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Biomedical subjects

T Hayakawa

Publications and source records attributed to T Hayakawa.

At least 163 records · Page 9Linked to original sources
PubMed2000

Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion.

In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.

Animals↗
PubMed2000

On the mechanism of plasmin-induced platelet aggregation. Implications of the dual role of granule ADP.

Plasmin-induced platelet aggregation has been considered to be a cause of reocclusion after thrombolytic treatment with plasminogen activators. However, little is known regarding the mechanism and regulation of plasmin-induced platelet aggregation. In this study, we demonstrated that plasmin causes the degranulation of platelets, and that ADP released from granules plays a crucial role in the induction of platelet aggregation. This conclusion is supported by results showing that both ADP antagonists and ADPase can inhibit the effect of plasmin on platelets. We also demonstrated that pretreatment of platelets with ADP makes the platelets more sensitive to plasmin, and plasmin-induced platelet aggregation is, therefore, observed at lower concentrations where no aggregation occurs in quiescent platelets. In other words, it is thought that ADP potentiates the plasmin-induced aggregation. The effect of ADP was inhibited by N(6)-[2-(methylthio)-ethyl]-2-(3,3, 3-trifluoropropyl)thio-5'-adenylic acid, monoanhydride with dichloromethylenebisphosphonic acid (AR-C69931), a selective antagonist for the P2T(AC) subtype of P2 receptor, but not by the P2Y1 receptor-selective antagonist adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS). The P2X1 receptor agonist alpha, beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) did not mimic the action of ADP. These data indicate that ADP potentiates plasmin-induced platelet aggregation via the P2T(AC) receptor. In addition, epinephrine, a typical G(i) agonist against platelets, could potentiate the plasmin-induced platelet aggregation, suggesting that the signal via the G(i) protein is involved in potentiating the plasmin-induced platelet aggregation, ADP is secreted from platelet granules, and concomitantly works in conjunction with plasmin in a P2T(AC) receptor-mediated manner.

Adenosine Diphosphate↗
PubMed2000

Possible role of hepatocyte growth factor/scatter factor and activin A produced by the target organ in liver metastasis.

The molecular mechanism of organ-specific metastasis to the liver remains largely unknown. However, it is conceivable that paracrine growth factors produced by a target organ induce migration and proliferation of malignant cells to that organ, and this is the cause of organ-specific metastasis. In this study, we investigated the effect of hepatocyte growth factor/scatter factor (HGF/SF) and activin A, which are known to be produced by the liver, on the motility and growth of liver-metastatic cell line FBJ-LL. HGF/SF and activin A induced motility synergistically, but they did not affect the proliferation of FBJ-LL cells. Expression of the HGF/SF receptor, the c-met gene, and the activin-receptor type IA, type IB, and type IIA genes in FBJ-LL cells was detected by reverse transcription polymerase chain reaction. These findings suggest that both HGF/SF and activin A promote organ-specific metastasis to the liver by induction of migration through their specific receptors on liver-metastatic cells.

Activin Receptors, Type I↗
PubMed2000

Long-term replication of Epstein-Barr virus-derived episomal vectors in the rodent cells.

Plasmids containing the origin of replication, oriP, of the Epstein-Barr virus (EBV) and EBV nuclear antigen-1 genes replicate extrachromosomally in primate cells. However, these plasmids have been believed not to replicate in rodent cells. We demonstrate here that these plasmids can replicate in some types of rodent cells over a long period. This result should offer not only the new insight into the mechanisms of species-specific replication of EBV, but also the possibility that an EBV-based vector can be used for gene transfer experiments in non-primate cells and an animal experiment regarding human gene therapy.

3T3 Cells↗
PubMed2000

Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini.

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.

Amylases↗
PubMed2000

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells.

We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.

3T3 Cells↗
PubMed2000

Paradoxical actions of the NO donor SIN-I on single laryngeal motoneurons in the nucleus ambiguus.

In order to study the pharmacological action of the NO donor SIN-1 upon laryngeal motoneurons, we focally iontophoresed SIN-1 onto single motoneurons identified by antidromic stimulation of the recurrent nerve. SIN-1 produced opposing effects on motoneuron activities. Of 34 identified motoneurons, 38.2% showed a statistically significant decrease (decreasing group), while 29.4% showed an increase in their antidromic response to SIN-1 application (increasing group), respectively. The remaining neurons did not show any change (no change group). When the antidromic spike amplitude during the precontrol period was compared between the three groups, both the increasing and decreasing groups exhibited larger antidromic spikes than those measured in the no change group. The results are discussed in terms of dual pharmacological actions of NO.

Action Potentials↗
PubMed2000

Site-specific integration of adeno-associated virus-based plasmid vectors in lipofected HeLa cells.

