The role of nitric oxide signaling in renoprotective effects of hydrogen sulfide against chronic kidney disease in rats: Involvement of oxidative stress,autophagy and apoptosis

The interplay between H₂S and nitric oxide (NO) is thought to contribute to renal functions. The current study was designed to assess the role of NO in mediating the renoprotective effects of hydrogen sulfide in the 5/6 nephrectomy (5/6 Nx) animal model. Forty rats were randomly assigned to 5 experimental groups: (a) Sham; (b) 5/6 Nx; (c) 5/6Nx+sodium hydrosulfide-a donor of H ₂S, (5/6Nx+sodium hydrosulfide [NaHS]); (d) 5/6Nx+NaHS+ L -NAME (a nonspecific nitric oxide synthase [NOS] inhibitor); (e) 5/6Nx+NaHS+aminoguanidine (a selective inhibitor of inducible NOS [iNOS]). Twelve weeks after 5/6 Nx, we assessed the expressions of iNOS and endothelial NOS (eNOS), oxidative/antioxidant status, renal fibrosis, urine N-acetyl-b-glucosaminidase (NAG) activity as the markers of kidney injury and various markers of apoptosis, inflammation, remodeling, and autophagy. NaHS treatment protected the animals against chronic kidney injury as depicted by improved oxidative/antioxidant status, reduced apoptosis, and autophagy and attenuated messenger RNA (mRNA) expression of genes associated with inflammation, remodeling, and NAG activity. Eight weeks Nω-nitro-l-arginine methyl ester ( L -NAME) administration reduced the protective effects of hydrogen sulfide. In contrast, aminoguanidine augmented the beneficial effects of hydrogen sulfide. Our finding revealed some fascinating interactions between NO and H ₂S in the kidney. Moreover, the study suggests that NO, in an isoform-dependent manner, can exert renoprotective effects in 5/6 Nx model of CKD.     

Role of small GTP binding proteins in the growth-promoting and antiapoptotic actions of gastrin

G17 has growth promoting and antiapoptotic effects on the AR4-2J pancreatic acinar cell line. We previously reported that whereas MAPK regulates G17-stimulation of AR4-2J cell proliferation, Akt mediates the antiapoptotic action of G17. We examined the signal-transduction pathways mediating G17 stimulation of AR4-2J cell growth and survival. G17 activated the small GTP binding proteins Ras, Rac, Rho, and Cdc42. Transduction of the cells with adenoviral vectors expressing dominant negative Akt, Ras, Rho, and Cdc42 but not dominant negative Rac inhibited AR4-2J cell proliferation and survival. Both exoenzyme C3 from Clostridium botulinum (C3), a toxin known to inactivate Rho, and PD98059, a MAPK inhibitor, reversed G17 inhibition of AR4-2J cell apoptosis. G17 induction of Akt activation was reduced by >60% by both dominant negative Ras and Rho and by 30% by dominant negative Cdc42. In contrast, G17-stimulated MAPK activation was blocked by >80% by dominant negative Ras but not by dominant negative Rho and Cdc42. Similar results were observed in the presence of C3. Dominant negative Rac failed to affect G17 induction of both Akt and MAPK, whereas it inhibited sorbitol by almost 50% but not G17-stimulated activation of p38 kinase. Thus G17 promotes AR4-2J cell growth and survival through the activation of multiple GTP binding proteins, which, in turn, regulate different protein kinase cascades. Whereas Ras activates Akt and MAPK, Rho and Cdc42 appear to regulate Akt and possibly other as yet unidentified kinases mediating the growth-stimulatory actions of G17.     

