Biomedical subjects

M Nishikawa

Publications and source records attributed to M Nishikawa.

At least 55 records · Page 3Linked to original sources

PubMed2001

Protein kinase C-catalyzed phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet secretion.

Protein kinase C (PKC)-potentiated inhibitory phosphoprotein of myosin phosphatase (CPI) was detected in human platelets. Like smooth muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC inhibited the activity of myosin phosphatase containing the PP1delta catalytic subunit and the 130-kd myosin-binding subunit (MBS). Treatment of intact platelets with thrombin or the stable thromboxane A(2) analog STA(2) resulted in increased phosphorylation of both CPI and MBS at Thr-696, whereas phorbol myristate acetate (PMA) and the Ca(++) ionophore ionomycin only induced CPI phosphorylation. PMA induced slow adenosine triphosphate (ATP) secretion of fura 2-loaded platelets with no change in cytosolic Ca(++). The PMA-induced increase in CPI phosphorylation preceded phosphorylation of 20-kd myosin light chain (MLC(20)) at Ser-19 and ATP secretion. The PKC inhibitor, GF109203X, inhibited PMA-induced phosphorylation of CPI and MLC(20) with similar IC(50) values. These findings suggest that the activation of PKC by PMA induces MLC(20) phosphorylation by inhibiting myosin phosphatase through phosphorylation of CPI. STA(2)-induced MLC(20) phosphorylation was also diminished but not abolished by GF109203X, even at high concentrations that completely inhibited STA(2)-induced CPI phosphorylation. A combination of the Rho-kinase inhibitor Y-27632 and GF109203X led to a further decrease in STA(2)-induced MLC(20) phosphorylation, mainly because of a significant inhibition of MBS phosphorylation at Thr-696. Inhibition of STA(2)-induced ATP release by Y-27632, GF109203X, or both appeared to correlate with the extent of MLC(20) phosphorylation. Thus, CPI phosphorylation by PKC may participate in inhibiting myosin phosphatase, in addition to the Rho-kinase-mediated regulation of myosin phosphatase, during agonist-induced platelet secretion. (Blood. 2001;97:3798-3805)

Adenosine Triphosphate↗

PubMed2001

Nonviral vectors in the new millennium: delivery barriers in gene transfer.

Development of an efficient method for introducing a therapeutic gene into target cells in vivo is the key issue in treating genetic and acquired diseases by gene therapy. To this end, various nonviral vectors have been designed and developed, and some of them are in clinical trials. The simplest approach is naked DNA injection into local tissues or systemic circulation. Physical (gene gun, electroporation) and chemical (cationic lipid or polymer) approaches have also been utilized to improve the efficiency and target cell specificity of gene transfer by plasmid DNA. After administration, however, nonviral vectors encounter many hurdles that result in diminished gene transfer in target cells. Cationic vectors sometimes attract serum proteins and blood cells when entering into blood circulation, which results in dynamic changes in their physicochemical properties. To reach target cells, nonviral vectors should pass through the capillaries, avoid recognition by mononuclear phagocytes, emerge from the blood vessels to the interstitium, and bind to the surface of the target cells. They then need to be internalized, escape from endosomes, and then find a way to the nucleus, avoiding cytoplasmic degradation. Successful clinical applications of nonviral vectors will rely on a better understanding of barriers in gene transfer and development of vectors that can overcome these barriers.

Animals↗

PubMed2001

Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages.

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.

Animals↗

PubMed2001

Targeting superoxide dismutase to renal proximal tubule cells inhibits mitochondrial injury and renal dysfunction inuduced by cisplatin.

We recently reported the synthesis of a cationic superoxide dismutase (SOD) derivative (AH-SOD) that rapidly and selectively accumulates in and around proximal tubule cells and effectively dismutes superoxide radicals in situ. The present study revealed that administration of cis-diamminedichloroplatinum(II)-elicited oxidative stress in renal mitochondria, decreased the renal expression of Bcl-x, released cytochrome c from mitochondria to cytosol, and induced apoptosis and renal dysfunction by a mechanism that was inhibited by AH-SOD. These results suggest that targeting SOD to proximal tubule cells protects renal function and permits the administration of fairly high doses of nephrotoxic anticancer agents, such as cisplatin, without causing renal injury.

Animals↗

PubMed2001

Hemispheric lateralization in the cortical motor preparation for human vocalization.

