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Biomedical subjects

T Hanafusa

Publications and source records attributed to T Hanafusa.

At least 163 records · Page 9Linked to original sources
PubMed1991

Sequence variations in the envelope protein of the hepatitis C virus: comparison with partial cDNA sequence of a new variant virus obtained by the polymerase chain reaction.

It has been reported that the envelope region located at the 3' portion of the structural protein coding region is one of the most variable regions at both nucleotide and amino acid sequence levels in the hepatitis C virus (HCV) genome. We cloned HCV cDNA fragments of an envelope protein coding region (HCVNK), which were derived from serum of a Japanese patient with hepatocellular carcinoma and were amplified by polymerase chain reaction. After determining the nucleotide sequence, deduced amino acid sequence of the envelope protein region was compared with those of six HCV strains already published (HCJ1, HCVUS, HCJ4, HCVJH, HCVJ and HCVBK). Homology analysis among the strains revealed that the seven strains were classified into two subtypes; a US subtype (HCJ1 and HCVUS) and a Japanese subtype (HCJ4, HCVJH, HCVJ, HCVBK and HCVNK), since percentage homologies between two subtypes (70.3-77.3%) were significantly lower than those within each subtype (83.9-93.5%). Detailed analysis of the amino acid sequences also indicates that the region at aa246-aa258, tentatively named intersubtype variable region-1, may distinguish the US subtype from the Japanese subtype.

Amino Acid Sequence↗
PubMed1991

Nicotinamide and 3-aminobenzamide reduce interferon-gamma-induced class II MHC (HLA-DR and -DP) molecule expression on cultured human endothelial cells and fibroblasts.

We investigated the effects of nicotinamide and 3-aminobenzamide, known as inhibitors of poly (ADP-ribose) synthetase, on the expression of interferon- gamma (IFN-gamma)-induced class I and II major histocompatibility complex (MHC) molecules on the surface of cultured human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (HDF). Indirect immunofluorescent staining on HUVEC and HDF was performed using monoclonal antibodies against class I MHC (HLA-A,B,C) and class II MHC (HLA-DR, HLA-DP and HLA-DQ) molecules, and then the expression of these molecules was determined using a fluorescence flow cytometry. Human recombinant IFN-gamma (100 U/ml) increased the expression of HLA-A,B,C molecules, and induced the expression of HLA-DR molecules and, to a lesser extent, of HLA-DP on both HUVEC and HDF. HLA-DQ molecules were not induced by IFN-gamma on either cell type. Nicotinamide and 3-aminobenzamide in the concentration greater than or equal to 1 mM reduced the IFN- gamma -induced expression of HLA-DR and HLA-DP on both HUVEC and HDF, whereas neither agent in the concentration of up to 10 mM affected the IFN- gamma -induced increase in HLA-A,B,C molecule expression. These data suggest that nicotinamide and 3-aminobenzamide suppress antigen presenting function of class II MHC positive endothelial cells and fibroblasts at the site of tissue inflammation.

Animals↗
PubMed1991

Demonstration of islet cell antibodies in apparently non-insulin dependent diabetic patients; a marker for the later development of insulin dependency.

One hundred and ten diabetic patients who were apparently non-insulin dependent at 1984 were followed up for 5 years from 1984 to 1989. Islet cell antibodies (ICA) of the patients were tested in 1987. Eleven patients were positive for ICA and 99 were negative. There was no significant difference in age, sex, duration of diabetes, and HbA1c levels between ICA-positive and negative groups. Six of 11 (54.5%) patients in ICA-positive group developed insulin-requiring state in the period from 1984 to 1989, while only 5 of 99 (5.1%) patients in ICA negative group became insulin-requiring. Glucagon tolerance test (1 mg i.v.) was performed on 8 patients who developed insulin-requiring state; among them 4 patients were ICA-positive and other 4 patients were ICA negative. The serum C-peptide response to intravenous glucagon injection was markedly decreased in 3 of the 4 ICA-positive patients, and only mildly decreased in all the 4 ICA-negative patients. The markedly decreased C-peptide response indicates that these ICA-positive subjects had developed insulin-dependency. We conclude that the presence of ICA in apparently non-insulin dependent diabetics indicates a high risk for developing insulin-dependency.

