Differential effects of theasinensins and epigallocatechin-3-O-gallate on phospholipid bilayer structure and liposomal aggregation.

(-)-Epigallocatechin-3-O-gallate (EGCg), the major catechin responsible for the health-enhancing and disease-preventive effects of green tea, is susceptible to auto-oxidation at physiological pH levels. However, whether the oxidized EGCg resulting from its oral consumption possesses any bioactive functions remains unclear. This study presents a differential analysis of intact and oxidized EGCg regarding their interactions with phosphatidylcholine liposomes, serving as a simple biomembrane model. In the presence of ascorbic acid, pre-oxidized EGCg induced liposomal aggregation in a dose-dependent manner, whereas intact EGCg did not. Toxicity evaluation using calcein-loaded liposomes revealed that liposomal aggregation is associated with minimal membrane damage. Through fractionation of the oxidized EGCg sample, the fraction containing theasinensins showed high liposomal aggregation activity. Overall, these results suggest that oxidatively condensed EGCg dimers may stimulate various cells by altering the plasma membrane in a manner different from that of EGCg monomers.

Evidence of increases of phytol and chlorophyllide by enzymatic dephytylation of chlorophylls in smoothie made from spinach leaves.

Phytol is a diterpene alcohol found abundantly in nature as the phytyl side chain of chlorophylls. Free form of phytol and its metabolites have been attracting attention because they have a potential to improve the lipid and glucose metabolism. On the other hand, phytol is unfavorable for those who suffering from Refsum's disease. However, there is little information on the phytol contents in leafy vegetables rich in chlorophylls. This study indicated that raw spinach leaves contain phytol of 0.4-1.5 mg/100 g fresh weight. Furthermore, crude enzyme extracted from the leaves showed the enzyme activities involved in dephytylation of chlorophyll derivatives and they were high at mild alkaline pH and around 45°C, and lowered at 55°C or above. Under the optimum pH and temperature for such enzymes determined in the model reaction using the crude enzyme, phytol content in the smoothie made from raw spinach leaves increased with an increase of chlorophyllide, another reaction product. Comparison between the increased amounts of phytol and chlorophyllide showed that the enzymatic dephytylation of chlorophylls was critically responsible for the increase of phytol in the smoothie. PRACTICAL APPLICATION: Phytol, which is released by the enzymes related to chlorophyll metabolism in plants, has been investigated because of its potential abilities to improve the lipid metabolism and blood glucose level. In contrast to such health benefits, they are known to be toxic for patients suffering from Refsum's disease. This research for the first time reports the phytol content in raw spinach leaves and that phytol can be increased in the smoothie made from spinach leaves by the action of endogenous enzymes on chlorophyll derivatives under a certain condition. These results help control phytol content in the smoothies.

Gentamicin Pharmacokinetics and Optimal Dosage in Infant Patients: A Case Report and Literature Review.

Gentamicin is an aminoglycoside antibiotic that is mostly used for the pediatric population. While the pediatric population is classified into neonates, infants, children, and adolescents based on developmental or maturational changes, infants are often overlooked in research. Three infant cases receiving gentamicin are presented to illustrate the pharmacokinetics and optimum dosage of gentamicin. Three infant patients received gentamicin (5.6-7.5 mg/kg/day) for urinary tract infections (UTIs) or bacteremia caused by Enterobacter aerogenes. The trough (Cmin) and peak (Cpeak) concentrations of gentamicin were 0.2-1.8 and 8.9 mg/L, respectively. The Cmin of a patient receiving gentamicin at 9.0 mg/kg/day was 3.3 mg/L, and the patient showed a decrease in urinary volume. The other two patients fully recovered from the infection and did not experience any adverse events. Additionally, we reviewed three studies regarding infant patients receiving gentamicin. The studies used gentamicin therapy for Gram-negative pathogen infections and UTIs caused by Escherichia coli and Enterococcus faecalis. The Cmin and Cpeak of patients receiving gentamicin at 2.2-7.5 mg/kg/day were 0.58-2.15 mg/kg and 4.67-8.88 mg/L, respectively. All patients were cured without any adverse events. Gentamicin dosages below 7.5 mg/kg/day may be effective and safe for use in infant patients. However, the optimal dosing regimen of gentamicin in infant patients is controversial, and limited data are available.

