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K Hotta

Publications and source records attributed to K Hotta.

At least 55 records · Page 3Linked to original sources
PubMed2000

Gastric adenomas and superficial adenocarcinomas display distinct patterns of mucin carbohydrate and core protein expression.

AIMS: To investigate the histogenetic relationship between gastric epithelial neoplasms we studied differences in expression of mucin carbohydrate antigens and mucin core protein, in normal and metaplastic gastric mucosa, and in gastric adenomas and superficial adenocarcinomas. METHODS AND RESULTS: We generated four monoclonal antibodies, including HGM72/75 against human gastric mucin and HCM14/21 against human colonic mucin, and investigated immunoreactivities of these antibodies and MUC2 protein expression in normal and metaplastic gastric mucosa, adenomas (15 samples) and superficial adenocarcinomas (intestinal-type, 77; diffuse-type, 59 samples). HGM72 reacted with mucous neck cells of the fundic glands and with pyloric glandular cells whereas HGM75 stained foveolar cells and metaplastic goblet cells. Weak binding of HCM14/21 and strong staining with MUC2 were found in metaplastic goblet cells. Binding of HGM75, HCM14, MUC2, but not HGM72 was high in adenomas. An equivalent staining with HGM72 and HGM75 with low expression of MUC2 and HCM14 was shown in intestinal-type carcinomas while the diffuse-type demonstrated more strong reactivity with HGM75 than with HGM72, MUC2 and HCM14. Little binding of HCM21 was observed in any specimens. CONCLUSIONS: This study demonstrates that adenomas predominantly have a intestinal phenotype, but both types of adenocarcinomas retain some cells with a gastric phenotype during the early steps of neoplastic development.

Adenocarcinoma↗
PubMed2000

Augmentative effects of L-cysteine and methylmethionine sulfonium chloride on mucin secretion in rabbit gastric mucous cells.

BACKGROUND: Our previous study showed that L-cysteine (Cys) and methylmethionine sulfonium chloride (MMSC) inhibited ethanol-induced gastric mucosal damage and increased the amount of surface mucin in rats. This study examined whether Cys and MMSC augmented mucin secretion and changed distribution of mucin vesicles ultrastructurally in mucous cells by using primary cultured mucous cells from rabbit glandular stomach. Changes in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) and in levels of cytosolic free Ca2+ were investigated by treatment with Cys and MMSC. METHODS: Mucin content was measured by an enzyme-linked lectin assay. Transmission electron micrography was used to examine ultrastructural distribution of mucin granules. The amount of cAMP or levels of free Ca2+ were measured by enzyme immunoassay or by fura-2. 16,16-Dimethyl prostaglandin E2 (dmPGE2) or ATP was used as the positive control. RESULTS: L-Cysteine and MMSC increased mucin secretion and decreased cellular mucin content. The same was noted for dmPGE2. Accelerated mucin granule movements toward the plasma membrane were shown by these agents. Intracellular cAMP increased with exposure to dmPGE2 for 20 min, while neither Cys nor MMSC increased cAMP. No increase in cytosolic free Ca2+ levels occurred after treatment with Cys or MMSC, but an increase was induced 10 s after the addition of ATP. CONCLUSIONS: The present findings indicate that the increase in mucin secretion by Cys and MMSC was not mediated through the cAMP or Ca2+ signal transduction pathway, but might occur through non-receptor-mediated mechanisms.

Animals↗
PubMed2000

Mutation in bombesin receptor subtype-3 gene is not a major cause of obesity in the Japanese.

Bombesin receptor subtype-3 (BRS-3) is one of the candidate genes of obesity. The mice lacking BRS-3 have been shown to develop mild obesity. These mice also showed hypertension and impaired glucose metabolism, supporting these mice as a good model for human obesity. We screened 104 Japanese obese men (BMI > 26.4, 26.5-44.1) to investigate whether there is any genetic defect in BRS-3 gene. The DNA fragments containing each exon of BRS-3 gene were amplified by polymerase chain reaction (PCR) and were directly sequenced. No mutation, nor polymorphism was found in the coding region of BRS-3, suggesting that mutation of this gene is not a major cause of obesity in humans.

