Basal expression studies of cystatins during specific growth stages of wheat spikes for defining their possible role in differential and stage dependent immunity against Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>)

Two genotypes showing differential immunity against Karnal bunt (Tilletia indica) were used to investigate the role of three members of cystatin gene family in growth stage dependent immunity in wheat (Triticum aestivum L.). Three members of cystatin gene family (WC1, WC2, and WC4) were cloned and sequenced. Analysis of sequenced data showed that there was 76–99% nucleotide and protein sequence identity between different genes of the wheat cystatin. In silico amino acid sequence analysis revealed the presence of a conserved signature pattern of residues and also the functional domains were presumed to be actively involved in imparting cysteine protease inhibition capability. The semi-quantitative and quantitative levels of these members were measured by means of RT-PCR, northern blotting, western blotting, and by ELISA techniques. The members of cystatin gene family were expressed in both resistant (HD 29) and susceptible genotypes (WH 542); however, the expression level was significantly (P < 0.001) higher in resistant compared to susceptible genotype at all the stages of wheat spikes. The patterns of expression of WC2, WC4 were similar except in the levels in S₁ and S₂ stages as it remained constant (P > 0.05) in contrary to WC1 family whose expression gradually increased from S_v to S₂ stage. According to the intensity of the detected band in RT PCR, northern blot and western blot, WC1 family seems to be expressed more than the other gene families. The immunoassay results further showed that WC1 protein was abundantly expressed in resistant genotype and high expression was observed at the S2 stage as compared to susceptible genotype (P < 0.001) suggesting that low level of expression of WC1 in S2 stage is responsible for KB infection. The results of the present study clearly indicate the role of cystatin gene family in differential and stage dependent immunity against KB.     

Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.     

Isolation, identification and application of novel bacterial consortium TJ-1 for the decolourization of structurally different azo dyes

A novel bacterial consortium (TJ-1), which could decolorize Acid Orange 7 (AO7) and manyother azo dyes, was developed. In TJ-1 three bacterial strains were identified as Aeromonas caviae, Proteus mirabilis and Rhodococcus globerulus by 16S rRNA gene sequence analysis. AO7 decolorization was significantly higher with the use of consortium as compared to the use of individual strains, indicating complementary interactions among these strains. AO7 decolorization was observed under microaerophilic condition in the presence of organic carbon source. Either yeast extract (YE) alone or a combination of YE and glucose resulted in much higher decolorization of AO7 as compared to glucose alone, peptone or starch. Kinetic studies with different initial AO7 concentrations showed that more than 90% decolorization could be achieved even at 200mg/l within 16h. Fed-batch studies showed that AO7 decolorization required 10h during the first cycle and 5h in the second and third cycles, showing that bacterial cells could be used for multiple cycles. The consortium also decolorized fifteen other azo dyes individually as well as a simulated wastewater containing a mixture of all the sixteen azo dyes, thus, conferring the possibility of application of TJ-1 for the treatment of industrial wastewaters.     

Life cycle changes during 12 successive generations of laboratory colonisation of the leafroller Cnephasia jactatana (Walker) (Lepidoptera: Tortricidae) on artificial diet

Abstract

Close monitoring of the lepidopteran leafroller Cnephasia jactatana under laboratory colonisation revealed few distinct effects of successive rearing on artificial diet on the life cycle. The second laboratory generation had a prolonged development time and altered sex synchronism in pupation and eclosion patterns. Some deleterious changes were observed in later generations, including decreases in fertility, egg hatch and sperm motility, failure of mating adults to separate, and pupal and adult malformations. These changes were not adaptive, but were due to incompatibility with the general purpose diet (GPD) used; they were absent under sub-colonisation on a sheepnut-bean based diet (SBD). Success in the laboratory colonisation of C. jactatana is attributed to a random mating protocol, choice of environmental conditions representing the wild habitat, and a rapid rate of population growth.     

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance,Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Background

Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress.

Methodology

The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.

Results

The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.

Conclusions

Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.     