Autoantibodies↗
PubMed1991

Lipopolysaccharide augments HLA-A,B,C molecule expression but inhibits interferon-gamma-induced HLA-DR molecule expression on cultured human endothelial cells.

The effect of bacterial lipopolysaccharide (LPS) on the expression of class I and II major histocompatibility complex (MHC) molecules on the surface of cultured human umbilical vein endothelial cells (HUVEC) was determined by indirect immunofluorescent staining followed by flow cytometric analysis. LPS at concentrations higher than 0.01 micrograms/ml augmented class I MHC (HLA-A,B,C) expression on HUVEC in a concentration-dependent manner. Optimal augmentation, approximately sixfold compared with control, was seen with 10 micrograms/ml of LPS. Time-course experiments indicated that the augmentation was maximal on Day 4. In contrast, LPS had no effect on the induction of class II MHC (HLA-DR) molecules and at concentrations higher than 0.01 micrograms/ml inhibited the interferon-gamma(IFN-gamma)-induced class II MHC expression. The inhibition was about 60% at the concentration of 100 micrograms/ml of LPS. Interleukin-1 (IL-1) had a similar effect as LPS on class I and II MHC expression. However, LPS appeared to affect MHC expression directly and not through production of IL-1 or cyclo-oxygenase pathway products, since anti-IL-1 antibodies or an inhibitor of cyclo-oxygenase pathway products, indomethacin, failed to reverse the effects of LPS. These data stress the role of LPS as a direct modulatory factor of class I and II MHC expression on endothelial cells during the development of immune and inflammatory response against Gram-negative bacteria.

Antibodies↗
PubMed1990

Examination of islets in the pancreas biopsy specimens from newly diagnosed type 1 (insulin-dependent) diabetic patients.

We attempted to examine the immunopathological change of the pancreatic islets of newly diagnosed Type 1 (insulin-dependent) diabetic patients and thereby to obtain useful information for the therapy of the patients. For this purpose, pancreas biopsy under laparoscopy was performed 2-4 months after the onset of Type 1 diabetes in seven newly diagnosed patients. All biopsies were performed safely without any complications. Immunohistochemical examination of the biopsy specimens revealed a marked decrease of insulin-containing cells, preservation of glucagon-containing cells, and various degrees of expression of MHC class I and class II antigens in islet cells and in endothelial cells within and around the islets. Signs of active autoimmune phenomena, e.g. lymphocytic infiltration or immunoglobulin deposition in islets, were not detected in any of these patients by light microscopical evaluation. We conclude that pancreas biopsy under laparoscopy has shown various immunological changes in the islets of newly diagnosed Type 1 diabetic patients. Pancreas biopsy, however, may not be suitable under the present protocol for the selection of patients for immunotherapy because of problems including sampling errors.

Adult↗
PubMed1990

Preventive effect of a new immunosuppressant FK-506 on insulitis and diabetes in non-obese diabetic mice.

We investigated the effect of an immunosuppressant FK-506 on histological change of islets, the onset of diabetes, and the change of spleen cell subsets in female non-obese diabetic mice. Mice administered intraperitoneally with FK-506 from 5 to 20 weeks of age showed marked suppression of mononuclear cell infiltration (insulitis) at 10 weeks of age. Among the subsets of the spleen cells, a significant decrease in the population of Thyl.2-positive T cells (pan-T), L3T4-positive T cells (mainly helper/inducer), and Lyt2-positive T cells (mainly suppressor/cytotoxic) was observed in FK-506-treated mice. Furthermore, glucose tolerance of the mice at 15 weeks of age was clearly improved. Cumulative incidence observed up to 40 weeks of age was 86% in control mice and 23% in FK-506-treated mice (p less than 0.01). These data indicate that FK-506 has a preventive effect on insulitis and diabetes by the suppression of cell-mediated autoimmunity in non-obese diabetic mice.

Animals↗
PubMed1990

Interferon-gamma reduces actin filaments and inhibits thyroid-stimulating hormone-induced formation of microvilli and pseudopods in mouse monolayer thyrocytes.

