Biomedical subjects

T Tsuchiya

Publications and source records attributed to T Tsuchiya.

At least 271 records · Page 15Linked to original sources

PubMed1996

Cloning and sequencing of the gene for Na+/H+ antiporter of Vibrio parahaemolyticus.

A gene encoding an Na+/H+ antiporter was cloned from vibrio parahaemolyticus into the plasmid pBR322 and expressed in Escherichia coli cells. The gene enabled mutant E. coli cells to grow in the presence of 0.2 M NaCl (or 10 mM LiCl). These cells were originally unable to grow under such conditions because of the lack of major Na+(Li+)/H+ antiporters. We detected Na+/H+ antiporter activity due to the gene in membrane vesicles. The gene was sequenced and the deduced amino acid sequence was found to be 72% identical to the NhaB Na+/H+ antiporter of E. coli.

Amino Acid Sequence↗

PubMed1996

Sequence of a Na+/glucose symporter gene and its flanking regions of Vibrio parahaemolyticus.

The nucleotide sequence of an approximately 6 kbp segment of chromosomal DNA of Vibrio parahaemolyticus was determined. The nucleotide sequence revealed four open reading frames (ORFs) in this region. Hydropathy profiles of the deduced amino acid sequence of the ORFs indicate that ORF1 encodes a hydrophobic polypeptide with typical characteristics of a membrane transport protein. All other ORFs encode hydrophilic polypeptides. ORF1 showed significant amino acid sequence similarity to proteins of the SGLT (Na+/glucose symporter) family, and the amino acid sequence of ORF4 showed very high similarity to several bacterial transcriptional repressor proteins (GalR-LacI family). We observed elevated glucose transport activity in cells harboring a plasmid carrying the DNA region corresponding to ORF1, and the glucose transport was greatly stimulated by Na+. Thus, we believe that ORF1 encodes a Na+/glucose symporter.

Amino Acid Sequence↗

PubMed1996

Immobilization-induced stress activates neuronal nitric oxide synthase (nNOS) mRNA and protein in hypothalamic-pituitary-adrenal axis in rats.

The purpose of this study was to determine whether immobilization stress can cause changes in the enzyme activity and gene expression of neuronal nitric oxide synthase (nNOS) in the hypothalamus, pituitary, and adrenal gland in rats. NOS enzyme activity was measured as the rate of [3H]arginine conversion to citrulline, and the level of nNOS mRNA signal was determined using in situ hybridization and image analysis. NOS-positive cells were also visualized using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) histochemistry and by immunohistochemistry using an anti-nNOS antibody. A significant increase of NOS enzyme activity in the anterior pituitary, adrenal cortex, and adrenal medulla (1.5-, 3.5-, and 2.5-fold) was observed in the stressed animals (immobilization of 6 h) as compared to non-stressed control rats. Up-regulation of nNOS mRNA expression in anterior pituitary and adrenal cortex was already detectable after stress for 2 h with 1.5- and 2-fold increase, respectively. The nNOS mRNA signals in hypothalamic paraventricular nucleus (PVN) significantly increased after the stress for 6 h. This increase in NOS enzyme activity was confirmed using NADPH-diaphorase staining and immunostaining in the PVN and adrenal cortex. An increase of NOS enzyme activity in adrenal medulla after immobilization for 6 h posited by far longer than in the adrenal cortex and anterior pituitary. The present findings suggest that psychological and/or physiological stress causes NO release in hypothalamic-pituitary-adrenal (HPA) axis and in sympatho-adrenal system. It is suggested that NO may modulate a stress-induced activation of the HPA axis and the sympatho-adrenal medullary system. The different duration of stress-induced NOS activity in HPA axis and the adrenal medulla may suggest NO synthesis is controlled by separate mechanism in the two HPA and the sympatho-adrenal systems.

Animals↗

PubMed1996

Heart period variability in patients with variant angina.

To clarify how cardiac autonomic control is affected in variant angina, we analyzed heart period variability in 35 patients with variant angina and in 19 control subjects. Patients with variant angina were divided into 1-vessel (group S, n = 17) and multivessel spasm groups (group M, n = 18) according to the site(s) of ST elevation on the electrocardiogram during attacks. The 24-hour Holter electrocardiogram recorded 6 +/- 3 days after the treatment with calcium antagonist was analyzed to avoid the possible influence of spontaneous attacks. In 5 group M patients, the electrocardiogram recorded 1 month after the treatment was also analyzed. There was no difference in the number of spontaneous attacks between groups S and M. The standard deviation of all normal RR intervals (SDNN) and the percentage of differences between adjacent normal RR intervals >50 (pNN50) in variant angina were slightly but significantly lower than those in controls. There were no differences in other indexes between variant angina and controls. When the data were analyzed separately in groups S and M, averaged RR intervals (MN), SDNN, pNN50, high-frequency power, and low-frequency power in group M were significantly lower than those in group S and controls, and the ratio of low- to high-frequency power in group M was significantly higher than that in group S and controls. There was no difference in any index between group S and controls. All abnormal indexes in group M recovered to levels similar to those in controls 1 month after the treatment. In conclusion, depressed cardiac vagal control and sympathetic-dominant sympathovagal interaction were present in patients with variant angina, especially in those with multivessel spasm.

