Biomedical subjects

T Hoshino

Publications and source records attributed to T Hoshino.

At least 19 recordsLinked to original sources

PubMed1992

Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system.

The high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.

Adenosine Triphosphate↗

PubMed1992

The presence of concanavalin-A(Con-A)-like molecules on natural-killer (NK)-sensitive target cells: their possible role in swainsonine-augmented human NK cytotoxicity.

In the present study we examined the expression of concanavalin-A(Con-A)-like molecules on natural-killer (NK)-sensitive target cells and investigated their possible role in the human NK-cell phenomenon. The incubation of either peripheral-blood lymphocytes (PBL) or large granular lymphocytes (LGL) with swainsonine (SW), an inhibitor of mannosidase II, resulted in the augmentation of cytotoxicity against K562 leukemia cells. The enhanced cytotoxicity was associated with increased binding of fluorescein isothiocyanate-conjugated Con-A to SW-treated effector cells, and immunofluorescence staining of the target K562 cells using goat anti-Con-A antibody (Ab) showed a significant positive shift in the flow cytometric pattern. Electrophoretic separation and immunoblotting analysis revealed that 4 components with a molecular weight of approximately 95, 80, 60 and 50 kDa were recognized by anti-Con-A Ab from the detergent-extract of K562 cells. The addition of Con-A during the antibody incubation step of the Western blotting abolished their expression, thus excluding non-specific binding of the antibody. The addition of Con-A also strongly inhibited the cytotoxicity of SW-treated effector cells (PBL or LGL) against K562 cells, and this inhibition was abolished by 40 mM alpha-methyl-mannopyranoside (alpha-MM), which binds to Con-A. Furthermore, Con-A increased the binding frequency of SW-treated LGL to K562, in spite of the inhibited cytotoxicity, and this effect could be neutralized by the further addition of alpha-MM. Our results suggest that Con A-like molecules might play an important role in cell-cell interactions between SW-treated effector cells and NK target cells.

Adenosine Triphosphate↗

PubMed1992

Thrombin-induced shape change in human megakaryoblastic leukemic cells, MEG-01, is mediated by protein kinase C.

We investigated the intracellular processes of the shape change in human megakaryoblastic leukemic cells, MEG-01, by platelet agonists. Thrombin induced the formation of many pseudopods. This shape change was also induced by phorbol 12-myristate 13 acetate (TPA) and weakly by Ca2+ ionophore, A23187, but not by ADP, collagen, or epinephrine. Electron microscopy and FITC-labeled phalloidin staining revealed thick submembranous microfilament bundles in the pseudopods of the shape-changed cells by thrombin. Shape change was inhibited by cytochalasin B. Since Ca(2+)-dependent phosphorylation reactions play central role on the initiation of shape change of platelet, we examined the effects of protein kinase C (PKC) inhibitor, H-7, and myosin light chain (MLC) kinase inhibitor, ML-9, on the shape change of MEG-01 cells induced by thrombin, and observed that H-7 potently inhibited thrombin-induced shape change, while ML-9 did not. These results suggest that thrombin-induced reorganization of microfilaments and shape change of MEG-01 cells are mediated by PKC, but not by MLC kinase.

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

PubMed1992

Cell kinetics of medulloblastomas.

BACKGROUND: Cell kinetics and clinical behavior were correlated for 22 primary and 10 recurrent or metastatic medulloblastomas from 30 patients. METHODS: Twenty-six patients received bromodeoxyuridine (BUdR) intravenously during craniotomy; excised specimens were stained by the immunoperoxidase method. The BUdR labeling index (LI) was calculated. Four patients received a pulse of tritiated thymidine; LI was calculated autoradiographically. RESULTS: The mean LI for 32 tumors (11.7 +/- 1.3% standard error) indicated high proliferative potential. LI appeared higher in tumors from younger patients and those in the cerebellar hemisphere as compared with those in the cerebellar vermis. LI did not correlate with survival time. In six BUdR-labeled tumors labeled with iododeoxyuridine, the S-phase duration was 6.1-11.3 hours (mean, 8.0 +/- 0.8 hours); the potential doubling time was 25-82 hours. The actual doubling time, estimated in three cases from serial imaging, was 20-24 days. A cell loss factor of approximately 0.90 was assumed. CONCLUSION: A high LI may reflect rapid medulloblastoma growth, but survival seemed more dependent on sensitivity to radiation therapy.

