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Biomedical subjects

K Tsuji

Publications and source records attributed to K Tsuji.

At least 469 records · Page 26Linked to original sources
PubMed1991

Enhancement of murine blast cell colony formation in culture by recombinant rat stem cell factor, ligand for c-kit.

Mice with W mutations characterized by hypopigmentation, sterility, anemia, and mast cell deficiency have abnormalities in c-kit, a receptor with tyrosine kinase activity. Recently, the ligand for c-kit was cloned by investigators in several laboratories. Zsebo et al identified and cloned a gene for a cytokine termed stem cell factor (SCF) in the medium conditioned by buffalo rat liver cells, and this cytokine proved to be c-kit ligand. We have examined the effects of recombinant rat SCF (rrSCF) on colony formation from primitive hematopoietic progenitors in culture. rrSCF and erythropoietin (Ep) supported formation of granulocyte/macrophage (GM) colonies as well as a small number of multilineage and blast cell colonies from marrow cells of normal mice. We then examined the effects of rrSCF using marrow and spleen cells of mice that had been treated with 150 mg/kg 5-fluorouracil (5-FU). Unlike single factors, combinations of factors such as rrSCF plus interleukin-3 (IL-3), rrSCF plus IL-6, and rrSCF plus granulocyte colony-stimulating factor (G-CSF) markedly stimulated the growth of multilineage colonies. In contrast to these factor combinations and a combination of IL-3 and IL-6, a combination of rrSCF and IL-4 did not support multilineage colony formation. Mapping studies of the development of multipotential blast cell colonies further indicated that rrSCF, like IL-6, G-CSF, and IL-11, shortens the dormant period in which the stem cells reside. When we tested the effects of rrSCF using pooled blast cells, which are highly enriched for progenitors and are devoid of stromal cells, rrSCF plus Ep supported formation of only a few multilineage colonies, indicating that rrSCF itself is ineffective in support of the proliferation of multipotential progenitors. However, rrSCF supported formation of a significant number of neutrophil and neutrophil/macrophage colonies from pooled blast cells, indicating that rrSCF is able to support directly the proliferation of progenitors in neutrophil/monocyte lineages. c-kit ligand may play important roles in adult hematopoiesis.

Animals↗
PubMed1991

[A study of new retrievable expandable metallic stent].

The authors improved retrievable expandable metallic stent (REMS), instead of the nylon suture, the REMS was connected with the stainless steel thread. Under fluoroscopy the stainless steel thread of the shrinking body could be observed and was strong enough to retract the placed stent, then the stent could be removed by the stainless steel hook. We placed the REMS in the I.V.C. of canine, and removed them after one week placement, we could not remove them after two weeks placement. This study suggested that the REMS was considered to be useful and safe for clinical application.

Animals↗
PubMed1991

High-performance capillary electrophoresis of proteins. Sodium dodecyl sulphate-polyacrylamide gel-filled capillary column for the determination of recombinant biotechnology-derived proteins.

Fused-silica capillary columns were filled with sodium dodecyl sulfate-polyacrylamide gel and the column effluent was monitored at 214 nm using a commercially available high-performance capillary electrophoresis (HPCE) instrument to separate and rapidly quantify recombinant biotechnology-derived proteins. An excellent linear relationship (r greater than 0.999) exists between the peak migration time and the molecular weights of reference proteins in the range 10,000-100,000 and 40,000-200,000 dalton by use of the capillary columns filled with acrylamide gel at a T composition of 5% and 3%, respectively. The relative standard deviation (R.S.D.) of the peak migration time is ca. 1%. Theoretical plates of 5 X 10(5)-1 X 10(6) per metre are routinely being obtained. Calibration graphs of peak area versus weight of recombinant biotechnology-derived proteins are linear (r greater than 0.999) and the proteins may be quantified with an R.S.D. of ca. 3-7%. As little as 50 nmol of a protein may be quantified and an impurity peak of molecular weight ca. 1500 less than that of the parent compound (ca. 60,000 dalton) may be differentiated by HPCE with a gel-filled capillary column.

