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Biomedical subjects

H Okada

Publications and source records attributed to H Okada.

At least 739 records · Page 41Linked to original sources
PubMed1991

Somatic mutation in constant regions of mouse lambda 1 light chains.

To study the distribution of somatic mutation, we determined nucleotide sequences of rearranged lambda 1-chain genomic DNA from four hybridomas obtained from C57BL/6 mice that had been immunized with (4-hydroxy-3-nitrophenyl)acetyl-conjugated chicken gamma globulin. In total, 114 nucleotide substitutions were observed, with neither insertion nor deletion. Sixty-one mutations occurred in the variable-joining region genes (V lambda 1-J lambda 1) and 49 in joining-constant (J lambda 1-C lambda 1) introns. Although frequency decreased with distance from the V lambda 1-J lambda 1 coding region, somatic mutations occurred in the entire J lambda 1-C lambda 1 intron and even in the C lambda 1 region. We found four nucleotide substitutions in C lambda 1 genes, all of which were replacement mutations. Therefore, the mechanism responsible for somatic mutation is operative into the C lambda 1 exons. Nucleotide sequences of rearranged but inactive lambda 2-chain genes from two hybridomas were also examined and compared with those of lambda 1-chain genes. The clustering of replacement mutations in complementarity-determining regions in the inactive lambda 2-chain genes similar to the active lambda 1-chain genes suggested a mechanism that induces somatic mutation preferentially in this region even in the absence of antigenic selection.

Animals↗
PubMed1991

Preparation and kinetic properties of 5-ethylphenazine-glucose-dehydrogenase-NAD+ conjugate, a semisynthetic glucose oxidase.

5-Ethylphenazine-glucose-dehydrogenase-NAD+ conjugate (EP(+)-GlcDH-NAD+) was prepared by linking both poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to glucose dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.8, and that of the NAD+ moieties was 1.2. This conjugate is a semisynthetic enzyme having glucose oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following values of the kinetic constants: for the system with oxygen, the turnover number per subunit is 0.40 s-1 and Km for oxygen is 1.57 mM; and for the system with MTT, the turnover number is 0.11 s-1 and Km for MTT is 0.072 mM. The catalytic cycle of the semisynthetic oxidase has two catalytic steps: reduction of the NAD+ moiety by the active site of the glucose dehydrogenase moiety and oxidation of the NADH moiety by another catalytic site of the ethylphenazine moiety. The apparent intramolecular rate constants of these steps were estimated, and the values are as follows: 0.39 s-1 for the reductions of the NAD+ moiety, 2.2 s-1 and 0.12 s-1 for the oxidation of the NADH moiety in the systems with oxygen and with MTT, respectively, and 3.2 s-1 and 0.18 s-1 for the reduction of the ethylphenazine moiety in the systems with oxygen and with MTT, respectively. On the bases of these results, the following three rate-acceleration mechanisms of the semisynthetic glucose oxidase are discussed: high effective concentration, intramolecular coupling of successive catalytic reactions, and multiple connection between the two kinds of the catalytic sites.

Binding Sites↗
PubMed1991

[HLA-antigens in thyrotoxic patients with overt diabetes mellitus].

The incidence of thyrotoxicosis accompanied by overt diabetes has been reported to be 2 to 3%. Several workers have suggested the possible role of immunological and inherited factors in the occurrence of thyrotoxic patients with overt diabetes. We investigated, therefore, the clinical characteristics, backgrounds, and HLA antigens in thyrotoxic patients with overt diabetes. In nine thyrotoxic patients with overt diabetes (group DM) (3 men and 6 females, average age of 45.8 +/- 2.9 yr), mean levels of free-triiodothyronine (FT3) and free thyroxine (FT4) were 8.2 +/- 0.8 pg/ml and 4.9 +/- 0.4 ng/dl, respectively. Although these levels were extremely high, they were significantly lower than those levels in forty thyrotoxic patients without overt diabetes (group ND) (8 men and 32 females, average age 35.1 +/- 4.5 yr). Mean levels of both thyrotrophin receptor-antibody (TR-Ab) and thyroid simulating antibody (TS-Ab) in group DM were relatively lower than those in group ND. Mean titers of both antithyroid antibody (TGHA) and antimicrosomal antibody (MCHA) in group DM were also relatively lower than those in group ND, respectively. Regarding the clinical features in thyrotoxic patients with overt diabetes, mean duration of diabetes mellitus was 4.1 +/- 2.5 years with mean levels of fasting plasma glucose (FPG), HbA1c, and serum fructosamine 208.1 +/- 34.0 mg/dl, 10.6 +/- 0.6%, and 3.9 +/- 0.9 mmol/L, respectively. Seven patients in group DM frequently had ketosis or ketoacidosis in their histories, and they had been treated with insulin injection. However, the diabetic complications in group DM were moderate or severe.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Amino acid alterations essential for increasing the catalytic activity of the nylon-oligomer-degradation enzyme of Flavobacterium sp.

