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Biomedical subjects

K Inoue

Publications and source records attributed to K Inoue.

At least 937 records · Page 52Linked to original sources
PubMed1997

Influence of thrombopoietin on platelet activation in myeloproliferative disorders.

Although thrombopoietin itself does not influence platelet aggregation, it enhances platelet activation in response to certain agonists. We evaluated the effects of thrombopoietin on platelet activation using platelet-rich plasma from 16 patients with myeloproliferative disorders (MPD group) and 16 healthy volunteers (control group). Preincubation with thrombopoietin significantly enhanced platelet aggregation stimulated by ADP, collagen, or epinephrine in the MPD group as well as the control group. However, aggregation induced by 3 micro ADP or 16 microM epinephrine showed significantly less augmentation by thrombopoietin in the MPD group than in the control group. Thrombopoietin significantly shortened the lag time between the addition of 3 microM ADP or 16 microM epinephrine and initiation of secondary aggregation and the lag time between addition of 2 microg/ml collagen and initiation of aggregation in both groups. When platelet-rich plasma was used without adjustment of the platelet count, thrombopoietin itself induced aggregation in two patients. Hypoaggregation after addition of 0.5 microg/ml collagen was observed in seven out of nine patients with normal thrombopoietin levels and only one of six patients with high levels (P = 0.04). Enhancement of 0.5 microg/ml collagen-induced aggregation by thrombopoietin was seen in five out of nine patients with normal thrombopoietin levels and none of the six patients with elevated levels (P = 0.04). These results indicate that platelet activation by certain agonists is enhanced by thrombopoietin in patients with these diseases as well as in normal controls and that the serum thrombopoietin level may regulate the function of circulating platelets in vivo.

Enzyme-Linked Immunosorbent Assay↗
PubMed1997

A medical school for rural areas.

Jichi Medical School (JMS) was established in 1972 to supply graduates to rural areas where medical resources are scarce. JMS has several unique characteristics aimed at motivating graduates to work in a rural practice. These include financial aid for students, a home prefecture recruiting scheme, location in a non-urban area, management by prefectures and support from the national government. The achievements of JMS over the 24-year period since its foundation have been evaluated. A questionnaire has been mailed to all JMS graduates since the first year of graduation. Using a pro-active approach to follow-up, the return rate has been virtually 100%. The authors investigated annual changes in the distribution of the graduates as well as the present status of the graduates in 1995. At that time, JMS graduates were distributed all over Japan. Among the 1871 graduates, 792 (42%) were working in rural areas in 1995. Nine-hundred and twenty-four graduated in the period from the first to the ninth output of graduates. Among these, 858 (93%) completed the requisite 9 years of work contracted between JMS and the graduates, 620 (67%) had practised in the same prefecture, and 305 (33%) were still practising in a rural area. Although there are still improvements to be made, JMS has succeeded in achieving its aim of supplying doctors to rural areas. The recruiting system of JMS is an effective approach to overcoming the shortage of rural doctors, which has continued to be an unresolved global problem.

Education, Medical, Undergraduate↗
PubMed1997

Brain-derived neurotrophic factor increases the stimulation-evoked release of glutamate and the levels of exocytosis-associated proteins in cultured cortical neurons from embryonic rats.

Differentiation and survival of neurons induced by neurotrophins have been widely investigated, but little has been reported about the long-term effect of brain-derived neurotrophic factor (BDNF) on synaptic transmission. Among many steps of neurotransmission, one important step is regulated release of transmitters. Therefore, the release of glutamate and GABA from cortical neurons cultured for several days with or without BDNF was measured by an HPLC-fluorescence method. Although BDNF had little effect on the basal release of glutamate, high K(+)-evoked release was greatly increased by BDNF. BDNF also tended to increase evoked release of GABA. Recently, several proteins involved in the step of "regulated release" have been identified. Thus, the effect of BDNF on the levels of these proteins was then investigated. Neurons were cultivated with or without BDNF, collected, and electrophoresed for western blotting. BDNF increased levels of synaptotagmin, synaptobrevin, synaptophysin, and rab3A, which were known as vesicle protein. Levels of syntaxin, SNAP-25, and beta-SNAP were also increased by BDNF. In addition, the numbers of cored and clear vesicles in nerve terminals or varicosities were also increased by BDNF. These results raise the possibility that BDNF increases regulated release of neurotransmitters through the up-regulation of secretory mechanisms.

