Extraepitopic compensatory substitutions partially restore fitness to simian immunodeficiency virus variants that escape from an immunodominant cytotoxic-T-lymphocyte response

Selection for escape mutant immunodeficiency viruses by cytotoxic T lymphocytes (CTL) has been well characterized and may be associated with disease progression. CTL epitopes accrue escape mutations at different rates in vivo. Interestingly, certain high-frequency CTL do not select for escape until the chronic phase of infection. Here we show that mutations conferring escape from immunodominant CTL directed against an epitope in the viral Gag protein are strongly associated with extraepitopic mutations in gag in vivo. The extraepitopic mutations partially restore in vitro replicative fitness of viruses bearing the escape mutations. Constraints on epitope sequences may therefore play a role in determining the rate of escape from CTL responses in vivo.     

Effect of prenatal stress and gonadal hormone condition on depressive behaviors of female and male rats

Whether prenatal stress (PNS) and gonadal hormones may influence depressive behavior of rats in the forced swim test was investigated. In Experiment I, adult diestrous female rats had increased immobility, which is indicative of depression, but did not show any significant difference in the duration of struggling compared to intact adult males. In Experiment 2, the behavior of adult intact, castrated, or castrated dihydrotestosterone (DHT)- or estrogen (E2)-replaced offspring of dams that were restrained under lights for 45 min on gestational day 18 (PNS) or were not subjected to gestational stress (non-PNS, control condition) were compared. There were no effects of PNS, but DHT and E2 produced anti-depressant effects on behavior of male rats. Castration decreased struggling and increased immobility compared to intact rats. DHT or E2 replacement was able to partially reinstate struggling and immobility behavior but not to levels of intact males. In Experiment 3, behavior of PNS or control rats that were in proestrus or were ovariectomized and DHT, E2, or vehicle-replaced were compared. Ovariectomy decreased struggling and increased immobility compared to that of proestrous rats. E2 or DHT to control females increased anti-depressant struggling behavior compared to ovariectomized control or PNS rats administered vehicle, which demonstrated greater duration of struggling than did E2-primed, PNS rats. E2 or DHT administration decreased immobility of PNS and control females. These findings suggest that E2 and DHT have some anti-depressant effects but that modest PNS may alter E2's ability to alleviate some depressive behavior in female, but not male rats.     

Sensitive liquid chromatographic method using fluorescence detection for the determination of estradiol 3- and 17-glucuronides in rat and human liver microsomal incubations: formation kinetics

We have developed a sensitive and specific HPLC-fluorescence assay for the determination of estradiol-3-glucuronide and estradiol-17-glucuronide in human and rat liver microsomal incubations. The method utilizes a mobile phase comprised of acetonitrile and 50 mM ammonium phosphate buffer (35:65, v/v) that is pumped though a phenyl column at 1 ml/min; the run time is less than 15 min. Calibration curves for both metabolites were linear over the range 20-4000 pmol. The intra- and inter-day coefficients of variation were <6%. In both rat and human liver microsomes, the formation of estradiol-3-glucuronide displayed atypical kinetics (consistent with activation), while estradiol-17-glucuronide formation was consistent with classical Michaelis-Menten kinetics. Overall, the assay described is a sensitive and reproducible method for the determination of estradiol glucuronides in liver microsomal preparations.     

Genome dynamics in major bacterial pathogens

Pathogenic bacteria continuously encounter multiple forms of stress in their hostile environments, which leads to DNA damage. With the new insight into biology offered by genome sequences, the elucidation of the gene content encoding proteins provides clues toward understanding the microbial lifestyle related to habitat and niche. Campylobacter jejuni, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis , the pathogenic Neisseria, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus are major human pathogens causing detrimental morbidity and mortality at a global scale. An algorithm for the clustering of orthologs was established in order to identify whether orthologs of selected genes were present or absent in the genomes of the pathogenic bacteria under study. Based on the known genes for the various functions and their orthologs in selected pathogenic bacteria, an overview of the presence of the different types of genes was created. In this context, we focus on selected processes enabling genome dynamics in these particular pathogens, namely DNA repair, recombination and horizontal gene transfer. An understanding of the precise molecular functions of the enzymes participating in DNA metabolism and their importance in the maintenance of bacterial genome integrity has also, in recent years, indicated a future role for these enzymes as targets for therapeutic intervention.     

Bifunctional oligonucleotide probes synthesized using a novel CPG support are able to detect single base pair mutations.