Adolescent↗

PubMed1992

Identification of Ca2+/calmodulin-dependent protein kinase and endogenous substrate of Fusarium oxysporum.

Ca2+/calmodulin-dependent phosphorylation and cross-reactivity between anti-rat brain Ca2+/calmodulin-dependent protein kinase II (CaMK) antibody and partially purified CaMK from Fusarium oxysporum were detected in the component of high-molecular mass (M(r) greater than 100,000). In vitro, Ca2+/CaM-dependent phosphorylation of only a 16-kDa protein was detected. The 16-kDa protein was localized in the membrane fraction. Amino acid sequence of one of the peptides derived from partial hydrolysis of the 16-kDa protein had a high homology (65.5%) with the bovine transducin beta chain. It is assumed that the 16-kDa protein is an endogenous substrate of F. oxysporum CaMK.

Amino Acid Sequence↗

PubMed1992

Neurotransmitter release from synaptotagmin-deficient clonal variants of PC12 cells.

Synaptotagmin (p65) is an abundant synaptic vesicle protein of neurons and contains regions similar to the regulatory domain of protein kinase C. These domains are thought to be involved in calcium-dependent interaction with membrane phospholipids during exocytosis. To assess the functional role of synaptotagmin, synaptotagmin-deficient clonal variants of PC12 cells were isolated. All of the variant cells released catecholamine and adenosine triphosphate in response to elevated intracellular concentrations of calcium, which suggests that synaptotagmin is not essential for secretion of catecholamine and adenosine triphosphate from PC12 cells.

Adenosine Triphosphate↗

PubMed1992

The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen.

Immunoglobulin G fractions from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, immunoprecipitate 125I-labeled omega-conotoxin GVIA-labeled calcium channels solubilized from rat brain. A 58-kDa antigen was detected by probing Western blots of partially purified calcium channels with LEMS plasma and IgG and was shown to be the relevant antigen in omega-conotoxin receptor immunoprecipitation. Monoclonal antibody 1D12, produced by immunizing mice with synaptic membranes, has properties similar to these autoimmune IgGs in both immunoprecipitation and Western blotting assays. 1D12 antigen was purified by immunoaffinity chromatography and shown to bind LEMS IgG. The antigen was identified by screening a rat brain cDNA library with 1D12 and was found to have strong homology to the synaptic vesicle membrane protein synaptotagmin. Our results indicate therefore that these antibodies immunoprecipitate omega-conotoxin receptors by binding to synaptotagmin that is associated with calcium channels. We suggest that the interaction between synaptotagmin and the voltage-gated calcium channel plays a role in docking synaptic vesicles at the plasma membrane prior to rapid neurotransmitter release and that autoantibody binding to a synaptotagmin-calcium-channel complex may be involved in the etiology of LEMS.

Antibodies, Monoclonal↗

PubMed1992

Purification and properties of a decapping enzyme from rat liver cytosol.

A decapping enzyme has been purified about 2400-fold from rat liver cytosol. The decapping enzyme was shown to be fairly homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme had an apparent molecular weight of 110,000 and consisted of two equal subunits. The enzyme hydrolyzed m7Guo5'PPP5'Ado to m7GMP and ADP. Analysis of the products produced from radioactively capped oligonucleotides and intact mRNA having 3H-cap suggests that the enzyme can hydrolyze capped mono- to pentanucleotides (m7Guo5'PPP5'N (where N = 1-5 nucleotides)) but not intact mRNA. The existence of methyl group at the N7 position of guanosine moiety of cap structure was necessary for the action of the decapping enzyme. This was confirmed by the comparison of the rates of hydrolysis of m7Guo5'PPP5'Ado by the enzyme in the presence of various nucleotides. The activity of enzyme was slightly stimulated by Na+, K+, NH4+, Ca2+ and polyamines. Mg2+ and Mn2+ were without effect on the enzyme activity.

Animals↗

PubMed1992

Cell kinetic analysis of human brain tumors by in situ double labelling with bromodeoxyuridine and iododeoxyuridine.