Adeno-associated virus (AAV) integrates specifically into a site (AAVS1) on human chromosome 19q13.3-qter. Similarly, there is accumulating evidence that this site-specific integration occurs by transfection of AAV-based plasmid vectors. In order to further define the process of plasmid integration events, we constructed some AAV plasmids, introduced them into HeLa cells by lipofection, and isolated chromosomal integrants. One of such plasmids, pTH-5, contained the rep and neomycin-resistant (neo(r)) genes flanked by the 5'- and 3'-inverted terminal repeats of AAV and the hygromycin-resistant (hyg(r)) gene located in the plasmid backbone. Southern blot analysis revealed that among 36 G418-resistant (G418(r)) clones isolated, 22 (61%) showed site-specific integration into AAVS1. Further structural and functional analyses on the expression of the hyg(r) gene in the site-specific clones and the LacZ gene in clones generated with plasmid pTH-2 indicated that, together with the AAV sequence, the plasmid backbone was integrated into the AAVS1 site and thus the neo(r) and hyg(r) genes remained linked at high frequencies in the targeted integrants compared with random integrants. Sequence analysis of integration junctions between pTH-5 and AAVS1 revealed that the junctions occurred in the p5 promoter region of the plasmid while mainly in the partial cDNA coding region of the AAVS1 site. We also found that plasmid pTH-1 linearized in the backbone before lipofection gave a significantly lower frequency of site-specific integration (26%) than the circular form (60%). This finding may support the involvement of the double-stranded, circular form of infected AAV in the integration process. Our results may help to understand the process and mechanism of site-specific integration of lipofected AAV plasmid vectors.

Cation Exchange Resins↗
PubMed2000

Comparative studies on the analysis of glycosylation heterogeneity of sialic acid-containing glycoproteins using capillary electrophoresis.

Comparative studies concerning glycoform analysis of sialoglycoproteins by capillary electrophoresis were performed using a few separation modes hitherto reported. Glycoprotein samples examined in the present study were successfully separated to their respective glycoforms using surface-modified capillaries commercially available for capillary gas chromatography in the running buffer near their isoelectric points. The analysis times were less than 50 min and reproducibilities in migration times were excellent (less than 2.0% RSD for both run-to-run and day-to-day analyses). We present a method for the glycoform analysis of alpha1-acid glycoprotein in sera by simple pre-treatment as an application. The present technique will become one of the general methods for the evaluation of glycosylation heterogeneity of commercially available glycoprotein drugs.

Electrophoresis, Capillary↗
PubMed2000

Comparison of characteristics between patients with GB virus C/hepatitis G virus (GBV-C/HGV) RNA and those with GBV-C/HGV E2-antibody in patients with hemophilia.

We compared characteristics of patients with GB virus C/hepatitis G virus (GBV-C/HGV) RNA to those of patients with GBV-C/HGV E2-antibody. GBV-C/HGV RNA and GBV-C/HGV antibody were assayed in 83 persons with hemophilia using a reverse transcription-polymerase chain reaction and an enzyme-linked immunosorbent assay, respectively. GBV-C/HGV RNA was detected in 19 (22.9%) patients and GBV-C/HGV antibody was detected in 17 (20.5%). The background characteristics between the patient groups did not differ with respect to age, severity of hemophilia based on the frequency of use of blood product, and both the initial age at the first use and years since the first use of blood products. There were no differences in coinfection with hepatitis C virus (HCV) and/or human immunodeficiency virus, except that infection with HCV subtype 1a was more prevalent in patients with GBV-C/HGV RNA (P = 0.0229). Human lymphocyte antigen (HLA) typing was conducted in 18 patients with GBV-C/HGV RNA and 15 patients with GBV-C/HGV E2-antibody; 13 of the patients with GBV-C/HGV antibody had either HLA DQ7, DR15, or DR8, whereas only 4 of the patients with GBV-C/HGV RNA did (P < 0. 001). It is concluded that the presumed age at the time of GBV-C/HGV infection, the frequency of exposure to GBV-C/HGV, and the time since the GBV-C/HGV infection were not associated with recovery from infection with GBV-C/HGV. Coinfection with HCV subtype 1a may be related to persistent GBV-C/HGV viremia, whereas HLA DQ7, DR15, or DR8 may be related to the clearance of GBV-C/HGV after infection.

Adolescent↗
PubMed2000

IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector.

The internal ribosome entry site (IRES) has been widely used to coexpress heterologous gene products by a message from a single promoter. However, little is known about the efficiency of IRES-dependent second gene expression in comparison with that of first gene expression. This study was undertaken to characterize the relative expression of IRES-dependent second gene in a bicistronic vector, which was derived from the 5' untranslated regions of the encephalomyocarditis virus (EMCV). IRES-dependent second gene expression was compared with cap-dependent first gene expression in several cultured cell lines and in mouse liver in vivo. The expression of the IRES-dependent second gene ranged from 6 to 100% (in most cases between 20 and 50%) that of the first gene. Second gene expression in a plasmid without the IRES was 0.1-0.8% (with some exceptions) that of the first gene. These findings have important implications for the use of IRES, i.e., care should be taken regarding the decreased capacity of IRES-dependent downstream gene expression as well as in determining which gene should be positioned as the first or second gene in a bicistronic vector.

