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I Ohkubo

Publications and source records attributed to I Ohkubo.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of vitronectin-mediated haptotaxis and haptoinvasion of MG-63 cells by domain 5 (D5(H)) of human high-molecular-weight kininogen and identification of a minimal amino acid sequence.

We found that human kinin-free high-molecular-weight kininogen (kf-HK) significantly inhibited vitronectin-mediated migration (haptotaxis) and invasive potentiation (haptoinvasion) of osteosarcoma (MG-63) cells but that HK, LK, the common heavy chain of HK and LK, and the light chain (D6(H)) of HK had no inhibitory effect. Recombinant GST-D5(H) (histidine-rich region of HK) obtained from Escherichia coli. (BL21) also inhibited both haptotaxis and haptoinvasion to about 30% of the control level in a dose-dependent manner. These findings suggest that a specific region of D5(H) is responsible for the inhibition of cell haptotaxis and haptoinvasion. Among the seven synthetic peptides covering D5(H), peptide H(479)KHGHGHGKHKNKGK(493) (P-5) inhibited both haptotaxis and haptoinvasion in a dose-dependent manner, suggesting that P-5 could possibly be utilized to prevent primary and secondary metastases of tumor cells.

Amino Acid Sequence↗
PubMed2001

Analysis of MUC4 mucin expression in lung carcinoma cells and its immunogenicity.

BACKGROUND: MUC4 has been cloned from tracheobronchial mucosa cDNA and reportedly is highly expressed in some human malignancies, including lung carcinoma. However, little is known about molecular and biologic characteristics. The authors analyzed expression levels of MUC4 mRNA and protein in lung carcinoma cells and analyzed the immunogenicity of this mucin. METHODS: Nine cultured lung carcinoma cell lines and 29 tumor samples from patients with lung carcinoma were examined by Northern hybridization for MUC4 mRNA expression and by flow cytometry or an immunohistochemical staining for its protein expression. Sera from the patients were examined for their reactivity with MUC4 by enzyme-linked immunosorbent assay. RESULTS: Forty-four percent of the cell lines and 72% of the tumor samples showed high levels of MUC4 mRNA expression. Although MUC4 protein was not detected in any live carcinoma cell lines by flow cytometry using rabbit antisera reactive with the MUC4 core, pretreatment with paraformaldehyde and sialidase resulted in successful detection of the protein in 50% of the cell lines. An immunohistochemical study revealed that 67% of the tumors exhibited MUC4 protein expression without any digestion. In 29% of the patients, high levels of anti-MUC4 immunoglobulin M or immunoglobulin G were detected. CONCLUSIONS: MUC4 protein expression was elevated in lung carcinoma tissues because of the increase in its mRNA expression and deglycosylation on its core. This mucin is sufficiently immunogenic to elicit humoral and cellular immunity specific for MUC4 in patients with malignant disease. MUC4 is expected to be useful as a target antigen in immunotherapy for patients with carcinoma of the lung.

Biomarkers, Tumor↗
PubMed2001

Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase.

We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney >> testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.

Amino Acid Sequence↗
PubMed2001

Rat tripeptidyl peptidase I: molecular cloning, functional expression, tissue localization and enzymatic characterization.

We purified tripeptidyl peptidase I (TPP I) to homogeneity from a rat kidney lysosomal fraction and determined its physicochemical properties, including its molecular weight, substrate specificity and partial amino acid sequence. The molecular weight of the enzyme was calculated to be 280,000 and 290,000 by non-denaturing PAGE and gel filtration, respectively, and to be 43 000 and 46 000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of six identical subunits. The Km, Vmax, kcat and kcat/Km values of TPP I at optimal pH (pH 4.0) were 680 microM, 3.7 micromol x mg(-1) x min(-1), 33.1 s(-1) and 4.87 x 10(4) s(-1) x M(-1) for Ala-Ala-Phe-MCA, respectively. TPP I was significantly inhibited by PCMBS and HgCl2, and moderately by DFP. These findings also suggest that TPP I is an exotype serine peptidase that is regulated by SH reagent. TPP I released the tripeptide Arg-Val-Tyr from angiotensin III more rapidly than from Ala-Ala-Phe-MCA, and also released Gly-Asn-Leu from neuromedin B with the same velocity as from Ala-Ala-Phe-MCA. Angiotensin III and neuromedin B have recently been found to be good natural substrates for lysosomal TPP I. Furthermore, we determined the rat liver cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRTI-1, is composed of 2485 bp and encodes 563 amino acids in the coding region. By Northern blot analysis, the order for TPP I mRNA expression was kidney > or = liver > heart > brain > lung > spleen >> skeletal muscle and testis. In parallel experiments, the TPP I antigen was detected in various rat tissues by immunohistochemical staining.

