Biomedical subjects
Publications and source records attributed to Y Tsuchiya.
A new kind of potential oscillation with a short period of 7.5 s was found to occur in a water/oil/water system which consisted of an aqueous solution of surfactant, nitrobenzene and an aqueous solution of NaCI. A long period (approximately 10 min) oscillation previously reported was also found to be superposed on the new oscillation. The power spectrum of the short period oscillation has been measured.
We have produced human CEA transgenic mice which were found to express CEA mRNA in all tissues. By immunoblot analysis using anti-CEA polyclonal antibody, we also detected CEA protein in all tissues. However, the molecular size of CEA in the brain was different from that in other tissues, although the mRNA size was same and no deletion nor rearrangement was detected at the DNA level. Immunohistochemical analysis of the lung and the colon showed that the expression sites were the bronchial epithelial cells of the lung and the columnar epithelial cells of the colon. Interestingly, the expression of CEA protein in the transgenic mice was polarised to the luminal side of epithelial cells similar to the normal CEA expression in human tissues. We also detected cell surface expression of human CEA on thymocytes and spleen cells and CEA expression was greatly reduced by the phosphatidylinositol-specific phospholipase C (PI-PLC) treatment.
The effect of Clostridium perfringens alpha toxin on contraction induced by electric stimulation of isolated guinea-pig diaphragm was investigated. The toxin inhibited electrically stimulated contraction of the tissue in a dose- and incubation time-dependent manner. Tetrodotoxin resulted in no effect of the action of the toxin. Nifedipine dose-dependently delayed the action of the toxin, but verapamil and diltiazem did not. On the other hand, treatment of the toxin with N-acetylimidazole caused significant reduction of the inhibitory activity of the toxin on contraction, but did not cause significant loss of phospholipase C activity (PN activity) as measured by hydrolysis of p-nitrophenylphosphorylcholine. The data showed that the toxin impairs contraction of isolated guinea-pig diaphragm.
Familial cases (a grandfather, a father and a daughter) of the EEC syndrome (ectrodactyly, ectodermal dysplasia and cleft lip/palate) are reported. All of them have a balanced reciprocal translocation (46,XY or XX, t(7;9) (q11.21;p12) or (46,XY or XX, t(7;9) (p11.2;q12)), but no other members of the family have either the EEC syndrome or chromosome abnormalities. This indicates that one of the chromosome sites 7q11.21, 9p12, 7p11.2 and 9q12 is a candidate for gene locus of the EEC syndrome.
To facilitate early detection of carcinoma of the gallbladder, a comparative study of macroscopic forms and ultrasonographic (US) features of stage I (TNM staging system) gallbladder cancer was carried out retrospectively in 71 cases of such carcinomas that were resected surgically. Macroscopic forms of the lesions were classified into four types: pedunculated (eight cases), sessile (15 cases), superficial raised (25 cases), and flat (23 cases). Among 71 cases, only 20 were diagnosed preoperatively by means of US. US patterns of the lesions were also classified into four types: fungating mass, lumen filling, smooth raised, and thickening of mucosa. US features corresponded well to the macroscopic forms. No flat lesion was identified preoperatively. For early detection of carcinoma of the gallbladder, it seemed essential to pay careful attention to mild mucosal change, as more than 50% of early cancers did not show apparently protruding lesions.
Methimazole (MMI) increases thyroglobulin (Tg) mRNA levels in FRTL-5 rat thyroid cells. The increase reflects a transcriptional action of the antithyroid agent and is inhibited by cycloheximide, as is the transcriptional action of TSH. It takes several hours to be apparent, is maximal between 24-48 h, and is specific, in that thyroid peroxidase and beta-actin mRNA levels are not increased simultaneously. The increased mRNA levels are associated with increased recovery of immunoprecipitable Tg in the medium of cells exposed to [35S]methionine. The MMI effect appears to be independent of the action of TSH or its cAMP signal, since the MMI-induced increase in Tg mRNA levels is evident in cells treated with TSH or maintained in its absence and is associated not with increases in cAMP levels but, rather, under some circumstances with a decrease. The effect is evident under conditions in which the ability of insulin or insulin-like growth factor-I to increase Tg mRNA levels is already maximal. The MMI-induced increase is inhibited by concentrations of iodide associated with autoregulation of FRTL-5 rat thyroid cells, is inhibited but not mimicked by propylthiouracil, and is not altered by T3. The increase in Tg mRNA levels does not correlate with increased DNA synthesis as a function of MMI concentration either in cells treated with TSH or in those maintained in its absence. A concentration of MMI (5 mM) that increases Tg mRNA levels can also inhibit 8-bromo-cAMP- or phorbol ester-induced increases in [3H]thymidine incorporation into DNA.
