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Biomedical subjects

H Kawauchi

Publications and source records attributed to H Kawauchi.

At least 73 records · Page 4Linked to original sources
PubMed1994

Inhibition of cell proliferation by Rana catesbeiana and Rana japonica lectins belonging to the ribonuclease superfamily.

Two frog egg lectins [Rana catesbeiana lectin (SBL-C) and Rana japonica lectin] preferentially agglutinate a large variety of human and animal tumor cells but not blood cells, lymphocytes, or fibroblasts. These lectins belong to the superfamily of pyrimidine base-specific RNases. The two lectins bound to a heparin-Sepharose column and were eluted from the column by an increase of NaCl molarity. Both their tumor cell-agglutinating activity and RNase activity were inhibited by heparin, and also by polyamines, such as spermine. Both lectins inhibited P388 leukemia cell proliferation. The inhibitory activity of SBL-C was blocked by addition of heparin. SBL-C inhibited protein synthesis by P388 cells, but RNase A did not. No lectin-induced antiproliferative effect was observed after sialidase treatment of cells. The antiproliferative activity of SBL-C was also inhibited by ammonium chloride treatment. These results suggest that internalization of the lectins by lectin receptor (sialoglycoconjugate)-mediated endocytosis is followed by cell death due to inhibition of protein synthesis. Administration of SBL-C i.p. delayed time to death in mice receiving i.p. transplants of Sarcoma 180 and Mep II cells.

Agglutination↗
PubMed1994

Isolation of growth hormone and in vitro translation of mRNA isolated from pituitaries of the gilthead sea bream Sparus aurata.

Growth hormone (GH) polypeptide was purified from pituitary glands of the gilthead sea bream (Sparus aurata) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each stage of purification, fractions were monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting using anti-bonito GH antiserum. The molecular weight of the sea bream GH was estimated by SDS-PAGE to be 21 kDa when electrophoresed in the absence of beta-mercaptoethanol (nonreduced conditions) and 22 kDa when electrophoresed under reduced conditions (in the presence of 1% beta-mercaptoethanol). Pituitary RNA was used to direct cell-free translation. When specific immunoisolation from 35S-labeled proteins was conducted, using antisera against Sparus or tilapia GH, a larger prehormone was immunoprecipitated. The size of the pre-GH was estimated to be 27-28 kDa under reduced conditions and 26-27 kDa under nonreduced conditions, in agreement with the calculated molecular weight of Sparus pre-GH of 26,296 based on the deduced amino acid sequence of Sparus GH cDNA. The specificity of the immunoprecipitation reaction was demonstrated by the ability of recombinant tilapia GH to compete with the radioactively labeled translation product. No such competition was found after the addition of BSA. Our results demonstrate that the sea bream GH is similar in its size to other purified fish GHs and provide direct evidence for the synthesis of GH as a prepeptide, thus supporting the conclusions presented earlier by GH cDNA cloning.

Animals↗
PubMed1994

Development of a homologous radioimmunoassay for coho salmon insulin-like growth factor-I.

A specific homologous radioimmunoassay (RIA) for measurement of insulin-like growth factor-I (IGF-I) in plasma of salmonid and a few non-salmonid fish species was developed using recombinant coho salmon IGF-I (rsIGF-I) as tracer and standard, and antiserum against this peptide raised in rabbits. The minimum detection level of IGF-I was 1.5 ng/ml and linearity was obtained in a range from 1.5 to 23 ng/ml. No cross-reaction was detected in the salmon IGF-I RIA with mammalian growth factors, salmon pituitary hormones, salmon or mammalian insulin, or any peptide in rat plasma. Although salmon IGF-I has high sequence similarity to mammalian IGF-I, it did not cross-react with anti-human IGF-I serum in human RIA and serial dilutions of plasma from salmon were not parallel to the human IGF-I standards in this assay system. In contrast, dilution curves for plasma of salmonids, such as coho (Oncorhynchus kisutch), Atlantic (Salmo salar), and sockeye (O. nerka) salmon, rainbow trout (O. mykiss), some other teleost fish, such as tilapia (Oreochromis mossabmica), carp (Cyprus carpio), eel (Anguilla rostrata), Atlantic halibut (Hippoglossus hippoglossus), and agnathan, the sea lamprey (Petromyzon marinus), assessed in salmon IGF-I RIA were parallel to the rsIGF-I standards. Acid-ethanol extraction of plasma samples altered the molecular weight, but not the quantity, of immunoreactive IGF-I, implying that IGF-I binding proteins in salmon plasma do not affect the performance of the salmon IGF-I RIA. Gel filtration of nonacidified plasma on a Sephadex G-75 superfine column produced two immunoreactive IGF-I peaks of molecular weights of approximately > 70 k and 7 kDa, whereas acidification of plasma increased the relative amount of the 7-kDa peak (IGF-I) and the > 70-kDa peak disappeared. The recoveries of rsIGF-I added to extracted or nonextracted plasma were 97.4 and 94.9%, respectively. Inter- and intraassay coefficients of variation were 3.6 and 3.3%, respectively. Plasma IGF-I levels in coho salmon smolts were 117.4 +/- 19.1 ng/ml as compared to IGF-I levels in parr (45.3 +/- 2.5 ng/ml) or in adult fish (45.2 +/- 5.4 ng/ml) measured in the same assay. Injection of salmon growth hormone, but not prolactin or somatolactin, caused a significant and dose-dependent elevation of plasma IGF-I levels, while either fasting or injection of streptozotocin led to a significant decline in systemic IGF-I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Purification and characterization of gonadotropin I and II from pituitary glands of tuna (Thunnus obesus)

