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K Hotta

Publications and source records attributed to K Hotta.

At least 199 records · Page 11Linked to original sources
PubMed1992

A separating method for quantifying mucus glycoprotein localized in the different layer of rat gastric mucosa.

A method was devised for separating rat gastric mucosa into three layers each containing a different mucin species. The mucus gel (first layer) was removed by stirring the gastric mucosa in a solution of phosphate-buffered saline containing 2% N-acetylcysteine. The surface mucosa (second layer), rich in surface mucus cells, was then separated from the deep mucosa (third layer) containing mucus neck cells, by scraping with forceps. The effectiveness of this method was confirmed by light microscopical observation after GOCTS-PCS (dual staining by the galactose oxidase-cold thionin Schiff method and paradoxical concanavalin A method) and AB-PAS staining (dual staining with alcian blue and the periodic acid Schiff method). The fixed specimen of scraped mucus and cell debris was rich in AB-PAS and GOCTS positive mucus, but was hardly stained by PCS, indicating mucus derived from surface mucus cells to have been efficiently recovered from this preparation. The residual mucosa could be stained by PCS but hardly at all by AB-PAS or GOCTS. The lyophilized powder specimens obtained from the three different layers of rat gastric mucosa were used to extract and quantify mucus glycoprotein (mucin). This was done to examine changes in mucin content in the three layers of gastric mucosa one hour following the oral administration of 20% ethanol or 0.35 N hydrochloric acid, both mild irritants. Mucin content was noted to significantly increase in the first layer but hardly at all in the second layer. In the third layer, it decreased significantly by 0.35 N hydrochloric acid, but changed only slightly by 20% ethanol administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effect of 4-MET on bond strength and penetration of monomers into enamel.

The effect of 4-[2-(methacryloyloxy)ethoxycarbonyl]phthalic acid (4-MET) in self-curing acrylic resin initiated by BPO-amine on the bond strength to etched enamel was studied. Scanning electron microscopic (SEM) observations of resin-enamel interfaces were carried out to elucidate the bonding mechanism and the effect of 4-MET on the adhesion. 4-MET, which has both hydrophilic and hydrophobic groups, increased the bond strength of self-curing acrylic resin to etched enamel and improved the bonding stability. SEM pictures strongly suggested that 4-MET promoted interpenetration of monomers into enamel prism peripheries and their polymerization therein resulted in excellent adhesion to etched enamel.

Acid Etching, Dental↗
PubMed1992

Isolation and partial characterization of serine- and threonine-rich porcine gastric mucus glycopeptides.

1. Two subfractions from purified porcine gastric mucus glycopeptide were found to separate from each other by cesium chloride equilibrium centrifugation. The highest density fraction and two lower density fractions separated were designated VHD, HD and LD, respectively. A comparative study of these components was made. 2. The high and low density fractions, HD and LD, appeared almost the same or identical, while VHD differed completely from either of them in the following respects: (1) VHD exhibited strong alcian blue binding activity. (2) 57% of VHD bound to the DEAE-Toyopearl column equilibrated with 0.2 M NaCl. (3) VHD eluted from the Sephacryl S-400 column as a lower molecular subunit. (4) One third of the sialic acid as a minor component in VHD was constituted by N-glycolylneuraminic acid. (5) Carbohydrate composition showed typical mucus glycoprotein with slightly higher fucose content. (6) Amino acid compositions of the anionic components prepared from VHD showed the highest Ser/Thr ratio, 1.92 compared to 0.46 for LD and 0.62 for HD. (7) Oligosaccharide released from VHD by alkaline-sodium borohydride treatment was larger than that from HD or LD. 3. The above results indicate the minor component, VHD, separated from the major components, to be a quite similar but not identical component to the so-called sulfated mucus glycoprotein reported previously [Slomiany et al. (1972) J. biol. Chem. 247, 5062-5070].

Alcian Blue↗
PubMed1992

Analysis of porcine gastric mucus glycoprotein added to a culture medium of Streptomyces sp. OH-11242 as the only source of carbon.

