Ribosylation induced structural changes in Bovine Serum Albumin: understanding high dietary sugar induced protein aggregation and amyloid formation.

Non-enzymatic glycation of proteins is believed to be the root cause of high dietary sugar associated pathophysiological maladies. We investigated the structural changes in protein during progression of glycation using ribosylated Bovine Serum Albumin (BSA). Non enzymatic attachment of about 45 ribose molecules to BSA resulted in gradual reduction of hydrophobicity and aggregation as indicated by red-shifted tryptophan fluorescence, reduced ANS binding and lower anisotropy of FITC-conjugated protein. Parallely, there was a significant decrease of alpha helicity as revealed by Circular Dichroism (CD) and Fourier transformed-Infra Red (FT-IR) spectra. The glycated proteins assumed compact globular structures with enhanced Thioflavin-T binding resembling amyloids. The gross structural transition affected by ribosylation led to enhanced thermostability as indicated by melting temperature and Transmission Electron Microscopy. At a later stage of glycation, the glycated proteins developed non-specific aggregates with increase in size and loss of amyloidogenic behaviour. A parallel non-glycated control incubated under similar conditions indicated that amyloid formation and associated changes were specific for ribosylation and not driven by thermal denaturation due to incubation at 37 °C. Functionality of the glycated protein was significantly altered as probed by Isothermal Titration Calorimetry using polyphenols as substrates. The studies demonstrated that glycation driven globular amyloids form and persist as transient intermediates during formation of misfolded glycated adducts. To the best of our knowledge, the present study is the first systematic attempt to understand glycation associated changes in a protein and provides important insights towards designing therapeutics for arresting dietary sugar induced amyloid formation.

Zolmitriptan attenuates hepatocellular carcinoma via activation of caspase mediated apoptosis.

A preclinical study using DEN-induced HCC rat model was attempted to evaluate the antitumor potential of zolmitriptan (ZOL). The molecular insights were investigated using ELISA, qRT-PCR and Western blot techniques. The result confirmed that the HCC condition was developed in response to lower expressions of caspase 3 and 9 which, in turn, was due to the upstream regulation of iNOS, Bcl-xl and Bcl-2, and downstream regulation of eNOS, BAX, BAD and Cyt C. The treatment with ZOL caused the significant activation of caspase mediated apoptotic signals that could be responsible for its anti-HCC potential. Later, 1H NMR based serum metabolomics study confirmed that ZOL restored the perturbed metabolites associated with DEN-induced HCC. The antineoplastic potential of ZOL was found comparable or to some degree better than the marketed chemotherapeutics, 5-flurouracil.

Antimicrobial activities of actinomycetes isolated from unexplored regions of Sundarbans mangrove ecosystem.

BACKGROUND: New broad spectrum antimicrobial agents are urgently needed to combat frequently emerging multi drug resistant pathogens. Actinomycetes, the most talented group of microorganisms isolated from unexplored regions of the world may be the ultimate solution to this problem. Thus the aim of this study was to isolate several bioactive actinomycetes strains capable of producing antimicrobial secondary metabolite from Sundarbans, the only mangrove tiger land of the world. RESULTS: Fifty four actinomycetes were isolated and analyzed for antimicrobial activity against fifteen test organisms including three phytopathogens. Nine morphologically distinct and biologically active isolates were subjected to polyphasic identification study.16 s rDNA sequencing indicated eight isolates to reveal maximum similarity to the genus streptomyces, whereas one isolate presented only 93.57% similarity with Streptomyces albogriseolus NRRL B-1305(T). Seventy-one carbon sources and twenty-three chemical sources utilization assay revealed their metabolic relatedness. Among these nine isolates three specific strains were found to have notably higher degree of antimicrobial potential effective in a broader range including phyto-pathogenic fungus. Finally the strain SMS_SU21, which showed antimicrobial activity with MIC value of 0.05 mg ml(-1) and antioxidant activity with IC50 value of 0.242 ± 0.33 mg ml(-1) was detected to be the most potential one. True prospective of this strain was evaluated utilizing GC-MS and the bioactive compound responsible for antimicrobial activity was purified. CONCLUSION: Rare bioactive actinomycetes were isolated from unexplored heritage site. Antimicrobial compound has also been identified and purified which is active against a broad range of pathogens.

The Pathogen of the Great Barrier Reef Sponge Rhopaloeides odorabile Is a New Strain of Pseudoalteromonas agarivorans Containing Abundant and Diverse Virulence-Related Genes.

Sponge diseases have increased dramatically, yet the causative agents of disease outbreaks have eluded identification. We undertook a polyphasic taxonomic analysis of the only confirmed sponge pathogen and identified it as a novel strain of Pseudoalteromonas agarivorans. 16S ribosomal RNA (rRNA) and gyraseB (gyrB) gene sequences along with phenotypic characteristics demonstrated that strain NW4327 was most closely related to P. agarivorans. DNA-DNA hybridization and in silico genome comparisons established NW4327 as a novel strain of P. agarivorans. Genes associated with type IV pili, mannose-sensitive hemagglutinin pili, and curli formation were identified in NW4327. One gene cluster encoding ATP-binding cassette (ABC) transporter, HlyD and TolC, and two clusters related to the general secretion pathway indicated the presence of type I secretion system (T1SS) and type II secretion system (T2SS), respectively. A contiguous gene cluster of at least 19 genes related to type VI secretion system (T6SS) which included all 13 core genes was found. The absence of T1SS and T6SS in nonpathogenic P. agarivorans S816 established NW4327 as the virulent strain. Serine proteases and metalloproteases of the classes S8, S9, M4, M6, M48, and U32 were identified in NW4327, many of which can degrade collagen. Collagenase activity in NW4327 and its absence in the nonpathogenic P. agarivorans KMM 255(T) reinforced the invasiveness of NW4327. This is the first report unambiguously identifying a sponge pathogen and providing the first insights into the virulence genes present in any pathogenic Pseudoalteromonas genome. The investigation supports a theoretical study predicting high abundance of terrestrial virulence gene homologues in marine bacteria.