Antiapoptotic effects of vitamins C and E against cypermethrin‐induced oxidative stress and spermatogonial germ cell apoptosis

Toxicological studies have demonstrated the relation between use of agrochemicals and fertility issues within males. Thus, the present study aimed to elucidate the propensity of cypermethrin (CYP) in bringing testicular germ cell apoptosis and effective attenuation by vitamins C and E in caprines. Reproductive toxicity of CYP was evaluated using histomorphological, cytological, and biochemical changes in the testicular germ cells in dose‐dependent (1, 5, 10 μg/mL) and time‐dependent (4, 6, 8 h) manner. Histological and ethidium bromide/acridine orange fluorescence staining exhibited that vitamins C and E (0.5 and 1.0 mM) successfully diminished the CYP‐induced testicular germ cells apoptosis. CYP exposure along with vitamins C and E supplementation also resulted in significantly increased ferric reducing antioxidant power activity along with the antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione‐s‐transferase, and decreased lipid peroxidation in testicular germ cells. Thus, vitamins C and E ameliorated CYP‐induced testicular germ cell apoptosis, thereby preventing spermatogonial cells degeneration and male infertility.     

Duration-dependent effects of nicotine exposure on growth and AKT activation in human kidney epithelial cells

Exposure to nicotine is known to cause adverse effects in many target organs including kidney. Epidemiological studies suggest that nicotine-induced kidney diseases are prevalent worldwide. However, the impact of duration of exposure on the nicotine-induced adverse effects in normal kidney cells and the underlying molecular mechanism is still unclear. Hence, the objective of this study was to evaluate both acute and long-term effects of nicotine in normal human kidney epithelial cells (HK-2). Cells were treated with 1 and 10 µM nicotine for acute and long-term duration. The result of cell viability showed that the acute exposure to 1 µM nicotine has no significant effect on growth. However, the 10 µM nicotine caused significant decrease in the growth of HK-2 cells. The long-term exposure resulted in significantly increased cell growth in both 1 and 10 µM nicotine-treated groups. Analysis of cell cycle and expression of marker genes related to proliferation and apoptosis further confirmed the effects of nicotine. Additionally, the analysis of growth signaling pathway revealed the decreased level of pAKT in cells with acute exposure whereas the increased level of pAKT in long-term nicotine-exposed cells. This suggests that nicotine, through modulating the AKT pathway, controls the duration-dependent effects on the growth of HK-2 cells. In summary, this is the first report showing long-duration exposure to nicotine causes increased proliferation of human kidney epithelial cells through activation of AKT pathway.     

Atrazine-induced alterations in rat erythrocyte membranes: ameliorating effect of vitamin E

Erythrocytes are a convenient model to understand oxidative damage to the membranes induced by various xenobiotics. The objective of the present study was to investigate the propensity of atrazine to induce oxidative stress and its possible attenuation by vitamin E. Experimental animals were orally administered atrazine (300 mg kg(-1) body weight, daily) and vitamin E (100 mg kg(-1) body weight, daily) for a period of 7, 14, and 21 days. Erythrocyte membranes were prepared and analyzed for acetylcholinesterase (AChE) activity, lipid peroxidation (LPO), and lipid composition. Susceptibility of erythrocytes to atrazine exposure was further investigated in terms of morphological alterations by scanning electron microscopy (SEM). Results indicate that atrazine exposure caused a significant inhibition of AChE activity and induction of oxidative stress in terms of increased malondialdehyde (MDA) levels. Atrazine treatment significantly decreased total lipid, cholesterol, and phospholipid content of erythrocyte membranes. SEM revealed varying degrees of distortion depending on duration of atrazine exposure. However, administration of vitamin E ameliorated the oxidative stress and changes in the erythrocyte membranes induced by atrazine.     

Bio-histopathological studies of endometrium in repeat breeding buffaloes (Bubalus bubalis)

A total of 24 uterine biopsies were successfully obtained from 40 repeat-breeding buffaloes. The endometrium of 15 buffaloes showed marked neutrophilic infiltration in the epithelium, stroma and uterine glands. In some cases, lymphocyte and plasma cell infiltration, cystic dilatation of the glands and periglandular fibrosis indicated chronicity of the endometritis. Most of the animals (85.0%) had uterine cultures positive for Streptococci, Staphylococci, Gram positive variable cocci, Gram negative bacilli, Anthracoids, and Gram positive bacilli with a predominance of Corynebacteria. With the treatment based on in vitro drug sensitivity tests, 57.1% repeat-breeding buffaloes conceived.     

Mycobacterial PE_PGRS proteins contain calcium-binding motifs with parallel beta-roll folds

The PE_PGRS family of proteins unique to mycobacteria is demonstrated to contain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel beta-roll or parallel beta-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE PGRS proteins in the light of macrophage-pathogen interaction and pathogenesis is presented.