1. In the process of obtaining the degradation enzymes of mucus glycoprotein, porcine gastric mucus glycoprotein (PGM), added as the only source of carbon, was removed from the culture medium of Streptomyces sp. OH-11242 [Iwase et al. (1988) Biochem. biophys. Res. Commun. 151, 422-428] and analysed. 2. The amino acid and carbohydrate compositions of porcine gastric mucus glycoprotein (PGM-m) recovered from a culture medium were similar to those of original PGM. 3. However, the elution profile of PGM-m on Sephacryl S-400 differed from that of PGM and closely resembled that of performic acid-treated PGM or protease-treated PGM. 4. Either of these corresponded to the so-called subunit of approximately 550,000 in mol. wt, as reported by Scawen and Allen [(1977) Biochem. J. 163, 363-368]. 5. Performic acid treatment of PGM-m led to the production of a smaller unit (unit m) having a mol. wt of about 72,000. Separate treatment of different sized components prepared from PGM-m showed the above unit m to be produced from each molecule. 6. Thus, PGM-m is a molecule partly modified by various glycosidases including endo-alpha-N-acetylgalactosaminidase and exposure of the modified part to performic acid results in oxidation. 7. Production of unit m from both larger and smaller molecules indicates the part susceptible to performic acid to exist at regular intervals on the mucus glycoprotein molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1992

Analysis of glycoform of O-glycan from human myeloma immunoglobulin A1 by gas-phase hydrazinolysis following pyridylamination of oligosaccharides.

A comparative study was made on the glycoform of O-glycan from human myeloma immunoglobulin A1. By gas-phase hydrazinolysis, O-glycan was released from its hinge portion. The released oligosaccharide was pyridylaminated and separated by a two-dimensional analytical method of gel filtration and reverse-phase HPLC. Four major pyridylamino derivatives (P1-P4) were obtained. The neutral component (P4) among them was identified as Gal beta 1,3GalNAc-PA by cochromatography with an authentic standard pyridylamino sugar. The desialylation of the other components indicated the largest P1 and middle size P2 components possibly corresponded to a disialylated structure, NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAc-PA, and a monosialylated component, NeuAc alpha 2,3Gal beta 1,3GalNAc-PA, respectively. The structural assignment of P3 is still incomplete. Four similar components were also detected in bovine fetuin whose relative content (P1:P2: P3:P:4) was 16:43:19:22. The relative content (%) of P1-P4 (glycoform) in IgA1 from the healthy control was 10.1 +/- 3.3, 48.2 +/- 4.6, 7.0 +/- 2.6, and 34.7 +/- 4.5. The glycoform of O-glycan on IgA1 thus appears the same for any individual. Analysis of IgA1 myeloma protein indicated glycoforms distinct from those of the healthy controls. The relative content of these component could be classed as 2:8:0:90 (Type I, only one case designated as Kita), 5:24:3:68 (Type II, seven cases), and 9:41:5:45 (Type III, four cases). Thus, the results for IgA1 myeloma protein indicate that at least three glycoforms of O-glycan are possible for the IgA1 hinge structure. However, only one glycoform was found in the healthy controls.

Amination↗
PubMed1992

Effects of the M1 muscarinic receptor antagonist pirenzepine on gastric mucus glycoprotein in rats with or without ethanol-induced gastric damage.

Changes in gastric mucus glycoprotein (mucin) isolated from pirenzepine-treated rats with or without ethanol (50%)-induced gastric damage were studied. The prior administration of pirenzepine inhibited significantly and dose-dependently the occurrence of macroscopically observable hemorrhagic lesions induced by treatment with ethanol. The gastric mucosa was separated into the surface mucosa, including the mucus gel layer, and the deeper mucosa by mechanical scraping, and the mucin in each was isolated. In ethanol-treated animals the mucin content of the deep corpus mucosa was significantly reduced to 68% the control value. This reduction was inhibited by pretreatment with pirenzepine. In the surface mucosa mucin content was also reduced to 48% of control value by ethanol treatment, but pirenzepine pretreatment increased mucin content to 235% the control value. Total mucin content in the entire stomach essentially resumed the control level by pretreatment with 100 mg/kg of pirenzepine. A single oral administration of pirenzepine (100 mg/kg) caused no change in total mucin content, but mucin in the deep corpus mucosa selectively and significantly increased to 124% the control. These and the results of carbohydrate analysis of purified mucin indicate that pirenzepine administration possibly accelerates the secretion of accumulated deep corpus mucus, retains this deep mucus on surface mucosal mucus, and protects the gastric mucosa in ethanol-induced gastric damage. This may be related to the antiulcerogenic effects of pirenzepine.

