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Biomedical subjects

I Ohkubo

Publications and source records attributed to I Ohkubo.

At least 37 records · Page 2Linked to original sources
PubMed1998

Purification and characterization of the active-site-mutated recombinant human mu-calpain expressed in baculovirus-infected insect cells.

Recombinant human mu-calpain whose active site Cys-115 was substituted with Ser was expressed in insect cells using baculovirus system. The mutant mu-calpain, purified using an affinity-column of calpastatin oligopeptides, had no proteolytic activities of autolysis and caseinolysis. The large subunit of the mutant mu-calpain was processed from the 80 kDa form to the 76 kDa form by the wild type calpain, supporting the intermolecular cleavage mechanism of procalpain during activation. Fluorescence polarization analysis revealed that the mutant mu-calpain retained high affinity toward fluorescein-labeled calpastatin domain 1. Fragmentation of the full-length calpastatin by the wild type calpain was enhanced by pre-incubating the inhibitor with the mutant calpain. The recombinant mutant calpain was suggested to retain the integrity of the high ordered structure of the wild type calpain.

Animals↗
PubMed1998

Opposing effects of low and high molecular weight kininogens on cell adhesion.

High molecular weight kininogen (HK) blocks cell spreading but not cell attachment to surfaces coated with vitronectin and other ligands of beta3 integrins. We sought to learn the structural basis of this phenomenon. Monoclonal antibodies against the histidine-rich D5 domain in the light chain of 2-chain HK abolished the inhibitory effect of 2-chain HK on spreading of MG-63 osteosarcoma cells on vitronectin-coated tissue-culture plastic. The antibodies were effective only if incubated with 2-chain HK in solution and did not abolish the anti-cell-spreading effect of 2-chain HK that was pre-adsorbed to tissue-culture plastic. Exposure of an epitope in the histidine-rich domain was less when HK was adsorbed to tissue-culture plastic (oxidized polystyrene) than when it was adsorbed to ELISA plastic (untreated polystyrene). Loss of the epitope correlated with increased anti-cell-spreading activity of HK on tissue-culture plastic. The light chain of 2-chain HK containing D5 and that containing recombinant D5 both had anti-cell-spreading activity, but only when present in solution during adhesion assays. Pre-adsorption of recombinant D5 to tissue-culture plastic resulted in a surface on which adsorbed 2-chain HK had little anti-cell-spreading activity. Binding study revealed that HKa bound to immobilized vitronectin. The histidine-rich D5 domain of light chain of HK was identified as one of the binding sites of vitronectin, suggesting that the masking of the RGD cell-binding site of immobilized vitronectin is the molecular mechanism of anti-cell-spreading effect of HKa. In contrast, low molecular weight kininogen (LK), which lacks D5, augmented cell spreading on vitronectin-coated tissue-culture plastic. Thus, HK and LK have opposing effects on VN-dependent cell adhesion. The augmenting effect of LK was greater if LK was preincubated with cells or adsorbed to the surface at pH>7.0. Analysis of fragments of LK and antibody inhibition studies localized the cell-adhesion activity to the D3 domain that is common to LK and HK. These findings indicate that the D5 domain mediates the adsorption of HK or 2-chain HK to vitronectin substratum in anti-adhesive conformations, i.e., masking of the RGD cell-binding site of vitronectin. Such conformers inhibit cell spreading on vitronectin even though a cell-adhesion site is present in D3.

Antibodies, Monoclonal↗
PubMed1998

Isolation and characterization of an alanyl aminopeptidase from rat liver cytosol as a puromycin-sensitive enkephalin-degrading aminopeptidase.

Alanyl aminopeptidase (AAP-S) was purified to homogeneity from rat liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 100,000 on Sephacryl S-200 HR and to be 90,000 on SDS-PAGE in the presence of beta-mercaptoethanol. These findings suggested that the enzyme exists as a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Tyr- and Met-MCAs, and moderately hydrolyzed Arg-, Lys-, Leu-, Phe- and Lys-Ala-MCAs at pHs ranging from 7.5to 8.0. The enzyme also hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order for k(cat)/Km values of AAP-S at the optimal pH (pH 7.5) was Lys->Met->Arg->Ala->Leu->Phe->Tyr->Lys-Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, PCMBS, Zn2+, Cd2+, Co2+, Cu2+ and Hg2+, and puromycin. The amino acid sequence of the first 43 residues of the enzyme was determined as Pro1-Glu-Lys-Arg-Pro5-Phe-Glu-Arg-Leu-Pro10-Thr-Glu-Val-Ser-Pro 15-Ile-Asn-Tyr-Ser-Leu20-(Cys)-Leu-Lys-Pro-Asp25-Leu-Leu- Asp-Phe-Thr30-Phe-Glu-Gly-Lys-Leu35-Glu-Ala-Ala-Ala-Gln40 -Val-Arg-Gln-. This N-terminal amino acid sequence is almost identical with those of puromycin-sensitive enkephalin-degrading aminopeptidases in rat and human brains, and the mouse neuroblastoma cell line Neuro2A. These findings suggest that the AAP-S from rat liver cytosol is a puromycin-sensitive aminopeptidase. Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytosol of the rat liver cells.

