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Biomedical subjects

S Ogata

Publications and source records attributed to S Ogata.

At least 19 recordsLinked to original sources
PubMed1992

An active-site mutation (Gly633-->Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum.

Dipeptidyl peptidase IV (DPPIV), a serine protease expressed on the cell surface, is deficient in a Fischer rat substrain. Northern blot analysis showed no difference in the size and amount of DPPIV mRNA between normal (344/NC) and deficient (344/CRJ) rats. Cloning and sequencing of DPPIV cDNAs revealed a G to A transition at nucleotide 1897 in the cDNA sequence of 344/CRJ, which leads to substitution of Gly633-->Arg in the active-site sequence Gly629-Trp-Ser-Tyr-Gly633 determined for the wild-type DPPIV [Ogata, S., Misumi, Y., Takami, N., Oda, K., & Ikehara, Y. (1992) Biochemistry 31, 2582-2587]. Pulse-chase experiments with hepatocytes showed that the wild-type DPPIV was initially synthesized as a 103-kDa form with high-mannose-type oligosaccharides, which was processed to a mature form of 109 kDa with the complex type during intracellular transport. In contrast, the mutant DPPIV, although being synthesized as the 103-kDa form, was rapidly degraded without being processed to the mature form. Site-directed mutagenesis of the wild-type and mutant cDNAs and their transfection/expression in COS-1 cells confirmed that the single substitution of Gly633-->Arg is sufficient to cause the rapid intracellular degradation of DPPIV. Immunoelectron-microscopic observations showed that the mutant DPPIV was detectable only in the endoplasmic reticulum (ER), in contrast to the distribution of the wild-type DPPIV in the Golgi complex and on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Mucin gene expression in colonic tissues and cell lines.

Complementary DNA clones encoding four different mucin core peptides have been isolated. However, the expression of these mucin genes in the colon has not been systematically studied. The present investigation used Northern blot analysis to study the expression of MUC1, MUC2, MUC3, and MUC4 mRNA in paired normal and cancerous colonic tissues, and nine colon cancer cell lines. Results were correlated with the clinicopathological features of the tumors and with the immunohistochemical expression of several carbohydrate tumor-associated antigens that may reside on mucins. MUC1 mRNA was expressed in all colonic tissues, and levels in paired normal and cancer tissues were similar in most cases. MUC2 and MUC3 mRNAs were expressed in both normal and cancer tissues, but levels were often decreased in the cancers. MUC4 mRNA was present in normal mucosa with comparable or sometimes greater expression in cancers. There was no apparent correlation between the expression of any particular mucin gene or pattern of mucin genes and the site, stage, or histological type of tumor. In addition, the expression of mucin-associated carbohydrate antigens did not correlate with any individual mucin gene or group of mucin genes. In colon cancer cell lines all four MUC genes were expressed rather weakly or not at all. These results indicate that the human colon expresses a broad repertoire of mucin genes which are differentially regulated in malignancy. Whether this differential regulation of mucin genes affect the behavior of the tumor and results in the altered glycosylation commonly seen in these requires further investigation.

Aged↗
PubMed1992

Mucins bearing the cancer-associated sialosyl-Tn antigen mediate inhibition of natural killer cell cytotoxicity.

