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Biomedical subjects

Y Yoshida

Publications and source records attributed to Y Yoshida.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition by a novel azole antifungal agent with a geranyl group on lanosterol 14 alpha-demethylase of yeast.

AFK-108 (1-[2-(2,4-dichlorophenyl)-2-((2E)-3,7-dimethylocta-2,6- dienyloxy)ethyl]-1H-imidazole) is a new imidazole derivative characterized by a geranyl substituent showing strong antifungal activity. Azole antifungal agents are known to be potent inhibitors of lanosterol 14 alpha-demethylase (P450(14)DM) of fungi. The role of the geranyl group of AFK-108 on interaction of AFK-108 with the target was studied by using Saccharomyces cerevisiae P450(14)DM as the model enzyme. AFK-108 and some of its derivatives bound to oxidized P450(14)DM with one-to-one stoichiometry and inhibited the demethylase activity. AFK-108 derivatives having the longer farnesyl or the shorter prenyl group showed lower affinity than AFK-108 for the enzyme. AFK-108 caused 100% inhibition at the equivalent concentration to P450(14)DM in the reaction mixture (0.07 microM), while the farnesyl derivative inhibited the activity by 60% at the same concentration. AFK-108 interfered with the binding of CO to the ferrous P450(14)DM. However, the interfering effect of the prenyl derivative was lower than that of AFK-108. Another AFK-108 derivative having the saturated 3,7-dimethyloctyl group was also a weaker inhibitor than AFK-108. These experiments suggest that the geranyl group of AFK-108 interacts with the substrate binding site of P450(14)DM that recognises the side chain of the substrate. AFK-108 is the first example of an azole derivative interacting with the side chain recognising region of the substrate binding site of P450(14)DM.

Antifungal Agents↗
PubMed1992

Structural analysis of the interaction between the side-chain of substrates and the active site of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast.

The role of the side-chain of lanosterol in the enzyme-substrate interaction of yeast P-450(14)DM (lanosterol 14 alpha-demethylase) was analyzed with lanosterol derivatives having functional groups on the side-chain. Purified P-450(14)DM from Saccharomyces cerevisiae catalyzed 14 alpha-demethylation of 26-hydroxylanosterol and 25-hydroxy-24,25-dihydrolanosterol with a lower activity than lanosterol and 24,25-dihydrolanosterol. This enzyme demethylated the (Z)-24-ethylidene-24,25-dihydrolanosterol with a low rate, but did not metabolize the E-isomer. The apparent Km of 26-hydroxylanosterol was 10.8 microM, which was higher than that of lanosterol, but lower than that of 24,25-dihydrolanosterol. On the other hand, competition experiments suggested that the affinity of 25-hydroxy-24,25-dihydrolanosterol and (Z)-24-ethylidene-24,25-dihydrolanosterol for P-450(14)DM was significantly lower than that of 24,25-dihydrolanosterol. Integration of the present results with the preceding ones (Aoyama, Y., Yoshida, Y., Sonoda, Y. and Sato, Y. (1991) Biochim. Biophys. Acta, 1081, 262-266 and Aoyama, Y. and Yoshida, Y. (1991) Biochem. Biophys. Res. Commun., 178, 1064-1071) suggests that yeast P-450(14)DM recognizes two parts of the side-chain, the structure around C-24 and the terminal fork consisting of C-25, C-26 and C-27.

Binding Sites↗
PubMed1992

Action of ebselen as an antioxidant against lipid peroxidation.

