Fungus-derived hydroxyl radicals kill hepatic cells by enhancing nuclear transglutaminase.

We previously reported the importance of induced nuclear transglutaminase (TG) 2 activity, which results in hepatic cell death, in ethanol-induced liver injury. Here, we show that co-incubation of either human hepatic cells or mouse primary hepatocytes derived from wild-type but not TG2-/- mice with pathogenic fungi Candida albicans and C. glabrata, but not baker's yeast Saccharomyces cerevisiae, induced cell death in host cells by enhancing cellular, particularly nuclear, TG activity. Further pharmacological and genetic approaches demonstrated that this phenomenon was mediated partly by the production of reactive oxygen species (ROS) such as hydroxyl radicals, as detected by a fluorescent probe and electron spin resonance. A ROS scavenger, N-acetyl cysteine, blocked enhanced TG activity primarily in the nuclei and inhibited cell death. In contrast, deletion of C. glabrata nox-1, which encodes a ROS-generating enzyme, resulted in a strain that failed to induce the same phenomena. A similar induction of hepatic ROS and TG activities was observed in C. albicans-infected mice. An antioxidant corn peptide fraction inhibited these phenomena in hepatic cells. These results address the impact of ROS-generating pathogens in inducing nuclear TG2-related liver injuries, which provides novel therapeutic targets for preventing and curing alcoholic liver disease.

Laminar shear stress upregulates endothelial Ca²âº-activated K⁺ channels KCa2.3 and KCa3.1 via a Ca²âº/calmodulin-dependent protein kinase kinase/Akt/p300 cascade.

In endothelial cells (ECs), Ca²âº-activated K⁺ channels KCa2.3 and KCa3.1 play a crucial role in the regulation of arterial tone via producing NO and endothelium-derived hyperpolarizing factors. Since a rise in intracellular Ca²âº levels and activation of p300 histone acetyltransferase are early EC responses to laminar shear stress (LS) for the transcriptional activation of genes, we examined the role of Ca²âº/calmodulin-dependent kinase kinase (CaMKK), the most upstream element of a Ca²âº/calmodulin-kinase cascade, and p300 in LS-dependent regulation of KCa2.3 and KCa3.1 in ECs. Exposure to LS (15 dyn/cm²) for 24 h markedly increased KCa2.3 and KCa3.1 mRNA expression in cultured human coronary artery ECs (3.2 ± 0.4 and 45 ± 10 fold increase, respectively; P < 0.05 vs. static condition; n = 8-30), whereas oscillatory shear (OS; ± 5 dyn/cm² × 1 Hz) moderately increased KCa3.1 but did not affect KCa2.3. Expression of KCa2.1 and KCa2.2 was suppressed under both LS and OS conditions, whereas KCa1.1 was slightly elevated in LS and unchanged in OS. Inhibition of CaMKK attenuated LS-induced increases in the expression and channel activity of KCa2.3 and KCa3.1, and in phosphorylation of Akt (Ser473) and p300 (Ser1834). Inhibition of Akt abolished the upregulation of these channels by diminishing p300 phosphorylation. Consistently, disruption of the interaction of p300 with transcription factors eliminated the induction of these channels. Thus a CaMKK/Akt/p300 cascade plays an important role in LS-dependent induction of KCa2.3 and KCa3.1 expression, thereby regulating EC function and adaptation to hemodynamic changes.

Ecto-5'-nucleotidase, CD73, is an endothelium-derived hyperpolarizing factor synthase.

OBJECTIVE: Adenosine dilates human coronary arteries by activating potassium channels in an endothelial cell-independent manner. Cell surface ecto-5'-nucleotidase (CD73) rapidly dephosphorylates extracellular adenosine 5'-monophosphate to adenosine. We tested the hypothesis that coronary vasodilation to adenine nucleotides is mediated by an endothelial CD73-dependent, extracellular production of adenosine that acts as an endothelium-derived hyperpolarizing factor. METHODS AND RESULTS: Videomicroscopy showed that adenine nucleotides, but not inosine, potently dilated and hyperpolarized human coronary arteries independent of nitric oxide, prostacyclin, and classical endothelium-derived hyperpolarizing factors, whereas endothelial denudation, adenosine receptor antagonism, adenosine deaminase, or CD73 blockers reduced vasodilations. Liquid chromatography-electrospray ionization-mass spectrometry revealed adenosine accumulation in perfusates from arteries in the presence of adenosine 5'-diphosphate. CD73 was localized on the cell surface of endothelial cells, but not of vascular smooth muscle cells, and its deficiency suppressed vasodilation of mouse coronary arteries to adenine nucleotides and augmented vasodilation to adenosine. Adenosine dose-dependently dilated and hyperpolarized human coronary arteries to a similar extent as adenosine 5'-diphosphate. CONCLUSIONS: Coronary vasodilation to adenine nucleotides is associated with endothelial CD73-dependent production of extracellular adenosine that acts as an endothelium-derived hyperpolarizing factor by relaxing and hyperpolarizing underlying vascular smooth muscle cells via activating adenosine receptors. Thus, CD73 is a novel endothelium-derived hyperpolarizing factor synthase in human and mouse coronary arteries.

