Cystathionine β-synthase and cystathionine γ-lyase double gene transfer ameliorate homocysteine-mediated mesangial inflammation through hydrogen sulfide generation

Elevated level of homocysteine (Hcy) induces chronic inflammation in vascular bed, including glomerulus, and promotes glomerulosclerosis. In this study we investigated in vitro mechanism of Hcy-mediated monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) induction and determined the regulatory role of hydrogen sulfide (H?S) to ameliorate inflammation. Mouse glomerular mesangial cells (MCs) were incubated with Hcy (75 μM) and supplemented with vehicle or with H?S (30 μM, in the form of NaHS). Inflammatory molecules MCP-1 and MIP-2 were measured by ELISA. Cellular capability to generate H?S was measured by colorimetric chemical method. To enhance endogenous production of H?S and better clearance of Hcy, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) genes were delivered to the cells. Oxidative NAD(P)H p47(phox) was measured by Western blot analysis and immunostaining. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH?-terminal kinase (JNK1/2) were measured by Western blot analysis. Our results demonstrated that Hcy upregulated inflammatory molecules MCP-1 and MIP-2, whereas endogenous production of H?S was attenuated. H?S treatment as well as CBS and CSE doubly cDNA overexpression markedly reduced Hcy-induced upregulation of MCP-1 and MIP-2. Hcy-induced upregulation of oxidative p47(phox) was attenuated by H?S supplementation and CBS/CSE overexpression as well. In addition to that we also detected Hcy-induced MCP-1 and MIP-2 induction was through phosphorylation of ERK1/2 and JNK1/2. Either H?S supplementation or CBS and CSE doubly cDNA overexpression attenuated Hcy-induced phosphorylation of these two signaling molecules and diminished MCP-1 and MIP-2 expressions. Similar results were obtained by inhibition of ERK1/2 and JNK1/2 using pharmacological and small interferring RNA (siRNA) blockers. We conclude that H?S plays a regulatory role in Hcy-induced mesangial inflammation and that ERK1/2 and JNK1/2 are two signaling pathways involved this process.     

Uncoupling of eNOS causes superoxide anion production and impairs NO signaling in the cerebral microvessels of hph-1 mice

J. Neurochem. (2012) 122, 1211-1218. ABSTRACT: In this study, we used the GTP cyclohydrolase I-deficient mice, i.e., hyperphenylalaninemic (hph-1) mice, to test the hypothesis that the loss of tetrahydrobiopterin (BH(4) ) in cerebral microvessels causes endothelial nitric oxide synthase (eNOS) uncoupling, resulting in increased superoxide anion production and inhibition of endothelial nitric oxide signaling. Both homozygous mutant (hph-1(-/-) ) and heterozygous mutant (hph-1(+/-) mice) demonstrated reduction in GTP cyclohydrolase I activity and reduced bioavailability of BH(4) . In the cerebral microvessels of hph-1(+/-) and hph-1(-/-) mice, increased superoxide anion production was inhibited by supplementation of BH(4) or NOS inhibitor- L- N(G) -nitro arginine-methyl ester, indicative of eNOS uncoupling. Expression of 3-nitrotyrosine was significantly increased, whereas NO production and cGMP levels were significantly reduced. Expressions of antioxidant enzymes namely copper and zinc superoxide dismutase, manganese superoxide dismutase, and catalase were not affected by uncoupling of eNOS. Reduced levels of BH(4) , increased superoxide anion production, as well as inhibition of NO signaling were not different between the microvessels of male and female mice. The results of our study are the first to demonstrate that, regardless of gender, reduced BH(4) bioavailability causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature.     

Engineering sugarcane cultivars with bovine pancreatic trypsin inhibitor (aprotinin) gene for protection against top borer (Scirpophaga excerptalis Walker)

