Search PubMed⌕ Search

Biomedical subjects

H Sinohara

Publications and source records attributed to H Sinohara.

At least 19 recordsLinked to original sources
PubMed2001

Molecular cloning and sequence analysis of C57BL/6 mouse contrapsin cDNA.

Contrapsin is a member of the serpin superfamily and inhibits trypsin much more strongly than alpha1-antiproteinase. Mouse and rat contrapsins, however, have similarity in sequence to human alpha1- antichymotrypsin. In order to test the hypothesis that reactive site regions of contrapsin family evolved under strong selective pressure, cDNA sequence of C57BL/6 mouse contrapsin was determined and compared with that of ICR mouse. The cDNA sequence of C57BL/6 mouse contrapsin was found to contain an open reading frame encoding polypeptide consisting of 418 amino acid residues. The work reported in this paper shows that the reactive site is not hypervariable as compared with the rest of molecule.

Amino Acid Sequence↗
PubMed2000

Molecular evolution in the hypervariable regions of fetuin: comparison between human and African green monkey fetuin.

Sequences of fetuin cDNA and its deduced amino acid residues from the African green monkey cell line Vero were found to differ by 7.3% and 12.9%, respectively, from the corresponding human sequences. Most amino acid substitutions were clustered within a small segment of the third domain (D3). Calculations of nonsynonymous and synonymous nucleotide substitution rates suggest that this small segment was mutated under positive selection. cDNAs encoding alpha1-antitrypsin, beta-actin and the sequences of intron 4 of alpha1-antitrypsin gene in human liver and Vero cells were also investigated. The results substantiated the positive selection imposed on the D3 segment.

Actins↗
PubMed1999

Occurrence of heavy chain of 7S IgM half-molecule whose NH2-terminal sequence is identical with that of kappa light chain sequence in patients with Waldenstrom macroglobulinemia.

We report a rare case of a half molecule 7S IgM (HM 7SIgM) consisting of a unique mu heavy chain and kappa light chain found in blood and urine samples from a patient with primary Waldenstrom macroglobulinemia. A 64kDa abnormal immunoglobulin was detected in serum and urine by immunoblot method, and purified by a two-dimensional SDS-PAGE after separation from IgG and albumin fractions on gel filtration. NH2-terminal amino acid sequence analysis of the heavy chain revealed that residues 1-20 were identical to those of the NH2-terminal region of kappa light chain derived from the same patient. This sequence was then followed by a sequence that could not be identified by a computer homology search on the protein database. Using polypeptide segments obtained from the unique mu chain by digestion with endopeptidase, we identified a sequence spanning from residue 127 in the variable region of the known mu chain to residue 19 in the known CH1 domain and a sequence spanning from residues 67-82 in the heavy chain variable region class II. From these results, we concluded that the 64 kDa protein was an abnormal half molecule 7S IgM consisting of a kappa light chain and a unique mu heavy chain of 35 kDa polypeptide in which the NH2-terminal 20 amino acids were replaced by 20 amino acids derived from the sequence of kappa light chain in the NH2-terminal region.

Amino Acid Sequence↗
PubMed1999

Guinea pig alpha 1-microglobulin/bikunin: cDNA sequencing, tissue expression and expression during acute phase.

cDNA encoding alpha 1-microglobulin/bikunin (AMBP) was amplified from guinea pig (Cavia porcellus) liver mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods, cloned and sequenced. The deduced amino acid sequence was found to be homologous to the sequence of AMBP of other mammals (69-76% amino acid identity). It has two Kunitz-type trypsin inhibitor domains in the bikunin part as reactive sites, one in the N-terminal region and another in the C-terminal region. The N-terminal inhibitor domain sequence is well-conserved, but the P1 residue of the C-terminal inhibitor domain sequence was found to be Gln rather than Arg, a residue highly conserved in the AMBP of seven other mammals examined to date. By RT-PCR and nested PCR, AMBP mRNA was detected not only in liver tissue, previously known to be a site of its synthesis, but also in pancreas, stomach, small intestine, colon, lung, spleen, kidney, testis, skeletal muscle, and leukocytes, but not in brain or heart. We examined the AMBP mRNA levels in guinea pig liver by RT-PCR, comparing normal levels and those in a state of inflammation. The mRNA levels, however, did not significantly change.