Amino Acid Sequence↗
PubMed1998

Estrogen receptor gene polymorphism and generalized osteoarthritis.

OBJECTIVE: Generalized osteoarthritis (OA) is a common disease, more often affecting women than men. Although genetic factors are known to be the major risk factor, the genes related to its development are as yet undetermined. We investigated the association between estrogen receptor gene polymorphisms and generalized OA. METHODS: Pvu II and Xba I restriction fragment length polymorphisms (RFLP) of estrogen receptor gene were analyzed in 65 Japanese patients with generalized OA and in 318 healthy control women. The RFLP were represented as Pp(Pvu II) and Xx(Xba I), with capital and small letters signifying the absence and the presence of restriction sites, respectively. RESULTS: Estrogen receptor genotype PpXx, with the combination of the Pvu II and Xba I RFLP, is a significant risk factor for the disease subset (odds ratio 1.86, 95% confidence interval 1.03-3.24; p = 0.039). The association of this genotype was more obvious in patients with severe radiographic changes and in younger patients. CONCLUSION: Some variant of the estrogen receptor gene is a genetic marker for generalized OA. How this genotype affects development of the disease remains to be elucidated.

Age Factors↗
PubMed1997

Alanyl aminopeptidase from human seminal plasma: purification, characterization, and immunohistochemical localization in the male genital tract.

Alanyl aminopeptidase (AAP) was purified to homogeneity from human seminal plasma. The calculated molecular weight of the purified enzyme was approximately 137,000+/-5,000 from light scattering, 140,000 (main) and 137,000 (minor) from non-denatured PAGE and 153,000 from SDS-PAGE in the absence or presence of 2-mercaptoethanol (2-ME). These findings suggest that the enzyme is monomeric in form in human seminal plasma. The enzyme hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order of Kcat/Km values of AAP at optimal pH (pH 7.5) was Ala- > Lys-Ala- > or = Met- > Leu- > Phe- > Arg- > or = Arg-Arg- > Lys- > Gly-MCAs. AAP was potently inhibited by bestatin, leuhistin, actinonin, amastatin, and 1,10-phenanthroline. These findings suggest that AAP is an aminopeptidase. We determined that the amino acid sequence of the first 22 residues of the enzyme was Ser1-Thr-Thr-Pro-Ser5-Ala-Ser-Ala-Thr-Thr10-Asn-Pro-Al a-Ser-Ala15-Thr-Thr-Leu-Asp-Gln20-Ser-Lys-. This sequence was completely coincident with that downstream of the transmembrane site of human intestinal alanyl aminopeptidase N (CD13). We also isolated cDNA encoding AAP from human prostate cDNA library, sequenced its structure, and confirmed human seminal plasma AAP to be identical with alanyl aminopeptidase N. We postulated that native human seminal plasma alanyl aminopeptidase is released into the seminal plasma after the specific site is cleaved by elastase or an elastase-like enzyme. The enzyme level in human seminal plasma determined by single radial immunodiffusion was 5.2+/-3.2 mg/100 ml (mean+/-SD, n=20) in individuals 20-47 years of age. AAP was immunohistochemically stained in the luminal site-cell membrane of epithelial cells in the prostatic gland and ductuli efferentes of the testis.

Adult↗
PubMed1996

Dipeptidyl peptidase II from porcine seminal plasma: purification, characterization, and its homology to granzymes, cytotoxic cell proteinases (CCP 1-4).

Dipeptidyl peptidase II (DPP II) was purified to homogeneity from porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approx. 185,000 and 200,000 on Superdex 200 column chromatography and non-denatured PAGE, respectively, and to be 58,000 and 61,000 on SDS-PAGE in the absence and presence of beta-mercaptoethanol (beta-ME), respectively. These findings suggested that the enzyme is composed of three identical subunits. The enzyme rapidly hydrolyzed the substrates Lys-Ala-MCA and Gly-Pro-MCA at acidic pH. The Km and V(max) values of DPP II at optimal pH (pH 6.0) were 1330 microM and 2.9 mumol/mg per min for Gly-Pro-MCA, and 360 microM and 1.43 mumol/mg per min for Lys-Ala-MCA, respectively. It was strongly inhibited by diisopropylphosphofluoride (DFP), and moderately by 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). These findings suggest that DPP II is a serine peptidase. Furthermore, the enzyme activity was also strongly inhibited by copper ions. The amino-acid sequence of the first 41 residues of the enzyme was determined as Ala1-Ser-Pro-Pro-Glu-Pro-Gly-Phe-Arg- Glu10-Val-Tyr-Phe-Glu-Gln-Leu-Leu-Asp-His-Phe20-Asn-Phe-Glu- Arg-Phe- Gly-Lys-Lys-Thr-Phe30-Arg-Gln-Arg-Phe-Leu-Val-Ser-Asp-Lys-Phe40 -Trp. This sequence showed homology (11.6-30.2%) to the N-terminal amino-acid sequences of cytotoxic cell proteinases (CCP 1-4), granzymes. Other properties of DPP II including pH optimum, pH stability, and heat stability were characterized.

