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Biomedical subjects

K Okuda

Publications and source records attributed to K Okuda.

At least 523 records · Page 29Linked to original sources
PubMed1991

Evaluation of a new human immunodeficiency virus (HIV) antibody detection method based on time-resolved fluoroimmunoassay.

We have developed a time-resolved fluoroimmunoassay for the detection of anti-HIV antibody using purified virus, recombinant gp120 or a synthetic peptide derived from gp41. The results of time-resolved fluoroimmunoassay correlated well with those obtained by enzyme immunoassay and with the results of immunoblotting. This time-resolved fluoroimmunoassay has sufficient sensitivity and specificity for clinical use.

Fluoroimmunoassay↗
PubMed1991

Internalization of the granulocyte-macrophage colony-stimulating factor receptor is not required for induction of protein tyrosine phosphorylation in human myeloid cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts its biologic activities through binding to specific high-affinity cell surface receptors. After binding, the ligand/receptor complex is rapidly internalized in most hematopoietic cells. Using a human factor-dependent cell line, MO7, and normal human neutrophils, we found that the internalization is exquisitely temperature-dependent, such that ligand/receptor internalization does not detectably occur at 4 degrees C. Activation of the GM-CSF receptor has previously been shown to stimulate a number of postreceptor signal transduction pathways, including activation of a tyrosine kinase and activation of the serine/threonine kinase, Raf-1. The GM-CSF-stimulated increase in tyrosine kinase activity occurs rapidly at both 4 degrees C and 37 degrees C, and therefore is likely to be independent of receptor internalization. At 37 degrees C, the protein tyrosine phosphorylation was transient in MO7 cells, with maximum phosphorylation observed after 5 to 15 minutes, followed by a rapid decline. At 4 degrees C, the protein tyrosine phosphorylation of the same substrates was greater than at 37 degrees C, and no decline in substrate phosphorylation was observed for at least 90 minutes. In contrast to tyrosine phosphorylation, the activation and hyper-phosphorylation of Raf-1 observed at 37 degrees C in both MO7 cells and neutrophils was markedly diminished at 4 degrees C. These results indicate that at least one postreceptor signal transduction mechanism, activation of a tyrosine kinase, does not require ligand/receptor internalization, and indicate that receptor internalization may be a consequence, rather than the initiator, of signal transduction.

Bone Marrow Cells↗
PubMed1991

Molecular cloning and sequence analysis of cDNA encoding delta 4-3-ketosteroid 5 beta-reductase of rat liver.

A cDNA clone encoding delta 4-3-ketosteroid 5 beta-reductase was isolated from rat liver cDNA libraries using antibodies specific for the enzyme and oligonucleotides as probes. The cDNA contained 981-base pair open reading frame encoding 327 amino acid residues (Mr 37,376) and an unusually long 3'-untranslated region rich in AT sequence in the total length of 3189 base pairs. The predicted amino acid sequence contains the sequences similar to the putative NADPH- and steroid-binding regions.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a cytochrome P-450 from rat kidney mitochondria that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3.

A cytochrome P-450 that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3 (P-450cc24: P-450cholecalciferol24) was purified to electrophoretic homogeneity from the kidney mitochondria of female rats treated with vitamin D3 (Ohyama, Y., Hayashi, S., and Okuda, K. (1989) FEBS Lett. 255, 405-408). The molecular weight was 53,000, and its absorption spectrum showed peaks characteristic of cytochrome P-450. The turnover number was 22 min-1 and the specific content was 2.8 nmol/mg protein. The N-terminal amino acid sequence, Arg-Ala-Pro-Lys-Glu-Val-Pro-Leu-, is different from the N-terminal sequence of any other cytochrome P-450s so far reported. Upon reconstitution with the electron-transferring system of the adrenal mitochondria, the enzyme showed a high activity in hydroxylating 25-hydroxyvitamin D3 as well as 1 alpha,25-dihydroxyvitamin D3 at position 24. However, the purified enzyme hydroxylated neither vitamin D3 nor 1 alpha-hydroxyvitamin D3. The enzyme was also inactive toward xenobiotics. The enzyme hydroxylated 25-hydroxyvitamin D3 at position 24 but not at 1 alpha, indicating that the enzyme is distinct from that catalyzing 1 alpha-hydroxylation. The reaction followed Michaelis-Menten kinetics, and the Km value for 25-hydroxyvitamin D3 was 2.8 microM. Both vitamin D3 and 1 alpha-hydroxyvitamin D3 inhibited the 24-hydroxylation of 25-hydroxyvitamin D3 in a competitive, concentration-dependent manner. 25-Hydroxyvitamin D3 24-hydroxylase activity was significantly inhibited by 7,8-benzoflavone, ketoconazole, and CO, whereas it was only slightly inhibited by aminoglutethimide, metyrapone, and SKF-525A. Mouse antibodies raised against the cytochrome P-450 inhibited the reaction about 70% and reacted with the P-450cc24 in immunoblotting but did not react with other kinds of cytochrome P-450 in rat liver microsomes and mitochondria.

