Terpenoid bioactive compound from <Emphasis Type="Italic">Streptomyces rochei</Emphasis> (M32): taxonomy,fermentation and biological activities

The present study emphasized the production of biologically active terpenoid compound from Streptomyces rochei M32, which was isolated from Western Ghats ecosystem, South India. The presence of resistant genes like mecA, vanA of Staphylococcus aureus and bla _SHV, bla _TEM of Pseudomonas aeruginosa was confirmed by molecular studies. The isolated compound from Streptomyces rochei M32 inhibited wide range of standard and clinical drug resistant pathogens and enteric pathogens. The rice bran supplemented basal medium influenced the active compound production on 8th day of fermentation and yielded 1875 mg of crude extract from 10 g of rice bran substrate. Purification and characterization of crude ethyl acetate extract was achieved by preparative thin layer chromatography. The active fraction was identified as terpenoid class compound by chemical screening. Based on the results of spectral studies (NMR, LC–MS, FTIR, etc.), the active compound was tentatively identified as 1, 19-bis (3-hydroxyazetidin-1-yl) nonadeca-5, 14-diene-1, 8, 12, 19-tetraone with molecular weight 462.41 g/mol. Minimum inhibitory concentration value ranges between 7.6 and 31.2 µg/mL against test organisms was observed. The cytotoxicity results on cervical cancer (HeLa) cell line showed IC₅₀ value of 2.034 µg/mL. The corresponding compound is not previously reported from any microbial resources.     

Electrophoretic variants of alpha 2u-globulin in the livers of adult male rats: a possible polymorphism

Two-dimensional polyacrylamide gel electrophoresis has been used to examine the microsomal fractions from the livers of 32 adult male Alpk/AP (Wistar-derived) rats for the presence of alpha 2u-globulin variants of differing isoelectric point. Three major such isoelectric variants are described. Different combinations of these three forms were found in the population examined, with one-half of the animals expressing all three variants in approximately equal proportions and with one variant being present in all the animals examined. An understanding of the relevance of such different alpha 2u-globulin profiles to the individual animal must now await the assignment of a biological role for alpha 2u-globulin.     

Purification, properties, and immunochemical localization of a receptor for intrinsic factor-cobalamin complex in the rat kidney

High levels of receptor for intrinsic factor-cobalamin (vitamin B12) were detected in human, canine, and rat kidneys. The ratio of specific activity (picomoles/mg of protein) for kidney relative to intestine was 116, 20, and 797, respectively, in these species. The receptor was purified about 3000-fold from 200 g of rat kidney with a recovery of 16% and exhibited a single band on nondenaturing gel electrophoresis. Quantitative amino acid analysis of the receptor gave a value of 457,310 g of amino acid/mol of intrinsic factor-cobalamin binding activity. The pure receptor revealed an Mr of 430,000, as assessed by filtration with Bio-Gel A-5m. Treatment with papain resulted in the production of active monomers of Mr to about 205,000-210,000. Electrophoresis in the presence of sodium dodecyl sulfate confirmed the monomer Mr to be 230,000. The monomer receptor did not reveal the presence of any further subunits upon reductive alkylation. Following cyanogen bromide cleavage the kidney receptor revealed three peptides of Mr 115,000, 60,000, and 54,000. The pI of these peptides was 5.17, 6.17, and 6.17, respectively. Western blot analysis using antiserum raised to the receptor demonstrated a protein with an Mr of 175,000 and 230,000 for intestinal and kidney membrane receptors, respectively. Immunologically, the rat kidney receptor was identical to the rat ileal receptor but was distinct from the canine ileal receptor. Ultrastructural localization revealed the presence of the receptor in the apical surface membrane of proximal tubular cells of the kidney and absorptive cells of the ileum. The kidney is the best source for obtaining this receptor in reasonable quantities.     

