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Biomedical subjects

S Yamano

Publications and source records attributed to S Yamano.

At least 109 records · Page 6Linked to original sources
PubMed1989

cDNA cloning and sequence and cDNA-directed expression of human P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression of the IIB mRNAs in human liver.

A cDNA designated hIIB1, representing the entire coding sequence of a P450 in the IIB gene family, was isolated from a human liver lambda gt11 library by using the rat IIB1 cDNA as a probe. The hIIB1 protein, deduced from the cDNA sequence, contained 491 amino acids, had a calculated molecular weight of 56,286, and displayed 76% amino acid similarity with the rat IIB1 protein. Expression of this cDNA, using the vaccinia virus system, yielded a P450 that had a reduced CO-binding spectrum with an absorption maximum of 452 nm. The expressed human enzyme was able to catalyze the deethylation of 7-ethoxy-coumarin. Total RNA from 13 livers was probed for levels of hIIB mRNA. Two livers had high levels, four contained moderate levels, and eight contained very low, or no detectable, mRNA. These data suggest either that defective hIIB1 genes exist in humans or that the hIIB1 gene is regulated and variably induced in our liver specimens. To search for mutant mRNA transcripts, libraries were constructed from livers expressing low levels of hIIB1 mRNA. A cDNA, designated hIIB2, was isolated that was identical with the hIIB1 cDNA except for the presence of an unusual alteration of the DNA near its 5' end corresponding to the putative exon 4. This alteration was caused by a deletion of 29 bp and an insertion of 44 bp of nonhomologous DNA. This sequence replacement occurs at the junction of the third and fourth exons as predicted from the structure of the rat IIB1 gene, suggesting that a faulty splice might have given rise to the variant hIIb2 transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Cytochrome P-450 hPCN3, a novel cytochrome P-450 IIIA gene product that is differentially expressed in adult human liver. cDNA and deduced amino acid sequence and distinct specificities of cDNA-expressed hPCN1 and hPCN3 for the metabolism of steroid hormones and cyclosporine.

Immunoblotting analysis of human liver microsome preparations revealed that human cytochrome P-450 PCN1 (hPCN1, Mr approximately 52,000) was expressed in each of 40 individual specimens examined. In about 10-20% of the livers, an immunologically related protein having a lower electrophoretic mobility (Mr approximately 52,500) was also detected. A single liver was found that expressed only the lower mobility protein, designated hPCN3, and RNA isolated from this liver was used to construct a lambda gt11 library. The library was screened with an hPCN1 cDNA probe resulting in the isolation of a unique full-length cDNA that was sequenced and shown to encode hPCN3. The deduced amino acid sequence of this cDNA contained 502 residues, a calculated molecular mass of 57,115 daltons, and displayed 84% similarity with hPCN1. The deduced amino-terminal sequence of hPCN3 was identical to that of HFLa, a major cytochrome P-450 expressed in human fetal liver that is immunologically cross-reactive with several family III cytochrome P-450s. hPCN1 and hPCN3 cDNAs were expressed in Hep G2 cells using a vaccinia virus expression system and shown to encode active enzymes, both characterized by reduced CO-binding spectra with lambda max at 450 nm. Enzymatic analysis revealed that both cytochrome P-450s were similarly active in catalyzing oxidation of the calcium channel blocking drug nifedipine. Both enzymes also catalyzed 6 beta-hydroxylation of the steroid hormones testosterone, progesterone, and androstenedione, although hPCN1 exhibited several-fold higher expressed activity than hPCN3. Several minor oxidation products of these steroids (e.g. 15 beta-hydroxytestosterone), comprising up to approximately 20% of the total metabolites, were formed by hPCN1 but not hPCN3, indicating that hPCN3 is a more highly regiospecific monooxygenase catalyst with steroid substrates. Clear differences were also detected in their catalytic activities toward the immunosuppressive drug cyclosporine, with two hydroxylated metabolites (M1 and M17) and one demethylated metabolite (M21) formed by hPCN1 but only one metabolite (M1) formed by hPCN3. These studies establish that hPCN3 is a newly described cytochrome P-450 that is differentially expressed in the adult human population and that has overlapping substrate specificity compared to hPCN1 for metabolism of steroid and drug substrates.

Amino Acid Sequence↗
PubMed1989

S-100 protein-immunoreactive cells in the bovine ovary.

