Enhanced production of fibrinolytic enzyme by a new Xanthomonas oryzae IND3 using low-cost culture medium by response surface methodology

Cardiovascular diseases (CVDs) cause high mortality throughout the world. Existing fibrinolytic agents are highly expensive and have many side effects. Microbial fibrinolytic enzymes are very much considered as novel therapeutic candidate for the treatment of CVDs. Reports on fibrinolytic enzyme from Xanthomonas sp. is lacking. This study reports fibrinolytic enzymes from Xanthomonas oryzae IND3 as it shows hyperactivity on fibrin-agarose plates. This organism utilized various agro-industrial wastes for enzymes production. Among all, cow dung enhanced more enzyme production, hence it was used as the low-cost substrate for statistical optimization of fibrinolytic protease in Solid state fermentation. Response surface methodology was employed to optimize the factors and enhanced yield by 4-fold. The interactions among the variables, viz, sucrose, yeast extract, and pH of the medium were investigated using Central Composite Design (CCD). The predicted fibrinolytic enzyme activity was 2340 U/g, and the observed fibrinolytic enzyme activity was 2294?±?12.8?U/g. The fibrinolytic enzyme degraded blood clot in vitro completely. This study is the first report on statistical optimization of fibrinolytic enzyme production in SSF from Xanthomonas sp. The crude extract has immense activity on proteinaceous wastes. The production of fibrinolytic protease using the low-cost substrate could reduce the production cost of enzyme.     

Regulated Extracellular Choline Acetyltransferase Activity— The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway

Acetylcholine (ACh), the classical neurotransmitter, also affects a variety of nonexcitable cells, such as endothelia, microglia, astrocytes and lymphocytes in both the nervous system and secondary lymphoid organs. Most of these cells are very distant from cholinergic synapses. The action of ACh on these distant cells is unlikely to occur through diffusion, given that ACh is very short-lived in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), two extremely efficient ACh-degrading enzymes abundantly present in extracellular fluids. In this study, we show compelling evidence for presence of a high concentration and activity of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT) in human cerebrospinal fluid (CSF) and plasma. We show that ChAT levels are physiologically balanced to the levels of its counteracting enzymes, AChE and BuChE in the human plasma and CSF. Equilibrium analyses show that soluble ChAT maintains a steady-state ACh level in the presence of physiological levels of fully active ACh-degrading enzymes. We show that ChAT is secreted by cultured human-brain astrocytes, and that activated spleen lymphocytes release ChAT itself rather than ACh. We further report differential CSF levels of ChAT in relation to Alzheimer’s disease risk genotypes, as well as in patients with multiple sclerosis, a chronic neuroinflammatory disease, compared to controls. Interestingly, soluble CSF ChAT levels show strong correlation with soluble complement factor levels, supporting a role in inflammatory regulation. This study provides a plausible explanation for the long-distance action of ACh through continuous renewal of ACh in extracellular fluids by the soluble ChAT and thereby maintenance of steady-state equilibrium between hydrolysis and synthesis of this ubiquitous cholinergic signal substance in the brain and peripheral compartments. These findings may have important implications for the role of cholinergic signaling in states of inflammation in general and in neurodegenerative disease, such as Alzheimer’s disease and multiple sclerosis in particular.     

Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava

Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.     

Insights of antidiabetic,anti-inflammatory and hepatoprotective properties of antimicrobial secondary metabolites of corm extract from Caladium x hortulanum

Medicinal plants have therapeutic potential and are used worldwide to treat various diseases. In this study, the corm of Caladium x hortulanum was extracted with various solvents and implied the availability of phytochemicals such as flavonoids, alkaloids, tannins, steroids, phenols, glycosides, saponins and terpenoids. The solvent extracts of the corm showed antibacterial and antifungal activity with the growth inhibition zone ranged 0–24?mm. The isolation of phytochemicals was carried out using gel column chromatography, Thin Layer Chromatography followed by High Performance Liquid Chromatography. Gas Chromatography and Mass Spectrophotometry analysis was used to determine the phytochemicals. The corm extract showed potent antidiabetic activity on Hep G2 cell lines and CCl₄ induced toxicity was elucidated. This possessed antiinflammatory property on murine monocyclic macrophage cell line RAW 264.7 showed 45.85?±?1.8% inhibition of cyclooxygenase activity. The corm extract showed hepatoprotective activity. The CCl₄ incorporated Hep G2 cells showed 19.629?±?1.5% viability, whereas viability increased as 78.82?±?1.9% at 100?µg/ml of extract.     

