Long-term effect of Trigonella foenum graecum and its combination with sodium orthovanadate in preventing histopathological and biochemical abnormalities in diabetic rat ocular tissues

Trigonella foenum graecum seed powder (TSP) and Sodium Orthovanadate (SOV) have been shown to demonstrate antidiabetic effects by stabilizing glucose homeostasis and carbohydrate metabolism in experimental type-1 diabetes. However their efficacy in controlling histopathological and biochemical abnormalities in ocular tissues associated with diabetic retinopathy is not known. The purpose of this study was to investigate the comparative efficacy of individual as well as combination therapy of TSP and SOV in 8 weeks diabetic rat lens and retina. Retinas and lenses were taken from control, alloxan-induced diabetic rats and diabetic rats treated separately with insulin, 5%TSP, SOV (0.6 mg/ml) and a combined dose of SOV (0.2 mg/ml) and 5%TSP for 60 days. Control and each experimental group had six rats. Alterations in the activities of enzymes HK (hexokinase), AR (aldose reductase), SDH (sorbitol dehydrogenase), G-6-PD (glucose-6-phosphate dehydrogenase), GPx (glutathione peroxidase), GR (glutathione reductase) and levels of metabolites like sorbitol, fructose, glucose, MDA (malondialdehyde) and GSH (reduced glutathione) were measured in the cytosolic fraction of lenses besides measuring blood glucose levels and glycosylated haemoglobin. Histopathological abnormalities were studied in the lens using photomicrography and retina using transmission electron microscopy. Blood glucose, glycosylated haemoglobin levels and polyol pathway enzymes AR and SDH increased significantly causing accumulation of sorbitol and fructose in the diabetic lens and treatment with SOV and TSP significantly (p < 0.05) decreased these to control levels. Similarly, SOV and TSP treatments modulated the activities of HK, G-6-PD, GPx and GR in the rat lens to control values. Ultrastructure of the diabetic retina revealed disintegration of the inner nuclear layer cells with reduction in rough endoplasmic reticulum and swelling of mitochondria in the bipolar cells; and these histopathological events were effectively restored to control state by SOV and TSP treatments. In this study SOV and TSP effectively controlled ocular histopathological and biochemical abnormalities associated with experimental type-1 diabetes, and a combination regimen of low dose of SOV with TSP demonstrated the most significant effect. In conclusion, the potential of SOV and TSP alone or in low dose combination may be considered as promising approaches for the prevention of diabetic retinopathy and other ocular disorders.     

Combined treatment of sodium orthovanadate and Momordica charantia fruit extract prevents alterations in lipid profile and lipogenic enzymes in alloxan diabetic rats

Momordica charantia Linn., commonly called bitter gourd, is a medicinal plant used in the Ayurvedic system of medicine for treating various diseases including diabetes mellitus. Sodium orthovanadate (SOV) is also well-known insulin mimetic and an antidiabetic compound. Our laboratory has been using reduced doses of SOV along with administration of herbal extracts to alloxan diabetic rats and has established this combination as a good antihyperglycemic agent. The present study was undertaken to investigate the effects of treatment of Momordica fruit extract (MFE) and sodium orthovanadate, separately and in combination, on serum and tissue lipid profile and on the activities of lipogenic enzymes in alloxan induced diabetic rats. The results show that there was a significant (p < 0.01) increase in serum total lipids, triglycerides and total cholesterol levels after 21 days of alloxan diabetes. In the liver and kidney of diabetic rats the levels of total lipids and triglycerides also increased significantly (p < 0.01) while levels of total cholesterol decreased significantly (p < 0.01 and p < 0.05, respectively). The lipogenic enzymes showed decreased activity in the diabetic liver, while in kidney they showed an increased activity. When compared with the controls these changes were significant. The treatment of alloxan diabetic rats with MFE and SOV prevented these alterations and maintained all parameters near control values. Most effective prevention was however observed in a combined treatment of Momordica with a reduced dose of SOV (0.2%). The results suggest that Momordica fruit extract and SOV exhibit hypolipidemic as well as hypoglycemic effect in diabetic rats and their effect is pronounced when administered in combination. (Mol Cell Biochem 268: 111–120, 2005)     

Characterization of the effects of metabolic inhibitors, ATPase inhibitors and a potassium-channel blocker on stomatal opening and closing in isolated epidermis of Commelina communis L.

