Cardiac troponin T forms a tetramer in vitro

Cardiac troponin T (cTnT) is a myofibrillar protein essential for calcium-dependent contraction. This property has led to functional studies of developmentally expressed cTnT isoforms and mutants identified in patients with hypertrophic cardiomyopathy. The release of cTnT into the serum following myocardial infarction has led to the development of antibody-based assays for measuring cTnT serum concentration. We examined the behavior of cTnT in solution. Recombinant human cTnT3, the dominant isoform in the adult human heart, was used. The protein was pure and functional, as demonstrated by SDS-PAGE and surface plasmon resonance. cTnT3 was found to bind specifically and in a concentration-dependent manner to cTnC. Routine size exclusion chromatography suggested a higher-than-expected MW for cTnT. Using analytical ultracentrifugation, we found cTnT3 in solution to be mainly in the form of a tightly bound tetramer at concentrations as low as 4 micromol/L. Our sedimentation velocity and transmission electron microscopy results indicate that the tetramer's shape is elongated rather than globular. CTnT's self-association in solution is an important consideration in the design and interpretation of experiments with the aim of understanding the biochemical and biophysical properties of cTnT, its isoforms, and its mutants.     

Gene selection, alternative splicing, and post-translational processing regulate neuroligin selectivity for beta-neurexins

Neuroligins 1-4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with beta-neurexin. Both neuroligins and beta-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the beta-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of beta-neurexin and neuroligin suggested a 2:2 complex formation. The recognition properties of individual neuroligins toward beta-neurexin-1 (NX1beta), along with the influence of their splice inserts, were explored by surface plasmon resonance and affinity chromatography. Different neuroligins display a range of NX1beta affinities spanning more than 2 orders of magnitude. Whereas splice insert 4 in beta-neurexin appears to act only as a modulator of the neuroligin/beta-neurexin association, splice insert B in neuroligin-1 (NL1) is the key element regulating the NL1/NX1beta binding. Our data indicate that gene selection, mRNA splicing, and post-translational modifications combine to give rise to a controlled neuroligin recognition code with a rank ordering of affinities for particular neurexins that is conserved for the neuroligins across mammalian species.     

The growth-suppressive function of the polycomb group protein polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain

Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.     

From nest to nest--influence of ecology and reproduction on the active period of adult Gombe chimpanzees

The time spent between sleeping periods, which is called the active period, has to accommodate all essential activities, including feeding, resting, social behavior, and reproduction. To minimize costs in terms of, e.g., predation risk, suboptimal foraging, or sleep deficiency, the active period of diurnal animals should be less than or equal to the daylight period. Thus, the active period of an animal should be shaped by local environmental conditions as well as by metabolic and reproductive demands. Chimpanzees, which exhibit reduced predator pressure and a flexible fission-fusion society, were chosen as a model to explore these links. We investigated the influence of sex, female reproductive status, dominance rank, and season on the duration of the active period of adult chimpanzees at Gombe National Park, Tanzania (1975-1992). Sexually nonreceptive females had shorter active periods compared to males, while receptive females had even longer active periods than males. Dominance rank did not influence the duration of the active period of nonreceptive females, but high- and middle-ranking males had shorter active periods compared to low-ranking males. Nonreceptive females exhibited longer active periods during the dry season than in the wet season. No seasonal effect was discovered for males, perhaps because they already had long active periods in the wet season. Nonreceptive females seem to be able to accommodate all essential activities in the daylight period available, probably because they live less socially than males. Thus, the active period does not reflect differences in female competitive abilities, but does reflect such differences in males. The duration of the active period appears to be a simple, reliable tool for exploring basic responses and constraints in animal societies.     

Divergence of multimodular polyketide synthases revealed by a didomain structure

The enoylreductase (ER) is the final common enzyme from modular polyketide synthases (PKSs) to be structurally characterized. The 3.0 ?-resolution structure of the didomain comprising the ketoreductase (KR) and ER from the second module of the spinosyn PKS reveals that ER shares an ～600-?(2) interface with KR distinct from that of the related mammalian fatty acid synthase (FAS). In contrast to the ER domains of the mammalian FAS, the ER domains of the second module of the spinosyn PKS do not make contact across the two-fold axis of the synthase. This monomeric organization may have been necessary in the evolution of multimodular PKSs to enable acyl carrier proteins to access each of their cognate enzymes. The isolated ER domain showed activity toward a substrate analog, enabling us to determine the contributions of its active site residues.     