Animals↗
PubMed1991

Structural analysis of the gene encoding rat cholesterol alpha-hydroxylase, the key enzyme for bile acid biosynthesis.

The gene encoding cholesterol 7 alpha-hydroxylase (P450VIIA) was isolated from rat genomic DNA. The gene spanned about 11 kilobases and contained six exons. Blotting analysis of genomic DNA and complete matching of restriction maps of several isolated genomic clones indicated that there appeared to be only one gene in the rat genome. The putative transcription initiation site was present 61 base pairs upstream from the ATG codon. The typical TATA sequence and CCAAT promoter element were found at 24 and 47 base pairs upstream from the transcription initiation site, respectively. Alignment of several P450 proteins showed that the cholesterol 7 alpha-hydroxylase gene shared location of introns with none of the other P450 genes except for intron 5, which was in the same position as intron 10 of the gene encoding P450IVA1. The alignment also indicated that the distal helix of cholesterol 7 alpha-hydroxylase contained an asparagine in place of the well conserved threonine that is postulated to be involved in the O2 binding site. Unusual residues, Asn-126 and Thr-442, were also found at the sites where all other P450s have positively charged amino acids, which are considered to be involved in interaction with heme propionate. These replacements may be related to the unique function and unusual lability of the hydroxylase. Analysis of evolutionary distance between the cholesterol 7 alpha-hydroxylase gene and other known P450 genes indicated that yeast P450LIA is most closely related to P450VIIA. This finding suggests that the cholesterol 7 alpha-hydroxylase gene is an evolutionarily old P450 gene.

Amino Acid Sequence↗
PubMed1991

Expression of rat liver vitamin D3 25-hydroxylase cDNA in Saccharomyces cerevisiae.

The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D3 25-hydroxylase, cytochrome P450LMT25, was expressed under the control of the yeast alcohol dehydrogenase I promoter and terminator in Saccharomyces cerevisiae AH22 cells. The transformed yeast cells produced a P450LMT25 protein with an almost similar apparent molecular weight as compared with that of the authentic mature enzyme. The expression level of the P450LMT25 hemoprotein was about 5 x 10(4) molecules per cell as determined by reduced CO-difference spectra. The mitochondrial fraction prepared from the transformed yeast cells exhibited both 25-hydroxylase activity toward 1 alpha-hydroxyvitamin D3 and 27-hydroxylase activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in a reconstituted system containing bovine adrenodoxin and NADPH-adrenodoxin reductase.

Alcohol Dehydrogenase↗
PubMed1991

Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase.

A cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase (P450cc24) was isolated from a rat kidney cDNA library using specific antibodies to the enzyme. The isolated cDNA was 3.2 kbp long and contained a 1542-bp open reading frame encoding 514 amino acids. The deduced amino acid sequence contained a presequence typical of mitochondrial enzymes in the N-terminal region. The amino acid sequence shows less than 30% similarity to those of any other cytochrome P450s so far reported and, therefore, P450cc24 constitutes a novel family of P450. COS-7 cells transfected with the cDNA produced a protein that was reactive with the antibodies and catalyzed NADPH-dependent 24-hydroxylation of 25-hydroxyvitamin D3 in the presence of adrenodoxin and NADPH-adrenodoxin reductase. Using the cDNA as a probe we demonstrated that the increase of 24-hydroxylation activity caused by administration of vitamin D3 into rats was accompanied by an increase of the mRNA.

Amino Acid Sequence↗
PubMed1991

Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-1 in a human factor-dependent myeloid cell line.