The sialosyl-Tn (STn) antigen is a mucin-associated carbohydrate antigen expressed by a variety of adenocarcinomas. In the colon, expression of this antigen has been associated with a poor prognosis, independent of tumor stage or histology. The present study was performed to determine whether this adverse clinical outcome might be due to an interaction between STn-positive mucin and natural killer (NK) cell cytotoxicity. Ovine submaxillary mucin (OSM), a mucin highly rich in STn antigen, partially inhibited NK cell cytotoxicity against K562 target cells, but only at high concentrations. Low concentrations of OSM were not inhibitory but became markedly inhibitory in the presence of ammonium ions. Two other STn-positive submaxillary mucins also markedly inhibited NK cytotoxicity when combined with ammonium ions. Removal of sialic acid from OSM reversed the OSM/ammonium-mediated inhibition of NK cell activity. Unlike the submaxillary mucins, two mucins derived from human breast and lung cancer cells which lack the STn antigen, did not inhibit NK cell activity in this system. Likewise, four other non-mucin glycoproteins which lack STn expression did not inhibit NK cells despite having levels of sialic acid that were, in some cases, comparable to submaxillary mucin. These results indicate that mucins bearing the cancer-associated STn antigen can effectively inhibit NK cell cytotoxicity in the presence of ammonium ions. While this NK cell inhibition is likely to be caused by ammonium, mucin markedly enhances this effect, thereby implicating a novel immunomodulatory property of mucin.

Ammonium Chloride↗
PubMed1992

Identification of the active site residues in dipeptidyl peptidase IV by affinity labeling and site-directed mutagenesis.

The active site of dipeptidyl peptidase IV (DPPIV) was examined by chemical modification and site-directed mutagenesis. Purified DPPIV was covalently modified with [3H]diisopropyl fluorophosphate (DFP). The radiolabeled DPPIV was digested with lysyl endopeptidase, and the peptides were separated by high-performance liquid chromatography. A single 3H-containing peptide was obtained and analyzed for amino acid sequence and radioactivity distribution. A comparison of the determined sequence with the predicted primary structure of DPPIV [Ogata, S., Misumi, Y., & Ikehara, Y. (1989) J. Biol. Chem. 264, 3596-3601] revealed that [3H]DFP was bound to Ser631 within the sequence Gly629-Trp-Ser-Tyr-Gly633, which corresponds to the consensus sequence Gly-X-Ser-X-Gly proposed for serine proteases. To further identify the essential residues in the active-site sequence, we modified the DPPIV cDNA by site-directed mutagenesis to encode its variants. Expression of the mutagenized cDNAs in COS-1 cells demonstrated that any single substitution of Gly629, Ser631, or Gly633 with other residues resulted in the complete loss of the enzyme activity and DFP binding. Although substitution of Trp630----Glu or Tyr632----Phe caused no effect on the enzyme activity, that of Tyr632----Leu or Gly abolished the activity. These results indicate that the sequence Gly-X-Ser-(Tyr)-Gly is essential for the expression of the DPPIV activity.

Affinity Labels↗
PubMed1992

The DNA sequence of gamma-glutamyltranspeptidase gene of Bacillus subtilis (natto) plasmid pUH1.

The gamma-glutamyltranspeptidase (gamma-GTP) gene of Bacillus subtilis (natto) plasmid designated pUH1, which is responsible for polyglutamate production, has been cloned and the nucleotide sequence determined. The sequence contains a single open-reading frame stretching for 1260 bp with a relative molecular mass of 49,356. Putative -35 and -10 sequences, TTCAAA and TATTAT, were observed as the consensus sequence for the promoter recognized by the sigma 43 RNA polymerase of B. subtilis, and the ribosome binding site, the sequence of which was AACGAG, was complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The amino acid sequence of the gene with the segment of putative protein C403 of staphylococcal plasmid pE194 indicates homology, whereas that with Escherichia coli and mammalian gamma-GTPs does not show any similarity at all.

Amino Acid Sequence↗
PubMed1992

Epitope homology between bacterial heat shock protein and self-proteins in the host cell.

Mouse monoclonal antibodies (MAbs) showing distinct reactivity against the 60-kilodalton (kDa) antigen heat shock protein of Yersinia enterocolitica, designated cross-reacting protein antigen (CRPA), have previously been established. The reactivities of these MAbs (5C3 and 3C8) against mouse and human host cells were studied by Western blotting and flow cytometric analysis. The results indicated that epitopes on the bacterial 60-kDa heat shock protein are present on various molecules in mouse spleen cells and human B cells. An epitope recognized by MAb 5C3 was expressed on the mouse and human host cell surface, and an epitope recognized by MAb 3C8 was also expressed on the human host cell surface.

