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Biomedical subjects

I Kato

Publications and source records attributed to I Kato.

At least 289 records · Page 16Linked to original sources
PubMed1994

High-performance liquid chromatography of glycopeptides and oligosaccharides on graphitized carbon columns.

High-performance liquid chromatography of carbohydrate materials on graphitized carbon columns (GCC) has some advantages over other types of chromatography. Oligosaccharides and glycopeptides with few amino acids are barely retained on reversed-phase columns even under high salt or low pH conditions, but can be retained effectively on a graphitized carbon column. Moreover, elution of GCC requires concentrations of organic solvents lower than that required for normal-phase columns. The usefulness of graphitized carbon columns is exemplified by the following results: (i) Man9GlcNAc2 with only Asn or Asn-Phe (derived from soybean agglutinin) was not retained by a C18 reversed-phase column, but could be separated on a GCC with a gradient of 10-45% CH3CN in 30 min. (ii) Ribonuclease B glycopeptides obtained by Pronase digestion could be separated on GCC with a gradient of 10-30% CH3CN, but they were not retained on a C18 reversed-phase column even with water as eluent. (iii) Oligosaccharides released from ribonuclease B by endo-beta-N-acetylglucosaminidase were separated from each other and peptides on GCC with a linear gradient of 10 mM NH4OH to 10 mM NH4OH-12.5% CH3CN in 50 min at 70 degrees C. Silica-based columns do not allow such an alkaline eluent. (iv) Chito-oligosaccharides (DP 1-9) are well separated within 40 min on GCC with a gradient (10 mM NH4OH-10 mM NH4OH with 25% CH3CN) at 50 degrees C. Chito-oligosaccharides could not be separated by high-performance anion exchange columns such as Carbopac PA-1.

Acetylglucosaminidase↗
PubMed1994

Rapid and practical detection of beta-globin mutations causing beta-thalassemia by fluorescence-based PCR-single-stranded conformation polymorphism analysis.

We report a useful method for daily clinical examination for the diagnosis of thalassemia. We applied a fluorescence-based image analyser to a non-radioisotopic PCR-single-stranded conformation polymorphism (SSCP) analysis to detect mutations in the beta-globin gene. PCR primers were labelled with rhodamine X and the amplified fragments from the beta-globin gene were resolved by non-denaturing polyacrylamide gel electrophoresis. After loading, the glass plate was set in the image analyser and scanned with a green laser. We detected four common mutations in exon I and two major mutations in intron 1 of the beta-globin gene isolated from patients with beta-thalassemia. Moreover, to discriminate mutations and natural polymorphisms, we used primers including one base mismatch at the polymorphic site, which can substitute the polymorphic site by a constant base in the PCR amplified fragment. This fluorescence-based system was simple to operate and results were obtained rapidly as clear image data. Therefore, once the optimal conditions of the electrophoresis are determined, this system will be suitable for daily clinical use, especially for screening of molecular defects and for the prenatal diagnosis of genetic disorders.

Base Sequence↗
PubMed1994

The association of the insulin resistance syndrome with impaired glucose tolerance and NIDDM in the Japanese general population: the Hisayama study.

To elucidate the risk factors for initiating glucose intolerance, the relevant factors were explored in a cross-sectional survey conducted in a sample population aged 40-79 years old selected from a Japanese community, Hisayama, Japan in 1988. A 75-g oral glucose tolerance test was used to classify 1,073 men (72.5% of the entire population in the same age range) and 1,407 women (80.5%) into normal, impaired glucose tolerance and diabetes mellitus groups. In all age and sex groups with normal glucose tolerance, the sum of fasting and 2-h post-load insulin values varied widely and demonstrated significant positive correlations with triglycerides, body mass index, waist-hip ratio, systolic and diastolic blood pressure, while it negatively correlated to HDL cholesterol (p < 0.05). Insulin resistance was presumed to develop in normal glucose tolerance subjects with hyperinsulinaemia. The sum of the insulin concentrations, triglycerides, body mass index, waist-hip ratio and blood pressure levels was significantly associated with impaired glucose tolerance in all age and sex groups after adjustment for age (p < 0.05) and was also related to diabetes in either all or some age and sex groups, respectively (p < 0.05). It was shown that glucose intolerance in the general population was associated with the factors related to insulin resistance. These cross-sectional data, therefore, support the hypothesis that insulin resistance is the primary defect in the development of glucose intolerance in the Japanese general population. However, a further prospective study is still needed in order to confirm this hypothesis.

