NADPH production from NADP+ with a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans

Summary A convenient and efficient method of NADPH production from NADP⁺ has been established using a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans IFO 3172. Using airdried cells of the bacterium, the optimum conditions for NADPH production were examined, including the cell and glucose concentrations, NADP⁺ concentration, pH, buffer and reaction temperature. Under suitable conditions, the conversion ratio and the amount of NADPH accumulated reached about 100% and 73 mg/ml of the reaction mixture, respectively, after 1-h reaction. Intact cells of the bacterium also showed high NADPH production even in the reaction mixture without a surfactant. The addition of Triton X-100 to the reaction mixture and freeze-thawing treatment of intact cells enhanced the production. The NADPH production method was further improved by using cells of the bacterium immobilized by entrapment in a -carrageenan gel lattice. The immobilized cells had almost the same enzymatic properties as the air-dried cells. The conditions for the continuous production of NADPH with an immobilized cell column were also investigated. NADPH was produced in a good yield (about 95%) with this continuous process.     

Ripening of fleshy fruit: Molecular insight and the role of ethylene

Development and ripening in fruit is a unique phase in the life cycle of higher plants which encompasses several stages progressively such as fruit development, its maturation, ripening and finally senescence. During ripening phase, several physiological and biochemical changes take place through differential expression of various genes that are developmentally regulated. Expression and/or suppression of these genes contribute to various changes in the fruit that make it visually attractive and edible. However, in fleshy fruit massive losses accrue during post harvest handling of the fruit which may run into billions of dollars worldwide. This encouraged scientists to look for various ways to save these losses. Genetic engineering appears to be the most promising and cost effective means to prevent these losses. Most fleshy fruit ripen in the presence of ethylene and once ripening has been initiated proceeds uncontrollably. Ethylene evokes several responses during ripening through a signaling cascade and thousands of genes participate which not only sets in ripening but also responsible for its spoilage. Slowing down post ripening process in fleshy fruit has been the major focus of ripening-related research. In this review article, various developments that have taken place in the last decade with respect to identifying and altering the function of ripening-related genes have been described. Role of ethylene and ethylene-responsive genes in ripening of fleshy fruit is also included. Taking clues from the studies in tomato as a model fruit, few case studies are reviewed.     

Increased endogenous H2S generation by CBS, CSE, and 3MST gene therapy improves ex vivo renovascular relaxation in hyperhomocysteinemia

Hydrogen sulfide (H(2)S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H(2)S is produced as a metabolite of homocysteine (Hcy) by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H(2)S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H(2)S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H(2)S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H(2)S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H(2)S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.     

Involvement of gibberellins in breaking bud dormancy in euphorbia crinkle mosaic virus-infected stem cuttings ofEuphorbia pulcherrima willd.

Euphorbia pulcherrima, an ornamental plant, exhibits severe systemic viral infection. It grows vegetatively during summer and is dormant in winter. During the dormant period the buds on healthy stem cuttings remained dormant or rarely formed cyathia while the buds on virus-infected stem cuttings grew into leafy shoots and never became dormant. Quantitative estimation has revealed that breaking of bud dormancy in virus-infected stem cuttings may be regulated by markedly higher GA-like activity in them throughout the period than in their corresponding healthy stem cuttings.     

Diversity analysis of dairy and non-dairy strains of Lactococcus lactis ssp. lactis by multilocus sequence analysis (MLSA)

The genetic diversity of 31 identified strains of Lactococcus lactis ssp. lactis isolated from different dairy and non-dairy sources were investigated at gene level using multilocus sequence analysis (MLSA) and PCR-RFLP based on the differences in four selected partial protein coding gene sequences: araT, encoding aromatic amino acid-specific aminotransferase; dtpT, encoding di/tri peptide transporter; yueF, encoding non-proteolytic protein, peptidase, M16 family; and pdhA, encoding pyruvate dehydrogenase E1 component α-subunit. A set of seven test strains from different isolation sources and one reference strain, L. lactis ssp. lactis NCDC 094, were analyzed by MLSA. The strains showed distinct diversity among themselves and exhibited a greater percent similarity with reference strains L. lactis ssp. lactis CV56 (CP002365.1), IL1403 (AE005176.1), and KF147 (CP001834.1) in comparison with L. lactis ssp. cremoris NZ9000 (CP002094.1), MG1363 (AM406671.1), and SK11 (CP00425.1). The MLSA revealed one distinct genomic lineage within strains exclusively of L. lactis ssp. lactis. This analysis also revealed no source-wise genetic relationship in the test strains analyzed. Further, PCR-RFLP of araT, dtpT, yueF and pdhA also characterized the single genomic lineage exclusively of L. lactis ssp. lactis within a total of 24 test strains.     

MSC Therapy Attenuates Obliterative Bronchiolitis after Murine Bone Marrow Transplant

Rationale

Obliterative bronchiolitis (OB) is a significant cause of morbidity and mortality after lung transplant and hematopoietic cell transplant. Mesenchymal stromal cells (MSCs) have been shown to possess immunomodulatory properties in chronic inflammatory disease.

Objective

Administration of MSCs was evaluated for the ability to ameliorate OB in mice using our established allogeneic bone marrow transplant (BMT) model.