Animals↗
PubMed1992

Sequence analysis of replication origin of plasmid pLS11 of Bacillus subtilis IFO 3022.

The structure of a 1.6-kb SphI-HindIII DNA sequence necessary and sufficient for the replication of a 8.6-kb plasmid pLS11 of Bacillus subtilis IFO 3022, which is responsible for gamma-polyglutamate production, has been characterized by using a trimethoprim (Tmp)-resistance gene derived form B. subtilis TTK24 chromosomal DNA as a selective marker. The 1.6-kb DNA sequence contains a rep gene encoding the protein (333 amino acids) essential for initiation of replication and a possible origin of replication. The predicted REP protein of pLS11 has an overall homology with the REP proteins of pUH1 (74.8% identity), pBAA1 (92.8%), and pFTB14 (78.7%) in Bacillus spp., pLP1 (42.1%) and pLAB1000 (36.3%) in Lactobacillus spp., and pUB110 (35.3%) and pC194 (37.4%) in Staphylococcus aureus, but has not any similarity with the REP protein of the staphylococcal plasmid pT181.

Amino Acid Sequence↗
PubMed1991

Cloning, structure and expression of cDNA for mouse contrapsin and a related protein.

A cDNA clone (lambda MC-2) for contrapsin, a serine-proteinase inhibitor, was isolated from a lambda ZAP mouse liver cDNA library. The 1.6 kb cDNA insert of lambda MC-2 contained an open reading frame that encodes a 418-residue polypeptide (46,970 Da), in which a signal peptide of 21 residues was identified by comparison with the N-terminal sequence of the purified protein. The predicted structure (MC-2) also contained other peptide sequences determined by Edman degradation. Four potential sites for N-linked glycosylation were found in the molecule, accounting for the difference in molecular mass between the predicted form and the purified protein (63 kDa). Further screening of the cDNA library with an EcoRI-EcoRI fragment (510 bp) of lambda MC-2 as a probe yielded another cDNA clone (lambda MC-7), which encodes a 418-residue polypeptide (MC-7) with a calculated mass of 47,010 Da. MC-2 showed 83% similarity at the amino acid level to MC-7, in contrast with 44% similarity to alpha 1-proteinase inhibitor. The possible reactive site (P1-P'1) for serine proteinase is suggested to be Lys-Ala for MC-2 and Ser-Arg for MC-7. Northern-blot analysis revealed that both MC-2 and MC-7 mRNAs have the same size of 1.8 kb and are markedly induced in response to acute inflammation. Construction of the expression plasmids pSVMC-2 and pSVMC-7 and their transfection into COS-1 cells demonstrated that pSVMC-2 directs the synthesis of a 63 kDa form whereas pSVMC-7 expresses a 56 kDa form. The difference in molecular mass between the two may be explained by the fact that the MC-7 sequence contains three potential sites for N-glycosylation, one site less than that of MC-2.

Amino Acid Sequence↗
PubMed1991

Activation by intracellular calcium of a potassium channel in cardiac sarcoplasmic reticulum.

The effects of low (pCa 7.5 to 3) concentrations of intracellular calcium ion on a single potassium channel in the sarcoplasmic reticulum of canine heart ventricular muscle were investigated using a planar lipid bilayer technique. The low concentrations were obtained by mixing EGTA and calcium chloride. By varying the pCa of the cytoplasmic face between 3 to 7.5, two novel effects were observed. First, an increase in the intracellular Ca2+ concentration produced an increase in the unit current amplitude of open states; the voltage-current relationship was ohmic at these concentrations. Second, an increase in the Ca2+ concentration increased the open probability. Both these effects of Ca2+ were dose-dependent, and were consistently observed in all channels tested. Thus, the SR potassium channel observed appears to belong to the class of Ca2(+)-activated potassium channels.

Animals↗
PubMed1991

Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1.

