Young Hye Kwon

Temporal and spatial dynamics of appressorium formation inUromyces appendiculatus

Appressorium development in Uromyces appendiculatus was studied to establish temporal and spatial parameters possibly important in signaling mechanisms in this fungus. It was determined that urediospore germling apices sensed inductive topographies, e.g., precisely shaped ridges 0.5 μm high by 2.0 μm wide, within 4 minutes after initial contact. The area in the germling that senses such topographies was located on the substrate side of the cell and within 6.0 μm of the apex. These conclusions were based on observed changes in the rate of germling growth and nuclear migration as well as the timing of apical tip swelling that began 4 minutes after contact. DNA synthesis during appressorium development occurred only after the first nuclear division and was probably in preparation for the second mitosis that normally occurs in the substomatal vesicle. Treatments with the DNA synthesis inhibitors hydroxyurea and actinomycin D reduced the DNA content of individual nuclei only after nuclear division. These results indicate that the nuclei in urediospore germlings were already in the G2 phase when germination began. Formation of a septum during appressorium development was initiated 45–50 minutes after contact of the germling with the ridge and was completed within 60 minutes after all of the cytoplasm had entered into the swollen apex.

Cysteine metabolism in periportal and perivenous hepatocytes: Perivenous cells have greater capacity for glutathione production and taurine synthesis but not for cysteine catabolism

Summary. Hepatocyte preparations highly enriched in cells from either the periportal or the perivenous zone of the liver acinus were prepared using a digitonin/collagenase perfusion method. Five enzymes of cysteine metabolism were assayed in both periportal and perivenous preparations. The ratios of periportal to perivenous activity were 0.76, 0.60, 0.81, 1.62, and 1.01 for cysteine dioxygenase, cysteinesulfinate decarboxylase, γ-glutamylcysteine synthetase, cystathionase, and asparate (cysteinesulfinate) aminotransferase, respectively. Only cysteinesulfinate decarboxylase activity was significantly different between periportal and perivenous cells. In incubations with 2 mmol/L [35S]cysteine, total cysteine catabolism ([35S]taurine plus [35S]sulfate) between periportal and perivenous cells was not different, which is consistent with the observation of similar cysteine dioxygenase activity across the hepatic acinus. Consistent with the lower cysteinesulfinate decarboxylase activity in periportal cells, 16% of the total catabolism of [35S]cysteine in periportal cells resulted in taurine synthesis compared to 28% in perivenous cells. A lower rate of [35S]glutathione synthesis was observed in periportal cells compared to perivenous cells, but γ-glutamylcysteine synthetase activity was not significantly different between perivenous and periportal cells. Cysteinesulfnate decarboxylase can be added to the list of enzymes whose activities are markedly enriched in perivenous cells.

Dietary Supplementation of Genistein Alleviates Liver Inflammation and Fibrosis Mediated by a Methionine-Choline-Deficient Diet in db/db Mice.

Nonalcoholic fatty liver disease is a complex disorder which includes simple steatosis, steatohepatitis, fibrosis and ultimately cirrhosis. Previous studies have reported that genistein, a soy phytoestrogen, attenuates steatohepatitis induced in obese and type 2 diabetic models. Here we investigated the effect of dietary genistein supplementation (0.05%) on nonalcoholic steatohepatitis (NASH) development induced by a methionine-choline-deficient (MCD) diet in db/db mice. MCD-diet-fed mice exhibited a significantly lower body weight and a higher degree of steatohepatitis with increased oxidative stress, steatosis, inflammation, stellate cell activation, and mild fibrosis. Although genistein did not inhibit hepatic steatosis, we observed that oxidative stress, endoplasmic reticulum stress, and AMP-dependent kinase inactivation were alleviated by genistein. Genistein also down-regulated the augmented gene expressions associated with hepatic inflammation and fibrosis. Therefore, these results suggest that genistein may protect MCD-diet-mediated NASH development by suppressing lipid peroxidation, inflammation, and even liver fibrosis in db/db mice.

