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Biomedical subjects

G Tamura

Publications and source records attributed to G Tamura.

At least 217 records · Page 12Linked to original sources
PubMed1985

Treatment with an ascochlorin derivative, AS-6 increases 45Ca2+ binding on the plasma membrane of adipocytes in db/db mice.

Genetically obese diabetic mice (db/db) have greatly diminished 45Ca2+ binding on the plasma membranes of the adipocytes (45-55%) compared with their lean littermates. Treatment for 1 week with a diet admixture of AS-6 (0.1% in the diet) significantly restored the binding to a level comparable to the lean littermates. The addition of AS-6 in vitro had no effect on the binding, which eliminates the possibility that AS-6 is a Ca2+ ionophore. The results suggest that AS-6 treatment enhances the Ca2+ binding by causing structural alteration(s) in the membranes.

Adipose Tissue↗
PubMed1985

Effect of oral treatment with a new hypoglycemic agent, AS-6, on the metabolic activities of adipocytes in db/db mice: a comparative study.

The mechanism of a new hypoglycemic agent, AS-6, was comparatively studied using the adipocytes from AS-6 treated and untreated genetically obese diabetic mice, db/db. the db/db mice were treated for 1 week with a diet admixture of AS-6 (0.1%). The treatment resulted in the following alterations in metabolic activities; AS-6 treatment increased 125I-insulin binding by 1.4-3.3 fold over the insulin range of 1-1000 microU/ml, the treatment increased the basal activities in 2-deoxyglucose uptake, and in CO2 generation and lipogenesis from U-(14C)-glucose compared with the db/db controls, the treatment partially restored insulin responsiveness in 2-DG uptake and CO2 generation, and 1 mU/ml of insulin greatly stimulated lipogenesis by 5.6 fold above the basal in the control adipocytes while AS-6 treatment changed the lipogenic response less stimulative to the insulin. The results suggest that AS-6 treatment significantly increases insulin binding to the adipocytes associating with an enhancement in glucose metabolism under basal and physiological concentrations of insulin.

Adipose Tissue↗
PubMed1985

The single exposure method for inhalation challenge with allergen.

We carried out inhalation challenge with a single exposure to a high concentration of allergen (the single exposure method) on 92 asthmatic patients. With the use of the 3 Hz forced oscillation technique, we could continuously and graphically estimate total respiratory resistance (Rrs) throughout the test. The subjects inhaled aerosolized house dust allergen (1:5 w/v) for 1 min during tidal breathing. As soon as Rrs reached twice that of baseline value or after 10 min from the start of allergen exposure, subjects inhaled aerosolized bronchodilator for 3 min. To evaluate the Rrs response curve to inhaled allergen, we defined provocation time (PT35-Grs) as the time from the start of allergen exposure to the point at which total respiratory conductance (Grs) decreased 35% from its baseline value and appearance time (Ta) as the time from the start of allergen exposure to the beginning of an increase in Rrs. We presume that Ta may reflect bronchial sensitivity to allergen, since Ta significantly correlated with a unit of allergen causing a decrease in FEV1 of 20% (r = 0.66; p less than 0.05) in the dosimeter method. We demonstrated that the single exposure method is comparable to the dosimeter method in evaluating bronchial responses to inhaled allergen and also demonstrated the reproducibility of bronchial response to inhaled allergen with the single exposure method.

Adolescent↗
PubMed1985

Synthesis and secretion of biologically active mouse interferon-beta using a Bacillus subtilis alpha-amylase secretion vector.

Mouse interferon-beta (IFN-beta) cDNA, whose signal sequence had been removed by BAL 31 digestion, was introduced into a Bacillus subtilis secretion vector constructed by using the promoter and signal sequence of the B. subtilis alpha-amylase gene. The resultant chimeric plasmids were transferred into B. subtilis 207-25. Four kanamycin-resistant transformants were selected by both colony hybridization and a new immunoblot method for secretory proteins. They secrete the proteins which cross-react with sheep anti-mouse IFN-beta serum into the culture medium. One of them expressed a high IFN-beta activity as assayed by the L cell and vesicular stomatitis virus system, while the other three showed weak or little IFN activities. Based on our previous study [Ohmura et al., Nucl. Acids Res. 12 (1984) 5307-5319], it was suggested that the secreted IFN molecules are hybrid proteins in which the NH2-terminal region consists of part of the alpha-amylase signal peptide. Nucleotide sequence analysis revealed that plasmid pTUB502, which expressed high IFN activity, is joined to the mouse IFN-beta gene from the codon position 6 of its mature protein. The other three plasmids, pTUB506, pTUB509, and pTUB519, contain the mouse IFN-beta gene from the codon positions 3, 1, and -5, respectively. The NH2-terminal region of the mouse IFN-beta seems to be closely related to its biological activity.

