Rosiglitazone transiently disturbs calcium homeostasis in monocytic cells

The PPARγ agonist Rosiglitazone exerts anti-hyperglycaemic effects by regulating the long-term expression of genes involved in metabolism, differentiation and inflammation. In the present study, Rosiglitazone treatment rapidly inhibited (5-30 min) the ER Ca²⁺ ATPase SERCA2b in monocytic cells (IC₅₀ = 1.88 μM; p < 0.05), thereby disrupting short-term Ca²⁺ homeostasis (resting [Ca²⁺]_cyto = 121.2 ± 2.9% basal within 1 h; p < 0.05). However, extended Rosiglitazone treatment (72 h) induced dose-dependent SERCA2b up-regulation, and restored calcium homeostasis, in monocytic cells (SERCA2b mRNA: 138.7 ± 5.7% basal (1 μM)/215.0 ± 30.9% basal (10 μM); resting [Ca²⁺]_cyto = 97.3 ± 8.3% basal (10 μM)). As unfavourable cardiovascular outcomes, possibly related to disrupted cellular Ca²⁺ homeostasis, have been linked to Rosiglitazone, this effect may be of clinical interest. In contrast, in PPRE-luciferase reporter-gene assays, Rosiglitazone induced non-dose-dependent PPARγ-dependent effects (1 μM: 152.5 ± 4.9% basal; 10 μM: 136.1 ± 5.1% basal (p < 0.05 for 1 μM vs. 10 μM)). Thus, we conclude that Rosiglitazone can exert PPARγ-independent non-genomic effects, such as the SERCA2b inhibition seen here, but that long-term Rosiglitazone treatment did not perturb resting [Ca]_cyto in this study.     

Mitochondria in gastric epithelial cells are the key targets for NSAIDs-induced injury and NGF cytoprotection

Gastric epithelial cells are important components of mucosal protection and targets of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced injury. Diclofenac (DFN) is one of the most widely used NSAIDs; however, even its short-term use can induce gastric erosions and ulcers. Nerve growth factor (NGF) has been reported to act not only on neuronal cells but also on endothelial cells; however, its action on gastric epithelial cells is unknown. This study was aimed to determine, whether NGF can protect gastric epithelial cells against DFN-induced injury, and to determine the underlying molecular mechanisms with a focus on mitochondria, survivin, and insulin-like growth factor 1 (IGF-1). Cultured normal rat gastric mucosal epithelial cells 1 (RGM1) were treated with phosphate-buffered saline (PBS; control), NGF (100 ng/mL) and/or DFN (0.25-1.00 mM) for 4 hours. We examined: (1) cell injury by confocal microscopy; (2) cell death/survival using Calcein AM live cell tracking dye; (3) mitochondrial structure and membrane potential function using MitoTracker in live cells; and (4) expression of NGF, its receptor - tropomyosin receptor kinase A (TrkA), survivin and IGF-1 by immunostaining. DFN treatment of RGM1 cells for 4 hours caused extensive cell injury, mitochondrial disintegration, reduced cell viability (from 94 ± 3% in controls to 14 ± 4% in 0.5 mM DFN-treated cells; P < 0.001), and expression of survivin and IGF-1. NGF treatment significantly increased survivin and IGF-1 expression by 41% and 75%, respectively versus PBS controls. Pretreatment with NGF before DFN treatment reduced mitochondrial damage and cell death by 73% and 82%, respectively versus treatment with DFN alone (all P < 0.001). This study also showed the presence of high-affinity TrkA receptors in the plasma membrane and mitochondria of RGM1 cells indicating novel actions of NGF.     

Differential scanning calorimetric studies on binding of N-acetyl-D-glucosamine to lysozyme

Differential scanning calorimetric (DSC) measurements were performed on the thermal denaturation of lysozyme and lysozyme complexed with N-acetyl-D-glucosamine (GlcNAc) at pH 5.00 (acetate buffer), 4.25 and 2.25 (Gly-HCl buffer). DSC data have been analyzed to obtain denaturation temperature T(d), enthalpy of denaturation DeltaH(D), heat capacity of denaturation DeltaC(pd) and cooperativity index eta. From these thermodynamic parameters, the binding constant K(L) and enthalpy of binding DeltaH(L), for the weak binding of lysozyme with GlcNAc have been determined. The values of K(L) and DeltaH(L) at pH 5.00 and 298 K are 42 +/- 4 M(-1) and -24 +/- 4 kJ mol(-1), respectively, and agree very well with the experimentally determined values from equilibrium and other studies. The binding constant has also been estimated by simulating the DSC curve with varying values of K(L) (T(d)) until it matches the experimental curve.     