To investigate the cortical information processing during the preparation of vocalization, we performed transcranial magnetic stimulation (TMS) over the cortex while the subjects prepared to produce voice in response to a visual cue. The control reaction time (RT) of vocalization without TMS was 250-350 msec. TMS prolonged RT when it was delivered up to 150-200 msec before the expected onset of voice (EOV). The largest delay of RT was induced bilaterally over points 6 cm to the left and right of the vertex (the left and right motor areas), resulting in 10-20% prolongation of RT. During the early phase of prevocalization period (50-100 msec before EOV), the delay induced over the left motor area was slightly larger than that induced over the right motor area, whereas, during the late phase (0-50 msec before EOV), it was significantly larger over the right motor area. Bilateral and simultaneous TMS of the left and right motor areas induced delays not significantly different from that induced by unilateral TMS during the early phase, but induced a large delay well in excess of the latter during the late phase. Thus, during the cortical preparation for human vocalization, alternation of hemispheric lateralization takes place between the bilateral motor cortices near the facial motor representations, with mild left hemispheric predominance at the early phase switching over to robust right hemispheric predominance during the late phase. Our results also suggested involvement of the motor representation of respiratory muscles and also of supplementary motor cortex.

Adult↗

PubMed2001

Somatic mutation of mitochondrial DNA in cancerous and noncancerous liver tissue in individuals with hepatocellular carcinoma.

Unlike other types of cancer, hepatocellular carcinoma (HCC) is usually preceded by chronic inflammation caused by viral infection. The mutation of mitochondrial DNA (mtDNA) in hepatocarcinogenesis associated with viral infection was investigated. Compared with control liver tissue, the frequency of mtDNA mutations was markedly increased in both noncancerous and cancerous liver specimens from individuals with HCC. The accumulation of mtDNA mutations in HCC tissue reflected the degree of malignancy. The frequency of mtDNA mutations in HCC tissue was also greater than that described previously for other types of tumors. These observations suggest that the repeated destruction and regeneration of liver tissue associated with chronic viral hepatitis lead to the accumulation of mtDNA mutations. The genetic instability that results in the high rate of mtDNA mutation in cancerous liver tissue is also consistent with the multicentric hepatocarcinogenesis detected clinically.

Carcinoma, Hepatocellular↗

PubMed2001

Column-switching high-performance liquid chromatography-electrospray ionization mass spectrometry for identification of heroin metabolites in human urine.

In order to prove heroin (DAM) use, a simple, rapid and sensitive analytical method has been established by combining semi-microcolumn HPLC, a column switching technique and electrospray ionization mass spectrometry (ESI-MS). Urine samples were directly introduced to the system, and endogenous urinary constituents were removed by using on-line column switching solid-phase extraction with a strong cation-exchange (SCX) cartridge column (2.0 mm I.D. x 10 mm). Heroin and its metabolites enriched on the top of the column were then successfully analyzed with excellent separation by use of a SCX semi-microcolumn (1.5 mm I.D. x 150 mm), accompanied by ESI mass spectral detection. The proposed conditions are as follows: mobile phase, 10 mM ammonium acetate (pH 6.0)-acetonitrile (30:70, v/v) (for main separation) and 30 mM ammonium acetate (for trapping); flow-rates, 120 microl/min (for main separation) and 200 microl/min (for trapping); capillary voltage, +4.5 kV; cone voltage, 50 V. Linear calibration curves were obtained in the selected ion monitoring (SIM) mode using protonated molecular ions (m/z 370 for DAM, m/z 328 for MAM and m/z 286 for MOR) over the concentration ranges from 10 to 1000 ng/ml for morphine (MOR) and 1-100 ng/ml for DAM and 6-acetylmorphine (MAM). The detection limits were 0.1-3 ng/ml. Upon applying the scan mode, 2-30 ng/ml were the detection limits. The present HPLC-ESI-MS method was successfully applied to the determination of opiates in users' urine samples.

Chromatography, High Pressure Liquid↗

PubMed2001

Observation of untreated patients with neuroblastoma detected by mass screening: a "wait and see" pilot study.

