Enhanced stability of alcohol dehydrogenase by non-covalent interaction with polysaccharides

Non-covalent interaction of alcohol dehydrogenase with polysaccharides was studied using three neutral and three anionic polysaccharides. The process of interaction of alcohol dehydrogenase with gum Arabic was optimized with respect to the ratio of enzyme to gum Arabic, pH, and molarity of buffer. Alcohol dehydrogenase–gum Arabic complex formed under optimized conditions showed 93 % retention of original activity with enhanced thermal and pH stability. Lower inactivation rate constant of alcohol dehydrogenase–gum Arabic complex within the temperature range of 45 to 60 °C implied its better stability. Half-life of alcohol dehydrogenase–gum Arabic complex was higher than that of free alcohol dehydrogenase. A slight increment was observed in kinetic constants (K _m and V _max) of gum Arabic-complexed alcohol dehydrogenase which may be due to interference by gum Arabic for the binding of substrate to the enzyme. Helix to turn conversion was observed in complexed alcohol dehydrogenase as compared to free alcohol dehydrogenase which may be responsible for observed stability enhancement.     

Molecular cloning,sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.     

Cytomorphological diversity in some selected members of Poaceae from Parvati Valley in Kullu district of Himachal Pradesh,India

The present work includes detailed male meiotic studies on 46 species of grasses falling into 59 accessions from different localities of Parvati Valley in Kullu district of Himachal Pradesh in the altitudinal range of 1,100 to 2,750 m. All the species have been studied cytologically for the first time from the study area. The meiotic chromosome count of n = 14 for Calamagrostis emodensis is the first ever chromosome report. Three species, namely Agrostis alba (n = 21), Avena byzantina (n = 21) and Bromus inermis (n = 14) have been studied cytologically for the first time from India. New intraspecific diploid/polyploid cytotypes have been reported for Arthraxon serrulatus (2n = 4x = 32), Iseilema laxum (2n = 12x = 60), Digitaria albudens (2n = 8x = 72), Festuca kashmiriana (2n = 2x = 14) and Stipa orientalis (2n = 2x = 20). The existence of variable number of B-chromosomes (2n = 60 + 0-5B) has been reported for the first time in the 12x cytotype of Iseilema laxum. Secondary associations of chromosomes in the tetraploid cytotype of Cymbopogon martini (n = 20) indicated its secondary polyploid nature. As many as 18 species showed various meiotic anomalies such as the phenomenon of cytomixis involving inter PMC migration of chromatin material, chromatin stickiness, interbivalent connections, abnormal spindle activity, presence of bridges and laggards during anaphases and telophases and abnormal sporads. These meiotic abnormalities consequently yielded sterile and heterogeneous-sized fertile pollen grains. The polyploidy and aneuploidy have played an active role in the evolution of grasses.     

Micronutrient Deficiency and Treatment Adherence in a Randomized Controlled Trial of Micronutrient Supplementation in ART-Na?ve Persons with HIV

Introduction

The MAINTAIN study is an on-going RCT comparing high-dose micronutrient and anti-oxidant supplementation versus recommended daily allowance (RDA) vitamins in slowing HIV immune deficiency progression in ART-naïve people with HIV infection.

Objective

We planned analysis of the first 127 participants to determine the baseline prevalence of serum micronutrient deficiencies and correlates, as well as tolerance and adherence to study interventions.

Methods

Participants receive eight capsules twice daily of 1) high-dose or 2) RDA supplements for two years and are followed-up quarterly for measures of immune deficiency progression, safety and tolerability. Regression analysis was used to identify correlates of micronutrient levels at baseline. Adherence was measured by residual pill count, self-report using the General Treatment Scale (GTS) and short-term recall HIV Adherence Treatment Scale (HATS).

Results

Prior micronutrient supplementation (within 30 days) was 27% at screening and 10% of study population, and was not correlated with baseline micronutrient levels. Low levels were frequent for carotene (24%<1 nmol/L), vitamin D (24%<40 nmol/L) and serum folate (20%<15 nmol/L). The proportion with B₁₂ deficiency (<133 pmol/L) was 2.4%. Lower baseline levels of B₁₂ correlated lower baseline CD4 count (r = 0.21, p = 0.02) with a 21 pmol/L reduction in B₁₂ per 100 cells/µL CD4. Vitamin D levels were higher in men (p<0.001). After a median follow-up of 1.63 years, there were 19 (15%) early withdrawals from the study treatment. Mean treatment adherence using pill count was 88%. Subjective adherence by the GTS was 81% and was moderately but significantly correlated with pill count (r = 0.29, p<0.001). Adherence based on short-term recall (HATS) was >80% in 75% of participants.