DNA Primers↗
PubMed2000

Humoral immunity against the proline-rich peptide epitope of the IgA1 hinge region in IgA nephropathy.

BACKGROUND: The human IgA1 hinge region is a unique mucin-like O-linked proline-rich glycopeptide, and its core peptide was found to be exposed aberrantly by the underglycosylation in IgA nephropathy (IgAN). We describe here the presence of humoral immunity against the IgA1 hinge peptide epitope in IgAN and evaluate the relationship between the underglycosylation of the IgA1 hinge region and humoral immunity. METHOD: The serum anti-IgA1 hinge peptide antibody (anti-alpha1HP ab) titre was measured and compared between the IgAN (n=37) and control groups (n=34) by enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide corresponding to the human IgA1 hinge region, PVPSTPPTPSPSTPPTPSPS, as an antigen. Next, to evaluate the relationship between the underglycosylation of the IgA1 hinge region and the humoral immunity, the reactivity of the serum IgG from the patients with IgAN against monoclonal IgA1 which had been digested enzymatically to remove the carbohydrates from the IgA1 hinge region was measured by ELISA. RESULTS: The anti-alpha1HP ab titre was significantly higher in the IgAN group than in the control group (OD value: IgG class, 0.564+/-0.344 vs 0. 331+/-0.154, P=0.0014; IgM class, 0.272+/-0.148 vs 0.141+/-0.072, P<0.0001) and it was positive in approximately 40% of the patients with IgAN. In addition, the reactivity of the serum IgG from the IgAN patients against the monoclonal IgA1 was found to be increased as the carbohydrates were enzymatically removed from the IgA1 hinge region (when native=100; asialo, 122+/-9.5; agalacto, 167+/-11.5; naked, 188+/-3.9). CONCLUSION: These results suggested that the peptide epitope of the IgA1 hinge region which was aberrantly exposed by underglycosylation could induce the humoral immune response in IgAN.

Adolescent↗
PubMed2000

Ultrasound biomicroscopy for examination of the sclerotomy site in eyes with proliferative diabetic retinopathy after vitrectomy.

PURPOSE: We evaluated the capability of ultrasound biomicroscopy (UBM) to predict fibrovascular proliferation at sclerotomy sites in eyes with postoperative vitreous hemorrhage due to proliferative diabetic retinopathy (PDR). METHODS: Ultrasound biomicroscopy was used for examining the sclerotomy sites in 13 eyes of 11 patients with PDR experiencing postoperative vitreous hemorrhage (PDR group). Thirty-nine sclerotomy sites (all entry sites of each eye) were examined before reoperation, and the UBM images were compared with findings obtained during revision of the vitrectomy. Thirteen eyes of 13 patients undergoing vitrectomy for nondiabetic diseases were used as controls and examined after vitrectomy. RESULTS: The UBM images were classified into the following four categories: A, tent; B, spheroid; C, trapezoid; and N, none. The findings were distributed as follows in the PDR group: category A, 18%; B, 5%; C, 56%; and N, 21 %; and as follows in the control group: category A, 28%; B, 5%; C, 5%; and N, 62%. In the PDR group, 11 of 12 sclerotomy sites disclosing fibrovascular proliferation possessed the trapezoidal image. Mean length of trapezoidal base was 2.49+/-0.97 mm and 1.51+/-0.75 mm in the groups with and without fibrovascular proliferation, respectively (P<0.01). The average relative reflectivity of the trapezoidal image against the sclera was 0.501+/-0.169 in the fibrovascular proliferation group and 0.891+/-0.183 in the fibrous ingrowth group (P<0.01). CONCLUSION: Ultrasound biomicroscopy is useful in detecting fibrovascular proliferation at sclerotomy sites because a large and low-reflecting trapezoidal UBM image is highly correlated to its presence.