Adult↗
PubMed1994

Sequence variation of human papillomavirus type 16 E7 in preinvasive and invasive cervical neoplasias.

Variation in the nucleotide sequence of the HPV 16 E7 gene in preinvasive cervical intraepitherial neoplasia (CIN) and invasive cervical carcinoma specimens was analyzed. Direct DNA sequencing of PCR-amplified products with primers different from those used for PCR with 5'-end labeling generated distinct sequence ladders with a low background, even in specimens containing relatively low copy numbers of HPV. Of 14 cervical neoplasias, 11 cases showed sequence diversity from prototype HPV16, and a total of 22 nucleotide exchanges were detected. Nine of these led to single amino acid exchanges: [Thr5] to [Lys5] in one case and [Asn29] to [Ser29] in eight cases. The [Ser29] E7 was distributed uniformly among invasive carcinomas and precancerous legions, and was also found in a normal cervix. The [Lys5] E7 and [Ser29] E7 had transforming potential similar to the prototype E7 assessed by cooperation with the activated ras gene in rat embryo fibroblasts.

Amino Acid Sequence↗
PubMed1994

Immunoenzymometric analysis for plasma von Willebrand factor degradation in diabetes mellitus using monoclonal antibodies recognizing protease-sensitive sites.

High-molecular-weight von Willebrand factor (vWf) multimers were separated from their smaller multimers by molecular-sieve chromatography and were measured by several sandwich enzyme-linked immunosorbent assays (ELISA) with monoclonal antibodies (moABs) against human vWf. The epitopes of these moABs were mapped partially using fragments generated by V-8 protease digests of native vWf. Large multimers were more immunoreactive with the sandwich ELISA using immobilized VW28-1 and enzyme-labeled VW92-3 than with any other ELISA. The epitopes recognized by these two moABs were sensitive to trypsin and plasmin digestion, and the other moABs appeared to be reactive with the extensively digested antigen. Relative reactivities of this ELISA to plasma vWf multimers in diabetes mellitus were significantly reduced compared to those in healthy subjects. These data demonstrate that molecular abnormalities of vWf circulating in diabetic patients may be caused by some plasma proteases that increase in the microcirculation of patients with diabetic vascular diseases.

Adult↗
PubMed1994

Effects of oral administration of Lactobacillus casei on antitumor responses induced by tumor resection in mice.

The effects of an oral administration of BLP, a preparation of viable Lactobacillus casei YIT 9018, upon tumor growth and the mitogenic responses of splenocytes from tumor-bearing mice were studied. BALB/c mice were insufficiently pre-immunized by resecting a Colon 26 tumor mass (primary tumor) grown for 5 days intradermally. Thereafter, mice were rechallenged by injecting Colon 26 tumor (secondary tumor) into the hind footpad. The secondary tumor grew progressively in control mice, but was markedly suppressed by oral administration with BLP at a dose of 100 or 200 mg/kg/day for 7 consecutive days. The suppression was a primary tumor-specific response. Viable L. casei, not heat-killed L. casei, could suppress the secondary tumor. Although the lymphoproliferative responses of splenocytes from the secondary tumor-bearing mice with T-cell mitogens (concanavalin A and phytohaemagglutinin) and cytokines (interleukin-1 and interleukin-2) were lower than those of normal mice, this suppression of mitogenic responses under tumor-bearing conditions was abolished by oral administration with BLP. Thus we concluded that oral BLP potentiated systemic immune responses that modified T-cell functions in tumor-bearing mice.

Adjuvants, Immunologic↗
PubMed1994

Soluble E-cadherin fragments increased in circulation of cancer patients.

