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K Hirokawa

Publications and source records attributed to K Hirokawa.

At least 19 recordsLinked to original sources
PubMed1996

An epitope on Ki antigen recognized by autoantibodies from lupus patients shows homology with the SV40 large T antigen nuclear localization signal.

OBJECTIVE: Epitopes on Ki antigen were analyzed using synthetic peptides, including KILT, a 16-mer peptide with an amino acid sequence homologous to the SV40 large T antigen nuclear localization signal (SV40 T NLS). METHODS: In addition to KILT, 4 synthetic peptides, all potential epitopes on Ki antigen according to computer analysis, were prepared and tested for reactivity with 49 anti-Ki-positive lupus sera by enzyme-linked immunosorbent assay. RESULTS: Eighteen sera reacted with KILT, but not with other peptides. The reaction of anit-Ki sera with KILT was specifically inhibited by recombinant Ki antigen. Eight of 49 anti-Ki sera reacted with a 7-mer synthetic peptide of SV40 T NLS, and the reaction was specifically inhibited by KILT. CONCLUSION: The 16-mer Ki peptide containing the sequence homologous to the SV40 T NLS is one of the antigenic epitopes recognized by anti-Ki antibodies in lupus sera.

Amino Acid Sequence↗
PubMed1996

Thymocyte-directed enhancement of apoptosis via soluble factor(s) derived from a cortical and a medullary thymic epithelial cell line.

Apoptosis of murine thymocytes was examined either in intact fetal thymus lobes or in thymus cell suspensions, both cultured alone or in the presence of either a cortical (TEC 1.4) or a medullary (TEC 2.3) thymic epithelial cell line. Both TECs induced a pronounced increase of apoptosis in 24-h cultivated single thymus cell suspensions but not in spleen or bone marrow cell cultures. Co-culture of thymocytes with murine fibroblasts did not enhance apoptosis of the thymus cells. A similar enhancement of thymocyte apoptosis was observed with dialysed culture supernatants derived from both TEC lines, the active component(s) having a molecular weight of > 30 kDa. In contrast, the cortical TEC 1.4 had a pronounced apoptosis inducing effect on intact fetal thymus lobes cultivated for six days, whereas the medullary TEC 2.3 had only a marginal influence. TEC 1.4 also induced a significant alteration in the ratio of CD4+CD8+ to CD4-CD8- cells. It is concluded that both the cortical and medullary epithelial cell lines are able to induce thymocyte apoptosis but that a large proportion of the cells within the intact thymus stroma is refractory to the respective signal(s) of the medullary epithelial cell line.

Animals↗
PubMed1995

Expression of ZAP-70 gene in the developing thymus and various nonlymphoid tissues of embryonic and adult mice.

The full length of cDNA encoding murine ZAP-70 was obtained from a cDNA library of embryonic mouse thymus. Northern blot analysis indicated that the expression of ZAP-70 mRNA was most pronounced in the thymus and spleen and also slightly in the brain. Analysis by in situ hybridization revealed that the mRNA was expressed not only in T cells in the thymus and spleen, but also in nerve cells of the brain. In the thymus, the distribution of positive cells was different between newborn and young adult mice. In the newborn thymus, positive signal in thymocytes was localized in the subcapsular layer as well as in the medulla, while in the young adult thymus, it was localized exclusively in the medulla. Furthermore, the in situ hybridization revealed that positive signal was also seen in the liver, intestine, and lung of embryonic mice. These findings have suggested that ZAP-70 plays an important role in growth, differentiation, and function of not only T cells but also nerve cells and several embryonic tissues.

Amino Acid Sequence↗
PubMed1995

Differential expression of protein tyrosine kinases in the cortical and medullary thymic epithelial cell lines.

