Effects of an intracellular Ca(2+) chelator on insulin resistance and hypertension in high-fat-fed rats and spontaneously hypertensive rats.

We explored the possibility that a sustained elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) may be a cellular abnormality common to both insulin resistance and hypertension. In high-fat diet (HFD) fed rats, the steady-state glucose infusion rate (GIR) during the euglycemic hyperinsulinemic clamp was reduced by 40% (P <.05) and mean arterial pressure (MAP) was elevated by 20 mm Hg (P <.01) in comparison to the normal chow-fed rats. Intravenous injection of 5,5'-dimethyl derivative of bis(o-aminophenoxy)ethane-N,N,N',N' tetraacetic acetoxymethyl ester (dimethyl-BAPTA/AM), an effective intracellular Ca(2+) chelator, 90 minutes before the clamp not only restored about 50% of the reduced GIR, but also normalized MAP in the HFD rats. The chelator injection also significantly increased GIR by 25% (P <.01) and reduced MAP about 30 mm Hg (P <.01) in the spontaneously hypertensive rats (SHR). In addition, we have recently shown in the HFD rats that an injection of dimethyl-BAPTA/AM normalizes elevated [Ca(2+)](i) in adipocytes. These results together demonstrate that lowering [Ca(2+)](i) simultaneously ameliorates both insulin resistance and hypertension and provide presumptive evidence that sustained high levels of [Ca(2+)](i) may play a common pathophysiologic role in these 2 diseases.

Improvement of insulin sensitivity by chelation of intracellular Ca(2+) in high-fat-fed rats.

It has been postulated that sustained high levels of intracellular calcium concentration ([Ca(2+)](i)) in the insulin target cells may cause insulin resistance. We evaluated this hypothesis by examining the effect of an intracellular Ca(2+) chelator, 5,5'-dimethyl derivative of bis (o-aminophenoxy) ethane-N,N,N',N' tetraacetic acetoxymethyl ester (dimethyl-BAPTA/AM), on insulin resistance. Insulin resistance was induced in rats by feeding a high-fat diet for 3 to 4 weeks. The whole body insulin sensitivity was determined by the steady state glucose infusion rate (GIR) under euglycemic hyperinsulinemic (6 mU x kg(-1) x min(-1)) clamps. Compared with control rats, the high-fat diet (HFD) fed rats showed significantly lower GIR (12.2 +/- 0.7 v 20.2 +/- 0.9 mg x kg(-1) x min(-1); P <.01). In the HFD rats, an intravenous injection of dimethyl-BAPTA/AM (6 mg/kg) 90 minutes before the clamps significantly increased GIR to 16.3 +/- 0.9 mg x kg(-1) x min(-1) (P <.02), reversing insulin resistance by about 50%; but this intervention had no effect in the controls. This increase in GIR by dimethyl-BAPTA/AM was observed without an increase in femoral artery blood flow, indicating that the chelator increased GIR directly through improving cellular responsiveness to insulin. The stimulatory effect of insulin on 2-deoxy glucose (2-DG) uptake by the isolated epididymal adipocytes was reduced by 35% in the HFD rats compared with the control rats (P <.01). Pretreatment of the HFD rats with dimethyl-BAPTA/AM restored 2-DG uptake to the level in the control rats. The direct measurement of [Ca(2+)](i) using fura-2/AM in isolated adipocytes showed that basal [Ca(2+)](i) was significantly higher in the HFD rats than in the control rats (145 +/- 11 v 112 +/- 9 nmol/L; P <.05). An injection of dimethyl-BAPTA/AM in the HFD rats lowered [Ca(2+)](i) to 127 +/- 11 nmol/L, which did not differ from the level in the control rats (P >.2). The present study clearly demonstrates that an injection of intracellular Ca(2+) chelator in the HFD rats reverses insulin resistance, as well as normalizes elevated [Ca(2+)](i) in the insulin target cells. The results strongly support that sustained high levels of [Ca(2+)](i) in the insulin target cells may play an important role in insulin resistance, at least in the HFD rats.

Rice 1Cys-peroxiredoxin over-expressed in transgenic tobacco does not maintain dormancy but enhances antioxidant activity.

Possible functions that have been proposed for the plant 1Cys-peroxiredoxin, include activity as a dormancy regulator and as an antioxidant. The transcript level of rice 1Cys-peroxiredoxin (R1C-Prx) rapidly decreased after imbibition of rice seeds, but the protein was detected for 15 days after imbibition. To investigate the function of this protein, we generated transgenic tobacco plants constitutively expressing the R1C-Prx gene. The transgenic R1C-Prx plants showed a germination frequency similar to control plants. However, the transgenic lines exhibited higher resistance against oxidative stress, suggesting that antioxidant activity may be its primary function.

Characterization of two fungal-elicitor-induced rice cDNAs encoding functional homologues of the rab-specific GDP-dissociation inhibitor.

By using the mRNA differential display approach to isolate defense signaling genes active at the early stage of fungal infection two cDNA fragments with high sequence homology to rab-specific GDP-dissociation inhibitors (GDIs) were identified in rice (Oryza sativa L.) suspension cells. Using polymerase-chain-reaction products as probes, two full-length cDNA clones were isolated from a cDNA library of fungal-elicitor-treated rice, and designated as OsGDI1 and OsGDI2. The deduced amino acid sequences of the isolated cDNAs exhibited substantial homology to Arabidopsis rab-GDIs. Northern analysis revealed that transcripts detected with the 3'-gene-specific DNA probes accumulated to high levels within 30 min after treatment with a fungal elicitor derived from Magnaporthe grisea. The functionality of the OsGDIs was demonstrated by their ability to rescue the Sec19 mutant of Saccharomyces cerevisiae which is defective in vesicle transport. The proteins, expressed in Escherchia coli, cross-reacted with a polyclonal antibody prepared against bovine rab-GDI. Like bovine rab-GDI, the OsGDI proteins efficiently dissociated rab3A from bovine synaptic membranes. Using the two-hybrid system, it was shown that the OsGDIs specifically interact with the small GTP-binding proteins belonging to the rab subfamily. The specific interaction was also demonstrated in vitro by glutathione S-transferase resin pull-down assay.