Amino Acid Sequence↗
PubMed2001

Zinc-alpha(2)-glycoprotein hinders cell proliferation and reduces cdc2 expression.

Zinc-alpha(2)-glycoprotein (Znalpha(2)gp) is widely distributed in body fluids and epithelia. Its expression in stratified epithelia increases with differentiation. We previously showed that Zn alpha(2)gp has ribonuclease activity, and that squamous tumor cells grown on a matrix of Znalpha(2)gp were growth-inhibited. Here we demonstrate, both by adding Znalpha(2)gp to the culture medium and, more unequivocally, by stably transfecting SiHa cells with Znalpha(2)gp cDNA, that the introduction of Znalpha(2)gp into SiHa tumor cells reduces proliferation. In response to Znalpha(2)gp, we find an accumulation of the cell population in G(2)/M by flow cytometry, paralleling the reduction of proliferation. In order to distinguish growth inhibition by cell cycle arrest from that produced by apoptosis or differentiation, we examine by RT-PCR how Znalpha(2)gp affects the expression of genes commonly used as markers of these properties. No changes are observed for PCNA, p53, c-myc, or bcl-2. Only cdc2 expression responds to Znalpha(2)gp, with a reduction of up to over a factor of two. Cdc2 is the only cyclin-dependent kinase regulating the G(2)/M transition without redundancy and is required as a rate-limiting step in the cell cycle. Its increased expression has been directly linked to increased proliferation and decreased differentiation of advanced tumors; conversely, its downregulation by Znalpha(2)gp might hinder tumor progression. J. Cell. Biochem. Suppl. 36: 162-169, 2001.

Blotting, Western↗
PubMed2000

Alteration of rat dipeptidyl peptidase III by site-directed mutagenesis: cysteine(176) is a regulatory residue for the enzyme activity.

To comprehend the importance of cysteine residues for the regulation of enzyme activity, we replaced each of seven cysteine residues in rat dipeptidyl peptidase III cDNA with alanine, glycine, or glutamic acid residue using site-directed mutagenesis. Each mutated cDNA was expressed in E. coli (BL21), and each expressed DPP III was purified to apparent homogeneity on SDS-polyacrylamide gel electrophoresis. Six of the mutant proteins had similar activity to that of the wild-type enzyme, whereas the activity of the Cys(176) --> Ala mutant enzyme was only 25-35% of that of the wild-type enzyme activity. This mutant enzyme was resistant against both PCMB and NEM. Furthermore, both Cys(176) --> Gly and Cys(176) --> Glu mutated enzymes showed no DPP III activity. These seven mutated enzymes contained significant amounts of zinc, as determined by atomic absorption spectrometry. The results indicate that Cys(176) is essential for the regulation of DPP III activity.

Amino Acid Substitution↗
PubMed2000

Molecular cloning and immunohistochemical localization of rat dipeptidyl peptidase III.

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS, and 82000 on non-denatured PAGE and 82000 on SDS-PAGE in the absence or presence of beta-ME. These findings suggest that the enzyme assumes a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Ala-Arg-MCA in a pH range of 7. 5 to 9.5. The K(in), K(cat) and K(cat)/K(m) values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 6.21x10(4) s(-1)M(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 3.62x10(4) s(-1)M(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS, NEM, beta-ME and iodoacetamide. Furthermore, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG, and we determined the cDNA structure and deduced the amino acid sequence. The cDNA designated as lambdaRDIII-11 is composed of 2640 bp of nucleotides in length and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH in structure, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. These findings suggest that DPP III is a metalloprotease that is probably regulated by SH modification. The DPP III antigen was extensively detected in the cytosol of various rat tissues by the immunohistochemical examination of the protein.

Amino Acid Sequence↗
PubMed2000

Puromycin-sensitive alanyl aminopeptidase from human liver cytosol: purification and characterization.

A cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human liver cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of liver cells and renal tubules, and was ubiquitously localized in various human tissues.

Aminopeptidases↗
PubMed2000

Enhancement of brush border membrane peptidase activity in rat jejunum induced by starvation.

