Jagtar Singh

Serine alkaline protease from a newly isolated Bacillus sp. SSR1

Abstract An extracellular serine alkaline protease produced by Bacillus sp. SSR1 was purified to homogeneity by Sephadex A-50 and Sepharose 6B column chromatography. The enzyme is a monomeric protein with a molecular weight of 29 and 35 kDa as estimated by SDS–PAGE and native PAGE respectively. The purified enzyme is stable in the alkaline pH range (8.0–11.0) and retains 100% activity at its optimum temperature of 40°C even after 300 min of incubation. The presence of CaCl 2 shifts the optimum temperature to 45°C and produces a 1.3-fold increase in its activity. The enzyme remains active and stable in various detergents and is strongly activated by metal ions (Fe 3+ , Ca 2+ , Na + )

Metagenomics: Concept, methodology, ecological inference and recent advances

Microorganisms constitute two third of the Earths biological diversity. As many as 99% of the microorganisms present in certain environments cannot be cultured by standard techniques. Culture‐independent methods are required to understand the genetic diversity, population structure and ecological roles of the majority of organisms. Metagenomics is the genomic analysis of microorganisms by direct extraction and cloning of DNA from their natural environment. Protocols have been developed to capture unexplored microbial diversity to overcome the existing barriers in estimation of diversity. New screening methods have been designed to select specific functional genes within metagenomic libraries to detect novel biocatalysts as well as bioactive molecules applicable to mankind. To study the complete gene or operon clusters, various vectors including cosmid, fosmid or bacterial artificial chromosomes are being developed. Bioinformatics tools and databases have added much to the study of microbial diversity. This review describes the various methodologies and tools developed to understand the biology of uncultured microbes including bacteria, archaea and viruses through metagenomic analysis.

Production and characterization of a thermostable β-galactosidase from Bacillus coagulans RCS3

A strain of Bacillus coagulans RCS3 isolated from ahot‐water spring produced significant β‐galactosidase activity at 10 days of growth in a flask. While enzyme production was maximum at 50 °C, the highest activity was at 65 °C, where the half‐life was 2 h. A 2 °C decrease in temperature increased the half‐life to 15 h without significantly changing the activity, suggesting that 63 °C is the temperature of preference compared with 65 °C for a combination of good activity and stability. The β‐galactosidase was also stable over pH 5–8, with peak activity at pH 6–7. It was strongly and competitively inhibited by the hydrolysis product galactose. Bivalent cations (Cu2+, Ni2+ and Hg2+) in the concentration range of 0.5–2.0 mM also inhibited enzyme activity. Both lactose solution and whey could be hydrolysed substantially within 36 h at 50 °C. The thermostability and pH‐stability and good hydrolytic capability make this enzyme potentially useful in the dairy industry.

A highly thermostable, alkaline CMCase produced by a newly isolated Bacillus sp. VG1

An extracellular, highly thermostable and alkaline CMCase was purified from Bacillus sp. VG1 using ion exchange and gel filtration chromatography. Enzyme was optimally produced in a medium containing 1.0% CMC and 0.5% tryptone. The purified CMCase had a pH optimum of 9–10 and a half life of 12 min even at 100 °C. The enzyme activity was reduced by Hg2+ and stimulated by Co2+, Na+ and K+. Various detergents and proteinases moderately inhibited the CMCase activity. The molecular weight studies showed a single band on SDS–PAGE.

