Genetic Polymorphisms in the Bovine Toll-Like Receptor 4 (TLR4) and Monocyte Chemo Attractant Protein-1(CCL2) Genes: SNPs Distribution Analysis in Bos indicus Sahiwal Cattle Breed

Toll-like receptor 4 gene (TLR4) that recognizes the Gram negative bacterial ligand LPS was sequenced in the Bos indicus Sahiwal cattle breed. Ninety four single nucleotide polymorphisms (SNPs) were detected within 10.8 kb gene region. Seventeen of the SNPs were in the coding regions and the one at position 9589(A > G) in exon3 resulted in an amino acid change from Valine to Isoleucine. These SNPs led to generation of 27 TLR4 gene haplotypes. All the Sahiwal animals studied presently showed the occurrence of the genotype CC at gene position 9662, which codes for the amino acid threonine at position 674 of the TLR4 protein, and which had been reported to be associated with lower somatic cell score and, therefore, a lower susceptibility to mastitis, in Taurus cattle. This nucleotide configuration of the Toll-like receptor 4 gene of the Bos indicus Sahiwal cattle breed could possibly indicate toward a lower susceptibility to mastitis in the Sahiwal animals. Monocyte chemo-attractant protein-1 (CCL2) gene encoding for small inducible cytokine A2 that belongs to the CC chemokine family was also sequence characterized in these Sahiwal animals. The CCL2 gene was observed to have 12 polymorphic sites in 3.3 kb region of which one SNP at position 2500 (A > G) in exon 3 resulted in amino acid change from Valine to Isoleucine at position 46 of the mature CCL2 peptide. Seventeen haplotypes of the CCL2 gene were predicted corresponding to 12 genotypes detected.     

Phylogenetic Relationships of the Marine Nematode Family Comesomatidae

The phylogenetic relationships of the Comesomatidae have remained unresolved at the family level because they have diagnostic morphological features of both the Monhysterida and the Chromadorida. A comparison of the partial sequence of 18S rDNA from representative taxa of Comesomatidae and of morphological data, analyzed in conjunction with molecular and morphological data from monhysterids and chromadorids, suggests a closer relationship of the Comesomatidae with Monhysterida than with Chromadorida.     

Anti-plasmodial action of de novo-designed, cationic, lysine-branched, amphipathic, helical peptides

ABSTRACT: BACKGROUND: A lack of vaccine and rampant drug resistance demands new anti-malarials. METHODS: In vitro blood stage anti-plasmodial properties of several de novo-designed, chemically synthesized, cationic, amphipathic, helical, antibiotic peptides were examined against Plasmodium falciparum using SYBR Green assay. Mechanistic details of anti-plasmodial action were examined by optical/fluorescence microscopy and FACS analysis. RESULTS: Unlike the monomeric decapeptides {(Ac-GXRKXHKXWA-NH2) (X = F,DeltaF) (Fm, DeltaFm IC50 >100 muM)}, the lysine-branched,dimeric versions showed far greater potency {IC50 (muM) Fd 1.5 , DeltaFd 1.39}. The more helical and proteolytically stable DeltaFd was studied for mechanistic details. DeltaFq, a K-K2 dendrimer of DeltaFm and (DeltaFm)2 a linear dimer of DeltaFm showed IC50 (muM) of 0.25 and 2.4 respectively. The healthy/infected red cell selectivity indices were >35 (DeltaFd), >20 (DeltaFm)2 and 10 (DeltaFq). FITC-DeltaFd showed rapid and selective accumulation in parasitized red cells. Overlaying DAPI and FITC florescence suggested that DeltaFd binds DNA. Trophozoites and schizonts incubated with DeltaFd (2.5 muM) egressed anomalously and Band-3 immunostaining revealed them not to be associated with RBC membrane. Prematurely egressed merozoites from peptide-treated cultures were found to be invasion incompetent. CONCLUSION: Good selectivity (>35), good resistance index (1.1) and low cytotoxicity indicate the promise of DeltaFd against malaria.     

Genotyping faecal samples of Bengal tiger <Emphasis Type="Italic">Panthera tigris tigris</Emphasis> for population estimation: A pilot study

Background

Bengal tiger Panthera tigris tigris the National Animal of India, is an endangered species. Estimating populations for such species is the main objective for designing conservation measures and for evaluating those that are already in place. Due to the tiger's cryptic and secretive behaviour, it is not possible to enumerate and monitor its populations through direct observations; instead indirect methods have always been used for studying tigers in the wild. DNA methods based on non-invasive sampling have not been attempted so far for tiger population studies in India. We describe here a pilot study using DNA extracted from faecal samples of tigers for the purpose of population estimation.

Results

In this study, PCR primers were developed based on tiger-specific variations in the mitochondrial cytochrome b for reliably identifying tiger faecal samples from those of sympatric carnivores. Microsatellite markers were developed for the identification of individual tigers with a sibling Probability of Identity of 0.005 that can distinguish even closely related individuals with 99.9% certainty. The effectiveness of using field-collected tiger faecal samples for DNA analysis was evaluated by sampling, identification and subsequently genotyping samples from two protected areas in southern India.

Conclusion

Our results demonstrate the feasibility of using tiger faecal matter as a potential source of DNA for population estimation of tigers in protected areas in India in addition to the methods currently in use.