The present study deals with an immunohistochemical localization of S-100 protein in the bovine ovary. Immunoreactivity for S-100 was observed in various types of cells, as well as in cells of the nervous system. The endothelial cells of arterial vessels, blood capillaries, and lymph vessels; the epithelial cells of ovarian cysts; and the oocytes of normal and atretic follicles showed an S-100 protein positivity. The immunoreactivity also was found in the epithelial cells of the rete ovarii. No cells other than these showed immunoreactivity to the anti-S-100 serum. S-100 protein can be a useful marker for providing information on ovarian function.

Animals↗
PubMed1989

Human NADPH-P450 oxidoreductase: complementary DNA cloning, sequence and vaccinia virus-mediated expression and localization of the CYPOR gene to chromosome 7.

The cDNA containing the full coding sequence of human NADPH-P450 oxidoreductase was isolated and completely sequenced. The cDNA contained 2398 base pairs, including 9 and 358 base pairs of 5' and 3' noncoding sequences, respectively. The human NADPH-P450 oxidoreductase protein deduced from the cDNA has 677 amino acids, with a calculated molecular weight of 76,656. The cDNA nucleotide and deduced amino acid sequences displayed 83 and 92% similarities, respectively, with those of the rat NADPH-P450 oxidoreductase. By use of somatic cell hybrids, the NADPH-P450 oxidoreductase gene was regionally localized to human chromosome 7 (7p15-q35). The levels of NADPH-P450 oxidoreductase protein and mRNA were analyzed in 13 human liver specimens and less than 3-fold variation was found among the different livers. The NADPH-P450 oxidoreductase cDNA was inserted into vaccinia virus and expressed in cell culture. The cDNA-expressed enzyme was active in reducing the electron acceptor cytochrome c. In addition, the NADPH-P450 oxidoreductase stimulated the enzymatic activity of vaccinia virus-expressed human P3(450) when both recombinant viruses were used to coinfect human cells in culture. An approximate equal mole level of NADPH-P450 oxidoreductase and P3(450) was required to achieve maximal activity for both ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase.

Animals↗
PubMed1989

Small-square (SS) net exists in trout muscle.

The small-square-net form (with the large-square-net form) was clearly observed in brown trout muscle in cross-square-net-section. Fresh adductor mandibularis and dorsal muscle strips, isometrically clamped to prevent contraction, were prefixed in 3% glutaraldehyde, 0.1 M cacodylate, pH 7.4, and post-fixed in 1% osmium tetroxide in veronal acetate buffer at 2 degrees C. The samples were dehydrated, embedded in epon-araldite, cross-sectioned, and stained with uranyl acetate and lead citrate for electron microscopic observation. The sections provided the first clear evidence of the small-square-net form, coexisting with the basket-weave pattern, in fish muscle. In previous experiments, we could not demonstrate the small-square-net pattern convincingly with guppy tissue (Yamaguchi et al., 1985a) which may be more susceptible to chemical or physical stress than is trout tissue. The existence of the diverse structural patterns in cross-sections of some, if not all, vertebrate striated muscle must be derived from a basic unit which although it may vary in size among species is structurally identical. The findings agreed with our unified model of narrow and wide Z-line structure (Yamaguchi et al., 1980; Yamaguchi et al., 1985a; Yamaguchi et al., 1985b).

Animals↗
PubMed1988

Polarity and length of actin filaments at the fascia adherens of the cardiac intercalated disk.

Digestion of canine and bovine intercalated disks with a calcium-activated protease (CAF) removes the electron-dense material similar to that found at the Z-line and presumably consisting primarily of alpha-actinin. The major filaments exposed by CAF are actin, and the polarity is away from the intercalated disk, as was confirmed by decoration with heavy meromyosin. The length of actin filaments associated with the fascia adherens region at the concave region is 1.2- to 2.2-fold that of actin filaments (I-filaments) in the sarcomere and varies depending on the interdigitation of the membrane at the cell junction. Actin filaments at the intercalated disk seem to be attached (or very close) to the membrane in a direct, rather than looping, manner.

Actins↗
PubMed1988

[Observation of the acrosome reaction of human spermatozoa using the combined preparation with triple stain technique and scanning electron microscopy].