The secretome of the plant pathogenic bacterium Erwinia chrysanthemi

Erwinia chrysanthemi causes soft-rot diseases of many plants by secreting a battery of enzymes which degrade the plant cell walls. We initiated a proteomic analysis to create a reference map of the E. chrysanthemi secretome. Extracellular proteins were isolated from E. chrysanthemi culture supernatants and resolved by two-dimensional electrophoresis. By analysis of mutants, Western blotting, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) 55 spots representing 25 unique proteins were identified. In uninduced conditions, we identified spots corresponding to the cellulase Cel5, the proteases PrtA, PrtB, and PrtC, the flagellin FliC, and some intracellular proteins whose presence probably resulted from spontaneous cell lysis. We identified another secreted protein, AvrL, homologous to an avirulence protein of Xanthomonas campestris. After culture in conditions inducing pectinase production, i.e., in the presence of galacturonate and plant extract, we identified spots corresponding to the endopectate lyases PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ, the pectin acetylesterases PaeX and PaeY, the pectin methylesterase PemA, and the polygalacturonase PehX. In the presence of other inducing compounds, we detected the rhamnogalacturonate lyase RhiE and the esterase FaeD. Analysis of mutants, altered for one type of secretion system, was performed to determine the targets of each system. The type I system Prt was necessary for the secretion of three proteases. No proteins secreted by the type III Hrp system could be detected in E. chrysanthemi supernatants. In addition to the already known substrates (eleven pectinases and one cellulase), this analysis revealed that the type II Out system mediates secretion of the esterase FaeD and of the Avr-like protein AvrL.     

Tetrathiomolybdate therapy protects against concanavalin a and carbon tetrachloride hepatic damage in mice

Tetrathiomolybdate, an anticopper drug, has been shown to protect mice against pulmonary fibrosis from bleomycin. Our hypothesis is that it does so by inhibiting fibrosis-inducing cytokines. Indeed, we have good evidence, not yet published, that tetrathiomolybdate inhibits pulmonary levels of transforming growth factor-beta and tumor necrosis factor-alpha expression in these bleomycin experiments. Herein, we evaluate tetrathiomolybdate's effectiveness in mitigating hepatitis and fibrosis in mice from the hepatotoxins, concanavalin A and carbon tetrachloride, and its inhibition of cytokines as a possible mechanism. In short-term experiments, concanavalin A elevated serum amino leucine transferase levels several fold, and tetrathiomolybdate completely prevented this increase. In additional experiments, tetrathiomolybdate therapy reversed the elevated serum transaminase levels despite continued concanavalin A injections, with nearly significant serum interleukin-1beta inhibition. Concanavalin A given for 12 weeks produced mild fibrosis, whereas concomitant tetrathiomolybdate treatment resulted in normal histology. Carbon tetrachloride given for 12 weeks resulted in very high serum amino leucine transferase levels, high serum transforming growth factor-beta levels, cirrhosis as seen histologically, and increase in liver hydroxyproline, a measure of fibrosis. Concomitant tetrathiomolybdate partially and significantly protected against increases in amino leucine transferase and transforming growth factor-beta, fully protected against the increase in hydroxyproline, and resulted in normal histology. In conclusion, tetrathiomolybdate protects against the hepatitis and fibrosis produced by these hepatotoxins, probably by inhibiting the excessive increase in inflammatory and fibrotic cytokines.     

Structure of an integrin-ligand complex deduced from solution x-ray scattering and site-directed mutagenesis

The structural basis of the interaction of integrin heterodimers with their physiological ligands is poorly understood. We have used solution x-ray scattering to visualize the head region of integrin alpha 5 beta 1 in an inactive (Ca2+-occupied) state, and in complex with a fragment of fibronectin containing the RGD and synergy recognition sequences. Shape reconstructions of the data have been interpreted in terms of appropriate molecular models. The scattering data suggest that the head region undergoes no gross conformational changes upon ligand binding but do lend support to a proposed outward movement of the hybrid domain in the beta subunit. Fibronectin is observed to bind across the top of the head region, which contains an alpha subunit beta-propeller and a beta subunit vWF type A domain. The model of the complex indicates that the synergy region binds on the side of the beta-propeller domain. In support of this suggestion, mutagenesis of a prominent loop region on the side of the propeller identifies two residues (Tyr208 and Ile210) involved in recognition of the synergy region. Our data provide the first view of a complex between an integrin and a macromolecular ligand in solution, at a nominal resolution of approximately 10 A.     