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.

Amino Acid Sequence↗
PubMed1991

Object representations in the early memories of sexually abused borderline patients.

OBJECTIVE: This study analyzed psychological representations in 58 subjects in order to achieve a better understanding of the relation between adult borderline personality disorder and reported histories of childhood sexual and physical abuse. METHOD: The subjects were 29 inpatients with borderline personality disorder diagnosed according to the Diagnostic Interview for Borderlines, 14 nonborderline inpatients with major depressive disorder according to the Research Diagnostic Criteria, and 15 normal comparison subjects recruited from the community and screened for the absence of psychopathology. Earliest memories were used as the source of mental representations in all subjects. The memories were reliably coded for malevolent affect tone, presence of deliberate injury, and effectiveness of helpers. Family histories of childhood sexual and physical abuse were obtained with the Familial Experiences Interview, a structured interview. Abuse histories for a subset of the subjects were corroborated by interviews with family members. RESULTS: A reported history of sexual abuse, but not a reported history of physical abuse, predicted the presence of extremely malevolent representations in these earliest memories as well as representations involving deliberate injury. These two kinds of representations also discriminated borderline patients who reported histories of sexual abuse from borderline patients who did not report sexual abuse. Mean affect tone (from malevolent to benevolent) did not, however, discriminate sexually abused or physically abused subjects. CONCLUSION: The results suggest that malevolent representations associated with the borderline diagnosis in previous research may be partially related to a history of childhood sexual abuse. Implications for the object relations theory of borderline personality disorder are noted.

Adolescent↗
PubMed1991

Characterization of a new human apolipoprotein A-I Yame by direct sequencing of polymerase chain reaction-amplified DNA.

A new genetic variant apolipoprotein (apo), A-I Yame, was discovered during screening for apoA-I genetic variants with isoelectric focusing gel electrophoresis. To investigate the structural abnormality of apoA-I Yame, we amplified the apoA-I gene isolated from the proband's peripheral blood leukocytes with the polymerase chain reaction (PCR) method and directly sequenced these PCR fragments. ApoA-I Yame was found to have aspartic acid (GAC) substituted by tyrosine (TAC) at residue 13. We also identified this substitution by an automated DNA sequencer. This substitution was confirmed with amino acid sequencing of the isolated apoA-I Yame by Immobiline gel electrophoresis. This combined method, direct PCR from genomic DNA-derived individual peripheral blood leukocytes and subsequent direct sequencing, can be used to identify the entire sequence of apoA-I in a short period of time. Furthermore, with this method, it is possible to identify both alleles in heterozygous individuals.

Alleles↗
PubMed1991

Molecular cloning and characterization of rat contrapsin-like protease inhibitor and related proteins.

A glycoprotein with Mr 63,000 purified from rat serum was found to inhibit trypsin activity but not chymotrypsin or elastase activity, resembling contrapsin purified from mouse serum. To obtain further information on the molecular structure, a cDNA clone (lambda CPi-21) for this contrapsin-like protease inhibitor was isolated from a rat liver cDNA library. The 1.6-kb cDNA insert contained an open reading frame that encodes a 416-residue polypeptide (CPi-21), in which the first 29 residues were suggested to comprise a signal peptide by comparison with the NH2-terminal sequence of the purified protein. The predicted structure also contained other peptide sequences determined by Edman degradation. Four potential N-linked glycosylation sites were found in the molecule, presumably accounting for the larger molecular mass of the mature form. Further screening of the cDNA library with a Pst-XbaI fragment (302 bp) of lambda CPi-21 as a probe yielded two other cDNA clones (lambda CPi-23 and lambda CPi-26), which encode 413-residue and 418-residue polypeptides, respectively. A comparison of their amino acid sequences revealed that CPi-21 has 89 and 71% homology with CPi-23 and CPi-26, respectively. The primary structure of each of the three proteins has about 70% homology with that of mouse contrapsin, in contrast to 43-46% homology with that of rat alpha 1-protease inhibitor. These results indicate that all the CPi proteins presented here belong to a subfamily of "serpins" of which mouse contrapsin was the first member to be identified.

