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Biomedical subjects

K Hotta

Publications and source records attributed to K Hotta.

At least 19 recordsLinked to original sources
PubMed1996

Construction of pRES18 and pRES19, Streptomyces-Escherichia coli shuttle vectors carrying multiple cloning sites.

We developed two Streptomyces-Escherichia coli shuttle vectors. The plasmid pRES102, consisting of the essential region of pRES1 and the thiostrepton resistance gene (tsr) fragment of pIJ702, was combined with the E. coli plasmid vector pUC18 or pUC19. The resulting shuttle vectors, designated pRES18 and pRES19, respectively, have relatively compact size (6.25 kb), low copy number, multiple cloning sites reciprocally arranged in opposite directions, and selection markers for both Streptomyces (tsr) and E. coli (beta-lactamase (bla) and beta-galactosidase (lacZ). These shuttle vectors are capable of carrying DNA fragments as long as 10 kb, of being maintained in S. griseus, S. lavendulae and S. lividans, and are compatible with pIJ702.

Cloning, Molecular↗
PubMed1996

Regulation of obese (ob) mRNA and plasma leptin levels in rhesus monkeys. Effects of insulin, body weight, and non-insulin-dependent diabetes mellitus.

We have cloned the rhesus monkey obese cDNA and have analyzed its expression in monkeys with a wide range of body weights (lean to very obese) and with or without non-insulin-dependent diabetes mellitus to examine the relationship of ob gene expression to obesity and non-insulin-dependent diabetes mellitus. The sequence of monkey ob protein, excluding the signal peptide, showed 91% identity with the human protein. We observed a significant correlation between the level of ob mRNA and body weight. We also found a significant relationship between ob mRNA and fasting plasma insulin concentration; however, insulin stimulation during a 100-140-min euglycemic/hyperinsulinemic clamp did not result in any changes in ob mRNA levels. Circulating levels of the ob gene product leptin were also significantly correlated with body weight. These results show that ob gene expression is related to body weight and is not acutely regulated by insulin.

Amino Acid Sequence↗
PubMed1996

Interaction of a GRB-IR splice variant (a human GRB10 homolog) with the insulin and insulin-like growth factor I receptors. Evidence for a role in mitogenic signaling.

We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor. We identified a human cDNA that is a splice variant of the human GRB10 homolog GRB-IR, which we term GRB10/IR-SV1 (for GRB10/GRB-IR splice variant 1). The protein encoded by the GRB10/IR-SV1 cDNA contains an SH2 domain and a pleckstrin homology domain. Cloning of a full-length human cDNA revealed a predicted coding sequence that was similar to the mouse GRB10 protein, although GRB10/IR-SV1 contained an 80-amino acid deletion. The GRB10/IR-SV1 cDNA is a splice variant of the GRB-IR cDNA such that GRB10/IR-SV1 contains an intact pleckstrin homology domain and a distinct amino terminus. The interaction of GRB10/IR-SV1 with the insulin receptor and the insulin-like growth factor I (IGF-I) receptor is mediated by the SH2 domain, and we show that glutathione S-transferase-SH2 domain fusion proteins interact specifically in vitro with the insulin receptor derived from mammalian cells. The GRB10/IR-SV1 SH2 domain also interacted with an approximately 135-kDa phosphoprotein from unstimulated cell lysates, an interaction that decreased after insulin stimulation. We present evidence that the GRB10/IR-SV1 protein plays a functional role in insulin and IGF-I signaling by showing that microinjection of an SH2 domain fusion protein inhibited insulin- and IGF-I-stimulated mitogenesis in fibroblasts, yet had no effect on mitogenesis induced by epidermal growth factor. Our findings suggest that GRB10/IR-SV1 may serve to positively link the insulin and IGF-I receptors to an uncharacterized mitogenic signaling pathway.

Adipose Tissue↗
PubMed1996

Peripheral alpha-linked N-acetylglucosamine on the carbohydrate moiety of mucin derived from mammalian gastric gland mucous cells: epitope recognized by a newly characterized monoclonal antibody.