Inhibitory effects of thiol compounds on theaflavin browning and structural analysis of the causative substances.

Theaflavin, a polyphenol responsible for the bright orange color and various bioactivities of black tea exudates, is susceptible to autoxidation at neutral and mild alkaline pH, changing its color to brown. In the presence of cysteine (Cys), glutathione (GSH), or N-acetyl cysteine (NAC), the browning of theaflavin solution was inhibited concomitantly with time-dependent decreases in the concentrations of both theaflavin and thiol group. The rank order of the decrease was Cys â‰… GSH > NAC, suggesting the relevance of the nucleophilic property of the thiol group to its reaction with theaflavin. LC-MS analysis of the reaction products indicated formation of novel derivatives that were mono- or di-molecular adducts of thiol compounds. We determined the chemical structures of theaflavin-Cys and theaflavin-GSH adducts by NMR and proposed the reaction mechanisms. It was found that the theaflavin-Cys adduct was not a simple adduct, to which a new cyclic structure was added.

Factors affecting serum phenobarbital concentration changes in pediatric patients receiving elixir and powder formulations.

BACKGROUND: Phenobarbital (PB) is commonly used as elixir and powder formulations in pediatric care. Its dose adjustment is performed based on individual drug concentration monitoring. Few studies have comprehensively analyzed the variation factors for serum PB concentration. In this study, we retrospectively investigated the factors that influence serum PB concentration and assessed the impacts of dosage formulation and administration route. METHODS: This retrospective cohort study covered clinical data from January 2007 to September 2019 at Mie University Hospital. The present study included 60 pediatric patients administered the elixir and powder of PB through oral route and enteral tube. Simple and multiple linear regression analyses were performed to identify the risk factors that affect the weight-corrected PB serum concentration/dose (C/D) ratio in pediatric patients. Six subgroups were also established according to the concomitant use of drugs that potentially inhibit PB metabolism, dosage formulation, and administration route to investigate the difference in the PB C/D ratio among the subgroups. RESULTS: A significant regression equation to predict the PB C/D ratio was found through simple and multiple linear regression analyses, with an adjusted coefficient of determination of 0.53 (p < 0.001). Further, the concomitant uses of valproic acid (VPA) or amiodarone, which were the only two drugs seen in this study as potential inhibitors of PB, was found to have the greatest effect on the PB C/D ratio (standardized partial regression coefficient (ß) = 0.543, p < 0.001). Furthermore, a significant difference in the PB C/D ratio was found between the subgroups classified by the concomitant use of VPA or amiodarone (p = 0.002). However, there were no significant correlations between the PB C/D ratio, dosage formulation, and administration route. CONCLUSIONS: The most influential factor on the PB C/D ratio was the concomitant use of VPA or amiodarone with PB. This result could provide an important perspective in pediatric drug therapy where elixir and powder formulations are administered via the oral route and enteral tube.

Aspartate racemase and D-aspartate in starfish; possible involvement in testicular maturation.

D-Aspartate, aspartate racemase activity, and D-aspartate oxidase activity were detected in tissues from several types of starfish. Aspartate racemase activity in male testes of Patiria pectinifera was significantly elevated in the summer months of the breeding season compared with spring months. We also compared aspartate racemase activity with the gonad index and found that activity in individuals with a gonad index ≥6% was four-fold higher than that of individuals with a gonad index <6%. The ratio of the D-form of aspartate to total aspartate was approximately 25% in testes with a gonad index <6% and this increased to approximately 40% in testes with a gonad index ≥6%. However, such changes were not observed in female ovaries. Administration of D-aspartate into male starfish caused testicular growth. These results indicate the possible involvement of aspartate racemase and D-aspartate in testicular maturation in echinoderm starfish.

