Search PubMed⌕ Search

Biomedical subjects

K Hirokawa

Publications and source records attributed to K Hirokawa.

At least 91 records · Page 5Linked to original sources
PubMed1996

Abnormality in the early signal transduction pathway is responsible for the impaired proliferative response and low K+ current in a T-cell clone by stimulation with anti-CD3 antibody.

Two T-cell clones were established from young and old C57BL/6 mice, respectively. The proliferative response to anti-CD3 stimulation was significantly greater in the young (YT5) than in the old (OT13) T cell clone. However, a similar high response was observed in both T-cell clones upon stimulation with phorbol myristate acetate (PMA) and ionomycin (INM). The calcium-dependent K+ current (IK(Ca)) was recorded with the patch-clamp method in these T-cell clones. With anti-CD3 stimulation, the amplitude of the outward K+ current was significantly lower in OT13 than in YT5 cells. With stimulation with PMA and INM, however, no significant difference in IK(Ca) was obtained between the two types of cells. The level of proliferative response of T-cells to mitogens was well reflected by the amplitude of IK(Ca). Some abnormality in the early pathway of signal transduction, which led to the intracellular Ca2+ influx, appears to be responsible for the impaired proliferation of the old T-cell clone.

Aging↗
PubMed1996

The receptor tyrosine kinase, Cek8, is transiently expressed on subtypes of motoneurons in the spinal cord during development.

Receptor tyrosine kinases (RTKs) play important roles in cellular proliferation, differentiation, and survival. We performed reverse transcriptase-polymerase chain reactions (RT-PCR) from enriched embryonic day 5 (E5) chick motoneurons by panning to identify RTKs involved in the early development of motoneuron. In situ hybridization revealed that Cek8, a member of the eph family, was specifically expressed on motoneurons at the brachial and lumbar segments of the spinal cord which innervate limb muscles, and disappeared after the naturally occurring cell death period (E6-E11). Immunohistochemistry using an anti-Cek8 monoclonal antibody showed the localization of Cek8 protein at the cell bodies and axonal fibers of motoneurons and muscles. The unique expression of Cek8 suggests its involvement in cellular survival or cell-cell interactions for specific subpopulations of developing motoneurons.

Amino Acid Sequence↗
PubMed1996

Clinical pharmacologists and drug regulation--future perspective in Japan.

1. The Japanese pharmaceutical administration system, and recommendations for the improvement of clinical trial methodology following the implementation of GCP guidelines in Japan are described with special reference to three recent developments in Japanese drug regulation. 2. The implementation of GCP guidelines has not been fully satisfactory and clinical trials in Japan still run into problems. The Ministry of Health and Welfare has recently established a Committee on Safety of Medicines after serious trouble with a new drug was experienced. The Committee released an interim report on the reform of clinical trials supervision and the system for examination of New Drug Applications. 3. ICH-GCP guidelines will be implemented in Japan in the next few years. The principal differences between Japanese GCP and ICH-GCP guidelines, and their effects on clinical trials in Japan with reference to the acceptability of ICH-GCP, are discussed. 4. The final proposals on the National Health Insurance drug pricing method and the appropriate use of drugs by the Central Social Insurance Medical Council are anticipated to have a significant impact on the drug market. The changes in pricing system, especially premiums for innovative drugs, and the implications for the drug market are considered. 5. In these changed circumstances, the importance of the role of clinical pharmacologists in the framework of the new drug regulations in Japan is reviewed.

Clinical Trials as Topic↗
PubMed1996

Critical proline residues of the cytoplasmic domain of the IL-5 receptor alpha chain and its function in IL-5-mediated activation of JAK kinase and STAT5.

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.

Animals↗
PubMed1996

Demonstration of a cross-talk between IL-2 and IL-5 in phosphorylation of IL-2 and IL-5 receptor beta chains.

We have examined phosphorylation mediated by cross-talk between growth signal pathways induced by IL-2 and IL-5. To analyze the phosphorylation process in the same cells, we established two sublines, T88-Mbeta1, which is a subline of a murine IL-5-dependent cell line, T88-M, by introduction of the human IL-2 receptor beta chain (IL-2Rbeta), and secondly CTLL-5Ralphabeta, which is a subline of a murine IL-2-dependent cell line, CTLL-2, by introduction of the murine IL-5 receptor alpha chain (IL-5Ralpha) and IL-5 receptor beta chain (IL-5Rbeta, betac) genes. Both T88-Mbeta1 and CTLL-5Ralphabeta expressed high-affinity receptors for IL-2 and IL-5, and proliferated in response to both factors. Tyrosine phosphorylation of IL-2Rbeta was induced by stimulation of T88-Mbeta1 with not only IL-2 but also IL-5. Anti-IL-2Rbeta-directed immune complexes from T88-Mbeta1 stimulated with IL-5 as well as with IL-2 contained an activated tyrosine kinase. However, stimulation with IL-5 but not IL-2 induced the tyrosine phosphorylation of IL-5Rbeta, betac, suggesting that IL-2 does not activate a tyrosine kinase which efficiently catalyzes the IL-5Rbeta molecule in response to IL-5. On the other hand, the detection of JAK1 and the other common set of phosphotyrosine-containing proteins after stimulation with either IL-5 or IL-2 suggests the existence of the same tyrosine phosphorylation pathways.

