Sequence analysis of UTR and coding region of kappa-casein gene of Indian riverine buffalo (Bubalus bubalis).

In this study, complete nucleotide as well as derived amino acid sequence characterization of water buffalo (Bubalus bubalis) kappa-casein gene has been presented. Kappa-casein cDNA clones were identified and isolated from a buffalo lactating mammary gland cDNA library. Sequence analysis of kappa-casein cDNA revealed 850 nucleotides with an open reading frame (ORF) of 573 nucleotides, encoding mature peptide of 169 amino acids. The 5' untranslated region (UTR) comprised 71 nucleotides, while 3' UTR was of 206 nucleotides. A total of 11 nucleotide and seven amino acid changes were observed in, buffalo (Bubalus bubalis) as compared to cattle (Bos taurus), sheep (Ovis aries) and goat (Capra hircus). Among these nucleotide changes, eight were unique in buffalo as they were fully conserved in cattle, sheep and goat. Majority of the nucleotide changes and all the amino acid changes; 14 (Asp-Glu), 19(Asp/Ser-Asn), 96(Ala-Thr), 126(Ala-Val), 128(Ala/Gly-Val), 156(Ala/Pro-Val) and 168(Ala/Glu-Val) were limited to exon IV. Three glycosylation sites, Thr 131, Thr 133 and Thr 142 reported in cattle and goat kappa-casein gene were also conserved in buffalo, however, in sheep Thr 142 was replaced by Ala. Chymosin hydrolysis site, between amino acids Phe 105 and Met 106, important for rennet coagulation process, were found to be conserved across four bovid species. Buffalo kappa-casein with the presence of amino acids Thr 136 and Ala 148 seems to be an intermediate of "A" and "B" variants of cattle. Comparison with other livestock species revealed buffalo kappa-casein sharing maximum nucleotide (95.5%) and amino acid (92.6%) similarity with cattle, whereas with pig it showed least sequence similarity of 76.0% and 53.2%, respectively. Phylogenetic analysis based on both nucleotide and amino acid sequence indicated buffalo kappa-casein grouping with cattle, while sheep and goat forming a separate cluster close to them. The non-ruminant species viz. camel, horse and pig were distantly placed, in separate lineages.     

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor that binds a variety of structurally and functionally unrelated ligands, including advanced glycation endproducts (AGEs), amyloid fibrils, amphoterin, and members of the S100 family of proteins. The receptor has been implicated in the pathology of diabetes as well as in inflammatory processes and tumor cell metastasis. For the present study, the extracellular region of RAGE (exRAGE) was expressed as a soluble, C-terminal hexahistidine-tagged fusion protein in the periplasmic space of Escherichia coli. Proper processing and folding of the purified protein, predicted to contain three immunoglobulin-type domains, was supported by the results of electrospray mass spectroscopy and circular dichroism experiments. Sedimentation velocity experiments showed that exRAGE was primarily monomeric in solution. Binding to several RAGE ligands, including AGE-BSA, immunoglobulin light chain amyloid fibrils, and glycosaminoglycans, was demonstrated using pull-down, dot-blot, or enzyme-linked microplate assays. Using surface plasmon resonance, the interaction of exRAGE with AGE-BSA was shown to fit a two-site model, with KD values of 88 nM and 1.4 microM. The E. coli-derived exRAGE did not bind the advanced glycation endproduct Nepsilon-(carboxymethyl)lysine, as reported for the cellular receptor, and the possible role of RAGE glycosylation in recognition of this ligand is discussed. This new RAGE construct will facilitate detailed studies of RAGE-ligand interactions and provides a platform for preparation of site-directed mutants for future structure/function studies.     

Behavioral responses to odorants in drosophila require nervous system expression of the beta integrin gene myospheroid