Animals↗
PubMed1992

Effect of rebamipide on mucus secretion by endogenous prostaglandin-independent mechanism in rat gastric mucosa.

The effect of rebamipide (2-(4-chlorobenzoylamino)-3-[2 (1H)-quinolinon-4-yl]-propionic acid, OPC-12759, CAS 11911-87-6), an anti-ulcer agent developed to enhance defensive factors in the gastric mucosa, on gastric mucus secretion was studied by a newly developed biochemical method to measure the gastric mucus glycoprotein content in rats. 1 h after intraperitoneal administration, rebamipide did not produce a significant change in the intramucosal mucus contents (surface mucosal layer and deep mucosal layers of corpus and antral regions) but significantly increased the content of soluble mucus, which recovered from the gastric contents, to about 160% of the control value. Since rebamipide has been shown to increase the biosynthesis of prostaglandins, indometacin was administered to the rats prior to rebamipide administration to examine whether the increase in prostaglandin biosynthesis contributes to the rebamipide-induced increase in gastric mucus secretion. The increase in the soluble mucus was not altered by the pretreatment with indometacin, thus indicating that rebamipide per se has a potential to increase the gastric mucus secretion by a mechanism that is not mediated by the endogenous prostaglandins. The effect of rebamipide on the gastric mucus secretion might contribute to heal and to prevent the recurrence of peptic ulcer diseases as well as to maintain the homeostasis of the gastric mucosa.

Alanine↗
PubMed1991

Rat-liver-type phosphofructokinase mRNA. Structure, tissue distribution and regulation.

We have cloned a full-length cDNA for rat-liver-type phosphofructokinase. The similarities of the rat liver-type phosphofructokinase mRNA to the human and mouse counterparts were 94% and 99% in their amino acid sequences and 88% and 94% in the nucleotide sequences of their coding regions, respectively. Rat liver-type phosphofructokinase mRNA was expressed in all tissues examined, but its level was regulated tissue-specifically. The nutritional and hormonal regulations of the mRNA in the liver were examined in comparison with those of two other key glycolytic enzymes, glucokinase and L-type pyruvate kinase. The level of liver-type phosphofructokinase mRNA was essentially unchanged by starvation (72 h) or diabetes. The mRNA level also did not change significantly on refeeding starved rats on a high carbohydrate diet, or treating diabetic ones with insulin. These results suggested that rat liver-type phosphofructokinase mRNA in the liver was not under control of diet or insulin, in contrast to glucokinase and L-type pyruvate kinase.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence and transcriptional start point of the kan gene encoding an aminoglycoside 3-N-acetyltransferase from Streptomyces griseus SS-1198PR.

We determined the nucleotide sequence of a 1794-bp BglII-BamHI fragment containing the kan gene (KmR) which encodes a novel aminoglycoside 3-N-acetyltransferase of Streptomyces griseus SS-1198PR. The sequence was found to contain four open reading frames (ORF) in the direction of transcription determined by low resolution S1 nuclease mapping. Except for the largest one, these ORF are unlikely to be the coding sequence of kan, because of their inability to confer kanamycin resistance. The largest ORF could encode a 30.8-kDa protein consisting of 284 amino acids (aa) with 93.3 mol% G + C in the third position of codons, and is preceded by potential ribosome-binding sites. Thus, the largest ORF was likely to represent the kan gene. The transcription start point of kan was located 84 bp upstream from the start codon. The -10 region showed similarity to the Escherichia coli consensus promoter motif, while no sequence similarity was found in the -35 region. The deduced aa sequence of kan showed 85% similarity with that of the aacC7 gene of Streptomyces rimosus forma paromomycinus [Lopez-Cabrera et al., J. Bacteriol. 171 (1989) 321-328] suggesting that these genes are derived from a common ancestral gene.

Acetyltransferases↗
PubMed1991

Structure of the entire human muscle phosphofructokinase-encoding gene: a two-promoter system.

