Wastewater-grown duckweed may be safely used as fish feed

Duckweed has been used for the treatment of wastewater and as fish feed. A comparative study was carried out to determine (i) the efficacy of duckweed in treating hospital-based wastewater and (ii) the level of the microbial contamination of fish fed on wastewater-grown duckweed. There were two groups of ponds where fish farming was done. In one group of ponds (control ponds), duckweed that was grown using artificial fertilizer was used as fish feed; in another group (study ponds), wastewater-grown duckweed was used as fish feed. The faecal contamination of water, duckweed, and fish from study and control ponds were monitored by faecal coliform estimation. The presence of enteric pathogens among handlers, water, duckweed, and fish samples was also examined. It was observed that the faecal coliform counts of raw wastewater were 4.7 Log10 CFU/mL, which was reduced to <1 Log10 CFU/mL after treating with duckweed. There was no significant difference (P > 0.05) in faecal coliform counts in water collected from duckweed ponds and fish ponds of study and control areas. The wastewater-grown duckweed did not pose any health hazard to the handlers. These results demonstrated that the wastewater-treated duckweed may be safely used as fish feed.     

Cardioprotection initiated by reactive oxygen species is dependent on activation of PKCepsilon

To examine whether cardioprotection initiated by reactive oxygen species (ROS) is dependent on protein kinase Cepsilon (PKCepsilon), isolated buffer-perfused mouse hearts were randomized to four groups: 1) antimycin A (AA) (0.1 microg/ml) for 3 min followed by 10 min washout and then 30 min global ischemia (I) and 2 h reperfusion (R); 2) controls of I/R alone; 3) AA bracketed with 13 min of N-2-mercaptopropionyl- glycine (MPG) followed by I/R; and 4) MPG (200 microM) alone, followed by I/R. Isolated adult rat ventricular myocytes (ARVM) were exposed to AA (0.1 microg/ml), and lucigenin was used to measure ROS production. Murine hearts and ARVM were exposed to AA (0.1 microg/ml) with or without MPG, and PKCepsilon translocation was measured by cell fractionation and subsequent Western blot analysis. Finally, the dependence of AA protection on PKCepsilon was determined by the use of knockout mice (-/-) lacking PKCepsilon. AA exposure caused ROS production, which was abolished by the mitochondrial uncoupler mesoxalonitrile 4-trifluoromethoxyphenylhydrazone. In addition, AA significantly reduced the percent infarction-left ventricular volume compared with control I/R (26 +/- 4 vs. 43 +/- 2%; P < 0.05). Bracketing AA with MPG caused a loss of protection (52 +/- 7 vs. 26 +/- 4%; P < 0.05). AA caused PKCepsilon translocation only in the absence of MPG, and protection was lost on the pkcepsilon(-/-) background (38 +/- 3 vs. 15 +/- 4%; P < 0.001). AA causes ROS production, on which protection and PKCepsilon translocation depend. In addition, protection is absent in PKCepsilon null hearts. Our results imply that, in common with ischemic preconditioning, PKCepsilon is crucial to ROS-mediated protection.     

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane “partner” proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.     

CB(1) antagonism restores hepatic insulin sensitivity without normalization of adiposity in diet-induced obese dogs

The endocannabinoid system is highly implicated in the development of insulin resistance associated with obesity. It has been shown that antagonism of the CB(1) receptor improves insulin sensitivity (S(I)). However, it is unknown whether this improvement is due to the direct effect of CB(1) blockade on peripheral tissues or secondary to decreased fat mass. Here, we examine in the canine dog model the longitudinal changes in S(I) and fat deposition when obesity was induced with a high-fat diet (HFD) and animals were treated with the CB(1) antagonist rimonabant. S(I) was assessed (n = 20) in animals fed a HFD for 6 wk to establish obesity. Thereafter, while HFD was continued for 16 additional weeks, animals were divided into two groups: rimonabant (1.25 mg·kg(-1)·day(-1) RIM; n = 11) and placebo (n = 9). Euglycemic hyperinsulinemic clamps were performed to evaluate changes in insulin resistance and glucose turnover before HFD (week -6) after HFD but before treatment (week 0) and at weeks 2, 6, 12, and 16 of treatment (or placebo) + HFD. Magnetic resonance imaging was performed to determine adiposity- related changes in S(I). Animals developed significant insulin resistance and increased visceral and subcutaneous adiposity after 6 wk of HFD. Treatment with RIM resulted in a modest decrease in total trunk fat with relatively little change in peripheral glucose uptake. However, there was significant improvement in hepatic insulin resistance after only 2 wk of RIM treatment with a concomitant increase in plasma adiponectin levels; both were maintained for the duration of the RIM treatment. CB(1) receptor antagonism appears to have a direct effect on hepatic insulin sensitivity that may be mediated by adiponectin and independent of pronounced reductions in body fat. However, the relatively modest effect on peripheral insulin sensitivity suggests that significant improvements may be secondary to reduced fat mass.     