The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp. KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e. Gly181----Asp (EII type) and His266----Asn (EII type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold.

Amidohydrolases↗
PubMed1991

Distribution and purification of aspartate racemase in lactic acid bacteria.

The distribution of aspartate racemase (EC 5.1.1.13) in various kinds of bacteria demonstrated that the enzyme occurs in lactic acid bacteria, such as Streptococcus species and Lactobacillus species. The enzyme from Streptococcus thermophilus IAM10064 was more thermostable than that from Streptococcus lactis IAM1198 which contained the enzyme most abundantly among the lactic acid bacteria we examined here. We purified the enzyme about 3400-fold to homogeneity from cell-free extract of S. thermophilus, which is composed of two identical subunits with a molecular weight of 28,000 as a homodimer. The enzyme utilizes specifically aspartate as a substrate, but not alanine and glutamate. Maximal reaction velocity was observed at 37 degrees C and around pH 8.0. The sequence of the NH2-terminal amino acids of the enzyme was determined to be Met-Glu-Asn-Phe-Phe-Ser-Ile-Leu-Gly-XXX-Met-Gly-Thr-Met-Ala-Thr-Glu-Ser- Phe-.

Amino Acid Isomerases↗
PubMed1991

Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus.

The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli.

Amino Acid Isomerases↗
PubMed1991

Preparation and kinetic properties of 5-ethylphenazine-poly(ethylene-glycol)-glutamate-dehydrogenase conjugate. A semisynthetic NADH oxidase.

5-Ethylphenazine-poly(ethylene glycol)-glutamate dehydrogenase conjugate (EP(+)-PEG-GluDH) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine to glutamate dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.7. This conjugate is a semisynthetic enzyme having NADH oxidase activity; the ethylphenazine moiety works as a catalytic group, and the coenzyme-binding site of glutamate dehydrogenase works as a substrate-binding site. The effects of the presence of the substrate-binding site near the catalytic group were studied by using EP(+)-PEG-GluDH. Before the preparation of the conjugate, the reactivity of NADH bound in the coenzyme-binding site toward the ethylphenazine moiety was estimated for glutamate and lactate dehydrogenases. The results show that the NADH molecule bound in the site of glutamate dehydrogenase reacts with EP(+)-PEG at a rate of 43% of that of free NADH, but the NADH molecule bound in lactate dehydrogenase does not react with 1-(3-carboxypropyloxy)-5- ethylphenazine. Therefore, glutamate dehydrogenase was used as the substrate-binding site of the semisynthetic NADH oxidase. The results of the kinetic analysis of the activity of EP(+)-PEG-GluDH show that the apparent turnover number of the active site is 0.38 s-1, which corresponds to the apparent intramolecular rate constant of the oxidation of NADH bound in the active site. The apparent effective concentration of bound NADH for the catalytic group of the ethylphenazine moiety is 0.33 mM. This means that the presence of the substrate-binding site near the catalytic group increases the local NADH concentration by at most 0.33 mM, and this is the rate-accelerating effect of the binding site.

Binding Sites↗
PubMed1991

Antisense sequences of antigenic peptides are found in MHC class II molecules.

The sequence of the well known antigenic peptide OVA 323-339 as well as other peptides containing reiterative motifs of the core region essential for binding to Class II MHC molecules was compared for homology with the DNA derived antisense peptide sequences corresponding to the alpha chain of the mouse IAd molecule. Homology was obtained within the sequence found in the transmembrane region of the molecule. Increased binding of the peptides containing the reiterative motifs based on the OVA 323-339 sequence may be due to the increased number of contact sites on IAd molecules which were identified as being complementary in a sense-antisense manner.