Chronic administration of quetiapine stimulates dorsal hippocampal proliferation and immature neurons of male rats, but does not reverse psychosocial stress-induced hyponeophagic behavior.

Quetiapine, an atypical antipsychotic, has been used for the treatment of several neuropsychiatric disorders. However, the underlying mechanism of the broad therapeutic range of quetiapine remains unknown. We previously reported that several aversive conditions affect dorsal/ventral hippocampal neurogenesis differentially. This study was aimed to elucidate the positive effects of chronic treatment with quetiapine on regional differences in hippocampal proliferation and immature neurons and behavioral changes under psychosocial stress using the Resident-Intruder paradigm. Twenty-three male Sprague-Dawley rats were intraperitoneally administered a vehicle or quetiapine (10 mg/kg) once daily for 28 days. Two weeks after starting the injections, animals were exposed to intermittent social defeat (four times over two weeks). The behavioral effects of stress and quetiapine were evaluated by the Novelty-Suppressed Feeding (NSF) test. The stereological quantification of hippocampal neurogenesis was estimated using immunostaining with Ki-67 and doublecortin (DCX). Chronic quetiapine treatment stimulated the Ki-67- and DCX-positive cells in the dorsal hippocampus, but not in the ventral subregion. The stress-induced changes in neurogenesis and hyponeophagic behavior were not reversed by repeated administration of quetiapine. Future study with additional behavioral tests is needed to elucidate the functional significance of the quetiapine-induced increase in dorsal hippocampal neurogenesis.

Involvement of butyrate in electrogenic K+ secretion in rat rectal colon.

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are synthesized from dietary carbohydrates by colonic bacterial fermentation. These SCFAs supply energy, suppress cancer, and affect ion transport. However, their roles in ion transport and regulation in the intracellular environment remain unknown. In order to elucidate the roles of SCFAs, we measured short-circuit currents (ISC) and performed RT-PCR and immunohistochemical analyses of ion transporters in rat rectal colon. The application of 30 mM butyrate shifted ISC in a negative direction, but did not attenuate the activity of epithelial Na+ channels (ENaC). The application of bumetanide, a Na+-K+-2Cl- cotransporter inhibitor, to the basolateral side reduced the negative ISC shift induced by butyrate. The application of XE991, a KCNQ-type K+ channel inhibitor, to the apical side decreased the ISC shift induced by butyrate in a dose-dependent manner. The ISC shift was independent of HCO3- and insensitive to ibuprofen, an SMCT1 inhibitor. The mucosa from rat rectal colon expressed mRNAs of H+-coupled monocarboxylate transporters (MCT1, MCT4, and MCT5, also referred to as SLC16A1, SLC16A3, and SLC16A4, respectively). RT-PCR and immunofluorescence analyses demonstrated that KCNQ2 and KCNQ4 localized to the apical membrane of surface cells in rat rectal colon. These results indicate that butyrate, which may be transported by H+-coupled monocarboxylate transporters, activates K+ secretion through KCNQ-type K+ channels on the apical membrane in rat rectal colon. KCNQ-type K+ channels may play a role in intestinal secretion and defense mechanisms in the gastrointestinal tract.

Intake of green-plant membrane with dietary oil suppresses postprandial hypertriglyceridemia in rats via promoting excretion of bile acids.

Green-plant membrane is a phytonutrient present in green leafy vegetables at high concentration. Postprandial increases in blood triglyceride levels result in insulin resistance and type 2 diabetes. Additionally, dietary life and eating order also affect postprandial hypertriglyceridemia. In this study, the effects of once-daily intake of green-plant membrane with dietary oil on postprandial hypertriglyceridemia were investigated in vitro and in vivo. In vitro, green-plant membrane bound hydrophobic bile acids but did not inhibit pancreatic lipase activity. Following the administration, green-plant membrane with dietary oil in rats, oral fat tolerance tests, increases in serum triglycerides levels were significantly reduced. Moreover, fecal total lipid and bile acid volumes were significantly increased in rats that administered 200 mg/mL green-plant membrane. These results suggest that green-plant membrane with dietary oil inhibits dietary fat absorption via promotion of bile acid excretion in feces and the effectiveness of eating green-plant membrane, such as green leafy vegetables, with meals.

