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Biomedical subjects

A Wada

Publications and source records attributed to A Wada.

At least 379 records · Page 21Linked to original sources
PubMed1988

Inhibition of bovine adrenocortical mitochondrial cytochrome P-450(11)beta-mediated reactions by imidazole derivatives and mineralocorticoid analogs.

The effects of several imidazole antimycotic agents, an imidazole and several mineralocorticoid analogs on the cytochrome P-450(11)beta-catalyzed 11 beta-hydroxylation of 11-deoxycorticosterone and aldosterone synthesis were examined. Ketoconazole, clotrimazole, miconazole and etomidate were found to be potent inhibitors of the reactions, causing 50% inhibition of the 11 beta-hydroxylase activity at concentrations between 10(-8) and 10(-7) M. The potency of etomidate as to the inhibition of aldosterone- and 18-hydroxycorticosterone-production was found to be almost equal to that in the case of 11 beta-hydroxylation. Spironolactone and other newly synthesized mineralocorticoid analogs were also found to inhibit the cytochrome P-450(11)beta-mediated reactions. The ID50 values of these drugs for inhibition of the 11 beta-hydroxylase activity were almost equal to those in the case of the aldosterone- and 18-hydroxycorticosterone-biosynthetic activities. The results of kinetical studies indicated that one of the mineralocorticoid analogs, Compound 23-0586, acts as a competitive inhibitor for the cytochrome P-450(11)beta-mediated reactions.

Adrenal Cortex↗
PubMed1988

Binding of [3H]phencyclidine to adrenal medullary cells: inhibition of 22Na influx, 45Ca influx, 86Rb efflux and catecholamine secretion caused by carbachol and veratridine.

In bovine adrenal medullary cells, phencyclidine inhibited carbachol-induced influx of 22Na, 45Ca and secretion of catecholamines in a concentration-dependent manner with a similar potency (IC50 7.0 microM). Phencyclidine also suppressed veratridine-induced influx of 22Na, 45Ca and secretion of catecholamines (IC50 60.0 microM). High K-induced 45Ca influx and catecholamine secretion were not affected by phencyclidine. In the cells preloaded with 86Rb (an alternative tracer for 42K), phencyclidine inhibited the efflux of 86Rb caused by carbachol (IC50 10.0 microM) or by veratridine (IC50 56.0 microM), but had no effect on high K-induced 86Rb efflux. [3H]Phencyclidine bound specifically to adrenal medullary cells, and binding was saturable, reversible and with two different equilibrium dissociation constants (4.3 and 77.4 microM). In a competition experiment, the specific binding of [3H]phencyclidine was not inhibited by carbachol, muscarine, D-tubocurarine, hexamethonium, tetrodotoxin, veratridine and scorpion venom. The present findings suggest: (1) phencyclidine does not inhibit voltage-dependent Ca channels and Ca-dependent K channels; (2) phencyclidine binds to two populations of sites, each of which is functionally linked to nicotinic receptor-ion channel complex and to voltage-dependent Na channels, and inhibits Na influx caused by carbachol and veratridine. Inhibition of Na influx by phencyclidine leads to the reduction of Ca influx, K efflux and catecholamine secretion caused by carbachol and veratridine.

Adrenal Medulla↗
PubMed1988

Probing stability and dynamics of proteins by protease digestion. II: Identification of the initial chymotryptic cleavage sites of homologous cytochromes c.

Three homologous cytochromes c from horse, rabbit and tuna were subjected to chymotryptic digestion and their initial cleavage sites were identified. The sites in oxidized cytochromes c are the COOH-terminal sides of Tyr-48, Phe-46 and Tyr-46 for horse, rabbit and tuna cytochromes c, respectively. The results show that the chymotrypsin attacks a single site in each protein; the sites are located at the almost identical position on the polypeptide chain. Through the time-course studies of digestion, it was found that the three cytochromes c have different chymotrypsin-susceptibility at the initial cleavage site in the order of horse less than rabbit less than tuna. Studies on chymotryptic digestion of tuna cytochrome c in the reduced form revealed that the haem-reduction does not alter the initial cleavage site but increases the resistance to the proteolysis at the site. The uniqueness of the initial cleavage site in each cytochrome c species suggests that the protease susceptibility reflects some overall properties of the protein. At the same time, it was clarified that the initial cleavage site is also affected by a neighboring region by the fact that another potential cleavage site is located near the site in question. In order to elucidate the initial cleavage site, several physical properties of tuna cytochrome c molecule deduced from the X-ray 3D structure, accessible surface area, temperature factor, effective hydrophobicity and electrostatic potential, were compared with the experimental results and it was concluded that these properties given by a residue have no direct relationship with the chymotrypsin susceptibility.

