PubMed Health⌕ Search

Biomedical subjects

Junji Nishimura

Publications and source records attributed to Junji Nishimura.

At least 37 records · Page 2Linked to original sources
PubMed2003

Sequence requirement for nuclear localization and growth inhibition of p27Kip1R, a degradation-resistant isoform of p27Kip1.

p27(Kip1R) is an isoform of p27(Kip1), having a distinct C-terminus. The sequences of p27(Kip1R) required for nuclear localization and growth inhibition were determined in HeLa cells using a green fluorescence protein (GFP) as a reporter molecule. Region 153-168 and residues K168 and I169 were determined to play a critical role in the nuclear localization of p27(Kip1R). Aliphatic amino acid was found to be a substitute for the basic residue in the typical nuclear localization signal, while its functional substitution was incomplete, thereby causing a significant cytoplasmic retention of p27(Kip1R). p27(Kip1R) is thus the first example of an atypical bipartite nuclear localization signal with aliphatic amino acid as a functional residue. Despite cytoplasmic retention, p27(Kip1R) inhibited the cell growth as well as p27(Kip1), while GFP alone had no effect. The mutants lacking an N-terminus containing the binding regions for cyclins and cyclin-dependent kinases also showed a significant degree of nuclear localization, but failed to inhibit cell growth. The growth inhibition by p27(Kip1R) as well as p27(Kip1) was thus suggested to originate in the common N-terminal region.

Amino Acid Sequence↗
PubMed2003

Expression of apoptosis-associated protein RCAS1 in macrophages of histiocytic necrotizing lymphadenitis.

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1), which is recognized by the 22-1-1 monoclonal antibody (MoAb) against human uterine adenocarcinoma cell line SiSo, has been identified on various kinds of cancer cells. RCAS1 appears to be an apoptosis-associated protein that induces apoptosis in activated T-cells and erythroid progenitor cells. We previously demonstrated that monocytes/macrophages express RCAS1. In the present study, we investigated RCAS1 expression by 22-1-1 MoAb in histiocytic necrotizing lymphadenitis (HNL), which is characterized by necrotic lesions consisting of T-cells undergoing apoptosis and macrophages in proliferation. Expression of RCAS1 was analyzed by immunohistochemical staining in 9 cases of HNL and in 9 cases of reactive lymphadenitis used as a control. The ratio of RCAS1+ cells to CD68+ cells (monocytes/macrophages) was significantly higher in the patients with HNL than in the patients with reactive lymphadenitis (P = .0002; paired t test). Our findings suggest that RCAS1 expressed on macrophages may play an important role in the induction of activated T-cell apoptosis in cases of HNL.

Adolescent↗
PubMed2003

The signaling pathways of erythropoietin and interferon-gamma differ in preventing the apoptosis of mature erythroid progenitor cells.

Interferon (IFN)-gamma is a survival factor for mature erythroid progenitor cells. To elucidate related survival mechanisms, we compared the role of phosphatidylinositol 3-kinase (PI3-kinase) in the survival signals of IFN-gamma and erythropoietin (EPO). Human erythroid colony-forming cells (ECFCs) purified from peripheral blood were used, and Ly294002 was used as a PI3-kinase inhibitor. Treating ECFCs with a high concentration of Ly294002 (50 micromol/L) in the presence of EPO and/or IFN-gamma reduced cell viability by inducing apoptosis. However, treating cells with a lower concentration of Ly294002 (10 micromol/L) did not affect the antiapoptotic function of IFN-gamma and abolished the antiapoptotic effect of EPO. Adding IFN-gamma or EPO induced Bcl-x expression in ECFCs, as determined by Western blotting, and expression was suppressed in the presence of Ly294002. We also examined the phosphorylation of the protein kinase Akt, the downstream target of PI3-kinase. EPO stimulation significantly increased the level of Akt phosphorylation, but IFN-gamma did not. These results suggest that IFN-gamma plays a role in preventing the apoptosis of erythroid progenitor cells by affecting Bcl-x expression, thereby reducing the disruption of the mitochondrial transmembrane potential via PI3-kinase pathways that are related to but distinct from the EPO pathway.