Expression of protein tyrosine kinase (PTK) was evaluated and compared between cortical and medullary thymic epithelial cell (TEC) lines, TEC 14C18 and TEC1C6, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) methods using degenerate primers for conserved sequence in catalytic domain of PTK revealed the expression of 13 kinds of PTKs in these cell lines. These PTKs included 6 receptor and 7 non-receptor types. In the next step, we compared the intensity of mRNA expression level of these kinases between cortical and medullary TEC lines. Tyrosine kinases of receptor type were differentially expressed between cortical and medullary TEC lines; e.g., mRNA expression of flg was observed more intensively in the cortical TEC line than in the medullary one, while mRNA expression of bek, met and sky was vice versa. On the other hand, expression of non-receptor tyrosine kinases, including four JAK-tyk family members, are comparable in both lines. These results suggested that these PTKs, especially of receptor type, might play an important role in the development of the thymic microenvironment.

Amino Acid Sequence↗
PubMed1995

Cell-free transmission of Fv-4 resistance gene product controlling Friend leukemia virus-induced leukemogenesis: a unique mechanism for interference with viral infection.

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). We previously demonstrated that mixed radiation bone marrow chimeras containing Fv-4r-bearing BALB/c-Fv-4Wr (C4W) bone marrow and Fv-4r-bearing C3H/He (C3H) bone marrow grafted into C3H recipient mice (C4W+C3H-->C3H) were resistant to Friend leukemia virus (FLV)-induced leukemogenesis, even when they contained as high as 70% C3H-derived cells. This indicates that FLV-sensitive C3H-derived cells are rendered refractory to infection and/or transformation with FLV when they coexist in mice with Fv-4r-bearing cells. To investigate the mechanism of Fv-4 resistance to FLV-induced leukemogenesis, we first examined the expression of Fv-4r env antigen in the peripheral blood mononuclear cells (PBMC) of these chimeras. The Fv-4r env antigen was present not only on C4W-derived cells, but also on Fv-4r-bearing C3H-derived cells in C4W+C3H-->C3H mixed bone marrow chimeras. The Fv-4r env antigen that binds to the cells surface of C3H cells was found in sera from normal C4W mice, C4W-->C3H chimeras, and C4W+C3H-->C3H mixed chimeras. The serum Fv-4r env antigen binds to ecotropic MuLV receptors, shown by specific binding to transfectant mink cells expressing ecotropic MuLV receptor, but not to parental mink cells. To determine whether the binding of Fv-4r env antigen to the putative MuLV receptors would block FLV infection, C3H thymocytes or spleen cells that had been preincubated with C4W serum were mixed with FLV and the subsequent production of MuLV specific antigens was examined. C3H thymocytes or spleen cells treated with C4W serum became refractory to binding by FLV. These results provide evidence that the Fv-4r env antigen is released from C4W-derived cells in vivo and binds to cells expressing surface receptors for ecotropic MuLV, thereby protecting them from infection with FLV. The implication of these findings for gene therapy of retrovirus-induced disease such as acquired immune deficiency syndrome (AIDS) is discussed.

Animals↗
PubMed1995

Differential effects of gonadectomy on thymic stromal cells in promoting T cell differentiation in mice.

Twenty-six week-old BDF1 mice were gonadectomized and grafted with thymus from irradiated (8.5 Gy) newborn, 6-week-old, or 26-week-old mice. One month later, grafted thymuses were recovered and examined in terms of thymocyte numbers, subpopulations and proliferative responses to Concananavlin A (Con A). The growth of the irradiated thymus was significantly higher in gonadectomized (Gx) than in sham-operated (Sham) mice and the magnitude of thymic growth was apparently age-dependent, as it was greater for newborns than for older mice. Con A response of thymocytes was also significantly higher in Gx mice than in Sham mice, and the magnitude of the response declined with advancing age of the thymus donors. Flow cytometric analysis revealed that a significant increase in the percentage of CD4+CD8- was observed in thymus grafts showing high Con A responses. However, this effect of Gx on the thymus graft was dependent on age of the thymus donor. Namely, newborn thymus grafts could grow equally well in both Gx and Sham recipients, whereas thymus grafts from 6- and 26-week-old mice could grow well only in Gx, but not in Sham recipients. The number of thymocytes was comparable in thymus grafts from 6- and 26-week-old mice, but the proliferative response to Con A was higher in the former than in the latter graft. Collectively, Gx appeared to promote immigration of thymocyte precursors into the thymus and to enhance proliferation and differentiation of thymocytes towards CD4+CD8- T cells, in an age-related manner.