The duality of teleost pituitary gonadotropins was established in an advanced marine fish, the tuna (Thunnus obesus). Two different molecular forms of gonadotropins, designated tGTH I and tGTH II, were isolated from an alcoholic extract of pituitary glands following ion-exchange chromatography and reversed-phase HPLC. Both tGTH I and tGTH II stimulated estradiol-17 beta and testosterone production in tuna ovarian follicles in vitro, although responses to tGTH II were significantly greater than those to tGTH I. Each gonadotropin consisted of alpha- and beta-subunits. tGTH I was stable in acidic conditions, whereas tGTH II dissociated into two subunits after acid treatment. Alpha subunits of tGTH I and tGTH II had identical amino acid sequences of 94 amino acid residues. The tGTH I beta and tGTH II beta consisted of 102 and 115 amino acid residues, respectively, and showed 35% sequence identity. tGTH I beta is structurally more similar to salmon GTH I beta than to salmon GTH II beta, whereas tGTH II beta is more similar to salmon GTH II beta. Thus it is evident that the tuna pituitary gland produces two chemically distinct gonadotropins.

Amino Acid Sequence↗
PubMed1994

Effect of S-carboxymethylcysteine on the clearance of middle ear effusion. An experimental study.

The therapeutic effect of S-carboxymethylcysteine (S-CMC) on otitis media with effusion was investigated by use of immune-mediated otitis media with effusion (OME) induced in chinchillas. The S-CMC was administered daily to individuals in two different doses (100 mg/kg and 200 mg/kg) for 2 weeks after the induction of immune-mediated otitis media. The severity of OME was evaluated by observation of the tympanic membrane under a surgical microscope, by tympanogram, and by histologic findings of the middle ear mucosa. Results of the study showed that the oral administration of S-CMC at a dose of 200 mg/kg of body weight is more effective than at a dose of 100 mg/kg in the clearance of middle ear effusions in experimental OME. An autoradiographic study using 14C-S-CMC revealed that orally administered S-CMC is rapidly absorbed by the digestive tract and transported to the tubotympanum via the circulation. The uptake and excretion by secretory cells were apparently greater in activated secretory cells. However, S-CMC does not act on the infiltrating cells to prevent the release of chemical mediators such as histamine and prostaglandin E2. This finding suggests that prophylactic use of S-CMC may not be anticipated.

Animals↗
PubMed1994

Pit-1/GH factor-1 involvement in the gene expression of somatolactin.

A presumed pituitary hormone, somatolactin (SL), belonging to the GH/PRL family, is produced in the intermediate lobe of teleost pituitary. The pituitary-specific transcription factor, Pit-1, is indispensable not only for the expression of mammalian GH and PRL genes, but for the development of GH- and PRL-producing cells present in the anterior lobe of the pituitary as well. Thus, in this study, examination was made of the possible involvement of Pit-1 in the intermediate lobe-specific expression of the SL gene. Using antibodies against chum salmon Pit-1, the presence of the 40-kilodalton Pit-1 protein in anterior and neurointermediate lobes of rainbow trout pituitary was demonstrated. By immunohistochemical examination, Pit-1 protein was shown to be located in the nuclei of SL-producing cells in the intermediate lobe. In experiments of cotransfection into HeLa cells, rat Pit-1 enhanced the promoter activity of SL gene, and a 0.5-kilobase upstream region from the transcriptional start site was sufficient for this enhancement. It follows from these results that Pit-1 protein may possibly be involved in SL gene expression as well as the development of SL-producing cells.

Animals↗
PubMed1994

[Partial purification and properties of deoxyribonucleases from eggs and liver of Xenopus laevis. Comparison with deoxyribonuclease II from bovine spleen].

