Effects of Biotin Deficiency on Biotinylated Proteins and Biotin-Related Genes in the Rat Brain.

Biotin is a water-soluble vitamin that functions as a cofactor for biotin-dependent carboxylases. The biochemical and physiological roles of biotin in brain regions have not yet been investigated sufficiently in vivo. Thus, in order to clarify the function of biotin in the brain, we herein examined biotin contents, biotinylated protein expression (e.g. holocarboxylases), and biotin-related gene expression in the brain of biotin-deficient rats. Three-week-old male Wistar rats were divided into a control group, biotin-deficient group, and pair-fed group. Rats were fed experimental diets from 3 wk old for 8 wk, and the cortex, hippocampus, striatum, hypothalamus, and cerebellum were then collected. In the biotin-deficient group, the maintenance of total biotin and holocarboxylases, increases in the bound form of biotin and biotinidase activity, and the expression of an unknown biotinylated protein were observed in the cortex. In other regions, total and free biotin contents decreased, holocarboxylase expression was maintained, and bound biotin and biotinidase activity remained unchanged. Biotin-related gene (pyruvate carboxylase, sodium-dependent multivitamin transporter, holocarboxylase synthetase, and biotinidase) expression in the cortex and hippocampus also remained unchanged among the dietary groups. These results suggest that biotin may be related to cortex functions by binding protein, and the effects of a biotin deficiency and the importance of biotin differ among the different brain regions.

Biochemical alterations in the palatal processes in fetuses of biotin-deficient mice.

To clarify the role of biotin in palatal formation, we investigated the effects of biotin deficiency on the development of palatal processes in mouse fetuses at midgestation. We also investigated protein expressions in the palatal processes. Pregnant mice were given either a biotin-deficient diet or a biotin-supplemented (control) diet from day 0 of gestation (dg 0). Some dams in the biotin-deficient group were changed to a biotin-supplemented diet on dg 12, 13 or 14. On dg 15, the palatal processes were dissected from these fetuses and their peptides were characterized using two-dimensional electrophoresis and liquid chromatography/tandem mass spectrometry (LC-MS/MS) system. Regarding Trasler's stage for the growth of the palatal processes in mouse fetuses on dg 15, the average stage of palatal development was 5.83 +/- 0.39 in the biotin-supplemented group, 5.39 +/- 0.66 in the dg 13-supplemented group, and 4.64 +/- 0.90 in the biotin-deficient group. The development of the palatal processes significantly increased in relation to the earlier day of biotin supplementation. In a protein analysis of palatal processes by isoelectro focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 19-kDa spot was confirmed around position at pI 6-7 in the biotin-supplemented group, but this protein was not present in either the biotin-deficient group or the dg 13-supplemented group. From the MS/MS database of peptides, adenosine diphosphate (ADP)-ribosylation factor 2 (arf2) and alpha-crystallin were detected in the mesenchyme of the palatal processes. It is suggested that the expression of these proteins may be downregulated by biotin deficiency, inducing the inhibited development of palatal processes.

Effects of acute ethanol administration on LPS-induced expression of cyclooxygenase-2 and inducible nitric oxide synthase in rat alveolar macrophages.

