Biomedical subjects

H Kawauchi

Publications and source records attributed to H Kawauchi.

At least 91 records · Page 5Linked to original sources

PubMed1993

Purification and characterization of Silurus asotus (catfish) roe lectin.

A rhamnose-binding lectin isolated from Silurus asotus (catfish) roe by DEAE-cellulose ion exchange and galactose-Sepharose affinity chromatographies predominantly agglutinated human type B and rabbit erythrocytes. S. asotus lectin (SAL) also agglutinated sarcoma 180 ascites carcinoma cells, but not AH109A cells. The most effective saccharide in hemagglutination inhibition assay was L-rhamnose. The monosaccharides possessing steric similarity to the hydroxyl group orientation at C2 and C4 of the pyranose ring structure of L-rhamnose, such as L-mannose and L-lyxose, were also effective. The molecular weight of SAL was determined to be 38000 by size exclusion chromatography on TSK gel G3000SW and 33000 by SDS-polyacrylamide gel electrophoresis under reducing conditions. SAL did not require a Ca2+ ion or free thiol group for its agglutination activity. The N-terminal 29 amino acid sequence was determined by a gas-phase sequencer as follows, ANMITCYGDVQKLHXETGLIIVKSXLYGR (X: not determined). It has no homology to the sequences of well known vertebrate lectins.

Agglutination↗

PubMed1993

Three rhamnose-binding lectins from Osmerus eperlanus mordax (olive rainbow smelt) roe.

Three rhamnose-binding lectins were purified from the roe of Osmerus eperlanus mordax (olive rainbow smelt) by affinity chromatography and ion-exchange chromatography. The apparent molecular weights of Osmerus eperlanus mordax lectin (OML) -1, -2 and -3 were 25000, 32000 and 26000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. On native PAGE, these three lectins showed different migration patterns (Rm value; 0.37, 0.53 and 0.66, respectively). OMLs agglutinated rabbit and human type B erythrocytes and sarcoma 180 cells, but not human type A and O erythrocytes and AH109A cells. The most effective monosaccharide inhibitor was L-rhamnose. L-Mannose and D-galactose were also good inhibitors. Furthermore, OML-induced hemagglutination was inhibited more strongly by melibiose or raffinose rather than lactose or lactulose. Therefore, OMLs are L-rhamnose/alpha-D-galactosyl type lectins. OMLs did not require a detergent, when extracted from crude material, and Ca2+, Mg2+, EDTA and dithiothreitol were not necessary for the OML-induced hemagglutination activities. The OMLs had similar N-terminal amino acid sequences.

Amino Acid Sequence↗

PubMed1993

Purification and some properties of ribonuclease from Xenopus laevis eggs.

A 122 kDa RNase from eggs of Xenopus laevis was purified by sequential chromatography on Sephadex G-75, DEAE-cellulose, heparin-Sepharose and TSK gel G3000SW columns, and gave a single 60 kDa band on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The RNase composed of two 60 kDa subunits is able to recognize pyrimidine bases specifically. The pH optimum of the RNase was 7.5 in Tris-HCl buffer. The enzyme activity was abolished by treatment at 80 degrees C for 5 min and pH 2 or 12 for 1 h. Since egg lectins with RNase activity obtained from Rana catesbeiana and R. japonica and bovine pancreatic RNase A show about 30% protein homology and these three proteins are 12-14 kDa heat-stable RNases, [K. Titani, K. Takio, M. Kuwada, K. Nitta, F. Sakakibara, H. Kawauchi, G. Takayanagi and S. Hakomori, Biochemistry, 26, 2189 (1987); Y: Kamiya, F. Oyama, R. Oyama, F. Sakakibara, K. Nitta, H. Kawauchi, Y. Takayanagi and K. Titani, J. Biochem. (Tokyo), 108, 139 (1990)], the data suggest that the X. laevis egg RNase is a unique protein compared with RNases from not only amphibians, but also mammals.

Amino Acids↗

PubMed1993

Structure of the channel catfish (Ictalurus punctatus) growth hormone gene and its evolutionary implications.