To obtain a tool to study the structural characterization and the detection of mucin derived from the gastric gland mucous cells, we developed a monoclonal antibody, designated HIK1083, against mucin purified from rat gastric mucosa. In an ELISA, HIK1083 reacted strongly with the mucin purified from a deep layer of the corpus and antrum but only slightly reacted with that obtained from the surface mucosal layer. The reaction of mucin and HIK1083 was inhibited by the oligosaccharides obtained by the alkaline borohydride reduction of antigenic mucin. Two purified oligosaccharide alditols reacting with the monoclonal antibody obtained from the antigenic mucin had one and two peripheral alpha-linked N-acetylglucosamine residues, respectively, according to the evidence from NMR spectroscopy. Moreover, among the commercially available p-nitrophenyl derivatives of monosaccharides, only p-nitrophenyl-N-acetyl-alpha-D-glucosaminide inhibited the reaction of this monoclonal antibody and the antigenic mucin in a concentration-dependent manner. These results, as well as the immunohistochemical observations, indicate that alpha-linked N-acetylglucosamine residues are specifically attached to the peripheral region of the carbohydrate moiety of the mucin synthesized in and secreted from the gastric-gland-type cells, and indicate that the monoclonal antibody HIK 1083 recognizes this structure.

Acetylglucosamine↗
PubMed1996

Interaction of the Rho family small G proteins with kinectin, an anchoring protein of kinesin motor.

The Rho family small G proteins are implicated in various cell functions, such as cell morphological change, cell motility, and cytokinesis. However, their modes of action in regulating these cell functions remain to be clarified. In the present study, we have isolated a cDNA encoding a protein which interacts with the GTP-bound form, but not with the GDP-bound form, of the Rho family members, including RhoA, Racl, and Cdc42, by the yeast two-hybrid method. This protein is kinectin, known to be a vesicle membrane anchoring protein of kinesin, which is an ATPase motor transporting vesicles along microtubules.

Binding Sites↗
PubMed1996

Stimulation of mucin biosynthesis in rat gastric mucosa by FRG-8813 and its structural analogs.

Certain chemical properties, which may determine the stimulatory actions of the new histamine H2 receptor antagonist, FRG-8813 (2-(furfurylsulfinyl)-N-(4-[4-(piperidinomethyl)-2-pyridyl]o xy-(Z)- 2-butenyl)acetamide), on mucin biosynthesis, were identified by considering the derivation of this drug using an organ culture system of the rat stomach. [3H]Glucosamine and [35S]sulfate incorporation was stimulated in the corpus region by FRG-8813 and its structural analog, compound A (N-[4-[[4- (piperidinylmethyl)pyridyl]-2-oxy]-(Z)-2-butenyl]phthalimide). The chronotropic response to histamine in the guinea pig right atria was suppressed by FRG-8813 in a concentration-dependent fashion. In contrast, compound A did not suppress the histamine-induced response. Ranitidine at 10(-4) M did not suppress the FRG-8813-induced increase in [3H]glucosamine incorporation into mucin. These results suggest that the pyridine derivative and amide structure are chemically important in FRG-8813 as a stimulant on mucus metabolism. Also, this effect is not directly due to histamine H2 receptor antagonism.

Acetamides↗
PubMed1996

Altered expression of carnitine palmitoyltransferase II in liver, muscle, and heart of mouse strain with juvenile visceral steatosis.

We conducted a quantitative study of the effect of carnitine deficiency on the mRNA level of carnitine palmitoyltransferase II in the liver, muscle and heart of mice with juvenile visceral steatosis, a strain that is systematically deficient in carnitine. The amount of carnitine palmitoyltransferase II mRNA was increased in liver and muscle of homozygotes, as compared with heterozygotes and normal controls, at 2, 4, and 8 wk of age. The mRNA levels of this enzyme were normalized after carnitine administration. The mRNA level of carnitine palmitoyltransferase II in the heart was increased only at 8 wk, and was not affected by carnitine administration. These results suggest that carnitine displays some effect on the mRNA level of the carnitine palmitoyltransferase II gene in liver and muscle, probably through fatty acid metabolic change.