BACKGROUND: Activation of alveolar macrophages acts as a primary defense mechanism of lung with immunologic and inflammatory processes. Incidence of respiratory distress syndrome (ARDS) has been reported to be higher in alcoholics than that in nonalcoholics. Both cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are induced by inflammatory stimuli such as LPS and cytokines and are involved in host defense. COX-2 and iNOS have been reported to play important roles in pathophysiology of ARDS. The aim of the present study was to elucidate whether acute ethanol administration to rats affects on COX-2 and iNOS expression in isolated alveolar macrophages. METHODS: Ethanol (4.5 g per kg body weight as a 20% solution) was intraperitoneally injected to male Wistar rats. At 2.5 hrs after the injection, alveolar macrophages were collected from rats by bronchoalveolar lavage and were stimulated with lipopolysaccharide (LPS, 1 mug/ml). Expression of COX-2 and iNOS and activation of MAPKs was evaluated by Western blotting. RESULTS: In alveolar macrophages isolated from ethanol-treated rats, LPS-stimulated production of both prostaglandin E2 and nitrite was significantly lower than that in macrophages isolated from vehicle-treated control rats. LPS-induced expression of both COX-2 and iNOS was significantly lower in macrophages from ethanol-treated rats than that in macrophages from the control rats, while expression of beta-actin was not different in these groups. LPS increased phosphorylation of both extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The levels of phosphorylated ERK and p38 were significantly lower in macrophages from ethanol-treated rats compared with those from the control rats. Treatment of macrophages with ethanol (100 - 400 mM) in vitro significantly inhibited expression of COX-2 in a concentration-dependent manner, while only a high concentration (400 mM) of ethanol significantly inhibited expression of iNOS. Ethanol also inhibited COX-2 expression in the presence of Tiron. Expression of COX-2 and iNOS was significantly inhibited by U0126 but not by SB203580. CONCLUSION: In rat alveolar macrophages, LPS-induced expression of COX-2 and iNOS is mediated by ERK MAPK but not by p38 MAPK. Acute ethanol administration to rats attenuates induction of both COX-2 and iNOS in alveolar macrophages by inhibiting phosphorylation of ERK.

Interactions of alcohol dehydrogenase to p-hydroxyacetophenone-sepharose and p-acetamidophenol-sepharose.

BACKGROUND: p-Hydroxyacetophenone (HAP)-sepharose is known to be an effective ligand for isolation of aldehyde dehydrogenase and chloramphenicol acetyltransferase. In this study, we investigated ligand specificities to alcohol dehydrogenase (ADH) using p-HAP-sepharose and p-acetamidophenol (AAP)-sepharose. METHODS: p-HAP and p-AAP were coupled to epoxy-activated sepharose and used as ligands for affinity chromatography to detect binding proteins. Fractions of affinity chromatography were collected and separated by SDS-PAGE. Commassie brilliant blue staining was carried out for detecting proteins. For determining protein sequences, polypeptide was separated by HPLC after cleavage of Lys-specific protease digestion. RESULTS: p-HAP ligands have the ability to bind to seven proteins, including class I ADH extracted from a rat cytosolic fraction as well as HLADH (horse liver ADH). p-HAP, p-AAP and benzaldehyde (10 mM each) could elute HLADH from the complex of HLADH-pHAP-sepharose column. On the other hand, p-AAP-sepharose has no ability to bind to HLADH, although three proteins from a rat liver cytosolic fraction were found to be able to bind to p-AAP-sepharose. CONCLUSIONS: p-HAP binds to ADH in a structure-specific manner and this binding is nonspecific to species of origin of ADH.

Enhancement of alcohol dehydrogenase activity in vitro by acetylsalicylic acid.

The interaction of acetylsalicylic acid with alcohol dehydrogenase was investigated. Horse liver alcohol dehydrogenase bound to a p-hydroxyacetophenone affinity column was eluted by acetylsalicylic acid. In vitro enzymatic activity of alcohol dehydrogenase in the presence of ethanol as a substrate was significantly increased by incubation with acetylsalicylic acid. These results suggest that acetylsalicylic acid has an affinity with alcohol dehydrogenase and enhances its activity.

Inhibitory action of ethanol on cyclooxygenase-2 expression through suppression of the extracellular signal-related kinase-mediated pathway in rat alveolar macrophages.

The purpose of the present study was to elucidate the effects of ethanol on expression of cyclooxygenase in alveolar macrophages. Rat alveolar macrophages were collected by bronchoalveolar lavage and stimulated by lipopolysaccharide. Lipopolysaccharide-induced cyclooxygenase-2 expression and prostaglandin E2 production were inhibited by ethanol (100-200 mM) in a concentration-dependent manner. Ethanol at 100-200 mM concentration-dependently inhibited cyclooxygenase-2 mRNA expression. Lipopolysaccharide-stimulated phosphorylation of both extracellular signal-related kinase and p38 mitogen-activated protein kinase was also significantly inhibited by ethanol (50-200 mM). Cyclooxygenase-2 expression was significantly inhibited by U0126 but was not affected by SB203580. In the presence of SB203580, lipopolysaccharide-induced cyclooxygenase-2 expression was also inhibited by ethanol (50-200 mM). On the other hand, cyclooxygenase-1 was expressed constitutively in alveolar macrophages and cyclooxygenase-1 expression was affected neither by lipopolysaccharide nor ethanol. Lipopolysaccharide-induced expression of inducible nitric oxide synthase in alveolar macrophages was not affected by ethanol at 50-200 mM. These results suggest that lipopolysaccharide-induced expression of cyclooxygenase-2 is mediated by extracellular signal-related kinase but not by p38 kinase and that ethanol selectively attenuates cyclooxygenase-2 expression mainly by inhibiting activation of extracellular signal-related kinase.