Animals↗
PubMed1991

Central nervous system action of basic fibroblast growth factor: inhibition of gastric acid and pepsin secretion.

To examine the effects of basic fibroblast growth factor (bFGF) on gastric secretion, the present study was carried out using pylorus-ligated rats. Intracisternally injected bFGF inhibited the secretion of both gastric acid and pepsin, and this gastric antisecretory action of bFGF was a dose-related response. On the other hand, the intraperitoneal injection of bFGF did not change gastric secretion. These results strongly suggested for the first time that bFGF, a growth factor that promotes the proliferation of various cell types, might also be a chemical messenger that is involved in the central regulation of gastric secretion. This biological action of bFGF may be considered as a novel nonmitogenic activity of this growth factor.

Animals↗
PubMed1991

HLA class II antigens and DNA restriction fragment length polymorphism in myasthenia gravis in Japan.

Human leukocyte phenotypes and genes in the HLA class II regions were studied in 46 Japanese patients with myasthenia gravis. When the HLA phenotypes of the patients with myasthenia gravis were compared with the controls, an increased frequency of HLA-DRw53 was observed in females less than 30 years of age. The genomic DNAs of the HLA-DRw53-positive patients and DRw53-positive controls were analyzed by using four complementary DNA probes for HLA class II genes. With DQB complementary DNA as the probe, a higher incidence of the 6.5-kb or 8.2-kb BamHI fragment was observed in the patients (76.0%) compared with the controls (19.0%). In contrast, no significant difference was observed between patients and controls when complementary DNAs for DRB, DQA, and DPB were used as probes. These results indicate that the genetic background of Japanese females with early-onset myasthenia gravis is different from other patients with myasthenia gravis, and that DQB genes can greatly influence the onset of myasthenia gravis.

Adolescent↗
PubMed1991

Association of the complement allele C4AQ0 with primary Sjögren's syndrome in Japanese patients.

We studied allotypes of the fourth component of complement (C4) and factor B in 76 patients with Sjögren's syndrome (SS) and in 63 normal subjects. C4A-null (C4AQ0) was found in 10 of 28 patients who had primary SS, compared with 1 of 63 control subjects (P less than 0.005). In contrast, no significant difference in the frequency of any C4 allotype was observed between patients with secondary SS and control subjects. An association of HLA-DRw53 with primary SS in Japanese patients has been reported. Since there is no linkage disequilibrium between DRw53 and C4AQ0, it is possible that at least 2 genes in the major histocompatibility complex may determine susceptibility to the development of primary SS in the Japanese population.

Autoimmune Diseases↗
PubMed1991

Murine mast cell colony formation supported by IL-3, IL-4, and recombinant rat stem cell factor, ligand for c-kit.

Recently, a novel cytokine designated stem cell factor (SCF) was isolated from medium conditioned by buffalo rat liver cells and proved to be the ligand for c-kit. We have examined the effects of recombinant rat SCF alone and in various combinations with interleukin-3 and interleukin-4 on murine mast cell colony formation in methylcellulose culture. As a source of connective tissue-type mast cells (CTMC), we used peritoneal mast cells. No individual factor supported colony formation by purified peritoneal mast cells. When cells were grown in combinations of two factors, significant mast cell colony growth was seen. When cells were grown in the presence of three factors, not only the number of colonies was increased but also the colonies were larger. Mast cells in these colonies contained safranin- and berberine sulfate-positive cells, but the proportions of positive and negative cells varied depending on the factor combinations. We then examined the effects of these factors on proliferation of bone marrow-derived mast cells (BMMC) by replating pooled mast cell colonies. As a single factor, only interleukin-3 supported mast cell colony formation. Combinations of two of the three factors supported mast cell colony formation. However, the most impressive synergism was seen again with the combination of the three factors. Not only was the number of colonies increased, but there was a significant increase in size. These results indicate that SCF is an important factor for the proliferation of both CTMC and BMMC.