BACKGROUND: Recent studies have indicated that mass screening for neuroblastoma detects tumors that otherwise would have regressed spontaneously without recognition. Therefore, we started an observation program for these patients to determine how frequently spontaneous regression occurs. PROCEDURE: Eighteen patients were detected by mass screening between June 1994 and December 1996. Eight of these cases matched the following criteria and entered the observation program: Stage I or II, less than 5 cm in diameter; no involvement of large vessels or organs; not difficult to resect; informed consent. If there were an increase in tumor size, an elevation of tumor markers, or evidence of metastasis, the tumor would be immediately resected. RESULTS: Five of the eight cases showed spontaneous regression. Although the remaining three tumors were resected 6-10 months after diagnosis, all patients survived without evidence of recurrence. CONCLUSIONS: At least 60% of neuroblastoma cases who entered our observation program regressed spontaneously.

Adrenal Gland Neoplasms↗

PubMed2001

Nitric oxide regulates mitochondrial respiration and functions of articular chondrocytes.

OBJECTIVE: Biologic effects of nitric oxide (NO) have been shown to increase under hypoxic conditions. Because the oxygen tension in joint cavities of patients with arthritis is fairly low, biologic effects of NO would be expected to be significantly large in these compartments. This study was undertaken to investigate the effects of NO on the energy metabolism and functions of articular chondrocytes under different oxygen tension conditions. METHODS: Articular chondrocytes from rabbits were cultured under various oxygen concentrations in the presence or absence of NO and NOC18, an NO donor. Cellular respiration was measured using a Clark-type oxygen electrode. Levels of ATP in the cells were determined according to the luciferin-luciferase method. Cellular synthesis of proteoglycans was determined by measuring the incorporation of radioactivity (derived from 35S-labeled SO4) into glycosaminoglycans. Expression of stress-related proteins was evaluated by Western blotting analysis using specific antibodies. RESULTS: Respiration and ATP synthesis of cultured chondrocytes were inhibited by NO, particularly under low oxygen concentrations. The presence of either NO or specific inhibitors of mitochondrial electron transport suppressed the synthesis of proteoglycans without affecting cell viability. When exposed to NO, cellular levels of heat-shock protein 70 (hsp70) and heme oxygenase 1 (HO-1) increased markedly. The presence of inhibitors of mitochondrial electron transport also increased cellular levels of hsp70 and HO-1. CONCLUSION: These results suggest that NO generated in the joint might inhibit energy metabolism and the synthesis of proteoglycans of chondrocytes, thereby modulating pathophysiologic processes occurring in patients with arthritis.

Adenosine Triphosphate↗

PubMed2001

NF-kappaB activation in peripheral blood monocytes/macrophages and T cells during acute Kawasaki disease.

Kawasaki disease (KD) is a febrile disease of childhood characterized by systemic vasculitis, and the levels of many proinflammatory cytokines are elevated in the serum at the acute stage. We investigated the activation of transcription factor NF-kappaB for genes that encode the proinflammatory cytokines in CD14+ monocytes/macrophages and CD3+ T cells in peripheral blood by means of Western blot and flow cytometric analyses. Western blot analysis demonstrated that NF-kappaB activation was more increased in CD14+ monocytes/macrophages than in CD3+ T cells in all children during the acute stage. Flow cytometric analysis revealed that NF-kappaB activation in CD14+ monocytes/macrophages was significantly higher than in CD3+ T cells at the acute stage (30.0 +/- 16.0% vs 11.4 +/- 5.0%, P < 0.01, Wilcoxon test). NF-kappaB activation in CD14+ monocytes/macrophages was significantly decreased after high-dose intravenous immunoglobulin therapy (P < 0.05). The present findings suggest that CD14+ monocytes/macrophages play an important role in cytokine production during acute KD.

Acute Disease↗

PubMed2001

Transfer of full-length Dmd to the diaphragm muscle of Dmd(mdx/mdx) mice through systemic administration of plasmid DNA.

Mutations in the gene encoding dystrophin, a large cytoskeletal protein in muscle, lead to Duchenne muscular dystrophy (DMD). Affected individuals often die of respiratory failure resulting primarily from diaphragm muscle degeneration. Here we report a new procedure to transfer the full-length dystrophin cDNA into the diaphragm muscle of Dmd(mdx/mdx) mice, which carry a mutation in the dystrophin gene (Dmd). Significant gene transfer was found after intravenous injection of naked plasmid DNA followed by a brief (eight second) occlusion of blood flow at the vena cava. This is the first demonstration of gene transfer into the diaphragm muscle through systemic administration of naked plasmid DNA. The approach has potential application for treatment of DMD.

