Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE

Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinesis, when their extracts were treated with E-64.     

Efficient expression of genetically engineered hepatitis B virus surface antigen P31 proteins in yeast

We have constructed plasmids that express modified hepatitis B virus surface antigen (HBsAg) P31-coding genes (M-P31c, d, e, f, and i) having various genetically engineered pre-S2 regions. The plasmids contain the GAPDH (gene coding for glyceraldehyde-3-phosphate dehydrogenase) promoter and the PGK (gene coding for 3-phosphoglycerate kinase) terminator, both isolated from sake brewing yeast, Saccharomyces cerevisiae Kyokai III. Expression levels of the modified HBsAg P31 proteins in yeast are greatly increased from 0.4% to 11.7% of total cell protein. However, the specific mRNAs are expressed at equal levels and the degradation rates of the modified P31 proteins do not vary significantly. Therefore, we considered that different expression levels of the modified P31 proteins are attributed to the changes of the post-translational efficiency. And it was suggested that the conformational stability of the N-terminal peptide (Met-1-Phe-46) in the endoplasmic reticulum membrane determines the expression level of modified P31 proteins.     

Identical properties of aldosterone and corticosterone binders and their presence in rat brain and kidney

The [3H]corticosterone binders from rat brain and kidney were characterized by binding affinity and chromatographies, and compared with the binders for [3H]aldosterone and [3H]triamicinolone acetonide. Corticosterone-binding globulin-like molecules at very high concentrations in crude extracts were completely eliminated by a DEAE-gel adsorption procedure. [3H]Aldosterone binder in the renal, DEAE-treated fraction was recovered in a single peak by gel-filtration chromatography and by ultracentrifugation in linear sucrose gradients, independent of hormone-binding and tungstate, a stabilizer of the binder. The Stokes' radius and sedimentation coefficient of the renal aldosterone binder were 6.6 nm and 9.3S, respectively, indicating an apparent molecular weight of 263,000. Corticosterone-preferring binder also existed in the DEAE-treated fraction. Both aldosterone and corticosterone binders were found in the brain and kidney preparations. Comparison among the binders showed identical values of Stokes' radius and elution pattern from DEAE-Toyopearl in a linear salt gradient regardless of the organ and the hormones. Scatchard analyses of [3H]aldosterone and [3H]corticosterone binding showed for each ligand only one group of high-affinity sites with the equivalent dissociation constants, 4-7 nM. The orders of steroids in competing for the two high-affinity sites were equivalent: corticosterone greater than or equal to aldosterone much greater than triamcinolone acetonide, and that for the triamcinolone acetonide binding was triamcinolone acetonide much greater than aldosterone greater than or equal to corticosterone. Hydroxyapatite column chromatography separated the aldosterone and corticosterone binders from the triamcinolone acetonide binder, but not the aldosterone binder from the corticosterone binder. It is concluded that aldosterone and corticosterone binders distinct from triamcinolone acetonide binder exist in rat brain and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of selenite on glucocorticoid receptor

Rat liver glucocorticoid receptor was partially purified and characterized for its hormone binding using selenite. Selenite at very low concentrations irreversibly inhibited the hormone binding. The concentration for half maximal inhibition was approximately 2.8 microM. The inhibition was restored by dithiothreitol. The receptor-hormone complex became considerably insensitive to the selenite inhibition. The receptor inhibited by selenite was eluted at the same position as the native receptor from DEAE ion exchange and gel filtration columns. The results suggest that at least four sulfhydryl groups are located in the hormone binding domain of the receptor making a cluster.     

Activation and maturation mechanisms of boar acrosin zymogen based on the deduced primary structure