The inhibitory activity of bovine pancreatic trypsin inhibitor (aprotinin), a natural polypeptide and a proteinase inhibitor, was demonstrated on gut proteinases of three lepidopteran borers of sugarcane using commercially available aprotinin. A synthetic gene coding for aprotinin, designed and codon optimized for better expression in plant system (Shantaram 1999), was transferred to two sugarcane cultivars namely CoC 92061 and Co 86032 through particle bombardment. Aprotinin gene expression was driven by maize ubiquitin promoter and the plant selection marker used was hygromycin resistance. The integration, expression and functionality of the transgene was confirmed by Southern, Western and insect bioassay, respectively. Southern analysis showed two to four integration sites of the transgene in the transformed plants. Independent transgenic events showed varied levels of transgene expression resulting in different levels (0.16–0.50%) of aprotinin. In in vivo bioassay studies, larvae of top borer Scirpophaga excerptalis Walker (Lepidoptera: Pyralidae) fed on transgenics showed significant reduction in larval weight which indicated impairment of their development. Results of this study show the possibility of deploying aprotinin gene for the development of transgenic sugarcane cultivars resistant to top borer.     

Insight into the structural requirements of thiophene-3-carbonitriles-based MurF inhibitors by 3D-QSAR,molecular docking and molecular dynamics study

The discovery of clinically relevant inhibitors against MurF enzyme has proven to be a challenging task. In order to get further insight into the structural features required for the MurF inhibitory activity, we performed pharmacophore and atom-based three-dimensional quantitative structure–activity relationship studies for novel thiophene-3-carbonitriles based MurF inhibitors. The five-feature pharmacophore model was generated using 48 inhibitors having IC₅₀ values ranging from 0.18 to 663?μm. The best-fitted model showed a higher coefficient of determination (R²?=?0.978), cross-validation coefficient (Q²?=?0.8835) and Pearson coefficient (0.9406) at four component partial least-squares factor. The model was validated with external data set and enrichment study. The effectiveness of the docking protocol was validated by docking the co-crystallized ligand into the catalytic pocket of MurF enzyme. Further, binding free energy calculated by the molecular mechanics generalized Born surface area approach showed that van der Waals and non-polar solvation energy terms are the main contributors to ligand binding in the active site of MurF enzyme. A 10-ns molecular dynamic simulation was performed to confirm the stability of the 3ZM6-ligand complex. Four new molecules are also designed as potent MurF inhibitors. These results provide insights regarding the development of novel MurF inhibitors with better binding affinity.     

TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury: ROLE OF MITOCHONDRIA*

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of G_Ca to G_Na of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca²⁺ uptake, ATP levels, and O₂ consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca²⁺ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca²⁺ uptake was likely due to both lower ψ_m and MCU activity. Similar to isolated myocytes, O₂ consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.     

Dynamic movement of the calcium sensor STIM1 and the calcium channel Orai1 in activated T-cells: puncta and distal caps

The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca(2+) influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca(2+) channel components to existing and newly forming immunological synapses.     

Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition

The statins, hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower serum cholesterol, exhibit myriad clinical benefits, including enhanced vascular integrity. One potential mechanism underlying increased endothelial cell (EC) barrier function is inhibition of geranylgeranylation, a covalent modification enabling translocation of the small GTPases Rho and Rac to the cell membrane. While RhoA inhibition attenuates actin stress fiber formation and promotes EC barrier function, Rac1 inhibition at the cell membrane potentially prevents activation of NADPH oxidase and subsequent generation of superoxides known to induce barrier disruption. We examined the relative regulatory effects of simvastatin on RhoA, Rac1, and NADPH oxidase activities in the context of human pulmonary artery EC barrier protection. Confluent EC treated with simvastatin demonstrated significantly decreased thrombin-induced FITC-dextran permeability, a reflection of vascular integrity, which was linked temporally to simvastatin-mediated actin cytoskeletal rearrangement. Compared with Rho inhibition alone (Y-27632), simvastatin afforded additional protection against thrombin-mediated barrier dysfunction and attenuated LPS-induced EC permeability and superoxide generation. Statin-mediated inhibition of both Rac translocation to the cell membrane and superoxide production were attenuated by geranylgeranyl pyrophosphate (GGPP), indicating that these effects are due to geranylgeranylation inhibition. Finally, thrombin-induced EC permeability was modestly attenuated by reduced Rac1 expression (small interfering RNA), whereas these effects were made more pronounced by simvastatin pretreatment. Together, these data suggest EC barrier protection by simvastatin is due to dual inhibitory effects on RhoA and Rac1 as well as the attenuation of superoxide generation by EC NADPH oxidase and contribute to the molecular mechanistic understanding of the modulation of EC barrier properties by simvastatin.