a phylogenetic context and data from extant taxa are used to constrain inferences concerning the soft-tissue structures in T. rex. Using this extant phylogenetic bracket, we are able to offer well-supported inferences concerning most of the hindlimb musculature in this taxon. We also refrain from making any inferences for certain muscles where the resulting optimizations are ambiguous. This reconstruction differs from several previous attempts and we evaluate these discrepancies. In addition to providing a new and more detailed understanding of the hindlimb morphology of T. rex--the largest known terrestrial biped--this reconstruction also helps to clarify the sequence of character-state change along the line to extant birds.