Association of mannose-binding lectin gene polymorphism with tuberculosis susceptibility and sputum conversion time

Mannose‐binding lectin (MBL) plays an important role in innate immunity. The effect of low MBL levels producing variants of MBL2 gene on tuberculosis (TB) has been controversial with some studies reporting it to confer protection against the disease, whereas others estimating a susceptibility relation. Other than conducting a case–control study to evaluate the role of MBL A/B polymorphism on TB, we conducted a longitudinal study to check whether this MBL variant can influence the host response to Mycobacterium tuberculosis infection. A total of 357 TB patients (286 pulmonary TB, 71 extrapulmonary (EP) TB) and 392 healthy controls belonging to same ethnicity were included in the study. We found the mutant allele ‘B’ allele confers a protective role against TB in our study population. This effect was absent in EP patients. On stratification on the basis of sex, the protective role of the ‘B’ allele was found to be limited to females only and males reported no significant difference. No effect of MBL A/B polymorphism on sputum conversion time was reported. We conclude that MBL ‘B’ allele is associated with protection against TB, but no influence was found on sputum conversion rate.

Characterization of ionically bound peroxidases from apple (Mallus pumilus) fruits.

Abstract Ionically bound peroxidases (POD) were salt extracted from the pulp of four Indian apple varieties, i.e., Golden delicious HP, Golden delicious JK, Red delicious, and Royal delicious. They were precipitated with chilled ethanol. Thermal treatments of partially purified enzymes were given from 40–70°C for 30 minutes. Golden delicious HP peroxidase showed thermostability at 60°C, while three other peroxidases were observed at 50°C. Phenolic compounds (i.e., caffeic acid, ferulic acids, p‐coumaric acid, protocatechuic acid) and metal ions (i.e., Cu2+ and Fe2+) activated all apple peroxidases. However, Mn2+ inhibited the peroxidases from Golden delicious HP, Golden delicious JK, and Red delicious, and a substantial increase was observed in Royal delicious peroxidase. Mg2+ inhibited the peroxidases from Golden delicious HP and Red delicious, but marginal activation was reported in peroxidases from Golden delicious JK and Royal delicious. Zn2+ established stimulation in Golden delicious HP and Golden delicious JK peroxidases, but inhibition was observed in peroxidases in Red delicious and Royal delicious.. Methionine, proline, tryptophan, and valine stimulated all four apple peroxidases, but cysteine showed inhibition in Golden delicious JK.

Protective role of IL-18 -137G/C polymorphism in a North Indian population with asthma: a pilot study.

BACKGROUND IL-18, a pleiotropic, pro-inflammatory cytokine that plays a major role in innate as well as acquired immunity, has been implicated in asthma etiology and this is the first study investigating the role of IL-18 -137G/C (rs 187238) promoter polymorphism in asthma pathogenesis in a North Indian population. METHODS A pilot study was conducted with a total of 824 subjects, out of which 410 were asthma patients including 323 patients suffering from allergic rhinitis and 414 healthy controls from regions of North India. Tetra-Primer Amplification Refractory Mutation System Polymerase Chain Reaction (Tetra-Primer ARMS PCR) was used for genotyping the IL-18 -137G/C polymorphism. RESULTS While the homozygous wild (GG) genotype was equally prevalent in asthma patients as well as control subjects (70.0%), the homozygous mutant (CC) genotype was more prevalent among the controls (8.0%) than in asthma patients (3.4%), which yielded a significant protection or decreased risk towards asthma. Statistical analysis revealed Odds Ratio (OR)=0.43 (95% CI=0.21-0.85), Chi2 (χ2)=6.93 and p-value=0.008 (p<0.005). Moreover, a few asthma phenotypic traits also revealed significant protective associations with the polymorphism. CONCLUSIONS The IL-18 -137G/C polymorphism confers a significant protection from asthma in the studied North Indian population. This is the first study to report the protective association of the polymorphism with the disease.

