The role of ZFP580, a novel zinc finger protein, in TGF-mediated cytoprotection against chemical hypoxia­induced apoptosis in H9c2 cardiac myocytes.

Zing finger protein 580 (ZFP580) is a novel Cys2-His2 zinc-finger transcription factor that has an anti-apoptotic role in myocardial cells. It is involved in the endothelial transforming growth factor­ß1 (TGF­ß1) signal transduction pathway as a mothers against decapentaplegic homolog (Smad)2 binding partner. The aim of the present study was to determine the involvement of ZFP580 in TGF­ß1­mediated cytoprotection against chemical hypoxia­induced apoptosis, using H9c2 cardiac myocytes. Hypoxia was chemically induced in H9c2 myocardial cells by exposure to cobalt chloride (CoCl2). In response to hypoxia, cell viability was decreased, whereas the expression levels of hypoxia inducible factor-1α and ZFP580 were increased. Pretreatment with TGF­ß1 attenuated CoCl2­induced cell apoptosis and upregulated ZFP580 protein expression; however, these effects could be suppressed by SB431542, an inhibitor of TGF­ß type I receptor and Smad2/3 phosphorylation. Furthermore, suppression of ZFP580 expression by RNA interference reduced the anti­apoptotic effects of TGF­ß1 and thus increased CoCl2­induced apoptosis. B­cell lymphoma (Bcl)­2­associated X protein/Bcl­2 ratio, reactive oxygen species generation and caspase­3 activation were also increased following ZFP580 inactivation. In conclusion, these results indicate that ZFP580 is a component of the TGF-ß1/Smad signaling pathway, and is involved in the protective effects of TGF­ß1 against chemical hypoxia­induced cell apoptosis, through inhibition of the mitochondrial apoptotic pathway.

[Effects of ZFP580 on ventricular remodeling after myocardial ischemia/reperfusion in rats].

OBJECTIVE: To investigate the relationship between zinc finger protein(ZFP580)and ventricular remodeling after myocardial is-chemia/reperfusion(I/R) injury in rats. METHODS: Seventy-two rats were divided into sham group and I/R groups which would be tested in se-ries time of 0.5 h, 1 h, 2 h, 4 h, 1 d,7 d,14 d,28 d after reperfusion to observe the expression of ZFP580 in rat myocardium. The H9C2 cells were cultured and treated with transforming growth factor-beta1 (TGF-ß 1) to establish cardiac hypertrophy in vitro model in series time of 0 h, 8h, 16 h and 24 h. The cardiomyocyte hypertrophy morphology was measured. The mRNA levels of atrial natriuretic peptide(ANP), myosin heavey chain beta(ß -MHC) and ZFP580 genes were quantified. The protein levels of MMP-3 and ZFP580 were quantified after H9C2 cells were transfected by lentiviral-mediated ZFP580 gene. RESULTS: Myocardial I/R injury model was successfully established. Myocardial tis-sue in rats had large area infarction, and myocardial cells were eosinophilic changed. The increased level of ZFP580 protein was observed in the cardiomyocytes around infarction zone. The expression of TGF-ß 1 in myocardium was up-regulated after myocardial I/R injury. TGF-ß 1 (5 ng/ml) treatment could induce cardiomyocyte hypertrophy in H9C2 cells. TGF-ß 1 treatment increased the cell size and mRNA levels of ANP andß -MHC genes (P < 0.05), which represent degree of cardiac hypertrophy. TGF-ß 1 treatment also increased the protein levels of ZFP580 in H9C2 cells (P < 0.05). In the H9C2 cells transfected by lentiviral-mediated gene, the protein level of MMP3 was decreased (P < 0.05). CONCLUSIONS: ZFP580 is probably related with ventricular remodeling after myocardial I/R injury by involving TGF-ß 1 induced cardiomyocyte hypertrophy and attenuating MMP-3 production.

Regulation of Hydroxylation and Nitroreduction Pathways during Metabolism of the Neonicotinoid Insecticide Imidacloprid by Pseudomonas putida.

Imidacloprid (IMI) is mainly metabolized via nitroreduction and hydroxylation pathways, which produce different metabolites that are toxic to mammals and insects. However, regulation of IMI metabolic flux between nitroreduction and hydroxylation pathways is still unclear. In this study, Pseudomonas putida was found to metabolize IMI to 5-hydroxy and nitroso IMI and was therefore used for investigating the regulation of IMI metabolic flux. The cell growth time, cosubstrate, dissolved oxygen concentration, and pH showed significant effect on IMI degradation and nitroso and 5-hydroxy IMI formation. Gene cloning and overexpression in Escherichia coli proved that P. putida KT2440 aldehyde oxidase mediated IMI nitroreduction to nitroso IMI, while cytochrome P450 monooxygenase (CYP) failed to improve IMI hydroxylation. Moreover, E. coli cells without CYP could hydroxylate IMI, demonstrating the role of a non-CYP enzyme in IMI hydroxylation. Thus, the present study helps to further understand the environmental fate of IMI and its underlying mechanism.