To study the role of interferon-gamma (IFN gamma), which is released from activated lymphocytes in the development of lymphocytic thyroiditis, we investigated the effects of IFN gamma on the morphology of mouse thyrocytes, using scanning electron microscopy. Thyrocytes were cultured in monolayers for 5-7 days and incubated with TSH (10 mU/ml) for 1 h before fixation. IFN gamma (200 U/ml) was added to the culture medium for 1 h to 5 days before TSH stimulation. Coculture of thyrocytes with IFN gamma for more than 24 h inhibited TSH-induced morphological changes in the thyrocytes; IFN gamma inhibited the increase in the number of and elongation of microvilli and the appearance of pseudopods. IFN gamma also reduced the number of actin filaments in thyrocytes, as confirmed by immunofluorescence photometry. These results suggest that IFN gamma inhibits the morphological response of thyrocytes to TSH stimulation by the reduction of actin filaments.

Actins↗
PubMed1990

Proteolysis of myosin and troponin in human myocardium of elderly subjects.

Actomyosin was prepared from human myocardium and its protein composition was examined by SDS-polyacrylamide gel electrophoresis. For some preparations, particularly actomyosin isolated from elderly subjects, a high molecular weight (HMW) band (identified as a breakdown product of myosin heavy chain) appeared, while the troponin-T subunit decreased. Myofibril associated protease (MFP) activity showed no significant difference as a function of proteolysis. In agreement with the proteolysis of troponin-T and myosin, the Ca2+ sensitivity of Mg2(+)-ATPase activity decreased while the extent of the stimulation of Ca2(+)-ATPase by N-ethylmaleimide remained unchanged. This type of proteolysis would affect the Ca2(+)-dependent regulation of muscle contraction but not the contractility per se.

Actomyosin↗
PubMed1989

Aspartic acid at position 57 of DQ beta chain does not protect against type 1 (insulin-dependent) diabetes mellitus in Japanese subjects.

HLA DQ beta chain, in particular amino acid at position 57, has been reported to contribute to susceptibility and resistance to Type 1 (insulin-dependent) diabetes mellitus in Caucasians. Resistance has been proposed to be conferred by aspartic acid at this position. To ascertain the association of HLA DQ beta and DR beta genes with Type 1 diabetes in Japanese subjects, ten Japanese Type 1 diabetic patients were investigated at DNA level. Genomic DNA was amplified by polymerase chain reaction, and dot blot analysis was carried out using the amplified DNA with allele specific oligonucleotide probes. All patients had aspartic acid at position 57 of at least one of their two DQ beta chains, and there was no significant difference of amino acids at the same position of DR beta chain in patients compared to control subjects. These data indicate that the protective role of aspartic acid at position 57 of DQ beta chain is less significant in Japanese compared with Caucasian subjects.

Aspartic Acid↗
PubMed1989

Isolation and characterization of Escherichia coli hag operator mutants whose hag48 expression has become repressible by a Salmonella H1 repressor.

The expression of an Escherichia coli K12 flagellin gene, hagA48, is insensitive to the Salmonella H1 repressor (rh1+). By selecting merodiploid cells H2-rh1on-off/F'hag48 for motility in the presence of anti-H48 serum, mutants which had escaped from inhibition by the serum because of repression of their hag48 expression by rh1+ were isolated. Their nucleotide sequences were examined in the region containing the promoter, the position of which was confirmed by S1 nuclease analysis of the transcriptional initiation site. The two independently isolated mutants had the same heptamer insertion AGACGAT at a site overlapping with the promoter sequence, creating a putative operator sequence homologous to Salmonella H1, but not to H2. Other candidates for operator mutants had reduced flagellar synthesis because of mutations between the transcriptional and translational initiation sites or in the structural gene. The sequence analysis also revealed a repetitive extragenic palindrome (REP) consensus sequence and a transcriptional terminator of hag48 in a small, functionally unknown open reading frame (ORF).

Amino Acid Sequence↗
PubMed1989

Demonstration of interleukin-1 beta on perifollicular endothelial cells in the thyroid glands of patients with Graves' disease.