Acute Disease↗
PubMed1991

[A case of acute myocardial infarction due to coronary spasm].

The patient was a 47 year-old man, who has been known to have effort angina since September 1989. His exercise stress ECG has revealed ST elevation in V2-V4 with maximum exercise. He experienced severe chest pain lasting for an hour on the way to his office in the early morning on November 16, 1989, and was admitted to our hospital. His ECG and laboratory findings indicated typical acute anteroseptal myocardial infarction, but the coronary arteriography (CAG) which was performed 7 hours after the onset showed no significant stenotic lesion. After administrating nitrate and calcium antagonist, he has had no attack of angina pectoris and his exercise stress test has revealed no ST-T changes on his ECG. 1 month later, while antianginal drugs were discontinued in order to perform an ergonovine stress test, the patient frequently complained of left anterior chest pain with remarkable ST elevation in precordial leads on his ECG. The CAG at chronic stage revealed that there was a 99% stenosis at Segment 6 of the left anterior descending artery (LAD) which was supplied with good collateral flow from the right coronary artery. The LAD was completely occluded at Segment 6 after intracoronary administration of ergonovine maleate 0.005 mg to the left coronary artery. After the intracoronary infusion of isosorbide dinitrate, there was no significant stenosis seen in the LAD except the minimum wall irregularity at Segment 6. These findings suggested that coronary spasm might play a major role of the occurrence of acute myocardial infarction in this case.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium Channel Blockers↗
PubMed1990

Membrane-anchoring domain of rat liver 5'-nucleotidase: identification of the COOH-terminal serine-523 covalently attached with a glycolipid.

The involvement of glycosylphosphatidylinositol (GPI) in membrane anchoring of 5'-nucleotidase was investigated by chemical analyses. 5'-Nucleotidase purified from rat liver microsomes was subjected to BrCN cleavage, hexane extraction, and high-performance liquid chromatography, resulting in the purification of a single fragment with Mr 2300. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Val-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser and characteristic components of GPI including ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. In addition, it was confirmed that digestion of 5'-nucleotidase with lysyl endopeptidase yielded a fragment containing the dipeptide Phe-Ser and the same GPI components as above. The sequences of the tetradeca- and dipeptides thus determined are identified at positions 510-523 and 522-523, respectively, in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing a hydrophobic amino acid sequence [Misumi, Y., Ogata, S., Hirose, S., & Ikehara, Y. (1990) J. Biol. Chem. 265, 2178-2183]. Taken together, these results indicate that the mature 5'-nucleotidase molecule lacks the predicted COOH-terminal peptide extension and is attached at serine-523 with GPI, which functions as the membrane anchor of 5'-nucleotidase.

5'-Nucleotidase↗
PubMed1990

Primary structure of human placental 5'-nucleotidase and identification of the glycolipid anchor in the mature form.

A cDNA was cloned coding for human placental 5'-nucleotidase. The 3547-bp cDNA contains an open reading frame that encodes a 574-residue polypeptide with calculated size of 63 375 Da. The NH2-terminal 26 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. four potential N-linked glycosylation sites are found in the molecule, accounting for a larger mass of the mature form (71 kDa). The predicted structure contains a hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. To confirm this possibility, we tried to isolate and characterize the membrane-anchoring domain of 5'-nucleotidase. BrCN-cleaved fragments of the protein were extracted with hexane and subjected to HPLC, resulting in purification of a single component of 2.3 kDa. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Val-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser, ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. The peptide sequence determined is identified at positions 510-523 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing the hydrophobic amino acid sequence. Thus, it is concluded that the mature 5'-nucleotidase lacks the predicted COOH-terminal peptide extension (524-548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5'-nucleotidase.

5'-Nucleotidase↗