Amino Acid Sequence↗
PubMed1996

FISH localization of human cytoplasmic actin genes ACTB to 7p22 and ACTG1 to 17q25 and characterization of related pseudogenes.

Human beta- and gamma-cytoplasmic actin genes (ACTB and ACTG1) were mapped to chromosomes 7p22 and 17q25, respectively, by fluorescence in situ hybridization (FISH). Four processed pseudogenes, beta-actin-related ACTBP9 and gamma-actin-related ACTGP1, ACTGP3, and ACTGP9, were isolated from human libraries. By PCR of somatic cell hybrid DNAs, ACTBP9 and two beta-actin-related pseudogenes (ACTBP7 and ACTBP8) were mapped to human chromosomes 18, 15, and 6, respectively. The gamma-actin-related pseudogenes were mapped by FISH to chromosomes 3q23 (ACTGP1), 20p13 (ACTGP3), and 6p21.1 (ACTGP9).

Actins↗
PubMed1995

Chromosomal mapping of the human smooth muscle actin gene (enteric type, ACTA3) to 2p13.1 and molecular nature of the hindIII polymorphism.

The human gene for smooth muscle actin (enteric type, ACTA3) has been isolated, and three overlapping clones, lambda HACTSG-17, -2, and -112, were used as probes for fluorescence in situ hybridization of human chromosomes. The gene was localized to chromosome 2p13.1. To clarify the molecular nature of the HindIII RFLP present in the first intron of the gene, the 1105-bp EcoRI-BamHI fragment contained in lambda HACTSG-17 was sequenced. PCR with primers designed from the determined sequence yielded either the 463- or the 439-bp product or both, using human DNA as template. The 463-bp product was cleavable with HindIII, but the 439-bp product was not. Comparison of their nucleotide sequences revealed that they differ in the presence/absence of a 24-bp sequence harboring a HindIII restriction site. Therefore, analysis of PCR products by size has been shown to be sufficient to detect the RFLP. The allelic frequency on 156 chromosomes was determined by PCR to be 45 (439 bp, corresponding to the formerly designated A1 allele):55 (463 bp, A2 allele) in the Japanese population.

Actins↗
PubMed1995

Human seminal plasma beta-microseminoprotein: its purification, characterization, and immunohistochemical localization.

beta-Microseminoprotein was very efficiently purified from human seminal plasma with only three steps including DEAE-Sephacel and Zinc-chelate Sepharose CL-6B column chromatography. The purified protein was a non-glycoprotein with a molecular weight (M(r)) of 19,000 and 17,000 on gel filtration and reduced SDS-PAGE, respectively. The protein gave six bands from M(r) 15,600 to 25,500 on non-reduced SDS-PAGE. The characterization including the molecular weight, amino acid sequence of N-terminus and concentrations in various body fluids is discussed. Furthermore, the immunohistochemical localization of the protein among various human tissues is demonstrated.

Amino Acid Sequence↗
PubMed1995

Human gene for beta-microseminoprotein: its promoter structure and chromosomal localization.

The gene for human beta-microseminoprotein (MSP, beta-inhibin, prostatic secretory protein94, or PSP94) was isolated. The nucleotide sequence of its upstream region (2,860 bp), exon 1 (35 bp), and a part of the following intron (218 bp) was determined. Transient transfection analysis on MSP-expressing (PC-3) and -nonexpressing (HepG2) cells using luciferase reporter plasmids suggested that the region from -2738 to -276 is not essential for the basal promoter activity, and that the regions from -275 to -207 and from -186 to -128 function in a cell-specific manner. The chromosome locus of the gene (MSMB) was determined through application of PCR to the DNAs of rodent-human somatic cell hybrids and also by the fluorescence in situ hybridization technique to be region q11.2 of chromosome 10.

Animals↗
PubMed1995

Anticardiolipin antibodies in patients with pregnancy loss induce factor Xa production in the presence of beta 2-glycoprotein I.