Animals↗
PubMed1997

Induction of hepatocyte growth factor activator messenger RNA in the liver following tissue injury and acute inflammation.

Hepatocyte growth factor activator (HGFA) is a serine protease that is responsible for localized activation of hepatocyte growth factor (HGF) in injured tissue. The activated HGF may be involved in regeneration of the injured tissue. HGFA is produced and secreted by the liver and circulates in the plasma as an inactive zymogen. In response to tissue injury, the HGFA zymogen is converted to the active form by limited proteolysis. In this study, we isolated a rat HGFA complementary DNA (cDNA) clone and analyzed the production of HGFA messenger RNA (mRNA) in response to tissue injury using this cDNA clone as a probe. The nucleotide sequence of the cDNA revealed that the amino acid sequences of rat and human HGFA showed a high degree of conservation in the regions of the characteristic domain structures, suggesting that rat and human HGFA are activated by a similar mechanism and have similar enzymatic activities in vivo. Tissue distribution analysis showed that the liver was the major site of rat HGFA mRNA synthesis. Moreover, the cells producing HGFA mRNA were identified as parenchymal liver cells. The level of HGFA mRNA increased in the liver after hepatotoxin or nephrotoxin treatment. This increase was also observed during acute inflammation induced by turpentine. These results suggest that the increase in production of HGFA mRNA in response to tissue injury is the result of an inflammatory response, and that HGFA is an acute phase protein.

Acute Disease↗
PubMed1997

The anti-inflammatory drug sodium salicylate inhibits nitric oxide formation induced by interleukin-1beta at a translational step, but not at a transcriptional step, in hepatocytes.

Recent evidence suggests that nitric oxide (NO) mediates cellular injury under the pathological conditions such as endotoxemia in the liver of rats. Regulation of NO production is crucial for improving the hepatic dysfunction. We have previously reported that, in cultured rat hepatocytes, a single cytokine interleukin-1beta (IL-1beta) stimulated a release of nitrite, an oxidation product of NO, into culture medium dose- and time-dependently. The objective of this study was to investigate an ability of the anti-inflammatory drug NaSA to affect the production of NO in hepatocytes. IL-1beta increased levels of inducible NO synthase (iNOS) messenger RNA (mRNA) with a maximal effect at 8 hours in primary cultures of rat hepatocytes. Nuclear factor-kappaB (NF-kappaB), that is an important nuclear factor protein in iNOS gene transcription in response to inflammatory mediators, also appeared in the nuclear fraction of hepatocytes 1 hour after addition of IL-1beta. Sodium salicylate markedly inhibited the NO formation induced by IL-1beta, but did not affect NF-kappaB activation and iNOS mRNA induction. Western blot analysis revealed that sodium salicylate (NaSA) blocked a step of iNOS protein synthesis. These findings indicate that NaSA may reduce hepatic injury by preventing the induction of NO formation in response to IL-1beta at the posttranscriptional step.

Animals↗
PubMed1997

Interleukin-6 is an autocrine growth factor for folliculostellate-like TtT/GF mouse pituitary tumor cells.

