Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.     

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana, was studied. [24-14C]-24-Dehydro-26-deoxy-5 beta-ranol (3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one) was chemically synthesized from [24-14C]cholic acid and incubated with bullfrog liver homogenate fortified with NADPH. 24-Dehydro-26-deoxy-5 beta-ranol was shown to be converted into both 26-deoxy-5 beta-ranol and 24-epi-26-deoxy-5 beta-ranol [(24S)- and (24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrols] in addition to 5 beta-ranol [(24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol], which is the major bile alcohol of the bullfrog. [24-3H]-26-Deoxy-5 beta-ranol and [24-3H]-24-epi-26-deoxy-5 beta-ranol were prepared from 24-dehydro-26-deoxy-5 beta-ranol by reduction with sodium [3H] borohydride and administered respectively to two each of four bullfrogs by intraperitoneal injection. After 24 h, labeled 5 beta-ranol was isolated from the bile of the bullfrogs that received [24-3H]-26-deoxy-5 beta-ranol. In contrast little if any radioactivity could be detected in 5 beta-ranol or its 24-epimer after administration of [24-3H]-24-epi-26-deoxy-5 beta-ranol.     

Identification of new bile alcohols, 5 beta-cholestane-3 alpha,7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha,7 alpha,26,27-tetrol in human gallbladder bile

The nature of cholestanetetrols present as the glucurono-conjugates in human gallbladder bile was studied. Glucurono-conjugated bile alcohols were isolated by ion exchange chromatography and, after enzymatic hydrolysis, were fractionated by reversed phase partition chromatography to give a fraction containing tetrahydroxy bile alcohols which was analyzed by gas-liquid chromatography and mass spectrometry. Along with the three previously identified bile alcohols, 5 alpha- and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,24-tetrols, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,26-tetrol, three new cholestanetetrols, possessing two hydroxyl groups in the ring system and two in the side chain, were detected in the tetrahydroxy bile alcohol fraction. These new bile alcohols were identified as 5 beta-cholestane-3 alpha, 7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha, 7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha,26,27-tetrol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of authentic standards prepared from chenodeoxycholic acid by partial synthesis.     

Synthesis of four diastereoisomers at carbons 24 and 25 of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid, intermediates of bile acid biosynthesis

The synthesis of four stereoisomers at C-24 and C-25 of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid is described. Pyridium chlorochromate oxidation of 3 alpha,7 alpha,12 alpha-triacetoxy-5 beta-cholan-24-ol (II) prepared from cholic acid (I) afforded 3 alpha,7 alpha,12 alpha-triacetoxy-5 beta-cholan-24-al (III) which was converted to a mixture of the four stereoisomers (IV-VII) by a Reformatsky reaction with ethyl DL-alpha-bromopropionate followed by alkaline hydrolysis. Separation of these isomers (IV-VII) was achieved by silica gel column chromatography, and subsequent reversed-phase partition column chromatography. The configurations at C-24 were elucidated by conversion of each isomer into (24R)- or (24S)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol (XII or XI) by Kolbe electric coupling, the C-24 configurations of which were determined by modified Horeau's method and 13C-nuclear magnetic resonance spectroscopy. The stereochemistries at C-25 were deduced by comparison of IV-VII with the products of the hydroboration followed by oxidation with alkaline hydrogen peroxide of (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid (XIII).     

Identification of bile alcohols in rat bile

Bile alcohols in rat bile were analyzed by gas-liquid chromatography-mass spectrometry. Six bile alcohols were newly identified as minor constituents in addition to 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, major bile alcohol of rat bile. The bile alcohols newly identified were 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, and 5 beta-cholestane-3 alpha,6 beta,7 beta,25,26-pentol. The biliary bile alcohols of the rat occurred mainly as the sulfuric acid esters and, in lesser amounts, as glucuronoconjugated and unconjugated forms. The amount of total bile alcohols was about 27.9 nmol in 1 ml of bile.     

Isolation and identification of bile salts conjugated with cysteinolic acid from bile of the red seabream, Pagrosomus major.

