Enhanced Production of Antimicrobial Compounds by Three Salt-Tolerant Actinobacterial Strains Isolated from the Sundarbans in a Niche-Mimic Bioreactor

A novel reactor system, the rotating disk bioreactor (RDBR), was used to mimic the niche environmental conditions of three salt-tolerant estuarine actinobacteria isolated from the Sundarbans region off the Bay of Bengal, designated MS310 (99% similar in its 16S rRNA gene sequence to Streptomyces parvallus), MS3/20 and MS1/7. The RDBR, operated at a rotational speed of one revolution per day, 50% submergence of discs, aeration rate of 1.0 vvm, and with a sucrose-containing medium, faithfully mimicked the intertidal estuarine habitat of these marine isolates, and supported biofilm formation and production of antimicrobial metabolites-in particular, actinomycin D by MS310. Onset of antibiotic production by MS310 occurs at 20 h in the RDBR compared to 55 h in a conventional stirred-tank bioreactor (STBR). Furthermore, peak antimicrobial activity is attained much earlier in the RDBR with MS310 (at 45 h) than that reported with a terrestrial strain of S. parvallus grown in a STBR (at 144 h). Peak antimicrobial activity of metabolites produced by MS1/7 and MS3/20 were also attained earlier in the RDBR (at 25 and 12 h, respectively) than in a STBR (at 80 and 28 h, respectively). Antibiotic synthesis in the three isolates, in general, appears to be associated with their growth. Overall, the RDBR may be considered the preferred alternative to the STBR for production of antimicrobials by biofilm-forming estuarine bacteria for its much higher surface/volume ratio, lower costs, and easy operability.     

Apoptosis induction in human leukemic cells by a novel protein Bengalin,isolated from Indian black scorpion venom: Through mitochondrial pathway and inhibition of heat shock proteins

Scorpion venom possesses protein toxins having numerous biological activities, some of which are potentially anticancerous. Previously we had reported antiproliferative activity of the venom of Indian black scorpion, Heterometrus bengalensis Koch. Here we have isolated and purified a novel protein named Bengalin (72 kDa) from the venom, responsible for antiproliferative and apoptogenic activities against human leukemic cells U937 (histiocytic lymphoma) and K562 (chronic myelogenous leukemia). N-terminal sequence of first 20 amino acids of Bengalin was G-P-L-T-I-L-H-I-N-D-V-H-A-A/R-F-E-Q/G-F/G-N-T. Bengalin induced cell growth inhibition at IC₅₀ values of 3.7 and 4.1 μg/ml for U937 and K562 cells respectively did not significantly affect normal human lymphocytes. Inhibition of U937 and K562 cell proliferation occurred by apoptosis as evidenced from damaged nuclei, cell cycle arrest at sub G1 phase, increase of early apoptotic cells, augmentation of DNA fragmentation and also a reduction of telomerase activity. Further insights revealed that Bax:Bcl2 ratio was elevated after Bengalin treatment. Moreover Bengalin elicited loss of mitochondrial membrane potential (MMP) which commenced cytochrome c release in cytosol, decreased heat shock protein (HSP) 70 and 90 expression, activated caspase-9, caspase-3 and induced poly(ADP-ribose) polymerase (PARP) cleavage. We have also determined that HSP70 and 90 inhibitions correlated with Bengalin induced antiproliferation, caspase-3 upregulation, apoptogenesis and increased DNA fragmentation. These results hypothesize that Bengalin might provide a putative molecular mechanism for their anticancer effect on human leukemic cells which might be mediated by mitochondrial death cascade. Inhibition of HSPs might also play a crucial role in induction of apoptosis.     

Exploration of amino alcohol derivatives as novel,potent, and highly selective sphingosine-1-phosphate receptor subtype-1 agonists

In pursuit of a potent and highly selective sphingosine-1-phosphate receptor agonists with an improved in vivo conversion of the precursor to the active phospho-drug, we have utilized previously reported phenylamide and phenylimidazole scaffolds to identify a selectivity enhancing moiety (SEM) and selectivity enhancing orientation (SEO) within both pharmacophores. SEM and SEO have allowed for over 100 to 500-fold improvement in selectivity for S1P receptor subtype 1 over subtype 3. Utility of SEM and SEO and further SAR study allowed for discovery of a potent and selective preclinical candidate PPI-4955 (21b) with an excellent in vivo potency and dose responsiveness and markedly improved overall in vivo pharmacodynamic properties upon oral administration.     