Utilization of vinasse for the production of polyhydroxybutyrate by Haloarcula marismortui.

Vinasse, a recalcitrant waste of the ethanol industry was employed for the production of polyhydroxyalkanoate (PHA) by the extremely halophilic archaeon, Haloarcula marismortui in shake flasks. The PHA was recovered by osmotic lysis of the cells and subsequent purification by sodium hypochlorite and organic solvents. Through UV-vis spectroscopy, differential scanning calorimetry, Fourier transform infrared, and nuclear magnetic resonance spectroscopy, the PHA was found to have characteristics very similar to that of the standard polyhydroxybutyrate (PHB) from Sigma. Inhibitory effect of polyphenols contained in vinasse was assessed by a quick and reliable cup-plate agar-diffusion method. Raw vinasse (10%) was utilized leading to accumulation of 23% PHA (of cell dry weight) and following an efficacious pre-treatment process through adsorption on activated carbon, 100% pre-treated vinasse could be utilized leading to 30% accumulation of PHB by H. marismortui. Maximum specific growth rate, specific production rate, and volumetric productivity attained using 10% raw vinasse were comparable to that obtained using a previously reported nutrient deficient medium (NDM), while the values with 100% pre-treated vinasse were higher than that determined using NDM medium. This is the first report of polyhydroxybutyrate production by a halophilic microorganism utilizing vinasse.

Heterotrophic nitrification by Achromobacter xylosoxidans S18 isolated from a small-scale slaughterhouse wastewater.

Heterotrophic carbon utilizing microbes were acclimatized in the laboratory by inoculating sludge collected from the waste discharge pond of a small-scale rural abattoir in India in a nutrient solution intermittently fed with glucose and ammonium chloride. Cultures of 10 well-developed isolates were selected and grown in a basal medium containing glucose and ammonium chloride. Culture supernatants were periodically analyzed for ammonium nitrogen (NH(4)(+)-N) and chemical oxygen demand (COD). Polyphasic taxonomic study of the most active nitrifier (S18) was done. Half saturation concentration (K(s)), maximum rate of substrate utilization (k), yield coefficient (Y) and decay coefficient (K(d)) were determined from the Lineweaver-Burk plot using the modified Monod equation. S18 was able to remove 97 ± 2% of (NH(4)(+)-N) and 88 ± 3% of COD. Molecular phylogenetic study supported by physiological and biochemical characteristics assigned S18 as Achromobacter xylosoxidans. Nitrification activity of A. xylosoxidans was demonstrated for the first time, while interestingly, the distinctive anaerobic denitrification property was preserved in S18. K (s) values were determined as 232.13 ± 1.5 mg/l for COD reduction and 2.131 ± 1.9 mg/l for NH(4)(+)-N utilization. Yield coefficients obtained were 0.4423 ± 0.1134 mg of MLVSS/mg of COD and 0.2461 ± 0.0793 mg of MLVSS/mg of NH(4)(+)-N while the decay coefficients were 0.0627 ± 0.0013 per day and 0.0514 ± 0.0008 per day, respectively. After a contact period of 24 h, 650 ± 5 mg/l solids were produced when the initial concentration of COD and NH(4)(+)-N were 1820 ± 10 mg/l and 120 ± 5.5 mg/l, respectively. This is the first report on the kinetic coefficients for carbon oxidation and nitrification by a single bacterium isolated from slaughterhouse wastewater.

ISOLATION AND CHARACTERIZATION OF CYANOBACTERIA POSSESSING ANTIMICROBIAL ACTIVITY FROM THE SUNDARBANS, THE WORLD'S LARGEST TIDAL MANGROVE FOREST(1).

Eight obligately halophilic, euryhaline cyanobacteria from intertidal soil were isolated in artificial seawater nutrients III (ASN-III) medium. Antimicrobial activity, 16S rRNA gene sequences, phenotypic characters as well as growth and antibiosis in response to variable salinity, temperature, phosphate concentration, and pH were studied. Minimum inhibitory concentrations (MIC) of the extracts against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and multiple drug-resistant clinical isolates ranged between 0.25 and 0.5 mg · mL(-1) . Cytotoxicity tests showed 73%-84% human colon adenocarcinoma (HT-29/C1) cell survival at MIC values, indicating that the extracts were nontoxic. Morphologically, six cyanobacteria were assigned to the Lyngbya-Phormidium-Plectonema (LPP) group B, and one each was assigned to Oscillatoria and Synechocystis genera. Glycerol, mannitol, and starch supported better photoheterotrophic growth than simpler mono- and disaccharides. No heterocyst formation was observed when grown under nitrogen-starved conditions. All isolates survived 7‰ salinity, grew at minimum 32‰ salinity, and showed sustained growth throughout 32‰-82‰ salinity but matured poorly in freshwater medium supplemented with 30.0 g · L(-1) NaCl. Antimicrobial production occurred only at 32‰ salinity. While four of the eight isolates demonstrated sustained growth at 37°C, maximum antimicrobial activity was obtained at 25°C. All strains showed maximum growth and antimicrobial elaboration at 0.04 g · L(-1) phosphate. All isolates thrived at pH 9.5; six grew at pH 4.5, though antimicrobial production occurred only at pH 7.5. Molecular phylogenetic analysis based on 16S rRNA gene sequences of the filamentous isolates validated the previous taxonomic affiliations established on morphological characteristics. This is the first study of antimicrobial-producing halophilic cyanobacteria from the mangroves.