Short-term inhibition of GABAergic IPSCs induced by association of pre- and postsynaptic activation in the neonatal hippocampus.

Activity-dependent plasticity including short and long-term depression accompanied by a reduction in transmitter release probability has been demonstrated in both inhibitory and excitatory synapses. In the neonatal hippocampus, repetitive postsynaptic depolarization is followed by presynaptic alterations of the efficacy of GABAAergic transmission. Both facilitation and inhibition have been observed, but the mechanisms underlying this plasticity have not yet been elucidated. In the present experiment, repetitive postsynaptic depolarization by itself did not cause marked alterations of spontaneous inhibitory postsynaptic currents (sIPSCs). Activation of presynaptic neurons by increasing extracellular K+ concentration ([K+]o) temporarily induced facilitation of sIPSCs, but successive repetitive depolarizations transiently reduced the current frequency. This newly discovered inhibition was expressed presynaptically, could not be induced by postsynaptic depolarization alone, and was facilitated by the activation of NMDA receptors. IPSC inhibition was suppressed using the antagonists of metabotropic glutamate receptors (mGluRs) or muscarinic ACh receptors (mAChRs). Furthermore, transient inhibition was reduced by an antagonist of the type 1 cannabinoid receptor (CB1 receptor). The effect of CB1 receptor agonist on the sIPSCs was potentiated by [K+]o elevation, implying a role for the [K+]o elevation other than the release of transmitters. These results show that weak postsynaptic activation, when combined with presynaptic activation, leads to an inhibition of GABAergic synapses in the neonatal hippocampus. This inhibition is mediated by a mechanism involving mGluRs, mAChRs, and CB1 receptors, and potentiated by NMDA receptor activation.

Involvement of intracellular transport in TREK-1c current run-up in 293T cells.

The TREK-1 channel, the TWIK-1-related potassium (K+) channel, is a member of a family of 2-pore-domain K+ (K2P) channels, through which background or leak K+ currents occur. An interesting feature of the TREK-1 channel is the run-up of current: i.e. the current through TREK-1 channels spontaneously increases within several minutes of the formation of the whole-cell configuration. To investigate whether intracellular transport is involved in the run-up, we established 293T cell lines stably expressing the TREK-1c channel (K2P2.1) and examined the effects of inhibitors of membrane protein transport, N-methylmaleimide (NEM), brefeldin-A, and an endocytosis inhibitor, pitstop2, on the run-up. The results showing that NEM and brefeldin-A inhibited and pitstop2 facilitated the run-up suggest the involvement of intracellular protein transport. Correspondingly, in cells stably expressing the mCherry-TREK-1 fusion protein, NEM decreased and pitstop2 increased the cell surface localization of the fusion protein. Furthermore, the run-up was inhibited by the intracellular application of a peptide of the C-terminal fragment TREK335-360, corresponding to the interaction site with microtubule-associated protein 2 (Mtap2). This peptide also inhibited the co-immunoprecipitation of Mtap2 with anti-mCherry antibody. The extracellular application of an ezrin inhibitor (NSC668394) also suppressed the run-up and surface localization of the fusion protein. The co-application of these inhibitors abolished the TREK-1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK-1c channels and the run-up. These findings clearly showed the involvement of intracellular transport in TREK-1c current run-up and its mechanism.

The Effect of Heat-acid Treatment on the Formation of Resistant Starch and the Estimated Glycemic Index in Potatoes.