Monoclonal antibodies were raised against human placental soluble E-cadherins and used in an immunoenzymometric assay to detect soluble E-cadherins in biological fluids. The E-cadherin assay was accurate enough to quantitate the concentration of soluble E-cadherin in the cell culture supernatants. Immunoreactive E-cadherins, identified as existing in the soluble form in normal serum, were shown to have apparent lower molecular mass (approximately 80 kDa) than intact molecules of E-cadherin. We found that the immunoreactive E-cadherin levels in the serum of the studied cancer patients were significantly elevated (mean +/- s.d. 3.80 +/- 2.36 micrograms ml-1, P < 0.0001) when compared with the normal levels (1.99 +/- 0.50 micrograms ml-1). We also found that serum E-cadherin levels in the 22 patients with gastric cancer (3.51 +/- 1.78 micrograms ml-1, P < 0.02) or the 11 patients with hepatocellular cancer (5.55 +/- 3.11 micrograms ml-1, P < 0.001) were significantly higher than those in the 26 diabetic patients (2.33 +/- 1.58 micrograms ml-1). Of the 54 cancer patients, 53.7% exhibited an elevated amount of soluble E-cadherin in serum. Thus, it is evident that soluble E-cadherin in circulation can be used as a prospective tumour marker that accurately reflects the progressive regeneration of E-cadherin at tumour sites, potentially induced by tumour-associated proteolytic degradation.

Adult↗
PubMed1994

Expression of recombinant mouse/human chimeric antibody specific to human GMP-140/P-selectin.

To construct a mouse/human chimeric antibody, we cloned the genomic DNAs for Ig from a murine hybridoma that produces PL7-6 monoclonal antibody specific to human P-selectin and expressed them in SP2/0 myelomas using a series of pSV2 vectors. Transfected cells that produce the mouse/human chimeric anti-human P-selectin antibody were geneticin-selected and screened by an immunoassay using immobilized antigen. The chimeric antibody, cPL-2R1, expressed by the resultant clone has the murine Ig variable region and the human Ig constant region. The native antibody PL7-6 and the chimeric antibody cPL-2R1 react equally with purified P-selectin and thrombin-stimulated platelets. Competitive inhibition tests demonstrated that the native antibody PL7-6 and the chimeric antibody cPL-2R1 had identical affinity for purified human P-selectin. Thus, although human IgG1 constant region was substituted for the murine counterpart in this chimeric antibody, its specificity and binding affinity for P-selectin was not altered. This chimeric antibody may prove useful when employed in combination with imaging reagents or therapeutic drugs for targeting activated platelets or endothelium in patients with thrombosis or intravascular inflammation.

Amino Acid Sequence↗
PubMed1994

A novel oligosaccharide, xylosyl beta 1-4xylosyl beta 1-(4-methylumbelliferone), synthesized by cultured human skin fibroblasts in the presence of 4-methylumbelliferyl-beta-D-xyloside.

4-Methylumbelliferyl-beta-D-xyloside (Xyl-MU) was added to the medium of cultured human skin fibroblasts. After incubation, the culture medium was pooled, concentrated with a lyophilizer, and dialyzed against distilled water. Then the Xyl-MU derivatives in the diffusate were purified by gel-filtration and HPLC. A novel Xyl-MU derivative was obtained, in addition to the previously reported Xyl-MU derivatives, Xyl-MU-induced glycosaminoglycan (GAG-MU), SA-Gal-Xyl-MU, GlcA-Xyl-MU, Gal-Gal-Xyl-MU, and Gal-Xyl-MU. This Xyl-MU derivative was subjected to carbohydrate composition analysis, enzyme digestion, Smith degradation and ion-spray mass spectrometric analysis, and the results indicated that it was Xyl beta 1-4Xyl beta 1-MU. Although the quantity of Xyl beta 1-4Xyl beta 1-MU synthesized by human skin fibroblasts increased with incubation time, its production was independent of that of the GAG-MU. Xyl-Xyl-MU is different from the intermediates in the regular pathway of GAG-MU biosynthesis initiated by added Xyl-MU, posing an interesting question as to its significance in GAG biosynthesis.

Carbohydrate Sequence↗
PubMed1994

Isolation and analysis of cDNA encoding a precursor of Canavalia ensiformis asparaginyl endopeptidase (legumain).

