Biomedical subjects

M Mizugaki

Publications and source records attributed to M Mizugaki.

At least 73 records · Page 4Linked to original sources

PubMed1998

Detection of human papillomavirus type 33 DNA in extragenital Bowen's disease with the polymerase chain reaction.

BACKGROUND: Data on the association of human papillomavirus (HPV) infection with extragenital Bowen's disease are very scarce. OBJECTIVE: To evaluate the prevalence of HPV infection in extragenital Bowen's disease showing histologically a number of koilocytes in the lesional epidermis, we studied formalin-fixed, paraffin-embedded tissues of 9 such cases. METHODS: HPV DNA was studied in such samples by the polymerase chain reaction (PCR) and in situ hybridization. RESULTS: Despite negative results with in situ hybridization, the PCR with HPV type 33 primer detected the presence of virus DNA in 2 out of 9 cases. CONCLUSION: As far as we know, this is the first report of the detection of HPV type 33 DNA in the lesional skin of extragenital Bowen's disease. The prevalence of HPV infection in extragenital Bowen's disease may be higher than expected, especially in cases histologically showing many koilocytes in the lesional epidermis.

Aged↗

PubMed1998

Extra-weak chemiluminescence produced by autoxidation of kampo extract preparations stored in heat-stressed conditions.

The purpose of this study was to evaluate the usefulness of extra-weak chemiluminescence (CL) measurement as a rapid method to estimate the stability of Kampo extract preparations. It was found that the Kampo drugs that emit little CL were stable, while those with higher CL were comparatively unstable with regard to the various stability markers, including change of coloration (browning), contents of specific ingredients, high molecular compounds, amino acids and sugars under various conditions of heat storage. Excellent correlation existed between the CL of Kampo drugs and the coloration (delta E* (ab)) and the other above-mentioned evaluation markers. From this investigation, it was deduced that the CL of Kampo drugs originates in the early stage of the Maillard reaction and reflects the stability of the preparations, and that CL is useful for estimating the stability of Kampo drugs.

Amino Acids↗

PubMed1997

Characterization of S-hexylglutathione-binding proteins of human hepatocellular carcinoma: separation of enoyl-CoA isomerase from an Alpha class glutathione transferase form.

Recent studies have revealed binding of mitochondrial enoyl-CoA isomerase (ECI) to S-hexylglutathione-Sepharose, an affinity matrix used for purification of glutathione transferases (GSTs), and the enzyme has been suggested to be identical with the Alpha class form of GST with a subunit molecular mass of about 30 kDa. In the present study, S-hexylglutathione-binding proteins of human hepatocellular carcinomas were characterized to examine their identity. Supernatant fractions of carcinoma and surrounding tissues were applied to an affinity column, and bound fractions were resolved into three proteins with subunit molecular masses/pI values of 33 kDa/7.0, 30 kDa/5.8 and 29 kDa/5.8 in addition to the well-characterized four GST subunits, A1, A2, M1 and P1, by two-dimensional gel electrophoresis. The proteins were further purified by chromatofocusing at pH 7.4-4.0. The 30 and 29 kDa proteins were eluted at pH 4.9 and by 1 M NaCl respectively, and could be clearly separated from each other. The 29 kDa protein exhibited a low but significant activity towards 1-chloro-2,4-dinitrobenzene (4.25 micromol/min per mg of protein) and reacted with anti-(GST A1-2) antibody, suggesting that it is a member of the GST Alpha class. The 30 kDa protein did not react with anti-GST antibodies and was identified as ECI by immunoblotting and N-terminal-amino-acid-sequencing analyses. The results thus indicated that the Alpha class GST form composed of the 29 kDa subunits and ECI are two different proteins. The 33 kDa protein was eluted from the chromatofocusing column at pH 7.0 and did not react with either anti-GST antibodies or antibodies against mitochondrial enzymes involved in the beta-oxidation of fatty acids. However, it exhibited a carbonyl reductase activity with menadione and ubiquinone, and amino acid sequences of its peptides cleaved by Staphylococcus aureus V8 proteinase were consistent with those reported for the enzyme. Thus this protein binding to S-hexylglutathione-Sepharose was identified as carbonyl reductase.

Aged↗

PubMed1997

Expressions of P-selectin- and HSP72-like immunoreactivities in rat brain after transient middle cerebral artery occlusion.