Deoxyribonucleases from eggs and the liver of Xenopus laevis were partially purified by DEAE-cellulose and heparin-Sepharose affinity column chromatographies. The fractions having egg and liver DNase activities were eluted on high performance liquid chromatography through TSK gel G3000SW at the molecular weights of 41.5 and 45 kDa, respectively. The frog DNases hydrolyzed a native DNA over a heat-denatured DNA, and also formed double-strand cuts not only in linear lambda-DNA but also in closed circular pBR322DNA. The pH optimum of the DNases was 4.5-5.0 in 50 mM acetate buffer. These enzyme activities were abolished by treatment at 80 degrees C for 5 min and pH 2, 3 or 12 for 1 h. The enzymes act in such a manner as deoxyribonuclease II (from bovine spleen)-type nuclease with respect to substrate specificity, optimum pH and cation dependence.

Animals↗
PubMed1993

Lungfish prolactin exhibits close tetrapod relationships.

This paper describes the isolation and the complete amino-acid sequence of prolactin (PRL) from the pituitary glands of African lungfish, Protoputerus aethiopicus. We purified the hormone from an alkaline extract of the pituitaries using a two-step chromatographic procedure by detecting specific immunoblot reactivity with rabbit antisera against salmon PRL. The lungfish PRL consists of 200 amino-acid residues. Sequence comparison revealed that the PRL shows 66% identities with amphibian, reptilian and bird PRLs, 57% with mammalian PRLs, and 38% with teleost (modern bony fish) PRLs. Moreover, the PRL contains three disulfide bonds homologous to those of tetrapod PRLs and differs from teleost PRLs which lack the amino-terminal disulfide bond. Thus, the structural features of lungfish PRL indicate a closer relationship to tetrapod PRLs than to teleost PRLs. All PRLs sequenced to date share 22 common amino acids, which may be important for the activities common to all PRLs.

Amino Acid Sequence↗
PubMed1993

cDNA cloning of the beta subunit of teleost thyrotropin.

cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human follitropin (42%), human lutropin (32%), and salmon gonadotropin (31-33%) beta subunits. The identification of TSH in addition to two gonadotropins (gonadotropins I and II) in the teleost fish suggests that the divergence of three kinds of glycoprotein hormones from an ancestral molecule took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods. Northern blot analysis showed that the expression of the rainbow trout TSH beta gene is specific to the pituitary gland and is significantly higher in immature fish than in mature fish, suggesting that TSH plays some role in the biological processes of immature fish.

Age Factors↗
PubMed1993

Agglutinins from aquatic insects--tumor cell agglutination activity.

Agglutinins were identified in whole body extracts of aquatic insects by means of murine tumor cell agglutination, using sarcoma 180 ascites, Ehrlich, and MM-46 cells. Screening revealed agglutinins in 5 of 10 of the larvae tested, and in 2 of 6 of the water-dwelling adult insects; Gerris paludum insularis and Gyrinus japonicus. Only the agglutinin from adult G. paludum also agglutinated human erythrocytes. An ascites tumor was converted into a solid form in vivo after administration of G. paludum agglutinin. The observation that these aquatic insect agglutinins preferentially agglutinate tumor cells has considerable implications in terms of anti-tumor effects such as inhibition of cell proliferation and metastasis.

Agglutination↗
PubMed1993

Alpha,N-acetyl beta-endorphin [1-8] is the terminal product of processing of endorphins in the melanotrope cells of Xenopus laevis, as demonstrated by FAB tandem mass spectrometry.

The major N-terminal acetylated endorphin of the pars intermedia of Xenopus laevis was purified and submitted to fast-atom bombardment tandem mass spectroscopy. The collisionally induced dissociation MS/MS spectrum of the [M+H]+ ion revealed sufficient fragment ions to determine unambiguously the identity of the peptide as alpha,N-acetyl beta-endorphin [1-8], the sequence of which was predicted on the basis of the nucleotide sequence of Xenopus POMC cDNA. The determination was confirmed by showing that the synthetic peptide of this structure had identical FAB tandem mass spectrometric characteristics as the endogenous endorphin. We conclude that alpha,N-acetyl beta-endorphin [1-8] is the terminal product of processing of endorphins in the melanotrope cell of Xenopus laevis.

Animals↗
PubMed1993

Flounder growth hormone: isolation, characterization, and effects on growth of juvenile rainbow trout.

A highly purified growth hormone (GH) was isolated from the pituitary of the Japanese flounder (Paralichthys olivaceus) following extraction with alkaline solution, gel filtration on Sephadex G-75, and high-performance liquid chromatography (HPLC) on a reverse-phase column. Flounder GH (flGH) has a molecular weight of 20 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and an isoelectric point of 7.1 by gel electrofocusing. The complete amino acid sequence was determined by Edman degradation of the peptide fragments generated by enzymatic digestion and chemical cleavage. The flGH comprises 173 amino acid residues with pyroglutamate at the N-terminus. Compared with other fish GHs, flGH has an uninterrupted deletion of 14 amino acid residues near the C-terminus. flGH is the smallest of growth hormones so far reported and is a potent stimulator of growth in juvenile rainbow trout.