Aged↗

PubMed1996

A 5'-(trifluoromethyl)anthracycline glycoside: synthesis of antitumor-active 7-O-(2,6-dideoxy-6,6,6-trifluoro-alpha-L-lyxo-hexopyranosyl) adriamycinone.

7-O-(2,6-Dideoxy-6,6,6-trifluoro-alpha-L-lyxo-hexopyranosyl)adriam ycinone (3), whose substituent at C-5' is a lipophilic trifluoromethyl group, has been prepared by coupling of 3,4-di-O-acetyl-2,6-dideoxy-6,6,6-trifluoro-alpha-L-lyxo-hexopyran osyl bromide (20) with 14-O-(tert-butyldimethylsilyl)adriamycinone under the Koenigs-Knorr conditions. The key step in this synthesis was the C-trifluoromethylation of 5-O-acetyl-2,3-di-O-benzyl-4-deoxy-aldehydo-L-erythro-pentose (10), derived from D-lyxose in 10 steps, with (trifluoromethyl)trimethylsilane in the presence of tetrabutylammonium fluoride, whereupon 1,1,1-trifluoro-L-arabino-hexitol (11) was obtained along with its 2-epimer. The synthetic product 3 showed remarkable antitumor activity in vivo in a low dose range compared to the analogs including doxorubicin. The fact may be ascribed to the presence of a trifluoromethyl group at C-5', suggesting the importance of the group in view of biological activity.

Animals↗

PubMed1996

Localization and synthesis of acetylcholine in human leukemic T cell lines.

In order to clarify the origin of acetylcholine (ACh) in human blood, we measured the content and synthesis activity of ACh in several human leukemic cell lines. The intracellular ACh content determined by a specific and sensitive radioimmunoassay in the human leukemic T cell lines, HSB-2, MOLT-3, and CEM, was 79.6, 36.2, and 9.5 pmol/10(6) cells, respectively. These values were 9-70-fold higher than those of other cell lines, including a helper T cell line, Jurkat. Stimulation of HSB-2 and MOLT-3 by phytohemagglutinin (PHA) increased both the intracellular content and release of ACh into the culture medium, but did not influence the intracellular content and release of ACh in CEM. ACh synthesis activity was found in all the T cell lines tested. Bromoacetylcholine (100 microM), a choline acetyltransferase (ChAT) inhibitor, and bromoacetyl-L-carnitine (100 microM), a carnitine acetyltransferase (CarAT) inhibitor, decreased ACh-synthesizing activity in MOLT-3, and HSB-2 and CEM, by about 50% and 30%, respectively, indicating that both ChAT, and to a lesser extent CarAt, are involved in ACh synthesis in T cells. These results suggest that T lymphocytes have the potential to synthesize and release ACh, which may play a role in regulating T cell-dependent immune responses.

Acetylcholine↗

PubMed1996

Properties of a Na+/galactose (glucose) symport system in Vibrio parahaemolyticus.

We have investigated galactose transport in a mutant strain of Vibrio parahaemolyticus that lacks a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system) and a trehalose-PTS. Cells of the V. parahaemolyticus actively transported D-galactose and Na+ greatly stimulated the transport. Maximum stimulation of D-galactose transport activity was observed at 10mM NaCl, and Na+ could be replaced with Li+. Addition of galactose to the cell suspension under anaerobic conditions elicited Na+ uptake. Therefore, we conclude that this organism accomplishes galactose transport by a Na+/solute symport mechanism. Judging from inhibition results, D-galactose, D-glucose and to a lesser extent alpha-D-fucose are substrates of this transport system. The Na+/galactose symport system exhibited a high affinity for D-galactose (Km: 40 microM) and showed a relatively lower affinity for D-glucose (Km: 420 microM), but the maximum velocities for galactose and glucose transport were almost same (about 52 nmol/min per mg protein). The Na+/D-galactose symport system was induced by either D-galactose or alpha-D-fucose, and repressed by D-glucose.