Betaine Alleviates Hypertriglycemia and Tau Hyperphosphorylation in db/db Mice

Betaine supplementation has been shown to alleviate altered glucose and lipid metabolism in mice fed a high-fat diet or a high-sucrose diet. We investigated the beneficial effects of betaine in diabetic db/db mice. Alleviation of endoplasmic reticulum (ER) and oxidative stress was also examined in the livers and brains of db/db mice fed a betaine-supplemented diet. Male C57BL/KsJ-db/db mice were fed with or without 1% betaine for 5 wk (referred to as the db/db-betaine group and the db/db group, respectively). Lean non-diabetic db/db+ mice were used as the control group. Betaine supplementation significantly alleviated hyperinsulinemia in db/db mice. Betaine reduced hepatic expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha, a major transcription factor involved in gluconeogenesis. Lower serum triglyceride concentrations were also observed in the db/db-betaine group compared to the db/db group. Betaine supplementation induced hepatic peroxisome proliferator-activated receptor alpha and carnitine palmitoyltransferase 1a mRNA levels, and reduced acetyl-CoA carboxylase activity. Mice fed a betaine-supplemented diet had increased total glutathione concentrations and catalase activity, and reduced lipid peroxidation levels in the liver. Furthermore, betaine also reduced ER stress in liver and brain. c-Jun N-terminal kinase activity and tau hyperphosphorylation levels were lower in db/db mice fed a betaine-supplemented diet, compared to db/db mice. Our findings suggest that betaine improves hyperlipidemia and tau hyperphosphorylation in db/db mice with insulin resistance by alleviating ER and oxidative stress.

Cysteine dioxygenase and γ-glutamylcysteine synthetase activities in primary cultured hepatocytes respond to sulfur amino acid supplementation in a reciprocal manner

Summary. Hepatocytes were cultured for 3 days as spheroids (aggregates) or as monolayers in basal medium and in sulfur amino acid-supplemented media. Cultured hepatocytes had low levels of cysteine dioxygenase (CDO) activity and normal levels of γ-glutamylcysteine synthetase (GCS) and cysteinesulfinate decarboxylase (CSDC) activities compared to freshly isolated cells. CDO activity increased and GCS activity decreased in a dose-response manner in cells cultured in either methionine- or cysteine-supplemented media. CSDC activity was not significantly affected by methionine supplementation. Changes in CDO and GCS were associated with changes in cysteine catabolism to taurine plus sulfate and in synthesis of glutathione, respectively. These responses are similar to those observed in liver of intact rats fed diets supplemented with sulfur amino acids. A near-maximal response of CDO or GCS activity was observed when the medium contained 1.0 mmol/L of methionine plus cyst(e)ine. Changes in CDO and GCS activities did not appear to be mediated by changes in the intracellular glutathione concentration. Cultured hepatocytes offer a useful model for further studies of cysteine metabolism and its regulation in response to sulfur amino acid availability.

Involvement of Endoplasmic Reticulum Stress in Palmitate-induced Apoptosis in HepG2 Cells

The results of recent studies indicate that high levels of free fatty acids (FFAs) and adipokines may be the main causes of non-alcoholic liver disease; however, the molecular mechanism that links FFAs to lipotoxicity remains unclear. In the present study, we treated HepG2 cells with FFA (either palmitate or oleate) to investigate the mechanisms involved in lipotoxicity in the liver cells. We also treated cells with palmitate in the presence of a chemical chaperone, 4-phenylbutyric acid (PBA), to confirm the involvement of ER stress in lipotoxicity. Palmitate significantly induced cytotoxicity in dose- and time-dependent manners. Apoptosis was also significantly induced by palmitate as measured by caspase-3 activity and DAPI staining. Palmitate led to increased expressions of the spliced form of X-box-protein (Xbp)-1 mRNA and C/EBP homologous transcription factor (CHOP) protein, suggesting activation of the unfolded-protein response. PBA co-incubation significantly attenuated apoptosis induced by palmitate. The above data demonstrate that high levels of palmitate induce apoptosis via the mediation of ER stress in the liver cells and that chemical chaperones act to modulate ER stress and accompanying apoptosis.