Adult↗
PubMed2000

Identification and characterization of the point mutation which affects the transcription level of the chromosomal 3-N-acetyltransferase gene of Streptomyces griseus SS-1198.

We determined the molecular basis for the enhanced expression of the aac(3)-Xa gene encoding an aminoglycoside 3-N-acetyltransferase in Streptomyces griseus. A C-->T substitution was identified at the putative promoter of the mutant gene. RNA analyses demonstrated that the substitution caused a marked increase in the production of the gene-specific transcripts. Therefore, it seemed very likely that the aac(3)-Xa gene was activated by the substitution resulting in the emergence of a stronger promoter.

Arylamine N-Acetyltransferase↗
PubMed2000

CGRP modulates mucin synthesis in surface mucus cells of rat gastric oxyntic mucosa.

We examined the effects of the calcitonin gene-related peptide (CGRP), including the possible participation of nitric oxide (NO), on mucin biosynthesis in the surface epithelium and remaining deep mucosa as well as the entire mucosa and compared the distribution of CGRP and NO synthase (NOS) using a combination of double immunofluorescence labeling and multiple dye filter. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing [(3)H]glucosamine and CGRP, with or without the NOS inhibitor. CGRP dose-dependently enhanced [(3)H]glucosamine incorporation into the corpus mucin but had no effect on antral mucin biosynthesis. The CGRP receptor antagonist, CGRP-(8-37), prevented the increase in (3)H-labeled corpus mucin. This stimulation of corpus mucin synthesis disappeared after removal of the surface mucus cell layer. CGRP activated the mucin biosynthesis in the surface mucus cells. In the full-thickness corpus mucosa, CGRP-induced activation was completely blocked by the NOS inhibitor. CGRP-immunoreactive fibers were intertwined within the surface mucus cell layer with type I NOS immunoreactivity. These results show that CGRP-stimulated mucin biosynthesis mediated by NO is limited to surface mucus cells of the rat gastric oxyntic mucosa.

Animals↗
PubMed2000

TNF-alpha increases transcription of Galpha(i-2) in human airway smooth muscle cells.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that has an important role in the regulation of airway smooth muscle tone and reactivity. We have shown previously that TNF-alpha upregulates the expression of Galpha(i-2) protein without significantly increasing G(s)alpha protein and enhances adenylyl cyclase inhibition by carbachol in cultured human airway smooth muscle cells (Hotta K, Emala CW, and Hirshman CA. Am J Physiol Lung Cell Mol Physiol 276: L405-L411, 1999). The present study was designed to investigate the molecular mechanisms by which TNF-alpha upregulates Galpha(i-2) protein in these cells. TNF-alpha pretreatment for 48 h increased the expression of Galpha(i-2) protein without significantly altering the Galpha(i-2) protein half-life (41.0 +/- 8.2 h for control and 46.8 +/- 5.2 h for TNF-alpha-treated cells). Inhibition of new protein synthesis by cycloheximide blocked the increase in Galpha(i-2) protein induced by TNF-alpha. Furthermore, TNF-alpha treatment for 12-24 h increased the steady-state level of Galpha(i-2) mRNA without significantly altering Galpha(i-2) mRNA half-life (9.0 +/- 0.75 h for control and 8.9 +/- 1.1 h for TNF-alpha-treated cells). The transcription inhibitor actinomycin D blocked the increase in Galpha(i-2) mRNA induced by TNF-alpha. These observations indicate that the increase in Galpha(i-2) protein induced by TNF-alpha is due to an increased rate of Galpha(i-2) protein synthesis, most likely as a consequence of the transcriptional increase in the steady-state levels of its mRNA.

Cell Count↗
PubMed2000

Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients.