Purification, characterizations of a snake guard seeds lectin with antitumor activity against Ehrlich ascites carcinoma cells in vivo in mice

A lectin was purified (designated as TCSL) from the Snake guard seeds with molecular mass of 56±2 kDa containing two subunits (34±1 and 22±1 kDa.). TCSL exhibited high agglutination activity at the temperature range 30 to 70°C and did not lose its activity between pH 3.0 to 12.0. The lectin was stable in the presence of denaturants and agglutinated mouse, goat, cow, chicken and human erythrocytes. TCSL did not show antifungal activity whereas it agglutinated six pathogenic bacteria and showed less toxicity against brine shrimp nauplii with the LC50 of 261±29 μg/ml. TCSL showed 28% and 72% inhibition against Ehrlich ascites carcinoma (EAC) cells in vivo in mice when administered 1 mg/kg/day and 2 mg/kg/day (i.p.) respectively for five days. TCSL enhanced the number of macrophages remarkably in the normal mice. The lectin reduced the tumor burden to 62% of EAC cells and significantly increased the hemoglobin and RBC. Treating the EAC bearing mice with TCSL at 2 mg/Kg/day for ten days with a monitoring of 20 days decreased the total WBC towards the normal level and it increased the life span by 39%.     

Ovotoxic effects of galactose involve attenuation of follicle-stimulating hormone bioactivity and up-regulation of granulosa cell p53 expression

Clinical evidence suggests an association between galactosaemia and premature ovarian insufficiency (POI); however, the mechanism still remains unresolved. Experimental galactose toxicity in rats produces an array of ovarian dysfunction including ovarian development with deficient follicular reserve and follicular resistance to gonadotrophins that characterize the basic tenets of human POI. The present investigation explores if galactose toxicity in rats attenuates the bioactivity of gonadotrophins or interferes with their receptor competency, and accelerates the rate of follicular atresia. Pregnant rats were fed isocaloric food-pellets supplemented with or without 35% D-galactose from day-3 of gestation and continuing through weaning of the litters. The 35-day old female litters were autopsied. Serum galactose-binding capacity, galactosyltransferase (GalTase) activity, and bioactivity of FSH and LH together with their receptor competency were assessed. Ovarian follicular atresia was evaluated in situ by TUNEL. The in vitro effects of galactose were studied in isolated whole follicles in respect of generation of reactive oxygen species (ROS) and expression of caspase 3, and in isolated granulosa cells in respect of mitochondrial membrane potential, expression of p53, and apoptosis. The rats prenatally exposed to galactose exhibited significantly decreased serum GalTase activity and greater degree of galactose-incorporation capacity of sera proteins. LH biopotency and LH-FSH receptor competency were comparable between the control and study population, but the latter group showed significantly attenuated FSH bioactivity and increased rate of follicular atresia. In culture, galactose increased follicular generation of ROS and expression of caspase 3. In isolated granulosa cells, galactose disrupted mitochondrial membrane potential, stimulated p53 expression, and induced apoptosis in vitro; however co-treatment with either FSH or estradiol significantly prevented galactose-induced granulosa cell p53 expression. We conclude that the ovotoxic effects of galactose involves attenuation of FSH bioactivity that renders the ovary resistant to gonadotrophins leading to increased granulosa cell expression of p53 and follicular atresia.     

The GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) is a sensor and a sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET (2) technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.     

Protein kinase C inhibits insulin-induced Akt activation in vascular smooth muscle cells.

Protein kinase C (PKC) activation, enhanced by hyperglycemia, is associated with many tissue abnormalities observed in diabetes. Akt is a serine/threonine kinase that mediates various biological responses induced by insulin. We hypothesized that the negative regulation of Akt in the vasculature by PKC could contribute to insulin resistant states and, may therefore play a role in the pathogenesis of cardiovascular disease. In this study, we specifically looked at the ability of PKC to inhibit Akt activation induced by insulin in cultured rat aortic vascular smooth muscle cells (VSMCs). Activation of Akt was determined by immunoblotting with a phospho-Akt antibody that selectively recognizes Ser473 phosphorylated Akt. A PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited insulin-dependent Akt phosphorylation. However, PMA did not inhibit platelet-derived growth factor (PDGF)-induced activation of Akt. We further showed that the PKC inhibitor, G06983, blocked the PMA-induced inhibition of Akt phosphorylation by insulin. In addition, we demonstrated that PMA inhibited the insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). From these data, we conclude that PKC is a potent negative regulator of the insulin signal in the vasculature, which indicate an important role of PKC in the development of insulin resistance in cardiovascular disease.