Animals↗
PubMed1991

Orientation of HLA-DNA gene and identification of a CpG island-associated gene adjacent to DNA in human major histocompatibility complex class II region.

We have constructed the detailed physical map of the HLA class II gene region by the pulsed field gel electrophoresis (PFGE) and cosmid walking technique. In this process, the DNA gene was found to be located telomeric to DPA1 with the 5'----3' orientation which is the same as the DPA1 and DPA2 genes, but opposite to the DQA1, DQA2 and DRA genes. This orientation is reverse to that of the counterpart gene in the rabbit major histocompatibility complex region. About 30 kb downstream from the DNA gene towards DOB, a CpG island characterized by clustered sites for rare cutting restriction enzymes and frequently associated with the 5' end of housekeeping genes was identified by PFGE and cosmid walking. From a complementary DNA (cDNA) library constructed from a Epstein-Barr virus-transformed B-cell line, a cDNA clone was isolated using the genetic probe from this CpG island. Its nucleotide sequences suggested that it represented a new non-HLA gene with a single copy which was of little genetic polymorphism and named NAT (DNA-associated transcript). Northern blot analysis showed that the NAT gene was expressed with a 4-kb transcript in all of tissues examined so far.

Base Sequence↗
PubMed1991

Severe acute graft-versus-host disease by HLA-DPB1 disparity in recombinant family of bone marrow transplantation between serologically HLA-identical siblings: an application of the polymerase chain reaction-restriction fragment length polymorphism method.

It has remained to be established that matching of the HLA-DP antigen plays a key role in bone marrow transplantation (BMT), mainly due to the difficulty of the primed lymphocyte test (PLT) method for DP typing. We previously reported an efficient technique for HLA class II genotyping, by digestion of polymerase chain reaction (PCR)-amplified genes with restriction fragment length polymorphisms (RFLP) endonucleases (PCR-RFLP method). DNAs from 46 recipients and corresponding donors in serologically HLA-identical sibling-BMT cases were DP typed by this PCR-RFLP method. Of the 46 cases, five (10.9%) were genetically DP mismatched (recombinant frequency between the DR-DQ and DP subregions was at least 2.7% per meiosis), providing an important opportunity to look at the effect of the disparity only seen in the DP antigen on BMT. Three of the four DP-mismatched BMT cases that could be evaluated developed severe acute graft-versus-host disease, suggesting that DP disparity played an important role in BMT.

Acute Disease↗
PubMed1991

HLA class II antigens are associated with Japanese pemphigus patients.

We investigated the HLA class II antigens in 30 Japanese cases of pemphigus, 17 cases of pemphigus vulgaris (PV) and 13 cases of pemphigus foliaceus (PF), by both serologic and restriction fragment length polymorphism (RFLP) analyses. We detected two major haplotypes susceptible to PV, i.e., DRw12-DQw7 and DRw6-DQw5. In contrast, DR2 was absent in PV. RFLP analyses showed that DRw6 PV patients had a disease-associated restriction fragment representing DQw5, the same association as that found in DRw6 Jewish PV patients. However, DRw12 Japanese PV patients had DQw7, whereas DR4 Jewish PV patients had DQw8. On the other hand, all 13 PF patients were serologically typed for DQw1, which could not be further subdivided into DQw5 by RFLP analyses. These results suggest that Japanese and Jewish PV patients may be immunogenetically closely related to each other, but Japanese PV patients appear to be immunogenetically different from Japanese PF patients.

Autoantibodies↗
PubMed1991

DNA damage in forestomach epithelium from male F344 rats following oral administration of tert-butylquinone, one of the forestomach metabolites of 3-BHA.

