Exogenous application of calcium chloride in wheat genotypes alleviates negative effect of drought stress by modulating antioxidant machinery and enhanced osmolyte accumulation

Plants use various mechanisms to cope with drought constraints at morphological, physiological, and biochemical level by means of different adaptive mechanisms. All organisms use a network of signal transduction pathways to control their metabolism and to adapt to the environment. Among these pathways, calcium (Ca²⁺) ions play an important role as a universal second messenger. Calcium has unique properties and universal ability to transmit diverse signals that trigger primary physiological actions in the cell in response to hormones, pathogens, and stress factors. Calcium plays a fundamental role in regulating the polar growth of cells and tissues and participates in plant adaptation to various stress factors. This study was conducted to examine the role of Ca²⁺ in ameliorating the adverse effect of drought stress responses in two contrasting wheat genotypes, HD 2733 (drought sensitive) and HD 2987 (drought tolerant), differing in their drought tolerance. The plants were treated with mannitol or Hoagland solution and then supplemented with CaCl₂ (10 mM). Measurements of seed germination, shoot growth, and chlorophyll content showed that calcium treatment increased all these factors in tolerant genotype (HD 2987) under induced stress condition. Drought stress reduced relative water content, osmolyte, and soluble sugar accumulation in both the genotypes, but CaCl₂ supplementation increased all the components under stress condition in HD 2987 as compared to HD 2733. The oxidative damage caused by induced stress was lower in HD 2987 compared to HD 2733 genotypes as assessed by their higher photosynthetic capacity and lower electrolyte leakage, malondialdehyde (MDA) content as well as H₂O₂ accumulation. Less accumulation of superoxide and H₂O₂ was also observed in HD 2987 genotype after CaCl₂ supplementation combined with mannitol treatment. In addition, the enhanced accumulation of calcium in the HD 2987 genotype is correlated with the higher activities of antioxidant enzymes than HD 2733 genotype under similar stress conditions. Our findings provide evidence of the protective role of exogenous calcium in conferring better tolerance against mannitol-induced drought stress in wheat genotypes, which could be useful as genetic stock to develop wheat tolerant varieties in breeding programs.     

Determination and quantification of asiaticoside in endophytic fungus from <Emphasis Type="Italic">Centella asiatica</Emphasis> (L.) Urban

Centella asiatica (L.) Urban is a highly considered medicinal plant owing to its secondary metabolites asiaticoside, madecassoside, asiatic acid, and madecassic acid. The asiaticoside, one of the most important constituents of the plant, is a triterpenoid saponin having memory enhancement property. Given its medicinal properties, we isolated and characterized endophytic fungi from this plant with the aim to screen these microorganisms for asiaticoside production. In total, we isolated 13 endophytic fungi from the leaves of the plant, out of which one of the isolates produced asiaticoside. This asiaticoside producing isolate was identified as Colletotrichum gloeosporioides by internal transcribed spacer-based rDNA sequencing. The presence of asiaticoside in ethyl acetate extract of C. gloeosporioides was confirmed by LC–MS. The production of asiaticoside measured in relation to incubation time and subculture generation revealed presence of 62.29?±?3.36 µg/100 mL of asiaticoside by C. gloeosporioides on the 15th day in first subculture generation followed by a decrease in subsequent generations. A similar trend was also shown by yield and growth curve of C. gloeosporioides. The asiaticoside production and yield were found to be positively correlated. This paper reported the production of asiaticoside by an endophytic fungus C. gloeosporioides for the first time. The present findings definitely provide an impetus to the production of asiaticoside by utilizing the endophytic source.

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Chemical compound studied in this article: Asiaticoside (PubChemCID: 108062)

     

Characterization of Programmed Death-1 Homologue-1 (PD-1H) Expression and Function in Normal and HIV Infected Individuals

Chronic immune activation that persists despite anti-retroviral therapy (ART) is the strongest predictor of disease progression in HIV infection. Monocyte/macrophages in HIV-infected individuals are known to spontaneously secrete cytokines, although neither the mechanism nor the molecules involved are known. Here we show that overexpression of the newly described co-stimulatory molecule, PD1 homologue (PD-1H) in human monocyte/macrophages is sufficient to induce spontaneous secretion of multiple cytokines. The process requires signaling via PD-1H as cytokine secretion could be abrogated by deletion of the cytoplasmic domain. Such overexpression of PD-1H, associated with spontaneous cytokine expression is seen in monocytes from chronically HIV-infected individuals and this correlates with immune activation and CD4 depletion, but not viral load. Moreover, antigen presentation by PD-1H-overexpressing monocytes results in enhanced cytokine secretion by HIV-specific T cells. These results suggest that PD-1H might play a crucial role in modulating immune activation and immune response in HIV infection.     

Cloning,Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome

The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6–7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.     

Novel expression system for Corynebacterium acetoacidophilum and Escherichia coli based on the T7 RNA polymerase-dependent promoter

The industrially important species of corynebacteria viz. Corynebacterium acetoacidophilum appear to be alternative hosts for recombinant protein production; despite many efforts, a strong promoter-based system in corynebacteria has not been established so far. Described here is a T7 promoter-based expression system which was functional in both gram-positive C. acetoacidophilum and gram-negative Escherichia coli in an external inducer independent manner. This is the very first report of a T7 expression system for Corynebacterium sp. Also, it is a useful addition in the existing T7 expression systems of E. coli.     

Production of recombinant proteins by microbes and higher organisms

Large proteins are usually expressed in a eukaryotic system while smaller ones are expressed in prokaryotic systems. For proteins that require glycosylation, mammalian cells, fungi or the baculovirus system is chosen. The least expensive, easiest and quickest expression of proteins can be carried out in Escherichia coli. However, this bacterium cannot express very large proteins. Also, for S–S rich proteins, and proteins that require post-translational modifications, E. coli is not the system of choice. The two most utilized yeasts are Saccharomyces cerevisiae and Pichia pastoris. Yeasts can produce high yields of proteins at low cost, proteins larger than 50 kD can be produced, signal sequences can be removed, and glycosylation can be carried out. The baculoviral system can carry out more complex post-translational modifications of proteins. The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells. Genetically modified animals secrete recombinant proteins in their milk, blood or urine. Similarly, transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.     

Functional Access to Neuron Subclasses in Rodent and Primate Forebrain

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