Apoptosis↗
PubMed2003

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells.

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.

Ammonium Chloride↗
PubMed2003

p16(INK4a) induces differentiation and apoptosis in erythroid lineage cells.

OBJECTIVE: Hematopoiesis is regulated by proliferation, differentiation, and death. p16(INK4a) has been reported to regulate apoptosis and differentiation of diverse cells, as well as arresting the cell cycle at G1 phase. The aim of this study is to explore the properties of p16 in apoptosis and differentiation of erythroid cells. METHODS: We transfected the INK4a gene to K562 cells, which defect the INK4a gene, and compared the effect of enforced expression of p16(INK4a) with that of various additives, topoisomerase I inhibitor (SN 38), interferon-alpha, phosphatidyl-inositol-3 kinase inhibitor (LY294002), and serum deprivation, which arrest cell cycle at different phases. We also investigated the role of p16(INK4a) in normal day-6 human erythroid colony-forming cells by transfecting the INK4a gene. RESULTS: p16(INK4a) induced cell cycle arrest at the G0/G1 phase, and promoted erythroid differentiation in viable K562 cells, but induced apoptosis in K562 cells with incomplete differentiation. The apoptosis induced by p16 was accompanied with downregulation of bcl-x and nuclear NF-kappaB. These findings were not observed in K562 cells treated with various additives. p16(INK4a) decreased the cell viability and promoted apoptosis in day-9 ECFC. CONCLUSION: We propose that p16(INK4a) plays a role in maintaining homeostasis during erythroid differentiation, and that the mechanisms for this effect are not confined to those inducing cell cycle arrest.

Apoptosis↗
PubMed2003

Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain.

The contraction of smooth muscle is regulated primarily by intracellular Ca2+ signal. It is well established that the elevation of the cytosolic Ca2+ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca2+ concentration and force development revealed that the alteration of the Ca2+-sensitivity of the contractile apparatus as well as the Ca2+ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca2+ concentration is considered to contribute to the major mechanisms regulating the Ca2+-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca2+ elevation is increased either by Ca2+-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca2+-dependent manner but also by multiple kinases in a Ca2+-independent manner, thus adding a novel mechanism to the regulation of the Ca2+-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.

Adenosine Triphosphatases↗
PubMed2003

Local antigen-driven oligoclonal expansion of B cells in the liver portal areas of patients with primary biliary cirrhosis.

BACKGROUND/AIMS: The antigen-driven clonal proliferation of B cells within target tissue has been reported in some autoimmune diseases. The purpose of this study was to examine the clonal characteristics of B cells in the liver portal area of primary biliary cirrhosis (PBC). METHODS: The liver portal area was microdissected from liver biopsy sections from two PBC patients. Genomic DNA was extracted and rearranged immunoglobulin heavy chain variable region (VH) genes were amplified and sequence analyzed. RESULTS: Sixteen VH sequences from portal area 1A of patient 1 had three different rearrangements. Nineteen VH sequences from portal area 1B of this patient had three different rearrangements. In three sequences from the portal area 1B, a stepwise accumulation of somatic mutations was observed. Between the sequences from the two portal areas, no common VH sequence was observed. In patient 2, 15 VH sequences from portal area 2A had three different rearrangements. Fourteen VH sequences from portal area 2B had two different rearrangements. One rearrangement was present both in the portal area 2A and portal area 2B. CONCLUSION: The oligoclonal B cell proliferation and stepwise accumulation of somatic mutations suggested that an antigen-driven B cell response had occurred in the portal area of PBC.

Aged↗
PubMed2003

Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells.

1 Using fura-2 fluorometry of [Ca(2+)](i) in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. 2 Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca(2+)](i) elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. 3 The response to PAR1-AP (SFLLRNP) was significantly (P<0.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaffected by the preceding stimulation with either trypsin or PAR2-AP (SLIGRL). The response to PAR2-AP was reduced by the preceding stimulation with trypsin and PAP2-AP. PAR1-AP attenuated the subsequent responses not only to thrombin and PAR1-AP but also to trypsin and PAR2-AP, while PAR2-AP specifically attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher affinity PAR1-AP (haPAR1-AP) (Ala-pF-Arg-Cha-HArg-Tyr-NH(2)) specifically attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was significantly (P<0.05) attenuated by the preceding stimulation with thrombin but not trypsin. 5 In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin-cleaved PAR1 was no longer responsive to thrombin.

Animals↗
PubMed2003

Re-entry of tumour B cells into the cycle of somatic mutation and isotype switching in follicular lymphoma.

Sequencing of the immunoglobulin (Ig) gene transcripts of the tumour B cells in lymph node (LN) and bone marrow (BM) from a follicular lymphoma (FL) patient associated with multiple myeloma identified two dominant clones. One of the clones, present in both LN and BM, had somatic mutations and extensive clonal diversity. Among the diversified clones, two dominant populations of identical sequences (group I and II) were present. Group II was a descendant population of group I and had nine more somatic mutations. Group I contained micro-, delta-, gamma- and alpha-expressing clones. Group II clones contained mainly micro- and delta-expressing clones. These findings showed that somatic mutation and isotype switching occurred repeatedly in this patient.

Aged↗
PubMed2003

Clonal analysis of B cells in the synovial membrane of patients with rheumatoid arthritis.

OBJECTIVE: To investigate the clonal characteristics of B cells in the synovial membranes of rheumatoid arthritis (RA) patients. METHODS: The clonality of B cells at separate sites of the synovial membrane and other tissues from RA patients were investigated by a reverse transcriptase-polymerase chain reaction with a VH framework 3 consensus primer and a subsequent single-strand conformation polymorphism analysis. RESULTS: Several dominant bands were observed in all synovial membrane samples and some of the dominants bands were common among the 2 or 3 separate regions of each synovial sample. The persistent existence of clonal B cells was observed in metachronous synovial fluid samples. CONCLUSION: Infiltrating B cells are oligoclonal and antigen-driven mechanisms may play a role in the generation of clonal B cells in RA synovium. The stable presence of B cell clones in synovial fluid suggests the involvement of these clones in the perpetuation of the chronic inflammation in RA.

Amino Acid Sequence↗
PubMed2003

Modulation of Ca2+ sensitivity regulates contractility of rabbit corpus cavernosum smooth muscle.

PURPOSE: We examined the role of the modulation of Ca2+ sensitivity for regulating the contractility of corpus cavernosum smooth muscle. MATERIALS AND METHODS: We applied simultaneous measurements of intracellular Ca2+ concentration and tension in fura-PE3 loaded intact strips and receptor coupled permeabilization by alpha-toxin. RESULTS: In intact fura-PE3 loaded strips the tension induced by 10 microM. phenylephrine was significantly greater than that produced by depolarization with 118 mM. K+, although the extent of intracellular Ca2+ concentration elevations was similar. During sustained contraction induced by 10 microM. phenylephrine the application of 10 microM. Y-27632 (a Rho kinase inhibitor) induced relaxation with a slight decrease in intracellular Ca2+ concentration, while the application of 3 microM. GF109203X (a protein kinase C inhibitor) induced relaxation without changing intracellular Ca2+ concentration. In alpha-toxin permeabilized strips 10 microM. phenylephrine induced a larger increase in force at a constant intracellular Ca2+ concentration and produced a leftward shift in the intracellular Ca2+ concentration-tension relationship, a response that was partially inhibited by pretreatment with Y-27632 or GF109203X. CONCLUSIONS: These results indicate that in rabbit corpus cavernosum smooth muscle phenylephrine induces contraction not only by increasing intracellular Ca2+ concentration, but also by increasing Ca2+ sensitivity of the contractile apparatus in a Rho kinase and protein kinase C dependent manner. Antagonism of Ca2+ sensitization pathways in the corpus cavernosum smooth muscle represents an alternate target for the treatment of erectile dysfunction.

Adrenergic alpha-Agonists↗
PubMed2003

[Successful treatment of refractory peripheral T-cell lymphoma by allogeneic peripheral blood stem cell transplantation].

A 40-year-old man was admitted to our hospital suffering from abdominal pain. He revealed marked splenomegaly, lymphadenopathy and pancytopenia. Lymph node biopsy showed diffuse proliferation of medium to large atypical lymphoid cells. Immunohistochemically the cells were positive for T-cell markers. He was diagnosed as having peripheral T cell lymphoma (PTCL) with involvement of the spleen and bone marrow. Because of refractoriness to combination chemotherapies and splenic irradiation, we performed allogeneic peripheral blood stem cell transplantation from an HLA-identical sister. Conditioning was consisted of conventional doses of total body irradiation and cyclophosphamide, and GVHD prophylaxis was performed with cyclosporine and methotrexate. After the patient developed grade III graft-versus-host disease, his splenomegaly improved dramatically. Although the effectiveness of allogeneic hematopoietic cell transplantation in the treatment of malignant lymphoma is controversial, we should consider this as a second-line therapy to refractory subsets of PTCL.

Adult↗
PubMed2003

[Changes in the contractility of the NIH3T3 fibroblast induced by the over-expression of CPI-17].

The role of protein kinase C (PKC) in the regulation of contraction has been controversial. Recently, CPI-17, a PKC-potentiated inhibitor protein of PP1, has been cloned and shown to be specifically expressed in SMC. In this study, we over-expressed CPI-17 and its mutants in NIH3T3 cells, which do not express CPI-17, and examined its effect on the contractile property. For the measurement of tension, NIH3T3 cells were collected by trypsinization, mixed with type I collagen and made into a ring preparation (reconstituted ring: RR). The isometric tension developments were measured using this RR and a force transducer. The application of phorbol dibutyrate (PDBu; 0.3 microM) to RR transfected by CPI-17 induced a contraction that reached 2-3 times greater that the 10% FBS-induced contraction, while PDBu relaxed the RR transfected with vector alone (control) or CPI-17 mutants (T38A and T38E). The PDBu-induced contraction of the CPI-17 transfected RR could be inhibited by 3 microM GF109203X (PKC inhibitor) but not by 3 microM Y27632 (rho kinase inhibitor). The application of PDBu during contraction induced by 1 microM bradykinin induced further contraction in CPI-17 transfected RR, while it induced a complete relaxation in control RR. These results indicated that CPI-17 is a molecular switch that reverses the PKC mediated effect on contraction. The limited expression of CPI-17 to SMC may explain the previous observation that the effects of PKC stimulation were quite different in SMC from those of other cell types.

Animals↗
PubMed2002

Clinical significance of telomerase activity in multiple myeloma.

BACKGROUND: The clinical course of patients with multiple myeloma varies, and therefore it is important to evaluate the disease state. We studied the telomerase activity of myeloma cells as a possible prognostic factor in such patients. METHODS: Twenty five samples from patients with multiple myeloma were studied. We purified myeloma cells in bone marrow samples according to the expression of surface antigens, CD38 and CD45. CD38+/CD45- or dim cells had morphologic characteristics of myeloma cells, with a purity exceeding 95%. The telomerase activity of myeloma cells was determined by a polymerase chain reaction-based telomeric repeat amplification protocol assay. Ki-67 positivity of the purified cells was determined by flow cytometry using anti-Ki-67 antibody. The relationship between telomerase activity and prognostic factors was also examined. RESULTS: A significantly high degree of telomerase activity was detected in subjects with a serum beta2-microglobulin level > or = 6 mg/dL or at Stage III (P = 0.002). The serum C-reactive protein, lactate dehydrogenase, and creatinine levels did not correlate with the telomerase activity, but this activity did significantly correlate with Ki-67 positivity and the percentage of plasma cells in the bone marrow (r = 0.561, P = 0.004, and r = 0.397, P = 0.049, respectively). The patients with high levels of telomerase activity were thus found to have a significantly short survival time after sampling (P = 0.035). CONCLUSIONS: The measurement of the telomerase activity in myeloma cells was found to be a reliable marker for the proliferating capacity and tumor mass in myeloma patients. The telomerase activity of myeloma cells may therefore be useful as a prognostic factor.

Adult↗
PubMed2002

Mechanism of down-regulation of L-type Ca(2+) channel in the proliferating smooth muscle cells of rat aorta.

The mechanism of down-regulation of L-type Ca(2+) channel (L-VOC) was investigated in rat aortic smooth muscle cells in primary culture. On culture days 3-5, the cells actively incorporated the 5-bromo-2'-deoxy-uridine (BrdU), and did not respond to K(+) depolarization nor express alpha(1C) subunit of L-VOC. At confluence on day 8, BrdU incorporation decreased, and the cells up-regulated alpha(1C) subunit mRNA, expressed alpha(1C) subunit protein at cell periphery, and responded to K(+) depolarization. Treating the proliferating cells on day 3 with serum-free media or 10 microM PD98059, a MAP kinase kinase inhibitor, for 2 days induced the expression of alpha(1C) subunit protein and the responsiveness to K(+) depolarization. However, the serum starvation, but not PD98059, decreased the BrdU incorporation and increased the alpha(1C) subunit mRNA. It is concluded that the expression of L-VOC is substantially suppressed in the proliferating cells due to two mechanisms; a MAP kinase-mediated post-transcriptional down-regulation and the transcriptional down-regulation by additional mitogenic signals.

Animals↗
PubMed2002

Eradication of virus-infected T-cells in a case of adult T-cell leukemia/lymphoma by nonmyeloablative peripheral blood stem cell transplantation with conditioning consisting of low-dose total body irradiation and pentostatin.

We describe the case of a 55-year-old man with adult T-cell leukemia/lymphoma (ATL) in first remission who underwent nonmyeloablative allogeneic peripheral blood stem cell transplantation with conditioning consisting of 4 courses of pentostatin and low-dose total body irradiation. Complete chimerism in peripheral blood was achieved on day 42 without severe myelosuppression. Concomitantly, the proviral DNA load for human T-cell leukemia virus I (HTLV-I) in peripheral blood mononuclear cells decreased below detectable limits and was still undetectable on day 270. This fact indicates that eradication of ATL cells is feasible by induction of an alloimmune response without high-dose chemoradiotherapy.

Adult↗
PubMed2002

The mechanisms for tachykinin-induced contractions of the rabbit corpus cavernosum.

1. This study was designed to investigate the mechanisms for the contractions induced by tachykinins (substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)) in the rabbit corpus cavernosum strips, using fura-PE3 fluorimetry and alpha-toxin permeabilization. 2. Tachykinins induced contractions in the rabbit corpus cavernosum in a concentration-dependent manner. The potency order was SP>NKA>NKB. 3. The tachykinin-induced contractions were enhanced by phosphoramidon (PPAD), an endopeptidase inhibitor, but not by N(omega)-nitro-L-arginine methylester (L-NAME). 4. The NK(1) receptor selective antagonist, SR 140333 significantly inhibited the tachykinin-induced contractions. Although the NK(2) receptor selective antagonist, SR 48968 alone did not influence the effects of tachykinins, it potentiated the inhibitory effect of SR 140333. The NK(3) receptor selective antagonist, SR142801 had no effect. 5. In the rabbit corpus cavernosum, tachykinins induced sustained increases in [Ca(2+)](i) and tension in normal PSS, while only small transient increases in [Ca(2+)](i) and tension were observed in Ca(2+)-free solution. 6. In alpha-toxin permeabilized preparations, tachykinins induced an additional force development at a constant [Ca(2+)](i). 7. These results indicated that in the rabbit corpus cavernosum: (1) Tachykinins induced contractions by increasing both the [Ca(2+)](i) and myofilament Ca(2+) sensitivity; (2) The tachykinin-induced [Ca(2+)](i) elevations were mainly due to the Ca(2+) influx; (3) Tachykinin-induced contractions were mainly mediated through the activation of NK(1) receptor expressed in the rabbit corpus cavernosum smooth muscle, and affected by the endopeptidase activity and (4) Tachykinins may thus play a role in controlling the corpus cavernosum tone.

Animals↗