Fifty-seven patients with brain tumors (29 gliomas, 23 meningiomas, 5 miscellaneous) received infusions of intravenous iododeoxyuridine (IUdR) and bromodeoxyuridine (BUdR) 1-5 hr apart shortly before tumor removal. Excised tumor specimens were stained sequentially for BUdR and IUdR. The percentage of BUdR-labelled cells was determined to establish the labelling index (LI), or S-phase fraction, and the ratio of cells labelled only with IUdR to cells labelled with BUdR or with BUdR and IUdR was determined to calculate the duration of S-phase (Ts) and the potential doubling time (Tp) of each tumor. The BUdR LIs varied from less than 1% to 20%, reflecting the malignancy of each tumor. Despite the difference in LIs, however, Ts was fairly uniform (mean +/- SD, 8.7 +/- 2.0 hr). Tp varied from 2 days to more than 1 month and correlated closely with the BUdR LIs (Tp = 23/LI0.93; r2 = 0.91). Double-labelling studies with IUdR and BUdR allow the S-phase fraction, Ts and Tp to be determined from a single biopsy specimen and thus provide more useful information on the growth characteristics of individual tumors than can be obtained by single-labelling studies with BUdR.

Brain Neoplasms↗

PubMed1992

Proliferative potential and outcome in pediatric astrocytic tumors.

In 43 pediatric patients (29 male, 14 female) with primary astrocytic tumors of the central nervous system (CNS), the correlation was evaluated between outcome and proliferative potential, measured by the bromodeoxyuridine (BrdU) labeling index (LI). Twenty-five patients had low-grade gliomas, 13 had anaplastic gliomas, and 5 had glioblastomas multiforme (GBM). All patients underwent surgery; 37 also had chemotherapy, radiation therapy, or both. The median BrdU LIs were less than 1% (range 0-9.3%) in low-grade gliomas, 2.3% (range 0-21.2%) in anaplastic gliomas, and 7.7% (range 0-21.3%) in GBM. Seven of eight patients with BrdU LI greater than 5% have died (median survival 29 weeks). Median survival has not been reached in patients with BrdU LI less than 1% (19/22 alive) or between 1% and 5% (12/13 alive) after median follow-up periods of 165 and 120 weeks, respectively. A high BrdU LI correlated with short survival (p = 0.0001); the association between malignant histology and short survival was weaker (p = 0.019). BrdU LI is therefore a significant predictor of outcome in patients with primary CNS astrocytomas and appears to be a stronger predictor than histology in patients with low-grade and anaplastic gliomas. More patients need to be studied to confirm these preliminary observations.

Adolescent↗

PubMed1992

Predicting the recurrence of ependymomas from the bromodeoxyuridine labeling index.

The usefulness of histopathological grading in predicting the prognosis of patients with ependymomas is controversial. To clarify the discrepancy between the histological malignancy and the prognosis of these tumors, we estimated the proliferative potential of 32 intracranial and intraspinal ependymomas and correlated the findings with the clinical behavior. Each patient received an intraoperative infusion of bromodeoxyuridine (BUdR, 200 mg/m2 i.v.) before tumor removal; the BUdR labeling index (LI), or percentage of BUdR-labeled cells, was determined immunohistochemically in excised specimens. The mean BUdR LI (+/- SD) of intracranial malignant ependymomas was 4.1 +/- 2.8%. Nonmalignant intracranial and intraspinal ependymomas and subependymomas had mean LIs of 1.5 +/- 0.9%, 1.1 +/- 0.3%, and less than 1%, respectively. Overall, 44% of the tumors recurred. There were no statistically significant differences in the recurrence rates of intracranial and intraspinal ependymomas, including subependymomas (43% and 44%, respectively), or of intracranial ependymomas with LIs greater than 1.0% and less than 1.0% (67% and 44%, respectively). However, the early recurrence rate (within 24 months after treatment) of tumors with LIs greater than 1.0% was higher than that of tumors with LIs of less than 1.0% (100% vs. 25%, P less than 0.05). The BUdR LI also showed a statistically significant inverse correlation with the time to recurrence. These findings indicate that BUdR LI reflects the proliferative potential of individual ependymomas and can be used to help predict the recurrence and estimate the prognosis of these tumors.

Adolescent↗

PubMed1992

Histochemical study of pituitary adenomas with Ki-67 and anti-DNA polymerase alpha monoclonal antibodies, bromodeoxyuridine labeling, and nucleolar organizer region counts.