Aging↗
PubMed1995

A novel type of cell death of lymphocytes induced by a monoclonal antibody without participation of complement.

A monoclonal antibody, RE2, raised by immunizing a rat with cell lysate of a mouse T cell clone, was found to directly kill interleukin 2-dependent T cell clones without participation of serum complement. Fab fragments of RE2 had no cytolytic activity, while the cross-linking of Fab fragments with anti-rat immunoglobulin reconstituted the cytotoxicity. The cytotoxicity was temperature dependent: the antibody could kill target cells at 37 degrees C but not at 0 degrees C. Sodium azide, ethylenediaminetetraacetic acid, and forskolin did not affect the cytolytic activity of RE2, while the treatment of target cells with cytochalasin B and D completely blocked the activity. This suggested that the cell death involves a cytoskeleton-dependent active process. Giant holes on the cell membrane were formed within 5 minutes after the treatment with RE2, as observed by scanning electron microscopy. There was no indication of DNA fragmentation nor swelling of mitochondria during the cytolysis, suggesting that the cell death is neither apoptosis nor typical necrosis. The antibody also killed T cell lymphomas and T and B cell hybridomas only when these cells were preactivated with concanavalin A, lipopolysaccharide, or phorbol myristate acetate. Preactivated peripheral T and B cells were sensitive to the cytotoxicity of RE2, while resting T and B cells were insensitive. These results provide evidence for a novel pathway of cell death of activated lymphocytes by membrane excitation.

Animals↗
PubMed1995

Cholinergic signals to and from the immune system.

This article reviews recent data from our laboratory towards the impact of the autonomous nervous system on mutual interactions between the immune system and the central nervous system. Using a pharmacological approach in rats it is shown that shifts in the adrenergic/cholinergic balance in vivo affect in vitro functions of the non-specific and specific immune system, whereby adrenergic and cholinergic stimulation in general have opposite effects. A high degree of integration appears to exist between cells of the immune system with the cholinergic system. Lymphocytes were found to react to acetylcholine, but are also able to produce and to degradate this neurotransmitter. In addition, changes in the cholinergic tonus were found to affect immune signaling to the brain and to protect thymocytes from apoptosis, possibly via a direct effect on thymic epithelial cells.

Adrenergic Agents↗
PubMed1995

The role of thymus in the aging of Th cell subpopulations and age-associated alteration of cytokine production by these cells.

Mouse CD4+ T cells were subdivided into two subpopulations, naive (CD44low CD45RBhigh) and memory (CD44high CD45RBlow) T cells, by flow cytometric analysis. Examination of spleen and peripheral blood of C57BL/6 mice of various ages revealed that there was a reciprocal age-associated change in these two subpopulations, i.e. naive T cells predominant in young mice decreased with age, while memory T cells increased. In order to investigate the role of the thymus in the age change of naive and memory T cells, we employed two experimental systems: radiation bone marrow chimeras constructed between young and old mice, and grafting of young or old thymus into nude mice. Data from these two experiments suggested that the young thymus has a greater ability to provide naive T cells than the old thymus, while the old thymus favors the maintenance of memory T cells rather than naive T cells. In reference to cytokine production by enriched naive and memory T cells, young naive T cells produced mainly IL-2 and young memory T cells mainly IL-4. On the other hand, in old mice, memory T cells produced twice as much IL-2 than naive T cells, although the level was significantly lower than that of young mice. In addition, old naive T cells produced twice as much IL-4 than old memory T cells. These results suggested a distinct age change in the profile of cytokine production and functional heterogeneity of two Th cell subpopulations.

