Purification and characterization of a novel β-galactosidase from Bacillus sp MTCC 3088

An extracellular β-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was harvested from the late stationary-phase of Bacillus sp MTCC 3088. The enzyme was purified 36.2-fold by ZnCl₂ precipitation, ion exchange, hydrophobic interaction and gel filtration chromatography with an overall recovery of 12.7%. The molecular mass of the purified enzyme was estimated to be about 484 kDa by gel filtration on a Sephadex G-200 packed column and the molecular masses of the subunits were estimated to be 115, 86.5, 72.5, 45.7 and 41.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the native enzyme, determined by polyacrylamide gel electrofocusing, was 6.2. The optimum pH and temperature were 8 and 60°C, respectively. The Michaelis–Menten constants determined with respect to o-NO₂-phenyl-β-D-galactopyranoside and lactose were 6.34 and 6.18 mM, respectively. The enzyme activity was strongly inhibited (68%) by galactose, the end product of lactose hydrolysis reaction. The β-galactosidase was specific for β-D anomeric linkages. Enzyme activity was significantly inhibited by metal ions (Hg²⁺, Cu²⁺ and Ag⁺) in the 1–2.5 mM range. Mg²⁺ was a good activator. Catalytic activity was not affected by the chelating agent EDTA. Journal of Industrial Microbiology & Biotechnology (2000) 24, 58–63. Received 09 February 1999/ Accepted in revised form 24 September 1999     

Arsenic Exposure and Outcomes of Antimonial Treatment in Visceral Leishmaniasis Patients in Bihar,India: A Retrospective Cohort Study

BackgroundIn the late twentieth century, emergence of high rates of treatment failure with antimonial compounds (SSG) for visceral leishmaniasis (VL) caused a public health crisis in Bihar, India. We hypothesize that exposure to arsenic through drinking contaminated groundwater may be associated with SSG treatment failure due to the development of antimony-resistant parasites.MethodsA retrospective cohort design was employed, as antimony treatment is no longer in routine use. The study was performed on patients treated with SSG between 2006 and 2010. Outcomes of treatment were assessed through a field questionnaire and treatment failure used as a proxy for parasite resistance. Arsenic exposure was quantified through analysis of 5 water samples from within and surrounding the patient’s home. A logistic regression model was used to evaluate the association between arsenic exposure and treatment failure. In a secondary analysis survival curves and Cox regression models were applied to assess the risk of mortality in VL patients exposed to arsenic.ResultsOne hundred and ten VL patients treated with SSG were analysed. The failure rate with SSG was 59%. Patients with high mean local arsenic level had a non-statistically significant higher risk of treatment failure (OR = 1.78, 95% CI: 0.7–4.6, p = 0.23) than patients using wells with arsenic concentration <10 μg/L. Twenty one patients died in our cohort, 16 directly as a result of VL. Arsenic levels ≥ 10 μg/L increased the risk of all-cause (HR 3.27; 95% CI: 1.4–8.1) and VL related (HR 2.65; 95% CI: 0.96–7.65) deaths. This was time dependent: 3 months post VL symptom development, elevated risks of all-cause mortality (HR 8.56; 95% CI: 2.5–29.1) and of VL related mortality (HR 9.27; 95% CI: 1.8–49.0) were detected.Discussion/ConclusionThis study indicates a trend towards increased treatment failure in arsenic exposed patients. The limitations of the retrospective study design may have masked a strong association between arsenic exposure and selection for antimonial resistance in the field. The unanticipated strong correlation between arsenic exposure and VL mortality warrants further investigation.     

Evidence that phosphate specific transporter is amplified in a fluoroquinolone resistant Mycobacterium smegmatis.

We reported in an earlier study that active efflux of drug has a predominant role in conferring resistance in a laboratory-generated ciprofloxacin-resistant mutant of Mycobacterium smegmatis. This mutant exhibited mRNA level overexpression, as well as chromosomal amplification, of the gene pstB, encoding the putative ATPase subunit of phosphate specific transport (Pst) system. We demonstrate here that this mutant shows enhanced phosphate uptake and that inactivation of pstB in the parental strain results in loss of high affinity phosphate uptake and hypersensitivity to fluoroquinolones. These findings suggest a novel role of the Pst system in active efflux, in addition to its involvement in phosphate transport.     

Column Test in Field Application Study Using Biostimulant Ball in Contaminated Coastal Sediment

This study consisted of a field column test to evaluate the effectiveness of the slow release and long-term outcomes of a biostimulant ball (BSB) in contaminated coastal sediment. The BSB was prepared using biostimulating agents consisting of sulphate, nitrate and acetate with uncontaminated dredged sediment, and cellulose acetate (CA) and polysulfone (PS). SEM images were captured to investigate the physical properties of the PS and CA-coated biostimulant ball, and doing so was observed to have a great influence in the release rate of the nutrients. The CA-coated BSB had large pores with an irregular shape inside and a honeycomb structure outside. The PS-coated BSB had no pores inside and had small pores outside the coating, and the release rate of the PS-coated BSB was slower with more long-term effects than the CA-coated BSB. The CA- and PS-coated BSB sample showed that the chemical oxygen demand (COD), total phosphorous (TP) and total nitrogen (TN) had been effectively reduced, and conversely, pH is not significantly affected when using the BSB. Moreover, the fractions of heavy metals (Cd, Zn, Cu, Pb, Cr and Fe) were highly influenced when using the PS-coated BSB added sample than when using the CA-coated BSB. Therefore, application of CA- and PS-coated BSB is a promising method for cleaning contaminated coastal sediment.     