Determination of the liver cytosolic proteins that bind to p-hydroxyacetophenone.

The purpose of the present study was to determine the proteins that bind to acetophenones in the liver. Immobilized p-hydroxyacetophenone (p-HAP) was used as a ligand of affinity chromatography. Analysis using sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that five polypeptides in the liver cytosolic fraction specifically bound to the p-HAP matrix. These polypeptides were digested with Lys-specific protease and used to generate peptide maps by reversed-phase high-performance liquid chromatography. Consequently, identification from a data base of protein sequences revealed that the five polypeptides were glycogen phosphorylase, cytosolic aldehyde dehydrogenase, adenosine kinase, class I alcohol dehydrogenase and glutathione S-transferase A2. In addition to p-HAP, acetylsalicylic acid also displayed a prominent ability to elute these five enzymes from the p-HAP affinity column loaded with the cytosolic fraction of the liver. Thus, p-HAP has affinities to the above liver enzymes and is a useful ligand for analysis of them.

Reduction of lipopolysaccharide-induced cyclooxygenase-2 expression in diabetic arteries.

In order to clarify the effects of diabetes mellitus on induction of cyclooxygenase (COX)-2 and subsequent prostaglandin production in blood vessels, we investigated lipopolysaccharide (LPS)-induced COX-2 induction and prostacyclin production in aortic strips isolated from streptozotocin-induced diabetic rats and vehicle-injected control rats. The rats at 10 weeks after injection of streptozotocin had significantly lower body weights and higher serum glucose levels compared with those in the control rats. LPS stimulation resulted in a marked increase in the release of prostacyclin from the aortic strips. This increase was abolished in the presence of indomethacin or cycloheximide but was not affected by removal of the endothelium. In diabetic aortae, both LPS-induced prostacyclin production and COX-2 induction were diminished compared with the control aortae. No significant difference in COX-1 expression was observed between diabetic and control aortae. The diminution of LPS-induced COX-2 expression was also observed in alveolar macrophages isolated from diabetic rats. These results suggest that COX-2 expression and subsequent prostacyclin production in response to LPS are selectively attenuated in diabetic blood vessels.

Decreased modulation by lipopolysaccharide of aortic smooth muscle contractility in streptozotocin-induced hyperglycemic rats.

Infection is a major complication of patients with diabetes, and endotoxemic shock is a serious complication during sepsis. The purpose of this study was to determine whether the action of bacterial lipopolysaccharide (LPS) on vasocontractility is altered in diabetic vessels. Diabetes was induced in 10-week-old Wistar rats by an intraperitoneal injection of streptozotocin. LPS-induced increase in cGMP (cyclic guanosine 3',5'-monophosphate) level was lower in aortae from streptozotocin-induced hyperglycemic (diabetic) rats than in those from vehicle-injected control rats, while LPS-induced nitric oxide production was not different in the diabetic and control aortae. Phenylephrine-induced contraction of diabetic aortae was lower than that of the control aortae. LPS treatment resulted in depression of contractile response to phenylephrine in both diabetic and control aortae, and the degree of depression was much lower in diabetic aortae. Treatment with N monomethyl l-arginine (l-NMMA) prevented diminution of phenylephrine-induced contraction of the aortae after LPS stimulation, and the degree of the preventive effect by l-NMMA was significantly lower in diabetic aortae than in the control aortae. Protein expression of inducible nitric oxide synthase detected by Western blot analysis was not different in the diabetic and control aortae. The decrease in cGMP production after LPS stimulation in diabetic aortae was not prevented by treatment of the aortae with superoxide dismutase but was partially prevented by that with Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid), a cell-permeable scavenger of reactive oxygen species. These results suggest that LPS-induced depression of vasocontractility is attenuated in diabetic aortae due to a decrease in nitric oxide-stimulated cGMP production, probably resulting from increased inactivation of inducible nitric oxide by excessive intracellular oxidative stress. It is concluded that contractility of aortae from streptozotocin-induced hyperglycemic rats may be less affected by LPS during endotoxemia.