Partial growth hormone deficiency was found in a 13-year-old Japanese boy who was born by breech delivery. Magnetic resonance imaging showed transection of the pituitary stalk and the presence of an ectopic posterior lobe located at the proximal stump of the transected stalk.
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A woman born in 1924 with colon cancer initially operated on approximately four years ago showed a second recurrence in the lung. Concomitant administration of high dose 5'-DFUR plus MMC was started in February, 1990. Chest X-ray examination six weeks after the start of this therapy revealed a remarkable decrease in size of the pulmonary metastatic focus. At 13 weeks after the start of this therapy, the pulmonary metastatic focus showed nearly complete disappearance. The condition above has been maintained until this writing, eight months after the start of this therapy. No recurrence has been observed in the liver and other organs either, while CEA has normalized from 17.6 ng/ml to 0.9 ng/ml. The therapy was discontinued after five courses based on the moderate loss of appetite and complete disappearance of the lung focus. No other side effect than the loss of appetite was detected, and could be safely treated at an outpatient clinic. The above findings suggested that this was an effective and safe therapy for pulmonary metastasis from colon cancers.
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NB-598, (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bith iophen-5-yl)methoxy]benzene-methanamine, was found to inhibit human microsomal squalene epoxidase (from Hep G2 cells) in a competitive manner. NB-598 inhibited cholesterol synthesis from [14C]acetate dose dependently in Hep G2 cells and increased the intracellular radioactivity of squalene. A single oral administration of NB-598 inhibited cholesterol synthesis from [14C]acetate in rats. Moreover, multiple oral administration of NB-598 to dogs decreased serum total and low density lipoprotein cholesterol levels and increased serum squalene levels. After termination of treatment, the reduced serum cholesterol and increased squalene levels returned to their control values.
Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma-carboxamide groups of protein-bound glutamine residues. These enzymes are involved in many biological phenomena. Transglutaminase reactions also have been shown to be suitable for applied enzymology. In this study, as a first step of studies to elucidate the structure/function relationship of transglutaminase, we constructed an expression plasmid, pKTG1, containing a cDNA of guinea-pig liver transglutaminase between the NcoI and PstI sites of an expression vector, pKK233-2, and produced the liver transglutaminase as an unfused protein in Escherichia coli. The purified recombinant enzyme was indistinguishable from natural liver transglutaminase in some structural properties such as molecular mass, amino acid composition, and amino- and carboxyl-terminal sequences. However, the alpha-amino group of the amino-terminal alanine residue of the recombinant transglutaminase was not acetylated as was that of the natural enzyme. Comparison of the recombinant enzyme with the natural one did not indicate significant differences in specific activity and apparent Km values for substrates in the histamine incorporation into acetyl alpha s1-casein. The sensitivity to activation by Ca2+ and the rate of catalyzed protein cross-linking were also similar between recombinant and natural transglutaminases. These results indicated that the N alpha-acetyl group in natural liver transglutaminase has not a particular role in the catalytic function of this enzyme.
During the storage of secretin in acid and neutral aqueous solutions, five degradation peptides (A1, A2, A3, A4, A5) and one degradation peptide (N1) were produced, respectively. They were isolated in pure form by HPLC, and the intramolecular structures were studied by a combination of amino acid analysis, enzymatic digestions, HPLC, and Fab-mass spectroscopy. Although the degradation peptides are composed of the same amino acids as secretin after acid hydrolysis (except A1 and A4 which are cleavage products S16-27 and S4-27, respectively), reversed-phase HPLC analysis of their digestive fragments with trypsin and alpha-chymotrypsin are different from those of secretin. By Fab-mass spectroscopy, the m/z values for the S1-6 fragments obtained from secretin, A2, and A3 were 663, 663, and 645, respectively. When S1-6 from A2 was treated with aminopeptidase M, a fragment obtained was identical with the synthetic beta-aspartyl3 S3-6, as determined by HPLC. The A2 and N1 peptides are completely the same based on various chemical analyses. The A3 peptide can also be rapidly degraded to secretin and beta-aspartyl3 secretin. Consequently, A1 and A4 are concluded to be the cleavage peptides of secretin, S16-27 and S4-27, respectively, A2 and N1 are concluded to be beta-aspartyl3 secretin, and A3 is concluded to be aspartoyl3 secretin.