Animals↗
PubMed1996

Establishment of a therapeutic model for retroviral infection using the genetic resistance mechanism of the host.

Resistance to retroviral infection is often regulated by multiple genes that control different aspects of the host-virus interaction. Genetically distinct inbred strains of mice differ in their susceptibility to retrovirus and have allowed the identification of several host-resistant loci that regulate the host defense mechanism to retroviral infection. Using the murine retrovirus infection system, a therapeutic model has been developed of retrovirus infection in association with the resistant mechanism of host genes. The most effective result achieved with the model was when using bone marrow transplantation of retrovirus-resistant cells with receptor interference function, which was genetically defined by the Fv-4 resistant gene. The possible application of these findings to the gene therapy of retrovirus-induced disease of humans is discussed.

Animals↗
PubMed1996

A novel gene that interferes with the phosphotransfer signal transduction mediated by the EnvZ osmosensor in Escherichia coli.

In Escherichia coli, expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity and other environmental stimuli. A two-component signal transduction system, mediated by EnvZ and OmpR, is crucially responsible for this osmotic regulation of the ompC and ompF genes. In this study, an E. coli gene was cloned, which interferes with expression of both the ompC and ompF genes at the level of transcription, provided that the cloned gene was introduced in E. coli cells by a multicopy plasmid. The gene product was identified as F107, which was previously characterized as a hypothetical protein in E. coli genome databases. F107 containing 107 amino acids appears to be highly hydrophobic, and has a sequence similarity to the eukaryotic type of cytochrome-c oxidase subunit III. The mechanism by which F107 inhibits transcription of ompC and ompF was examined extensively, mainly by using a set of envZ and ompR mutants. These results suggested that F107 interferes specifically with a function of the EnvZ osmosensory kinase. Possible mechanisms by which F107 affects the EnvZ function are discussed.

Amino Acid Sequence↗
PubMed1996

A cyanobacterial gene that interferes with the phosphotransfer signal transduction involved in the osmoregulatory expression of ompC and ompF in Escherichia coli.

Synechococcus sp. PCC7942 is a phototrophic cyanobacterium. In this study we cloned a Synechococcus gene that has a striking effect on the production of the Escherichia coli outer membrane proteins, OmpC and OmpF, provided that this gene was introduced by a multicopy plasmid into the heterologous cells. This multicopy gene in E. coli cells was able to specifically shut off the production of both OmpC and OmpF at the level of transcription. The nucleotides were sequenced for this gene, named sis, and its gene product was purified from E. coli to near homogeneity. A computer-aided search found that the deduced amino acid sequence consisting of 138 residues is novel, with no significant similarity to any other protein in the databases. Since the transcription of ompC and ompF is regulated by the regulatory factors EnvZ and OmpR, through phosphotransfer signal transduction, we explored the inhibitory effect of Sis in various genetic backgrounds as to envZ and ompR. In particular, the inhibitory effect of Sis was observed even in an DeltaenvZ background, but was not observed in a certain background in which the ompC and ompF transcription was supported by a mutant OmpR that can function in a phosphorylation-independent manner. These results suggested that the EnvZ kinase may not be the direct target of Sis, but rather that the process(es) concerning the phosphorylation and/or dephosphorylation of the OmpR protein may be affected by Sis. However, no direct effect of Sis was seen in an in vitro OmpR-phosphorylation assay with the purified OmpR and Sis proteins. Based on these results, possible functions of Sis are discussed with special reference to the phosphotransfer signal transduction in E. coli.

Amino Acid Sequence↗
PubMed1996

Distribution of Fv-4 resistant gene product in Friend leukemia virus-resistant Fv-4r mouse strain.

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). We demonstrated previously that the Fv-4 resistant (Fv-4r) gene product, Fv-4r env antigen, is released from Fv-4r-bearing BALB/c-Fv-4Wr (C4W) mouse-derived cells into serum in vivo and binds to cells expressing surface receptors for ecotropic MuLV, thereby protecting them from infection with Friend leukemia virus (FLV) by receptor interference. This unique resistance mechanism against retroviral infection might provide a possible therapeutic model system of human retroviral infection such as AIDS. To further investigate the Fv-4r gene action in vivo, we examined the distribution and character of Fv-4r env antigen in serum and systemic organs from C4W mice. The Fv-4r env antigen was immunohistochemically localized to the lympho-hematopoietic cells and exocrine glandular cells, such as those of the salivary gland and pancreas. Using immunoprecipitation followed by Western blotting, we determined two types of gp70-related Fv-4r env antigen in the serum of C4W mice, showing molecular weights of either 70-75 kDa and 80-85 kDa. When thymocytes from Fv-4 susceptible gene (Fv-4r)-bearing C3H mouse were mixed with C4W mouse serum, the 70-75k Da molecule of the C4W serum dominantly bound to C3H thymocytes and thus contributed to receptor interference function. Using immunoelectron microscopy, Fv-4r env antigen was mainly localized to the cell surface membrane of thymic lymphoid cells, while acinar cells of the salivary gland possessed Fv-4r env antigen in the endoplasmic reticulum (ER) as well as on the cell surface membrane. These data indicate that several glandular organs, as well as lymphohematopoietic organs of C4W mice, may contribute to the production of cell-free Fv-4r env antigen, resulting in protection of cells from infection with FLV by receptor interference.

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↗