The molecular mechanisms of the hepatoprotective effect of gomisin A against oxidative stress and inflammatory response in rats with carbon tetrachloride-induced acute liver injury.

Oxidative damage and inflammation are implicated in the pathogenesis of liver injury and fibrosis. In the present study, we investigated the molecular mechanism by which gomisin A conferred a hepatoprotective effect, focusing on its antioxidant and anti-inflammatory effects using rats with carbon tetrachloride (CCl(4))-induced acute liver injury. Pretreatment with gomisin A prior to the administration of CCl(4) markedly prevented an increase in alanine aminotransferase, aspartate aminotransferase, and histological hepatic lesions. Gomisin A was also associated with a decrease in hepatic lipid peroxidation, and increased superoxide dismutase activity, suggesting that gomisin A has an antioxidant effect. In addition gomisin A treatment ameliorated mRNA levels of CCl(4)-induced inflammatory mediators, including tumor necrosis factor-α, interleukin-1ß and inducible nitric oxide (NO) synthase, and the protein levels of transcriptional upregulator nuclear factor kappa B (NF-κB) and phospho-inhibitor of NF-κB (IκB). Furthermore, α-smooth muscle actin (α-SMA), a myofibroblast marker, was also inhibited by gomisin A treatment. These results suggest that gomisin A inhibits the oxidative stress and activation of NF-κB, leading to down-regulation of pro-inflammatory mediators and amelioration of fibrogenesis.

Nondestructive prediction of oren extract powder, a herbal medicine, in suppositories by chemometric near-infrared spectroscopy.

Near-infrared (NIR) spectroscopy combined with chemometrics has been utilized in predictions of natural medicine content without destroying samples. Suppositories (oren powdered extract content 0, 0.5, 1.0, 2.5, 10, 12.5, and 15%) were produced by mixing oren powdered extract with macrogol mixture consisting of 1 part macrogol 1500 and 2.5 parts macrogol 4000 at 54°C, and pouring the melt mixture into a plastic container. NIR spectra of the 10 prepared samples were recorded 10 times, and a total of 100 spectra were randomly divided into two data sets, one for calibration and the other for validation. The calibration model for the oren content of the suppository was calculated based on NIR spectra using a partial least-squares regression analysis after pre-treatment (smoothing and the multiplicative scatter correction). The relationship between the actual and predicted values for calibration and validation models had a straight line with correlation coefficients of 0.9936 and 0.9898, respectively. The regression vector result of the calibration model indicates that the peaks at 6945, 5747, and 5160 cm(-1) in the regression vector were consistent with those in oren powder extracts. NIR spectroscopy combined with chemometrics offers promise as a method of predicting the oren powder content in suppositories without destroying the samples.

Effects of bezafibrate in nonalcoholic steatohepatitis model mice with monosodium glutamate-induced metabolic syndrome.

Recently, we reported that monosodium glutamate-treated mice (MSG mice) developed severe hyperlipidemia and diabetes mellitus and several complications of obesity. MSG mice acquired fatty livers and subsequently underwent changes that are characteristic of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). In the present study, the effects of bezafibrate on obesity, diabetes mellitus, and NAFLD/NASH were examined in MSG mice. A single dose of MSG (4 mg/g) was administered subcutaneously to neonatal male mice within 24h of birth. Bezafibrate was mixed into the normal feed for 8 weeks. The weight and body mass index of MSG mice increased significantly despite the unchanged intake of food. Triglyceride and total cholesterol levels in blood, visceral adipose tissue, and interscapular adipose tissue rose significantly. In the livers of MSG mice, moderate centrilobular microvesicular steatosis, ballooning degeneration with Mallory bodies, and scattered infiltration of neutrophils and lymphocytes were observed. Centrilobular hepatocytes were 4-hydroxynonenal-positive in MSG mice. Bezafibrate ameliorated the severity of diabetes mellitus, hyperinsulinemia, and hyperlipidemia. Adiponectin and leptin concentrations in blood improved, and the accumulation of visceral fat was inhibited. The expression of acyl-CoA oxidase, a beta-oxidation gene, and carnitine palmitoyl transferase, which regulates lipid metabolism, increased markedly on administration of bezafibrate. The liver pathology in MSG mice also improved with bezafibrate; specifically, macro- and microvesicles in hepatocytes nearly disappeared, and NAFLD activity score improved. It is concluded that bezafibrate inhibits the accumulation of visceral fat, following amelioration of hyperlipidemia, in MSG-induced obese mice, due to improvements in diabetes mellitus, fatty liver, and NAFLD.