Ammonia↗
PubMed1996

Disordered expression of hepatic glycolytic and gluconeogenic enzymes in Otsuka Long-Evans Tokushima fatty rats with spontanteous long-term hyperglycemia.

Expression of key regulatory enzymes involved in glucose metabolism was studied in the livers of Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of non-insulin dependent diabetes mellitus. The activity and mRNA levels of glucokinase and L-type pyruvate kinase was increased in the liver of OLETF rats compared with control rats. There was no such remarkable change in liver-type phosphofructokinase. The activities of glucose-6-phosphatase and fructose-1,6-biphosphatase also increase despite high plasma levels of glucose and insulin. The activity of phosphoenolpyruvate carboxykinase did not show any significant change. The mRNA levels for fructose-1,6-biphosphatase, and phosphoenolpyruvate carboxykinase exhibited no marked changes. These results suggest that the expression of glucose-6-phosphatase and fructose-1,6-biphosphatase is disordered in OLETF rats.

Animals↗
PubMed1996

Establishment of monoclonal antibodies against carbohydrate moiety of gastric mucins distributed in the different sites and layers of rat gastric mucosa.

Eight monoclonal antibodies (MAbs), designated RGM21 approximately RGM42, were generated against mucin purified from the rat gastric mucosa. By applying ELISA, all of these MAbs were proved to react not only with the purified mucin, but also with the oligosaccharide mixture obtained from the antigenic mucin by alkaline borohydride treatment. Treatment of the mucin-attached ELISA well with trypsin, sodium periodate or galactose oxidase prior to the addition of the MAb was applied to characterize these MAbs. Histochemical observation indicated that all these MAbs were able to stain the formalin fixed-paraffin embedded sections of the rat gastroduodenal mucosa. Although each of these MAbs reacted with distinct mucus-producing cells localized in particular regions of the gastroduodenal mucosa, their staining specificity could generally be classified into four groups. These MAbs might be useful for estimating the physiological and pathological changes of mucins in the gastric mucosa.

Animals↗
PubMed1996

Mechanisms for cytoprotection by vitamin U from ethanol-induced gastric mucosal damage in rats.

A comparison was made of the effects of a nonsulfhydryl compound, vitamin U (methylmethioninesulfonium chloride, MMSC), and a sulfhydryl compound, cysteine (Cys), with regard to the inducement of acute gastric mucosal damage in the presence and absence of N-ethylmaleimide (NEM), a sulfhydryl-blocking reagent. The effects of MMSC, Cys, or NEM on gastric mucin content were examined using a newly developed biochemical method. MMSC and Cys inhibited mucosal damage due to 50% ethanol. The preinjection of NEM had no effect on cytoprotection of prostaglandins, but prevented the effects of Cys and MMSC. MMSC and Cys increased surface mucin content but lessened that of deep mucin. NEM decreased surface mucin and increased deep mucin. It thus follows that sulfhydryl compounds accelerate the secretion of deep mucin and accumulate surface mucin. The cytoprotective mechanism of MMSC may thus be mediated by sulfhydryl compounds, and the increase in surface mucosal mucin may possibly be related to cytoprotection.

Animals↗
PubMed1996

Disordered expression of glycolytic and gluconeogenic liver enzymes of juvenile visceral steatosis mice with systemic carnitine deficiency.

A quantitative study of the effect of carnitine deficiency on expression of glycolytic and gluconeogenic enzymes was performed using juvenile visceral steatosis mice which are systemically deficient in carnitine. The amounts of glucokinase and L-type pyruvate kinase mRNA were reduced in homozygotes, compared to heterozygotes and normal controls at 2 and 8 weeks. Liver-type phosphofructokinase, however, did not differ significantly. The abundance of fructose 1,6-bisphosphatase mRNA was unchanged at 2 and 8 weeks. The level of phosphoenolpyruvate carboxykinase mRNA was increased slightly at 2 weeks, but not at 8 weeks. A part of these changes could not be explained by the plasma glucose or insulin level. Carnitine administration restored the mRNA of these enzymes to normal levels. These results suggest that carnitine deficiency affects the expression of these liver enzymes.