     

Social bonds enhance reproductive success in male macaques

For animals living in mixed-sex social groups, females who form strong social bonds with other females live longer and have higher offspring survival [1-3]. These bonds are highly nepotistic, but sometimes strong bonds may also occur between unrelated females if kin are rare [2, 3] and even among postdispersal unrelated females in chimpanzees and horses [4, 5]. Because of fundamental differences between the resources that limit reproductive success in females (food and safety) and males (fertilizations), it has been predicted that bonding among males should be rare and found only for kin and among philopatric males [6] like chimpanzees [7-9]. We studied social bonds among dispersing male Assamese macaques (Macaca assamensis) to see whether males in multimale groups form differentiated social bonds and whether and how males derive fitness benefits from close bonds. We found that strong bonds were linked to coalition formation, which in turn predicted future social dominance, which influenced paternity success. The strength of males' social bonds was directly linked to the number of offspring they sired. Our results show that differentiated social relationships exert an important influence on the breeding success of both sexes that transcends contrasts in relatedness.     

Molecular detection and isolation of facultatively methylotrophic bacteria, including Methylobacterium podarium sp. nov., from the human foot microflora

This is the first study to demonstrate that diverse methylotrophic bacteria occur in the human foot microflora. Polymerase chain reaction (PCR) amplification of DNA from the soles and toe clefts of feet of five subjects indicated Methylobacterium strains to be present in all cases. Polymerase chain reaction amplification also showed the gene for the alpha-subunit of methanol dehydrogenase (mxaF) to be present in all samples. Two types of mxaF were recovered, one closest to that of Methylobacterium extorquens and the other most similar to that of Hyphomicrobium methylovorum. Numerous methylotrophic strains able to grow on methylamine were isolated with ease from the feet of nine volunteers. These were found by 16S rRNA analysis to be most closely related to Methylobacterium species, Brevibacterium casei, Pseudomonas strain NZ099 and P. migulae. Three strains from two subjects were of a novel species, Methylobacterium podarium sp. nov. This facultatively methylotrophic, obligately aerobic, pink-pigmented, non-motile rod grew with a wide range of multicarbon and one-carbon compounds including citrate, xylose, mono-, di-, and trimethylamine, dimethylsulphide, methanethiol, dimethylsulphoxide, dimethylsulphone and methanol.     

Modification of surface properties of biomaterials influences the ability of Candida albicans to form biofilms

Candida albicans biofilms form on indwelling medical devices (e.g., denture acrylic or intravenous catheters) and are associated with both oral and invasive candidiasis. Here, we determined whether surface modifications of polyetherurethane (Elasthane 80A [E80A]), polycarbonateurethane, and poly(ethyleneterephthalate) (PET) can influence fungal biofilm formation. Polyurethanes were modified by adding 6% polyethylene oxide (6PEO), 6% fluorocarbon, or silicone, while the PET surface was modified to generate hydrophilic, hydrophobic, cationic, or anionic surfaces. Formation of biofilm was quantified by determining metabolic activity and total biomass (dry weight), while its architecture was analyzed by confocal scanning laser microscopy (CSLM). The metabolic activity of biofilm formed by C. albicans on 6PEO-E80A was significantly reduced (by 78%) compared to that of biofilm formed on the nonmodified E80A (optical densities of 0.054 +/- 0.020 and 0.24 +/- 0.10, respectively; P = 0.037). The total biomass of Candida biofilm formed on 6PEO-E80A was 74% lower than that on the nonmodified E80A surface (0.46 +/- 0.15 versus 1.76 +/- 0.32 mg, respectively; P = 0.003). Fungal cells were easily detached from the 6PEO-E80A surface, and we were unable to detect C. albicans biofilm on this surface by CSLM. All other surface modifications allowed formation of C. albicans biofilm, with some differences in thearchitecture. Correlation between contact angle and biofilm formation was observed for polyetherurethane substrates (r = 0.88) but not for PET biomaterials (r = -0.40). This study illustrates that surface modification is a viable approach for identifying surfaces that have antibiofilm characteristics. Investigations into the clinical utility of the identified surfaces are warranted.     

Anchorage-dependent control of muscle-specific gene expression in C2C12 mouse myoblasts

Summary Our previous studies have demonstrated that expression of growth-associated genes is regulated by the adhesive state of the cell. To understand the role of cell adhesion in regulating the switch from growth to differentiation, we are studying the differentiation of mouse myoblasts into multinucleated contractile myotubes. In this report, we describe a novel means of culturing C₂C₁₂ myoblasts that permits an analysis of the role of cell adhesion in regulating the sequential induction of muscle-specific genes that control myogenesis. Suspension of an asynchronous, proliferating population of myoblasts in a viscous gel of methylcellulose dissolved in medium containing 20% serum induces growth arrest in G₀ phase of the cell cycle without a concomitant induction of muscle-specific genes. Reattachment to a solid substratum in 20% serum, 0.5nM bFGF, or 10 nM IGF-1 rapidly activates entry of the quiescent cells into G₁ followed by a synchronous progression of the cell population through into S phase. bFGF or IGF-1 added separately facilitate only one passage through the cell cycle, whereas 20% serum or the two growth factors added together support multiple cell divisions. Adhesion of suspended cells in DMEM alone or with 3 nM IGF-1 induces myogenesis as evidenced by the synthesis of myogenin and myosin heavy chain (MHC) proteins followed by fusion into myotubes. bFGF completely inhibits this differentiation process even in the presence of myogenic doses of IGF-1. Addition of 3 nM IGF-1 to quiescent myoblasts maintained in suspension culture in serum-free conditions does not induce myogenin or MHC expression. Thus, adhesion is a requirement for the induction of muscle gene expression in mouse myoblasts. The development of a muscle cell culture environment in which proliferating myoblasts can be growth arrested in G₀ without activating muscle-specific gene expression provides a means of analyzing the synchronous activation of either the myogenic or growth programs and how adhesion affects each process, respectively. Supported by training grant T32-HL07035