Conflicting results have been obtained in previous studies concerning the adaptation of intestinal blush border membrane enzymes to starvation. This study was designed to clarity the changes in these enzymes under starvation conditions, using a molecular biological approach. Sprague-Dawley rats were starved or given total parenteral nutrition (TPN) for 5 days. Rats allowed free access to food were used as controls. Changes in the activity and expression of jejunal brush border membrane enzymes were compared between three groups. In the starved group, aminopeptidase N and dipeptidyl peptidase IV activity was significantly elevated to 177% and 166%, respectively, of control values. In contrast, sucrase and maltase activity was significantly decreased. The activity of these peptidases also tended to be increased at the renal brush border membrane. Up-regulation of peptidase activity was not evident in the TPN group. Western and Northern blot analysis revealed that the changes in aminopeptidase N activity were attributable to increases in the protein and mRNA level. The activity and expression of brush border membrane peptidases in rat jejunum is up-regulated during starvation, and these changes are considered to be an effect of whole-body malnourishment, rather than an absence of luminal nutrition.

Aminopeptidases↗
PubMed2000

Identification of components of the intrafollicular bradykinin-producing system in the porcine ovary.

As a step in elucidating the biological role of plasma kallikrein (PK) present in the follicular fluid of mammalian ovaries, we examined pig ovary fluid to determine its constituent activators and substrates. Using the inactive precursor form of plasma kallikrein (prePK) as a substrate, we purified an enzyme capable of activating this protein. The prePK-activating enzyme was shown to be the active enzyme blood coagulation factor XIIa. We also isolated high molecular weight kininogen (HMW-K) from the same fluid. Incubation of HMW-K with the ovarian follicular fluid PK resulted in the production of the nanopeptide bradykinin (BK). Expression of prePK, blood coagulation factor XII, and HMW-K was examined by Northern blot analysis using ovary and liver poly(A)(+) RNA. All these transcripts were found in the liver, but none were found in the ovary. In addition, it was found that BK levels in the fluid derived from the small follicles were approximately 6 times higher than those from medium and large follicles. These results demonstrate the presence of a BK-producing system in the ovarian follicles and suggest the physiological importance of this peptide hormone in the early stages of follicular development and at ovulation.

Amino Acid Sequence↗
PubMed1999

Expression of genes for estrogen receptors alpha and beta in human articular chondrocytes.

OBJECTIVE: To investigate the gene expression of estrogen receptor (ER) alpha and ERbeta in human articular chondrocytes. METHODS: 16 articular cartilage specimens were obtained from 15 patients during surgery. Three of the specimens were from men and 13 from women; three from hip joints and 13 from knee joints; four were normal and 12 showed osteoarthritic cartilage. Total RNA was extracted from the articular chondrocytes and the expression of both ERalpha and ERbeta genes was investigated by the reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Gene expressions of ERalpha were detected in all specimens and those of ERbeta were found in 15 specimens by the RT-PCR method. There was a significant correlation between the amounts of ERalpha and ERbeta. Expression levels of both genes were significantly higher in men than in women. There were no significant differences in the expression levels of both ER genes between the hip and knee joint sites, nor between normal and osteoarthritic tissues. CONCLUSION: This study is to our knowledge the first to demonstrate the gene expression of both ERalpha and ERbeta in human articular chondrocytes. Since there are some functional differences between the two receptors, the effects of estrogen on cartilage metabolism should be elucidated by two different receptor mechanisms.

Adult↗
PubMed1999

Polymorphic CAG repeats of the androgen receptor gene and rheumatoid arthritis.

OBJECTIVE: In view of the possible role of androgens in the pathogenesis of rheumatoid arthritis (RA), this study investigated the association between repeat lengths of CAG microsatellites of the androgen receptor (AR) gene and RA. METHODS: The number of CAG repeats in exon 1 of the AR gene was determined in 90 men and 276 women with RA, as well as in 305 male and 332 female controls. RESULTS: The male RA patients tended to have shorter repeats than the male controls (22.5 versus 23.1, p=0.07), whereas the female RA patients had similar repeats to the female controls (22.7 versus 22.9, p=0.17). Patients of both sexes were divided into younger and older age at onset groups, and compared with younger and older controls. Younger onset male RA patients had significantly shorter CAG repeat lengths than the younger male controls (21.8 versus 23.2, p=0.007) or the older onset male RA patients (21.8 versus 23.2, p=0.04). Older onset male RA and both younger and older onset female RA patients had similar CAG repeat lengths when compared with their controls. Neither seropositivity nor rheumatoid nodule positivity had a significant relation with CAG repeat lengths. CONCLUSION: Shorter CAG repeats of the AR gene, presenting high levels of transactivation activity, are related to younger age onset male RA, suggesting the possible role of androgens as a modulating factor.