A DNA fragment of 1.6 kilo base pairs (kb), encoding part of the channel catfish (Ictalurus punctatus) growth hormone (GH) gene, was generated by the polymerase chain reaction (PCR) using 2 degenerate synthetic oligonucleotides (30 and 33 mer) derived from the N- and C-terminal amino acid sequences of the catfish GH polypeptide as amplification primers and with catfish genomic DNA as a template. This DNA fragment was used as a probe for the isolation of a catfish GH gene from a genomic library constructed in a lambda phage cloning vector, lambda Dash II. Three positive clones were isolated, and their complete nucleotide sequences were determined. Nucleotide sequences from clones 1 and 3 were identical, whereas clone 2 had 2 base substitutions. The gene spans approximately 3 kb and is comprised of 5 exons and 4 introns. The initiation codon, the termination codon, and the canonical polyadenylation sequence were identified. The amino acid sequence deduced from the predicted coding region of the gene is in agreement with that of the native GH polypeptide sequence. A sequence (TATAAAA) matching the TATA box consensus sequence was located at nucleotide positions -30 to -23. Furthermore, 2 sequences corresponding to the mammalian Pit-1/GHF-1 binding sites (consensus sequence TT[AA]TATNCAT) were identified in the 5' flanking region starting at positions -113 and -134. Another sequence (GTACCAGTGA) conserved among the GH genes of the channel catfish and other known animal species was also identified at position -220. The biological functions of this sequence remain to be determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Association of annexin V with prolactin in the rat anterior pituitary gland.

When pituitary extracts were subjected to non denaturing polyacrylamide gel electrophoresis, an unknown protein was found to associate with a proportion of the prolactin. This protein was dissociated from prolactin by sodium dodecyl sulfate. The protein was purified and sequenced. As the amino terminus was blocked, the amino acid sequences of three peptide fragments were determined. The obtained sequences of 41 amino acids were identical to partial sequences of a known protein, rat Annexin V. The molecular mass, 36 kDa, was also the same as the molecular weight of Annexin V. The existence of Annexin V mRNA in rat pituitary glands was also confirmed by polymerase chain reaction. These results show that Annexin V, a member of the calcium-dependent phospholipid binding proteins, is synthesized in the rat pituitary gland, and suggest its association with prolatin in the gland.

Animals↗

PubMed1992

The complete amino acid sequence of growth hormone from sturgeon (Acipencer guldenstadti).

The complete amino acid sequence of growth hormone (GH) from a chondrostean species, the sturgeon (Acipencer gludenstaditi), has been determined. Two variants of GH, termed GH I and GH II, were isolated from the pituitary by alkaline extraction, gel filtration on a Sephadex G-100 column, and reversed-phase high-performance liquid chromatography (rpHPLC) on a TSK gel ODS-120T column. The purified proteins were confirmed to be GHs by immunoblotting using bovine and chum salmon GH antisera. For determining of the primary structures, these GHs were digested with lysyl endopeptidase and cleaved with cyanogen bromide. The resulting fragments were separated by rpHPLC and subjected to sequence analysis on an automated gas-phase sequencer employing an Edman method. Both GHs consist of 190 amino acid residues, and contain two disulfide linkages at positions 52-163 and 180-188. The GHs differ from each other at only three positions. Sequence comparison with GHs from other vertebrates revealed that sturgeon GHs have greater sequence homology with tetrapod GHs (63-76%) than with teleost GHs (42-63%).

Amino Acid Sequence↗

PubMed1992

A novel tumor-associated protein: clinical significance of serum levels in various clinical conditions with special reference to gynecological malignant diseases.

A novel tumor-associated protein (TAP), that was originally detected immunologically through the use of a monospecific antiserum against a placental antigen, was quantified by means of the rocket technique of Laurell. Four hundred and fifty-seven serum samples were obtained from healthy female subjects (55), and patients with leiomyomas (162), benign ovarian tumors (78), pelvic endometriosis (45), cervical cancer (73), endometrial cancer (18) and ovarian cancer (26), respectively. Statistical analysis showed that TAP exhibited the closest relationship in ovarian cancer patients in whom the appearance of TAP and its high level were most prominent. The present preliminary study suggests the clinical usefulness of this protein as a clinical adjunct for the management of ovarian cancer.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1992

Chum salmon (Oncorhynchus keta) stanniocalcin inhibits in vitro intestinal calcium uptake in Atlantic cod (Gadus morhua).