DNA damage in the forestomach epithelium of male F344 rats was tested by alkaline elution assay following oral administration of 3-tert-butyl-4-hydroxyanisole (3-BHA) and its free metabolites. Although 1% 3-BHA and 0.001% tert-butylhydroquinone (BHQ) caused no detectable DNA damage, a tert-butylquinone (BQ) treated group showed a significantly higher elution rate than the corn oil and 3-BHA group. Dose-dependent induction of DNA damage in forestomach epithelium by 3-BHA metabolites was compared. BQ showed DNA damaging activity from 0.0001% (1 microgram/ml). On the other hand, BHQ showed the activity at greater than 100 times higher concentration than that of BQ. Six other quinones related to the BQ have been synthesized and examined in vivo for their activity in single strand breaks in forestomach epithelium DNA. The order of the DNA damage was not always correlated with the order of the first reduction potentials of the quinones. 2-Benzylthio-5-tert-butyl-p-benzoquinone (BQSBn-2), 2-phenyl-1,4-benzoquinone and 2,5-di-tert-butyl-p-benzoquinone had higher DNA damaging activity than BQ. The effects of ethylacrylate (EA) pretreatment on the DNA damage by BQ and BQSBn-2 were compared. The DNA damage by BQ was decreased by increasing the EA concentrations. On the other hand, BQSBn-2 had a DNA damaging activity following the EA pretreatment. In order to examine whether 3-BHA is metabolized in forestomach, 3-BHA and BQ levels in the mucosa of forestomach were measured by HPLC with an electrochemical detector. The concentrations of BQ in mucosa correlated with the 3-BHA dose levels from 0.01% to 0.1% 3-BHA. The concentration of BQ in mucosa was approximately 1/4000 of the concentration of 3-BHA in mucosa at the dose level of 1% 3-BHA. The present results indicate that BQ and its thiol conjugates are the active metabolites from 3-BHA in forestomach and may play an important role in inducing carcinogenicity of 3-BHA in F344 rats.

Acrylates↗
PubMed1991

Serum soluble CD4 and CD8 levels in Kawasaki disease.

The levels of soluble CD4 (sCD4) and sCD8 in serum correlate with the T cell subset activation and may be important in monitoring and characterizing disease processes during immunological diseases. We compared acute Kawasaki disease (KD) with anaphylactoid purpura (AP) and acute febrile viral infections, such as measles and infectious mononucleosis (IM), in terms of serum sCD4 and sCD8 levels. The levels of serum sCD4 and sCD8 were measured by a sandwich enzyme immunoassay. In addition, peripheral blood mononuclear cell subsets were analysed by single and two-colour flow-cytometric analyses in KD and IM patients. The levels of serum sCD4 and sCD8 were significantly elevated in patients during acute stages of KD, measles and IM, but not AP. Peripheral blood CD4+, CD8+ and also HLA-DR+ T cells count did not increase during the acute stage of KD; however, peripheral blood CD8+ and HLA-DR+ T cell counts were increased during the acute stage of IM. Our results suggest that there is a low level of activation of peripheral blood T cells during acute KD, or that infiltrated T cells in some local tissues of KD patients contribute to the elevated levels of serum sCD4 and sCD8.

Acute Disease↗
PubMed1991

Genetic differences shown by HLA typing among Japanese patients with euthyroid Graves' ophthalmopathy, Graves' disease and Hashimoto's thyroiditis: genetic characteristics of euthyroid Graves' ophthalmopathy.

Euthyroid Graves' ophthalmopathy (EO) is an ophthalmic disorder without persistent hyperthyroidism. To elucidate genetic differences among EO, Graves' disease (Gr) and Hashimoto's thyroiditis (H), we analysed HLA-A, B, C, DR, DQ, D and DP types in 23 Japanese EO patients, 88 Gr patients, 46 H patients and 186 control subjects utilizing assays of lymphocyte cytotoxicity and restriction fragment length polymorphism (RFLP). When compared with the control subjects, EO patients showed significant associations with HLA B40 (w61), DR9, DQw3, and Dw15 (P less than 0.01) and with HLA B12 and Cw1 (P less than 0.05). When allowance was made for the number of antigens tested, only DQw3 was significant. Significant differences were found between EO and Gr (DPw2), and between EO and H (Cw1) even after correction of P values. Comparisons between EO and related subgroups of Gr confirmed the heterogeneity of EO again. It is concluded from these results that EO is associated with different HLA types from Gr and H.