Factors affecting prevalence of overweight among 12- to 17-year-old urban adolescents in Hyderabad, India

Objective: The problem of overweight and obesity is not confined only to developed countries but is also widely prevalent in developing countries. The objective of this study was to assess the prevalence of overweight and obesity as defined by the International Obesity Task Force (IOTF) among school‐age children in Hyderabad, India, and identify its associated factors. Research Methods and Procedures: A cross‐sectional and institutional study, adopting a multistage stratified cluster sampling procedure, was carried out during 2003 on adolescents 12 to 17 years of age of both sexes from Hyderabad, India. Results: The overall prevalence of overweight was 6.1% [95% confidence interval (CI): 4.2, 8.0] among boys and 8.2% among girls (CI: 6.0, 10.4); 1.6% and 1.0% were obese, respectively. The prevalence was significantly higher (p < 0.05) among adolescents who watched television ≥3 h/d (10.4%) or belonged to a high socioeconomic background (14.9%, p < 0.001), whereas it was significantly lower among those participating regularly in outdoor games ≥6 h/wk (3.1%, p < 0.004) and household activities ≥3 h/d (4.7%, p < 0.001). The logistic regression analysis revealed that the prevalence of overweight was 4 times higher among the adolescents of high socioeconomic status [odds ratio (OR): 4.1; CI: 2.25, 7.52], 3 times higher in those not participating in outdoor games (OR: 2.75; CI: 1.56, 4.72), and 1.92 times higher in those watching television ≥3 h/d (OR: 1.92; CI: 1.16, 3.18). Discussion: This study confirmed the findings of earlier studies carried out in Western countries and emphasizes that regular physical exercise, doing household activities, regulated television viewing, and healthy eating behaviors could contribute to controlling overweight and obesity.     

Reproductive function for a C-terminus extended, male-transmitted cytochrome c oxidase subunit II protein expressed in both spermatozoa and eggs

Our previous study documented expression of a male-transmitted cytochrome c oxidase subunit II protein (MCOX2), with a C-terminus extension (MCOX2e), in unionoidean bivalve testes and sperm mitochondria. Here, we present evidence demonstrating that MCOX2 is seasonally expressed in testis, with a peak shortly before fertilization that is independent of sperm density. MCOX2 is localized to the inner and outer sperm mitochondrial membranes and the MCOX2 antibody's epitope is conserved across >65 million years of evolution. We also demonstrate the presence of male-transmitted mtDNA and season-specific MCOX2 spatial variation in ovaries. We hypothesize that MCOX2 plays a role in reproduction through gamete maturation, fertilization and/or embryogenesis.     

Nicotinic receptors that bind alpha-bungarotoxin on neurons raise intracellular free Ca2+.

Many populations of vertebrate neurons have a membrane component that binds alpha-bungarotoxin and cholinergic ligands. Despite the abundance of this component and its similarities to nicotinic receptors, its function has remained controversial. Using a fluorescence assay, we show here that activation of the component elevates the intracellular concentration of free Ca2+, demonstrating a receptor function for the toxin-binding component. Whole-cell voltage-clamp and intracellular recordings did not detect a significant current resulting from receptor activation, possibly because the currents were small or the receptors rapidly desensitized. The rise in intracellular free Ca2+ caused by the receptor was prevented by Ca2+ channel blockers. This suggests a signaling cascade likely to have important regulatory consequences for the neuron.     

