Iron uptake and transport by the carboxymycobactin-mycobactin siderophore machinery of Mycobacterium tuberculosis is dependent on the iron-regulated protein HupB

BioMetals - Iron-starved Mycobacterium tuberculosis utilises the carboxymycobactin-mycobactin siderophore machinery to acquire iron. These two siderophores have high affinity for ferric iron and...     

Migfilin and filamin as regulators of integrin activation in endothelial cells and neutrophils

Cell adhesion and migration depend on engagement of extracellular matrix ligands by integrins. Integrin activation is dynamically regulated by interactions of various cytoplasmic proteins, such as filamin and integrin activators, talin and kindlin, with the cytoplasmic tail of the integrin β subunit. Although filamin has been suggested to be an inhibitor of integrin activation, direct functional evidence for the inhibitory role of filamin is limited. Migfilin, a filamin-binding protein enriched at cell-cell and cell-extracellular matrix contact sites, can displace filamin from β1 and β3 integrins and promote integrin activation. However, its role in activation and functions of different β integrins in human vascular cells is unknown. In this study, using flow cytometry, we demonstrate that filamin inhibits β1 and αIIbβ3 integrin activation, and migfilin can overcome its inhibitory effect. Migfilin protein is widely expressed in different adherent and circulating blood cells and can regulate integrin activation in naturally-occurring vascular cells, endothelial cells and neutrophils. Migfilin can activate β1, β2 and β3 integrins and promote integrin mediated responses while migfilin depletion impairs the spreading and migration of endothelial cells. Thus, filamin can act broadly as an inhibitor and migfilin is a promoter of integrin activation.     

Recent admixture in an Indian population of African ancestry

Identification and study of genetic variation in recently admixed populations not only provides insight into historical population events but also is a powerful approach for mapping disease loci. We studied a population (OG-W-IP) that is of African-Indian origin and has resided in the western part of India for 500 years; members of this population are believed to be descendants of the Bantu-speaking population of Africa. We have carried out this study by using a set of 18,534 autosomal markers common between Indian, CEPH-HGDP, and HapMap populations. Principal-components analysis clearly revealed that the African-Indian population derives its ancestry from Bantu-speaking west-African as well as Indo-European-speaking north and northwest Indian population(s). STRUCTURE and ADMIXTURE analyses show that, overall, the OG-W-IPs derive 58.7% of their genomic ancestry from their African past and have very little inter-individual ancestry variation (8.4%). The extent of linkage disequilibrium also reveals that the admixture event has been recent. Functional annotation of genes encompassing the ancestry-informative markers that are closer in allele frequency to the Indian ancestral population revealed significant enrichment of biological processes, such as ion-channel activity, and cadherins. We briefly examine the implications of determining the genetic diversity of this population, which could provide opportunities for studies involving admixture mapping.     

SNPs in stress-responsive rice genes: validation, genotyping, functional relevance and population structure

ABSTRACT: BACKGROUND: Single nucleotide polymorphism (SNP) validation and large-scale genotyping are required to maximize the use of DNA sequence variation and determine the functional relevance of candidate genes for complex stress tolerance traits through genetic association in rice. We used the bead array platform-based Illumina GoldenGate assay to validate and genotype SNPs in a select set of stress-responsive genes to understand their functional relevance and study the population structure in rice. RESULTS: Of the 384 putative SNPs assayed, we successfully validated and genotyped 362 (94.3%). Of these 325 (84.6%) showed polymorphism among the 91 rice genotypes examined. Physical distribution, degree of allele sharing, admixtures and introgression, and amino acid replacement of SNPs in 263 abiotic and 62 biotic stress-responsive genes provided clues for identification and targeted mapping of trait-associated genomic regions. We assessed the functional and adaptive significance of validated SNPs in a set of contrasting drought tolerant upland and sensitive lowland rice genotypes by correlating their allelic variation with amino acid sequence alterations in catalytic domains and three-dimensional secondary protein structure encoded by stress-responsive genes. We found a strong genetic association among SNPs in the nine stress-responsive genes with upland and lowland ecological adaptation. Higher nucleotide diversity was observed in indica accessions compared with other rice sub-populations based on different population genetic parameters. The inferred ancestry of 16% among rice genotypes was derived from admixed populations with the maximum between upland aus and wild Oryza species. CONCLUSIONS: SNPs validated in biotic and abiotic stress-responsive rice genes can be used in association analyses to identify candidate genes and develop functional markers for stress tolerance in rice.     

Cell surface carbohydrates of Agrobacteriumtumefaciens involved in adherence during crown gall tumor initiation

N-acetyl-D-galactosamine and β-D-galactose, located in the O-antigenic region of $\text{Agrobacterium}$ lipopolysaccharide, are involved in host-bacterium adherence during crown gall tumor initiation as indicated by fluorescence spectroscopy studies.     

Natural mutations in a 2'-5' oligoadenylate synthetase transgene revealed residues essential for enzyme activity

Unlike other RNA polymerases, 2'-5' oligoadenylate synthetases, a family of interferon-induced enzymes, catalyze the formation of 2'-5', not 3'-5', phosphodiester bonds. Moreover, to be active, these proteins require double-stranded RNA as a cofactor. We have been identifying the specific residues of these proteins that impart their novel properties. Here, we report the identity of three such residues that underwent natural mutations in a transgenic mouse line. When deliberately introduced into recombinant proteins, each of these mutations rendered the protein enzymatically inactive. In an effort to understand the roles of these residues in enzyme activity, new mutants carrying other residues in one of these three sites were generated. Detailed characterization of the properties of the mutant proteins revealed that Lys 404 is needed for proper binding of the acceptor substrate, Pro 500 provides structural flexibility to the protein, and Ser 471 is probably required for its proper folding. This study illustrates the power of using natural mutations in transgenes as guides for studying structure-function relationships of proteins.     

Expression of Biologically Active β Subunit of Bovine Follicle-Stimulating Hormone in the Methylotrophic YeastPichia pastoris

Follicle-stimulating hormone (FSH), a pituitary gonadotropin, is a heterodimer composed of an α subunit, which is common to all the glycoprotein hormones, noncovalently associated with the hormone-specific β subunit. The objective of the present study is to develop a recombinant DNA expression system for the β subunit of FSH that can be applied to study structure–function relationships while producing large quantities of the hormone subunit for immunocontraceptive, clinical, and veterinary purposes. We report here the expression of biologically active bovine FSHβ (bFSHβ) in the methylotrophic yeastPichia pastoris.ThePichia-expressed FSHβ (pFSHβ) was secreted into the culture medium and was found to be immunologically very similar to pituitary-derived ovine FSHβ. Replacement of the cognate signal peptide with the yeast α mating factor signal peptide increased the level of expression from 230 ng/ml (cognate signal peptide) to 4 μg/ml (α mating factor signal peptide) of the culture supernatant. pFSHβ^His.tag(pFSHβ with six histidine residues at the C terminus) was purified to apparent homogeneity using one-step nickel affinity chromatography. The molecular weight of purified pFSHβ^His.tagwas approximately 22,000, which was slightly higher than that of the pituitary-derived ovine FSHβ. pFSHβ^His.tagcould assemble with the α subunit to yield a heterodimer capable of binding to the FSH receptors and also elicit biological response. These data show that pFSHβ^His.tagis properly folded and biologically active.