Biomedical subjects

T Tsuchiya

Publications and source records attributed to T Tsuchiya.

At least 217 records · Page 12Linked to original sources

PubMed1998

Molecular cloning, water channel activity and tissue specific expression of two isoforms of radish vacuolar aquaporin.

A major membrane intrinsic protein (VM23) in vacuoles of radish (Raphanus) tap root was investigated. The cDNAs for two isoforms of VM23, gamma- and delta-VM23, encode polypeptides of 253 and 248 amino acids, respectively. gamma- and delta-VM23 correspond to the gamma- and delta-TIP (tonoplast intrinsic protein) of Arabidopsis. The deduced amino acid sequences of the two VM23 isoforms were 60% identical. The amino-terminal sequence of gamma-VM23 showed agreement with the direct sequence of the purified VM23, suggesting that gamma-VM23 is the most abundant molecule among the VM23 isoforms. When mRNAs of gamma- and delta-VM23 were injected into Xenopus oocytes, the osmotic water permeability of oocytes increased 6-fold (60 to 200 microns s-1) of the control oocytes. The transcripts of both isoforms were detected in a high level in growing hypocotyls and young leaves, but delta-VM23 was not detected in seedling roots. Light illumination enhanced the transcription of two genes of VM23 in cotyledons and roots but suppressed their expression in hypocotyls the growth of which was inhibited by light. These findings suggest that the expression of VM23 is tightly related to cell elongation.

Amino Acid Sequence↗

PubMed1998

Slow potential-guided radiofrequency catheter ablation in atrioventricular nodal reentrant tachycardia: characteristics of the potential associated with successful ablation.

To examine the characteristics of Haïssaguerre's slow potential (SP) specific to effective catheter ablation of the slow pathway in AV nodal reentrant tachycardia, the properties of SP and its recording site were analyzed in 52 patients who underwent successful SP-guided ablation. The properties of SP included the ratio of the amplitude of SP to that of atrial potential (A)(SP/A), the SP duration, the interval between His-bundle potential (HP) and SP (HP-SP), the interval between A and SP (A-SP), the interval between SP and ventricular potential (V) (SP-V), and the ratio of A-SP to the interval between A and the V (A-SP/A-V). The SP recording site was determined by the ratio of the amplitude of A to that of V (A/V) and by the relative position of the ablation catheter on X ray (right anterior oblique projection), expressed as the ratio of the distance between the coronary sinus ostium and SP site to that between the coronary sinus ostium and HP recording site (relative SP position). Twenty-eight slow pathways were ablated with a single energy application, while the other 24 required applications > or = 2. In all successful applications, SP/A, SP duration, HP-SP, A-SP, SP-V, A-SP/A-V, A/V, and relative SP position were 51% +/- 25%, 28 +/- 5 ms, -11 +/- 9 ms, 57 +/- 25 ms, 68 +/- 13 ms, 46% +/- 9%, 15% +/- 13%, and 51% +/- 13%, respectively. A significant correlation was observed between the relative SP position and A-SP, and between the relative SP position and A-SP/A-V (r = 0.60 and 0.37, respectively), while it was not between the relative SP position and HP-SP, nor between the relative SP position and SP-V. When the characteristics of SP were comparatively analyzed between the effective and ineffective applications in 24 patients in whom applications > or = 2 were required, there was no difference observed in HP-SP, A-SP, SP-V, A-SP/A-V, and A/V. However, SP/A, SP duration, and the relative SP position in the effective applications were all greater than those in the ineffective ones (56% +/- 20% vs 35% +/- 18%, P < 0.001; 29 +/- 4 vs 26 +/- 5 ms, P < 0.01; and 52% +/- 15% vs 33% +/- 11%, P < 0.001, respectively). These results indicate that SP with an amplitude over a half of A amplitude and recorded at the mid-septum of the tricuspid annulus can be a marker for successful slow pathway ablation. Although the local atrial electrogram appears late as the SP recording site shifts to the lower position, the timing of SP relative to HP and V remained unchanged, suggesting that SP is independent of the local atrial activation.

Action Potentials↗

PubMed1998

NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli.