Self-injurious behavior is decreased by cyproterone acetate in adult male rhesus (Macaca mulatta)

Self-injurious behavior (SIB) presents a serious problem in laboratory macaques that cannot be socially housed for scientific reasons and among institutionalized children and adults where it is often associated with different forms of brain dysfunction. We have experienced limited success in reducing SIB in macaques by enhancing their environment with enrichment devices. Psychotropic drugs also help, but problems are associated with their use. Because sexual and aggressive behavioral problems in men have been treated with progestational drugs, we tested the efficacy of cyproterone acetate (CA, 5-10 mg/kg/week) on reducing SIB in 8 singly housed, adult male rhesus macaques. The main findings were: (1) SIB and other atypical behaviors were significantly reduced during CA treatment; (2) serum testosterone was significantly reduced during CA treatment; (3) cerebral spinal fluid (CSF) levels of 5HIAA and HVA, metabolites of serotonin and dopamine, respectively, declined significantly during CA treatment; (4) the duration of SIB positively correlated with levels of 5HIAA in CSF; but (5) sperm counts were not reduced during treatment. Thus, CA was a partially effective treatment (3 months) for adult male macaques whose behavioral problems include SIB. In summary, CA reduced SIB, overall aggression, serum testosterone, CSF 5HIAA, and CSF HVA. We hypothesized that the progestin activity of CA represses the hypothalamic gonadal axis and decreases testosterone, which in turn decreases SIB. In addition, we speculate that the decrease in 5HIAA and HVA in CSF may have been caused by progestins decreasing the activity of MAO. Therefore, the reduction of SIB may also be related to an increase in the availability of active monoamines in the CNS.     

Calcium uptake by bovine epididymal spermatozoa is regulated by the redox state of the mitochondrial pyridine nucleotides

Immature caput epididymal sperm accumulate calcium from exogenous sources at a rate 2- to 4-fold greater than mature caudal sperm. Calcium accumulation by these cells, however, is maximal in the presence of lactate as external substrate. This stimulation of calcium uptake by optimum levels of lactate (0.8-1.0 mM) is about 5-fold in caput and 2-fold in caudal sperm compared to values observed with glucose as substrate. Calcium accumulation by intact sperm is almost entirely mitochondrial as evidenced by the inhibition of uptake by rotenone, antimycin, and ruthenium red. The differences in the ability of the various substrates in sustaining calcium uptake appeared to be related to their ability to generate NADH (nicotinamide adenine dinucleotide). Previous reports have documented that mitochondrial calcium accumulation in several somatic cells is regulated by the oxidation state of mitochondrial NADH. A similar situation obtains for bovine epididymal sperm since calcium uptake sustained by site III oxidation of ascorbate in the presence of tetramethyl phenylenediamine and rotenone was also stimulated by NADH-producing substrates, including lactate, and inhibited by substrates generating NAD+ (nicotinamide adenine dinucleotide, oxidized form). Further, calcium uptake by digitonin-permeabilized sperm in the presence of succinate was stimulated when NADH oxidation was inhibited by rotenone. The compounds alpha-keto butyric, valeric, and caproic acids, which generate NAD+, inhibited the maximal calcium uptake observed in the presence of succinate and rotenone, and the hydroxy acids lactate and beta-hydroxybutyrate reversed this inhibition. These results document the regulation of sperm calcium accumulation by the physiological substrate lactate, emphasize the importance of mitochondria in the accumulation of calcium by bovine epididymal sperm, and suggest that the mitochondrial location of the isozyme LDH-X in mammalian sperm may be involved in the regulation of calcium accumulation.     

Phosphorylation-dependent interaction of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHA) with PADI6 following oocyte maturation in mice

Proteins in the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein family (YWHA; also known as 14-3-3) are involved in the regulation of many intracellular processes. We have examined the interaction of YWHA with peptidylarginine deiminase type VI (PADI6), an abundant protein in mammalian oocytes, eggs, and early embryos. Peptidylarginine deiminases catalyze the posttranslational modification of peptidylarginine to citrulline. PADI6 is associated with oocyte cytoplasmic sheets, and PADI6-deficient mice are infertile because of disruption of development beyond the two-cell stage. We found that PADI6 undergoes a dramatic developmental change in phosphorylation during oocyte maturation. This change in phosphorylation is linked to an interaction of PADI6 with YWHA in the mature egg. Recombinant glutathione S-transferase YWHA pull-down experiments and transgenic tandem affinity purification with liquid chromatography-mass spectrometry demonstrate a binding interaction between YWHA and PADI6 in mature eggs. YWHA proteins modulate or complement intracellular events involving phosphorylation-dependent switching or protein modification. These results indicate that phosphorylation and/or YWHA binding may serve as a means of intracellular PADI6 regulation.