Amino Acid Sequence↗
PubMed1991

Comparative studies of decay-accelerating factor and HLA-DR within the CD8-brightly positive population.

Decay-accelerating factor (DAF) is a membrane protein that inhibits C3 convertase activity of autologous complement at the cell surface. We found that DAF+ cells and DAF- (DAFlow, if any) cells are clearly separated from each other among CD8-brightly positive (CD8bright) cells. Using three-color fluorescence flow cytometry, we found that whereas the CD8bright DAF- population express HLA-DR (class II major histocompatibility complex antigens, and an activated T cell marker), the CD8bright DAF+ population does not. Therefore, among the CD8bright T cells, DAF and HLA-DR are mutually exclusive. In addition, the CD8bright DAF+ population proliferates in the presence of recombinant interleukin 2 (rIL2) while the CD8bright DAF- population does not. After a 4-day cultivation of peripheral blood lymphocytes in the presence of rIL2, expression of HLA-DR increased in the CD8bright DAF- population and expression of IL 2Rp55 (alpha chain, the receptor for IL2, and a marker of T cell activation) occurred in the CD8bright DAF+ population. Furthermore, C3 deposition occurred in the CD8bright HLA-DR+ population which lacks DAF when lymphocytes, that had been cultured for 3 days in the presence of rIL2, were incubated with fresh autologous serum. This result suggests that the absence of DAF on CD8bright HLA-DR+ T cells might play a role in permitting complement reaction on the cells in vivo and may be related to the regulation of T cell activation.

Adult↗
PubMed1991

Immunohistochemical demonstration of keratins 8 and 14 in benign tumours of the skin appendage.

The expression of keratins 8 and 14 was investigated immunohistochemically by the avidin-biotin-peroxidase (ABC) method using formalin-fixed paraffin-embedded specimens from 42 tumours of human skin appendages. Results were compared with the staining of 34 specimens from normal skin and skin appendages adjacent to the tumours. Keratin 14 was detected by the monoclonal antibody (mAb) 312C8-1, and was found in the basal cells of the epidermis, the outer root sheaths of hair follicles, and the peripheral cells of sebaceous glands. It was also detected in the inner and outer layers of cells in the ductal portion and the myoepithelial cells in the secretory portion of apocrine and eccrine sweat glands. Keratin 8 was detected by mAb 35BH11, and was present in the secretory cells of eccrine and apocrine sweat glands but not in myoepithelial or ductal cells. The pilosebaceous apparatus and the epidermis were uniformly negative. In benign skin appendage tumours, the staining patterns for both keratins generally resembled their distribution in the corresponding normal tissues. The demonstration of keratins 8 and 14 may be useful in the recognition, classification and diagnosis of skin appendage tumours.

Antibodies, Monoclonal↗
PubMed1991

Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae.

A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast, Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced in Saccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation in P stipitis were inducible. S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol by S. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

Aldehyde Reductase↗
PubMed1991

Bile evacuation induced by hypothalamic stimulation in dogs.

To investigate the role of the central nervous system in the cephalic phase of gallbladder emptying, the effects of electrical stimulation of the hypothalamus on the gallbladder and sphincter of Oddi and on bile flow rate were studied in anesthetized dogs. Stimulation of the anterior hypothalamic area induced gallbladder contraction and simultaneous relaxation of the sphincter without a significant increase in plasma cholecystokinin concentration. The bile flow rate was facilitated by stimulation of the same area. These responses were obtained even in dogs with cervical cord transection and were abolished by cervical vagotomy. The contraction response in the gallbladder was prevented by atropine, but a slight relaxation response in the sphincter remained. On the other hand, stimulation of the posterior hypothalamic area induced gallbladder relaxation and contraction or relaxation in the sphincter. The bile flow rate was decreased by stimulation of the same area. These responses were still obtained after vagotomy. These results indicate that the anterior hypothalamic area and the vagus nerves play an important role in gallbladder emptying in the cephalic phase.

Animals↗
PubMed1991

Round fingerpad sign: an early sign of scleroderma.