Within the pituitary, folliculostellate (FS) cells are considered to regulate the intercellular communication between endocrine cells by paracrine mechanisms. One of the possible paracrine factors involved, could be interleukin-6 (IL-6) which is produced by these cells. Since IL-6 has been shown to be a growth factor of pituitary cells, we have determined whether IL-6 can also influence FS cell proliferation. To test this, a FS cell-like mouse pituitary cell line (TtT/GF cells) was used that exhibits most characteristics of normal FS cells. Under serum-free conditions the proliferation of TtT/GF cells is critically dependent on the initial seeding density: cells seeded at low density do not grow at all whereas cells seeded at high density proliferate with maximal doubling times of 34 hrs. Mouse IL-6 (mIL-6) stimulated only low density cell cultures in a dose and time dependent manner. For cells seeded at high density, exogenously added IL-6 may have failed to stimulate growth because of endogenously produced mIL-6. Conditioned medium from TtT/GF cells, in which mIL-6 concentrations up to 1017 pg/ml were measured, stimulated the proliferation of TtT/GF cells, indicating an autocrine growth stimulatory mechanism. A neutralizing mIL-6 antibody partially suppressed the growth of TtT/GF cells seeded at high density and partially reduced the growth stimulatory activity of TtT/GF conditioned medium. Thus, IL-6 is one but not the only factor that is involved in the autocrine growth stimulatory loop. The relevance of this mechanism for normal FS cells and its physiological consequence needs to be elucidated.

Animals↗
PubMed1997

Endoscopic resection of rectal carcinoid tumors using aspiration lumpectomy.

BACKGROUND AND STUDY AIMS: Although carcinoid tumors have malignant potential, complete resection can be curative if the lesion is located within the submucosal layer. In this study, we evaluated the effectiveness of endoscopic aspiration lumpectomy for the treatment of small rectal carcinoid tumors. PATIENTS AND METHODS: Eight patients with rectal carcinoid tumors less than 10 mm in diameter and located within the submucosa were treated by aspiration lumpectomy. It was confirmed that none of the patients had any metastatic lesions before treatment. The procedure was carried out using an endoscope with a cylinder attached to the tip and a Teflon tube along its outer axis. The lesion was aspirated into the cylinder, grasped by a snare passed through the tube, and then resected. RESULTS: All lesions were excised completely, without any complications. The resected specimens were circular, with the lesions located in the center of the surrounding mucosa. The diameter of the specimens (mean +/- SD) was 18.3 +/- 6.2 mm. All tumors were histologically typical, and the lateral and bottom margins of the specimens were free of tumor cells. No recurrences or distant metastases were found in any of the patients during the mean observation period of 13.3 months. CONCLUSIONS: We conclude that aspiration lumpectomy is a useful and safe technique for the treatment of small rectal carcinoid tumors contained within the submucosa.

Carcinoid Tumor↗
PubMed1997

Trial of rural practice evaluation: mutual rotation by family physicians.

OBJECTIVES: We aimed to investigate the usefulness of mutual rotation by family physicians in providing an evaluation of rural medical practices. METHODS: Between June and October 1994, each of four family physicians rotated to the practices of the other three, where they worked as a transient locum for 4-5 days and evaluated each practice. They were field faculty physicians of the Department of Community and Family Medicine, Jichi Medical School, and based at general practices accredited for undergraduate and postgraduate training. Two school-based faculty physicians also participated in the study as spare members. RESULTS: The rotation was conducted four times to complete the mutual rotation programme. There was some difference in evaluation among the practices, which indicated the characteristics of the practices. The evaluation accorded relatively well among the participants. CONCLUSIONS: Mutual rotation by family physicians provides an objective and practicable evaluation of general practices and contribute to upholding their quality, which is crucial to medical education.

Faculty, Medical↗
PubMed1997

Real time observation of binding of herpes simplex virus type 1 (HSV-1) to Vero cells and neutralization of HSV-1 by sulfonated human immunoglobulin.