Adiponectin is a novel, adipose-specific protein abundantly present in the circulation, and it has antiatherogenic properties. We analyzed the plasma adiponectin concentrations in age- and body mass index (BMI)-matched nondiabetic and type 2 diabetic subjects with and without coronary artery disease (CAD). Plasma levels of adiponectin in the diabetic subjects without CAD were lower than those in nondiabetic subjects (6.6+/-0.4 versus 7.9+/-0.5 microg/mL in men, 7.6+/-0.7 versus 11.7+/-1.0 microg/mL in women; P<0.001). The plasma adiponectin concentrations of diabetic patients with CAD were lower than those of diabetic patients without CAD (4.0+/-0.4 versus 6.6+/-0.4 microg/mL, P<0.001 in men; 6.3+/-0.8 versus 7.6+/-0. 7 microg/mL in women). In contrast, plasma levels of leptin did not differ between diabetic patients with and without CAD. The presence of microangiopathy did not affect the plasma adiponectin levels in diabetic patients. Significant, univariate, inverse correlations were observed between adiponectin levels and fasting plasma insulin (r=-0.18, P<0.01) and glucose (r=-0.26, P<0.001) levels. In multivariate analysis, plasma insulin did not independently affect the plasma adiponectin levels. BMI, serum triglyceride concentration, and the presence of diabetes or CAD remained significantly related to plasma adiponectin concentrations. Weight reduction significantly elevated plasma adiponectin levels in the diabetic subjects as well as the nondiabetic subjects. These results suggest that the decreased plasma adiponectin concentrations in diabetes may be an indicator of macroangiopathy.

Adiponectin↗
PubMed2000

Reaction of various lectins to mucin derived from the different layers of rat gastric mucosa: comparison of enzyme-linked lectin binding assay with lectin histochemistry.

The purpose of this study was to demonstrate that the histochemical staining reactivities of lectins in rat stomach actually represent the gastric mucins, and to estimate the utility of the lectins for mucin histochemistry. In this paper, the lectin histochemistry was compared with an enzyme-linked lectin binding assay (ELLA) of the mucins derived from distinct regions and layers of the Sprague-Dawley rat stomach and it was examined to determine the definite binding and problematic binding of the conventional lectin. Among the 10 different biotinylated lectins, Canavalia ensiformis (ConA), Griffonia simplicifolia II (GS-II), Triticuin vulgaris (WGA), Ricinus communis I (RCA-I), Arachis hypogaea (PNA), Glycine max (SBA), Dolichos biflorus (DBA), Ulex europaeus I (UEA-I), Sambucus nigra (SNA) and Maackia amurensis II (MAL-II), examined in this study, GS-II, SBA, DBA, UEA-I, SNA and MAL-II bound clearly to the mucin of distinct regions and layers of the Sprague-Dawley rat stomach in agreement with the results of ELLA. Namely GS-II lectins preferentially bound to the mucin in the mucous neck cells of the corpus area. SBA and DBA clearly recognized the mucin in the covering epithelial mucous cells in the corpus and antral area. UEA-I was widely bound to all the mucin present in both the corpus and antrum. On the other hand, SNA and MAL-II could not react with the mucin obtained from the gastric mucosa but was specifically bound to the mucin purified from the mucous gel layer. These results suggested that the lectins described above are useful histochemical tools to recognize the mucus present in the different regions and layers of Sprague-Dawley rat gastric mucosa.

Animals↗
PubMed2000

Double stage activity in aminoglycoside antibiotics.