Amino Acid Sequence↗
PubMed1999

Some Bence-Jones proteins enter cultured renal tubular cells, reach nuclei and induce cell death.

Eighteen monoclonal Bence-Jones proteins (BJPs) were examined for their effects on cultured LLC-PK1 (porcine kidney proximal tubule) cells as well as for their amidase and DNase activities. Five proteins were found to enter the cell and to gain access to the nucleus without degradation of epitopes. Intranuclear BJPs ultimately induced DNA fragmentation and cell death. BJPs with relatively high amidase activity were cytotoxic. On the other hand, three of four BJPs with DNase activity had a cytocidal effect on cultured cells; the remaining BJP, which had a relatively high DNase activity but a very low amidase activity, failed to enter the cell and was not cytotoxic in vitro. These results suggest that catalytic and cytotoxic activities of some BJPs may make a significant contribution, in a substantial proportion of myeloma patients, to the development and/or deterioration of the disease.

Amidohydrolases↗
PubMed1999

cDNA sequencing of guinea pig alpha 2-HS glycoprotein, its expression in various tissues and acute phase expression.

cDNA encoding alpha 2-HS glycoprotein was amplified from guinea pig liver mRNA by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends, cloned and sequenced. By RT-PCR and nested PCR, alpha 2-HS glycoprotein mRNA was detected not only in liver tissue but also in pancreas, stomach, small intestine, colon, spleen, kidney, testis, skeletal muscle, brain, heart and leukocytes, but not in the lung. The alpha 2-HS glycoprotein mRNA levels in the liver were reduced to half at 48 h after subcutaneous injection of turpentine oil.

Acute Disease↗
PubMed1998

DNA-hydrolyzing activity of Bence Jones proteins.

Of 18 monoclonal Bence Jones proteins purified from urine of patients with multiple myeloma, 4 were found to have a DNA-nicking activity. In contrast, all Bence Jones proteins tested showed detectable amidase activity against carbobenzoxy-L-valyl-glycyl-L-arginine p-nitroanilide. No correlation between the two activities was found. Four patients excreting Bence Jones protein with the DNA-nicking activity showed somewhat severe symptoms, suggesting that this activity may be related to the progressive deterioration of clinical status.

Adult↗
PubMed1998

Cloning and sequencing of cDNA encoding haptoglobin, an acute phase protein in Syrian hamster, Mesacricetus auratus.

One of the most prominent acute phase proteins in Syrian hamster (Mesacricetus auratus) was identified as haptoglobin and cDNA encoding this protein was sequenced. The deduced amino acid sequence of the mature protein is 83.6, 80.5, 79.6, and 76.1% identical to those of mouse, rat, human (1 s isoform), and dog homologues, respectively. As compared with six known members of this family, including human haptoglobin-related protein, hamster haptoglobin had 11 unique substitutions and one unique codon deletion, that is, the corresponding residues have been conserved in all other members. This indicates that hamster haptoglobin gene has accumulated these unique mutations after the time of cricetid-murid split while the ancestral sequence has been conserved in all other species examined. Hamster haptoglobin, however, contains nine cysteine residues, all of which are found in conserved positions in primate and rodent homologues. Molecular phylogenetic trees of alpha- and beta-chains show that the alpha-chain is more divergent than the beta-chain and that the difference in genetic distance between canine and hamster alpha-chains is much greater than that of corresponding beta-chains.

Amino Acid Sequence↗
PubMed1998

Amidolytic and peptidolytic activities of immunoglobulin G present in sera from patients with rheumatoid arthritis, Sjogren's syndrome and systemic lupus erythematosus.

Polyclonal immunoglobulin G (IgG) from healthy subjects was found to be capable of hydrolyzing carbobenzoxy-Val-Gly-Arg-p-nitroanilide (a synthetic chromogenic substrate for trypsin) and D-Pro-Phe-Arg-p-nitroanilide (a substrate for plasma kallikrein). Statistically significant elevation of activity against the former substrate was found in patients with rheumatoid arthritis (RA), but not in patients with Sjogren's syndrome (SjS) or systemic lupus erythematosus (SLE). On the other hand, IgG samples from the patients with these three autoimmune diseases showed reduced activity against d-Pro-Phe-Arg methylcoumarinamide, although the differences were not statistically significant. Preliminary studies have shown that two out of three IgG samples from RA patients exhibited the activity of cleaving a pentapeptide, Gln-Arg-Arg-Ala-Ala, whereas virtually no cleavage of the same peptide was observed with IgG from healthy controls or from patients with SjS or SLE.