Characterization of glucose-6-phosphate dehydrogenase variants in the Sudan--including GdKhartoum, a hyperactive slow variant.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was characterized in blood samples of 94 male subjects in Sudan having deficient and non-deficient electrophoretic variants. They comprised 44 GdB, 17 GdA, 19 GdB-, 11 GdA- and 3 nondeficient (GdKhartoum) variants. Biochemical characteristics including enzyme activity, electrophoretic mobility, Km for glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP), heat stability and pH optimum of all the common and deficient variants were consistent with the reported characteristics of these variants. The GdKhartoum variant had 90% mobility in TEB buffer and 100% in phosphate buffer, 120% activity, Km of 130 +/- 49 microns for G6P and 0.8 +/- 0.2 microns for NADP, lowered thermostability and an optimum pH of 7.6. This variant was not inhibited by 15 mM maleic acid, 10 mM iodoacetate and dehydro-iso-androsterone. All other variants were inhibited by dehydro-iso-androsterone but uninhibited by maleic acid and iodoacetate.     

Chromosomal assignment of microsatellite loci in cotton

Microsatellite markers or simple sequence repeats (SSRs) represent a new class of genetic markers for cotton (Gossypium sp.). Sixty-five SSR primer pairs were used to amplify 71 marker loci and genotype 13 monosomic and 27 monotelodisomic cotton cytogenetic stocks. Forty-two SSR loci were assigned to cotton chromosomes or chromosome arms. Thirty SSRs were not located to specific chromosomes in this study. Nineteen marker loci were shown to occur on the A subgenome and 11 on the D subgenome by screening accessions of G. herbaceum (2n = 2x = 26 = 2A1) and G. raimondii (2n = 2x = 26 = 2D5). The aneuploid stocks proved to be very powerful tools for localizing SSR markers to individual cotton chromosomes. Multiplex PCR bins of the SSR primers and semiautomated detection of the amplified products were optimized in this experiment. Thirteen multiplex PCR bins were optimized to contain an average of 4 SSR primer pairs per bin. This provides a protocol for high-throughput genotyping of cotton SSRs that improves the efficiency of genetic mapping and marker-assisted programs utilizing SSR markers.     

Physio-biochemical and molecular assessment of Iron (Fe2+) toxicity responses in contrasting indigenous aromatic Joha rice cultivars of Assam,India

Iron (Fe) toxicity is one of the major abiotic stresses which limits the yield of lowland rice. This study aims to investigate the physiological, biochemical, and molecular aspects of two contrasting aromatic Joha rice, viz., Keteki and Kola Joha of Assam. Oxidative damage caused due to Fe²⁺ toxicity was quantitatively determined. Fe²⁺ toxicity in the growth medium increases the level of ROS and anti-oxidative enzyme activity. Along with the aforementioned damage caused due to Fe²⁺ toxicity, chlorophyll content decreases in both the rice varieties. Detection of Fe³⁺ and Fe²⁺ was also conducted by Perls’ Prussian and Turnbull blue method, respectively. In addition, spectrophotometric quantification of Fe²⁺ was determined by 2, 2′-Bipyridyl (Bpy). Above 2.5 mM, Fe²⁺ toxicity was found to be lethal in rice seedlings affecting their total growth and biomass. Gene expression analysis of iron-regulated transporter 1 (OsIRT1), Yellow Stripe-Like 15 (OsYSL15), and ferritin 1 (OsFer1) revealed the differential gene expression over a time period of Fe²⁺ toxicity. Our study suggested that the different parameters which are considered here can be helpful for the better understanding of how aromatic Joha rice performed under Fe²⁺ toxicity which can also help to reveal broader aspects that how gene players are involved in the iron homeostasis mechanism in Joha rice in coming future.

     

Enrichment of laccase production by Phoma herbarum isolate KU4 under solid-state fermentation by optimizing RSM coefficients using genetic algorithm

The process parameters were optimized to obtain enhanced enzyme activity from the fungus Phoma herbarum isolate KU4 using rice straw and saw dust as substrate under solid-state fermentation using Response surface methodology (RSM). Genetic algorithm was used to validate the RSM for maximum laccase production. Six variables, viz., pH of the media, initial moisture content, copper sulphate concentration, concentration of tannic acid, inoculum concentration and incubation time were found to be effective and optimized for enhanced production. Maximum laccase production was achieved by RSM at pH 5·0 and 86% of initial moisture content of the culture medium, 150 µmol l⁻¹ of CuSO₄, 1·5% tannic acid and 0·128 g inoculum g⁻¹ dry substrate inoculum size on the fourth day of fermentation. The highest laccase activity was observed as 79 008 U g⁻¹, which is approximately sixfold enhanced production compared to the unoptimized condition (12 085·26 U g⁻¹).