The role of an adhesion molecule such as P-selectin may be important in the pathogenesis of stroke. However, temporal, spatial and cellular profiles of the expression of such a protein have not been fully studied. Change of immunoreactive P-selectin was examined in rat brain after transient middle cerebral artery (MCA) occlusion in comparison with that of 72 kDa heat shock protein (HSP72) which is a well known marker of cell injury. Western blot analyses were performed to ensure the selective detection of immunoreactive P-selectin and HSP72 proteins with each antibody using brain samples before and after ischemia. Temporal, spatial and cellular changes of immunohistochemical expressions of P-selectin and HSP72 were evaluated with rat brain sections at 2 and 8 h, and 1, 3 and 7 days of reperfusion after 1 h of MCA occlusion (MCAO). Hematoxylin-eosin (HE) staining was performed to evaluate brain cell damage at 3 and 7 days of reperfusion. Western blot showed a single band at molecular weights of 140 and 72 kDa for P-selectin and HSP72, respectively, only after ischemia. No significant band was observed without primary antibody. P-selectin-like immunoreactivity was not normally present in rat brain sections. However, it was expressed mainly in the post-capillary venules of the cerebral cortex and caudate in the MCA territory with a peak at 8 h to 1 day. The expression was diminished by 3 days of reperfusion. An immunoreactive HSP72 was scarcely present in the cerebral cortex and caudate of the sham control brain. However, the protein was induced in neurons of the MCA territory. The HSP72 induction was gradually intensified from 8 h with peaks at 1 day in the cortex and at 3 days in the caudate. The immunoreactivity decreased by 7 days. Histopathological study with HE staining showed no evident cell damage at 3 and 7 days of reperfusion. The present results indicate that temporal, spatial and cellular differences were present in the expressions of immunoreactive P-selectin and HSP72 proteins. P-selectin was expressed from an earlier stage of reperfusion in post-capillary venules, and the expression became maximum at the same time both in the cerebral cortex and caudate. In contrast, HSP72 induction began later in neurons and reached maximum at a different time between the cortex and caudate.

Animals↗

PubMed1997

Postischemic expression of P-selectin immunoreactivity in rat brain.

Change of immunoreactive P-selectin was examined in rat brain after transient middle cerebral artery (MCA) occlusion (O) with anti-P-selectin monoclonal antibody using brain samples of sham control and after ischemia. Temporal, spatial, and cellular changes of immunohistochemical expressions of P-selectin were evaluated with rat brain sections at 2 and 8 h, 1, 3, and 7 days of reperfusion after 1 h of MCAO. Western blot showed a single band at molecular weight of 140 kDa for P-selectin after ischemia. P-selectin immunoreactivity was not normally present in rat brain sections. However, it was expressed mainly in the post-capillary venules of the cerebral cortex and caudate in the MCA territory with a peak at 8 h-1 day. The expression was diminished by 3 days of reperfusion. The present results indicate that P-selectin was expressed from an earlier stage of reperfusion in post-capillary venules, and the expression became maximum at the same time both in the cerebral cortex and caudate.

Animals↗

PubMed1997

Microdetermination of the thromboxane B3 gas chromatography-selected-ion monitoring using [18O]thromboxane B3 as an internal standard.

We devised a simple and effective purification for the microdetermination of thromboxane B3 (TXB3), a hydrolysis product of TXA3- [18O2]TXB3 was synthesized by the repeated base-catalyzed hydrolysis of methyl ester derivatives in [18O]water, to obtain an internal standard (IS) for the gas chromatography/selected ion monitoring (GC/SIM) of TXB3. The methyl ester (ME)-methoxime (MO)-dimethylisopropylsilyl (DMIPS) ether derivative was prepared, then GC/SIM was carried out by monitoring the ion at m/z 668 for TXB3 and that at m/z 672 for IS. A good linear response over the range of 10 pg approximately 10 ng was demonstrated. We were able to detect the levels of TXB3 in the medium of human erythroleukemia (HEL) cell cultured with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). This method can be used to determine 3-series thromboxane in biological samples.

Calibration↗

PubMed1997

Effects of haloperidol and cocaine pretreatments on brain distribution and kinetics of [11C]methamphetamine in methamphetamine sensitized dog: application of PET to drug pharmacokinetic study.

Repeated administration of methamphetamine (MAP) causes behavioral sensitization in animals. We previously reported that the maximum accumulation level of [11C]MAP in the MAP-sensitized dog brain was 1.4 times higher than that in the control. In behavioral studies, haloperidol (a dopamine D2 receptor antagonist) prevents MAP-induced behavioral sensitization, and cocaine (a dopamine reuptake blocker) has the cross-behavioral sensitization with MAP. In the present study, to elucidate the relation between the MAP-induced behavioral sensitization and the pharmacokinetics of MAP, we investigated the effects of haloperidol and cocaine pretreatments on brain regional distribution and kinetics of [11C]MAP using positron emission tomography (PET). A significant increase of [11C]MAP uptake into the sensitized dog brain was prevented by haloperidol and cocaine pretreatments. These pharmacokinetic changes were not due to the changes in the rate of MAP metabolism. These results suggest haloperidol and cocaine can change the cerebral pharmacokinetic profile of MAP in the behavioral-sensitized dog. The variations of MAP-accumulation may affect the development or expression of MAP-induced behavioral sensitization.

Animals↗

PubMed1997

Method for the simultaneous determination of Aconitum alkaloids and their hydrolysis products by gas chromatography-mass spectrometry in human serum.

This paper describes a newly developed method using gas chromatography-selected ion monitoring (GC-SIM) for the simultaneous determination of diester-diterpene type Aconitum alkaloids and their hydrolysis products in human serum. When the alkaloids were converted into their TMS derivatives, they produced a well defined peak on selected ion recording (SIR). Good linear response over the range of 100 pg to 7.5 ng was demonstrated for each alkaloid. When the alkaloids were spiked into human serum, good quantitative recovery was observed.

