Exercise-induced changes of MCT1 in cardiac and skeletal muscles of diabetic rats induced by high-fat diet and STZ

We hypothesized that a part of therapeutic effects of endurance training on insulin resistance is mediated by increase in cardiac and skeletal muscle mitochondrial lactate transporter, monocarboxylate transporter 1 (MCT1). Therefore, we examined the effect of 7 weeks endurance training on the mRNA and protein expression of MCT1 and MCT4 and their chaperon, CD147, on both sarcolemmal and mitochondrial membrane, separately, in healthy and type 2 diabetic rats. Diabetes was induced by injection of low dose of streptozotocin and feeding with high-fat diet. Insulin resistance was confirmed by homeostasis model assessment-estimated insulin resistance index and accuracy of two membranes separation was confirmed by negative control markers (glucose transporter 1 and cytochrome c oxidase. Real-time PCR and western blotting were used for mRNA and protein expression, respectively. Diabetes dramatically reduced MCT1 and MCT4 mRNA and their expression on sarcolemmal membrane whereas the reduction in MCT1 expression was less in mitochondrial membrane. Training increased the MCT1 mRNA and protein expression in both membranes and decreased insulin resistance as an adaptive consequence. In both tissues increase in CD147 mRNA was only parallel to MCT1 expression. The response of MCT1 on sarcolemmal and mitochondrial membranes was different between cardiac and skeletal muscles which indicate that intracellular lactate kinetic is tissue specific that allows a tissue to coordinate whole organism metabolism.     

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in syrian hamster hypothalamus

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degrees C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.     

Evaluating the Potential of an Antibody Against Recombinant OmpW Antigen in Detection of Vibrio cholerae by Surface Plasmon Resonance (SPR) Biosensor

Surface plasmon resonance (SPR), a highly sensitive and label-free optical biosensing technique, is a powerful tool for studying biomolecular interactions. An immunosensor for rapid, sensitive, and selective detection of Vibrio cholerae on the basis of SPR is reported. Recombinant OmpW antigen (a bacterial outer-membrane protein) of V. cholerae was expressed and purified and raising of polyclonal rabbit anti-OmpW was done. Antibodies were immobilized on a sensor surface and interactions between OmpW protein and the whole cell of V. cholerae with immobilized antibodies were studied in different experiments. The aim of this study was to evaluate the potential of anti-OmpW in detection of V. cholerae by developing an immunosensor based on SPR. The results showed high affinity interaction between OmpW and anti-OmpW (K _D = 2.4 ± 0.07 × 10⁻⁹ M) and SPR signals had a linear relationship with the number of V. cholerae ranging from 1 × 10² to 1 × 10⁷ cells/mL with limit of detection of 50 cells/mL. The specificity of the developed immunoassay was examined using some non-V. cholerae bacteria which did not produce any significant responses. This method is rapid, sensitive, and specific to target V. cholerae with a total analysis time of less than 60 min.

     

Exogenous application of free polyamines enhance salt tolerance of pistachio (Pistacia vera L.) seedlings

The protective effects of free polyamines (PAs) against salinity stress were investigated for pistachio seedlings (Pistacia vera cv. Badami-Zarand) in a controlled greenhouse. Seedlings were treated with 25, 50, 100 and 150 mM of salts including NaCl, CaCl₂ and MgCl₂. Foliar treatments of putrescine, spermidine (Spd) and spermine (Spm) (0.1 and 1 mM) were applied during the salinity period. Results showed that growth characteristics of pistachio seedlings decreased under salinity stress and the application of PAs efficiently reduced the adverse effects of salt stress. PAs reduced the severe effects of salt stress in pistachio seedlings neither by increasing the activities of peroxidase and ascorbate peroxidase nor by increasing the proline content but by increasing the activities of superoxide dismutase and catalase and decreasing the hydrogen peroxide (H₂O₂) activity. PAs treated seedlings showed a lower Na⁺:K⁺ ratio and Cl^? in leaves suggesting the role of PAs in balancing the ion exchange and better Na⁺:K⁺ discrimination under salt stress condition. These results showed the promising potential use of PAs especially Spm and Spd for reducing the negative effects of salinity stress and improving the growth of pistachio seedlings.     

Clinical grade cultivation of human Schwann cell, by the using of human autologous serum instead of fetal bovine serum and without growth factors

Clinical grade cultivation of human schwann cell by the utilization of human autologous serum instead of fetal bovine serum, and also avoiding any growth factors, can increase safety level of this procedure in cases of clinical cell transplantation. The aim of this study was demonstration of the feasibility of clinical grade schwann cell cultivation. In this experimental study after obtaining consent from close relatives we harvested 10 sural nerves from brain death donors and then cultured in 10 seperated culture media plus autologous serum. We also prepared autologous serum from donor??s whole blood. Then cultured cells were evaluated by S100 antibody staining for both morphology and purity. Cell purity range was from 97% to 99% (mean?=?98.11?±?0.782%). Mean of the cell count was 14,055.56?±?2,480.479 per micro liter. There was not significant correlation between cell purity and either the culture period or the age of donors (P?>?0.05). The spearman correlation coefficient for the cell purity with the period or the age of donors was 0.21 and 0.09, respectively. We demonstrated the feasibility of clinical grade schwann cell cultivation by the using of human autologous serum instead of fetal bovine serum and also without the using of growth factors. We also recommended all cell preparation facilities to adhere to the GMP and other similar quality disciplines especially in the preparation of clinically-used cell products.     

Biotransformation of methyl tert-butyl ether by human cytochrome P450 2A6

Methyl tert-butyl ether (MTBE) is widely used as gasoline oxygenate and octane number enhancer for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health are of major public concern. However, information on the metabolism of MTBE in human tissues is scarce. The present study demonstrates that human cytochrome P450 2A6 is able to metabolize MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and marker for exposure to MTBE. As CYP2A6 is known to be constitutively expressed in human livers, we infer that it may play a significant role in metabolism of gasoline ethers in liver tissue.     

A stretch of polybasic residues mediates Cdc42 GTPase-activating protein (CdGAP) binding to phosphatidylinositol 3,4,5-trisphosphate and regulates its GAP activity

The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions.