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Biomedical subjects

I Nakashima

Publications and source records attributed to I Nakashima.

At least 19 recordsLinked to original sources
PubMed2000

Mercuric chloride stimulates distinct signal transduction pathway for DNA synthesis in a T-cell line, CTLL-2.

Exposure of an interleukin-2 (IL-2)-dependent murine T-cell line (CTLL-2) to mercuric chloride in in vitro culture induced a low but definite level of DNA synthesis in the absence of exogenous IL-2, and further enhanced the IL-2-induced DNA synthesis. Addition of anti-IL-2 or anti-IL-4 antibody to the culture, which neutralized all of the IL-2 or IL-4 activity, respectively, never inhibited the mercuric chloride-mediated DNA synthesis. Correspondingly, no detectable level of IL-2, IL-4, and IL-15 mRNA was found in mercuric chloride-treated CTLL-2 cells in our test condition. Stimulation of CTLL-2 cells with IL-2 induced phosphorylation on extracellular signal-regulated kinases more intensively than on c-Jun NH2-terminal kinases (JNKs), and provoked tyrosine phosphorylation of Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). In contrast, by mercuric chloride stimulation, JNKs and c-Jun were preferentially phosphorylated, but no detectable level of phosphorylation was induced on JAKs and STATs. These findings provided a possibility that mercuric chloride promoted lymphocyte proliferation through a JNK-linked signal cascade in CTLL-2 cells, which differs from that triggered by IL-2.

Animals↗
PubMed2000

Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A.

We have previously reported the activation of Src by mercuric chloride based on the sulfhydryl modification. To evaluate the significance of cysteine residues in v-Src, we replaced each cysteine to alanine by oligonucleotide-directed mutagenesis and examined its effect on cell transformation. Of ten cysteine residues scattered over v-Src, four cysteines clustered in kinase domain, Cys483, Cys487, Cys496 and Cys498, were important for protein stability and cell transformation, whereas those in SH2 domain were dispensable. A single mutation in Cys498 yielded suppression of kinase activity and a temperature-sensitivity in anchorage independent growth. Double mutation either in Cys483/Cys487 or in Cys496/Cys498 yielded clear temperature-sensitivity in cell transformation and in stability of Src protein. Instability of Src protein was magnified by quadruple mutation in the cysteines, which decreased the half-life of Src to be less than one quarter of that of wild-type. In addition, both Cys483/Cyr487 and Cys496/Cys498 kinases became resistant to in vitro inactivation by herbimycin A, which directly inactivates v-Src in addition to its effect on HSP90. Taken together, our results strongly suggest that the cysteine clustered motif of v-Src are critical for protein stability, cell transformation and in vitro inactivation by herbimycin A.

Amino Acid Sequence↗
PubMed2000

Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase.

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.

Animals↗
PubMed2000

Ultraviolet light induces redox reaction-mediated dimerization and superactivation of oncogenic Ret tyrosine kinases.

The c-RET proto-oncogene encodes a receptor-type tyrosine kinase, and its mutations in the germ line are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B). Ret kinases are constitutively activated as a result of MEN2A mutations (Ret-MEN2A) or MEN2B mutations (Ret-MEN2B). Here we demonstrate that UV light (UV) irradiation induces superactivation of the constitutively activated Ret-MEN2A and Ret-MEN2B as well as activation of c-Ret. Before UV irradiation, small percentages of c-Ret (3-4%) and Ret-MEN2B (1-2%) and large percentages of Ret-MEN2A (30-40%) were dimerized through disulfide bonds. These dimerized Ret proteins were preferentially autophosphorylated, suggesting a close relation between up-regulated kinase activity and disulfide bond-mediated dimerization of Ret proteins. We found that UV irradiation promotes the disulfide bond-mediated dimerization of the Ret proteins, in close association with activation and superactivation of Ret kinases. UV irradiation also induced dimerization and activation of the extracellular domain-deleted mutant Ret (Ret-PTC-1). Interestingly, the levels of basic kinase activity and dimerization of Ret-PTC-1-C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-PTC-1 was replaced by alanine, were low and were not increased by UV irradiation. These results suggest that Ret-PTC-1 cysteine 376 is one of possibly multiple critical target amino acids of UV for Ret kinase activation. Overexpression of Cu/Zn superoxide dismutase in cells as a result of gene transfection prevented both the UV-mediated promotion of dimerization and the superactivation of Ret-MEN2A kinase. These results suggest that the UV-induced free radicals in cells attack intracellular domains of Ret to dimerize the kinase proteins for superactivation.

3T3 Cells↗
PubMed2000

4-hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death.

4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20-50 microM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.

Aldehydes↗
PubMed1999

Normal regulatory alpha/beta T cells effectively eliminate abnormally activated T cells lacking the interleukin 2 receptor beta in vivo.

Although interleukin 2 (IL-2) has been thought to be the most important cytokine for T cell growth, animals lacking IL-2 or a component of its receptor molecules have more expanded T cells with activated memory phenotype, indicating an indispensable role for the IL-2/IL-2 receptor system in regulating the size and activity of the T cell population. In this study, we investigated the possible mechanism of abnormal expansion of activated T cells in IL-2 receptor beta chain (IL-2Rbeta)(-/-) mice using the systems of bone marrow transplantation and T cell transfer. Here, we show that IL-2Rbeta(2/-) T cells in mice reconstituted with a mixture of IL-2Rbeta(2/-) and IL-2Rbeta(1/+) bone marrow cells did not develop into an abnormally activated stage, and that already activated IL-2Rbeta(2/-) T cells were effectively eliminated by IL-2Rbeta(1/+) T cells when both cells were cotransferred to T cell-deficient host mice. This regulation and/or elimination was dependent on T cells bearing alpha/beta type T cell receptor, especially on CD8(+) T cells and independent of the Fas-Fas ligand (FasL) system. IL-2Rbeta(1/+) T cells that eliminated activated IL-2Rbeta(2/-) T cells expressed FasL, perforin, granzyme B, and tumor necrosis factor alpha/beta. These results indicate a novel function of IL-2Rbeta that is necessary for the induction of regulatory T cells acting to eliminate activated T cells.

Animals↗
PubMed1999

Nitric oxide controls src kinase activity through a sulfhydryl group modification-mediated Tyr-527-independent and Tyr-416-linked mechanism.

c-Src kinase was activated when either murine NIH3T3 fibroblast cells or immunoprecipitated c-Src proteins were treated with nitric oxide generator, S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside. Nitric oxide (NO) scavenger hemoglobin and N(2)O(3) scavenger homocysteine abolished the SNAP-mediated c-Src kinase activation. Phosphoamino acid analysis and peptide mapping of in vitro labeled phospho-c-Src proteins revealed that SNAP promoted the autophosphorylation at tyrosine, which preferentially took place at Tyr-416. Peptide mapping of in vivo labeled c-Src kinase excluded the involvement of phospho-Tyr-527 dephosphorylation in the SNAP-mediated activation mechanism. Correspondingly, protein-tyrosine phosphatase inhibitor Na(3)VO(4) did not abolish the SNAP-mediated activation of Src kinase, and the constitutively activated v-Src kinase was also further up-regulated in activity by SNAP. SNAP, however, failed to up-regulate the kinase activity of Phe-416 mutant v-Src. 2-Mercaptoethanol or dithiothreitol, which should disrupt N(2)O(3)-mediated S-nitrosylation and subsequent formation of the S-S bond, abolished the up-regulated catalytic activity, and the activity was regained after re-exposing the enzyme to SNAP. Exposure of Src kinase to SNAP promoted both autophosphorylation and S-S bond-mediated aggregation of the kinase molecules, demonstrating a linkage between the two events. These results suggest that the NO/N(2)O(3)-provoked S-nitrosylation/S-S bond formation destabilizes the Src structure for Tyr-416 autophosphorylation-associated activation bypassing the Tyr-527-linked regulation.

3T3 Cells↗
PubMed1999

Protein kinase C-dependent anti-apoptotic mechanism that is associated with high sensitivity to anti-Fas antibody in ovarian cancer cell lines.

We compared the sensitivities to apoptosis via anti-Fas antibody of two human ovarian cancer cell lines, NOS4 and SKOV-3, both of which strongly express the Fas antigen on their cell surface. Treatment with anti-Fas antibody induced extensive DNA fragmentation in NOS4 cells but none in SKOV-3 cells. However; both cell lines underwent apoptosis in response to calcium ionophore A23187 or sphingomyelinase, demonstrating that the latter cell line is capable of DNA fragmentation. DNA fragmentation was not induced in either cell line by treatment with PKC activator PMA, however treatment with protein kinase C (PKC) inhibitor H-7 induced extensive DNA fragmentation in NOS4 cells, but again none in SKOV-3 cells. Protein kinase A inhibitor HA1004 treatment did not induce DNA fragmentation in either cell line. Correspondingly, treatment of cells with PMA before anti-Fas antibody or A23187 treatment partially inhibited induction of DNA fragmentation in NOS4 cells but not in SKOV-3 cells. Both NOS4 and SKOV-3 cell lines expressed isozymes of PKC at comparable levels. These results suggest the presence of a PKC-dependent anti-apoptotic mechanism in association with high sensitivity to anti-Fas antibody in these ovarian cancer cell lines.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein-I/ret transgenic mouse lines.

We examined the basis of the all or none difference in inducing melanocytic tumor development among three transgenic mouse lines (304, 192 and 242) to which the same promoter-enhancer (metallothionein-I) and oncogene (ret) were introduced. We initially demonstrated that both skin melanosis and Ret protein expression in skin, thymus and brain first became detectable before or immediately after birth in the mice of the tumor developing lines (304 and 192), whereas they became detectable a few days after birth in the mice of the non-tumor developing line (242) by Western blotting and immunohistochemical analysis. Interestingly, the Ret protein expression in skin developed rapidly after birth as a burst with peak levels on 0.5-1.5 day newborns of lines 304 and 192 and on 7.0-7.5 day-old mice of line 242. The levels of autophosphorylation of Ret kinase in skin were, however, invariable among these three transgenic mouse lines. The mice of line 242, but not those of lines 192 and 304, responded to Ret protein immunization by increased antigen-dependent lymphocyte proliferation and T-cell-mediated tumor growth suppression in vitro. Furthermore, ret-transgenic mice of line 242, but not line 304, rejected the subcutaneously transplanted tumors that had originally developed in a mouse of line 304. These results suggest that whether oncogene product-specific-tolerance is established or not to antitumor immunity may be decided by the dynamics of ret oncogene expression before and after delivery and this is the primary factor determining development or non-development of melanoma.

Animals↗
PubMed1999

Carbonyl compounds cross-link cellular proteins and activate protein-tyrosine kinase p60c-Src.

Glyoxal, a dicarbonyl compound, is produced under oxidative stress by the autoxidation of glucose and reacts with the protein amino group to form Schiff base. In vitro treatment of murine thymocytes and fibroblasts with glyoxal induced extensive tyrosine phosphorylation of multiple proteins, which was drastically inhibited by the addition of OPB-9195, an inhibitor of the carbonyl reaction with proteins. Glyoxal induced cross-linking of a number of cellular proteins, including glycosylphosphatidylinositol (GPI)-anchored cell surface Thy-1. We then demonstrated that treatment of cells with glyoxal promptly induced activation of non-receptor protein-tyrosine kinase c-Src, which was partially inhibited by OPB-9195. It is suggested from these results that carbonyl amine reaction quickly activates c-Src, possibly through cross-linkage of GPI-anchored proteins or putative specific receptors.

Animals↗
PubMed1999

Mercuric chloride induces increases in both cytoplasmic and nuclear free calcium ions through a protein phosphorylation-linked mechanism.

The mechanism of the lymphocyte stimulatory action of sulfhydryl group-reactive mercuric ions was studied with respect to its potential ability to induce a protein tyrosine phosphorylation-linked signal for mobilization of free Ca2+ into cytoplasm and nucleus of the cell. Exposure of human leukamic T cell line (Jurkat) cells to high (1 mM) and low (0.01 mM) concentrations of HgCl2 induced tyrosine phosphorylation of multiple proteins in a concentration-dependent manner. Confocal microscopy directly visualized the time course localization of Ca2+ inside the cells after exposure to HgCl2. The onset and level of Ca2+ mobilization following HgCl2 exposure were in parallel to those of protein tyrosine phosphorylation. Interestingly, by either concentration of HgCl2, Ca2+ was mobilized in both cytoplasm and nucleus almost simultaneously, and the level of Ca2+ mobilization in the nucleus was more than that in the cytoplasm. All the HgCl2-mediated Ca2+ mobilization was prevented by addition of protein kinase inhibitor staurosporin prior to HgCl2. These results suggest that heavy metal stress triggers a protein tyrosine phosphorylation-linked signal that leads to a nuclear event-dominant Ca2+ mobilization.

Calcium Signaling↗
PubMed1999

Different serum soluble Fas levels in patients with allergic rhinitis and bronchial asthma.