The growth potential of 65 pituitary adenomas was determined by histochemical analysis with Ki-67 and anti-DNA polymerase alpha monoclonal antibodies, bromodeoxyuridine (BrdUdR) labeling, and counts of argyrophilic nucleolar organizer regions (Ag-NORs). The mean proliferating cell indices (PCIs) determined by Ki-67 and anti-DNA polymerase alpha and the BrdUdR labeling index (LI) were generally very low [1.0 +/- 0.2%, 1.1 +/- 0.2%, and 0.5 +/- 0.1% (+/- SE), respectively]. Apart from adrenocorticotropic hormone-positive adenomas, which had significantly higher indices, there were no statistically significant differences in the indices among the other subtypes of pituitary adenomas. Recurrent tumors had higher Ki-67 and DNA polymerase alpha PCIs and BrdUdR LIs (3.6%, 4.2%, 1.4%) than primary tumors (0.8%, 0.8%, 0.3%; P less than 0.005). The number of Ag-NORs did not correlate significantly with any of the three indices. The mean number of Ag-NORs was higher in nonfunctioning adenomas than in functioning adenomas (2.04 vs 1.66, P less than 0.005); among prolactin-positive adenomas, those treated preoperatively with bromocriptine had more Ag-NORs than untreated tumors (1.75 vs 1.57, P less than 0.005). These results suggest that the Ki-67 and DNA polymerase alpha PCIs and the BrdUdR LI predict the growth potential of individual pituitary adenomas, whereas the number of Ag-NORs appears to correlate with hormone production rather than with the proliferative potential.

Adenoma↗

PubMed1992

Myelopathy due to large veins draining recurrent spontaneous caroticocavernous fistula.

A 45-year-old woman developed a myelopathy 9 years after a spontaneous left caroticocavernous fistula (CCF) was treated by a trapping procedure, with clipping distal to the fistula and ligation of the common carotid artery. Spinal MRI and vertebral angiography revealed recurrence of the CCF and gigantic tortuous veins draining through the brain stem down to the midcervical spinal cord. The abnormal veins and myelopathy disappeared after surgical closure of the CCF.

Arteriovenous Fistula↗

PubMed1992

Physiologic capacity of well-developed collaterals in patients with isolated left anterior descending artery disease.

To assess the physiologic significance of well-developed collaterals, 34 patients, with isolated left anterior descending artery disease (LAD) and without overt prior myocardial infarction, underwent cardiac catheterization and exercise thallium-201 emission computed tomography. The patients were divided into 3 groups; 11 patients with 90% stenosis of the proximal LAD and without collaterals (group 1), 11 with 99% stenosis of the proximal LAD, and without collaterals (group 2) and 12 with a total occlusion of the proximal LAD which was completely filled by well-developed collaterals (group 3). On left ventriculography, shortening fractions of the anterior wall were significantly reduced in group 2 as compared to group 1 and 3 (group 1 vs group 2: p less than 0.01, group 2 vs group 3: p less than 0.05), which reflected the lower ejection fraction of group 2 (p less than 0.01 and p less than 0.05, respectively). The perfusion defects of the anterior wall on both the initial and the delayed images were severer in groups 2 and 3 than in group 1 (group 1 vs group 2 and group 1 vs group 3 on the initial image: p less than 0.01, for both, group 1 vs group 2 and group 1 vs group 3 on the delayed image: p less than 0.05, for both). However, recovery of the perfusion defects from the initial image to the delayed image was better in group 3 than in groups 1 and 2 (group 1 vs group 2 and group 1 vs group 3: p less than 0.05, for both). Therefore, coronary blood flow through well-developed collaterals was considered to be comparable to the flow through a diseased vessel with 90% stenosis at rest. During maximal exercise, blood flow through well-developed collaterals was considered to be comparable to the flow through a diseased vessel with 99% stenosis, although the blood flow through well-developed collaterals was considered to be better than that through 99% stenosis during the recovery period. These findings suggest that patients with well-developed collaterals must be treated like those with severe stenosis.

Aged↗

PubMed1992

Circulating myosin light chain I levels after coronary reperfusion: a comparison with myocardial necrosis evaluated from single photon emission computed tomography with pyrophosphate.