Age Factors↗
PubMed1995

The characterization of the monoclonal antibody Th-10a, specific for a nuclear protein appearing in the S phase of the cell cycle in normal thymocytes and its unregulated expression in lymphoma cell lines.

A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG2a and had kappa light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G1, G2 and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characterize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis. The nuclear protein(s) detected by Th-10a monoclonal antibody was mostly 95 kDa and also 83 kDa polypeptide. The results also indicated that the 95 kDa nuclear protein was phosphorylated in vivo.

Animals↗
PubMed1995

Changes in glutathione in gastric mucosa of gastric ulcer patients.

To clarify the role of glutathione (GSH), an antioxidative substance, and its association with the gastric mucosal defense mechanism, we examined the gastric mucosal GSH and GSH-dependent enzymes in gastric ulcer patients. The subjects of this study were 10 patients with active ulcer on the lesser curvature side in the lower part of the gastric body and 11 normal controls. At the time of gastric endoscopy, gastric mucosal specimens were obtained by routine forceps biopsy from a site several millimeters apart from the ulcer margin, and these specimens were used for measurement of GSH, glutathione peroxidase (GSH-Px), glutathione-S transferase (GST) and gamma-glutamyl transpeptidase (gamma-GTP). The GSH level and the GSH-Px and GST activities in the patients with gastric ulcer were lower than those in the control group. The GSH-Px activity at the healed stage after treatment was increased compared with the pre-treatment value, while the GSH level was not markedly increased and the GST and gamma-GTP activities were similar to the pre-treatment value. The GSH level and GSH-Px activity remained decreased in the non-responder group. These results suggest that gastric mucosal GSH and GSH-dependent enzymes are closely related to the etiology and course of gastric ulcer.

Adult↗
PubMed1995

Characterization of intermediate T-cell receptor cells expanding in the liver, thymus and other organs in autoimmune lpr mice: parallel analysis with their normal counterparts.

Autoimmune MRL-lpr/lpr (lpr) mice were previously demonstrated to have an abnormal proliferation of intermediate T-cell receptor (TCR) cells of extrathymic origin in the liver. Despite this situation, thymectomy in lpr mice resulted in amelioration of autoimmune disease. To understand the underlying mechanism, we investigated associated T-cell differentiation in the thymus and other organs of these mice. When the disease was evoked, T cells with extrathymic properties, i.e. intermediate TCR-alpha beta cells expressing double-negative (DN) CD4-8- phenotype and interleukin-2 (IL-2) receptor beta-chain, became prominent not only in the liver, but also in the thymus. Such thymic T cells mainly resided in the medulla. A small-scale localization of such T cells was seen in the thymic medulla even in normal control mice. There was a heterogeneity among intermediate TCR cells in terms of the composition of DN cells and the expression of CD2 and B220 antigens, depending on the organs and the sites in the same organ. Intermediate TCR cells in the liver, thymus and autoimmune target organs (e.g. kidney) contained a high proportion of the active form (CD2+B220-), while intermediate TCR cells accumulating in peripheral organs, the spleen and lymph nodes, were mainly of the inactive form (CD2-B220+). The active form had an ability to proliferate in response to IL-2 and SEB, whereas the inactive form did not. The present results suggest that the proliferation of intermediate TCR cells occur at multiple sites; this may explain the effect of thymectomy, namely, the retarded onset of disease, in lpr mice.

Animals↗
PubMed1995

Proliferative activity and p53 over-expression of ovarian epithelial tumors.