Animals↗

PubMed2001

Bronchiolitis obliterans organizing pneumonia (BOOP) with suspected liver graft-versus-host disease after allogeneic bone marrow transplantation.

We report on a patient with chronic myelogenous leukemia who developed bronchiolitis obliterans organizing pneumonia (BOOP) after allogeneic bone marrow transplantation (BMT). A 19-year-old Japanese male complained of dry cough and dyspnea 7 months after BMT. The chest X-ray and computed tomography revealed patchy infiltrates bilaterally. Lung function test, lung biopsy and bronchoalveolar lavage were consistent with the diagnosis of BOOP. The patient also suffered from suspected graft-versus-host disease (GVHD) of the liver, after discontinuation of cyclosporine. Furthermore, prednisolone proved effective against the BOOP and the liver dysfunction. These findings indicate that BOOP is a possible pulmonary manifestation of chronic GVHD, and that immunological mechanisms may have effected the onset of BOOP after BMT in this case.

Adult↗

PubMed2001

Leptomeningeal contrast enhancement in moyamoya: its potential role in postoperative assessment of circulation through the bypass.

Leptomeningeal contrast enhancement (LMCE) is one of the MRI features of moyamoya. Its clinical significance, however, is not elucidated. Our purpose was to characterise LMCE on MRI and to evaluate its role in the assessment of circulation through a surgically established bypass in moyamoya. We studied 16 patients with idiopathic moyamoya (seven males, nine females, includingt four children, aged 7 to 54 years, mean 24 years) who underwent T1-weighted MRI before and after intravenous contrast medium. The presence of LMCE, its intensity and anatomical distribution, catheter angiographic findings, and relation of LMCE to the bypass surgery were assessed. More LMCE was seen in the cerebrum in most patients with moyamoya than in normal controls. LMCE in the brain stem and cerebellum was minimal, similar to that seen in the controls. LMCE was less prominent following surgery than before operation or in patients who did not undergo surgery. In three patients examined both before and after operation LMCE became less prominent following bypass surgery. As LMCE becomes less prominent after "effective" bypass surgery, this may be used for evaluation of effectiveness of surgery in moyamoya.

Adult↗

PubMed2001

Distal stump of an occluded intracranial vertebral artery at the vertebrobasilar junction mimicking a basilar artery aneurysm.

The distal stump of an occluded intracranial vertebral artery (VA) can mimic a basilar artery aneurysm of the vertebrobasilar junction. Their differentiation is crucial to establishing the appropriate treatment. We report two cases with occlusion of the distal stump of the VA due to atherosclerosis and arterial dissection. Magnetic resonance images with three-dimensional constructive interference in steady state sequences are useful in revealing the occluded segment as a continuous anatomical structure from the proximal VA to the basilar artery. This information may prevent unnecessary exploratory surgery for a suspected basilar artery aneurysm.

Adult↗

PubMed2001

Pigment epithelium-derived factor in the vitreous is low in diabetic retinopathy and high in rhegmatogenous retinal detachment.

PURPOSE: To report the levels of pigment epithelium-derived factor in the vitreous of patients with diabetic retinopathy, rhegmatogenous retinal detachment, and idiopathic macular hole. METHODS: Using enzyme-linked immunosorbent assay, we measured the levels of pigment epithelium-derived factor in the vitreous of 34 eyes of 33 patients who underwent vitrectomy for the treatment of diabetic retinopathy (17 eyes of 16 patients), rhegmatogenous retinal detachment (10 eyes), and idiopathic macular hole (seven eyes). RESULTS: The vitreal concentration of pigment epithelium-derived factor was 1.15 +/- 0.23 microg/ml (mean +/- standard error) in eyes with diabetic retinopathy, 3.28 +/- 0.69 microg/ml in rhegmatogenous retinal detachment, and 1.71 +/- 0.39 microg/ml in idiopathic macular hole. The pigment epithelium-derived factor level in rhegmatogenous retinal detachment was significantly higher than that in diabetic retinopathy (P =.0008) and idiopathic macular hole (P =.034). For eyes with diabetic retinopathy, the pigment epithelium-derived factor level was 0.88 +/- 0.21 microg/ml in proliferative diabetic retinopathy and 2.43 +/- 0.37 microg/ml in nonproliferative diabetic retinopathy (P =.0083). Additionally, the pigment epithelium-derived factor level in active diabetic retinopathy (0.70 +/- 0.22 microg/ml) was significantly lower than the level in inactive diabetic retinopathy (1.79 +/- 0.35 microg/ml; P =.018). CONCLUSIONS: These results suggest that pigment epithelium-derived factor inhibits angiogenesis and that lower levels of pigment epithelium-derived factor may be related to the angiogenesis in diabetic retinopathy and result in active proliferative diabetic retinopathy. The results also suggest that higher levels of pigment epithelium-derived factor in the eyes with rhegmatogenous retinal detachment may act as a neuroprotective agent for the detached retina.