Potatoes are generally regarded as high glycemic index (GI) foods. Resistant starch (RS) comprises the starch fraction that is not absorbed in the small intestine, thus controlling the glucose level and improving the intestinal environment. In this study, an analysis of the formation of RS of potato starch samples under different acetic acid-thermal treatment conditions was conducted. Additionally, the relationship between the rates of starch digestion, estimated GI (eGI), and the RS content was evaluated by employing in vitro enzymatic models. Compared with control samples, the RS content in the cold-stored samples after acid-boiling was higher, whereas that of samples after heating at 120 °C with acetic acid was decreased. The eGI was negatively correlated with the RS content in potatoes. Cold store after acid-boiling was effective in increasing the RS content. Furthermore, low eGI values may have resulted from higher levels of RS in potatoes.

Diversity of the bacterial community in Myanmar traditional salted fish yegyo ngapi.

The distribution and characterization of bacteria including lactic acid bacteria (LAB) in the traditional and popular salted fish yegyo ngapi in Myanmar were studied to clarify the contribution of these bacteria to the curing and ripening of this product. Samples of yegyo ngapi purchased from a market in Yangon were used. Most of the isolates obtained using de Man, Rogosa and Sharpe medium containing 10 % NaCl were identified as coccoid LAB on the basis of their basic phenotypic characteristics. From the results of 16S rRNA gene sequencing and PCR-restriction fragment length polymorphism analysis of this gene, most of the isolates were identified as the halophilic LAB Tetragenococcus muriaticus. Analyses of the 16S rRNA gene based on the clone library using DNA extracted from salted fish products were also performed. The results of these molecular-analysis-based techniques showed that spore-forming and non-spore-forming anaerobic bacteria including the genera Clostridium and Halanaerobium in addition to T. muriaticus were also frequently found in bacterial communities. These findings suggest that the anaerobic condition during curing and ripening resulted in bacterial communities composed of strictly anaerobic bacteria and halophilic LAB, and that these bacteria might also contribute to the manufacturing processes of this product. In addition, DNA sequences similar to that of Clostridium botulinum were found in the clone library analysis. Therefore, despite no reports of botulism poisoning from the region where the samples were taken, closer surveillance should be carried out from the viewpoint of food safety.

Molecular characterization of Tps1 and Treh genes in Drosophila and their role in body water homeostasis.

In insects, trehalose serves as the main sugar component of haemolymph. Trehalose is also recognized as a mediator of desiccation survival due to its proposed ability to stabilize membranes and proteins. Although the physiological role of trehalose in insects has been documented for decades, genetic evidence to support the importance of trehalose metabolism remains incomplete. We here show on the basis of genetic and biochemical evidence that the trehalose synthesis enzyme Tps1 is solely responsible for the de novo synthesis of trehalose in Drosophila. Conversely, a lack of the gene for the trehalose hydrolyzing enzyme Treh causes an accumulation of trehalose that is lethal during the pupal period, as is observed with Tps1 mutants. Lack of either Tps1 or Treh results in a significant reduction in circulating glucose, suggesting that the maintenance of glucose levels requires a continuous turnover of trehalose. Furthermore, changes in trehalose levels are positively correlated with the haemolymph water volume. In addition, both Tps1 and Treh mutant larvae exhibit a high lethality after desiccation stress. These results demonstrate that the regulation of trehalose metabolism is essential for normal development, body water homeostasis, and desiccation tolerance in Drosophila.

Resistant starch suppresses postprandial hypertriglyceridemia in rats.

Postprandial increase in blood triglyceride levels is an independent risk factor for coronary artery disease, and dietary resistant starch (RS) is increasingly being considered for its contribution to disease prevention. Specifically, RS has beneficial effects on of the glycemic index, diabetes, cholesterol levels, and weight management. However, the effects of once-daily intake of RS on postprandial hypertriglyceridemia remain poorly characterized. In this study, the effects of a single administration of cornstarch-derived RS on postprandial increases in blood triglyceride levels were investigated in rats using oral fat tolerance/loading tests. Following the administration of lipid meals, increases in serum triglycerides levels were significantly reduced in rats fed corn oil containing 500mg/mL RS. Moreover, fecal lipid volumes and wet weights following lipid meals were significantly greater in rats fed corn oil containing 500mg/mL RS than in the corn oil only group, confirming the inhibition of dietary fat absorption. Finally, a significant positive correlation was observed between fecal lipid contents and wet weights in rats administered RS. These results suggest that RS intake with dietary fats induces defecation and confirm results of recent reports on the health-promoting potential of once-daily RS intake.