Primate life histories and dietary adaptations: a comparison of Asian colobines and macaques

Primate life histories are strongly influenced by both body and brain mass and are mediated by food availability and perhaps dietary adaptations. It has been suggested that folivorous primates mature and reproduce more slowly than frugivores due to lower basal metabolic rates as well as to greater degrees of arboreality, which can lower mortality and thus fecundity. However, the opposite has also been proposed: faster life histories in folivores due to a diet of abundant, protein-rich leaves. We compared two primate taxa often found in sympatry: Asian colobines (folivores, 11 species) and Asian macaques (frugivores, 12 species). We first described new data for a little-known colobine (Phayre's leaf monkeys, Trachypithecus phayrei crepusculus) from Phu Khieo Wildlife Sanctuary, Thailand. We then compared gestation periods, ages at first birth, and interbirth intervals in colobines and macaques. We predicted that heavier species would have slower life histories, provisioned populations would have faster life histories, and folivores would have slower life histories than frugivores. We calculated general regression models using log body mass, nutritional regime, and taxon as predictor variables. Body mass and nutritional regime had the predicted effects for all three traits. We found taxonomic differences only for gestation, which was significantly longer in colobines, supporting the idea of slower fetal growth (lower maternal energy) compared to macaques and/or advanced dental or gut development. Ages at first birth and interbirth intervals were similar between taxa, perhaps due to additional factors (e.g., allomothering, dispersal). Our results emphasize the need for additional data from wild populations and for establishing whether growth data for provisioned animals (folivores in particular) are representative of wild ones.     

Analysis of putative inhibitors of anthelmintic resistance mechanisms in cattle gastrointestinal nematodes

Effects of the cytochrome P450 inhibitor piperonyl butoxide and the P-glycoprotein inhibitor verapamil on the efficacy of ivermectin and thiabendazole were studied in vitro in susceptible and resistant isolates of the cattle parasitic nematodes Cooperia oncophora and Ostertagia ostertagi. The effects of combined use of drug and piperonyl butoxide/verapamil, respectively, were investigated in the Egg Hatch Assay, the Larval Development Assay and the Larval Migration Inhibition Assay. The effects of piperonyl butoxide and verapamil as inhibitors of thiabendazole and ivermectin responses were particularly marked for larval development, where both inhibitors were able to completely eliminate all differences between susceptible and resistant isolates. Even the lowest concentrations of anthelmintics used in combination with inhibitors caused complete inhibition of development. Differences and/or similarities among responses in different isolates were only obtained in the two other assays: in the Egg Hatch Assay piperonyl butoxide caused a shift in concentration–response curves obtained with thiabendazole to the left for all isolates tested, changing relative differences between isolates. In contrast, an effect of verapamil in the Egg Hatch Assay was only apparent for benzimidazole-resistant isolates. In the Larval Migration Inhibition Assay only ivermectin was tested and piperonyl butoxide shifted the concentration–response curves for all isolates to the left, again eliminating differences in EC₅₀ values between susceptible and resistant isolates. This was not the case using verapamil as an inhibitor, where curves for both susceptible and benzimidazole-resistant isolates shifted to the left in Ostertagia isolates. In Cooperia the picture was more complex with ivermectin-resistant isolates showing a larger shift than the susceptible isolate. Single nucleotide polymorphisms in the β-tubulin isotype 1 gene were investigated. Significantly increased frequencies of resistance-associated alleles were observed for the codons 167 and 200 in one benzimidazole-resistant isolate but not in an isolate selected for benzimidazole resistance at an early stage of selection.     

Discrimination of Gastrointestinal Nematode Eggs from Crude Fecal Egg Preparations by Inhibitor-Resistant Conventional and Real-Time PCR

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles – only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.