Abstract. The specific effects of hypoxia and various inhibitors on stomatal opening in the light and closing in the dark were characterized in isolated epidermis from Commelina communis L. Reducing the guard cell metabolism with hypoxia and the uncoupler carbonyl cyanide-m-chloro-phenyl-hydrazone, CCCP, respectively, inhibited both stomatal opening and closing. Stomatal closing was very efficiently blocked by CCCP and this effect could be readily reversed by washing out the inhibitor. The authors were unable to inhibit stomatal opening with ATPase-inhibitors, without also affecting closing. Orthovanadate, up to 2 mol m⁻³, affected neither opening nor closing. Dicyclohexylcarbodiimide, DCCD, and diethylstilbestrol, DES, inhibited opening as well as closing to about 50%. The K⁺ -channel blocker tetraethylammonium chloride, TEA-Cl, inhibited both stomatal opening and closing, as did phenyl acetic acid, PAA, a compound considered to interfere with blue light induced stomatal opening. The results are discussed in the view that the uncontrolled K⁺ leakage from the guard cells is low, that K⁺ efflux during stomatal closing, as well as K⁺ influx during opening, occurs through specific K⁺-channels and that ATP and/or a membrane potential seems to be needed to keep these channels open.     

Downregulation of PTEN by sodium orthovanadate protects the myocardium against ischemia/reperfusion injury after chronic atorvastatin treatment

Acute statin treatment has been reported to be critical in protecting the cardiac cells against ischemia/reperfusion injury by activating PI3K/Akt signal pathway. In vitro rat myocardial ischemia/reperfusion model, chronic statin treatment led to upregulation of phosphatase and tensin homolog (PTEN). This has been potentially indicated the correlation in PTEN and protective effect of statin on myocardium. In this current study, we evaluated the role of sodium orthovanadate a nonspecific inhibitor to PTEN and its correlation with atorvastatin on protecting myocardium against ischemia/reperfusion injury. We found a long-term statin treatment could increase the PTEN level, and this process was counteracted in the presence of sodium orthovanadate. However, the phosphotyrosine level was not affected by this statin. Besides, this process was mediated by Akt signaling since phosphorylated Akt level was altered by statin and sodium orthovanadate treatment. In a conclusion, this study showed a potential mechanism underlying PTEN-induced attenuation in long-term statin’s therapeutic effect, which provided the new insight into the synergic role of PTEN and atorvastatin in protecting cardiac cells against ischemia/reperfusion injury.     

The high turnover Drosophila multidrug resistance-associated protein shares the biochemical features of its human orthologues

DMRP, an ABC transporter encoded by the dMRP/CG6214 gene, is the Drosophila melanogaster orthologue of the “long” human multidrug resistance-associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6, and MRP7/ABCC10). In order to provide a detailed biochemical characterisation we expressed DMRP in Sf9 insect cell membranes. We demonstrated DMRP as a functional orthologue of its human counterparts capable of transporting several human MRP substrates like β-estradiol 17-β-d-glucuronide, leukotriene C₄, calcein, fluo3 and carboxydichlorofluorescein. Unexpectedly, we found DMRP to exhibit an extremely high turnover rate for the substrate transport as compared to its human orthologues. Furthermore, DMRP showed remarkably high basal ATPase activity (68-75 nmol P_i/mg membrane protein/min), which could be further stimulated by probenecid and the glutathione conjugate of N-ethylmaleimide. Surprisingly, this high level basal ATPase activity was inhibited by the transported substrates. We discussed this phenomenon in the light of a potential endogenous substrate (or activator) present in the Sf9 membrane.     