We found that cells of Vibrio parahaemolyticus possess an energy-dependent efflux system for norfloxacin. We cloned a gene for a putative norfloxacin efflux protein from the chromosomal DNA of V. parahaemolyticus by using an Escherichia coli mutant lacking the major multidrug efflux system AcrAB as the host and sequenced the gene (norM). Cells of E. coli transformed with a plasmid carrying the norM gene showed elevated energy-dependent efflux of norfloxacin. The transformants showed elevated resistance not only to norfloxacin and ciprofloxacin but also to the structurally unrelated compounds ethidium, kanamycin, and streptomycin. These results suggest that this is a multidrug efflux system. The hydropathy pattern of the deduced amino acid sequence of NorM suggested the presence of 12 transmembrane domains. The deduced primary structure of NorM showed 57% identity and 88% similarity with that of a hypothetical E. coli membrane protein, YdhE. No reported drug efflux protein in the sequence databases showed significant sequence similarity with NorM. Thus, NorM seems to be a novel type of multidrug efflux protein. We cloned the ydhE gene from E. coli. Cells of E. coli transformed with the cloned ydhE gene showed elevated resistance to norfloxacin, ciprofloxacin, acriflavine, and tetraphenylphosphonium ion, but not to ethidium, when MICs were measured. Thus, it seems that NorM and YdhE differ somehow in substrate specificity.

Amino Acid Sequence↗

PubMed1998

In vitro synthesis of multicopy single-stranded DNA, using separate primer and template RNAs, by Escherichia coli reverse transcriptase.

A minor population of wild strains of Escherichia coli contains a retron, a retroelement responsible for the synthesis of multicopy single-stranded DNA (msDNA). The retron is a genetic element consisting of the gene for reverse transcriptase (RT) and the msr-msd region under a single promoter. A single RNA transcript from the msr-msd region serves not only as a template but also as a primer for msDNA synthesis. Here, using a cell-free system with purified RT from retron Ec73, we examined whether the reaction can occur in a bimolecular reaction with use of separately expressed msr and msd transcripts. DNA sequencing of the cell-free product revealed that the sequence of the 5'-end region was identical to that of msDNA-Ec73, indicating that the cDNA synthesis was primed from the 2'-OH group of the specific internal G residue of the primer RNA, identical to the branching G residue in the RNA molecule of msDNA-Ec73. The present results raise an intriguing possibility for a role of bacterial retrons in vivo, the possibility that cellular mRNAs can be converted into cDNAs in retron-harboring cells if the mRNAs contain a sequence complementary to the sequence directly upstream of the branching G residue of the msr RNA transcript.

DNA, Bacterial↗

PubMed1998

Mutants of Citrobacter freundii that transport and utilize melibiose.

We have isolated mutants of Citrobacter freundii that can grow on melibiose. Inducible alpha-galactosidase activity and melibiose transport activity were detected in the mutant cells but not in the wild-type cells. We detected a DNA region which hybridized with melB (the gene for the melibiose transporter) DNA of Escherichia coli in the chromosomal DNA of wild-type C. freundii. Protons, but not sodium ions, were found to be the coupling cations for melibiose (and methyl-beta-D-thiogalactoside) transport in the mutant cells.

Biological Transport↗

PubMed1998

A putative multisubunit Na+/H+ antiporter from Staphylococcus aureus.

We cloned several genes encoding an Na+/H+ antiporter of Staphylococcus aureus from chromosomal DNA by using an Escherichia coli mutant, lacking all of the major Na+/H+ antiporters, as the host. E. coli cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na+/H+ antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na+/H+ antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na+/H+ antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na+/H+ antiporter. We observed a large Na+ extrusion activity, driven by respiration in E. coli cells harboring the plasmid carrying the genes. The Na+ extrusion was sensitive to an H+ conductor, supporting the idea that the system is not a respiratory Na+ pump but an Na+/H+ antiporter. Introduction of the plasmid into E. coli mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.

Amino Acid Sequence↗

PubMed1998

Cloning and expression of the gene for the Na+-coupled serine transporter from Escherichia coli and characteristics of the transporter.