Proliferative activity (PA) and p53 over-expression were assessed in 68 cases of primary ovarian epithelial tumors of variable grades of malignancy by immunohistochemical methods using antibodies to PCNA (proliferative cell nuclear antigen) and p53 molecule. First, we compared the values in benign tumors, tumors of borderline malignancy and malignant tumors. Malignant tumors possessed higher PA value (26.8 +/- 14.7%) than benign tumors (1.6 +/- 1.4%), and tumors of borderline malignancy exhibited an intermediate value (11.4 +/- 5.1%). Intranuclear accumulations of p53 product were more frequently observed in malignant tumors (16/32, 50%) than in tumors of borderline malignancy (3/9, 33%). None of the benign tumors exhibited p53 over-expression. In malignant tumors, PA correlated well with the histologic grade of tumors, although the histologic type and stage of the diseases did not correlate significantly with PA. Then, we focussed on the cases of malignant ovarian cancer composed of heterogeneous lesions showing a different grade of malignancy; invasive lesion (IL), non-invasive lesion (NIL), and benign appearing lesion (BAL). PA of NIL was almost identical with that of IL (19.6 +/- 13.5% vs. 23.3 +/- 12.6%), while BAL showed significantly lower PA (2.1 +/- 3.0%, p < 0.001) than the above two lesions and was similar to that of benign tumors. Furthermore, p53 over-expression was never observed in BAL, even in cases in which IL and/or NIL showed p53 over-expression. Thus, the results indicated that histologically benign-appearing lesions of malignant ovarian epithelial tumors possessed a biologic character similar to benign tumors. Collectively, all these findings would support the concept that a part of malignant ovarian epithelial tumors would be derived from benign tumors through progressive transformation.

Adenocarcinoma, Clear Cell↗
PubMed1994

JAK3 Janus kinase is involved in interleukin 7 signal pathway.

Interleukin (IL) 7 is an important cytokine regulating both T and B cell development and inducing the formation of lymphokine-activated killer cells and cytolytic T lymphocytes. This study reports the role of JAK family kinases in the IL-7 signalling pathway in a T cell clone. The results have shown that out of 4 members of JAK family tyrosine kinases (JAK1, JAK2, JAK3 and Tyk2), only JAK3 was tyrosine-phosphorylated and activated in cells of a T cell clone by stimulation with IL-7. Furthermore, STAT1 alpha (STAT, the signal transducers and activators of transcription) and p44 of MAPK (mitogen-activated protein kinases) were tyrosine phosphorylated by IL-7 stimulation, indicating that the two signal pathways might be involved in IL-7 signal transduction.

Animals↗
PubMed1994

Cholinergic stimulation modulates apoptosis and differentiation of murine thymocytes via a nicotinic effect on thymic epithelium.

Effects of cholinergic agonists/antagonists on apoptosis and differentiation of murine thymus cells were investigated in two in vitro models. Treatment with 10(-7) M carbachol was found to counteract the effects of a cortical thymus epithelial cell line (TEC 1.4), on apoptosis and the ratio of CD4 CD8 DP/DN cells in cocultured fetal thymus lobes. A medullary line (TEC 2.3) did not influence apoptosis in fetal thymus lobes. Both TEC lines had the same strong apoptotic effect on thymus cells in suspension, but only the effect of TEC 1.4 was counteracted by carbachol. This cholinergic influence on TEC 1.4 cells is mediated via nicotinic cholinergic receptors, since d-tubocurarine, but not atropine, effectively blocked the effect of carbachol. The results suggest that cholinergic signals to thymic epithelial cells may have regulatory influence on thymic differentiation and selection processes.

Animals↗
PubMed1994

Presence of functional cyclic AMP responsive element in the 3'-untranslated region of the human thrombomodulin gene.

We characterized the transcriptional regulatory function of the 3'-untranslated region of the thrombomodulin gene by transient transfection assays in human umbilical endothelial cells. Deletion analyses of the 3'-untranslated region indicated that the region containing the consensus sequence of the cyclic AMP (cAMP) responsive element (position 2092) showed an increased transcriptional activity in response to cAMP. Gel-shift analysis showed that a band representing the fragment containing position 2092 was retarded when incubated with nuclear extracts from the cells treated with cAMP. In addition, the region downstream of the cAMP responsive element was found to function negatively in the gene expression. These results indicate that the 3'-untranslated region has a functional cAMP responsive element and plays an important role in the regulation of the thrombomodulin gene expression.

Base Sequence↗