Adult↗

PubMed2001

Development of gene drug delivery systems based on pharmacokinetic studies.

A series of pharmacokinetic studies following systemic or local administration for the development of delivery systems for gene drugs, such as plasmid DNA and oligonucleotides, are reviewed. The pharmacokinetics of gene drugs after intravenous injection into mice was evaluated based on clearance concepts. Pharmacokinetic analysis revealed that the overall disposition characteristics of the gene drug itself were determined by the physicochemical properties of its polyanionic DNA. Based on these findings, liver cell-specific carrier systems via receptor-mediated endocytosis were successfully developed by optimizing physicochemical characteristics. On the other hand, the pharmacokinetics of gene drugs after intratumoral injection were assessed in a tissue-isolated tumor perfusion system. The relationship between the physicochemical properties of gene drug delivery systems and intratumoral pharmacokinetics was determined and the therapeutic effect was also discussed in relation to pharmacokinetics. Collectively, it was demonstrated that a rational design of gene drug delivery systems that can control their in vivo disposition is possible by means of pharmacokinetic studies at whole body, organ and cellular levels.

Animals↗

PubMed2001

Accumulation of beta-catenin in the cytoplasm and the nuclei during the early hepatic tumorigenesis.

Hepatocellular carcinomas (HCCs) are thought to develop as well-differentiated tumors and progress to less-differentiated tumors. However, the genetic changes underlying the development and progression of HCCs are not well understood. Recent studies have shown frequent beta-catenin gene activation in HCCs by somatic alterations involving exon 3, resulting in the activation of the Wnt/Wingless signal transduction pathway. However, the exact process in which activation of Wnt/Wingless signal transduction pathway occurs during hepatic tumorigenesis remains to be elucidated. The aim of the present study was to investigate at what stage of hepatocellular tumorigenesis this pathway was activated. Altered expression of beta-catenin was investigated immunohistochemically with special reference to the grade of histological differentiation in 41 HCCs and eight dysplastic nodules. Mutational analysis of the beta-catenin gene with single-strand conformation polymorphism method and polymerase chain reaction amplification was related with the expression of this protein. beta-Catenin was expressed in the cytoplasm and the nuclei in three cases among eight dysplastic nodules, in four cases among 20 well differentiated HCCs, in five cases among 15 moderately differentiated HCCs, and one case among six poorly differentiated HCCs, respectively. Expression of beta-catenin in the cytoplasm and the nuclei was associated in one case with mutation and two cases without mutation for beta-catenin gene among 11 screened HCCs. It was concluded that beta-catenin was accumulated in the cytoplasm and the nuclei in pre-cancerous lesions of the liver and might contribute, at least in part, to hepatic tumorigenesis.

Journal Article↗

PubMed2001

Docosahexaenoic acid improves long-term potentiation attenuated by phospholipase A(2) inhibitor in rat hippocampal slices.

1. We investigated the possible involvement of phospholipase A(2) (PLA(2)) and its products in long-term potentiation (LTP) in the CA1 neurotransmission of rat hippocampal slices. 2. Inhibitors of Ca(2+)-independent PLA(2) (iPLA(2)) prevented the induction of LTP without affecting the maintenance phase of LTP whereas Ca(2+)-dependent PLA(2) inhibitors were virtually ineffective, which suggests a pivotal role of iPLA(2) in the initiation of LTP. 3. We then investigated the effect of docosahexaenoic acid (DHA) and arachidonic acid (AA) on BEL (bromoenol lactone, an iPLA(2)-inhibitor) -impaired LTP, and found that either DHA or AA abolished the effect of BEL. However, DHA did not restore BEL-attenuated LTP when applied after the tetanus. DHA per se affected neither the induction nor maintenance of LTP. Linoleic acid had no effects, either. 4. These results suggest that DHA is crucial for the induction of LTP and that endogenously released DHA during tetanus is sufficient to trigger the formation of LTP.

Animals↗