Identification of bile alcohols in human bile

Human gallbladder bile was examined for bile alcohols. Following isolation and hydrolysis, the bile alcohols were analyzed by capillary gas-liquid chromatography-mass spectrometry. The following bile alcohols were identified with certainty by direct comparison with reference standards: 5 beta-cholane-3 alpha,-7 alpha,23,24-tetrol; 5 beta-cholane-3 alpha,7 alpha,12 alpha,24-tetrol; 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol; 27-nor-5 beta-cholest-25-ene-3 alpha,7 alpha,-12 alpha,24-tetrol; 3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one; 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol; 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol; 5 beta-cholestane-3 alpha,7 alpha,24-triol; 5 beta-cholestane-3 alpha,7 alpha,25-triol; 5 beta-cholestane-3 alpha,7 alpha,26-triol; 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol; (24R)- and (24S)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentols; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,-25,26-pentol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26,27-pentol; 26-methoxy-5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol. There also existed two norcholestanetetrols and three cholestanetetrols with two hydroxyl substituents on the nucleus and two in the side chain. The human biliary bile alcohols occurred mainly as sulfate esters and in lesser amounts as glucuronoconjugated and unconjugated forms. The amount of total bile alcohols was about 0.9 mg (0.7-1.2 mg) in 1 g of bile solid, or 0.16 mumol (0.07-0.24 mumol) in 1 ml of gallbladder bile.     

Isolation and characterization of cDNA clones corresponding to the genes expressed preferentially in floral organs of Arabidopsis thaliana

Seventeen cDNA clones of genes corresponding to mRNAs expressed preferentially in floral organs of Arabidopsis thaliana were obtained by differential screening of a flower bud cDNA library, and classified into five groups (1A, 17A, 1B, 4B and 5B) by cross-hybridization and restriction analysis. Sequence analysis revealed that the 1A-1 and 17A-1 clones encode vegetative storage proteins (VSPs). The VSP mRNAs were detected in a small amount in leaves and increased to a limited level by wounding. Both 1B-1 and 5B-1 clones were homologous to transmembrane protein cDNAs. The protein encoded by 4B-1 clone contained a proline-rich region, but no homologous proteins were found in databases.     

Retention of nickel oxide (green) aerosol in rat lungs by long-term inhalation

Wistar male rats were exposed to green nickel oxide [NiO(G)] aerosols (mass median aerodynamic diameter, 0.6 μm) for 7 h/d, 5 d/wk for up to 12 mo. The average exposure concentration was controlled at 0.3 mg/m³ and 1.2 mg/m³ during the exposure period. Some rats were sacrificed just after the 3- and 6-mo exposures, and others were sacrificed after the termination of the 12-mo exposure. There were no differences in body weight gain between NiO(G) exposure groups and controls. The lung weights in the exposed rats were heavier than those in the control ones. Nickel concentrations in lungs of exposure groups were much higher than those of controls. The nickel concentrations in liver, kidney, spleen, and blood slightly increased with the passing of the exposure time. The nickel content in rat lungs during the 12-mo exposure was estimated theoretically. The estimated values agreed with the experimental data. This result shows that the clearance rate increases with the decreasing of the particle diameter.     

Bile salts of the coelacanth, Latimeria chalumnae

Bile salts of the coelacanth, Latimeria chalumnae, Smith, have been analyzed and shown to have three bile alcohols, latimerol, 5 alpha-cyprinol, and 5 alpha-cholestane-3 beta, 7 alpha,-12 alpha,25,26-pentol, two C24 bile acids, chenodeoxycholic acid and cholic acid, one C26 bile acid, probably 3 beta, 7 alpha, 12 alpha-trihydroxy-27-nor-5 alpha-cholestan-26-oic acid, and two C27 bile acids, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid and 3 beta,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid as determined by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry.     

Twisting of the second transmembrane alpha-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states

To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis. Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA). EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively. When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel. Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix. In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier. These residues correspond to those modified with EMA in the yeast carrier in the c-state. Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation. On the basis of the results, the roles of TM2 in the transport function of AAC were discussed.     

Detection of hydrolytic activity of trypsin with a fluorescence-chymotryptic peptide on a TLC plate

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.     

Complex formation of CD23/surface immunoglobulin and CD23/CD81/MHC class II on an EBV-transformed human B cell line and inferable role of tetraspanin

CD23, a low-affinity IgE receptor, is a type II transmembrane protein having a C-type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co-exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L-KT9 cells (an Epstein-Barr virus (EBV)-transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti-CD23 and anti-Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross-linking of CD23 with anti-CD23 and its second antibody as compared with the case of stimulation by anti-CD23 alone. The cross-linking of CD23 with anti-CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L-KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.     

Anti-lipid deposition effect of HMG-CoA reductase inhibitor, pitavastatin, in a rat model of hypertension and hypercholesterolemia

Since the rat is an atherosclerosis-resistant species, the study of atherosclerosis using rats is limited. The present study was undertaken to develop an atherosclerotic model in rats, to investigate the effect of nitric oxide (NO) inactivation and hyperlipidemia, and to evaluate the effect of pitavastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitor, on NO inactivation and on hyperlipidemia-induced changes in the cardiovascular system. Four-month-old male spontaneously hypertensive hyperlipidemic rats (SHHR) and Sprague-Dawley (SD) rats were used to study 1) the effect of the period of treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/L) on high fat diet (HFD)-treated SHHR and SD rats, and 2) the effect of pitavastatin (Pit, 0.3 mg/kg/day) on the changes in the aorta of L-NAME- and HFD-treated SHHR and SD rats. L-NAME administration for 1 month then HFD feeding for 2 months markedly increased the deposition of lipids and the thickness of the endothelium in SHHR. Continuous L-NAME treatment with HFD produced severe injury and stripped of endothelium in both strains. The plasma total cholesterol of L-NAME + HFD-treated and L-NAME + HFD + Pit-treated SHHR was significantly higher than that of control SHHR. Lipid deposition, however, was comparatively less in the aorta of L-NAME + HFD + Pit-treated SHHR. The concentration of cholesterol in the aorta of control SHHR was significantly lower than that in the aorta of L-NAME + HFD-treated SHHR, whereas that of L-NAME + HFD + Pit-treated SHHR was the same as that in control SHHR. These data indicated that Pit blocked lipid deposition in the aorta of L-NAME + HFD treated SHHR without changing plasma lipid profiles. In conclusion, NO inactivation and HFD induce lipid deposition in the endothelium, and the HMG-CoA reductase inhibitor blocks the deposition in SHHR.