Chum salmon (oncorhynchus keta) stanniocalcin was purified, partially identified and tested for bioactivity in an assay on the intestinal calcium uptake in a marine teleost (Gadus morhua). Basic ethanol extraction, ion exchange chromatography, gel filtration and reverse-phase high-performance liquid chromatography resulted in the isolation of a homogeneous glycoprotein that appears as a 46-kDa product under nonreducing conditions and as a 23-kDa product under reducing conditions after sodium dodecylsulphate-polyacrylamide gel electrophoresis. The glycoprotein is likely to be a homodimer composed of two subunits of 23 kDa each. Further characterization indicates homology to Australian eel, sockeye salmon, coho salmon and rainbow trout stanniocalcin, and the glycoprotein is thus concluded to be stanniocalcin. Stanniocalcin-like immunoreactivity was demonstrated in the corpuscles of Stannius of the Atlantic cod, with a specific antiserum raised against purified chum salmon stanniocalcin. The physiological importance and the biological activity of chum salmon stanniocalcin was tested by evaluating its effect on intestinal calcium uptake by the Atlantic cod in vitro. The intestine was perfused, both vascularly and through the intestinal lumen, and the calcium mucosa-to-serosa flux was measured using 45Ca2+ as a tracer. Stanniocalcin decreased the intestinal calcium uptake in a dose-related manner by 13.5% and 22.4% at doses of 2.2 and 10.9 nM stanniocalcin, respectively. The results establish the intestine as a target organ for stanniocalcin in marine teleosts.

Amino Acid Sequence↗

PubMed1992

Properties of common carp gonadotropin I and gonadotropin II.

Two gonadotropins, GtH I and GtH II, were extracted with 35% ethanol-10% ammonium acetate, pH 6.1, from female common carp pituitary glands and purified by ion-exchange chromatography on a DE-52 column followed by gel filtration on a Sephadex G-75 column. Molecular weights of GtH I and GtH II as determined by SDS-PAGE were 45,000 and 35,000, respectively. Both GtHs dissociate into two subunits following reduction with beta-mercaptoethanol. These subunits contain different N-terminal amino acids (Tyr and Gly for GtH I; Tyr and Ser for GtH II). GtH I was acid stable and did not dissociate into subunits following treatment with 0.1% trifluoroacetic acid; GtH II readily dissociated into subunits by this treatment. GtH I and GtH II have distinct elution profiles on reverse-phase HPLC. The N-terminal amino acid sequence of the beta-subunit of GtH II was identical to that of common carp maturational GtH described by other workers suggesting that GtH I is a newly identified molecule. This was supported by radioimmunoassay analysis. GtH II and a common carp maturational GtH preparation (F11 cGtH; Peter et al., 1982, J. Interdiscipl. Cycle Res. 13, 229-239) had similar immunological activity in tests with antisera to the beta-subunit of maturational GtH whereas GtH I had low (less than 6%) cross-reactivity. GtH I, GtH II, and F11 cGtH were equipotent in tests with antisera to the alpha-subunit of maturational GtH suggesting these molecules contain a similar alpha-subunit. In vitro bioassays using goldfish revealed that GtH I and GtH II share the same spectrum of biological activities causing stimulation of ovarian and testicular steroidogenesis and induction of oocyte final maturation. The demonstration of two chemically distinct GtHs in common carp is similar to what has been described for chum and coho salmon.

Amino Acid Sequence↗

PubMed1992

Maturational gonadotropin from the African catfish, Clarias gariepinus: purification, characterization, localization, and biological activity.

A gonadotropic hormone of the African catfish, Clarias gariepinus, was was purified and chemically characterized. Its biological activity was tested and its localization in the gonadotropic cells of the pituitary demonstrated. An ethanolic extract of 500 pituitaries of adult male and female African catfish was subjected to ion-exchange chromatography on DE-52. The 31- to 38-kDa fraction was further purified on Sephadex G-75. On rpHPLC over an ODS 120T column two major components appeared as single bands after SDS-PAGE. From the amino acid composition and sequence analysis of these fractions, compared with those of salmon and carp GTH II-alpha and salmon GTH II-beta it was concluded that they represent catfish GTH alpha- and II-beta-subunits. The biological activity of the complete hormone (the 31- to 38-kDa fraction from the G-75 column) was tested on the production of 11 beta-hydroxyandrostenedione and 17 alpha-hydroxy-20 beta-dihydroprogesterone by catfish testis in vitro. Polyclonal antibodies were raised against the purified beta-subunit. Immunocytochemical study using these showed them to bind specifically to hypophysial gonadotropic cells. To date only one form of GTH has been demonstrated in the African catfish.

Amino Acid Sequence↗

PubMed1992

A rapid procedure for the isolation of bioactive growth hormone.