The action of ebselen (2-phenyl-1,2-benzoisoselenazol-3(2H)-one) as an antioxidant was studied under various conditions to clarify how it prevents oxidative damage. It did not react with diphenylpicrylhydrazyl nor did it suppress the oxidation of methyl linoleate in acetonitrile solution or in aqueous dispersions induced by free radical initiator, suggesting that ebselen does not act as a potent radical scavenging antioxidant. On the other hand, it suppressed the oxidation of methyl linoleate emulsions in aqueous dispersions induced by iron. It also suppressed the spontaneous oxidation of rat brain and liver homogenates, but it did not suppress the oxidation of these homogenates induced by a free radical initiator. It was also found that ebselen reduced the fatty acid hydroperoxides to their corresponding alcohols and this reaction was enhanced by the presence of glutathione. These results suggest that ebselen acts as an antioxidant by reducing hydroperoxides, but that it does not act as a radical-scavenging antioxidant.

Animals↗
PubMed1992

Active site topology of Saccharomyces cerevisiae lanosterol 14 alpha-demethylase (CYP51) and its G310D mutant (cytochrome P-450SG1).

Incubation of phenyldiazene (PhN = NH) with lanosterol 14 alpha-demethylase, a cytochrome P-450 enzyme (CYP51) that oxidatively removes the 14 alpha-methyl group of lanosterol, results in the appearance of a 478-nm band indicative of phenyl-iron complex formation. In situ oxidation of the phenyl-iron complex by ferricyanide yields exclusively the N-phenylprotoporphyrin IX regioisomer with the phenyl group on the nitrogen of pyrrole ring C (NC). The biphenyl-iron complex formed in the analogous reaction of the enzyme with biphenyldiazene similarly rearranges on treatment with ferricyanide to the NC regioisomer of N-biphenylprotoporphyrin IX. The active site cavity must therefore be at least 10 A high directly above the iron atom and pyrrole ring C of the heme group, and lanosterol binds to the enzyme in the region above pyrrole ring C. Phenyl-iron complex formation is not detected spectroscopically with cytochrome P-450SG1, a catalytically inactive G310D mutant of lanosterol 14 alpha-demethylase in which the sixth iron coordination site is thought to be occupied by an imidazole ligand. Nevertheless, oxidation of the phenyldiazene-treated enzyme with ferricyanide provides the NA and NC regioisomers of N-phenylprotoporphyrin IX in a 40:60 ratio. The single amino acid substitution in cytochrome P-450SG1 thus causes a conformational change that retracts the amino acid residues that cover pyrrole ring A and moves an imidazole ligand into the active site.

Binding Sites↗
PubMed1992

Conalbumin-conjugated silica gel, a new chiral stationary phase for high-performance liquid chromatography.

A new chiral stationary phase using conalbumin (from chicken egg white) was developed for high-performance liquid chromatography. Chiral resolution of racemic azelastine, an antiallergic drug, was achieved on a conalbumin-conjugated silica gel column. The effects of the pH, the concentration of organic solvents and salts in the mobile phase, and the temperature on the capacity factor and resolution of racemic azelastine were examined. This column shows good stability and can separate optical isomers with an aqueous mobile phase. It should be very useful in studies on pharmacokinetics and in clinical chemistry.

Chromatography, High Pressure Liquid↗
PubMed1992

Oxidation of methyl linoleate in aqueous dispersions induced by copper and iron.

The oxidations of methyl linoleate micelles in aqueous dispersions induced by copper and iron have been studied, aiming specifically at elucidating the action of the copper ion in the chain initiation. Sodium dodecyl sulfate (SDS) and tetradecyltrimethylammonium bromide (TTAB) were used as anionic and cationic surfactants, respectively, in order to see the effect of the electric charge of the micelle surface. Both copper and iron induced the oxidations of methyl linoleate micelles by decomposing lipid hydroperoxide contained initially in methyl linoleate, tert-butyl hydroperoxide, or hydrogen peroxide added to the aqueous phase. The rate of oxidation induced by cupric ions was proportional to the first power of methyl linoleate concentration and to the half power of both cupric ion and hydroperoxide concentrations, suggesting that the oxidation was initiated by the peroxyl and alkoxyl radicals formed in the decomposition of hydroperoxide by copper. The formation of alkoxyl radicals was confirmed by its trapping with a spin trap. The rate of oxidation was dependent on the type of surfactant. Methyl linoleate containing a very small amount of hydroperoxide was oxidized by copper in the SDS system, but the rate of its oxidation was negligible when TTAB was used. However, the addition of tert-butyl hydroperoxide induced the oxidation even in the TTAB system. Hydroperoxyl and hydroxyl radicals formed in the SDS system induced the oxidation, but those formed in the TTAB system did not. It was shown that the effect of radicals on the initiation of lipid peroxidation depends on the type of radicals and site of radical formation.