We describe a clinical skin sign in scleroderma termed round fingerpad sign. This term refers to disappearance of the peaked contour on fingerpads and replacement with a hemisphere-like fingertip contour; this change is especially apparent on the ring fingers. A positive round fingerpad sign was found in 72 of 72 ring fingers in 36 patients with progressive systemic sclerosis in 69 of 72 ring fingers in patients with mixed connective tissue disease, and in 24 of 24 ring fingers of patients with Raynaud's phenomenon and sclerodactyly. In contrast, a negative round fingerpad sign was seen in 240 of 240 fingers in normal women (controls). The high sensitivity of this sign is noteworthy. A positive round fingerpad was seen in sign not only typical scleroderma patients but also in groups with less severe skin sclerosis (i.e., in those patients with mixed connective tissue disease or those with Raynaud's phenomenon and sclerodactyly). The sign is a new and useful clinical marker for the early diagnosis of scleroderma.

Adolescent↗
PubMed1991

Immunohistochemical alterations of C cells in sheep treated with vitamin D.

Hyperplasia and hypertrophy of C cells were demonstrated in sheep with hypercalcaemia induced by administration of vitamin D3 (2 million I.U. per day). After treatment with vitamin D3 for 10, 20 or 30 days, serum calcium values increased to 10.28, 11.86 and 10.44 mg per dl, respectively, compared to a normal concentration of around 9 mg per dl. Immunohistochemical reactions of calcitonin, chromogranin A and calcitonin gene-related peptide (CGRP) decreased, whereas intense neurone-specific enolase (NSE) immunoreactivity was noted in C cells. Immunohistochemical staining with anti-calcitonin, anti-chromogranin A, anti-CGRP and anti-NSE antisera was useful to demonstrate the functional state of stimulated C cells in sheep with hypercalcaemia.

Animals↗
PubMed1991

C cell hyperplasia and carcinoma developing in sheep with experimentally-induced lymphosarcoma.

Two cases of C cell hyperplasia and one case of C cell carcinoma of the thyroid glands were bilaterally recognized in 11 sheep with experimentally-induced lymphosarcoma. The serum calcium concentration in the C cell carcinoma case was slightly increased above the normal concentration of around 9 mg per dl. Bilateral C cell hyperplasia also developed in the thyroid lobes of the C cell carcinoma case. Immunohistochemically, hyperplastic C cells and tumour cells were positive for calcitonin, calcitonin gene-related peptide, chromogranin A and neurone-specific enolase. No amyloid deposition nor multiple endocrine neoplasia was demonstrated in any of the cases. Ultrastructurally, many secretory granules were observed in the cytoplasm of neoplastic cells constituting the C cell carcinoma and in the hyperplastic C cells.

Animals↗
PubMed1991

Pharmacokinetics of once-a-month injectable microspheres of leuprolide acetate.

The pharmacokinetic parameters of leuprolide acetate, a potent analogue of LH-RH, were determined in rats and dogs after i.v. and s.c. dosing with leuprolide solution. The effective human dose of once-a-month injectable microspheres of leuprolide was estimated to be about 3.2 to 8.1 mg analogue/month using these parameters. After microsphere injection at three different doses in rat serum leuprolide concentrations were sustained for over 4 weeks, and the AUCs and mean serum levels were linearly correlated with the dose. The serum levels and urinary excretion of the analogue in rats after repeated s.c. injection of the microspheres every 4 weeks exhibited similar profiles after each injection; no changes of the absorption and excretion of the analogue after the repeated injection could be demonstrated. The serum levels of the analogue metabolite (M-I) were 21% of the intact form 3 hr after injection of the microspheres but very low at the steady state after 1 to 4 weeks.

Animals↗
PubMed1991

Sustained pharmacological activities in rats following single and repeated administration of once-a-month injectable microspheres of leuprolide acetate.

Once-a-month injectable microspheres of leuprolide acetate prepared with copoly(DL-lactic/glycolic acid) using an in-water drying method were assessed for duration of the analogue release and pharmacological effects in rats after a single or repeated injection. The periodic challenge test revealed that a single injection of the microspheres caused a dramatic and persistent suppression of the ability of the pituitary-gonadal system to secrete gonadotropin and testosterone for over 5 weeks. The complete recovery of these functions was observed 10 weeks after the injection. The repeated injection of the microspheres at intervals of 2 or 4 weeks achieved persistent suppression of steroidogenesis after an initial transient flare-up and beneficially avoided the "acute-on-chronic response." This depot formulation is expected to assure patient compliance and produce stronger therapeutic effects than the daily solution.

Animals↗