We have isolated cDNA clones encoding boar acrosin, a serine protease participating in the initial stage of fertilization, from boar testis lambda gt11 cDNA libraries. Nucleotide sequencing of the overlapping clones indicates that the composite cDNA inserts contain 1,391 base pairs coding for a 5'-untranslated region, an open reading frame, a stop codon, a 3'-untranslated region, and a poly(A)+ tail. A polyadenylation signal, AATAAA, is located 33 bases upstream from the start of the poly(A)+ tail. The amino acid sequence deduced from the cDNAs shows that boar acrosin is initially synthesized as a prepro-protein with a 16-residue signal peptide at the NH2 terminus. This signal sequence is followed by a 399-residue sequence corresponding to the acrosin zymogen. COOH-terminal sequence analysis of boar sperm 55-kDa proacrosin and its processed forms indicates that the mature acrosin molecule contains 322 amino acid residues in two polypeptide chains, a 23-residue light chain and a 299-residue heavy chain, with a combined molecular mass of 35,735 Da, and that the 55-kDa proacrosin molecule has 14-, 18-, and 43-residue segments as COOH-terminal extensions that are removed during proacrosin maturation. The COOH-terminal 43-residue segment is rich in proline residues, including an unusual repeat of 23 consecutive prolines. The deduced amino acid sequence of boar acrosin shows a high degree of identity with major portions of other serine proteases, including the active site region and the location of cysteine residues. We conclude that boar acrosin is synthesized as a single-chain polypeptide with the regions corresponding to the light and heavy chains covalently connected by two disulfide bonds, and that the single-chain molecule is autoactivated by cleavage of the Arg23-Val24 bond after removal of the COOH-terminal 14-residue segment, resulting in the formation of the light and heavy chains. This two-chain molecule is then converted to the mature enzyme by removal of the COOH-terminal 18- and 43-residue segments.     

Multiple-dosing effects of benzo[a]pyrene in the mouse bone marrow micronucleus test

Multiple-dosing effects of benzo[a]pyrene (B[a]P) in the micronucleus test were studied using CD-1 male mice. Mice were treated orally once, twice or 3 times with 250, 500, 1000 or 2000 mg/kg, at 24-h intervals. Bone marrow cells were sampled 24 h after the last administration. The present study indicated that the incidence of polychromatic erythrocytes with micronuclei significantly increased more in the group of animals that received B[a]P twice than in those receiving it one or 3 times. The dose of 500 mg/kg B[a]P yielded the greatest response of any dose regimen.     

Effects of the Histamine H3-Agonist (R)-α-Methylhistamine and the Antagonist Thioperamide on Histamine Metabolism in the Mouse and Rat Brain

To study the feedback control by histamine (HA) H3-receptors on the synthesis and release of HA at nerve endings in the brain, the effects of a potent and selective H3-agonist, (R)-alpha-methylhistamine, and an H3-antagonist, thioperamide, on the pargyline-induced accumulation of tele-methylhistamine (t-MH) in the brain of mice and rats were examined in vivo. (R)-alpha-Methylhistamine dihydrochloride (6.3 mg free base/kg, i.p.) and thioperamide (2 mg/kg, i.p.), respectively, significantly decreased and increased the steady-state t-MH level in the mouse brain, whereas these compounds produced no significant changes in the HA level. When administered to mice immediately after pargyline (65 mg/kg, i.p.), (R)-alpha-methylhistamine (3.2 mg/kg, i.p.) inhibited the pargyline-induced increase in the t-MH level almost completely during the first 2 h after treatment. Thioperamide (2 mg/kg, i.p.) enhanced the pargyline-induced t-MH accumulation by approximately 70% 1 and 2 h after treatment. Lower doses of (R)-alpha-methylhistamine (1.3 mg/kg) and thioperamide (1 mg/kg) induced significant changes in the pargyline-induced t-MH accumulation in the mouse brain. In the rat, (R)-alpha-methylhistamine (3.2 mg/kg, i.p.) and thioperamide (2 mg/kg, i.p.) also affected the pargyline-induced t-MH accumulation in eight brain regions and the effects were especially marked in the cerebral cortex and amygdala. These results indicate that these compounds have potent effects on HA turnover in vivo in the brain.     

Gastrointestinal flora of cotton rats

The gastrointestinal (GI) flora of cotton rats was examined. No lactobacilli were detected in any part of the GI tract. Anaerobes, including Peptococcaceae, Bacteroidaceae, bifidobacteria and eubacteria, were the predominant bacteria in the stomach, small intestine, caecum and faeces. Aerobes and facultative anaerobes, including Enterobacteriaceae and streptococci, were detected at low numbers and very low frequency of occurrence in all parts of the GI tract. Sixty-one isolates of bifidobacteria were recovered from the stomach, small intestine, caecum and faeces of cotton rats. They were identified as Bifidobacterium animalis, B. pseudolongum biovar a and b. The study showed that the GI flora of cotton rats seem to be very different from the GI flora in other rodents.