We cloned a gene (sstT) for the Na+/serine symporter from the chromosome of Escherichia coli by using a low-copy-number vector and sequenced it. According to the deduced amino acid sequence, the transporter (SstT) consists of 414 amino acid residues. Hydropathy analysis suggested that the SstT protein possesses 9, instead of 12, hydrophobic domains.

Amino Acid Transport Systems↗

PubMed1998

Modification of LC phenotype and suppression of contact hypersensitivity response by stress.

BACKGROUND: Stress is thought to exacerbate a number of diseases, some of which are skin disorders. Epidermal Langerhans' cells play a major role in cutaneous immune reactions. OBJECTIVE: The effects of two types of stress on the cutaneous immune system were to be assessed in mice. METHODS: Mice received stress by immobilization or housing at various population densities. Epidermal sheets were stained for I-A molecules (a member of class II major histocompatibility complex) and analyzed with a confocal-laser- scanning microscope. Contact hypersensitivity reaction to 2,4,6-trinitrochlorobenzene was elicited in mouse ears. RESULTS: The cell density, intensity of I-A expression, and number of dendrites were decreased as the population density increased. Elicitation of contact hypersensitivity was suppressed in mice that received either population or immobilization stress. Increased I-A expression and number of dendrites were observed in adrenalectomized compared to sham-operated mice. The population-dependent suppression of contact hypersensitivity reaction was not observed in adrenalectomized mice. After incubation with serum from mice that received either immobilization stress or population stress, the expression of I-A molecules on a XS52 Langerhans' cell-like cell line was reduced. CONCLUSION: Stress affected the cutaneous immune system. There were indications that adrenergic hormones played a role in the regulation of the system.

Adrenalectomy↗

PubMed1998

pH-dependent growth retardation of Escherichia coli caused by overproduction of Na+/H+ antiporter.

The overproduction of Na+/H+ antiporter NhaA in Escherichia coli caused growth retardation. Quantities and the activity of the antiporter were studied for their causative roles in terms of this retardation. We constructed a series of nhaA-expression plasmids differing in their transcriptional and translational efficiencies. Low-level nhaA expression complemented the defect of an nhaA mutant and allowed the mutant to survive on a high-NaCl or high-LiCl medium. However, when the production of NhaA was strongly induced by the combination of a stronger promoter, an efficient translational initiation signal and a high copy number plasmid, the growth of the cells carrying the plasmid was severely retarded. This growth retardation correlated well with the amount of NhaA protein produced from the plasmids. Surprisingly, the growth retardation caused by overproduction of NhaA was enhanced more extensively at an alkaline pH than at a neutral pH, in which the antiporter activity was stimulated. However, these retardations were also observed for mutant NhaA antiporters without the activity. These results indicated that the growth retardation was due to an overproduction of the antiporter rather than its increased antiporter activity, and also affected by a pH-dependent change in NhaA, possibly its structural change.

Cell Division↗

PubMed1998

Preparation and characterization of everted membrane vesicles from cells of Staphylococcus aureus.

We developed a method for the preparation of everted membrane vesicles from cells of Staphylococcus aureus. The cells were first treated with ampicillin to weaken the peptidoglycan layer, then the cells were passed through a French press cell. The resulting vesicles were roughly 0.1 microm in diameter, judging from electron microscopic observations. We detected fairly high membrane-bound ATPase activity in the membrane vesicles. We observed respiratory-driven quenching of quinacrine fluorescence, which indicates that inward H+ transport took place. These results indicate that the vesicles are everted. We characterized the membrane-bound ATPase. We also detected Na+/H+ antiport, erythromycin/H+ antiport and chloramphenicol/H+ antiport activities in the membranes of S. aureus.

Adenosine Triphosphatases↗

PubMed1998

Alteration in levels of unsaturated fatty acids in mutants of Escherichia coli defective in DNA replication.