A real time biomolecular interaction assay system involving an optical sensor was applied to the quantitative analysis of the binding of herpes simplex virus type 1 (HSV-1) to Vero cells, and the neutralization antibody titer against this virus with a commercially available sulfonated human immunoglobulin preparation. The virus titers in a viral solution and the neutralizing antibody titer in the human immunoglobulin preparation could be successfully estimated in a short time with this system without any difficult cytopathic effect analysis of cultured cells.

Animals↗
PubMed1997

Nanomolar quantification and identification of various nitrosothiols by high performance liquid chromatography coupled with flow reactors of metals and Griess reagent.

Nitrosothiols (RS-NOs) appear to be critically involved in various signal transduction mechanisms. We describe here a specific and highly sensitive quantification method for RS-NOs by using high performance liquid chromatography (HPLC) combined with a flow reactor system. RS-NOs were applied to an HPLC system of C18-reverse phase or a gel filtration column and eluted with 10 mM sodium acetate buffer (pH 5.5) plus 0.5 mM diethylenetriamine pentaacetic acid with or without either 0-7% methanol or 0.15 M NaCl. The eluate from the HPLC column was mixed with a solution containing 1.75 mM HgCl2 or 1.75 mM CuSO4 for RS-NO decomposition in a reaction coil via a three-way connector. NO2- generated via the metal-induced RS-NO decomposition was then reacted with Griess reagent, which was infused through a second three-way connector, yielding a diazo-compound detected at 540 nm. In a separate experiment, a copper particle-loaded column was used for RS-NO degradation instead of the metal-ion flow reactor. In all RS-NOs tested, i.e., nitrosoglutathione (GS-NO), nitroso-L-cysteine, and nitrosoalbumin, the nitroso- group was converted to NO2- by the Hg2+-reaction system as well as copper-loaded column, and the recovery was almost 100%. The Cu2+-solution flow reaction system, however, yielded only 30% recovery of RS-NOs as NO2-. Also, the RS-NOs could be identified at nanomolar concentrations: detection limit, 3.0 nM in a 150-microl aliquot. These RS-NOs showed well-resolved elution profiles even in the presence of NO2- and NO3-. More importantly, biological generation of GS-NO was quantitatively demonstrated with RAW264 cells in culture incorporating free GSH in the medium. In conclusion, our novel RS-NO assay will be useful to examine the formation and functions of RS-NOs in biological systems.

Animals↗
PubMed1997

Localization of the phosphatidylserine-binding site of glyceraldehyde-3-phosphate dehydrogenase responsible for membrane fusion.

In this study, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phosphatidylserine (PS)-binding protein and localized the putative PS-binding site involved in the membrane fusion induced by this enzyme. In an attempt to identify the PS-binding proteins, we raised polyclonal antibodies against a 15-amino-acid synthetic peptide (amino acid residues 390-403 of phosphatidylserine decarboxylase), which was shown to bind specifically to PS. One polyclonal antibody, designated aPSD-2, crossreacted with GAPDH, and its binding to GAPDH was inhibited by PS but not by other phospholipids such as phosphatidylethanolamine and phosphatidylinositol. Kinetic analysis of GAPDH binding to phospholipid membranes by measuring surface plasmon resonance showed that GAPDH associated with the phospholipid membrane containing PS rapidly (k[on] =2.8 X 10(4) M(-1) X s[-1]) and dissociated extremely slowly (k[off]=5.9 X 10(-5) s[-1]), giving a low dissociation constant (KD=2.6nM). GAPDH bound less effectively to membranes without PS with a dissociation constants of 0.2 microM. GAPDH-induced vesicle fusion was also inhibited by aPSD-2, suggesting that this antibody recognizes the putative PS-binding site on GAPDH involved in the enzyme-induced membrane fusion. Chemical fragmentation of GAPDH with cyanogen bromide followed by separation and sequence analysis of the reactive peptide resulted in the identification of a single reactive peptide with the sequence of amino acid residues 45-103 of GAPDH. Analysis of aPSD-2 binding to synthetic peptides derived from the corresponding region localized the antibody-binding site to amino acid residues 70-94 of GAPDH. Both the 25-amino-acid synthetic peptide (amino acid residues 70-94 of GAPDH) and polyclonal antibody raised against this peptide inhibited GAPDH-induced membrane fusion, suggesting that these amino acid residues play a crucial role in this membrane fusion process.