A two-step isolation method is described for the purification of growth hormone and prolactin from rat pituitaries. Following alkaline extraction, the sample was chromatographed on Sephadex G-75. Growth hormone and prolactin, which eluted in the same gel filtration fraction, were separated on a reversed-phase HPLC column. The isolated proteins migrated to the same position as rat growth hormone and prolactin standards. Further confirmation that the purified proteins were indeed growth hormone and prolactin was provided by immunoblotting using several mammalian antisera and N-terminal amino acid sequencing. The purified rat growth hormone was shown to be biologically active when tested in the hypophysectomized rat body weight gain assay. Although rat prolactin remains to be tested, it seems likely that this hormone would also have biological activity. The present procedure is of general applicability and particularly useful when only small amounts of pituitary glands are available for extraction.

Amino Acid Sequence↗

PubMed1992

Stimulation of hepatic thyroxine 5'-deiodinase activity in rainbow trout (Oncorhynchus mykiss) by Pacific salmon growth hormone.

1. Growth hormone extracted from Pacific salmon pituitaries (sGH) was injected intra-peritoneally into rainbow trout to determine sGH effects on plasma levels of thyroxine (T4), 3,5,3'-triiodothyronine (T3) and properties of the hepatic 5'-deiodinase enzyme (5'-D) responsible for T4 to T3 conversion. 2. After 24 hr, sGH (0.1 or 0.5 microgram/g) did not alter the plasma T4 level or 5'-D, Km, but elevated plasma T3 and especially 5'-D Vmax. 3. Thus sGH, like the previously tested human GH, acutely enhances the potential for extra-thyroidal T3 production in trout by increasing the functional level of hepatic 5'-D, but without changing the plasma T4 level.

Animals↗

PubMed1992

Isolation and characterization of glycosylated and non-glycosylated prolactins from alligator and crocodile.

Two molecular forms of prolactin (PRL), glycosylated and non-glycosylated, were isolated from pituitary glands of two reptiles, alligator and crocodile. The reptilian PRLs were extracted under alkaline conditions from the precipitate obtained after pituitaries were first extracted with 0.25 M sucrose, 1 mM NH4HCO3, pH 6.3. Purification was performed by ion exchange chromatography on DE-52, gel filtration on Sephadex G-75 superfine, and reversed phase high performance liquid chromatography. Two forms of both alligator and crocodile PRL, designated PRLI and PRLII, with molecular weights of 26,000 and 24,000 were isolated. Alligator and crocodile PRLI and PRLII were stained specifically in immunoblots with anti-sea turtle PRL and anti-ostrich PRL. Sequence analysis revealed that both forms of alligator and crocodile PRLs consisted of 199 amino acid residues with a glycosylation consensus sequence (Asn-Ala-Ser) at position 60 in alligator and crocodile PRLs with a molecular weight of 26,000 (PRLI). In contrast, Thr was substituted for Asn at position 60 in the PRLs with a molecular weight of 24,000 (PRLII). The sequences of alligator PRLs differed from crocodile PRLs only in position 134: Val for alligator PRLs and Ile for crocodile PRLs. There is a high degree of structural conservation between the reptilian PRLs isolated in this study and avian PRL; each showed 92% sequence identity with chicken PRL and 89% with turkey PRL.

Alligators and Crocodiles↗

PubMed1992

Distribution of immunocompetent cells in the endolymphatic sac.

To better understand the role of immunocompetent cells in the defense mechanism of the inner ear, the distribution patterns of those cells were investigated in the endolymphatic sac (ES) of mice maintained in three different conditions: germ-free (GF), specific pathogen-free (SPF), and conventional (CV). In another experiment, the recruitment of lymphocyte subsets was examined in the ES of SPF rats undergoing a perilymphatic antigen challenge after systemic presensitization. In the ES of GF mice, no immunocompetent cells were found. In the ES of SPF and CV mice, cells positive for IgG, IgA, IgM, and Lyt-1 were present in much smaller numbers than in the nasal mucosa. Cells positive for Lyt-2 were not seen in the ES of any mice. In the ES of rats that underwent a perilymphatic antigenic stimulation after a systemic presensitization, B lymphocyte subsets (positive for IgG, IgA, IgM) were mobilized in increased numbers, and T cell subsets (helper/inducer and suppressor) were also found 1 week after perilymphatic antigen challenge. These results taken together suggest that the ES is not originally equipped to possess immunocompetent cells and mount an immune response, but that once it has been activated with the inner ear antigenic stimuli, the ES can be the active site of a local immune response of the inner ear.

Animals↗

PubMed1992

Experimental otitis media with effusion induced by middle ear effusion.