Amino Acid Sequence↗
PubMed1988

Characteristic features of thermal stability map of DNA in Escherichia coli and eukaryotic genes.

Distribution of double-helix thermal stability of Escherichia coli and eukaryotic DNAs was analyzed. The results confirmed the previous propositions based on the study of the stability distribution in phage DNAs: (1) stability fluctuation appears near the boundaries of protein coding regions (PCRs) and non protein coding regions (NPCRs); (2) PCRs have less fluctuation than NPCRs. The present analysis also revealed that the local G + C content is lower in the beginning of PCRs of E. coli than the average G + C content of PCR and that deviations in the amino acid composition and the third letter usage PCRs are involved in the low G + C content; the biological meaning of this is discussed in relation to mRNA structure.

Base Composition↗
PubMed1988

A theoretical study of the dielectric constant of protein.

The dielectric properties of a protein molecule were investigated by calculating a 'local dielectric constant' with the aid of normal mode analysis. This local dielectric constant was calculated from the electronic polarization of atoms and the orientational polarization of local dipoles. The former was obtained from atomic polarizations of the whole atoms in a protein molecule. The latter was determined from the fluctuation-dissipation theorem. The degree of dipole fluctuation was calculated from the positional fluctuation of each atom obtained by the normal mode analysis. Assuming a minimum volume for a continuum model, the resulting local dielectric constants ranged from 1 to 20 inside the protein.

Electric Conductivity↗
PubMed1988

Effects of protonophores on the synthesis of catecholamines and the intracellular pH in cultured bovine adrenal medullary cells.

The protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) stimulated the synthesis of 14C-catecholamines from [14C]tyrosine in cultured bovine adrenal medullary cells. The stimulatory effect of CCCP but not of FCCP was partially dependent on extracellular Ca2+. CCCP but not FCCP increased the influx of 45Ca2+ to the cells. When cells were incubated with either CCCP or FCCP (0.01-0.2 microgram/ml), the intracellular pH fell from 7.2 to 6.3-6.5 and catecholamine synthesis increased. Tyrosine hydroxylase activity in a soluble fraction prepared from cultured adrenal medullary cells was measured after incubation of the cells with FCCP or CCCP. Although FCCP did not affect the activity of the enzyme, CCCP caused a stable activation of it which was dependent on extracellular Ca2+. Since the optimal pH of soluble tyrosine hydroxylase is around 6.0 in adrenal medullary cells, FCCP may increase the synthesis of catecholamines by shifting the intracellular pH toward it. In addition to this mechanism, CCCP may enhance the synthesis of catecholamines by a Ca2+-dependent mechanism.

Adrenal Medulla↗
PubMed1988

[A case report of long-term survival in advanced gastric cancer with multiple liver metastases treated with mitomycin C and tegafur therapy after palliative gastrectomy].

A 64-year-old male with advanced gastric cancer with multiple liver metastasis was treated with intensive chemotherapy including mitomycin C and tegafur after palliative gastrectomy. The patient was medicated with 30 mg iv of mitomycin C after surgery and 1,000 mg ir or 1,200 mg po of tegafur for one year for induction therapy and 400-600 mg po of tegafur for long-term maintenance therapy. All metastatic lesions in liver were completely eliminated three years after the start of treatment. As to acute and chronic toxicity, slight leukopenia was noted in the treatment, but the patient was found to show a good quality of life because of less toxicity. The patient has been healthy with no evidence of malignancy at nine years and nine months after the surgery. The total cumulative dose of tegafur in this treatment was 1,141.0 g, which is probably the largest dose ever reported in Japan, suggesting that the combination of mitomycin C and tegafur is indicated as a modality for advanced gastric cancer with liver metastasis.