Conclusion

Micronutrient levels in asymptomatic HIV+ persons are in keeping with population norms, but micronutrient deficiencies are frequent. Adherence levels are high, and will permit a valid evaluation of treatment effects.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00798772","term_id":"NCT00798772"}}NCT00798772     

LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.     

Antibody-mediated targeting of liposomes to red cells in vivo

Covalent attachment of anti-rat erythrocyte F(ab')2 to liposomes specifically enhanced their binding to rat erythrocytes in vivo and reduced their uptake by the liver. Furthermore, at least 20-30% of the cell-bound liposomes delivered their contents to the cells. Besides, the liposome binding did not affect the survival time of the target cells at least up to 3 h in the blood circulation. These results demonstrate for the first time that liposomes can be successfully targeted to cells other than liver cells in vivo.     

Differential catalytic efficiency of allelic variants of human glutathione S-transferase Pi in catalyzing the glutathione conjugation of thiotepa.

Alkylating agents are extensively used in the treatment of cancer. The clinical usefulness of this class of anticancer drugs, however, is often limited by the emergence of drug-resistant tumor cells. Increased glutathione (GSH) conjugation through catalysis by GSH S-transferases (GSTs) is believed to be an important mechanism in tumor cell resistance to alkylating agents. In the present study, we report that the allelic variants of human Pi class GST (hGSTP1-1), which differ in their primary structures at amino acids in positions 104 and/or 113, exhibit significant differences in their activity in the GSH conjugation of alkylating anticancer drug thiotepa. Mass spectrometry revealed that the major product of the reaction between thiotepa and GSH was the monoglutathionyl-thiotepa conjugate. While nonenzymatic formation of monoglutathionyl-thiotepa was negligible, the formation of this conjugate was increased significantly in the presence of hGSTP1-1 protein. The hGSTP1-1-catalyzed GSH conjugation of thiotepa was time and protein dependent and followed Michaelis-Menten kinetics. The catalytic efficiency of hGSTP1-1(I104, A113) variant was approximately 1.9- and 2.6-fold higher compared with hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113) isoforms, respectively. The results of the present study indicate that the hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to thiotepa, and that subjects homozygous for the hGSTP1-1(I104,A113) allele, which is most frequent in human populations, are likely to be at a greater risk for developing GST-mediated resistance to thiotepa than heterozygotes or homozygotes with valine 104 background.     

Loss of resistin ameliorates hyperlipidemia and hepatic steatosis in leptin-deficient mice

Resistin has been linked to components of the metabolic syndrome, including obesity, insulin resistance, and hyperlipidemia. We hypothesized that resistin deficiency would reverse hyperlipidemia in genetic obesity. C57Bl/6J mice lacking resistin [resistin knockout (RKO)] had similar body weight and fat as wild-type mice when fed standard rodent chow or a high-fat diet. Nonetheless, hepatic steatosis, serum cholesterol, and very low-density lipoprotein (VLDL) secretion were decreased in diet-induced obese RKO mice. Resistin deficiency exacerbated obesity in ob/ob mice, but hepatic steatosis was drastically attenuated. Moreover, the levels of triglycerides, cholesterol, insulin, and glucose were reduced in ob/ob-RKO mice. The antisteatotic effect of resistin deficiency was related to reductions in the expression of genes involved in hepatic lipogenesis and VLDL export. Together, these results demonstrate a crucial role of resistin in promoting hepatic steatosis and hyperlipidemia in obese mice.     

Production of scleroglucan from Sclerotium rolfsii MTCC 2156

Scleroglucan, a neutral homopolysaccaride consisting of a linear chain of beta-D-(1-3)-glucopyranosyl and beta-D-(1-6)-glucopyranosyl groups, was produced by pure culture fermentation from Sclerotium rolfsii MTCC 2156 by submerged culture. Fermentation process was optimized in two steps. In the first step, one-factor-at-a-time method was used to investigate the effects of medium constituents such as carbon and nitrogen sources. In the second step, concentration of medium components was optimized using an L16-orthogonal array method. In all, 10 different carbon sources and eight different nitrogen sources were evaluated. Maximum yield of 16.58 g/l was obtained in a medium containing sucrose as a carbon source and sodium nitrate and yeast extract as nitrogen source.