Methods

Mice were lethally conditioned and received allogeneic bone marrow without (BM) or with spleen cells (BMS), as a source of OB-causing T-cells. Cell therapy was started at 2 weeks post-transplant, or delayed to 4 weeks when mice developed airway injury, defined as increased airway resistance measured by pulmonary function test (PFT). BM-derived MSC or control cells [mouse pulmonary vein endothelial cells (PVECs) or lung fibroblasts (LFs)] were administered. Route of administration [intratracheally (IT) and IV] and frequency (every 1, 2 or 3 weeks) were compared. Mice were evaluated at 3 months post-BMT.

Measurements and Main Results

No ectopic tissue formation was identified in any mice. When compared to BMS mice receiving control cells or no cells, those receiving MSCs showed improved resistance, compliance and inspiratory capacity. Interim PFT analysis showed no difference in route of administration. Improvements in PFTs were found regardless of dose frequency; but once per week worked best even when administration began late. Mice given MSC also had decreased peribronchiolar inflammation, lower levels of hydroxyproline (collagen) and higher frequencies of macrophages staining for the alternatively activated macrophage (AAM) marker CD206.

Conclusions

These results warrant study of MSCs as a potential management option for OB in lung transplant and BMT recipients.     

Microtubule-dependent movement of late endocytic vesicles in vitro: requirements for Dynein and Kinesin

Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.     

Seasonal effects of melatonin on ovary and plasma gonadotropin and vitellogenin levels in intact and pinealectomized catfish, Clarias batrachus (Linn)

Effects of daily administration of melatonin for 15 days were evaluated with respect to ovarian activities and plasma gonadotropin (GtH II) and vitellogenin (Vg) levels in intact (INT) and pinealectomized (Px) female catfish, C. batrachus, during preparatory (April), prespawning (May and June), spawning (July) and post-spawning (September) periods. Px (saline control groups) caused a stimulatory effect during preparatory (with respect to Vg synthesis and incorporation) and prespawning (with respect to Vg synthesis) periods whereas no effect was observed during spawning and post-spawning periods with respect to the reproductive parameters studied. During April, melatonin-treatment significantly decreased plasma GtH II levels and percentage of vitellogenic oocytes without any significant changes in plasma Vg levels and gonadosomatic index (GSI). During early prespawning period, in May, 50microg melatonin brought about a significant reduction in plasma GtH II levels in INT group, whereas 100microg caused a decrease in all parameters; on the other hand, in Px groups both dose levels proved to be inhibitory. In June (late prespawning period) melatonin-treatment could not bring about any change in GSI and plasma Vg levels compared to the control groups regardless of Px but plasma GtH II and mean number of yolky oocytes were significantly reduced in melatonin-treated INT group. During spawning period (July) melatonin inhibited the GSI, mean number of yolky oocytes and plasma GtH II levels without affecting plasma Vg levels. In September (post-spawning period), melatonin did inhibit both GSI and plasma GtH II levels. The results, thus, indicate that melatonin showed variable effects (inhibitory and/or no effect) to GSI, mean number of yolky oocytes and plasma Vg levels but a consistent inhibiton of plasma GtH II levels indicating that melatonin may control the reproduction by blocking the GtH II release from the pituitary via affecting the hypothalamo-hypophysial axis.     

Replication of Association Between a Common Variant Near Melanocortin‐4 Receptor Gene and Obesity‐related Traits in Asian Sikhs

Recent genome‐wide association studies (GWAS) in Asian Indians reported strong associations of variants near melanocortin‐4 receptor (MC4R) and MLX interacting protein‐like (MLXIPL) genes with insulin resistance and several obesity‐related quantitative traits (QTs). Here, we evaluated the association of two variants (rs12970134 and rs4450508) near MC4R and a nonsynonymous (Gln241His) variant (rs3812316) in MLXIPL gene with type 2 diabetes (T2D) and obesity‐related QTs in our case–control cohort (n = 1,528; 745 T2D cases and 783 controls) from a Sikh population from North India. We have successfully replicated the association of MC4R (rs12970134) with BMI (P = 0.0005), total weight (WT) (P = 0.001), and waist circumference (WC) (P = 0.001). These associations remained significant after controlling for multiple testing by applying Bonferroni's correction. However, our data did not confirm the association of rs3812316 in the MLXIPL gene with triglyceride (TG) levels. These observations demonstrate that the genetic variation in MC4R locus can have a moderate contribution in the regional fat deposition and development of central obesity in Asian Indians.     

Evaluation of Fermentation Efficiency of Yeast Strains and their Effect on Quality of Young Wines

The yeast has important role in fermentation of wine grapes and wine quality. The fermentation of wine grapes affect by efficiency of particular yeast strain, sugar content, pH, available temperature, etc. To evaluate the efficiency of yeast strains (Premier Cuvee, RS-1, RS-2, RS-3 and natural), present study was conducted on two wine grape varieties viz.; Sauvignon Blanc (White) and Cabernet Sauvignon (Red). Efficiency of yeast strains was evaluated in terms of conversion rate of sugar into alcohol. As per recorded data, strain RS-3 (Pichia kudriavzevii) was found more efficient than other strains in fermentation of Cabernet Sauvignon with efficiency of 84.4 per cent but in case of Sauvignon Blanc, the commercial culture Premier Cuevee was found superior over RS-3. The quality parameters of young wines of both the varieties were also affected by the used strains. Considering the efficiency and impact on various parameters of wines, local strain, i.e., RS-3 was found at par with commercial culture (Premier Cuvee). The RS-3 strain has potential to produce quality wines. However, studies on effects of RS-3 strain on some specific quality parameters of wines like varietal aroma compounds, flavours etc. are needed.