Adenocarcinoma, Papillary↗
PubMed1988

Temperature gradient DNA-probe column chromatography: as tools for detection and purification of particular DNAs and RNAs.

We have developed the temperature-gradient DNA-probe column chromatography as the new method for detecting and purifying particular DNAs or RNAs accurately. The method has high discrimination resolution, and can detect a single-base change in sample base sequences. The method also has high performances in purification of particular DNAs or RNAs, which has been demonstrated by purification of bovine mitochondria serine t-RNA. None of other method can succeed in complete purification of this t-RNA molecule. The present method can be applied not only to molecular biology as basic tools for purifying and picking-up particular sequences but also to the medical field as diagnostic tools.

Base Sequence↗
PubMed1987

Effects of alpha 2-adrenergic agonists on carbachol-stimulated catecholamine synthesis in cultured bovine adrenal medullary cells.

We examined the effects of alpha 2- and alpha 1-adrenergic agonists on synthesis of catecholamines in cultured bovine adrenal medullary cells. Clonidine, an alpha 2-adrenergic agonist, inhibited carbachol-stimulated synthesis of [14C]catecholamines from [14C]tyrosine in a concentration-dependent manner. Clonidine also inhibited carbachol-induced uptake of 45Ca2+ into cells at concentrations similar to those that inhibited the synthesis of [14C]catecholamines. Other alpha 2-adrenergic agonists, oxymetazoline and guanfacine, also strongly inhibited carbachol-stimulated synthesis of [14C]catecholamines. alpha 1-Adrenergic agonists, phenylephrine and norfenefrine, did not affect the synthesis. Tyrosine hydroxylase (EC 1.14.16.2) activity in a soluble fraction of cultured bovine adrenal medullary cells was assayed after gel filtration on a Sephadex G-25 column. Stimulation of the cells with carbachol increased the activity of tyrosine hydroxylase. Clonidine, oxymetazoline, and guanfacine all suppressed the carbachol-induced increase in activity of tyrosine hydroxylase in the cells. These results suggest that alpha 2-adrenergic agonists inhibit carbachol-stimulated synthesis of catecholamines by suppression of tyrosine hydroxylase activity, probably through the inhibition of Ca2+ uptake. However, the involvement of alpha 2-adrenoceptors in the inhibitory effects of alpha 2-agonists on catecholamine synthesis is still unsettled, since yohimbine failed to antagonize the inhibitory effect of clonidine on the synthesis in cultured bovine adrenal medullary cells.

2-Hydroxyphenethylamine↗
PubMed1987

Activation of tyrosine hydroxylase by micromolar concentrations of calcium in digitonin-permeabilized adrenal medullary cells.

In digitonin-permeabilized bovine adrenal medullary cells, Ca2+ (0.1-1.0 microM) caused an activation of tyrosine hydroxylase which was dependent on the presence of ATP. This Ca2+-induced activation of the enzyme was observed even in the presence of optimal concentration of either cyclic AMP or 12-O-tetradecanoylphorbol-13-acetate (TPA) which by itself increased the enzyme activity. Calmodulin inhibitors, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W-7), had little effect on the Ca2+-evoked activation of enzyme. These results suggest that micromolar concentrations of Ca2+ activate the activity of tyrosine hydroxylase probably through a Ca2+-dependent phosphorylation in digitonin-permeabilized adrenal medullary cells although the protein kinase(s) responsible for it still remains to be determined.

Adenosine Triphosphate↗
PubMed1987

Spectroscopic studies on histone-DNA interactions. I. The interaction of histone (H2A, H2B) dimer with DNA: DNA sequence dependence.