PROBLEM: Anticardiolipin antibodies (aCL) are commonly associated with recurrent pregnancy loss, though the mechanism is uncertain. Some investigators have indicated that aCL may be directed at a complex made up of cardiolipin and a blood anticoagulant, beta 2-glycoprotein I (beta 2GPI). We therefore investigated the effects of beta 2GPI-dependent aCL IgG enriched fractions, isolated from sera of patients with pregnancy losses, on blood coagulation. METHOD: beta 2GPI-dependent aCL were prepared from sera of three women with second trimester pregnancy losses, by cardiolipin affinity column chromography, following by anti-beta 2GPI affinity column chromatography. The effects of beta 2GPI and beta 2GPI-dependent aCL on the activation of factor X in vitro were examined. RESULTS: beta 2GPI inhibited the activation of factor X and beta 2GPI-dependent aCL blocked this inhibitory effect in a dose dependent manner. CONCLUSION: These results imply the possibility of beta 2GPI-dependent aCL induce hypercoagulation or thrombus by blocking the inhibitory effect of beta 2GPI on activation of factor X, which may result in pregnancy loss.

Abortion, Habitual↗
PubMed1994

Zn-alpha 2-glycoprotein is a novel adhesive protein.

An amino acid sequence (Arg-Gly-Asp-Val) specifically associating with cell adhesion between cells and extracellular matrices was found on the human Zn-alpha 2-glycoprotein (Zn alpha 2gp) molecule. Although other mammalian cell lines such as breast carcinoma and melanoma did not, SMKT R-3 cells (human renal cell carcinoma) but not the kidney cell lines (Vero and COS7) preferentially attached and spread on a tissue culture plate coated with either blood plasma Zn alpha 2gp or seminal plasma Zn alpha 2gp. The spreading of SMKT R-3 cells on Zn alpha 2gp required divalent cations such as Mn2+ and Mg2+, and this spreading was inhibited by synthetic peptides such as RGDS, LRGDV and ELRGDV. These findings suggested that the RGDV region mainly interacted with the cell surface integrins to regulate cell attachment and spreading.

Amino Acid Sequence↗
PubMed1994

Structure and expression of rat and mouse mRNAs for Zn-alpha 2-glycoprotein.

Rat and mouse cDNAs for Zn-alpha 2-glycoprotein (Zn alpha 2gp) were isolated from liver libraries (lambda gt11) and compared with the human one. The lengths of cDNA inserts analyzed were 1,233 and 1,273 nucleotides for rat and mouse, respectively. The deduced amino acid sequences suggested that rat and mouse Zn alpha 2gp proteins consist of 279 and 290 amino acid residues in the mature form, respectively. They have 59.4% (rat) and 58.6% (mouse) identities in amino acid sequence with the human counterpart, and between rat and mouse the identity is 88.5%. Among the three domains, domain B is best conserved; the identities are 74.7, 73.6, and 95.6% between human and rat, human and mouse, and rat and mouse, respectively. Four cysteine and eight tryptophan residues are all conserved, and two of the three asparagine residues that carry a glycan in the human protein are conserved. Analysis of rat tissues by Northern blot suggested that its mRNA is expressed in liver, and, to a much lesser extent in submandibular gland, lung, kidney, and stomach. A more detailed study by in situ hybridization demonstrated that some epithelial cells of renal tubules and the isthmus and the neck zone cells of gastric fundic glands express Zn alpha 2gp mRNA.

Animals↗
PubMed1994

Dipeptidyl peptidase IV from porcine seminal plasma: purification, characterization, and N-terminal amino acid sequence.

Dipeptidyl peptidase IV (DPP IV) was purified to homogeneity from porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approximately 290,000 on PAGE in the absence of sodium dodecyl sulfate (SDS) and 310,000 on Sephacryl S-300 HR column chromatography, and to be 115,000 and 105,000 on SDS-PAGE in the absence and presence of beta-mercaptoethanol. The enzyme is suggested to be composed of three identical subunits. The enzyme rapidly hydrolyzed the substrate Gly-Pro-MCA, and weakly the substrate Lys-Ala-MCA. It was strongly inhibited by diisopropylphosphofluoridate (DFP), and moderately by both phenylmethyl-sulfonyl fluoride (PMSF) and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). It was also strongly inhibited by zinc ion. The amino acid sequence of the first 18 residues of the enzyme was Asn-Lys-Gly-Thr-Asp-Asp-Ala-Ala-Ala-Asp-Ser-Arg-Arg- Thr-Tyr-Thr-Leu-Thr-. This sequence was highly homologous to the sequences in the rear of the transmembrane site of human and rat liver DPP IVs and mouse thymus DPP IV. The native DPP IV is suggested to be released into the seminal plasma after the cleavage of the hydrophobic N-terminal domain by chymotrypsin-like or pepsin-like enzymes. Other properties of DPP IV including kinetic parameters, pH stability and heat stability were characterized.

Amino Acid Sequence↗