The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor-dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.

Cell Line↗
PubMed1991

In vitro penetration of zona pellucida of salt-stored bovine oocytes before and after maturation by frozen-thawed spermatozoa.

Bovine oocytes, before and after maturation in culture, were stored in PBS with 2 M-(NH(4))(2)SO(4) + 0.1% dextran or 2 M-(NH(4))(2)SO(4) + 40 mM-Hepes + 0.5% dextran and were inseminated with frozen-thawed spermatozoa in BO medium with caffeine (5 mM) and heparin (10 mug/ml). The penetration rates of mature oocytes were very low (19 to 24%) and not significantly different between the two salt solutions in which the oocytes were stored for 2 to 89 days. Significantly lower (P < 0.01) penetration rates were observed in immature (7 to 8%) than in mature (20 to 21%) oocytes stored in the two solutions. The synergistic effect of caffeine and heparin was observed in the penetration rate of fresh mature oocytes but not in the stored oocytes, indicating the difficulty of assessing sperm capacitation and/or acrosome reaction of salt-stored mature bovine oocytes under the present condition. Using 0.1% protease the solubility of the zonae decreased in salt-stored but not in fresh oocytes, but there was no significant difference between the immature and mature oocytes regardless of storage in the salt solutions. It appears from these results that some alteration was induced in the nature of zona glycoprotein by ammonium sulfate solution.

Journal Article↗
PubMed1991

Synthesis and examination of antigenicity of four hormonogenic sites and two non-hormonogenic sites of human thyroglobulin.

Four peptides (HTg-1, 1-10; HTg-2, 2547-2558; HTg-4, 2592-2603; HTg-6, 2737-2748) which contain hormonogenic acceptor tyrosine (Tyr) residues and two control peptides (HTg-3, 2582-2591; HTg-5, 2687-2694) of human thyroglobulin (Tg) were synthesized, radioiodinated and their binding with serial anti-human Tg antisera which had been raised in two rabbits (TG-1, TG-2) tested. Although increased binding of each of the six peptides was observed, HTg-4 and HTg-2 had higher binding whereas HTg-1 and HTg-6 showed lower binding with the immune gamma globulin from both rabbits. Each of the six peptides was iodinated with inorganic iodine (127I) using the chloramine-T method and the inhibitory activity of each peptide on the interaction between 125I-T4 and anti-Tg antibodies was tested. At the same time, Tg obtained from a normal thyroid tissue (NTg, iodine content 0.38%) and from a Hürthle cell adenoma (CTg, iodine content 0.000%) were also tested for inhibition of 125I-T4 binding. 125I-T4 binding with rabbit anti-Tg antisera was displaced not only by NTg and CTg but also by three out of four hormonogenic peptides. Among the three peptides, HTg-2 had the highest inhibitory activity, inhibiting 125I-T4 binding to the extent of 21.5% (TG-1) and 16.0% (TG-2). Two control peptides (HTg-3, HTg-5) did not inhibit 125I-T4 binding with anti-Tg antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Multi-functional property of rat liver mitochondrial cytochrome P-450.

To solve the problem of whether a common enzyme catalyzes both 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 (a synthetic compound used therapeutically for vitamin D-deficient diseases) in rat liver mitochondria, enzymological and kinetic studies were performed. A cytochrome P-450 was purified from female rat liver mitochondria based on these catalytic activities and it was found that the two enzyme activities accompanied each other at all purification steps. The 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation activity of the final preparation had a turnover number of 36 min-1, and the value of the corresponding 1 alpha-hydroxyvitamin D3 25-hydroxylation activity was 1.4 min-1. When the enzyme was partially denatured by heating at different temperatures, both enzyme activities declined in a parallel fashion. Treatment of the enzyme with N-bromosuccinimide decreased both enzyme activities in a similar manner. 5 beta-Cholestane-3 alpha,7 alpha,12 alpha-triol competitively inhibited 25-hydroxylation of 1 alpha-hydroxy-vitamin D3 and vice versa. From these results it was concluded that 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 1 alpha-hydroxyvitamin D3 25-hydroxylation are catalyzed by a common enzyme in rat liver mitochondria.

Animals↗
PubMed1991

Human T-lymphotropic virus type 1-associated myelopathy and primary sclerosing cholangitis.