Age Factors↗
PubMed1996

Effects of traditional herbal medicine on gastric mucin against ethanol-induced gastric injury in rats.

The effect of the traditional herbal medicine, Rikkunshi-to and its component crude drugs, Zingiberis Rhizoma and Glycyrrhizae Radix, on the gastric mucin was studied using a method developed to separate and quantify the mucin localized in the different layers of rat gastric mucosa. The oral administration of spray-dried extract to Rikkunshi-to (1000 mg/kg), Zingiberis Rhizoma (500 mg/kg) and Glycyrrhizae Radix (500 mg/kg) significantly prevented gastric mucosal damage induced by 70% ethanol in rats. In ethanol-treated rats the mucin content of the deep mucosa was reduced, and the reduction of the deep corpus mucin content was significantly inhibited by pretreatment of Rikkunshi-to and Zingiberis Rhizoma. Rikkunshi-to and Glycyrrhizae Radix pretreatment increased the surface mucin content by 140 and 146%, respectively. The effect on the gastric mucin by each drug differed in the different layers of the gastric mucosa.

Administration, Oral↗
PubMed1996

Abundance of Gal beta 1,3GalNAc in O-linked oligosaccharide on hinge region of polymerized IgA1 and heat-aggregated IgA1 from normal human serum.

Gas-phase hydrazinolysis was used to analyze the glycoform of the O-linked oligosaccharide of human serum IgA1. In our previous report, only one glycoform was obtained from the IgA1 of healthy individuals. However, it was found to be composed of heterogeneous IgA1 components having mutually different glycoforms. First, the IgA1 was separated into two subfractions having different affinities toward jacalin. Among them, the high-affinity subfraction was mainly composed of polymerized IgA1. Comparative study of the carbohydrate chain showed a relative abundance of Gal beta 1,3GalNAc in the polymerized form. A simultaneous analysis of the N-glycan of these subfractions was also carried out. Three major components, two biantennary and one triantennary oligosaccharides, were obtained from both subfractions and the relative contents of these components were almost the same. On the other hand, IgA1 was artificially polymerized by heating at 63 degrees C for 2 h. The heat-stable IgA1 was separated from the heat-aggregated material on a Sephacryl S-300 column. The obtained heat-stable IgA1 (approximately 20%) was not further aggregated by more heating under the same conditions. The heat-stable IgA1 contained a much higher amount of the sialylated Gal beta 1,3GalNAc. Thus, it was shown that the degree of completeness of the hinge O-linked oligosaccharide might be correlated with the stability and polymerization process of the IgA1 molecule.

Acetylgalactosamine↗
PubMed1996

Estimation of the number of O-linked oligosaccharides per heavy chain of human serum IgA1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the hinge glycopeptide.

In our previous study, gas-phase hydrazinolysis was used to analyze the glycoform of the O-linked oligosaccharide of human serum IgA1. All O-linked oligosaccharide chains are known to be present in the hinge portion. However, the number of O-linked oligosaccharide chains on IgA1 remained unclear. In order to determine the number of linked sugar chains, we applied matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) to the hinge glycopeptide prepared from human serum IgA1. MALDI-TOFMS did not show clear peaks, probably due to the microheterogeneity of the structure of each sugar chain. However, elimination of peripheral sialic acid and galactose residues by sequential treatment with neuraminidase and beta-galactosidase gave clear mass spectra with several sharp peaks. On the basis of these spectra, we conclude that IgA1 prepared from normal human serum carries different numbers of sugar chains. There are two major populations, one contains five GalNAc residues and the other four GalNAc residues. On the other hand, the hinge glycopeptide prepared from myeloma IgA1 was composed mainly of one population containing four GalNAc residues. Earlier, we reported incomplete glycosylation of IgA1 isolated from the serum of an IgA1 myeloma patient. In this experiment, the presence of four O-linked oligosaccharides per heavy chain of IgA1 from a myeloma patient was found. The reason why only four out of five sites on the hinge glycopeptide were fully glycosylated in the IgA1 from the IgA1 myeloma patient is not clear.