Biological Transport↗

PubMed1996

Characteristics of slow conduction zone demonstrated during entrainment of idiopathic ventricular tachycardia of left ventricular origin.

Idiopathic ventricular tachycardia (VT) with the right bundle branch block pattern and left-axis deviation has been shown to be due to reentry, but the property of the slow conduction zone within the reentry circuit is little understood. In 7 patients (mean VT cycle length [CL]: 361 +/- 49 ms), rapid pacing from the right ventricular outflow tract was performed during VT while recording electrograms at the early activation site in the left ventricle and at the right ventricular apex; also, conduction times from the pacing site to these recording sits (St-A and St-B intervals, respectively) were measured. Both constant fusion (except for the last paced beat) and progressive fusion were seen in all patients, indicating VT entrainment. The left ventricular site was captured orthodromically with an St-A of 394 +/- 57 ms at the pacing CL of 351 +/- 47 ms during entrainment, while the right ventricular apex was captured directly with an St-B interval of 63 +/- 19 ms. The St-A interval was gradually prolonged with the shortening of the pacing CL, whereas the St-B interval remained unchanged. VT was interrupted in all patients at the pacing CL of 279 +/- 39 ms. The effects of intravenous lidocaine (1 mg/kg) and verapamil (1 mg) were examined in 5 and 7 patients, respectively. Neither drug terminated VT but the VT-CL was increased to 369 +/- 57 ms after lidocaine (p <0.05) and to 413 +/- 69 ms after verapamil (p <0.05) (p <0.05 vs after lidocaine). The St-A interval was significantly increased after lidocaine (p <0.05) and after verapamil (p <0.05), while the St-B interval remained unchanged. A significant correlation between changes in St-A interval and VT-CL after verapamil was noted (p <0.001). In conclusion, the slow conduction zone of this VT shows tachycardia-dependent conduction delay, and the mechanism of this slow conduction involves mainly calcium channel-dependent conduction and partly depressed sodium channel-dependent conduction.

Adolescent↗

PubMed1996

Reconstitution of the F1-ATPase activity from purified alpha, beta, gamma and delta or epsilon subunits with glutathione S-transferase fused at their amino termini.

Systems for overexpression and purification of active alpha, beta and gamma subunits of Escherichia coli H(+)-ATPase were established. The alpha and beta subunits recovered as soluble form were purified by hydroxyapatite column chromatography. Since the gamma subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate. By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active gamma subunit for reconstitution of the F1-ATPase activity with the purified alpha and beta subunit was obtained. The delta and epsilon subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography. These fused proteins (delta-GST and epsilon-GST) were incubated with the purified alpha, beta and gamma subunits and applied to affinity chromatography. The alpha beta gamma delta-GST and alpha beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins. These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the active complex. The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly. The delta-GST bound the gamma subunit significantly, and the alpha and beta subunits very weakly.

Base Sequence↗

PubMed1996

Strain of passive elements during force enhancement by stretch in frog muscle fibres.

1. The force enhancement during and after stretch (0.15 micron per sarcomere) was studied during fused tetani of single fibres isolated from the anterior tibialis muscle of Rana temporaria (0.5-3.6 degrees C; sarcomere length, 2.05-2.65 microns). Changes in length were recorded simultaneously from the fibre as a whole (puller movement) and from marked segments (approximately 0.5 mm in length) of the same fibre. 2. The residual force enhancement after stretch (recorded at the end of a long tetanus) was found to be linearly related to the slow component of tension rise during the stretch ramp. 3. The fibres were released to shorten against a very small load at different times after stretch (load clamp). The shortening records derived after a preceding stretch exhibited a larger and steeper initial transient than that recorded in an isometric tetanus without stretch. The excess length change (LS; nanometres per half-sarcomere) recorded during the initial transient increased with the amplitude of stretch and was linearly related to the force enhancement produced by the stretch (FE; % of maximum tetanic tension) according to the following regression: LS = 0.200 FE + 8.65 (P < 0.001). The length changes recorded from the whole fibre agreed well with measurements from individual segments. 4. Slack-test measurements confirmed the existence of a large initial transient phase when the fibre was released to shorten after a preceding stretch. The excess length change determined from the slack tests agreed closely with the values derived from load-clamp recordings. 5. The results support the view that stretching a muscle fibre during tetanus leads to strain of elastic elements and, presumably, to variation of filament overlap due to non-uniform distribution of the length change within the fibre volume. Regions with greater filament overlap are likely to generate the long-lasting extra force referred to as 'residual force enhancement after stretch'. The elastic elements recruited during stretch can be presumed to play an essential part in this process by supporting regions in which the filament overlap has been reduced during the stretch ramp. Recoil of these elastic elements is responsible for the excess length change that is recorded during the initial transient after release as described under point 3.