5' Untranslated Regions↗
PubMed2000

Oral uracil and Ftorafur plus leucovorin: pharmacokinetics and toxicity in patients with metastatic cancer.

PURPOSE: To assess the pharmacokinetics of Ftorafur (tegafur, FT), 5-fluorouracil (5-FU), and uracil in 31 cancer patients who were enrolled in phase I studies of oral uracil and FT (UFT). The correlation between pharmacokinetic parameters and toxic effects of UFT was evaluated. METHODS: Uracil and FT were orally administered in a 4:1 molar ratio at FT doses of 200-400 mg/m2 per day. Patients also received leucovorin at 150 mg/day. Daily doses were divided into three doses and administered at 8-h intervals for 28 consecutive days. Plasma FT concentrations were measured by high-performance liquid chromatography, and plasma 5-FU and uracil concentrations were determined using gas chromatography-mass spectrometry. National Institutes of Health Common Toxicity Criteria were used for assessment of toxicity. RESULTS: The concentrations of FT, 5-FU, and uracil showed wide interpatient variations. Maximum plasma concentrations (Cp(max)) of all three compounds were achieved in 0.3 to 4.0 h. At the various study doses, the terminal half-life (t 1/2beta) of FT ranged from 3.9 to 5.9 h, the area under the concentration-versus-time curve (AUC0-6h) ranged from 16,220 to 52,446 (ng/ml)h, the total clearance (ClT) ranged from 100 to 175 ml/min, and the steady-state volume of distribution (Vd(ss)) ranged from 18.3 to 28.7 l. The 5-FU generated from FT had an apparent distribution half-life (t 1/2alpha) and an apparent elimination half-life (t 1/2beta) of 0.3-1.3 h and 4.9-7.0 h, respectively. The AUC0-6h of 5-FU ranged from 120 to 325 (ng/ml)h. Uracil had a t 1/2alpha of 0.2-0.5 h and the level quickly returned to the endogenous level. The AUC0-6h for uracil ranged from 605 to 3764 (ng/ml)h, the ClT ranged from 3225 to 7748 ml/min, and the Vd(ss) ranged from 341 to 1354 l. The Cp(max) and AUC0-6h of both FT and uracil were significantly correlated with FT doses (P-values of 0.0244 and 0.0112) and with uracil doses (P-values of 0.0346 and 0.0083), respectively. In addition to interpatient variations, intrapatient variations were also observed in six patients who had pharmacology studies done on days 1 and 26+/-2 at the same study dose. We found that the repeated treatment with UFT caused cumulative increases in the values of Cp(max), Ctrough, and AUC0-6h of FT and 5-FU. The major toxic effects observed were diarrhea and nausea and vomiting. The occurrence of these toxic effects correlated significantly with the Cp(max) and AUC0-6h of 5-FU. CONCLUSIONS: The pharmacology studies showed that FT and uracil were readily absorbed orally and that FT was rapidly converted to 5-FU. The preliminary findings suggest that determination of plasma levels of 5-FU after oral administration of UFT may help predict subsequent toxic effects.

Tegafur↗
PubMed2000

Insulin alters cardiac muscle creatine kinase activity.

The expression of mRNAs of creatine kinase (CK)-B and CK-M has been show to be affected by insulin, and myocardial CK-MB activity is suppressed in insulin-deficient rats. We investigated the dose-related effect of insulin on CK-MB activity in cardiac and skeletal muscles. Male Wistar rats were divided into three groups: (1) control group, (2) diabetic group, injected with 65 mg/kg streptozotocin for 4 weeks, and (3) atenolol group, administered 30 mg/kg per day atenolol. Each group was further divided into three subgroups and administered either saline, or 20 (Ins 20) or 30 (Ins 30) U/kg per day insulin. After 3 weeks, the isoenzyme activity of CK and lactate dehydrogenase (LDH) in the left ventricle of the heart (LV) and the major pectoral muscle (PM) was measured. Serum insulin increased and plasma glucose decreased in Ins 20 and Ins 30, dose-dependently, in all three groups. Both CK-MB and -BB activity in LV increased dose-dependently with insulin treatment in the control, diabetes, and atenolol groups, although these changes did not occur in skeletal muscles. CK-MB in LV correlated with the serum insulin levels in all rats, while no correlation was found in the skeletal muscles. These findings suggest that insulin possibly regulates the distribution of CK isoenzymes in rat heart muscle, and that the effect of insulin is not due to the sympathetic drive induced by hypoglycemia.