An inter‐species protein–protein interaction network across vast evolutionary distance

In cellular systems, biophysical interactions between macromolecules underlie a complex web of functional interactions. How biophysical and functional networks are coordinated, whether all biophysical interactions correspond to functional interactions, and how such biophysical‐versus‐functional network coordination is shaped by evolutionary forces are all largely unanswered questions. Here, we investigate these questions using an “inter‐interactome” approach. We systematically probed the yeast and human proteomes for interactions between proteins from these two species and functionally characterized the resulting inter‐interactome network. After a billion years of evolutionary divergence, the yeast and human proteomes are still capable of forming a biophysical network with properties that resemble those of intra‐species networks. Although substantially reduced relative to intra‐species networks, the levels of functional overlap in the yeast–human inter‐interactome network uncover significant remnants of co‐functionality widely preserved in the two proteomes beyond human–yeast homologs. Our data support evolutionary selection against biophysical interactions between proteins with little or no co‐functionality. Such non‐functional interactions, however, represent a reservoir from which nascent functional interactions may arise.     

Genotyping antibiotic producing fluorescent pseudomonads to select effective rhizobacteria for the management of major vanilla diseases

The genotypic diversity that occurs in antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, the diversity of 2,4-diacetylphloroglucinol (DAPG) and phenazine-1-carboxylic acid (PCA) producingPseudomonas sp. from different vanilla plantations of three different regions were studied. Out of eighty-five isolates of rhizobacteria from native soils of vanilla plantations, forty-four were positive to PCR amplification of a 560 bp fragment, which is specific of the genusPseudomonas. Among the forty-four isolates, twenty-one exhibited more antimicrobial activity against fungal pathogensviz., Fusarium oxysporum f. sp.vanillae (Tucker) Gordon,Phytophthora meadii McRae andColletotrichum vanillae underin vitro conditions. Twenty-one and sevenPseudomonas showed positive amplification for DAPG and phenazine respectively. An analysis of the level of biodiversity with DAPG and PCA producers at the species level was performed by comparing the restriction patterns of the genus specific 16S–23S ribosomal DNAs (rDNAs) amplified by PCR. This allowed us to cluster the isolates into four different amplified rDNA restriction analysis (ARDRA) groups. Eventually, it was found that some of the DAPG and PCA producers, which originated from different locations, fall under the same cluster. Bacterisation of vanilla plants with antibiotic producing fluorescent pseudomonads isolates resulted in a significant reduction of major diseases in vanilla both in glasshouse and field conditions.     

Solid-phase Parallel Synthesis of 4-β-D-Ribofuranosylpyrazolo[4,3-d]pyrimidine Nucleosides

The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-(methyl)-1H and 2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H,6H)-dione (5) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of TMS triflate provided two novel protected nucleosides 6 and 7. The structures of 6 and 7 were assigned by 1H and 2D NMR experiments. Nucleosides 6 and 7 were then transformed to the key intermediates 12 and 15 respectively. Reaction of 12 and 15 with MMTCl resin in the presence of 2,6-lutidine afforded the necessary scaffolds B and C. Different amines (96) were introduced selectively by nucleophilic substitution on scaffolds B and C using solid-phase parallel semi-automated synthesizer. Cleavage of the products from the solid support with 30% HFIP in a parallel fashion yielded nucleoside libraries simultaneously, and they were analyzed and characterized by high-throughput LC-MS.     

Erythropoietin increases bioavailability of tetrahydrobiopterin and protects cerebral microvasculature against oxidative stress induced by eNOS uncoupling

This study was designed to determine whether treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Wild‐type and GTP cyclohydrolase I (GTPCH‐I)‐deficient hph1 mice were administered EPO (1000 U/kg/day, s.c., 3 days). Cerebral microvessels of hph1 mice demonstrated reduced tetrahydrobiopterin (BH4) bioavailability, increased production of superoxide anions and impaired endothelial NO signaling. Treatment of hph1 mice with EPO attenuated the levels of 7,8‐dihydrobiopterin, the oxidized product of BH4, and significantly increased the ratio of BH4 to 7,8‐dihydrobiopterin. Moreover, EPO decreased the levels of superoxide anions and increased NO bioavailability in cerebral microvessels of hph1 mice. Attenuated oxidation of BH4 and inhibition of endothelial NO synthase uncoupling were explained by the increased expression of antioxidant proteins, manganese superoxide dismutase, and catalase. The protective effects of EPO observed in cerebral microvessels of hph1 mice were also observed in GTPCH‐I siRNA‐treated human brain microvascular endothelial cells exposed to EPO (1 U/mL or 10 U/mL; 3 days). Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of BH4 and endothelial NO in the cerebral microvascular endothelium.