Recently, asparaginyl endopeptidase was purified to homogeneity from jack bean (Canavalia ensiformis) seeds, and its NH2-terminal amino acid sequence was determined for 25 residues [Abe, Y. et al. (1993) J. Biol. Chem. 268, 3525-3529]. On the basis of this sequence information, we searched for seed cDNAs encoding this enzyme. Seven clones were obtained and sequenced. By combining four of them, we obtained a cDNA for a precursor of the enzyme containing the reported NH2-terminal sequence. The other three clones seemed to be for precursors of its isozymes. When the deduced amino acid sequences of these enzyme precursors were compared with those in the GeneBank, EMBL, and NBRF databases, only one protein was found with a homologous sequence. It was a precursor of hemoglobinase from a blood fluke (Schistosoma mansoni). Significant homology was observed only in the range of sequence for the mature form. Although hemoglobinase and asparaginyl endopeptidase behave as cysteine proteinases, their protein natures are distinct from either papain-type proteinases or clostripain. Various plant seeds have been reported to contain asparaginyl endopeptidases. This may be the first report, however, that deals with the primary structure of such a proteinase.

Amino Acid Sequence↗
PubMed1994

Overproduction of Thermus aquaticus DNA polymerase and its structural analysis by ion-spray mass spectrometry.

We cloned the pol gene from the Thermus aquaticus YT-1 strain into a plasmid vector and constructed a high-level expression system of the gene in Escherichia coli. Six codons in the translational start region were changed to simple AT-type codons or codons which are most frequently used in E. coli by the genetic engineering techniques with retention of the amino acid sequence of the native enzyme. The modified pol genes were expressed under the lac promoter of pUC-type plasmid and 266,418 units of activity was obtained in a sonicated and heated crude extract from 2 g of E. coli cells bearing one of the recombinant plasmids, pTAQ9. Highly purified protein was subjected to structural analysis using a protein sequencer and an ion-spray mass spectrometer combined with reversed-phase HPLC (LC-MS). The primary structure of the DNA polymerase was identical with the amino acid sequence deduced from the nucleotide sequence of the pol gene as far as examined (about 95% of the sequence); though, several regions where small peptides of less than 5 residues were produced by lysyl endopeptidase digestion could not be sequenced.

Amino Acid Sequence↗
PubMed1994

Inhibitory effect of recombinant fibronectin polypeptides on the adhesion of liver-metastatic lymphoma cells to hepatic sinusoidal endothelial cells and tumor invasion.

We have investigated the inhibitory mechanism of the initial arrest of L5178Y-ML25 lymphoma cells in a target organ (liver) by using recombinant fibronectin fragments with cell- and/or heparin-binding domains (C-274, H-271 or the fusion fragment CH-271). Pretreatment of hepatic sinusoidal endothelial (HSE) cell monolayers with lymphoma cells or their conditioned medium for 4 to 6 h resulted in the enhancement of lymphoma cell adhesion to HSE cell monolayer. The increased tumor adhesiveness was completely abolished by preincubation of the conditioned medium with anti interleukin-1 beta monoclonal antibody (mAb). Synthetic sialyl Le(x) (SLe(x)) as a ligand for endothelial cell leukocyte adhesion molecule-1 (ELAM-1) adhesion receptor and anti ELAM-1 mAb blocked the conditioned medium-induced enhancement of tumor-endothelial cell interaction, while pretreatment of the activated HSE cell monolayer with anti vascular cell adhesion molecule-1 (VCAM-1) mAb did not affect the enhanced tumor cell adhesion. These results indicate that tumor cell interaction with the stimulated HSE cells is mediated by ELAM-1 molecules on HSE cells. However, the expression of SLe(x) and SLe(a) on the tumor surface was not observed by flow cytometric analysis. ELAM-1-mediated enhancement of tumor cell adhesion to HSE monolayer was also inhibited in a concentration-dependent manner by CH-271 fusion polypeptide or the sulfated chitin derivative sulfated carboxymethyl-chitin, which can bind to the heparin-binding domain of CH-271. In addition, CH-271 inhibited not only tumor-endothelium interaction but also tumor cell invasion into reconstituted basement membrane Matrigel in vitro.

Animals↗
PubMed1994

GD2 ganglioside-specific monoclonal antibody reacts with murine cytotoxic T lymphocytes reactive with FBL-3N erythroleukaemia.