Experimental otitis media with effusion was induced in chinchillas by middle ear effusion, which was induced by an injection of immune complex into the tympanic cavity. To elucidate the pathogenesis of otitis media with effusion, cytologic and biochemical findings of the effusion and histopathology of the middle ear mucosa of effusion-induced chinchillas were compared with those of experimental otitis media with effusion induced by different procedures; eustachian tube obstruction, intratympanic inoculation of endotoxin, and immune reaction. No significant differences were seen in cytology, biochemistry, and histopathology among OMEs induced by these procedures. However, middle ear effusions, when compared with the corresponding sera, were proven to contain higher amounts of histamine and prostaglandin E2. These findings seem to demonstrate that middle ear effusion containing a large number of inflammatory mediators is essential for induction and prolongation of inflammatory reaction in the middle ear.

Animals↗

PubMed1992

Eel insulin: isolation, characterization and stimulatory actions on [35S]sulphate and [3H]thymidine uptake in the branchial cartilage of the eel in vitro.

Our previous studies have shown that mammalian and salmon insulins stimulate sulphate uptake by cultured eel cartilage, suggesting the possible involvement of insulin in the regulation of cartilage matrix synthesis. In the present study, homologous eel insulin was isolated and characterized, and its effects on cartilage matrix synthesis and DNA synthesis were examined in vitro. Insulin was extracted from eel pancreas with acid-ethanol, and subsequently purified by isoelectric precipitation at pH 5.3, gel filtration on Sephadex G-50, and reversed-phase high-performance liquid chromatography. The amino acid composition and complete sequence (50 residues) of eel insulin revealed high homology to teleostean and mammalian insulins. The isolated eel insulin produced a more pronounced and longer lasting hypoglycaemic effect than bovine insulin in the eel. Homologous eel insulin, like bovine insulin-like growth factor (IGF-I) and insulin, stimulated sulphate uptake by cultured eel cartilage in a dose-dependent manner (16-1000 ng/ml). Combination experiments using maximal concentrations of bovine IGF-I (250 ng/ml) and increasing amounts of eel insulin (10-250 ng/ml) showed no additive effects of insulin on sulphate uptake, suggesting that insulin and IGF-I may share a common mechanism(s) of action. Eel insulin and bovine IGF-I also enhanced thymidine incorporation by eel cartilage in a dose-dependent manner (4-1000 ng/ml); eel insulin was equipotent with bovine IGF-I. These results suggest that insulin, like IGF-I, may exert direct growth-promoting actions in branchial cartilage of the eel.

Amino Acid Sequence↗

PubMed1992

Somatolactin, a novel pituitary protein: purification and plasma levels during reproductive maturation of coho salmon.

Somatolactin (SL), a newly discovered fish pituitary protein belonging to the GH/prolactin family, was isolated from coho salmon (Oncorhynchus kisutch). Antibodies were raised to purified coho SL, and a homologous radioimmunoassay was developed and validated. The assay was specific for SL as indicated by the absence of cross-reactivity with coho salmon GH, gonadotrophins I and II and less than 0.2% cross-reaction to prolactin. Serial dilutions of plasma and pituitary extracts from Oncorhynchus species including coho salmon, chinook salmon and rainbow trout were parallel to the coho salmon SL standard curve. Displacement curves for dilutions of Atlantic salmon (Salmo salar) plasma, but not pituitary extract were parallel to the standards. Plasma levels of SL were measured in coho salmon throughout the final year of reproductive maturation. During the period of gonadal growth, plasma SL levels increased and were highly correlated to oestradiol levels in females and 11-ketotestosterone levels in males. Peak levels of SL were observed at the time of final maturation and spawning in both sexes. It is hypothesized that SL may regulate some physiological aspect of reproduction.

Amino Acid Sequence↗

PubMed1992

Chemical identification of catfish growth hormone and prolactin.

Isolation and primary structure of growth hormone (GH) and prolactin (PRL) from the pituitary gland of catfish (Ictalurus punctatus) are described. Alkaline extract of the pituitary glands was fractionated by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE-cellulose, and reversed-phase high-performance liquid chromatography on Octadecyl silica ODS. Catfish GH and PRL were identified by Western blotting with antisera against chum salmon GH and PRL. The catfish GH consists of 178 residues and is the most similar to carp GH, with sequence identity of 77%, although there is an uninterrupted deletion of 10 amino acid residues that corresponds to carp GH (90-99). The PRL is composed of 187 residues, which also exhibits the highest identity (79%) with carp PRL. Sequence identity between catfish GH and PRL is only 27%.

Amino Acid Sequence↗