We have recently shown that three types (A,B, and C) of mRNA species are transcribed from a single gene encoding human muscle phosphofructokinase (hPFK-M) through alternative splicing [Nakajima et al., Biochem. Biophys. Res. Commun. 166 (1990) 637-641]. To determine its complete structure and elucidate the mechanism of alternative RNA splicing, we isolated the hPFK-M gene, which spans about 30 kb, and contains 24 exons. Transcription start points were observed for both exon 1 and exon 2 by S1 nuclease protection assay and primer extension. Motifs of an Sp1-binding site were observed in the upstream region of exon 1 (promoter 1). A TATA-box-like sequence and a CAAT-box-like sequence were identified in the upstream region of exon 2 (promoter 2). Reporter assay revealed that the promoter 1 region was functional both in HeLa cells and myoblastic clonal cells, and that the promoter 2 region was active only in myoblastic cells. Motifs of M-CAT known as a muscle-specific enhancer, were observed in the promoter 2 region. These results indicated that the hPFK-M gene contains at least two promoter regions, facilitating the expression of the heterogeneous gene transcripts in a cell-type-specific manner.

Base Sequence↗
PubMed1991

A new method of separation and quantitation of mucus glycoprotein in rat gastric mucus gel layer and its application to mucus secretion induced by 16,16-dimethyl PGE2.

A method was established for recovering the mucus gel layer of rat gastric mucosa without damage to underlying surface epithelium. The mucus gel was solubilized by stirring the gastric mucosa in a solution of N-acetylcysteine (NAC), a mucolytic agent. Optimal mucus gel solubilization was possible by treatment with 2% NAC for 5 minutes at room temperature. Mucus glycoprotein was quantitatively extracted and measured from the mucus gel sample obtained by the NAC treatment. This treatment caused no damage to surface epithelial cells, as observed by a light microscope. Besides NAC, pronase solution was also adequate for solubilizing the mucus gel layer without any damage to the surface epithelium. However, extraction and measurement of mucus glycoprotein from the pronase-treated mucus gel sample was not possible due to contamination by high molecular hexose-containing substances which were eluted along with the mucus glycoprotein from the column of Bio-Gel A-1.5m. This NAC method was used to examine changes in mucus glycoprotein content in the mucus gel at one hour following the oral administration of 16,16-dimethyl prostaglandin E2. A significant increase in mucus glycoprotein of the gel was brought about by the prostaglandin treatment. Thus, the present method was suitable for estimating the amount of mucus secreted in to the mucus gel layer.

Animals↗
PubMed1991

[Pharmacological effects of the novel dopamine uptake inhibitor 1-[2-[bis(4-fluorophenyl)-methoxy]ethyl]-4-(3-phenylpropyl) piperazine dihydrochloride (I-893) on the central nervous system].

The effects of 1-[2-[bis (4-fluorophenyl)methoxy]ethyl]-4-(3- phenylpropyl) piperazine dihydrochloride (I-893) on the central nervous system were behaviorally and electroencephalographically investigated. Intraperitoneally injected I-893 (5-10 mg/kg) dose-dependently increased spontaneous motor activity in mice, but repeated injections did not affect the increase in the locomotor activity. In reserpinized mice, spontaneous motor activity was not increased by oral I-893. In alpha-MPT-treated mice, the motor activity was lower than that in vehicle-treated animals with intermediate doses (10-40 mg/kg, p.o.) of I-893, but there was no difference between the two groups with high doses. In rats with unilaterally 6-OHDA-induced lesion of the nigrostriatal pathway, I-893 induced circling behavior toward the lesioned side. Haloperidol-induced catalepsy in rats was reduced by I-893. Tremorine-induced tremor in mice was inhibited by I-893. The effect was not altered in the mice treated with I-893 for 10 days. Oral I-893 induced stereotypy in rats, but it did not affect methamphetamine-induced stereotypy. Hypnosis induced by barbiturates was antagonized by I-893. In rats treated with I-893 for 6 days, pentobarbital-induced sleep was not different from that in vehicle-treated animals on the day after the final treatment. Intravenous I-893 altered EEGs in the cerebral cortex and amygdala nucleus to low voltage and fast waves and altered hippocampal EEG to theta waves in immobilized rabbits. These results suggest that I-893 inhibits re-uptake of dopamine released by exocytosis and indirectly has dopaminergic effects.