Adrenergic beta-Antagonists↗
PubMed2000

Monosynaptic inputs from the nucleus tractus solitarii to the laryngeal motoneurons in the nucleus ambiguus of the rat.

The cricothyroid (CT) and the posterior cricoarytenoid (PCA) muscles in the larynx are activated by the laryngeal motoneurons located within the nucleus ambiguus; these motoneurons receive the laryngeal sensory information from the nucleus tractus solitarii (NTS) during respiration and swallowing. We investigated whether the neurons in the NTS projected directly to the laryngeal motoneurons, and what is the synaptic organization of their nerve terminals on the laryngeal motoneurons using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) was injected into the CT muscle or the PCA muscle, the anterogradely WGA-HRP-labeled terminals from the NTS were found to directly contact the retrogradely CT-HRP-labeled dendrites and soma of both the CT and the PCA motoneurons. The labeled NTS terminals comprised about 4% of the axosomatic terminals in a section through the CT motoneurons, and about 9% on both the small (PCA-A) and the large (PCA-B) PCA motoneurons. The number of labeled axosomatic terminals containing round vesicles and making asymmetric synaptic contacts (Gray's type I) was almost equal to that of the labeled terminals containing pleomorphic vesicles and making symmetric synaptic contacts (Gray's type II) on the CT motoneurons. The labeled axosomatic terminals were mostly Gray's type II on the PCA-A motoneurons, while the majority of them were Gray's type I on the PCA-B motoneurons. These results indicate that the laryngeal CT and PCA motoneurons receive a few direct excitatory and inhibitory inputs from the neurons in the NTS.

Animals↗
PubMed2000

Nationwide epidemiological survey of chronic pancreatitis in Japan.

The aim of this study was to estimate the number of patients treated for chronic pancreatitis in 1994 in Japan and to explore the clinico-epidemiological features of chronic pancreatitis. Two surveys were conducted. Stratified random sampling was used to select departments in which patients with chronic pancreatitis were treated, and two different questionnaires were administered to obtain relevant information. From the first survey, the total number of patients treated for chronic pancreatitis in Japan in the year 1994 was estimated as 32,000 (95% confidence interval, 25,000-39,000). Clinico-epidemiological features, based on the 2,523 patients reported from the second survey, were subsequently clarified. The sex ratio (male/female) of the patients was 3.5. Alcoholic pancreatitis was the most common type in males (68.5%), and idiopathic pancreatitis in females (69.6%). Compared with the findings in the last survey in 1985, the proportion of patients with alcoholic pancreatitis has decreased slightly, from 58.7% to 55.5%, while that of idiopathic chronic pancreatitis has increased in both males and females. Patients diagnosed by advanced techniques such as computed tomography (CT) and endoscopic retrograde cholangiopancreatography (ERCP) accounted for 68.1% of the total. The number of patients with chronic pancreatitis treated in 1994 in Japan, was estimated as 32,000, with an overall prevalence rate of 45.4 per 100,000 population in males and 12.4 per 100,000 population in females.

Adolescent↗
PubMed2000

Molecular cloning of a novel human gene (SIRP-B2) which encodes a new member of the SIRP/SHPS-1 protein family.

A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1,735 base pairs and has a predicted open reading frame (ORF) encoding 354 amino acids. It possesses a putative signal sequence, a long extracellular domain, a transmembrane region, a short intracellular domain, and no catalytic domain, which is highly homologous to signal-regulatory protein (SIRP)-beta suggesting that it seems to be a new member of the SIRP family. Polymerase chain reaction (PCR)-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 20p13 near the marker D20S906.

Amino Acid Sequence↗
PubMed2000

Transpapillary biopsy in gallbladder disease.

BACKGROUND: Endoscopic biopsy is routine in gastrointestinal disease; similarly, histopathologic diagnosis is desirable in gallbladder disease. In this study we examined the clinical usefulness and the problems associated with transpapillary gallbladder biopsy. METHODS: Transpapillary gallbladder biopsy was attempted in 9 patients with gallbladder disease. After inserting a catheter sheath into the gallbladder using a guidewire via the transpapillary route without sphincterotomy, we inserted a biopsy forceps into the lumen of the sheath up to the gallbladder lumen and obtained specimens. RESULTS: We could obtain sufficient specimens for histopathologic diagnosis in 8 of 9 cases (88.9%). Of the 8 successful cases, targeted specimens were obtained in 7 (87.5%). Diagnostic accuracy with respect to malignant versus benign disease was 100% (2 of 2) and 83.3% (5 of 6), respectively. There were no complications. CONCLUSION: Transpapillary gallbladder biopsy is a clinically useful technique because it facilitates histopathologic diagnosis and therefore guides the choice of therapy.

Biopsy↗