Amino Acid Sequence↗
PubMed1996

ROM7/BEM4 encodes a novel protein that interacts with the Rho1p small GTP-binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the deltabem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the deltabem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.

Base Sequence↗
PubMed1996

Reactivity of glomerular and serum IgA1 to jacalin in IgA nephropathy.

To analyze O-linked oligosaccharides (O-glycans) in the hinge region of IgA1 in IgA nephropathy (IgAN), the reactivity of IgA1 to jacalin, which specifically binds to O-glycans, was investigated. Initially, renal biopsy specimens from 5 patients with IgAN and 3 patients with other renal diseases were investigated in an immunofluorescence study with jacalin, monoclonal antihuman IgA1 and IgA2 antibodies. All of the renal biopsy specimens of IgAN and none of other renal diseases were positively stained by both FITC-labeled jacalin and monoclonal anti-IgA1 antibody. The glomerular staining patterns of FITC-jacalin were similar to those of the monoclonal anti-IgA1 antibody. IgA2 was negative in all specimens. Based on the positive reactivity of deposited IgA1 to jacalin, the binding ability of serum IgA1 to jacalin was evaluated by inhibition assay using D-galactose in patients with IgAN (n = 58), other primary glomerulonephritides (PGN) (n = 41), and healthy controls (n = 52). The frequencies of the patients with serum IgA1 having a high affinity for jacalin were significantly greater in IgAN (19/58, 32.8%) compared with the healthy controls (2/52, 3.8%) and other PGN (4/41, 9.8%). These results suggested that the increased reactivity of O-glycan(s) in the IgA1 hinge region to jacalin is due to an unusual glycosylation of serum IgA1 in IgAN.

Adult↗
PubMed1996

Association of asialo-galactosyl beta 1-3N-acetylgalactosamine on the hinge with a conformational instability of Jacalin-reactive immunoglobulin A1 in immunoglobulin A nephropathy.

The O-glycans in Jacalin-binding immunoglobulin A1 (IgA1) were released by gas-phase hydrazinolysis and were fractionated by gel filtration and reversed-phase HPLC. Four peaks (P1 to P4) were obtained. There was a significant shift from Peak 2 (monosialylated Gal beta 1-3GalNAc) to Peak 4 (asialo-Gal beta 1-3GalNAc) in the IgA-nephropathy group compared with the negative control group (P < 0.05). One of the functions of the carbohydrate side chains is to stabilize the three-dimensional structure of the glycopeptides. In order to evaluate the stability of the Jacalin-binding IgA1 molecule, the increase in turbidity as a consequence of the increase of the aggregated IgA1 level under the condition of high temperature (63 degrees C) was observed. The increase in the turbidity was significantly higher in the IgA nephropathy group compared with the negative control group (21.7 vs. 5.9% at 150 min, P < 0.02). From a sample of IgA1 solution that had originated from a pooled normal serum, heat-tolerant (nonaggregated) and intolerant (aggregated) IgA1 molecules were separated by gel filtration. The heat-intolerant IgA1 had lower amounts of sialic acid (27.4 micrograms/mg IgA1) than the tolerant IgA1 (37.6 micrograms/mg IgA1). Further, the analysis of the O-glycans released from another IgA1 sample by the hydrazinolysis revealed that the ratio of the asialo-Gal beta 1-3GalNAc/total Gal beta 1-3GalNAc in the heat-in-tolerant IgA1 (27.2%) was increased compared with that in the heat-tolerant IgA1 (18.2%). From these results, it was suggested that unusual glycosylation on the hinge region of Jacalin-binding IgA1 induced an insufficient conformational stiffness to the hinge peptide, resulting in the aggregation of the IgA1 molecule in IgA nephropathy.

Acetylgalactosamine↗