Integrins are cell adhesion molecules that mediate numerous developmental processes in addition to a variety of acute physiological events. Two reports implicate a Drosophila beta integrin, betaPS, in olfactory behavior. To further investigate the role of integrins in Drosophila olfaction, we used Gal4-driven expression of RNA interference (RNAi) transgenes to knock down expression of myospheroid (mys), the gene that encodes betaPS. Expression of mys-RNAi transgenes in the wing reduced betaPS immunostaining and produced morphological defects associated with loss-of-function mutations in mys, demonstrating that this strategy knocked down mys function. Expression of mys-RNAi transgenes in the antennae, antennal lobes, and mushroom bodies via two Gal4 lines, H24 and MT14, disrupted olfactory behavior but did not alter locomotor abilities or central nervous system structure. Olfactory behavior was normal in flies that expressed mys-RNAi transgenes via other Gal4 lines that specifically targeted the antennae, the projection neurons, the mushroom bodies, bitter and sweet gustatory neurons, or Pox neuro neurons. Our studies confirm that mys is important for the development or function of the Drosophila olfactory system. Additionally, our studies demonstrate that mys is required for normal behavioral responses to both aversive and attractive odorants. Our results are consistent with a model in which betaPS mediates events within the antennal lobes that influence odorant sensitivity.     

Mineralization of 4-aminobenzenesulfonate (4-ABS) by Agrobacterium sp. strain PNS-1

A bacterial strain, PNS-1, isolated from activated sludge, could utilize sulphanilic acid (4-ABS) as the sole organic carbon and energy source under aerobic conditions. Determination and comparison of 16S r DNA sequences showed that the strain PNS-1 is closely related to the species of Agrobacterium genus. Growth on 4-ABS was accompanied with ammonia and sulfate release. TOC results showed complete mineralization of sulphanilic acid. This strain was highly specific for 4-ABS as none of the sulphonated aromatics used in the present study including other ABS isomers were utilized. Strain PNS-1 could, however, utilize all the tested monocyclic aromatic compounds devoid of a sulfonate group. No intermediates could be detected either in the growth phase or with dense cell suspensions. Presence of chloramphenicol completely inhibited 4-ABS degradation by cells pregrown on succinate, indicating that degradation enzymes are inducible. No plasmid could be detected in the Agrobacterium sp. Strain PNS-1 suggesting that 4-ABS degradative genes may be chromosomal encoded.     

Genetic diversity studies of Kherigarh cattle based on microsatellite markers

We report a genetic diversity study of Kherigarh cattle, a utility draught-purpose breed of India, currently declining at a startling rate, by use of microsatellite markers recommended by the Food and Agriculture Organization. Microsatellite genotypes were derived, and allelic and genotypic frequencies, heterozygosities and gene diversity were estimated. A total of 131 alleles were distinguished by the 21 microsatellite markers used. All the microsatellites were highly polymorphic, with mean (±s.e.) allelic number of 6.24 ±1.7, ranging 4–10 per locus. The observed heterozygosity in the population ranged between 0.261 and 0.809, with mean (±s.e.) of 0.574 ±0.131, indicating considerable genetic variation in this population. Genetic bottleneck hypotheses were also explored. Our data suggest that the Kherigarh breed has not experienced a genetic bottleneck in the recent past.     

Secretory factors of human neuroblastoma (IMR-32) and human glioblastoma (U87MG) cell lines induce neurite outgrowths in PC12 cells

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.     

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) interacts with PELP1 and activates MAPK

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1) (also known as the modulator of nongenomic activity of estrogen receptor) plays a role in genomic functions of the estrogen receptor via histone interactions and in nongenomic functions via its influence on the MAPK-Src pathway. However, recent studies have shown that differential compartmentalization of PELP1 could play a crucial role in modulating the status of nongenomic signaling by using molecular mechanisms that remain poorly understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is an early endosomal protein that plays a role in regulating the trafficking of growth factor-receptor complexes through early endosomes. By using a yeast two-hybrid screen, we identified HRS as a novel PELP1-binding protein providing evidence of a physiologic interaction between HRS and PELP1. The noted HRS-PELP1 interaction was accompanied by inhibition of the basal coactivator function of PELP1 upon estrogen receptor transactivation. HRS was found to sequester PELP1 in the cytoplasm, leading to the activation of MAPK in a manner that is dependent on the epidermal growth factor receptor but independent of the estrogen receptor, Shc, and Src. In addition, stimulation of MAPK and the subsequent activation of its downstream effector pathway, Elk-1, by HRS or PELP1 were found to depend on the presence of endogenous PELP1 or HRS. Furthermore, HRS was overexpressed and correlated well with the cytoplasmic PELP1, increased MAPK, and EGFR status in breast tumors. These findings highlight a novel role of HRS in up-regulating MAPK, presumably involving interaction with PELP1.