We report the first case of human T-lymphotropic type 1-associated myelopathy (HAM) linked with primary sclerosing cholangitis (PSC). The patient, a 44-year-old woman, also suffered from ulcerative colitis and polyarthritis. Two years and nine months after the clinical onset of PSC, the coexistence of HAM was shown by neurological manifestations characterized by slowly progressive symmetrical myelopathy, predominantly involving pyramidal tracts, and positive antibodies to human T-lymphotropic virus type 1 in serum as well as in cerebrospinal fluid.

Adult↗
PubMed1991

Adherence to experimental pellicle of rough-type lipopolysaccharides from subgingival plaque bacteria.

The adherence of lipopolysaccharides (LPSs) from periodontal disease-associated bacteria to saliva-coated hydroxyapatite (S-HA) and serum-coated HA beads was examined by chromogenic Limulus activity (toxicolor test). Phenol-water extracted LPS preparations from Bacteroides intermedius, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Eikenella corrodens, and rough-type LPS from Escherichia coli adhered to S-HA and serum-coated beads and agglutinated human erythrocytes. The adhered LPSs to S-HA and serum-coated HA beads were not removed by vigorous washing with distilled water. LPSs from Bacteroides (Porphyromonas) gingivalis strains and wild-type E. coli did not adhere to S-HA, serum-coated HA beads or show hemagglutinating activity. SDS-PAGE patterns stained with silver stain showed that LPSs adhered to S-HA, and serum-coated HA beads and erythrocytes possessed a distinct fast-migrating band similar to rough-type LPS. B. gingivalis LPSs possessed slow-migrating and repeating ladder bands similar to wild-type LPS.

Bacterial Adhesion↗
PubMed1991

[An epidemiological survey of group A, B, C & G hemolytic streptococci isolated from the Throat of Diabetic Children Streptococcicosis Study Group].

The incidence of group A, B, C & G hemolytic streptococci residing in the throat of children with diabetes was surveyed. The survey was carried out in August, 1982 and August, 1983. Included in the surveys were 136 diabetic children. The survey was also carried out with healthy school children at the same term as the contrast. The detection rate of the streptococci among the diabetic children was significantly higher than the healthy school children. A great portion of streptococci detected from these diabetic children was classified into group B, on the other hand, from the healthy school children, group A. Although from healthy school children, BIII, BIa and several other types were isolated, from diabetic children only BIa and BIII were isolated. The detection rate of the streptococci among the diabetic children was significantly higher in the early days in camp for improvement of diabetes than in the last day. In the latter case the detection rate was similar to that among healthy children. In the other words following antidiabetic therapy as diabetes improved the detection rate were also improved. Among these 136 diabetic children examined 31 gave positive cultures at both surveys each year. Serogroups or serotypes in each year examinations, isolates, were identical in 26 cases. Most of these groups were group B and the types were Ia and III.

Diabetes Mellitus↗
PubMed1991

[A study on hemolytic streptococci (group A, B, C and G) isolated from throat of the middle-aged and advanced-aged--especially as compared with elementary school children].

Hemolytic streptococci were isolated from throat of middle-aged and advanced-aged and these organisms were classified into groups A, B, C and G. 1) Persons 15 to 39 years old were included in one group and persons from 40 years of age upward were divided into 5 groups every 10 years. Comparison of these groups were done. Generally, group B organisms were most often isolated. The detection rate of group B organisms was higher in the older age group. The detection rates of group A and G organisms were approximately equal, but both organisms were isolated from a few persons. No organism of group C was isolated from males, while only two strains belonged to group C organism were isolated from females. 2) A number of strains of group A hemolytic streptococci were isolated from school children, particularly in the lower classes. But the proportion of group B organism to isolated streptococci showed an increase in upper classes, noticeably in females. 3) For middle-aged and advanced-aged, blood samples were obtained simultaneously on examination of the throat, and ASO value and ASK titer were determined. ASO values were higher in persons infected with groups A, C or G organisms than in persons infected with group B organism or no hemolytic streptococcus. ASK titers revealed a similar results to ASO values, though the relation between ASK titers and isolated hemolytic streptococci was less positive.

Adult↗
PubMed1991

Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.

Amino Acid Sequence↗