Amino Acid Sequence↗
PubMed1997

Heterologous expression and subcellular localization of pumpkin seed tonoplast intrinsic proteins (TIP) in yeast cells.

Two tonoplast intrinsic proteins (TIP) of pumpkin seeds, pMP23 and MP28, were expressed in yeast cells under control of the GAL1 promoter, and the subcellular localization of the proteins was analyzed. The pMP23 and MP28 stably accumulated in the yeast vacuolar membrane when the proteins were expressed in the proteinase A-deficient strain (pep4), which lacks the activities of vacuolar proteases. However, pMP23 and MP28 did not accumulate in the wild-type strain; the expressed pMP23 and MP28 were degraded in a proteinase A-dependent manner. These results indicate that pMP23 and MP28 are transported to the vacuolar membrane when expressed in yeast.

Cloning, Molecular↗
PubMed1997

Relationship between CYP2C9 and 2C19 genotypes and tolbutamide methyl hydroxylation and S-mephenytoin 4'-hydroxylation activities in livers of Japanese and Caucasian populations.

Genomic DNA was isolated from livers of 39 Japanese and 45 Caucasians and the genotypes of CYP2C9 and 2C19 genes were determined with PCR methods using synthetic oligonucleotide primers. Liver microsomes were also prepared from these human samples and activities for tolbutamide methyl hydroxylation and S-mephenytoin 4'-hydroxylation were determined. The single base mutation of C416T (Arg144Cys) in CYP2C9 was detected in 22% of Caucasians but not in Japanese samples. Another single base mutation at A1061C (Ile359Leu) in the CYP2C9 gene was found with frequencies of about 8% in both races. We did not detect any individuals who have either homozygous Cys144/Cys144 or Leu359/Leu359 CYP2C9 variant nor both heterozygous Cys144-Ile359 and Arg144-Leu359 CYP2C9 variant in the human samples examined. The CYP2C19m2 genetic polymorphism was found only in Japanese people, while CYP2C19m1 type was determined in both races, with higher incidence in Japanese than in Caucasian population. Immunoblotting analysis of human liver microsomes suggested that CYP2C9 is a major component of the human CYP2C enzyme pool; it accounted for approximately 20% of total P450 in liver microsomes of both human populations. The levels of CYP2C19 protein were determined to be about 0.8% and 1.4% of total P450 (mean) in Japanese and Caucasians, respectively. We did not detect CYP2C19 protein in liver microsomes of humans who were genotyped for CYP2C19 gene as m1/m1, m1/m2, and m2/m2 variants but detected CYP2C9 protein in all of the samples examined. Good correlations were found between levels of CYP2C9 and activities of tolbutamide methyl hydroxylation (r = 0.77) and between levels of CYP2C19 and activities of S-mephenytoin 4'-hydroxylation (r = 0.86) in liver microsomes of the human samples examined. Tolbutamide methyl hydroxylation activities were lower in human samples with the Leu359 allele of CYP2C9 than those with the Cys144 allele and wild-type (Arg144-Ile359); the former type showed slightly higher K(m) values. When calculated on P450 basis, liver microsomes of individuals having m1/m1, m1/m2, and m2/m2 types of CYP2C19 had very low catalytic activities for S-mephenytoin 4'-hydroxylation. These results provide useful comparisons for pharmacokinetic and toxicokinetic models of some of the clinically used drugs that are oxidized by CYP2C proteins in humans.

Aryl Hydrocarbon Hydroxylases↗
PubMed1997

Neuroleptic malignant syndrome and hydroxylase gene mutations: no association with CYP2D6A or CYP2D6B.