Mechanism of inhibitory action of ethanol on inducible nitric oxide synthesis in macrophages.

Effects of ethanol in vitro on inducible nitric oxide (NO) production in RAW 264.7 macrophages were investigated. Adding ethanol (100-600 mM) to the incubation medium simultaneously with lipopolysaccharide (LPS) concentration-dependently inhibited LPS-induced NO production without being cytotoxic. This inhibitory effect of ethanol on NO production was almost abolished when ethanol was added to the medium 12 h after the start of incubation with LPS, implying that ethanol inhibits the induction of inducible NO synthase (iNOS). Both LPS-induced protein and mRNA expression of iNOS were inhibited by ethanol (100-600 mM) concentration-dependently. LPS-induced activation of signal transducer and activator of transcription-1 (STAT-1) was inhibited by ethanol (100-400 mM). On the other hand, LPS-induced translocation of nuclear factor-kappaB (NF-kappaB) was not affected significantly by 100-600 mM ethanol. When cells were exposed to ethanol for 72 h before LPS stimulation, the inhibitory effect of ethanol on subsequent NO production was significantly attenuated compared with that in control cells pretreated with vehicle for 72 h, suggesting the development of tolerance to the inhibitory action of ethanol. These results suggest that ethanol inhibits inducible NO production, probably by inhibiting STAT1 activation. Tolerance to this inhibitory action of ethanol is produced after chronic exposure.

Involvement of decreased myo-inositol transport in lipopolysaccharide-induced depression of phosphoinositide hydrolysis in vascular smooth muscle.

The mechanism underlying lipopolysaccharide (LPS)-induced depression of phosphoinositide (PI) hydrolysis was investigated using rat aortas. In LPS-pretreated aortas, the 5-hydroxytryptamine-stimulated accumulation of inositol monophosphate and incorporation of exogenous myo-inositol into PIs were significantly less than those in control aortas. Both sodium-myo-inositol cotransporter (SMIT) and phosphatidylinositol transfer protein (PITP) genes were constituently expressed in rat aortas. The mRNA level of SMIT was remarkably lower in LPS-pretreated aortas, while that of PITP mRNA was not affected by LPS. These results suggest that LPS-induced depression of SMIT expression is involved in inhibition of agonist-stimulated PI hydrolysis by LPS.

Diverse effects of ethanol on the pathway of inducible prostaglandin E2 production in macrophages.

The effects of ethanol on inducible prostaglandin production in RAW macrophages were investigated. Indomethacin (1 microM) or cycloheximide (1 microM) abolished prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS, 1 microg/ml). Ethanol at concentrations from 100 mM to 600 mM concentration-dependently inhibited inducible PGE2 production, while ethanol only at higher concentrations (400 mM or more) showed cytotoxity to the cells. Cyclooxygenase-2 (COX-2) activity, estimated by transformation of exogenous arachidonic acid into PGE2, was not affected by ethanol (100-400 mM). LPS-induced expression of COX-2 mRNA was inhibited by ethanol (50-400 mM). On the other hand, protein expression of COX-2 by LPS was significantly increased by ethanol (100-400 mM). Ethanol alone at concentrations up to 600 mM did not induce expression of COX-2 protein. In a medium containing arachidonic acid (1 microM), ethanol at a low concentration (100 mM) did not significantly affect LPS-induced PGE2 production. These results suggest that ethanol shows diverse effects on the pathway of inducible PGE2 production in macrophages. Finally, ethanol may suppress utilization of arachidonic acid, resulting in reduction of inducible PGE2 production. Further study is needed to elucidate the mechanism of dissociation of ethanol effects on protein and mRNA expression.