Fourteen different aminoglycoside antibiotics (AGs) were challenged with aminoglycoside acetyltransferases (AACs) of actinomycete origin in order to examine their 'double stage activity' that is arbitrarily defined as antibiotic activity retainable after enzymatic modification. In kanamycin (KM)-group AGs tested [KM, dibekacin (DKB), amikacin and arbekacin (ABK)], ABK retained activity after acetylations by AAC(3), AAC(2') and AAC(6'). DKB also retained a weak activity after acetylation by AAC(2'). In gentamicin (GM)-group AGs tested [GM, micronomicin, sisomicin (SISO), netilmicin (NTL) and isepamicin], GM, SISO and NTL retained activites after acetylation by AAC(2'). In neomycin (NM)-group AGs tested [ribostamycin, NM, paromomycin], NM retained activity after acetylation by AAC(6') and AAC(2'). None of astromicin (ASTM)-group AGs tested (ASTM and istamycin B) retained activity after acetylation by AAC(2') and AAC(6'). The activities of acetylated ABK derivatives by AAC(3) and AAC(2') were distinctively high, compared to the others. Streptomyces lividans TK21 containing the cloned aac genes were markedly sensitive to AGs that retained activities after acetylation, indicating the substantial effect of 'double stage activity'.

Acetylation↗
PubMed2000

[Comparison of scleral buckling and vitrectomy for superior retinal detachment caused by flap tears].

PURPOSE: We compared the surgical results of vitrectomy and scleral buckling for uncomplicated superior retinal detachment caused by flap tears. SUBJECTS AND METHODS: Included in the study were 225 phakic eyes of 225 patients undergoing primary surgery from January 1990 to December of 1996 for superior retinal detachment caused by flap tears; all eyes had been followed for longer than six months after surgery. The choice of procedures was based on each surgeon's preference. The cases were evaluated retrospectively and the surgical outcome and the rate of complications compared between the two groups of eyes. RESULTS: Initial and final anatomical success rate were 92% and 100% after each procedure. Redetachment after the first procedure was due to new retinal breaks in 5 eyes, reopening of original breaks in 2 eyes of vitrectomy cases, and malpositioned buckle in 11 eyes of scleral buckling cases. Proliferative vitreoretinopathy occurred in 3 eyes of vitrectomy cases. CONCLUSION: Primary vitrectomy was as successful as scleral buckling for superior rhegmatogenous retinal detachment. Although a high incidence of postoperative cataract formation was a major drawback, vitrectomy had some advantages over to scleral buckling.

Adult↗
PubMed2000

[Colony PCR for rapid detection of antibiotic resistance genes in MRSA and enterococci].

We examined colony PCR that does not use the prepared template DNA but intact cells as the template DNA source for the rapid, simple and reproducible detection of antibiotic resistance genes in strains of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus. Two factors turned out to be important; i.e. the transfer size of bacterial cells and the use of DNA Polymerase with high performance. In practice, the tip of a toothpick was lightly touched with a colony on agar plates followed by the PCR mixture (20 microliters) containing KOD-plus-DNA Polymerase and multiplex primers. The cell transfer size ranged from 10(3) to 10(4) cfu and 30 cycles of PCR (95 degrees C, 30 sec.-->50 degrees C, 30 sec.-->72 degrees C, 60 sec.) following the first heating (95 degrees C, 3 min.) resulted in good amplification of the target regions of mecA and aac(6')/aph(2") that are responsible for the resistance to methicillin and arbekacin, respectively.

Aminoglycosides↗
PubMed1999

Human serum immunoglobulin G3 subclass bound preferentially to asialo-, agalactoimmunoglobulin A1/Sepharose.

As we reported before, exoglycosidase treatment of human serum IgA1 changed it to a sticky molecule. In order to examine the presence of the specific binding protein to the sticky IgA1 in human serum, IgA1, asialo-IgA1 (IgA1-S) and asialo-, agalacto-IgA1 (IgA1-SG)/Sepharose column chromatography of normal human serum was carried out. Purified hinge glycopeptide (HGP33) prepared from IgA1 was used for the preparation of HGP/Sepharose. A portion of the serum protein was bound to those columns and eluted with the buffer containing 1.0 M NaCl. About four times the amount of protein was eluted from the IgA1-SG/Sepharose column than that from IgA1/Sepharose. Most of the eluted protein was IgG, and the IgG1 and IgG3 subclasses but neither IgG2 nor IgG4 was dominant. Under the lower salt concentration, a portion of IgG was also bound to the HGP-SG/Sepharose column. The obtained results coincide well with the previous report of the co-present of IgG1 and IgG3 with deposited IgA1 in IgA nephropathy patients (Aucouturier, P., et al. (1989) Clin. Immunol. Immunopathol. 51, 338-347). Thus, the results solved the question of why the IgG3 was co-present with deposited IgA1 in IgA nephropathy patients.