A novel multifunctional controlled pore glass, MF-CPG (Fig. 1), has been synthesized and used to incorporate 3' terminal primary aliphatic amines into synthetic oligonucleotides. MF-CPG consists of a unique succinic acid linking arm which possesses both a masked primary amine for label attachment and a dimethoxytrityl protected hydroxyl for nucleotide chain elongation. Using MF-CPG, we have devised a simple and convenient technique to attach non-radioactive labels to the 3' terminus of oligonucleotides. Bifunctional probes can then be constructed by 32P labeling the 5' terminus with T4 kinase and gamma 32P-ATP. Using such bifunctional oligonucleotide probes in conjunction with polymerase chain reaction (PCR) amplification, we were able to detect single base substitutions in a target segment of the human H-ras protooncogene employing either functionality. Our technique thus expands the potential applications for oligonucleotides as hybridization probes.     

Mitochondrial haplotypes and the New Zealand origin of clonal European Potamopyrgus, an invasive aquatic snail

The small aquatic snail Potamopyrgus antipodarum is an important invading species in Europe, Australia and North America. European populations are generally believed to derive from accidental introductions from New Zealand, probably dating back to the mid-19th century. We have employed mitochondrial DNA sequences to test the proposed New Zealand origin of European Potamopyrgus, and to learn more about its genealogical history. Using a 481-bp region of the 16S ribosomal RNA gene, we identified 17 distinct haplotypes among 65 snails from New Zealand. In marked contrast, only two haplotypes were found across all European samples, which cover a large geographical area. Importantly, these two haplotypes are shared with snails from the North Island of New Zealand. Due to sampling limitations we cannot rule out a South Island origin for one of the haplotypes, but our results clearly demonstrate the New Zealand origin of European populations. The marked divergence among the two European haplotypes implies the successful colonization by two distinct mitochondrial lineages, which is consistent with previous data based on nuclear markers.     

Odour boosts visual object approach in flies

Multisensory integration is synergistic—input from one sensory modality might modulate the behavioural response to another. Work in flies has shown that a small visual object presented in the periphery elicits innate aversive steering responses in flight, likely representing an approaching threat. Object aversion is switched to approach when paired with a plume of food odour. The ‘open-loop’ design of prior work facilitated the observation of changing valence. How does odour influence visual object responses when an animal has naturally active control over its visual experience? In this study, we use closed-loop feedback conditions, in which a fly''s steering effort is coupled to the angular velocity of the visual stimulus, to confirm that flies steer toward or ‘fixate’ a long vertical stripe on the visual midline. They tend either to steer away from or ‘antifixate’ a small object or to disengage active visual control, which manifests as uncontrolled object ‘spinning’ within this experimental paradigm. Adding a plume of apple cider vinegar decreases the probability of both antifixation and spinning, while increasing the probability of frontal fixation for objects of any size, including a normally typically aversive small object.     

Fly flight: a model for the neural control of complex behavior.

Flies exhibit a repertoire of aerial acrobatics unmatched in robustness and aerodynamic sophistication. The exquisite control of this complex behavior emerges from encoding intricate patterns of optic flow, and the translation of these visual signals into the mechanical language of the motor system. Recent advances in experimental design toward more naturalistic visual and mechanosensory stimuli have served to reinforce fly flight as a key model system for understanding how feedback from multiple sensory modalities is integrated to control complex and robust motor behaviors across taxa.     

Splicing factor SRp30c interaction with Y-box protein-1 confers nuclear YB-1 shuttling and alternative splice site selection

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.     

Modelling expected trout ranges under current and future water temperature regimes in the Eastern Cape,South Africa

Different values have resulted in conflicts between anglers and conservation lobbies in the management of trout in South Africa. Key to the conflict is the demarcation of boundaries to areas in which brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss currently occur, or are likely to establish following stocking for angling. To provide a longer-term perspective on these areas, we developed models to link salmonid biological thermal thresholds to elevation. These, when applied spatially using a digital elevation model with a probability of occurrence model, provided the basis for estimating potentially available thermal habitat for these two cold water species. Here, we acknowledge that other variables (stocking history; river connectivity) also play a role in understanding trout distributions. Using a simple scenario of an increase in mean daily water temperatures of 2 °C, we demonstrated that both brown and rainbow trout are likely to exhibit considerable range reductions in the future. Because it is possible that these range restrictions will result in an increasing desire to introduce trout into areas above their current distribution limits for the maintenance of angling opportunities, conservation managers should prioritise these areas, with management interventions seeking to understand what will help to limit introductions.