PTPH1 immunohistochemical expression and promoter methylation in breast cancer patients from India: A retrospective study

Protein Tyrosine Phosphatase H1/Protein Tyrosine Phosphatase Non receptor Type 3 (PTPH1/PTPN3) is upregulated and/or mutated in glioma, ovarian, gastric, and colorectal cancers. Previous studies have documented that PTPH1-associated breast cancers exhibit enhanced sensitivity to tamoxifen and tyrosine kinase inhibitors through dephosphorylation of ER and epidermal growth factor receptor, respectively. Owing to the key role that PTPH1 plays as a biomarker in predicting the response of chemotherapeutic drugs and lack of studies on Indian breast cancer patients, the present study investigated PTPH1 protein expression and its relationship to clinical features, ER/PR/HER2/neu statuses, and methylation of promoter in breast cancer tissues (n = 67) among Indian population by immunohistochemistry and methylation specific polymerase chain reaction. PTPH1 expression was upregulated in 58.21% (39/67) and downregulated in the rest of tumor specimens, and it correlated with ER, PR, and HER2/neu statuses with p values of <0.0001, 0.0113, and 0.0448, respectively. Additionally, we found that the 2 kb region upstream of PTPH1 gene harbored CpG sites within, and was ubiquitously methylated in breast cancer (n = 13), colon cancer tissue (n = 1), uterine cancer tissue (n = 1), normal breast tissue (n = 1) in addition to Hela and MCF7 cell lines. In conclusion, our data showed a strong correlation of the PTPH1 status with the ER and ubiquitous nature of PTPH1 promoter methylation at specific CpG sites irrespective of cancer types and protein expression. Our findings underscore the clinical relevance of PTPH1 expression in Indian patients and warrant additional studies to explore the importance of ubiquitously methylated promoter at specific CpG sites in upstream of the PTPH1 gene.     

Plant Growth-Promoting Chitinolytic Paenibacillus elgii Responds Positively to Tobacco Root Exudates

Bacterial strains from chitin/chitosan-rich soils, from two industries, were screened for their chitinolytic, antifungal, and mineral phosphate solubilization abilities. The isolate SMA-1-SDCH02, positive for all three properties, was selected and identified as Paenibacillus elgii based on morphological and biochemical characters and supported by 16S rRNA gene sequence analysis. P. elgii enhanced the growth of groundnut in terms of shoot height, root length, total chlorophyll, and fresh and dry weight when applied alone or in combination with chitosan. The plant growth-promoting activity of P. elgii was seen in tobacco in a specially designed gnotobiotic setup indicating its capability to promote growth of at least groundnut and tobacco. Metabolite changes in the bacteria, studied using attenuated total reflectance-infrared (ATR-IR) spectroscopy, revealed split bands of amide I at the 1659- and 1636-cm⁻¹ regions when grown in minimal media amended with tobacco root exudates. The difference in ATR-IR bands in the presence of tobacco root exudates indicated production of compounds with differences in functional groups.     

Identification of kernel iron- and zinc-rich maize inbreds and analysis of genetic diversity using microsatellite markers

Development of micronutrient enriched staple foods is an important breeding goal in view of the extensive problem of ‘hidden hunger’ caused by micronutrient malnutrition. In the present study, kernel iron (Fe) and zinc (Zn) concentrations were evaluated in a set of 31 diverse maize inbred lines in three trials at two locations – Delhi (Kharif 2007 & 2008) and Hyderabad (Rabi 2007–08). The ranges of kernel Fe and Zn concentrations were 13.95–39.31 mg/kg and 21.85–40.91 mg/kg, respectively, across the three environments. Pooled analysis revealed significant genotype × environment (G × E) interaction in the expression of both the micronutrient traits, although kernel Fe was found to be more sensitive to G × E as compared to kernel Zn. Seven inbred lines, viz., BAJIM-06-03, DQPM-6, CM212, BAJIM-06-12, DQPM-7, DQPM-2 and CM129, were found to be the most stable and promising inbred lines for kernel Zn concentration, while for kernel Fe concentration, no promising and stable genotypes could be identified. Analysis of molecular diversity in 24 selected inbred lines with phenotypic contrast for the two kernel micronutrient traits, using 50 SSR markers covering the maize genome, revealed high levels of polymorphisms (214 SSR alleles; mean PIC value?=?0.62). The phenotypically contrasting and genetically diverse maize inbred lines identified in this study could be potentially utilized in further studies on QTL analysis of kernel micronutrient traits in maize, and the stable and most promising kernel micronutrient-rich maize genotypes provide a good foundation for developing micronutrient-enriched maize varieties suitable for the Indian context.     

Role of PKCζ‐NADPH oxidase signaling axis in PKCα‐mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C‐ζ (PKC‐ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC‐α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ, NADPH oxidase, and PKC‐α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC‐ζ and NADPH oxidase attenuate AngII induced increase in PKC‐α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ and NADPH oxidase activities. Overall, PKCζ‐NADPH oxidase‐PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII‐mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.