Copper↗
PubMed1992

The 4 beta-methyl group of substrate does not affect the activity of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast: difference between the substrate recognition by yeast and plant sterol 14 alpha-demethylases.

Interaction of obtusifoliol and 24,28-dihydroobtusifoliol with yeast lanosterol 14 alpha-demethylase (P-450(14)DM) was studied to elucidate the role of the 4 beta-methyl group of substrate. P-450(14)DM of Saccharomyces cerevisiae catalyzed 14 alpha-demethylation of obtusifoliol. Apparent Vmax of obtusifoliol demethylation (15.4 nmol/min/nmol P-450) was similar to that of 24-methylene-24,25-dihydrolanosterol demethylation and was a little higher than those of lanosterol and 24,25-dihydrolanosterol demethylations. Apparent Km for obtusifoliol (12.0 microM) was higher than those for lanosterol and 24-methylene-24,25-dihydrolanosterol but was lower than that for 24,25-dihydrolanosterol. 24,28-Dihydroobtusifoliol was a very poor substrate for yeast P-450(14)DM. These facts suggest that the 4 beta-methyl group of sterol slightly affects the activity of yeast P-450(14)DM, while hydrogenation of a double bond in the sterol side-chain considerably impairs the activity. This finding is a contrast to the fact that the plant P-450(14)DM could not catalyze demethylation of sterols having 4 beta-methyl group, but favorably interacts with sterols having saturated side chain.

Cholestadienols↗
PubMed1992

Effects of various bile acids and their sodium salts on development of pepsinogen-altered pyloric glands in rats.

Effects of dietary bile acids and their sodium salts on the development of pepsinogen-altered pyloric glands (PAPG) were examined in male WKY/N Crj rats initially given a single dose of 160 mg/kg body weight of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by gastric intubation. From week 3 the animals were administered basal diet containing 0.5% supplements of cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) or their sodium salts (Na-C, Na-DC and Na-CDC), or 5% ascorbic acid (ASA) or its salt (Na-AS) for 18 weeks. The concentration of DCA and Na-DC was reduced to 0.3% from week 12. At week 20, animals were killed and the numbers of immunohistochemically-demonstrated PAPG were determined. Values were significantly higher with Na-C and Na-CDC than with the corresponding parent acids, and in the Na-C case PAPG development was greater than with MNNG alone. In addition, Na-CDC itself induced the numbers of PAPG significantly. These results suggest that bile salts are possible intrinsic promoters of gastric carcinogenesis. They were without effect, however, on forestomach lesions.

Animals↗
PubMed1992

N-nitrosoheptamethyleneimine-induced pulmonary and esophageal carcinogenesis and effects of concomitant treatment with bleomycin in rats.

The combination effects of bleomycin with N-nitrosoheptamethyleneimine (NHMI) or dihydroxy-di-N-propylnitrosamine (DHPN) on pulmonary carcinogenesis were investigated. Male F344 rats were given NHMI (20 or 40 ppm) or DHPN (200 ppm) in the drinking water and intraperitoneally injected with bleomycin (1 mg/kg) once a week for 18 weeks and then killed at week 24. Many rats treated with NHMI died before the termination of the experiment due to toxicity or development of advanced esophageal carcinomas, considered to be the main cause of death. Detailed histological examination performed on rats killed at week 24 revealed no statistically significant effects of bleomycin on NHMI or DHPN induction of neoplastic lesions in the lung or esophagus, although pulmonary carcinomas were only found in two rats treated with NHMI plus bleomycin. Under the present experimental conditions, NHMI exerted stronger carcinogenic activity in the esophagus than in the lung, and no obvious modifying effects of simultaneously administered bleomycin were evident on NHMI- or DHPN-induced pulmonary carcinogenesis.