BACKGROUND: The pathogeneses of allergic rhinitis and bronchial asthma are believed to be closely mutually related because of the similar dynamics of allergy-inducing cells and molecules and clinical overlap. In this study, we compared these diseases in the dynamics of cell apoptosis-regulating molecules. METHODS: Allergic rhinitis patients (n=36), bronchial asthma patients (n=22), and healthy subjects (n=32) were subjected to measurement of serum (soluble Fas) (sFas) levels during the stable and attack disease phases by a sandwich enzyme-linked immunosorbent assay. RESULTS: Serum sFas levels in patients with allergic rhinitis during the attack phase were significantly lower (P<0.0001) than those in healthy individuals. There were no differences between them during the attack and stable disease phases. In contrast, serum sFas levels in patients with bronchial asthma during the attack phase were higher (P<0.0005) than those in healthy individuals. Interestingly, the levels during the attack phase were lower (P<0.002) than those during the stable phase. CONCLUSIONS: Our results suggest a different pathogenesis for allergic rhinitis and bronchial asthma at the cell apoptosis-linked step.

Apoptosis↗
PubMed1999

Elevated soluble tumor necrosis factor receptor levels in seasonal allergic rhinitis patients.

In this study, we examined the symptom scores and tumor necrosis factor-alpha (TNF-alpha), p55 soluble tumor necrosis factor receptor (sTNFR1), and p75 soluble tumor necrosis factor receptor (sTNFR2) levels in the sera and nasal epithelial lining fluids (ELF) of 20 patients with Japanese cedar pollinosis from the pre- to the postseason period, and compared the results with those of 10 nonallergic control subjects. The symptom scores of the allergic subjects were significantly (P<0.01) higher than those of the nonallergic subjects during the early stage and mid-stage of the season. There were no statistical differences between the allergic and nonallergic subjects in the TNF-alpha levels in sera and ELF from the pre- to the postseason. In the allergic subjects, however, the levels of sTNFR1 and sTNFR2 in ELF were significantly elevated during the early stage (P<0.05) and mid-stage (P<0.01) of the season, whereas those in sera did not change from the pre- to the post-season period. The levels of TNF-alpha in ELF were more than 10 times higher than those in sera, whereas the levels of sTNFR1 and sTNFR2 in ELF were less than half of those in sera in the allergic and nonallergic subjects. These results suggest that sTNFR1 and sTNFR2 may play a role in the pathogenesis of nasal allergic reaction.

Adult↗
PubMed1999

Differential regulation of MMP-9 and TIMP-2 expression in malignant melanoma developed in metallothionein/RET transgenic mice.

We recently established a metallothionein-I(MT)/RET transgenic mouse line in which skin melanosis, benign melanocytic tumor and malignant melanoma develop stepwise. Malignant melanoma cells but not benign melanocytic tumor cells had metastatic ability in transgenic mice. In the present study, we investigated the expression of several matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), including MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MT1-MMP, TIMP-1 and TIMP-2, in these tumors. Western and northern blot analyses revealed that malignant transformation of melanocytic tumors developed in MT/RET transgenic mice accompanied with upregulation of MMP-9 and downregulation of TIMP-2. Expression of other MMP and TIMP genes examined was very low or undetectable in both benign and malignant tumors. Since activation of MMP-9 in malignant tumors was detected by gelatin zymography, these results suggest that imbalance of expression of the MMP-9 and TIMP-2 genes might be associated with metastatic ability of melanoma cells developed in MT/RET transgenic mice.

Animals↗
PubMed1999

Clinical and MRI study of brain stem and cerebellar involvement in Japanese patients with multiple sclerosis.

OBJECTIVES: To investigate the clinical and MRI features of brain stem and cerebellar lesions in Japanese patients with multiple sclerosis. METHODS: A retrospective study of 66 consecutive Japanese patients with multiple sclerosis (42 women and 24 men) was done by reviewing the medical records and MRI films. Forty nine patients were diagnosed as having clinically definite multiple sclerosis and 17 patients as having clinically probable multiple sclerosis according to Poser's criteria. Prevalence rates of each brain stem and cerebellar manifestation and frequency and distribution of MRI lesions in these patients were studied. RESULTS: Forty three patients (65%) had one or more infratentorial manifestations. Cranial nerves were clinically involved in 28 patients (42%), and most of the lesions were identified by MRI. Among them, manifestations of facial, trigeminal, and abducens nerves were relatively common. Cerebellar ataxia was found in 20 patients (30%). The MRI study showed that the lesions responsible for ataxia in these patients were mainly found in the cerebellar peduncles, but cerebellar hemispheric lesions were detected in only four patients (6.4%). CONCLUSION: The low frequency (6.4%) of the cerebellar MRI lesions in these patients is in sharp contrast with the figures reported for white patients with multiple sclerosis (50%-90%). Racial and genetic differences may have an influence on the susceptibility of each part of the CNS to demyelination in multiple sclerosis.

Asian People↗