Ganglioside expression on cytotoxic T lymphocytes induced against the mouse erythroleukaemia line FBL-3N, was analysed, compared with that of naive T lymphocytes in the thymus, lymph nodes and spleen. Although normal uncultured and cultured T lymphocytes expressed no GD2, GD3 or GM2 gangliosides, cytotoxic T cells with CD4+CD8-, CD4-CD8+, and CD4-CD8- phenotypes reacted with anti-GD2 monoclonal antibody with various intensities. The reactivity with anti-GD2 was associated with the intensity of cytotoxic activity as analysed using cytotoxic T lymphocyte (CTL) clones established from the bulk CTLs of each phenotype. An increase of the mRNA expression of GM2/GD2 synthase gene was demonstrated by Northern blot hybridization using RNA extracted from thymocytes, spleen cells, Con A-treated spleen cells and various types of CTL cells. These results indicated that GD2 ganglioside expression might be associated with the functional differentiation of murine T lymphocytes.

Animals↗
PubMed1994

Sudden cerebral hypoperfusion enhances respiratory suppression in hypoxic newborn piglets.

The relationship between sudden cerebral hypoperfusion and sudden respiratory suppression was investigated in newborn piglets. A total of 11 experiments on five newborn piglets were divided into three groups: normoxic, hyperoxic and hypoxic. The sudden cerebral hypoperfusion was created by manual compression of the Arteria carotis communis and Vena jugularis interna. Respiratory suppression was calculated by measuring the duration of apnea and the percent reduction of respiratory amplitude. The means of apnea duration in normoxic and hyperoxic groups were 6.6 +/- 0.8 and 1.2 +/- 2.4 s, respectively, while two experiments on the hypoxic group showed 8.4 s and complete cessation of respiration in one of them. The per cent reductions of respiratory amplitude in the normoxic and hyperoxic groups were 23.3 +/- 8.5 and 17.6 +/- 11.7%, respectively. In the hypoxic group, one of two experiments showed a 22.7% reduction, which was equivalent to that of the normoxic group. We conclude that sudden cerebral hypoperfusion enhances respiratory suppression, especially when it coincides with hypoxia. The phenomenon may be one of the possible causes for a certain type of sudden infant death syndrome. Further study will be undertaken to elucidate the direct effects of the mechanism of cerebral hypoperfusion on respiratory suppression.

Animals↗
PubMed1994

A clinical trial of Azelastine in recurrent aphthous ulceration, with an analysis of its actions on leukocytes.

Azelastine (azelastine hydrochloride) was orally administered to 43 patients with recurrent aphthous ulcers (RAU), and its clinical effects and in vitro influence on leukocytes were examined. During at least 6 months after drug treatment, no oral ulcers occurred in 7 patients, and an improvement of the oral condition was exhibited in all except 4 of the remaining patients. The frequency of occurrence of RAU was significantly reduced, from 1.7 +/- 0.9 to 0.9 +/- 0.5 times/month, and ulcer duration and oral irritation were also significantly reduced from 12.5 +/- 2.5 and 7.5 +/- 2.4 days to 9.8 +/- 2.6 and 5.3 +/- 2.4 days, respectively. Neutrophils from patients treated with Azelastine generated a suppressed volume of superoxide (O2-). Suppression of O2- generation and chemiluminescence by in vitro Azelastine was also confirmed to occur in a dose-dependent manner. Furthermore, the production of TNF-alpha and GM-CSF in lymphocytes was suppressed in the presence of Azelastine, and the drug protected sheep red blood cells and epithelial tumor cell lines against hydrogen peroxide impairment and hypotonic shock. These clinical and experimental results lead to the conclusion that improvement of RAU by Azelastine depends on the protection of cell membranes and the suppression of leukocyte-function, including reactive oxygen generation.

Adolescent↗
PubMed1994

Detection of alcohol-tolerant hiochi bacteria by PCR.

We report a sensitive and rapid method for detection of hiochi bacteria by PCR. This method involves the electrophoresis of amplified DNA. Nucleotide sequences of the spacer region between 16S and 23S rRNA genes of 11 Lactobacillus strains were identified by analysis of PCR products. Five primers were designed by analysis of similarities among these sequences. A single cell of Lactobacillus casei subsp. casei could be detected when purified genomic DNA was used as the template. When various cell concentrations of L. casei subsp. casei were added to 50 ml of pasteurized sake and the cells were recovered, the detection limit was about one cell. No discrete band was observed in electrophoresis after PCR when human, Escherichia coli, mycoplasma, Acholeplasma, yeast, or mold DNA was used as the template.

Bacteriological Techniques↗