The combination of triple stain technique (TST) and scanning electron microscopy (SEM) was used to investigate the reliability of TST in observing the acrosome reaction. The TST preparation was followed by SEM to observe the same spermatozoa by both methods. We compared the results obtained with TST at various pH of rose Bengal solution to those with SEM. 1. In our method, the acrosome reaction on the same spermatozoa was easily examined by both TST and SEM. 2. The fine surface structure of the sperm head was not affected by the TST procedure, because the percentages of acrosome non-reacted (ANR) and acrosome reacted (AR) sperm were similar when spermatozoa from the same semen were examined by SEM or SEM after TST (23.0%, 77.0% VS. 24.5%, 75.5%, respectively). 3. When results obtained with TST at various pH of rose Bengal solution were compared to those of SEM after TST, the accuracy was 70.5% at pH 4.3, 95.9% at pH 5.3 and 61.8% at pH 6.3. Sensitivity and specificity were 100%, 21.4% at pH 4.3, 94.5%, 97.6% at pH 5.3 and 38.7%, 97.5% at pH 6.3 respectively. From these results, it can be concluded that TST with pH 5.3 rose Bengal solution is a reliable and practical method for detecting the acrosome reaction of human spermatozoa.

Acrosome↗
PubMed1987

Mutant regulatory subunit of 3',5'-cAMP-dependent protein kinase of yeast Saccharomyces cerevisiae.

Four mutants with amino acid substitution(s) at or near the putative phosphorylation site (Arg142 Arg143 Thr144 Ser145) of the regulatory subunit of cAMP-dependent protein kinase were obtained by site-directed mutagenesis. Three mutants, BCY1A1a145 (Ser145 to Ala), BCY1His143 (Arg143 to His) and BCY1Asn144, Ala145 (Thr144 to Asn and Ser145 to Ala) complemented a bcy1 mutant, whereas BCY1Gly143 (Arg143 to Gly) did not. In addition, mutant, BCY1Asn144, Ala145 exhibited a dominant cold-sensitive phenotype, which can be most easily explained by the functional alteration of the regulatory subunit of cAMP-dependent protein kinase by the mutations. Analyses of these mutant genes revealed that phosphorylation of the regulatory subunit is not a prerequisite for the regulation of the cAMP-dependent protein kinase activity in responding to the cAMP level.

Cyclic AMP↗
PubMed1986

Guinea-pig liver morphine 6-dehydrogenase as a naloxone reductase.

Elution profiles of guinea-pig liver naloxone reductase and morphine 6-dehydrogenase on Matrex green A, Sephadex G-100 and DEAE-cellulose (DE32) column chromatography used sequentially in the purification procedure were identical. The ratios of the two enzyme activities were almost constant throughout all the purification steps. The two enzymes were similarly more stable at pH 6.0 than at pH 8.0 on storage at 4 degrees. The reversible inactivation of the two enzymes by the removal of 2-mercaptoethanol from the enzyme solution was the same. Inhibitory effects of lithocholic acid, CuSO4, quercitrin, phenylarsine oxide, and prostaglandin E1 on the two enzymes were almost the same. These results indicated that naloxone reductase is identical to morphine 6-dehydrogenase in the guinea-pig liver. For the reduction of naloxone, the enzyme utilized either NADPH or NADH as cofactor, and pH optima were 6.8 with NADPH and 6.2 with NADH. The Km values for NADPH and NADH were 6.5 and 2.2 microM respectively. The Vmax values for naloxone were 1.2 units/mg protein with NADPH and 0.5 unit/mg protein with NADH. The Km values for naloxone were 0.27 mM with NADPH and 0.44 mM with NADH. The reaction product formed by the enzyme was identified as 6 alpha-naloxol by thin-layer and gas-liquid chromatographic analyses. Accordingly, it is clear that the enzyme catalyzes the stereospecific reduction of naloxone to form the 6 alpha-hydroxyl congener.

Alcohol Oxidoreductases↗
PubMed1985

Blocking of human fertilization in vitro by sera with sperm-immobilizing antibodies.

Serum samples with sperm-immobilizing antibody activity from six women were examined for ability to block sperm-egg interaction by a zona penetration test where human follicular ova matured in vitro were used. Exposure of spermatozoa from a fertile healthy donor to the sera impaired binding to and penetration through the zona pellucida of the spermatozoa completely in five cases and incompletely in one case. Successful fertilization in vitro was achieved by using fetal cord serum instead of autoserum of the patient included in the in vitro fertilization and embryo transfer program. These results suggest that interference with sperm-egg interaction may be an additional mechanism of infertility that is caused by antisperm antibodies.

Adult↗
PubMed1985

Purification and characterization of guinea pig liver morphine 6-dehydrogenase.

Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, has been purified about 440-fold from the soluble fraction of guinea pig liver with a yield of 38%. The purified enzyme was a homogeneous protein on polyacrylamide gel disc electrophoresis and isoelectric focusing. The molecular weight and isoelectric point of the enzyme were 29,000 and 7.6, respectively. The enzyme utilizes both NAD and NADP as a cofactor, and the Km values were 0.12 mM for NAD and 0.42 mM for NADP. The Vmax values for morphine were 588 milliunits/mg of protein (with NAD) and 1600 milliunits/mg of protein (with NADP). The Km values for morphine were 0.12 mM (with NAD) and 0.49 mM (with NADP). The enzyme also exhibited activity for morphine-related compounds: nalorphine, normorphine, codeine, and ethylmorphine; however, 7,8-saturated congeners such as dihydromorphine and dihydrocodeine were poor substrates. The enzyme was inactivated by removal of 2-mercaptoethanol from the enzyme solution. The inactivated enzyme was rapidly recovered by the addition of 2-mercaptoethanol. Phenylarsine oxide and CdCl2 (dithiol modifiers) inhibited competitively toward cofactor binding and noncompetitively toward morphine binding. These results suggest that the enzyme possesses the essential thiol groups, probably vicinal dithiol, at or near the cofactor-binding site. Using the partially purified enzyme, 8-(2-hydroxyethylthio)dihydromorphinone was isolated as the product and identified by UV, mass, and NMR spectra. It was confirmed that morphinone proposed as the dehydrogenation product was nonenzymatically and covalently bound to 2-mercaptoethanol. Accordingly, the isolated morphinone-2-mercaptoethanol conjugate must be formed by two steps: enzymatic production of morphinone from morphine and then nonenzymatic binding of 2-mercaptoethanol to morphinone.

Alcohol Oxidoreductases↗
PubMed1985

Production of monoclonal antibodies to porcine zona pellucida and their inhibition of sperm penetration through human zona pellucida in vitro.

Two hybridoma cell lines producing murine monoclonal antibodies to antigens common to the zona pellucida (ZP) of pigs and humans were obtained by immunization of mice with solubilized porcine zona antigen. Indirect immunofluorescence tests showed that both these monoclonal antibodies stained the entire layer of porcine ZP but stained different regions of human ZP, one staining the entire layer and the other only the outer surface. At high concentrations, these two monoclonal antibodies directed against antigens common to porcine and human ZP prevented sperm binding and penetration into human ZP in vitro, whereas a monoclonal antibody directed against an antigen restricted to porcine ZP did not have these inhibitory effects. It is concluded that human and porcine ZP share at least two antigens with different locations in the ZP, and that these influence or are essential for interaction of human sperm with the ZP. These results provide a rationale for using porcine ZP clinically as a vaccine for human immunocontraception.

Animals↗
PubMed1985

[Correlation between serum prolactin secretion patterns and the ability of fertilization and cleavage of human oocyte (effects of transient hyperprolactinemia on fertilization and cleavage of human oocyte)].

To assess the effect of serum prolactin (PRL) on the fertilization and cleavage of oocytes, its secretion patterns in normoprolactinemic women (28 cases, 35 cycles) who had undergone IVF-ET because of bilateral tubal occlusion and/or severe oligospermia, and the relationship between its levels and the fertilization and cleavage rate were studied. Three serum PRL secretion patterns were observed. In the TIP-O (Transient Increase in serum PRL in the Ovulatory phase) group (7 cycles), the serum PRL was significantly increased in the ovulatory phase compared with the follicular phase. In the TIP-OL (TIP at the Ovulatory and mid-Luteal phase) group (11 cycle), it was significantly increased in both the ovulatory and the mid-Luteal phases, but in the NON-TIP group (17 cycles) there was no significant increase in any phase. The fertilization rate of the TIP-OL group (60.0%) was lower than that of the NON-TIP Group (89.5%) and the TIP-O group (87.5%). The cleavage rate of the TIP-O group (50.0%) was rather lower, and that of TIP-OL group (30.0%) was significantly (p less than 0.05) lower than that of the NON-TIP group (78.9%). These results suggest that transient increase in serum PRL may suppress the ability of fertilization and cleavage of human oocytes.

Adult↗
PubMed1984

Measurement of urinary thiamine propyl disulfide metabolites as an index of liver function.

Thiamine propyl disulfide (TPD) was orally administered in patients with liver disease to measure the main metabolite, 2-hydroxypropyl methyl sulfone (2HPMS), in urine, for the test of liver function. The amount of urinary excretion of 2HPMS decreased in proportion to the degree of severity of liver disease, with intimate correlation with various tests reflecting hepatic reserve function (p less than 0.01). Phenobarbital (PB), one of the inducers of hepatic microsomal drug metabolizing enzymes scarcely influenced the results of this test. In patients with ordinary liver disease without remarkable disturbance of intestinal absorption and renal excretory function, this method appears to be clinically applicable.

Acute Kidney Injury↗