Animals↗
PubMed1985

Synthesis and secretion of human epidermal growth factor by Escherichia coli.

A synthetic gene for human epidermal growth factor (hEGF) was joined to a sequence encoding the signal peptide of Escherichia coli alkaline phosphatase. This hybrid gene was placed under the control of the alkaline phosphatase gene (phoA) promoter in a recombinant plasmid, which was used to transfect E. coli. The hybrid protein that was expressed in host cells under conditions of phosphate limitation was processed accurately during the secretion process, and mature hEGF was recovered in the periplasmic fraction. On the other hand, no EGF was detected in the periplasmic space when the synthetic hEGF gene was not accompanied by the phoA signal sequence.

Alkaline Phosphatase↗
PubMed1985

Secretion of human interferon-alpha induced by using secretion vectors containing a promoter and signal sequence of alkaline phosphatase gene of Escherichia coli.

We constructed a new vector containing the promoter and the signal sequence of E. coli phoA gene, the structural gene for the periplasmic alkaline phosphatase. One of the most useful characteristics of this vector is the unique HindIII restriction site located just at the end of the phoA signal sequence. This restriction site was generated by oligonucleotide-directed site-specific mutagenesis without changing the amino acid sequence of the signal peptide. Any kind of foreign structural gene can be easily inserted into the HindIII site by using synthetic oligonucleotides to construct a hybrid gene which has neither an extra sequence nor a deletion between the phoA signal sequence and the foreign structural gene. Human alpha-interferon gene was inserted into this HindIII site. When this hybrid gene was expressed under the control of the phoA promoter region, a low but significant activity was recovered in the cold water wash of the cells after an osmotic shock procedure.

Alkaline Phosphatase↗
PubMed1985

Transfer of DNA killer plasmids from Kluyveromyces lactis to Kluyveromyces fragilis and Candida pseudotropicalis.

Killer plasmids pGKL1 and pGKL2 of double-stranded linear DNAs were transferred from Kluyveromyces lactis to strains of Kluyveromyces fragilis and Candida pseudotropicalis. The resultant killer strains produced 17-fold and 6-fold larger amounts of killer toxin than K. lactis did, respectively. The killer toxin produced by each species appeared to be a glycoprotein.

Bacterial Toxins↗
PubMed1985

Measurement of SRS-A activities in plasma of asthmatic patients.

Ethanol extracts of arterial blood from 20 of 29 asthmatic patients during attacks produced sustained contractions of the guinea pig ileum. These contractions were inhibited by FPL 55712, a specific antagonist of slow reacting substance of anaphylaxis (SRS-A). SRS-A activity was significantly higher during attack than during remission. Reverse-phase high pressure liquid chromatography (HPLC) was performed on two extracts which had shown high SRS-A activity with bioassay. One extract showed double peaks, which had retention times identical to those of leukotriene (LT) C4 and LTD4. The other extract showed a single peak, which had a retention time identical to that of synthetic LTC4.

Adult↗
PubMed1985

The participation of the subepithelial airway receptor in the bronchoconstriction of monkeys.

In order to elucidate the mechanism of the bronchial response of monkeys to constrictive stimuli, the effects of pretreatment with aerosolized lidocaine, atropine, and isoproterenol were studied. Treatment with aerosolized lidocaine and atropine not only extinguished the cough reflex, but also decreased bronchial susceptibility to aerosolized methacholine. Moreover, the pretreatment with lidocaine blocked the histamine challenge, but atropine did not. The aerosolized lidocaine decreased the bronchoconstriction induced with methacholine, but not with histamine. Pretreatment with over 1% of isoproterenol did not extinguish the cough reflex. However, this dose of isoproterenol did suppress an increase in Rrs due to the challenge of both methacholine and histamine. Isoproterenol also decreased the bronchoconstriction due to the previous challenge by both methacholine and histamine. The antagonistic effects of lidocaine, atropine, and isoproterenol on the broncho-constriction of monkeys are discussed in relation to the subepithelial receptor.

Aerosols↗
PubMed1985

An ascochlorin derivative, AS-6, reduces insulin resistance in the genetically obese diabetic mouse, db/db.