Chromatography, Liquid↗
PubMed1999

Study of the relationship between sticky human serum IgA1 and its O-glycan glycoform.

The high-affinity IgA1 toward jacalin was mostly composed of aggregated IgA1 and abundantly contained the asialo disaccharide, Galbeta1,3GalNAc, in the O-linked oligosaccharide in the hinge region [Journal of Biochemistry 120, 92-97 (1996)]. Meanwhile, the removal of sialic acid from IgA1 accelerated the aggregation of the IgA1 molecule [J. Am. Soc. Nephrol. 9, 2048-2054 (1998)]. In order to examine the nature of such a sticky IgA1, affinity chromatography using asialo-IgA1 (deSIgA1)-Sepharose was carried out. Seventeen percent of normal human serum IgA1, 27% of asialo-IgA1 (IgA1-S), and 48% of asialo-, agalacto-IgA1 (IgA1-SG) were bound to the column. Removal of the N-acetylgalactosamine residue from IgA1-SG resulted in a decreasing affinity toward deSIgA1-Sepharose. Thus, the binding ability toward the column was the highest for the IgA1-SG among the deglycosylated IgA1s. On the other hand, heat treatment of IgA1 accelerated the aggregation but decreased its binding ability toward the column. Such heat denaturation probably destroys the structure of the binding site. Since the enzymatic removal of the N-glycan sugar chains did not induce the aggregation and exhibited no effect on the binding, the incomplete O-linked sugar chain on the hinge portion should be directly related to the sticky characteristics of the IgA1 molecule. The binding was non-covalent and not strong because the asialo-, agalacto-hinge glycopeptide was eluted slightly slower than the native one from the column and the bound IgA1 was dissociated in the presence of 1 M NaCl.

Chromatography, Affinity↗
PubMed1999

Brachyury downstream notochord differentiation in the ascidian embryo.

The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential Ci-Bra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation.

Animals↗
PubMed1999

Mutual separation of hinge-glycopeptide isomers bearing five N-acetylgalactosamine residues from normal human serum immunoglobulin A1 by capillary electrophoresis.

Immunoglobulin A1 (IgA1) from normal human serum is known to have O-linked sugar chains, sialylated Galbeta1,3GalNAc, in the hinge portion. In order to reduce the microheterogenity of the sugar chain, the hinge glycopeptide prepared from IgA1 was sequentially treated with neuraminidase and beta-galactosidase. The asialo-, agalacto-hinge glycopeptide (HGP-SG) composed of a 33-mer peptide (HP33) and N-acetylgalactosamine (GalNAc) residues was obtained. The HGP-SG was separated into three major peaks, A, B and C, by high-performance liquid chromatography (HPLC). Each glycopeptide fraction was further separated by capillary electrophoresis (CE). Peaks A, B and C with HPLC abundantly contained HP33 bearing five and six N-acetylgalactosamine residues (HGP33-5,6GN), HGP33-4,5GN and HGP33-3,4GN, respectively. Among these glycopeptide peaks, only the HGP33-5GN peak was partly split into two peaks based on the CE analysis - HGP33-5GN-alpha and -beta. The glycopeptide, HGP25-5GN shortened by the thermolysin digest of HGP33-SG was also well separated into the alpha and beta forms by CE analysis. No differences in their mass and peptide portion were observed between HGP25-5GN-alpha and -beta. Therefore, the obtained result might indicate that HGP25-5GN-alpha was an isomer of HGP25-5GN-beta differing in its stereospecific structure of the peptide portion and/or the attachment site of the GalNAc residue.

Acetylgalactosamine↗