Diversity analysis of selected rice landraces from West Bengal and their linked molecular markers for salinity tolerance

Study of genetic diversity in crop plants is essential for the selection of appropriate germplasm for crop improvement. As salinity posses a serious environmental challenge to rice production globally and especially in India, it is imperative that the study of large collections of germplasms be undertaken to search for salt tolerant stocks. In the present study, 64 indica germplasms were collected from different agro-climatic zones of West Bengal, India, from the Himalayan foothills in the northern part down to the southern saline belt of the state keeping in view the soil characteristics and other edaphic factors prevailing in the region. Salt tolerance parameters were used to screen the large set of germplasms in terms of root-shoot length, fresh-dry weight, chlorophyll content, Na+/K+ ratio and germination potential in presence of salt. Standard evaluation score or SES was calculated to find out tolerant to sensitive cultivar. Twenty-one SSR markers, some associated with the Saltol QTL and others being candidate gene based SSR (cgSSR) were used to study the polymorphism of collected germplasm. A wide diversity was detected among the collected germplasms at the phenotypic as well as molecular level. Of the 21 SSR markers, 15 markers were found to be polymorphic with 88 alleles. Based on phenotypic and biochemical results, 21 genotypes were identified as salinity tolerant, whereas 40 genotypes turned out to be salt susceptible. The present study shows that apart from the established salt tolerant lines, several other landraces like Bonkanta, Morisal, Ghiosh, Patni may be the source of salt tolerant donor in future breeding programs.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.     

Phospholipases as possible virulence factor in Vibrio parahaemolyticus

Phospholipase C activity was elevated in pathogenic Vibrio parahaemolyticus isolated from patients. Phospholipase A activity was more pronounced in the nonpathogenic V. parahaemolyticus strains isolated from water. Extracts of the strains containing phospholipase C and A activity but no thermostable direct haemolysin (TDH) were capable of producing lesions in guinea pig skin indicating the presence of a toxic factor other than TDH. It is suggested that the toxic factor may be phospholipase C since the purified enzyme from Clostridium perfringens produced a similar reaction in guinea pig skin.     

以基因枪介导获转ps1—barnase基因的工程雄性不育水稻植株

以ps1-barnase(brn)为目的基因,pHcintG(PG)为选择/标记基因进行共转化,以PDS-1000-氦气基因枪介导,将brn及PG基因转化到水稻台北309及秋光的核DNA中,得到了转ps1-barnase基因的工程雄性不育植株。以悬浮细胞作为基因枪轰击的靶材料,转化植株再生频率较初级愈伤组织的为高。转brn基因植株的其他主要性状与供体亲本无显著差异,但却表现不育。其不育的程度在不同的植株之间表现不同。在转brn基因植株中观察到全不育(占全部brn阳性植株的40.6％)、高不育(占15.6％)及半不育的个体(占43.7％)。全不育的转基因植株自交完全不能结实(结实率为零),除个别植株外,花粉完全不被I-KI染色;而人工授以正常的花粉则可以获得杂交种子。而brn基因的阴性植株及未进行转化的对照植株则完全可育,表明转基因植株之雄性不育乃brn基因所致。结果表明,brn基因在水稻中是完全可以正常表达的,其表达的时期推测在花粉母细胞减数分裂前至花粉形成之间的整个时期。     

HIV-1 Vpr Modulates Macrophage Metabolic Pathways: A SILAC-Based Quantitative Analysis

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Enhanced conjugation of Candida rugosa lipase onto multiwalled carbon nanotubes using reverse micelles as attachment medium and application in nonaqueous biocatalysis

Three liquid phases (viz. aqueous, nonaqueous, and reverse micelles) were scrutinized as medium for attachment of the enzyme Candida rugosa lipase (CRL) onto multiwalled carbon nanotubes (CNTs). The nanotubes were functionalized to attain carboxyl and amino groups on their surfaces before enzyme conjugation. Transmission electron microscopy and Fourier transformation infrared spectroscopic studies were used for characterization of the nanotubes during the course of functionalization. High enzyme loadings associated with the functionalized CNTs were observed when reverse micelles were used as the attachment medium. In addition, high activity in terms of ester synthesis in organic solvents was also observed while using those preparations. The nanobioconjugates prepared using reverse micelles were found to be highly sturdy and exhibited appreciable operational stability of around 95 ± 3% at 20th cycle (in case of carboxylated nanotubes) and 90 ± 5% at 10th cycle (in case of aminated nanotubes) for esterification. This shows the potential application of reverse micelles as the attachment medium for surface active enzymes such as CRL onto CNTs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:828–836, 2014     

Specific Localization of β-Arrestin2 in Myenteric Plexus of Mouse Gastrointestinal Tract

β-arrestin2 is a key molecule involved in signaling and internalization of activated G protein-coupled receptors including µ-opioid receptors (MOR). Previously we have shown that decreased expression of β-arrestin2 upon chronic morphine is associated with the development of opioid tolerance in the gastrointestinal tract. However, the localization of β-arrestin2 within the gastrointestinal wall is not known. In this study we found that β-arrestin2 is localized in the soma of a select group of neurons in the myenteric ganglia but not in smooth muscle. The density of β-arestin2 was significantly higher in the ileum than the colon. We identified four variants of β-arrestin2 in the ileum, with ARRB-005 and ARRB-013 being the most abundant. Further, the current study utilized multiple-labeling immunofluorescence to characterize the chemical coding of neurons expressing β-arrestin2 in the murine myenteric plexus and the co-localization of MOR₁ and β-arrestin2. β-arrestin2 co-localized with choline acetyltransferase and calretinin. In contrast, β-arrestin2 neither co-localized with substance P, nitric oxide synthase nor calbindin. Genetic deletion of β-arrestin2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (-log M EC₅₀: wild type = 5.8 vs. β-arrestin2 knockout = 5.9). Our findings suggest specificity in the localization of β-arrestin2 in the myenteric plexus within MOR₁-expressing neurons and provide a relation for direct intracellular crosstalk between MOR₁ receptor activation and β-arrestin2 signaling in the myenteric neurons. β-arrestin2 deletion does not directly alter basal enteric cholinergic neuronal function.     

Epicatechin ameliorates ionising radiation-induced oxidative stress in mouse [corrected] liver

The current study was intended to evaluate the hepatoprotective effect of Epicatechin (EC) against radiation-induced oxidative stress, in terms of inflammation and lipid peroxidation. Swiss albino mice were administered with EC (15 mg/kg body weight) for three consecutive days before exposing them to a single dose of 5-Gy (60)Co gamma (γ) irradiation. Mice were necropsied and livers were taken for immunohistochemistry, western blot analysis and biochemical tests for the detection of markers of hepatic oxidative stress. Nuclear translocation of nuclear factor kappa B (NF-κB) and lipid peroxidation were increased whereas the activities of superoxide dismutase (SOD) and catalase (CAT), reduced glutathione (GSH) content and ferric reducing antioxidant power (FRAP) were diminished upon radiation exposure compared to control. Translocation of NF-κB from cytoplasm to nucleus and lipid peroxidation were found to be inhibited whereas an increase in SOD, CAT, GSH and FRAP was observed in the mice treated with EC prior to irradiation. Thus, pre-treatment with EC offers protection against γ-radiation induced hepatic alterations.     

Overexpression of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) in HepG2 cells leads to increased hyaluronan synthesis and cell proliferation by up-regulation of cyclin D1 in AKT-dependent pathway

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.