We previously reported that mutations in the dnaA gene which encodes the initiator of chromosomal DNA replication in Escherichia coli caused an alteration in the levels of unsaturated fatty acids of phospholipids in membranes. In this study, we examined fatty acid compositions in other mutants which are defective in DNA replication. As in the case of temperature-sensitive dnaA mutants, temperature-sensitive dnaC and dnaE mutants, which have defects in initiation and elongation, respectively, of DNA replication showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) compared with the wild-type strain, especially at high temperatures. On the other hand, temperature-sensitive mutants defective in cellular processes other than DNA replication, such as RNA synthesis and cell division, did not show a lower level of unsaturation of fatty acids compared with the wild-type strain. These results suggest that the inhibition of DNA replication causes a lower level of unsaturation of fatty acids in Escherichia coli cells.

Bacterial Proteins↗

PubMed1998

Decrease of the obese gene expression in bovine subcutaneous adipose tissue by fasting.

The expression of mRNA of leptin, the product of the obese gene, in bovine adipose tissue was analyzed by a lysate RNase protection assay. The mRNA level was significantly decreased by food deprivation for two days and partially recovered after 3 hr of refeeding, indicating that obese gene expression in the ruminant was regulated by feeding.

Adipose Tissue↗

PubMed1998

Evidence for the essential role of myosin subfragment-2 in the ATP-dependent actin-myosin sliding in muscle contraction.

The role of myosin subfragment-2 (myosin S-2) in muscle contraction was studied by using an in vitro motility assay system in which the ATP-dependent sliding between myosin-coated polystyrene beads and actin filament arrays (actin cables) of giant algal cells were recorded under constant external loads provided with a centrifuge microscope. With antibody to myosin S-2 below 0.3 mg/ml, the maximum "isometric" force generated by myosin molecules on the bead decreased markedly, but the unloaded bead-sliding velocity along actin cables did not change appreciably, indicating a decrease in the number of myosin molecules interacting with actin cables. The antibody at 0.3-1.5 mg/ml decreased not only the maximum isometric force, but also the unloaded bead-sliding velocity in a dose-dependent manner. With the antibody at 1.5-3 mg/ml, the beads eventually stopped moving to remain attached to actin cables. These beads could be readily detached from actin cables with very small centrifugal forces, indicating very weak actin-myosin linkages. The antibody had no effect on rigor actin-myosin linkages formed before the antibody application. These results are consistent with the view that myosin S-2 plays an essential role in muscle contraction.

Actins↗

PubMed1998

Troglitazone reduces plasma leptin concentration but increases hunger in NIDDM patients.

OBJECTIVE: Troglitazone, which improves peripheral insulin resistance of experimental diabetic animals and diabetic patients, affects ob gene expression in the adipose tissue of rodents. The present study was undertaken to examine a hypothesis that clinical administration of troglitazone may reduce circulating leptin levels and affect eating behavior in NIDDM patients. RESEARCH DESIGN AND METHODS: Troglitazone was administered at a dosage of 200 mg twice daily for 12 weeks in 20 poorly controlled NIDDM patients. Chronological changes in glycemic control, serum lipids, immunoreactive leptin (IRL) levels, and BMI were measured. Body fat weight was also assessed by bioelectric impedance. RESULTS: Troglitazone significantly decreased fasting plasma glucose, serum immunoreactive insulin, and HbA1c levels. Serum levels of IRL and triglyceride were significantly reduced by troglitazone administered for 4, 8, and 12 weeks. Troglitazone administration significantly increased the BMI in NIDDM patients, and two-thirds of the patients complained of increased hunger after the start of troglitazone administration. CONCLUSIONS: Troglitazone significantly reduces circulating leptin levels at clinical doses. It may affect the eating behavior of poorly controlled NIDDM patients through the improvement of glycemic control and/or the reduction of circulating leptin.

Blood Glucose↗

PubMed1998

Effect of vitamin E on contact sensitization responses induced by 2,4-dinitrochlorobenzene in mice.