Binding of the histone (H2A, H2B) dimer with chicken erythrocyte DNA has been studied by salt-titration spectroscopy in equilibrium conditions. The circular dichroism of DNA near 275 nm is depressed by the interaction with (H2A, H2B) at low concentrations of salt. The depression increases with increasing amounts of (H2A, H2B), and reaches a plateau at an (H2A, H2B) to DNA ratio of 1.5 (w/w), at which one (H2A, H2B) dimer occupies 28 base-pairs of DNA. The fluorescence emission intensity of the tyrosine residues in (H2A, H2B) is depressed by the H2A, H2B)-DNA interaction. When the DNA-(H2A, H2B) complex is titrated with NaCl, these two signals show transitions with increasing ionic strength of the buffer, whose normalized transition curves agree well. The midpoint of the transition is about 0.42 M-NaCl for a sample with a DNA concentration of 0.05 mg/ml and an (H2A, H2B) to DNA ratio of 0.4 (w/w). The fluorescence titration curves have been analyzed to obtain the binding constant for the (H2A, H2B) dimer with DNA. The sample concentration dependence of the titration profiles is consistent with the model of non-cooperative binding of (H2A, H2B) dimer to DNA. The titration profiles are reversible. The obtained binding constant for the (H2A, H2B) dimer with chicken erythrocyte DNA at 20 degrees C (pH 7.6), as a function of the ionic strength, I, is as follows: log10K = -14.9 log10(I)-1.2. The change of enthalpy delta H accompanied by the binding of the (H2A, H2B) dimer is nearly equal to zero, within an error of +/- 1.4 kcal/mol (1 cal = 4.184 J). DNA sequence dependence of the stability of DNA-(H2A, H2B) interactions is observed using reconstituted materials of synthetic DNAs. A decreasing stability of the interaction is observed following the order: the duplex of poly[(dA)-(dT)] greater than chicken erythrocyte DNA or the copolymer duplex of poly(dA).poly(dT) greater than the duplex of poly[(dG)-(dC)]. The difference in free energy of the association of the (H2A,H2B) dimer between the two copolymers is 0.8 kcal/mol.

Animals↗
PubMed1987

Spectroscopic studies on histone-DNA interactions. II. Three transitions in nucleosomes resolved by salt-titration.

The multiple-step transitions in DNA-histone interactions in chicken erythrocyte nucleosomes with increasing ionic strength are resolved by salt-titration spectroscopy. Both the circular dichroism of the DNA and the fluorescence of the histones in nucleosomes change during the titration process with concentrations of NaCl from 0.1 M to 2.5 M. By differentiating the titration curves, three distinct peaks corresponding to three structural transitions are observed. The two peaks near 0.95 M and 1.45 M-NaCl are common to the circular dichroism and fluorescence curves. The circular dichroism curve has another peak near 0.55 M-NaCl. Because the derivative of the fluorescence titration curve for the DNA-(H3, H4) complex has only one peak near 1.45 M-NaCl, that peak is attributed to the dissociation of the histone dimer (H3, H4). The peak near 0.95 M-NaCl corresponds to the dissociation of the dimer (H2A, H2B) from the DNA-(H3, H4) complex, as shown by binding experiments of (H2A, H2B) to the DNA-(H3, H4) complex at the salt concentration near this peak. The peak near 0.55 M-NaCl reflects some inner-core structural change. As the change of the circular dichroism signal is reversible, salt-titration spectroscopy is applicable to equilibrium studies of the physical chemical properties of DNA-histone interactions. By the assumption of a non-co-operative model, the binding constant for the chicken erythrocyte (H2A, H2B) dimer to the DNA-(H3, H4) complex is calculated as 2.8 X 10(6) M-1 at 1.0 M-NaCl (20 degrees C, pH 7.6). The DNA sequence dependence of the stability of the DNA-(H3, H4) interaction is observed in the salt-titration profiles of reconstituted material. Decreasing stability of the interaction of (H3, H4) is observed following the order: poly[(dG)-(dC)] much greater than chicken erythrocyte DNA greater than poly[(dA)-(dT)]. It is concluded that histones (H3, H4) have a different DNA sequence dependence from histones (H2A, H2B).

Animals↗