Adult↗
PubMed1999

Dipeptidyl peptidase III from rat liver cytosol: purification, molecular cloning and immunohistochemical localization.

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in the absence or presence of beta-mercaptoethanol. These findings suggest that the enzyme exists in a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Gly-Arg-MCA in the pH range of 7.5 to 9.5. The Km, k(cat) and k(cat)/Km values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 62.1 s(-1) x nM(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 36.2 s(-1) x nM(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. These findings suggest that DPP III is an exo-type peptidase with characteristics of a metallo- and serine peptidase. For further information on the molecular structure, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRDIII-11, is composed of 2640 bp and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the cytosol of various rat tissues by immunohistochemical examination.

Amino Acid Sequence↗
PubMed1998

Regulation of Zn-alpha2-glycoprotein-mediated cell adhesion by kininogens and their derivatives.

MC3T3-E1 (mouse osteoblast-like) cells adhered to a tissue culture plate coated with human Zn-alpha2-glycoprotein (Znalpha2gp). The adhesion of MC3T3-E1 cells to Znalpha2gp was inhibited by synthetic peptides such as RGDS and ELRGDV, and by antibody against vitronectin receptor. These findings suggested that the RGD region of Znalpha2gp interacts with the vitronectin receptor (alphavbeta3) on the MC3T3-E1 cell surface. Furthermore, we found that the common heavy chain of both HMW- and LMW-kininogens accelerated the Znalpha2gp-mediated MC3T3-E1 cell adhesion. Among the three domains of the common heavy chain of both kininogens, domain 3 promoted the cell adhesion by up to 200%. Among the nine synthetic peptides covering domain 3, the peptide, N334AEVYVVPWEKKIYPTVN351 accelerated in a dose-dependent manner the Znalpha2gp- and vitronectin (VN)-mediated MC3T3-E1 cell adhesion. These findings suggested that a defined region of domain 3 is responsible for the acceleration of cell adhesion.

3T3 Cells↗
PubMed1998

The expression of beta-microseminoprotein gene is regulated by cAMP.

The promoter of the human gene for beta-microseminoprotein (MSP) was analyzed by transient transfection assay using LNCaP cells. The region between -2738 and -207 was not essential for the basal promoter activity, but regions from -206, -108, and -83 were necessary. Forskolin induced promoter activity to 3- to 14-fold when constructions containing the possible cAMP response element (CRE, -60 to -53) were used, but did not do so when it was deleted from them. In a gel-mobility shift assay using a probe corresponding to -68 to -48, the shifted band was competed with a consensus CRE, and supershifted when the anti-CREB-1 antibody was included. Forskolin added to LNCaP cells increased MSP mRNA to 6-fold within 12 h. These results suggest that the expression of MSP gene is regulated by cAMP via the CRE present in the promoter. Hormones such as (-)-epinephrine or vasoactive intestinal polypeptide also increased MSP mRNA to 2- to 3-fold.

Colforsin↗
PubMed1998

Purification and characterization of a tumor lipid-mobilizing factor.

Cancer patients with weight loss showed urinary excretion of a lipid-mobilizing factor (LMF), determined by the ability to stimulate lipolysis in isolated murine epididymal adipocytes. Such bioactivity was not detectable in the urine of cancer patients without weight loss or in normal subjects. The LMF was purified using a combination of ion exchange, exclusion, and hydrophobic interaction chromatographies to give a single component of apparent Mr 43,000, which showed homology in amino acid sequence with human plasma Zn-alpha2-glycoprotein. Both substances showed the same mobility on denaturing and nondenaturing gels and the same chymotrypsin digestion pattern, both stained heavily for carbohydrate, and they showed similar immunoreactivity. Polyclonal antisera to human plasma Zn-alpha2-glycoprotein was also capable of neutralization of the bioactivity of human LMF in vitro. Using competitive PCR to quantify expression of Zn-alpha2-glycoprotein, we found that only those tumors that were capable of producing a decrease in carcass lipid expressed mRNA for Zn-alpha2-glycoprotein. These results provide strong evidence to suggest that tumor production of Zn-alpha2-glycoprotein is responsible for the lipid catabolism seen in cancer patients.

Adipocytes↗