The effect of vitamin E on the contact sensitization responses induced in mice by 2,4-dinitrochlorobenzene (DNCB) was studied. Mice were fed a vitamin E-adequate or a vitamin E-deficient diet for 5 weeks. The amounts of thiobarbituric acid-reactive substances in the spleens and draining auricular lymph nodes of mice were decreased by dietary vitamin E. Dietary vitamin E prevented lipid peroxidation in the spleens and lymph nodes of mice. Contact sensitization develops in two phases, induction (sensitization) and elicitation. Following sensitization to DNCB on ears, draining lymph node responses, i.e., lymph node weight, total lymph node cell number and in vitro lymph node cell proliferation as assessed by [3H]methyl thymidine incorporation, were examined. These responses, activated by DNCB, were lower in the mice fed a vitamin E-deficient diet as compared with those of the mice fed a vitamin E-adequate diet. In the elicitation phase, lymphocytes from sensitized mice respond to the antigen and blastogenate in vitro. The blastogenesis of spleen lymphocytes in the DNCB-sensitized mice was decreased by vitamin E deficiency, which was enhanced by exogenously adding vitamin E. It was found that vitamin E deficiency decreases the contact sensitization responses to DNCB in mice, but responses were restored by exogenous vitamin E. In conclusion, vitamin E may participate in the lymphocyte responses to contact allergens through scavenging reactive oxygen species.

Animals↗

PubMed1998

[Pulmonary cryptococcosis exhibiting diffuse multiple nodular shadows].

We report a case of pulmonary cryptococcosis showing diffuse multiple nodular shadows in all lung fields. A 39-year-old woman with no immunological abnormalities was admitted with complaints of cough and sputum. She had experienced measles 4 weeks prior to admission. Chest x-ray films revealed diffuse nodular opacities throughout the lung fields, a finding suggestive of metastatic lung cancer. Detailed examinations, including transbronchial lung biopsy, were not conclusive. A diagnosis of pulmonary cryptococcosis was made on the basis of findings from video-assisted thoracoscopic biopsy. Primary pulmonary cryptococcosis usually appears as a solitary nodule or limited infiltration. Immunologically compromised hosts commonly demonstrate various abnormal shadows, such as the multiple nodular shadows observed in our patient. It has been reported that measles infection can cause temporary immune suppression. Secondary immunodeficiency resulting from the preceding infection with measles could explain the unusual chest x-ray findings in this case.

Adult↗

PubMed1998

[Simultaneous analysis and detection of pesticides in fresh fruits and vegetables by HPLC and GC].

A method was established for simultaneous determination of pesticide residue in fresh fruits and vegetables by HPLC and GC. CH3CN extraction/NaCl partiton method was used in order to recover hydrophilic pesticide such as acephate, methamidophos. Dimethoate and methamidophos from okra and DDVP from strawberry were detected by GC. On the other hand confirmation method by GC and GC/MS was studied for peaks detected by HPLC with UV and/or FL detector. OPP, TBZ, imazalil chlorpyrifos etc. in citrus fruits were detected by the proposed method.

Chromatography, Gas↗

PubMed1998

[Treatment of children with non-Hodgkin's lymphoma with CCLSG NHL 855/890 protocols long-term outcome and incidence of secondary malignancies].

We report here on treatment results of consecutive CCLSG NHL studies (NHL855, 1985-1989; NHL890, 1989-1996). The NHL855 protocol consisted of an induction phase of five drugs (VCR, PRD, CPM, DXR, and high-dose MTX) and a maintenance phase of 7 drugs. The probabilities of EFS at 7 years were 78% (SE, 10%) for the patients with localized disease, and 38% (SE, 7%) for those with advanced disease. In the NHL 890 protocol, the patients were assigned to two different treatment groups according to their histology and received different consolidation therapy; non-lymphoblastic subtype was treated almost identically to NHL855 while LASP and VP-16 were newly added for the lymphoblastic subtype. The 7-year EFS improved to 91% (SE, 6%) for localized disease, and 61% (SE, 6%) for advanced disease. A remarkable improvement was particularly evident for lymphoblastic type with mediastinal mass. Optional trial of high-dose sequential chemotherapy and peripheral blood progenitor cell auto grafting resulted in an unfavorable outcome. The 7-year EFS according to main histological subgroups were as follows: 84% (10%) for large cell type, 67% (11%) for Burkitt's-type, 58% (10%) for lymphoblastic type. Secondary cancer occurred in two of the 163 patients studied. Both patients were AML (M0/M4) and MLL rearrangement was detected in the M4 case.

Adolescent↗