In order to clarify the mechanism of secretin degradation in aqueous solutions, the formation of degradation products from secretin, aspartoyl3 secretin and beta-aspartyl3 secretin was investigated; the stabilities of these three peptides were investigated as well. Aspartoyl3 secretion and beta-aspartyl3 secretin, degradation peptides produced during the storage of secretin in aqueous solutions, were isolated by preparative reversed-phase HPLC (RP-HPLC). The amounts of secretin and its two degradation peptides resulting from storage of secretin in various buffer solutions (pH 2.3 to 10.0, mu = 0.5 M, 60 degrees C) were determined by analytical RP-HPLC. Secretin and the isolated degradation peptides were stored separately in various aqueous buffer solutions resulting in the degradation of each peptide. A mixture of secretin and its degradation or cleavage peptides was formed in each solution. The observed degradation rates for each peptide approximately followed first-order kinetics. The pH-rate profiles for conversion of secretin and beta-aspartyl3 secretin were similar, while that for aspartoyl3 secretin was different from these two. Aspartoyl3 secretin was more stable than the others at pH 2.3 to 4.0, but it was easily degraded between pH 5.0 and 10.0. Investigation of aspartoyl3 secretin degradation showed that its degradation was related to the pH value of the solution, and that hydroxide ion catalyzes the ring opening of the aspartoyl peptide. Secretin was most stable in pH 7.0 buffer solution and more stable in acidic solutions than in alkaline solutions. Secretion was mainly degraded through the following pathways: cleavage peptides reversible secretin in equilibrium aspartoyl peptide in equilibrium beta-aspartyl peptide vector cleavage peptides.
Hydrolysis and acyl migration studies on L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (1, NB-355), which produced long-lasting plasma L-dopa levels after oral dosing, have been conducted. Compound 1 exists as pure 4-O-pivaloyl-L-dopa in the solid state, but it converts rapidly to a mixture of the 3- and 4-O-isomers in solution. The rate of acyl migration increased with increases in pH and temperature, and the content of the 4-O-isomer in the equilibrium state was 53-59%. The hydrolysis rate of 1 to L-dopa (6) also increased with increases in pH and temperature, and accelerated steeply at neutral and alkaline pH. The rapid hydrolysis at neutral pH was not observed with O-pivaloyl-L-tyrosine (3), di-O-pivaloyl-L-dopa (4), or L-dopa methyl ester (5). Because of this chemical lability, 1 was hydrolyzed in rat plasma far faster than the other tested catechol esters. However, in rat intestinal homogenate at pH 6.0, 1 was hydrolyzed at the slowest rate among the tested esters, predominantly by a diisofluorophosphate (DFP)-sensitive esterase. Thus, 1 showed a unique in vitro profile on hydrolysis and acyl migration due to the existence of a neighboring hydroxyl group. The stability of 1 in the intestine might be essential for the long-lasting plasma L-dopa profile after oral dosing of 1.
An excitatory potential generated in response to stimulation by a current pulse has been studied on an artificial membrane composed of a Nuclepore filter impregnated with sorbitan monooleate. An excitatory potential response accompanying a fall in the resistance of the membrane was found to occur above a threshold value of the pulse height for current stimulation. The relation between the pulse width of current stimulation and the threshold for the generation of an excitatory potential response has also been examined.
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In order to study the structure-activity relationships of dynorphin A-(1-8) amide [Dyn(1-8)-NH2], 20 analogues were synthesized by the solution method. Their biological activities were determined in the three bioassays [guinea pig ileum (GPI), mouse vas deferens (MVD), and rabbit vas deferens (RVD)] and in the mouse tail-pinch test after intravenous administration. Some analogues that showed interesting activity in the bioassays and/or in the analgesic tests were further characterized in mu-, delta-, and kappa-representative binding assays. The obtained data indicate that modification of the enkephalin segment to give metabolically stable analogues with high affinity and selectivity for the kappa receptor is strictly limited and that introduction of MeArg in position 7 protects the Arg6-Arg-7 bond from enzymatic degradation without potency drop and change of opioid receptor selectivity. [MeTyr1,MeArg7,D-Leu8]Dyn(1-8)-NHEt (18) [IC50 (nM) = 0.3 (GPI), 7.4 (MVD), and 2.6 (RVD); tail pinch ED50 (mg/kg) = 0.75] showed opioid activity similar to that of dynorphin A in the three bioassays and relatively high kappa-receptor selectivity in the binding assays and produced a 2.5-fold more potent analgesic effect than morphine. [D-Cys2-Cys5,MeArg7,D-Leu8]Dyn(1-8)-NHEt (20) showed a 40-60-fold more potent opioid activity than 18 in the three bioassays and produced a 3.4-fold more potent analgesic effect than 18. In the binding assays, however, 20 showed higher affinity for mu and delta receptors than for the kappa receptor.