Application of an antibody biochip for p53 detection and cancer diagnosis

Detection of the p53 tumor suppressor gene is important in early cancer diagnostics because alterations in the gene have been associated with carcinogenic manifestations in several tissue types in humans. We have developed an antibody-based detection instrument, the biochip, to detect the presence of the anti-p53 antibody in human serum. The design of this highly integrated detector system is based on miniaturized phototransistors having multiple optical sensing elements, amplifiers, discriminators, and logic circuitry on an IC board. The system utilizes laser excitation and fluorescence signals to detect complex formation between the p53 monoclonal antibody and the p53 antigen. Recognition antibodies are immobilized on a nylon membrane platform and incubated in solutions containing antigens labeled with Cy5, a fluorescent cyanine dye. Subsequently, this membrane is placed on the detection platform of the biochip and fluorescence signal is induced using a 632.8-nm He-Ne laser. Using this immuno-biochip, we have been able to detect binding of the p53 monoclonal antibody to the human p53 cancer protein in biological matrices. The performance of the integrated phototransistors and amplifier circuits of the biochip, previously evaluated through measurement of the signal output response for various concentrations of fluorescein-labeled molecules, have illustrated the linearity of the microchip necessary for quantitative analysis. The design of this biochip permits sensitive, selective and direct measurements of a variety of antigen-antibody formations at very low concentrations. Furthermore, the acquisitions of the qualitative and quantitative results are accomplished rapidly, in about 15 min. These features demonstrate the potential of this antibody-based biochip for simple, rapid and early biomedical diagnostics of cancer.     

Oleate and linoleate enhance the growth-promoting effects of insulin-like growth factor-I through a phospholipase D-dependent pathway in arterial smooth muscle cells

Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis.     

Mycobacterium tuberculosis promotes apoptosis in human neutrophils by activating caspase-3 and altering expression of Bax/Bcl-xL via an oxygen-dependent pathway

In addition to direct bactericidal activities, such as phagocytosis and generation of reactive oxygen species (ROS), neutrophils can regulate the inflammatory response by undergoing apoptosis. We found that infection of human neutrophils with Mycobacterium tuberculosis (Mtb) induced rapid cell death displaying the characteristic features of apoptosis such as morphologic changes, phosphatidylserine exposure, and DNA fragmentation. Both a virulent (H37Rv) and an attenuated (H37Ra) strain of Mtb were equally effective in inducing apoptosis. Pretreatment of neutrophils with antioxidants or an inhibitor of NADPH oxidase markedly blocked Mtb-induced apoptosis but did not affect spontaneous apoptosis. Activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis, but it was markedly augmented and accelerated during Mtb-induced apoptosis. The Mtb-induced apoptosis was associated with a speedy and transient increase in expression of Bax protein, a proapoptotic member of the Bcl-2 family, and a more prominent reduction in expression of the antiapoptotic protein Bcl-x(L). Pretreatment with an inhibitor of NADPH oxidase distinctly suppressed the Mtb-stimulated activation of caspase-3 and alteration of Bax/Bcl-x(L) expression in neutrophils. These results indicate that infection with Mtb causes ROS-dependent alteration of Bax/Bcl-x(L) expression and activation of caspase-3, and thereby induces apoptosis in human neutrophils. Moreover, we found that phagocytosis of Mtb-induced apoptotic neutrophils markedly increased the production of proinflammatory cytokine TNF-alpha by human macrophages. Therefore, the ROS-dependent apoptosis in Mtb-stimulated neutrophils may represent an important host defense mechanism aimed at selective removal of infected cells at the inflamed site, which in turn aids the functional activities of local macrophages.