Adenoma↗
PubMed1992

Effect of acyl derivatives of 4,4,4-trifluoro-1-phenyl-1,3-butanedione on 2-acetylaminofluorene-induced glutathione S-transferase positive foci in the rat liver.

Acyl derivatives of 4,4,4-trifluoro-1-phenyl-1,3-butanedione (TFPB), 1-benzoyl-2-trifluoromethyl-2-acetoxyethene (BTAE), and 1-benzoyl-2-trifluoromethyl-2-(4-methylthio)benzoyloxyethene (BTME), were synthesized and investigated for inhibition of tRNA binding by N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), and induction of glutathione S-transferase placental form (GST-P) positive foci in the rat liver by 2-acetylaminofluorene (2-AAF). Male F344 rats were given BTAE or BTME intraperitoneally and 2-AAF by intragastric intubation. Two weeks following the treatment, the rats were maintained on the diet containing 0.05% phenobarbital for an additional 6 weeks and then killed. Development of GST-P positive foci was not affected by concomitant treatment with BTAE or BTME. These two compounds inhibited the in vitro binding of N-AcO-AAF to tRNA. Thus, although these diacylmethane derivatives had the in vitro inhibitory activity, they did not inhibit tumor-initiating activity of 2-AAF in the rat liver.

2-Acetylaminofluorene↗
PubMed1992

DNA ploidy of primary hepatocellular carcinoma and pulmonary metastases.

To better comprehend the differences in deoxyribonucleic acid (DNA) content between a primary hepatocellular carcinoma (HCC) and pulmonary metastatic nodules, tissue specimens taken at autopsy of 25 patients who had not received any drugs to treat the malignancy were examined using microspectrophotometry. The DNA distribution patterns were classified into Types I-III, and low (Types I and II) or high (Type III) ploidies, according to DNA distribution. Changes in the DNA content from high to low ploidies, namely DNA ploidy reduction from the primary lesion to pulmonary metastatic lesions, was evident in 9 of the 25 patients (36%), and changes from low to high were noted in 2 of the 25 patients (8%). The remaining 14 (56%) showed no evidence of changes in the DNA ploidy pattern. Reduction of DNA ploidy seen in HCC and its metastatic lesions in the lung may be one of the aspects of clonal evolution or selection mechanisms during the progression of tumor growth and metastasis.

Autopsy↗
PubMed1992

An experimental study on the treatment of hydrofluoric acid burns.

A 20% solution of hydrofluoric acid (HF) was applied to the skin of rats and a biomedical observation of the tissues and sera was made. Flushing with running water was effective for HF burns. By applying 2.5% calcium gluconate jelly, concentrations of fluoride in the urine and the tissues surrounding the injured region were reduced. Thus, the results proved that irrigation with running water and jelly applications were evaluated as the most effective therapy among various methods tested for HF burns.

Animals↗
PubMed1992

Age variation in the upper limit of hearing.

The upper limit of hearing was measured in 6105 otologically normal ears of subjects ranging in age from 5 to 89 years. The results are as follows: in each age group from 5 to 59 years in both sexes, the upper limit of hearing showed an approximately normal distribution if a logarithmic scale was used for the upper limit of hearing axis. The mode of the distribution shifted to a lower frequency with increasing age. Over age 60 years, the distribution became much wider. Standard upper limit age curves were established by calculating 10th, 25th, 50th, 75th and 90th percentiles for each age group. From early childhood where no age variation was recognized in conventional audiometry, deterioration of the upper limit of hearing was already in progress. This deterioration was slight between ages 25 and 39 but at ages over 40 it was accelerated and led to so-called presbycousis. The upper limit of hearing was found to be one of the best parameters for showing the quantitative age-related changes in hearing.