Entamoeba histolytica: an ecto-phosphatase activity regulated by oxidation-reduction reactions

In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.     

Prosurvival and proapoptotic functions of ERK1/2 activation in murine thymocytes in vitro

The extracellular signal-regulated kinases 1/2 (ERK1/2) are serine/threonine-selective protein kinases involved in proliferation and differentiation of cells, including thymocytes. The requirement of ERK1/2 for thymocyte differentiation and maturation has been well established; however, their role in regulating thymocyte survival and apoptosis has not been resolved.Here, we asked whether ERK1/2 affected thymocyte survival in vitro in response to apoptotic stimuli. The results show that phorbol 12-myristate 13-acetate (PMA) treatment (with or without ionomycin) and serum starvation (s/s) induced sustained ERK1/2 activation in murine thymocytes. Importantly, pharmacological treatment of thymocytes with the MEK inhibitor UO126 revealed that PMA-induced ERK1/2 activation was proapoptotic, whereas serum starvation-induced ERK1/2 activation inhibited apoptosis and promoted cell survival. While basal MEK activity was required for both s/s- and PMA-induced ERK1/2 activation, MEK activity increased only in response to PMA. The results show that the suppression of ERK1/2 phosphatases was responsible for s/s-induced sustained ERK1/2 activation. Unexpectedly, neither s/s-induced proapoptotic nor PMA-induced anti-apoptotic functions of ERK1/2 depended on the Bcl-2 family phosphoprotein Bim_EL, which was previously implicated in thymocyte apoptosis. Lastly, etoposide treatment of immature thymocytes induced both p53 and ERK1/2 activation, but ERK1/2 activity did not affect the phosphorylation and stabilization of p53. Thus, ERK1/2 has a dual role in promoting cell survival and cell death in thymocytes in the context of different stimuli.     

Vanadate affects nuclear division and induces aberrantly-shaped cells during subsequent cytokinesis in Tetrahymena

Sodium orthovanadate at 0.1-5.0 mM affected cell proliferation of Tetrahymena in a dose-dependent manner. At 1 h the cell increment was 76-12% of the control (100%), but after lag periods in 1-5 mM the growth rate remained at 76% of control in 0.1 mM vanadate and at 64-61% of control in 0.2-5.0 mM vanadate. Endocytosis was affected in both a time- and dose-dependent manner; an increasing number of cells did not form vacuoles. Cell motility increased initially in 0.1 mM vanadate but decreased later as it did in 0.5-2.0 mM vanadate where the proportion of immobile cells increased with time. Cell divisions occurred at all concentrations but macronuclear elongation was disturbed and subsequent cytokinesis resulted in daughter cells containing the entire G2 macronucleus, a large or small portion of it, or no nucleus at all. Moreover, odd cell shapes appeared with time. The size of the cell and nucleus increased but there was great variation with disturbed cytoplasm/nucleus ratios. Treated cells had dilated rough endoplasmic reticulum that included dense material, presumed to be vanadate, which was not seen in control cells. Scant amounts of dense material were found in dense granules, small vacuoles, and abundantly in contractile vacuoles. It is argued that interference with proper microtubular function is the main effect of vanadate.     

Phototropism of Pellia: Evidence for Mediation by Auxin-stimulated Acid Efflux

Flash induced 685 nm fluorescence emission of preilluminated and dark kept Chlamydobotrys stellata has been measured under conditions of CO₂-deprivation. The difference in fluorescence intensity between dark kept and preilluminated cells is taken as a measure for the reduced state of the primary stable electron acceptor of photosystem II, Q, at the given intensity of preillumination. CO₂ removal from growing cultures of this alga for 15 min diminishes photosynthetic electron transport at the oxidizing side of this photosystem. Prolonged CO₂-absence influences also its reducing side. Measurements of flash induced oxygen yields support the conclusion that both sides of photosystem II are affected in the absence of bicarbonate.     

Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating

CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.