Molecular cloning, expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage.

A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isolated from a leaf cDNA library of Chinese cabbage. The predicted amino acid sequence of C2C-Prx has 2 conserved cysteines and several peptide domains present in most of the 2Cys-Prx subfamily members. It shows the highest sequence homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%). Southern analysis using the cDNA insert of C2C-Prx revealed that it consists of a small multigene family in Chinese cabbage genome. RNA blot analysis showed that the gene was predominantly expressed in the leaf tissue of Chinese cabbage seedlings, but the mRNA was generally expressed in most tissues of mature plant, except roots. The expression of C2C-Prx was slightly induced by treatment with H2O2 (100 microM) or Fe3+/O2/DTT oxidation system, but not by ABA (50 microM) or GA3 (10 microM). The C2C-Prx is encoded as a preprotein of 273 amino acids containing a putative chloroplast-targeting signal of 65 amino acids at its N-terminus. The N-terminally truncated recombinant protein (deltaC2C-Prx) migrates as a dimer in a non-reducing SDS-polyacrylamide gel and as a monomer in a reducing condition. The deltaC2C-Prx shows no immuno cross-reactivity to antiserum of the yeast thiol-specific antioxidant protein, and vice versa. The deltaC2C-Prx prevents the inactivation of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxidation system. In the yeast thioredoxin system containing thioredoxin reductase, thioredoxin, and NADPH, the deltaC2C-Prx exhibits peroxidase activity on H2O2.

Cloning and expression of a new isotype of the peroxiredoxin gene of Chinese cabbage and its comparison to 2Cys-peroxiredoxin isolated from the same plant.

A cDNA encoding a newly identified isotype of peroxiredoxin (Prx) was isolated from a Chinese cabbage flower bud cDNA library and designated CPrxII. Database searches using the predicted CPrxII amino acid sequence revealed no substantial homology to other proteins with the exception of the yeast type II Prx with which CPrxII shares 27.8% sequence identity. Recombinant CPrxII expressed in Escherichia coli was able to protect glutamine synthetase from inactivation in a metal-catalyzed oxidation system and to reduce H2O2 with electrons provided by thioredoxin. This specific antioxidant activity of CPrxII was about 6-fold higher than that of 2Cys-Prx of the same plant. In contrast to 2Cys-Prx, which is predominantly expressed in leaf tissue of cabbage seedlings, CPrxII is highly expressed in root tissue as revealed by Northern and Western blot analyses. The CPrxII gene exists as a small multigene family in the cabbage genome.

Identification of antigenic differences between the phosphorylated and nonphosphorylated forms of the E7 protein of human papillomavirus type 16.

To analyze the antigenic properties of the human papillomavirus type 16 E7 oncoprotein, two monoclonal antibodies, VD6 and IB10, that have different reactivities to the E7 protein were generated. While the VD6 antibody reacted strongly with E7 protein in CaSki cell extracts, the other antibody, IB10, showed much weaker reactivity with E7. This reactivity increased in a dose-dependent manner in the presence of the casein kinase II-specific inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole). Antigenic site estimation and an in vitro phosphorylation assay, using bacterially expressed E7 protein, demonstrated that the weak reactivity of IB10 was related to the phosphorylation status of the E7 protein. Phosphorylation of E7 reduced considerably the reactivity of IB10 but did not affect the reactivity of VD6, which reacts with the N-terminal portion of E7. In immunoprecipitation (IP) assays, IB10 precipitated weakly the E7 protein from CaSki cell extracts. Together, these data suggest that unphosphorylated E7 protein shows distinct antigenic character compared to its phosphorylated form under denaturing conditions; however, under native conditions, the phosphorylated and nonphosphorylated E7 proteins have some antigenic cross-reactivity.

Purification and characterization of an antifungal PR-5 protein from pumpkin leaves.

A 28-kDa antifungal PR-5 protein (PLTP) was purified from pumpkin leaves to homogeneity by using ammonium sulfate fractionation, a regenerated chitin column, and reversed-phase column chromatographies on butyl-Toyopearl and HPLC C18 columns. Analysis of 14 N-terminal amino acid sequences of PLTP shows 100% sequence identity to those of two PR-5 proteins, NP24 from tomatoes and AP24 from tobacco. The identical sequence also exhibited high amino acid sequence homology to that of an osmotin-like protein (OLP; 71%) from tobacco cells and thaumatin (64%), a sweet-tasting protein of Thaumatococcus danielli Bench. When the PLTP was immuno-blotted with antiserum raised against the tobacco OLP, the OLP antibody specifically cross-reacted with the PLTP, suggesting that they share several common epitopes in their tertiary structure of the proteins. The purified PLTP rapidly lyzed hyphal tips of Neurospora crassa at a concentration greater than 200 nM and significantly inhibited the fungal growth of Fusarium oxysporum in an agar-disc plate at a concentration greater than 2 microM. It also shows a synergistic effect with nikkomycin, a chitin synthase inhibitor, for the growth inhibition of Candida albicans.