Amino Acid Sequence↗
PubMed1993

Isolation of prolactin and growth hormone from the pituitary of the holostean fish Amia calva.

Pituitaries from adult male and female Amia calva (Order Holostei) were acid extracted and fractionated by gel filtration column chromatography and reversed-phase high performance liquid chromatography. This two-step isolation procedure yielded homogeneous pools of Amia prolaction (PRL) and growth hormone (GH). The amino acid composition of both purified polypeptides was determined. Primary sequence analysis of the first 22 positions at the N-terminal of Amia PRL revealed that this region has 63% sequence identity with eel PRL-1. The N-terminal region of Amia PRL lacks the disulfide bridge which is characteristic of tetrapod PRLs. Primary sequence analysis of the first 24 positions at the N-terminal of Amia GH revealed that this region has 62% sequence identity with eel GH and 54% sequence identity with both blue shark GH and sea turtle GH. Based on N-terminal analysis, it appears that Amia PRL and GH are more closely related to teleost PRLs and GHs than they are to tetrapod PRLs and GHs.

Amino Acid Sequence↗
PubMed1993

The primary structure of sturgeon prolactin: phylogenetic implication.

The complete amino acid sequence of prolactin (PRL) from a chondrostean species, the sturgeon (Acipenser gueldenstaedti), has been determined. Sturgeon PRL was isolated from the pituitary glands by gel filtration on a Sephadex G-25 column and high-performance liquid chromatography on a reverse-phase column following acid-acetone extraction. Sturgeon PRL was identified by immunoblot reactivity using antisera against salmon and ovine PRL. It consists of 204 amino acid residues, which is the largest among known PRLs, and contains three disulfide bonds corresponding to those of tetrapod PRLs. Sequence comparison with PRLs from other vertebrates revealed that sturgeon PRL has slightly higher sequence identities (35-46%) with teleost PRLs than with tetrapod PRLs (30-40%). These structural characteristics imply that an ancestor of the ray-finned fishes had PRL with three disulfide bonds and at some point after divergence of Chondrostei, the disulfide bond in the amino-terminus of PRL was lost.

Amino Acid Sequence↗
PubMed1993

Ribonuclease activity of sialic acid-binding lectin from Rana catesbeiana eggs.

Sialic acid-binding lectin (SBL) isolated from Rana catesbeiana eggs is a basic protein which agglutinates a large variety of tumour cells and has an amino acid sequence homologous to that of human angiogenin and pancreatic ribonuclease (RNase). Although SBL and angiogenin lack the Cys-65-Cys-72 disulphide bond of pancreatic RNase, the locations of the other three disulphide bonds are similar among the three molecules. SBL was found to exhibit RNase activity, as well as catalytic properties resembling those of bovine RNase A in some respects. For example, SBL hydrolyses poly(uridylic acid) and poly(cytidylic acid) as substrates, and prefers the former. RNase A and angiogenin are strongly inhibited by human placental RNase inhibitor, whereas the RNase activity and tumour cell agglutination activity of SBL are not affected by this inhibitor.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of two distinct gonadotropins, GTHI and GTHII, from bonito (Katsuwonus plelamis) pituitary glands.

Two distinct glycoproteins homologous to chum salmon GTHI and GTHII were isolated from the pituitary glands of a marine fish, the bonito (Katsuwonus plelamis), and characterized by amino acid sequence analysis in order to obtain additional evidence for duality of teleost GTHs. Glycoproteins were extracted from the pituitary glands, and intact GTHI and GTHII, consisting of two distinct subunits, were purified by ion-exchange chromatography on DEAE-cellulose, rpHPLC on Asahipak C4P-50 in alkaline buffer, and gel filtration on Superdex 75. The association of the subunits was stable in GTHI (39 kD) and unstable in GTHII (30 kD) in acidic conditions. Immunoblotting revealed that antisera against beta subunits of chum salmon GTHs reacted with GTHII, but not with GTHI. In addition, none of the GTHs was stained with antiserum against human TSH beta. Sequence analysis demonstrated that bonito GTHI beta is homologous to salmon GTHI beta with 43% sequence identity, and bonito GTHII beta is homologous to salmon GTHII beta with 67% identity. Sequence identity between bonito GTHI beta and GTHII beta was only 28%. Thus, it is evident that the bonito pituitary gland produces two chemically distinct gonadotropins homologous to chum salmon GTHs.

Amino Acid Sequence↗