Animals↗

PubMed1996

Studies on the mechanisms of tumorigenesis induced by polyetherurethanes in rats: leachable and biodegradable oligomers involving the diphenyl carbamate structure acted as an initiator on the transformation of Balb 3T3 cells.

The tumor-initiating activities of the methanol extracts of polyetherurethanes (PEUs) were first detected in the presence of 12-0-tetradecanyl-phorbol-13-acetate (TPA) using Balb 3T3 transformation assay. A model hard segment of PEUs, 4,4'-di(ethoxycarboamide) diphenylmethane (MDU), showed initiating activity, while chemical moieties other than the hard segment were shown to be negative in the test. The transformation assay was carried out using glass dishes half coated with two different PEUs, PU4 and PU8. In the presence of TPA, the transforming activities correlated with the tumorigenic potential in the rat implantation study on the coated surface of PEUs, but not on the uncoated glass area. From these results it was concluded that initiation was caused by the hard-segment moiety such as MDU structure derived not only from the leachable extracts but also from the biodegradable substances by the direct interaction of cells with the coated materials.

3T3 Cells↗

PubMed1996

Psoas abscess complicating Crohn's disease: report of two cases.

We report herein the cases of two patients with Crohn's disease complicated by a psoas abscess. The first patient was a 29-year-old man who underwent definitive surgery after acute inflammation had been controlled by nutritional therapy. In the second patient, a 37-year-old man, the abscess required drainage under local anesthesia prior to surgery. Both patients have been free from recurrence of any abdominal symptoms for about 2 years since undergoing surgery. Although psoas abscess is still regarded as a rare complication, with the increasing prevalence of Crohn's disease in Japan it has become one of the most important complications requiring surgical intervention.

Adult↗

PubMed1996

Evaluation of skin sensitization potential of nickel, chromium, titanium and zirconium salts using guinea-pigs and mice.

Contact sensitization capacity of four metal salts, nickel sulphate (NiSO4), potassium dichromate (K2Cr2O7), titanium chloride (TiCl4) and zirconium chloride (ZrCl4), was evaluated using guinea-pigs and mice. In the guinea-pig sensitization tests, we set up an injection concentration to 1% for all chemicals, and changed the challenge concentration. Guinea-pigs were sensitized with NiSO4, K2Cr2O7 and TiCl4. Among the test metal salts, K2Cr2O7 showed the highest sensitization rate and strongest skin reactions. ZrCl4 did not cause any sensitization responses under our experimental conditions. Minimum challenge concentration to cause a skin response was < 0.25% for K2Cr2O7, 0.5% for NiSO4 and 2% for TiCl4, respectively. A sensitive mouse lymph node assay (SLNA) also determined NiSO4 and K2Cr2O7 as a sensitizer. In the SLNA, TiCl4 caused mild lymph node responses, but was classified as a non-sensitizer as well as ZrCl4. Considering these results, the order of sensitization potential was K2Cr2O7 > NiSO4 > ZrCl4. NiSO4- and K2CrO7-sensitized animals did not show any reactions to ZrCl4 and TiCl4. No cross-reaction among these metal salts was found.

Administration, Topical↗

PubMed1996

Epidermal cell proliferative activity assessed by proliferating cell nuclear antigen (PCNA) decreases following immobilization-induced stress in male Syrian hamsters.

The effects of immobilization-induced stress on epidermal cell proliferative activity, assessed by the proliferating cell nuclear antigen (PCNA) positive ratio, were examined in male Syrian hamsters. In non-stressed animals, PCNA-positive cells were identified frequently in the epidermis in pinna, abdomen, dorsum and scrotum. However, in animals stressed for 4 consecutive days (6 h/day) PCNA-positive cells were significantly reduced in all areas. In stressed animals, the plasma levels of cortisol, corticosterone, epinephrine, and norepinephrine, which are inhibitory to epidermal cells, increased, but plasma testosterone, which is stimulatory to epidermal cells, decreased. The present findings suggest that psychological or physiological stress influences proliferative activity, a fundamental cutaneous function, by inducing changes in the neuroendocrine system.

Animals↗

PubMed1996

Marked increases in neuronal nitric oxide synthase (nNOS) mRNA and NADPH-diaphorase histostaining in adrenal cortex after immobilization stress in rats.