An ascochlorin derivative, AS-6, is a new hypoglycemic agent orally active in both obese hyperinsulinemic and insulin-deficient diabetic animal models. AS-6, when given as a 0.025-0.2% admixture in the diet, dose-dependently ameliorated polydipsia, polyuria, and glycosuria in the genetically obese diabetic mouse, C57BL/KsJ db/db, while neither insulin nor tolbutamide showed any beneficial effects. The amelioration by AS-6 was associated with a marked decrease in serum glucose and triglyceride. The effects persisted at least 10 wk, accompanied by a steady decrease in drinking water consumption. The chronic treatment prevented pancreatic islet degeneration, e.g., degranulation of the beta-cells, basophilic appearance of the exocrine border around the islets, and small round cell infiltration. The isolated islets from AS-6-treated mice released much more insulin in response to glucose than those from untreated controls. A significant correlation between serum immunoreactive insulin and glucose/triglyceride from both treated and untreated mice suggests that AS-6 restores sensitivity and responsiveness to insulin to the mice. In fact, the combined treatment with insulin synergistically decreased serum glucose by 50% below AS-6 treatment alone. Furthermore, the epididymal fat pad slices from AS-6-treated db/db mice increased CO2 generation and lipogenesis over the untreated controls, and the glucose metabolic rate (CO2 generation plus lipogenesis from U-[14C]-glucose) in the slices and the serum glucose level inversely correlated at r = 0.8799.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Inhalation provocation test with house dust allergen using tidal breathing.

Using the modified 3-Hz oscillation method, the inhalation challenge test was performed with house dust allergen upon subjects while tidal breathing throughout the test. Their total respiratory resistance (Rrs) was continuously monitored with the inhalation of saline as a control, and 250-, 50-, and 10-fold diluted allergen solution to the inhalation of aerosolized metaproterenol. The starting point of the induced bronchoconstriction was checked and as soon as Rrs increased up to twice the base-line value, the subjects inhaled the aerosolized metaproterenol. From the dose-response curve of Rrs, the dose of inhaled allergen (minimum dose) as bronchial sensitivity was determined; also the decreasing rate of respiratory conductance, which was calculated from the slope of elevation of Rrs, as bronchial reactivity. It was found that the more diluted allergen required for a positive skin reaction, the more likely the patients were to have a subsequent positive bronchial inhalation challenge to house dust allergen. There was a relationship between the increased bronchial reactivity and the increased RAST score. These results indicate that this inhalation challenge test is specific, safe, and time-saving.

Airway Resistance↗
PubMed1984

Structural and functional abnormalities in the adipocyte plasma membrane from db/db mouse, and the effect on the abnormalities of oral treatment with AS-6.

The protein bands of adipocyte plasma membranes from the genetically obese diabetic mice C57BL/KsJ db/db (db/db mice) showed slight but significant changes compared with their lean littermates. The treatment for 1 week with a new antidiabetic agent, AS-6, caused the changes to revert toward the condition in the lean littermates. In the absence of insulin, the plasma membrane and mitochondria mixture (P3 fraction) of the lean littermates densely labeled 55000 and 57000 dalton protein bands by phosphorylating with (a-32P)-ATP, whereas the labeling was less in the P3 from AS-6 treated and untreated db/db mice. Insulin inhibited phosphorylation of these bands in P3 from the lean littermates and untreated db/db mice, while the hormone enhanced the labeling in AS-6 treated db/db mice compared with the basal condition without insulin. Ca2+ greatly enhanced the labeling in all three groups, whereas Mg2+ mimicked the insulin action diminishing the labeling of these bands in the lean and untreated db/db groups. However, Mg2+ enhanced the phosphorylation in the P3 from AS-6 treated db/db mice compared with the basal condition.

Adenosine Triphosphate↗
PubMed1984

Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.

Amino Acid Sequence↗
PubMed1984

Characterization of a novel killer toxin encoded by a double-stranded linear DNA plasmid of Kluyveromyces lactis.