Increase in the titer of lentiviral vectors expressing potassium channels by current blockade during viral vector production.

BACKGROUND: High titers of lentiviral vectors are required for the efficient transduction of a gene of interest. During preparation of lentiviral the vectors, the protein of interest is inevitably expressed in the viral vector-producing cells. This expression may affect the production of the lentiviral vector. METHODS: We prepared lentiviral vectors expressing inwardly rectifying potassium channel (Lv-Kir2.1), its dominant-negative form (Lv-Kir-DN), and other K(+) channels, using the ubiquitously active ß-actin and neuron-specific synapsin I promoters. RESULTS: The titer of Lv-Kir-DN was higher than that of Lv-Kir2.1, suggesting a negative effect of induced K(+) currents on viral titer. We then blocked Kir2.1 currents with the selective blocker Ba(2+) during Lv-Kir2.1 production, and obtained about a 5-fold increase in the titer. Higher extracellular K(+) concentrations increased the titer of Lv-Kir2.1 about 9-fold. With a synapsin I promoter Ba(2+) increased the titer because of the moderate expression of Kir2.1 channel. Channel blockade also increased the titers of the lentivirus expressing Kv1.4 and TREK channels, but not HERG. The increase in titer correlated with the K(+) currents generated by the channels expressed. CONCLUSION: In the production of lentivirus expressing K(+) channels, titers are increased by blocking K(+) currents in the virus-producing cells. This identifies a crucial issue in the production of viruses expressing membrane channels, and should facilitate basic and gene therapeutic research on channelopathies.

A pore forming peptide from spider Lachesana sp. venom induced neuronal depolarization and pain.

BACKGROUND: Arachnoid venoms contain numerous peptides with ion channel modifying and cytolytic activities. METHODS: We developed a green fluorescent protein (GFP)-based assay that can monitor the changes in currents through overexpressed inwardly rectifying K(+) channels (Kir2.1), in which GFP expression was increased by blockade of Kir2.1 current. Using this assay, we screened venom of many spider species. A peptide causing GFP decreasing effect was purified and sequenced. Electrophysiological and pain-inducing effects of the peptide were analyzed with whole-cell patch-clamp recordings and hot-plate test, respectively. RESULTS: Among venoms we screened, soluble venom from Lachesana sp. decreased the GFP expression. Purification and sequencing of the peptide showed that the peptide is identical to a pore-forming peptide purified from Lachesana tarabaevi venom. Whole cell patch-clamp recordings revealed that the peptide had no effect on Kir2.1 current. Instead, it induced a current that was attributable to the pore-formation of the peptide. The peptide was selectively incorporated into hyperpolarized, i.e., Kir2.1 expressing, cells and for this reason the peptide decreased GFP expression in our Kir2.1 assay. The pore-formation positively shifted the reversal potential and induced burst firings in the hippocampal neurons in a synaptic current-independent way. The application of the Lachesana sp. peptide induced pain-related behavior in mice. CONCLUSIONS: The peptide, which was found in Lachesana sp. venom, formed pores and thereby depolarized neurons and induced pain. GENERAL SIGNIFICANCE: Our data suggested an additional physiological role of the pore-forming peptides.

Flies without trehalose.