The changes of neuronal nitric oxide synthase (nNOS) mRNA expression in the rat adrenal gland following immobilization-induced stress were examined by the in situ hybridization technique. To introduce immobilization stress, the animals were wrapped with flexible wire gauze for 6 h. In the adrenal medulla and cortex, signals for nNOS mRNA were detected. In the adrenal medulla, the difference between non-stressed and stressed animals was not clear. In the adrenal cortex, the expression of nNOS mRNA markedly increased (2.5-fold) in the stressed animals. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) histochemistry also showed an increase of staining in adrenal cortex after stress. This suggests that nitric oxide (NO) is involved in stress-induced activation in adrenal cortex function.

Adrenal Cortex↗

PubMed1996

Quantitative analysis of cutaneous calcitonin gene-related peptide content in response to acute cutaneous mechanical or thermal stimuli and immobilization-induced stress in rats.

The effects of various stimuli on restricted skin areas or immobilization-induced stress on the calcitonin gene-related peptide (CGRP) content in rat skin were examined by radioimmunoassay (RIA) and immunohistochemistry. Various stimuli were delivered to the shaven skin of the medial thigh by pinching, brushing, or contact with a glass tube containing hot (50 degrees C) or ice-water for 2 min. To induce immobilization stress, animals were placed in the prone position and wrapped with flexible wire gauze at room temperature. The cutaneous CGRP content determined by RIA as well as the number of CGRP-immunoreactive nerve fibers of the skin were significantly higher at sites stimulated by pinching or ice-water compared to non-stimulated areas within the same animals. However, after brushing, hot water stimulation or any period (2 min, 30 min, 2 h, 6 h, or 3 days x 6 h) of immobilization stress, no differences in cutaneous CGRP content were observed. Plasma corticosterone levels increased after immobilization stress of 30 min or greater, but plasma CGRP level did not change after any period of immobilization stress. These data suggest that some forms of cutaneous stimulation cause a rapid rise in CGRP content in the skin, while emotional stress does not influence the cutaneous CGRP content.

Animals↗

PubMed1996

HLA class II DNA typing in Buerger's disease.

To analyze the genetic factors involved in the pathogenesis of Buerger's disease, the polymorphisms of HLA class II genes (HLA-DP, -DQ and -DRB) were investigated in 36 patients using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. HLA class I (B54 and B52) and HLA class II (DQ alleles) genes were also typed by polymerase chain reaction-sequence-specific oligonucleotide probe (PCR SSOP) method to confirm alleles. PCR-RFLP typing revealed that the frequencies of HLA class II genes, DRB1*0405, DQB1*0401, DQA1*03 and DPB1*0501, were significantly increased in the patients with Buerger's disease compared with the healthy controls. Further, DNA typing confirmed that HLA-B54 was associated with Buerger's disease. These results suggested that the haplotype of HLA-B54-DRB1*0405-DQB1*0401- DQA1*03-DPB1*0501 was associated with Buerger's disease in Japanese population.

Adult↗

PubMed1996

Decreased connexin32 and a characteristic enzyme phenotype in clofibrate-induced preneoplastic lesions not shared with spontaneously occurring lesions in the rat liver.

Two different types of focal preneoplastic lesions, tentatively named Type I and II lesions, were recognized in the liver of rats chronically treated with clofibrate for 104 weeks. Type I lesions were characterized by mostly negative glucose-6-phosphate dehydrogenase (G6PD) activity (6 out of 10, 60%) and positive expression of succinate dehydrogenase (10 out of 10, 100%), in addition to the previously documented complete lack of expression of glutathione S-transferase, placental form (GST-P) and gamma-glutamyl transpeptidase (GGT). Furthermore, most importantly, Type I lesions exhibited a clear decrease in immunohistochemically demonstrated connexin32 (Cx32) spot counts on their hepatocyte membranes, similarly to nitrosamine-induced lesions. In contrast, Type II lesions, mostly small in size and positively expressing GST-P and/or GGT and G6PD, similarly to their previously reported nitrosamine-induced counterparts, did not exhibit a significant decrease in Cx32 count. In addition, spontaneously occurring lesions, again sharing the same enzyme phenotype, did not show a decrease in Cx32. The results indicate that: (i) a clear distinction between the two lesions, with Type I being involved in clofibrate-induced tumors and Type II being more likely to be spontaneous in nature; (ii) a decrease in Cx32 is closely linked to lesion development and possibly stage of progression, irrespective of the enzyme phenotype and the applied carcinogen; (iii) the unaltered condition of Cx32 may suggest a slow growing or non-progressive nature.

Animals↗