A novel killer toxin, encoded by a double-stranded linear DNA plasmid pGK l-1 (5.4 MDa) in Kluyveromyces lactis IFO 1267 was purified 320 000-fold from the culture broth of yeast. The toxin was obtained in an electrophoretically homogeneous state with a yield of 24% by hydroxyapatite column chromatography, chromatofocusing and polyacrylamide gel electrophoresis. The purified toxin was dissociated into two subunits with molecular masses of 27 kDa and above 80 kDa, as estimated by Laemmli's sodium dodecylsulfate gel electrophoresis; the exact composition ratio of the two subunits remains unestablished. The isoelectric point was between 4.4 and 4.8. As compared with the reported narrow pH range of action and instability of k1 killer toxin encoded by a double-stranded RNA plasmid of Saccharomyces cerevisiae, the K. Lactis toxin was effective with sensitive strains of S. cerevisiae in a relatively wider pH range between 4 and 8; it was stable for several months at pH 6.0 when stored below -20 degrees C. In contrast to the simple protein nature of the k1 killer toxin with a molecular mass of 11.47 kDa, the K. lactis toxin maintained a mannoprotein nature, as it was absorbed by a ConA-Sepharose column and eluted by methyl alpha-D-mannoside. The growth inhibitory activity of K. lactis toxin was enhanced 2-35-fold by the presence of 4-60% glycerol.

Ascomycota↗
PubMed1984

A Bacillus subtilis secretion vector system derived from the B. subtilis alpha-amylase promoter and signal sequence region, and secretion of Escherichia coli beta-lactamase by the vector system.

A secretion vector system in Bacillus subtilis was constructed from the alpha-amylase promoter and signal sequence coding region of an alpha-amylase hyperproducing strain, B. subtilis NA64, and the major part of the plasmid pTUB4 which was derived from pUB110. When an Escherichia coli beta-lactamase gene, lacking its own promoter and signal sequence coding region, was introduced into the secretion vector system, beta-lactamase was expressed in B. subtilis. In addition, more than 95% of the enzyme synthesized was secreted into the culture medium via the secretion vector system. Secreted beta-lactamase crossreacted with rabbit antiserum raised against the E. coli enzyme.

Bacillus subtilis↗
PubMed1984

Flavin and iron-sulfur containing ferredoxin-linked glutamate synthase from spinach leaves.

Ferredoxin-dependent glutamate synthase (native enzyme) [EC 1.4.7.1] of spinach has been purified to homogeneity in the presence of 2-oxoglutarate and sodium chloride and the properties of the enzyme have been studied. The molecular weight of the enzyme was estimated to be 140,000 by gel filtration. Subunit analysis by SDS-gel electrophoresis yielded a single protein band whose molecular weight was about 170,000. This purified enzyme showed a flavo-protein-like absorption spectrum having maxima at 279 and 438 nm with shoulders at 415 and 460 nm and a broad band around 360 nm. Fluorometric data indicated the presence of 2 mol of flavin per mol of the enzyme. Preliminary paper chromatography results indicated the presence of FAD and FMN in the purified enzyme. The enzyme also contained 4 mol of acid-labile sulfide and 4 g-atoms iron per mol of enzyme. In the absence of 2-oxoglutarate and/or sodium chloride, the purified enzyme was separated by either DE-52 cellulose chromatography or gel filtration with Ultrogel AcA 34 into two molecular forms (modified enzymes) with considerable inactivation. When reduced methyl viologen plus ferredoxin was used as the electron donor, the purified (native) enzyme showed high ferredoxin-dependent activity with a specific activity of 100 units/mg protein. Methyl viologen-dependent activity was negligible in the absence of ferredoxin. Kinetic properties and results of ESR studies were described. The results indicate that ferredoxin-linked glutamate synthase of spinach leaves is an iron-sulfur flavoprotein.

Amino Acid Oxidoreductases↗
PubMed1984

Induction of rat hepatic and intestinal alkaline phosphatase activity produced by bile from bile duct-ligated animals.

Bile duct ligation caused a threefold elevation of not only hepatic but also intestinal alkaline phosphatase. The increase was transient in intestinal mucosa and peaked at 12 h. Hepatic phosphatase levels reached a plateau by 36 h. Intraperitoneal injection of bile from ligated animals into normal rats produced qualitatively similar results. Studies were performed to further characterize the induction of phosphatase activity in both tissues. The addition of bile from ligated animals to organ explants of liver and intestine produced a two- and threefold rise in activity, respectively. Therefore, neural and hormonal factors are not essential in producing this induction. The increased activity produced in vitro in both tissues was due to heat-stable and dialyzable factor(s) and was blocked by inhibitors of protein synthesis. In the intestine activity occurred over the entire brush border as demonstrated by electron microscopic histochemistry. In the liver, increased activity was noted over the entire plasma membrane and was not localized to the canalicular membrane. Tunicamycin treatment of liver and intestinal explants markedly suppressed the induced phosphatase activity. Simultaneous treatment with protease inhibitors restored some of the phosphatase activity. These findings suggest that glycosylation of the alkaline phosphatase might provide at least partial protection from proteolysis.

Alkaline Phosphatase↗