This study was performed to assess the influence of coronary reperfusion on the serial serum myosin light chain (LC)I levels and to evaluate the relationship between the peak LCI level and the infarct size calculated from single photon emission computed tomography (SPECT) with technetium-99m pyrophosphate (Tc-99m PYP) in 11 patients who underwent coronary reperfusion. Blood was drawn before reperfusion, immediately after reperfusion, and once a day for 14 days, to estimate the time course of serum LCI release. The infarct size estimated by Tc-99m PYP ranged from 7.3 to 62.4 ml. The LCI levels obtained before reperfusion were less than 2.5 ng/ml but those obtained immediately after reperfusion were much higher. The value ranged from 2.7 to 9.7 ng/ml and that expressed as a percentage of peak LCI (% peak LCI) ranged from 19 to 83%. Collateral circulation, reperfusion arrhythmia and the degree of residual stenosis had no influence upon the % peak LCI. The correlation between peak LCI levels and SPECT-determined infarct size was good, with a correlation of 0.76 (p less than 0.01, regression line by least squares method y = 3.31 + 1.53x). Early serum LCI might be influenced by coronary reperfusion but the peak LCI value reflected acute myocardial necrosis in patients who underwent coronary reperfusion.

Adult↗

PubMed1992

Circadian fluctuations of tissue plasminogen activator antigen and plasminogen activator inhibitor-1 antigens in vasospastic angina.

To elucidate the circadian variation of fibrinolytic components in vasospastic angina, plasma levels of tissue plasminogen activator antigen (t-PA), free plasminogen activator inhibitor antigen (free PAI-1), t-PA/PAI-1 complex, and total PAI-1 were measured in venous plasma samples. Samples were taken every 6 hours (6:00 AM, noon, 6:00 PM, and midnight) for 24 hours in 14 patients with vasospastic angina, in 9 patients with exertional angina, and in 19 normal subjects. Twenty-four-hour Holter monitoring (Holter monitor, Del Mar Avionics, Irvine, Calif.) was also carried out in all subjects. All of the fibrinolytic components showed circadian variation, with a peak level at 6:00 AM in every study group except for the t-PA/PAI-1 complex in the group of patients with exertional angina. The values for all or the fibrinolytic components at each sampling time were higher in patients with coronary artery disease than in normal subjects. In particular, the mean value of free PAI-1 at 6:00 AM in patients with vasospastic angina was significantly higher than that in normal subjects and that in patients with exertional angina. This value of free PAI-1 in patients with vasospastic angina was closely associated with the duration of ischemic attacks. These results suggested that the circadian fluctuation of fibrinolytic components may be an important factor that leads to coronary thrombosis at the time of coronary spasm, especially in the early morning.

Acetylcholine↗

PubMed1992

Mitotic activity of cells in the fibrous zone of the rat mandibular condyle.

To investigate cell renewal in this fibrous zone, Wistar rats at 3 and 6 weeks of age were injected intraperitoneally with colchicine (1 mg/kg) three times, at 10:00, 13:00 and 16:00 h, and mitotic cells were examined after killing the rats at 18:00 h. Colchicine induced a significant increase in the number of arrested metaphases in the fibrous and the proliferating cell zone. The mitotic rates (per 1000 cells per h) in the fibrous zone at 3 weeks of age were 0.426 for the superficial layer and 0.266 for the deeper layer. These findings suggest that the fibrous zone of young rats grew with the proliferation of the cells within this zone. Age-associated histological changes in the fibrous zone were related to changes in mitotic rates within that zone.

Aging↗

PubMed1992

Intraduodenal osmolality directly enhances insulin secretion in the rat.

To elucidate the direct effect of an intestinal osmolality on insulin release, we investigated the insulin response to intra-duodenal infusion of mannitol in rats. After the anesthesia with intraperitoneal pentobarbital sodium, one milliliter of mannitol solution (10% or 20%) was infused into the duodenum. Portal and femoral blood insulin concentrations significantly increased at 30, 60, and 120 min after intra-duodenal infusion of mannitol, although the blood glucose level did not change. Subcutaneous pre-administration of propranolol (0.4mg/kg) or metoprolol (25mg/kg) completely abolished this phenomenon. These results suggest that intestinal osmolality can directly enhance insulin secretion and that beta 1-adrenergic mechanism is involved in this phenomenon.

Adrenergic beta-Antagonists↗