To examine a possible association between debrisoquine 4-hydroxylase gene mutations and neuroleptic malignant syndrome, we assessed frequencies of wild type and A and B mutant alleles of the CYP2D6 gene in 24 patients with a history of neuroleptic malignant syndrome, 50 patients with neuroleptic-treated schizophrenia but no history of neuroleptic malignant syndrome, and 50 healthy controls. Allele frequencies did not differ significantly between these groups. Homozygotes for CYP2D6A and for CYP2D6B, which indicate a poor-metabolizer phenotype for the CYP2D6 substrate, were not detected among the neuroleptic malignant syndrome cases. This result indicates no excess of poor CYP2D6 metabolizers in neuroleptic malignant syndrome. The aetiology of neuroleptic malignant syndrome is not explainable in terms of CYP2D6 gene mutations.

Alleles↗
PubMed1997

Regional differences in calcium sensitivity in the guinea-pig intestine.

The effect of the Ca(2+)-channel antagonist nicardipine on the basal tone of six segments (duodenum, jejunum, ileum, proximal colon, distal colon and rectum) of the guinea-pig intestine has been investigated in muscle preparations. Nicardipine reduced the basal tone of the proximal and distal colon but not of the duodenum, jejunum, ileum and rectum. Similarly, when each segment was incubated in Ca(2+)-free medium, the basal tone of the proximal and distal colon, but not that of the other four segments, was reduced. The reduced basal tone recovered after cumulative addition of Ca2+ in both colon preparations. The basal tone of the distal colon was partly reduced by tetrodotoxin, atropine and clonidine. Conversely, L-type Ca(2+)-channel antagonists (Cd2+, verapamil and nicardipine), but not the T-type Ca(2+)-channel antagonist Ni2+, completely reduced the basal tone of the distal colon. These results indicate that in the regulation of basal tone there are additional regional differences in the effect of Ca2+ influx into the cells from the extracellular fluid which might involve L-type-like Ca2+ channels and might partly be because of neuronal factors.

Adrenergic alpha-Agonists↗
PubMed1997

Lipopolysaccharide and the glycoside ring of staurosporine induce CD14 expression on bone marrow granulocytes by different mechanisms.

We established previously that lipopolysaccharide (LPS) can induce the expression of LPS-binding sites on bone marrow cells (BMC). We now report that staurosporine (STP), a glycosylated indolocarbazole alkaloid with potent inhibitory activity for various protein kinases, can induce the same effect. With both agents, the newly expressed LPS receptor was found to be CD14. The STP-induced effect was independent of its protein kinase inhibitory activity because several other protein kinase inhibitors, such as the indolocarbazole K-252a, the bisindolylmaleimide RO-31-8220, the perylenequinone calphostin C, and the isoquinolinesulfonamide H7, did not induce CD14 expression. The observation that the STP analog K-252a with an identical polyaromatic aglycon moiety was inactive yet the analog UCN-01 with an identical glycoside ring was active suggests that the induction of CD14 expression is triggered by the sugar moiety of STP. Three lines of evidence show that the mechanism of CD14 expression induced by STP differs from that induced by LPS: (i) unlike LPS, STP can stimulate BMC from LPS-unresponsive C3H/HeJ mice, (ii) LPS and STP effects are additive at a saturating dose of LPS, and (iii) the protein kinase inhibitor K-252a inhibits the LPS-induced but not STP-induced stimulation. Therefore, our findings show that both a protein kinase-dependent (LPS-induced) and a protein kinase-independent (STP-induced) mechanism can lead to the expression of the LPS receptor CD14 on BMC. We also found that the STP-induced stimulation of BMC is modulated by cyclosporin A, vinblastine, and verapamil. This observation may suggest that the inducible effect of STP could be initiated by its interaction with P-glycoprotein, a membrane pump with drug efflux function that plays a critical role in the multidrug resistance of cancer cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