Adult↗
PubMed1991

HLA-DQB1 genotyping by a modified PCR-RFLP method combined with group-specific primers.

We previously reported a simple technique for HLA-DQB genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). However, this method has some problems in that some heterozygotes cannot be discriminated from each other. Furthermore, concomitantly amplified product derived from the DQB2 gene by the primers used previously also obstructs precise DQB1 genotyping. To resolve these problems, we have developed two different pairs of specific primers for selective amplification of the DQB1 gene and also used restriction endonucleases which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA-DQB1 alleles, making reading of RFLP band patterns much easier. The second exon of the DQB1 gene was selectively amplified by DQw1 group-specific primers and/or DQw2,3,4 group-specific primers using genomic DNAs from 70 HLA-homozygous B-cell lines and 50 healthy Japanese. Of the seven DQw1-associated DQB1 alleles, six alleles could be defined by digestion of 6 restriction enzymes, although DQB1*0602 and DQB1*0603 could not be discriminated from each other because of unavailability of suitable enzymes. Similarly, all of the six DQw2,3,4-associated DQB1 alleles could be defined by digestion of 5 restriction enzymes. Using this modified PCR-RFLP method, complete DQB1 genotyping of all heterozygotes is possible except for discrimination between DQB1*0602 and 0603. Thus this method is simpler and more practical for a routine DNA typing than the PCR-SSO method or our previous PCR-RFLP method.

Alleles↗
PubMed1991

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping.

We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacrylamide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, FokI, MboII and Mn1I can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, FokI, DdeI, BsaJI, BssHII, Cfr13I, RsaI, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.

Alleles↗
PubMed1991

Assignment of human porphobilinogen deaminase to 11q24.1----q24.2 by in situ hybridization and gene dosage studies.

In situ hybridization and gene dosage-effect studies were conducted to determine the detailed chromosomal location of the gene encoding human porphobilinogen deaminase (PBGD). Red cell PBGD activity was normal in one patient with monosomy for 11q24.2----qter but was increased 1.5 times in another patient with trisomy for 11q22.2----qter. The cDNA probe for PBGD was found to be specifically hybridized to band 11q24. These results suggest that the gene for PBGD is localized within the region 11q24.1----q24.2.

Abnormalities, Multiple↗
PubMed1991

Dual effect of protein kinase C on the induction of DNA synthesis by colcemid in G0-arrested human diploid fibroblasts.

G0-arrested human diploid fibroblasts, TIG-1, was stimulated to induce DNA synthesis by serum, epidermal growth factor (EGF), colchicine, colcemid, or 12-O-tetradecanoylphorbol-13-acetate (TPA). The induction of DNA synthesis was mediated by protein kinase C (PKC) when stimulated with TPA but not when stimulated with other agents. When TPA-stimulated cells were immediately treated with colcemid, induction of DNA synthesis was reduced. This reduction diminished when colcemid was added more than 6 h after TPA treatment. Conversely, when colcemid-stimulated cells were treated with TPA, induction of DNA synthesis was also reduced. This reduction was enhanced when the interval between the addition of two stimulants was extended. PKC-deprivation abolished both stimulatory and inhibitory effects of TPA on DNA synthesis. Staurosporine blocked an induction of DNA synthesis by TPA but appeared to be ineffective on the inhibitory action of TPA on DNA synthesis by colcemid. These results suggest that the inhibitory effect of TPA on the induction of DNA synthesis by colcemid is mediated by down regulation-sensitive and staurosporine-insensitive PKC.

Alkaloids↗