High risk association of IL-4 VNTR polymorphism with asthma in a North Indian population

BACKGROUND A case-control study was conducted to evaluate the role of IL-4 VNTR polymorphism in asthma that has been associated with various inflammatory diseases worldwide. This is the first case-control study conducted in India, investigating the role of IL-4 VNTR polymorphism in asthma pathogenesis. METHODS A case-control study was performed with a total of 824 adult subjects, inducting 410 asthma patients and 414 healthy controls from North India. The genotypes were identified by polymerase chain reaction. RESULTS Statistical analysis for the IL-4 VNTR polymorphism revealed that the Rp1 allele was significantly associated with asthma with OR=1.47, 95% CI (1.11-1.94) and p=0.005. The Rp1/Rp1 homozygous mutant genotype posed a high risk towards asthma with OR=2.39, 95% CI (0.96-6.14) and p=0.040. The Rp2/Rp1 heterozygous genotype also posed a risk towards asthma with OR=1.39, 95% CI (1.00-1.94) and p=0.040. Most of the phenotypic traits were significantly associated with the disease. CONCLUSIONS IL-4 VNTR polymorphism is a high risk factor for asthma in the studied North Indian population.

Role of TLR4 C>1196T (Thr399Ile) and TLR4 A>896G (Asp299Gly) polymorphisms in a North Indian population with asthma: a case-control study.

Toll‐like receptor 4 (TLR4) is the most important TLR among the pattern recognition receptors which recognizes lipopolysaccharide of gram‐negative bacteria. They identify a highly conserved structure of microbes called pathogen‐associated molecular patterns and activate immune and inflammatory responses that have been shown to be involved in the pathogenesis of asthma. The role of TLR4 gene polymorphisms in asthma was detected in a total of 964 individuals, including 483 healthy controls and 481 asthma patients from a North Indian population. The genotyping was carried out using polymerase chain reaction–restriction fragment length polymorphism method. Statistical analysis revealed that the heterozygous genotype as well as the mutant (T) allele of the TLR4 C>1196T (Thr399Ile) polymorphism shows resistance towards asthma with OR = 0.70, 95% CI (0.49–0.99), P corrected value = 0.046 and OR = 0.72, 95% CI (0.52–0.98), P corrected value = 0.039, respectively. However, no association was found between the TLR4 A>896G (Asp299Gly) polymorphism and asthma patients (P > 0.05). This is the first study conducted in India conferring TLR4 (Thr399Ile) polymorphism resistance towards asthma, while lack of association was found between TLR4 (Asp299Gly) polymorphism and asthma in the studied North Indian population.

Antiproliferative potential of a novel parthenin analog P16 as evident by apoptosis accompanied by down-regulation of PI3K/AKT and ERK pathways in human acute lymphoblastic leukemia MOLT-4 cells

Leukemia is one of the deadliest types of cancer. Lack of effective treatment strategies has resulted in an extensive quest for new therapeutic molecules against it. This study explores the molecular mechanism of anticancer activity of P16, a semisynthetic analog of parthenin, against the human acute lymphoblastic leukemia MOLT-4 cells. P16 displayed antiproliferative activity in different cancer cell lines; however, MOLT-4 cells showed highest sensitivity for P16 with IC50 value of 0.6μM. Further studies revealed that P16 induced cell death by apoptosis. It caused mitochondrial stress, which was mediated by the translocation of Bax from cytosol to mitochondria and release of cytochrome c into the cytosol and consequent activation of caspase-9. However, P16 was also able to activate caspase-8, thus involving both extrinsic and intrinsic pathways of apoptosis. Further, activation of caspase-3 led to cleavage of its target proteins PARP-1 and ICAD, which resulted in apoptotic DNA damage. P16 induced apoptosis was accompanied by the down-regulation of important leukemic cell survival proteins like pAKT (S473), pAKT (T308), pP70S6K, pCRAF, and pERK1/2. However, inhibition of caspases by Z-VAD-FMK reversed the down-regulatory effect of P16 on pAKT (S473) and pP70S6K, as evident by the cell viability assay and flow cytometric analysis but this inhibition did not completely reverse the antiproliferative effect of P16, thereby indicating the role of additional factors apart from caspases in P16 induced apoptosis in MOLT-4 cells. Owing to its antiproliferative potential against leukemia cells, P16 can further be explored as an effective therapeutics against leukemia.