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.

Spironolactone, but not enalapril, potentiates hypoxia-inducible factor-1 alpha and Ets-1 expression in newborn rat kidney.

Hypoxia is regarded as an important physiological factor that controls nephrogenesis. We investigated whether the renin-angiotensin-aldosterone system (RAAS) affects hypoxia-related target genes in developing kidneys. Newborn rat pups were treated with enalapril (30 mg/kg/d) or spironolactone (200 mg/kg/d) for 7 days. Tissue hypoxia was assessed by the uptake of a hypoxyprobe-1, pimonidazole (200 mg/kg), and the expression of hypoxia-responsive genes. In the enalapril group, hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, and Ets-1 protein expression were not changed, compared to the control group. In the spironolactone group, HIF-1alpha and Ets-1 protein expression were significantly increased by immunoblots and immunohistochemistry, whereas HIF-2alpha protein expression was not changed, compared to the control group. In the enalapril group, the immunoactivity of pimonidazole was not significantly different from that of the controls. However, in the spironolactone group, pimonidazole staining demonstrated that the cortex and medulla underwent severe hypoxia. In summary, our data showed that aldosterone inhibition in the developing kidney augmented the hypoxic responses, and up-regulated the expression of key mediators of hypoxia including HIF-1alpha and Ets-1. Angiotensin II inhibition did not affect hypoxia-related alterations in the developing kidney. The components of RAAS may differentially modulate renal hypoxia and its related target genes in the developing rat kidney.