Adult↗
PubMed1998

Various forms of rabbit plasma alpha-1-antiproteinase.

Amino acid sequencing of the ficin-derived C-terminal fragments of alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) prepared from rabbit plasma revealed the presence of the E isoform, which had been confirmed in the cDNA library in addition to the F and S-1 forms. The S-2 form was identified in inflamed rabbit plasma. The ficin digest of human plasma alpha-1-antitrypsin resulted in a major fragment of the M type. Multiple forms of alpha-1-antiproteinase in the rabbit plasma implicate the unknown functions other than the inhibition of neutrophil elastase.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of rabbit plasma alpha-1-antiproteinase E.

Alpha-1-antiproteinase E, the fourth isoform of rabbit alpha-1-antiproteinase (alpha-1-antitrypsin) having a glutamic acid at the reactive center, has been purified from the plasma by sequential chromatography on hydroxyapatite and anion-exchange columns. The E form of alpha-1-antiproteinase formed a complex with trypsin, chymotrypsin, elastase, plasmin and pancreatic kallikrein as judged by SDS-PAGE. The E form inhibited elastase in a stoichiometric manner and chymotrypsin moderately, but the inhibition of trypsin was gradual. The F form inhibited trypsin most effectively followed by chymotrypsin and elastase. N-chlorosuccinimide reduced the elastase inhibitory activity of the E form, while the F form was more effectively inactivated by the oxidant.

Animals↗
PubMed1997

Sequencing of cDNA encoding serum albumin and its extrahepatic synthesis in the Mongolian gerbil, Meriones unguiculatus.

We have sequenced serum albumin cDNA from liver of the Mongolian gerbil, Meriones unguiculatus. The deduced amino acid sequence showed 82.6% and 73.6% identity with the corresponding proteins from rats and humans, respectively. Identical cDNA was detected in pancreas by reverse transcription followed by polymerase chain reaction (RT-PCR). Further amplification of cDNA by nested PCR revealed the presence of the same cDNA in the brain and kidney. These results indicate that serum albumin is expressed in some extrahepatic tissues. In rats, an albumin-related 70-kDa protein (P70) has been proposed to be associated with cobalt-induced epilepsy (Onozuka et al. (1995) Neurochem. Res., 20, 901-905). We intensively searched for a P70-like protein in the brain of an epilepsy-prone gerbil strain, MGS/Idr, by the RT-PCR and nested PCR using several pairs of primers based on the albumin cDNA sequence. However, we found only mRNA for albumin itself.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and sequencing of cDNA encoding plasma countertrypin, a member of mammalian fetuin family, from the Mongolian gerbil, Meriones unguiculatus.

Complementary DNA clones coding for countertrypin were isolated from a liver cDNA library of the Mongolian gerbil, and sequenced. They contained one open reading frame encoding 348 amino acid residues, which were assigned to consist of an 18-residue signal peptide and a 330-residue mature protein. The amino acid sequence was about 74% identical with mouse countertrypin and rat fetuin, 60% with bovine fetuin, and 55% with human alpha2HS glycoprotein, indicating that this protein belongs to the mammalian fetuin family. The members of this family are known to consist of three domains, i.e., two tandemly arranged cystatin domains (D1 and D2) and an unrelated domain (D3) located at the C-terminal region. When compared with the other members of this family, D3, especially its N-terminal half, varies greatly with deletion or insertion as well as nucleotide substitutions even among three rodent species, i.e., gerbil, rat, and mouse. The sequence comparison also suggests that the conformation of human alpha2HS glycoprotein differs greatly from that of other members of this family. A molecular phylogenetic tree of 7 members, constructed on the basis of the synonymous substitution rate of D1 and D2, shows that the gerbil gene diverged prior to the separation of mouse and rat.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and sequencing of cDNAs encoding three heavy-chain precursors of the inter-alpha-trypsin inhibitor in Syrian hamster: implications for the evolution of the inter-alpha-trypsin inhibitor heavy chain family.