Bile salts present in gallbladder of wild and cultured red seabream, Pagrosomus major, a marine teleost were analyzed. The bile from wild red seabream was found to contain two previously unknown bile salts along with two known bile salts, taurocholate and taurochenodeoxycholate. Isolation of each bile salt was performed by column chromatography. Fast atom bombardment mass spectra of the unknown bile salts showed the molecular ions (M-H)- of m/z 544 and 528 which are shifted 30 mass units upfield compared to those (m/z 514 and 498) of taurocholate and taurochendeoxycholate, respectively; this is consistent with the presence of cysteinolic acid (mol wt 155) instead of taurine (mol wt 125). Enzymatic hydrolysis of the bile salts released cholic acid and chenodeoxycholic acid, respectively, and an amino acid that was identified as D-cysteinolic acid by direct comparison with an authentic sample. From these results, the bile salts in the bile of wild red seabream were identified as the conjugates of cholic acid and chenodeoxycholic acid with cysteinolic acid. 1H- and 13C-magnetic resonance spectra of the bile salts were also consistent with the proposed structure. The cysteinolic acid conjugates were found only in wild and not in cultured red seabream; this distinction seems to result from differences in dietary cysteinolic acid.     

Absolute configuration at carbon 23 of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol excreted by patients with cerebrotendinous xanthomatosis

Absolute configuration at C-23 of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol, one of the bile alcohols isolated from the patients with cerebrotendinous xanthomatosis, was unequivocally determined as 23S by conversion of a key intermediate, (23S)-5 beta-cholest-25-ene-3 alpha,7 alpha,12 alpha,23-tetrol to either the bile alcohol of known absolute configuration, (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrol, or the naturally occurring 23,25-pentol.     

Injury due to extravasation of thiopental and propofol: Risks/effects of local cooling/warming in rats

Inadvertent leakage of medications with vesicant properties can cause severe necrosis in tissue, which can have devastating long-term consequences. The aim of this study was to evaluate the extent of extravasation injury induced by thiopental and propofol, and the effects of cooling or warming of local tissue on extravasation injury at macroscopic and histopathologic levels. Rats were administered intradermally thiopental (2.5 mg/100 µL) or propofol (1.0 mg/100 µL). Rats were assigned randomly to three groups: control (no treatment), cooling and warming. Local cooling (18–20 °C) or warming (40–42 °C) was applied for 3 h immediately after agent injection. Lesion sizes (erythema, induration, ulceration, necrosis) were monitored after agent injection. Histopathology was evaluated in skin biopsies taken 24 h after agent injection. Thiopental injection induced severe skin injury with necrosis. Peak lesions developed within 24 h and healed gradually 18–27 days after extravasation. Propofol induced inflammation but no ulceration, and lesions healed within 1–2 days. Local cooling reduced thiopental- and propofol-induced extravasation injuries but warming strongly exacerbated the skin lesions (e.g., degeneration, necrosis) induced by extravasation of thiopental and propofol. Thiopental can be classified as a “vesicant” that causes tissue necrosis and propofol can be classified as an “irritant”. Local cooling protects (at least in part) against skin disorders induced by thiopental and propofol, whereas warming is harmful.     

Estrogen regulates hepcidin expression via GPR30-BMP6-dependent signaling in hepatocytes

Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E(2)) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E(2)-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E(2) and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E(2)-induced hepcidin expression. BMP6 expression induced by E(2) was abolished by GPR30 silencing. Finally, both E(2) and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E(2) and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.     

Isolation and characterization of 39 microsatellite loci in the endangered Japanese loach Leptobotia curta

A microsatellite-enriched genomic library was obtained for the endangered Japanese loach Leptobotia curta, and 39 dinucleotide markers were successfully isolated and characterized. These markers had between one and nine alleles, with expected heterozygosity ranging from 0 to 0.839, in a population from the Lake Biwa-Yodo River system of Japan. Linkage equilibrium was observed in most loci, and only one locus showed significant deviation from Hardy-Weinberg equilibrium. These microsatellite markers will be useful for genetic diversity studies of wild and captive L. curta populations.