Adolescent↗
PubMed1992

Human glial fibrillary acidic protein (GFAP): molecular cloning of the complete cDNA sequence and chromosomal localization (chromosome 17) of the GFAP gene.

We isolated three glial fibrillary acidic protein (GFAP) cDNA clones from a glioma cell line, U-251 MG. One clone isolated from a U-251 MG cDNA library was long, but lacked both ends. Using poly(A)+ RNA and primers synthesized according to the sequence of this clone, we used the polymerase chain reaction-assisted rapid amplification of cDNA ends (PCR-RACE) method, which is a strategy to isolate cDNA ends, and obtained cDNA clones for the 5' and 3' ends. From the sequences of these overlapping clones, the complete nucleotide sequence of human GFAP cDNA was established. The start (ATG) and the stop (TGA) signals were seen at nucleotide positions 15 and 1311, respectively, and divided the entire sequence of 3027 bp into 14 bp of 5' non-coding, 1296 bp of coding and 1717 bp of 3' non-coding regions. Using cDNA probes made from both the coding and the 3' non-coding regions, Northern blot hybridization was performed with two different stringencies on RNAs from human and rodent brains and human GFAP-positive and -negative cells. It was shown that the 3' non-coding region probe was more specific for human GFAP than the coding region probe which was specific only under higher stringency conditions. This was also suggested by homology analysis of the sequence with those of various intermediate filament proteins. Based on these findings, we performed spot blot hybridization of sorted human chromosomes and Southern blot hybridization of PCR-amplified DNAs of a panel of hamster-human somatic cell hybrids and localized the human GFAP gene to chromosome 17.

Amino Acid Sequence↗
PubMed1992

Serum fluoride as an indicator of occupational hydrofluoric acid exposure.

To define the relationship between ionic fluoride concentration in the serum of workers and the amount of hydrofluoric acid (HF) in the work environment, pre-and postshift serum and urine samples of 142 HF workers and 270 unexposed workers were examined. The maximum and minimum concentrations of HF in the air in each workshop varied from the mean by less than 30%. The preexposure levels of serum and urinary fluoride in HF workers were higher (P < 0.001) than the control values. This suggests that fluoride excretion from the body continues for at least 12h. The postshift serum and urinary fluoride concentrations of these workers were significantly higher (P < 0.001) than the preshift concentrations. A good correlation (r = 0.64) was obtained between postshift serum fluoride and postshift urine fluoride. There was a linear relationship between mean serum fluoride concentration and HF concentration in the workshop. A mean fluoride concentration of 82.3 micrograms/l with a lower fiducial limit (95%, P = 0.05) of 57.9 micrograms/l was estimated to correspond to an atmospheric HF concentration of 3 ppm. This is the maximum allowable environmental concentration recommended by the Japanese Association of Industrial Health, and it is also the threshold limit value suggested by the American Conference of Governmental Industrial Hygienists. The results demonstrate that exposure to HF can be monitored by determining the serum fluoride concentration.

Adolescent↗
PubMed1992

Distribution of 72-kDa heat-shock protein in rat brain after hyperthermia.

The distribution of the 72-kDa heat-shock protein (hsp72) in rat brain, 24 h following in vivo transient hyperthermia (41.5 degrees C, 15 min), was studied using immunohistochemistry (n = 22). Tissue sections were also stained with hematoxylin and eosin, and with an anti-glial fibrillary acidic protein to evaluate neuronal and astrocytic response to transient hyperthermia, respectively. hsp72 was observed in glia and endothelial cells throughout brain. hsp72 was also found in neurons located in the: dentate gyrus, habenula, and hypothalamus, granular layer of the cerebellum and the olfactory area. Our data indicate, that hyperthermia causes neuronal expression of hsp72, particularly in cerebral neuronal populations which control the neuroendocrine stress response.

Animals↗