We examined the presence of interleukin-1 (IL-1) in the thyroid glands of 20 patients with Graves' disease and 10 control subjects, using anti-IL-1 monoclonal antibodies in conjunction with immunohistochemical techniques. In 13 (65%) Graves' disease patients, IL-1 beta, but not IL-1 alpha, was demonstrated on the capillary endothelial cells (EC) around follicles (perifollicular EC). However, the EC of arteries and veins were negative for both IL-1 beta and IL-1 alpha. In the control group, none of the thyroid glands showed positive staining for IL-1 beta or IL-1 alpha. There was a significant correlation (P less than 0.025) between the presence of IL-1 beta on perifollicular EC and the presence of serum antimicrosomal antibodies. These results provide an important insight into the involvement of IL-1 beta in the development of Graves' disease.

Adolescent↗
PubMed1989

In vivo phosphorylation states and kinase activities of transforming p60c-src mutants.

To further investigate regulation of p60src by tyrosine phosphorylation, the in vivo phosphorylation states and kinase activities of transforming mutants derived from p60c-src were examined. One set of six in vitro recombinants encoded chimeric proteins containing the carboxyl terminus of chicken p60c-src (including Tyr 527) in combination with various isolated amino acid substitutions present in p60v-src. Another set of seven viral isolates were randomly selected from soft-agar colonies infected with replicating retroviruses that originally encoded normal p60c-src. Results show that mutations at multiple sites throughout amino- and carboxy-terminal domains are sufficient to alter the in vivo phosphorylation state and kinase activity of p60c-src. Moreover, all of the transforming p60c-src mutants exhibited elevated in vivo kinase activities, as assayed by immune blot analysis of cellular proteins using antibodies to phosphotyrosine, which correlated well with enhanced auto-phosphorylation at Tyr 416. By contrast, Tyr 527 phosphorylation in the transforming mutants varied from low to high levels that were correlated to a limited extent with levels of in vivo kinase activities. Taken together, results suggest that, in addition to complete dephosphorylation of Tyr 527, other mechanisms contribute to p60c-src activation.

Animals↗
PubMed1988

Exaggerated insulin secretory response in patients with insulinomas to midaglizole, a drug with alpha 2-adrenergic blocking activity.

Midaglizole is a new alpha 2-adrenergic blocking agent which increases insulin release from normal pancreatic islets. We studied its effect in four patients with insulinomas. In three patients oral administration of 150 mg of midaglizole caused a large increase in serum insulin and a corresponding decrease in plasma glucose. The magnitude of the response cannot exclude the possibility that midaglizole has direct beta cell stimulatory activity. Two of the three patients had operations, and their insulin responses to midaglizole became normal after removing the tumours. In contrast, midaglizole did not stimulate insulin secretion in the fourth patient. A midaglizole stimulation test might be useful in screening patients with insulinomas.

Adenoma, Islet Cell↗
PubMed1988

Induction of insulitis by adoptive transfer with L3T4+Lyt2- T-lymphocytes in T-lymphocyte-depleted NOD mice.

To clarify the pathogenesis of insulitis in the nonobese diabetic (NOD) mouse, an animal model for human insulin-dependent diabetes mellitus, T-lymphocyte-depleted NOD mice (B mice) were adoptively transferred with spleen and lymph node cells from cyclophosphamide-treated NOD mice after separating the cells with monoclonal antibodies against various T-lymphocyte surface antigens plus complement. Light-microscopic and immunohistochemical studies were also performed to investigate the lymphocytic infiltrations. The incidence of insulitis detected in B mice was much lower when compared with that of the lesion naturally occurring in the NOD mouse. However, higher incidence of insulitis was inducible in B mice by transferring unfractionated lymphoid cells from NOD mice. When the Thy1+ cell-depleted fraction was transferred into the B mice, no increase in the incidence of insulitis was observed. The Lyt1+ or L3T4+ cell-eliminated fraction was also unable to transfer insulitis. Conversely, donor cells depleted of Lyt2+ components successfully induced insulitis in the recipient B mice. These data were consistent with the immunohistochemical study, which showed that the main phenotype of the cells infiltrating the islets was L3T4+. These results suggest the importance of L3T4+Lyt2- T-lymphocytes in the pathogenesis of insulitis in NOD mice.

Animals↗