Living organisms adapt to environmental changes through metabolic homeostasis. Sugars are used primarily for the metabolic production of ATP energy and carbon sources. Trehalose is a nonreducing disaccharide that is present in many organisms. In insects, the principal hemolymph sugar is trehalose instead of glucose. As in mammals, hemolymph sugar levels in Drosophila are regulated by the action of endocrine hormones. Therefore, the mobilization of trehalose to glucose is thought to be critical for metabolic homeostasis. However, the physiological role of trehalose as a hemolymph sugar during insect development remains largely unclear. Here, we demonstrate that mutants of the trehalose-synthesizing enzyme Tps1 failed to produce trehalose as expected but survived into the late pupal period and died before eclosion. Larvae without trehalose grew normally, with a slight reduction in body size, under normal food conditions. However, these larvae were extremely sensitive to starvation, possibly due to a local defect in the central nervous system. Furthermore, Tps1 mutant larvae failed to grow on a low-sugar diet and exhibited severe growth defects on a low-protein diet. These diet-dependent phenotypes of Tps1 mutants demonstrate the critical role of trehalose during development in Drosophila and reveal how animals adapt to changes in nutrient availability.

Regulation of rat hepatic α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase, a key enzyme in the tryptophan- NAD pathway, by dietary cholesterol and sterol regulatory element-binding protein-2.

PURPOSE: Nicotinic acid is one of the older drugs used to treat hyperlipidemia, the greatest risk factor of coronary heart disease. Nicotinic acid is also a precursor of the coenzyme nicotinamide adenine dinucleotide (NAD). In mammals, α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) plays a key role in NAD biosynthesis from tryptophan. However, the relationship between ACMSD and cholesterol metabolism has not been clarified enough yet. The present study was performed to make clear the relationship between ACMSD and cholesterol metabolism using hypercholesterolemic rats and rat primary hepatocytes. METHODS: Male Sprague-Dawley rats were fed a diet containing cholesterol for 10 days to induce hypercholesterolemia. The NAD levels in the plasma and liver and hepatic ACMSD activity were determined. In vitro study, the expression of ACMSD and the transcriptional factors that regulate cholesterol metabolism were determined using rat primary hepatocytes treated with cholesterol and 25-hydroxycholesterol or simvastatin, a statin medication, by quantitative real-time PCR analysis and Western blotting analysis. RESULTS: The hepatic NAD level of the hypercholesterolemic group was significantly higher than the control, and the hepatic ACMSD activity of this group was significantly suppressed. There was a significant negative correlation between the hepatic ACMSD activity and liver cholesterol levels. Additionally, in primary rat hepatocytes treated with cholesterol and 25-hydroxycholesterol or simvastatin, ACMSD gene and protein expression was subjected to sterol-dependent regulation. This gene expression changed in parallel to sterol regulatory element-binding protein (SREBP)-2 expression. CONCLUSION: These results provide the first evidence that ACMSD is associated with cholesterol metabolism, and ACMSD gene expression may be upregulated by SREBP-2.

Gram-negative bacterial lipopolysaccharide stimulates activin a secretion from human amniotic epithelial cells.

Activin A is involved in inflammation. The present study was performed to clarify if lipopolysaccharide, a component of Gram-negative bacteria, stimulates activin A secretion from human amniotic epithelial cells and to determine if activin A plays a role in amnionitis. Fetal membranes were obtained during elective cesarean sections performed in full-term pregnancies of patients without systemic disease, signs of premature delivery, or fetal complications. Amniotic epithelial cells were isolated by trypsinization. The activin A concentrations in the culture media were measured by enzyme-linked immunosorbent assay, and cell proliferation was assessed by 5-bromo-2'-deoxyuridine incorporation. Amniotic epithelial cells secreted activin A in a cell density-dependent manner, and lipopolysaccharide (10 µ g/mL) enhanced the secretion at each cell density. Lipopolysaccharide (10-50 µ g/mL) also stimulated activin A secretion in a dose-dependent manner. Contrary to the effect of activin A secretion, lipopolysaccharide inhibited cell proliferation in amniotic epithelial cells. The present study suggests that lipopolysaccharide stimulation of activin A secretion may be a mechanism in the pathogenesis of amnionitis.