Complementary DNAs encoding precursors of the three heavy chains (HC1, HC2, HC3) of the inter-alpha-trypsin inhibitor in Syrian hamster liver were sequenced. The deduced amino acid sequence of the HC1 precursor was 87, 82, and 79% identical with those of the HC1 precursors from mouse, man and pig, respectively. The HC2 and HC3 precursors showed similar degrees of sequence identity with the corresponding human and mouse HC precursors. When the hamster HC1 precursor was compared with its own HC2 and HC3 precursors, however, even the most highly conserved segment consisting of 565 amino acid residues, i.e., about 2/3 of the whole molecule, showed only about 35 and 65% sequence identity, respectively. Essentially the same results were obtained on the intra-species comparisons of three subfamilies in man and mouse. Thus, the interspecies conservation of a given HC subfamily is much greater than the similarity between the three different HC subfamilies within a given species. These results suggest that (i) higher vertebrates possess three HC genes which have been evolving independently of each other under purifying selection; (ii) the diversification of the three HC subfamilies, for which the middle regions of the molecules were mainly responsible, occurred before eutherian radiation; and (iii) each HC subfamily may have unique function(s), although at present virtually nothing is known about the functional differences between the three HC subfamilies.

Alpha-Globulins↗
PubMed1996

Cystatin-like domain of mouse countertrypin, a member of mammalian fetuin family, is responsible for the inhibition of trypsin. Evidence from site-directed mutagenesis.

Members of mammalian fetuin family, such as human alpha 2 HS glycoprotein and bovine fetuin, consist of three domains, two tandemly arranged cystatin-like domains and an unrelated domain, but they have no inhibitory activity against cysteine proteinases. We found that countertrypin, a novel trypsin inhibitor, is mouse counterpart of human alpha 2 HS glycoprotein, and that human alpha 2 HS glycoprotein and bovine fetuin which were prepared without use of ethanol are capable of inhibiting trypsin (Yamamoto, K. and Sinohara, H. (1993) J. Biol. Chem, 268, 17750-17753). In the present study, cDNA encoding countertrypin was isolated and sequenced, and evidence is presented, based on the site-directed mutagenesis, that lysine-231 in the second cystatin domain is the P1 site for trypsin inhibition.

Amino Acid Sequence↗
PubMed1996

Inter-alpha-trypsin inhibitor and its related proteins in Syrian hamster urine and plasma.

Urinary excretion of trypsin inhibitor increased after injection of a carcinogen, N-nitrosobis(2-oxopropyl)amine, into Syrian hamsters. Two inhibitors were purified to apparent homogeneity from urine collected during the course of the carcinogenesis experiment. Their complete amino acid sequences were determined by Edman degradation of the intact proteins and partially degraded fragments. One corresponded to a hamster liver cDNA clone that hybridized with human bikunin probe [Ide et al, (1994) Biochim, Biophys. Acta 1209, 286-292], except that the protein sequence lacked C-terminal serine and the other was trypstatin, the C-terminal half of the bikunin molecule. Three proteins containing covalently linked bikunin were also identified in pooled blood plasma. They were all dissociated into heavy and light chains by treatment with chondroitinase ABC or 50 mM NaOH, but not by heating at 100 degrees C in the presence of sodium dodecyl sulfate and dithiothreitol, N-terminal amino acid sequence analyses of the native chains and partially degraded fragments thereof revealed that these proteins are (i) human-type inter-alpha-trypsin inhibitor, consisting of heavy chains 1 and 2 and bikunin, (ii) bovine-type inter-alpha-trypsin inhibitor, consisting of heavy chains 2 and 3 and bikunin, and (iii) pre-alpha-trypsin inhibitor, consisting of heavy chain 3 and bikunin. Heterodimer of bikunin/heavy chain 1 or bikunin/heavy chain 2 was not detected. These results suggest that the composition, and hence function, of the inter-alpha-trypsin inhibitor family differs considerably from species to species.

Alpha-Globulins↗