Animals↗
PubMed1991

Pyrrolomycin group antibiotics inhibit substance P-induced release of myeloperoxidase from human polymorphonuclear leukocytes.

In order to search for microbial modulators of the activity of neuropeptide, we established a screen based on substance P (SP)-induced myeloperoxidase (MPO) release from human polymorphonuclear leukocytes (PMN). SP induced MPO release in a dose-dependent manner at concentrations ranging from 1 approximately 10 x 10(-4) M. In comparison at 1 x 10(-4) M, induction was also observed with SP derivatives but not with other neuropeptides such as neurokinin and enkephalin. Based on this, we searched for microbial inhibitors against SP-induced MPO release. An actinomycete metabolite designated HS3, which turned out to be identical with dioxapyrrolomycin or A1-R2081, and structurally related pyrrolomycins were found to inhibit SP-induced MPO release. In addition, these compounds inhibited the f-Met-Leu-Phe (FMLP)-induced MPO release from PMN. Pyrrolomycin derivatives with an N-methylated pyrrole ring showed, however, a selective inhibition of the SP-induced MPO release. This was in contrast to results with aseanostatin P5 which selectively inhibited FMLP-induced MPO release.

Actinomycetales↗
PubMed1990

Tissue specificity in expression and alternative RNA splicing of human phosphofructokinase-M and -L genes.

Mode of the expression of phosphofructokinase (PFK) -M and -L genes was examined in various human tissues including muscle, placenta, liver, kidney, pancreas, stomach and reticulocytes. The gross level of mRNA expression of PFK-M and -L genes was estimated by Northern analysis. Polymerase chain reaction was used to detect mRNA expressed at low levels in these tissues. Tissue-specific expression of alternatively spliced PFK-M gene transcripts was also determined by polymerase chain reaction. The results indicated that alternative splicing of PFK-M gene transcripts was controlled in a tissue-specific manner.

Base Sequence↗
PubMed1990

Genetic defect in muscle phosphofructokinase deficiency. Abnormal splicing of the muscle phosphofructokinase gene due to a point mutation at the 5'-splice site.

The genetic defect in muscle phosphofructokinase deficiency (type VII glycogenosis, Tarui disease) was investigated. Six cDNAs for muscle phosphofructokinase, including a full-length clone, were isolated from a non-amplified library of muscle from a patient. By sequence analysis of these clones, a 75-base in-frame deletion was identified. The rest of the sequence was identical to that of the normal cDNA, except for a silent base transition at position 516 (ACT (Thr) to ACC (Thr]. The deletion was located in the 3'-terminal region of exon 13 (numbered with reference to the rabbit muscle phosphofructokinase gene (Lee, C.-P., Kao, M.-C., French, B.A., Putney, S.D., and Chang, S.H. (1987) J. Biol. Chem. 262, 4195-4199]. Genomic DNA of the patient was amplified by polymerase chain reaction. Sequence analysis of the amplified DNA revealed a point mutation from G to T at the 5'-end of intron 13. This mutation changed the normal 5'-splice site of CAG:GTATGG to CAG:TTATGG. A cryptic splice site of ACT:GTGAGG located 75 bases upstream from the normal splice site was recognized and spliced in the patient.

Base Sequence↗
PubMed1990

Analysis of mucin-derived oligosaccharides by medium-pressure gel-permeation and amino-plate thin-layer chromatography conducted in conjunction.

Oligosaccharides present in mucin were labeled by reduction with NaB3H4 and separated by gel-permeation chromatography with a Toyopearl HW-40S column using 0.1 M pyridine acetate, pH 5.0, as the solvent. Each fraction was further analyzed by thin-layer chromatography (TLC) on a Funagel AMP plate, a glass plate precoated with 3-aminopropyl-bonded silica. Acetonitrile/10 mM triethylamine acetate (3/2, by volume) served as the solvent. The sites of oligosaccharides on the TLC plate could be determined according to size, anionic charge, and sugar composition. They could thus be "mapped" on the plate. In this manner, the distribution of oligosaccharides on bovine submaxillary mucin and rat gastric mucin was determined. Each radiolabeled oligosaccharide in newly synthesized rat gastric mucin, metabolically labeled with [14C]glucosamine or [35S]sulfate, was also identified by this method.

Animals↗