Serum 25-Hydroxyvitamin D Status and Longitudinal Changes in Weight and Waist Circumference: Influence of Genetic Predisposition to Adiposity

Studies of the relationship between serum 25-hydroxyvitamin D (25(OH)D) and changes in measures of adiposity have shown inconsistent results, and interaction with genetic predisposition to obesity has rarely been examined. We examined whether 25(OH)D was associated with subsequent annual changes in body weight (ΔBW) or waist circumference (ΔWC), and whether the associations were modified by genetic predisposition to a high BMI, WC or waist-hip ratio adjusted for BMI (WHR_BMI). The study was based on 10,898 individuals from the Danish Inter99, the 1958 British Birth Cohort and the Northern Finland Birth Cohort 1966. We combined 42 adiposity-associated Single Nucleotide Polymorphisms (SNPs) into four scores indicating genetic predisposition to BMI, WC and WHR_BMI, or all three traits combined. Linear regression was used to examine the association between serum 25(OH)D and ΔBW or ΔWC, SNP-score × 25(OH)D interactions were examined, and results from the individual cohorts were meta-analyzed. In the meta-analyses, we found no evidence of an association between 25(OH)D and ΔBW (-9.4 gram/y per 10 nmol/L higher 25(OH)D [95% CI: -23.0, +4.3; P = 0.18]) or ΔWC (-0.06 mm/y per 10 nmol/L higher 25(OH)D [95% CI: -0.17, +0.06; P = 0.33]). Furthermore, we found no statistically significant interactions between the four SNP-scores and 25(OH)D in relation to ΔBW or ΔWC. Thus, in view of the narrow CIs, our results suggest that an association between 25(OH)D and changes in measures of adiposity is absent or marginal. Similarly, the study provided evidence that there is either no or very limited dependence on genetic predisposition to adiposity.     

Microbial Waste Biomass for Removal of Chromium(VI) from Chrome Effluent

ABSTRACT

Microbial waste biomass, a by-product of the fermentation industry, was developed as a biosorbent to remove hexavalent chromium (Cr) from the acidic effluent of a metal processing industry. In batch sorption, 100% Cr(VI) removal was achieved from aqueous solution in 30 min contact at pH 4.0–5.0. The Cr(VI) sorption equilibrium was evaluated using the Langmuir and Freundlich models, indicating the involvement of ion exchange and physicochemical interaction. Fourier transform infrared (FTIR) analysis revealed the presence of amine, hydroxyl, and imine functional groups present on the surface of microbial biomass that are involved in Cr binding. In a continuous sorption system, 95 mg L^?1 of Cr(VI) was adsorbed before the column reached a breakthrough point of 0.1 mg L^?1 Cr(VI) at the column outlet. An overall biosorption capacity of 12.6 mg Cr(VI) g^?1 of dry microbial waste was achieved, including the partially saturated portion of the dynamic sorption zone. Insignificant change in metal removal was observed up to 10 cycles. In pilot-scale studies, 100% removal of Cr(VI) was observed up to 5 weeks, and the method was found to be cost-effective, commercially viable, and environmentally friendly, as it does not generate toxic chrome sludge.     

A role for calcium in stabilizing transport vesicle coats.

Calcium has been implicated in regulating vesicle fusion reactions, but its potential role in regulating other aspects of protein transport, such as vesicle assembly, is largely unexplored. We find that treating cells with the membrane-permeable calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), leads to a dramatic redistribution of the vesicle coat protein, coatomer, in the cell. We have used the cell-free reconstitution of coat-protomer I (COPI) vesicle assembly to characterize the mechanisms of this redistribution. We find that the recovery of COPI-coated Golgi vesicles is inhibited by the addition of BAPTA to the cell-free vesicle budding assay. When coatomer-coated membranes are incubated in the presence of calcium chelators, the membranes "uncoat," indicating that calcium is necessary for maintaining the integrity of the coat. This uncoating is reversed by the addition of calcium. Interestingly, BAPTA, a calcium chelator with fast binding kinetics, is more potent at uncoating the coatomer-coated membrane than EGTA, suggesting that a calcium transient or a calcium gradient is important for stabilizing COPI vesicle coat. The primary target for the effects of calcium on coatomer recruitment is a step that occurs after ADP-ribosylation factor binding to the membrane. We suggest that a calcium gradient may serve to regulate the timing of vesicle uncoating.