SILAC-based proteomic analysis reveals that salidroside antagonizes cobalt chloride-induced hypoxic effects by restoring the tricarboxylic acid cycle in cardiomyocytes.

Hypoxic status alters the energy metabolism and induces cell injury in cardiomyocytes, and it further triggers the occurrence and development of cardiovascular diseases. Our previous studies have shown that salidroside (SAL) exhibits anti-hypoxic activity. However, the mechanisms remain obscure. In the present study, we successfully screened 92 different expression proteins in CoCl2-induced hypoxic conditions, 106 different expression proteins in the SAL-mediated anti-hypoxic group were compared with the hypoxic group using quantitative proteomics strategy, respectively. We confirmed that SAL showed a positive protective function involving the acetyl-CoA metabolic, tricarboxylic acid (TCA) cycle using bioinformatics analysis. We also demonstrated that SAL plays a critical role in restoring the TCA cycle and in protecting cardiomyocytes from oxidative injury via up-regulation expressions of PDHE1-B, ACO2, SUCLG1, SUCLG2 and down-regulation of MDH2. SAL also inhibited H9c2 cell apoptosis by inhibiting the activation of pro-apoptotic molecules caspase 3 and caspase 9 as well as activation of the anti-apoptotic molecular Bcl-2. Additionally, SAL also improved mitochondrial membrane potential (ΔΨm), reduced reactive oxygen species (ROS) and intercellular Ca(2+) concentration ([Ca(2+)]i) accumulation and inhibited the excessive consumption of ATP in H9c2 cells.

Degradation of the neonicotinoid insecticide acetamiprid via the N-carbamoylimine derivate (IM-1-2) mediated by the nitrile hydratase of the nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333.

The metabolism of the widely used neonicotinoid insecticide acetamiprid (ACE) has been extensively studied in plants, animals, soils, and microbes. However, hydration of the N-cyanoimine group in ACE to the N-carbamoylimine derivate (IM-1-2) by purified microbes, the enzyme responsible for this biotransformation, and further degradation of IM-1-2 have not been studied. The present study used liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy to determine that the nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333 transforms ACE to IM-1-2. CGMCC 7333 cells degraded 65.1% of ACE in 96 h, with a half-life of 2.6 days. Escherichia coli Rosetta (DE3) overexpressing the nitrile hydratase (NHase) from CGMCC 7333 and purified NHase converted ACE to IM-1-2 with degradation ratios of 97.1% in 100 min and 93.9% in 120 min, respectively. Interestingly, IM-1-2 was not further degraded by CGMCC 7333, whereas it was spontaneously hydrolyzed at the N-carbamoylimine group to the derivate ACE-NH, which was further converted to the derivative ACE-NH2. Then, ACE-NH2 was cleaved to the major metabolite IM-1-4. IM-1-2 showed significantly lower insecticidal activity than ACE against the aphid Aphis craccivora Koch. The present findings will improve the understanding of the environmental fate of ACE and the corresponding enzymatic mechanisms of degradation.

Co-metabolic transformation of the neonicotinoid insecticide imidacloprid by the new soil isolate Pseudoxanthomonas indica CGMCC 6648.

A new imidacloprid (IMI) degrading bacterium Z-9 (deposited number CGMCC 6648) was isolated and identified as Pseudoxanthomonas indica by 16S rRNA gene analysis. Two metabolites were identified as olefin and 5-hydroxy IMI by liquid chromatography-mass spectrometry and nuclear magnetic resonance analysis. P. indica CGMCC 6648 degraded 70.1% of IMI (1.22 mmol L(-1)) and formed 0.93 mmol L(-1) 5-hydroxy IMI and 0.05 mmol L(-1) olefin IMI in 6 days and in the presence of 100 mmol L(-1) glucose. The half-life of IMI degradation was 3.6 days. P. indica CGMCC 6648 transforms IMI via a co-metabolism mechanism and different carbohydrates have significant effects on 5-hydroxy IMI formation, whereas different organic acids have substantial effects on olefin IMI production. Lactose is the best co-substrate for IMI degradation and 5-hydroxy IMI formation with 0.77 mmol L(-1) degraded and 0.67 mmol L(-1) formed in 48 h, respectively. Pyruvate is the best co-substrate for olefin IMI formation with 0.17 mmol L(-1) produced in 96 h for all carbon sources tested. Pyruvate significantly stimulates the conversion of 5-hydroxy IMI to olefin IMI, whereas glucose slightly inhibits this reaction. P. indica CGMCC 6648 rapidly degrades IMI and forms olefin IMI, which may enhance its potential for biodegradation of IMI and increase its insecticidal activity, which can decrease the IMI dosage required.