Aconitine↗

PubMed1997

Determination of ascorbic acid in human vitreous humor by high-performance liquid chromatography with UV detection.

PURPOSE: Ascorbic acid (AA) accumulates in vitreous at a concentration several times higher than in plasma. It has been suggested that AA may serve as an antioxidant that protects ocular tissues from free radical attack. There are many reports about the concentration of AA in ocular tissues. However, AA in adult human vitreous humor has not been determined. We measured concentrations of AA from pathologic human vitreous samples and compared the results. METHODS: AA was measured by high-performance liquid chromatography (HPLC) with UV detection. Human vitreous humor was collected from patients undergoing pars plana vitrectomy. RESULTS: AA was quantified in vitreous humor of proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), macular hole (MH), idiopathic premacular fibrosis (PMF), and Terson syndrome (Terson). The concentrations of AA were 120.9 +/- 36.3 micrograms/ml (mean +/- SD), 129.8 +/- 36.6, 311.5 +/- 126.7, 446.9 +/- 154.2 and 406.0 +/- 22.0, respectively. There was no significant difference between the PDR and the PVR groups (unpaired t-test). Patients with PDR and PVR showed significantly lower concentrations of AA than those with MH, PMF, and Terson (p < 0.01). CONCLUSIONS: These findings suggest that increased oxidative stress may be produced in the ocular tissues of eyes with PDR and PVR, and AA appears to be consumed (oxidized) in performing its protective role.

Ascorbic Acid↗

PubMed1997

Alteration of DNA methylation levels in MRL lupus mice.

Recent reports suggest that DNA methylation is involved in the cause of autoimmune disease. We investigated the alteration of DNA methylation levels in lupus strains of mice, MRL/lpr as a model, which develop an age-dependent lymphadenopathy and autoimmune disease. DNA methylation levels of thymus and axillary lymph nodes in 20-week-old MRL/lpr mice, which are in an autoimmune disease state, were lower than those of 4-week-old MRL/lpr mice with no symptoms as yet. No significant changes were observed in MRL/+ strain mice, which seemed normal at least 20 weeks, while DNA methylation levels in the spleen of both strains of mice increased significantly from the age of 4 to 20 weeks. However, no significant changes of DNA methylation levels in peripheral blood were observed with ageing in MRL strains. Moreover, we clarified that administration of 5-azacytidine had a strong effect on longer survival of MRL/lpr mice and reduced DNA methylation levels in the axillary lymph nodes and spleen. The possible relevance of DNA methylation levels to the progression of autoimmune disease is discussed.

Animals↗

PubMed1997

Molecular cloning of the nemA gene encoding N-ethylmaleimide reductase from Escherichia coli.

Using the gene mapping membrane technique, we identified a gene (nemA) that encodes N-ethylmaleimide reductase in Escherichia coli. The open reading frame encodes a polypeptide of 365 amino acids with a molecular mass of 39,514 Da. The deduced amino acid sequence showed a high degree of homology (87% identical) with the pentaerythritol tetranitrate reductase of Enterobacter cloacae and the morphinone reductase of Pseudomonas putida (52% identical).

Amino Acid Sequence↗

PubMed1997

Phenobarbital molecularly imprinted polymer selectively binds phenobarbital.

Molecularly imprinted polymer (MIP) was prepared against phenobarbital using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross linking monomer. We analyzed the recognition properties of the phenobarbital MIP. In some organic solvents, imprinted polymer showed selective binding to phenobarbital. Two dissociation constants of binding were calculated by Scatchard plot analyses; Kd values were 1.8, 121.7 microM, and the number of binding sites was 8.3, 92.3 mumol/g MIP in toluene-heptane-acetic acid (25 : 75 : 1, v/v), respectively. The relationship between the binding affinity to phenobarbital MIP and the polarity of the solvent system, as well as the structure of the template molecule is also discussed.

Cross-Linking Reagents↗

PubMed1997

4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by N-ethylmaleimide-reductase of Yarrowia lipolytica.

We studied the steric course of the reaction catalyzed by the N-ethylmaleimide (NEM) reductase of Yarrowia (Candida) lipolytica (Y. lipolytica), using 4R-[4-2H1]NADPH and 4S-[4-2H1]NADPH as cofactors and N-ethylcitraconimide as substrate. Active substrates and inhibitors of NEM reductase and its subcellular distribution were also investigated to clarify the biochemical properties of this enzyme. NEM reductase catalyzes the reduction of N-ethylmaleimide to N-ethylsuccinimide with NAD(P)H as the cofactor. Several maleimide and cyclopentenone derivatives tested were also active substrates for NEM reductase of Y. lipolytica. Some pyrazolone derivatives, particularly 1-phenyl-5-pyrazolone, were found to be effective inhibitors of NEM reductase. Subcellular localization of NEM reductase was carried out using protoplast formation and differential centrifugation. Ninety-eight percent of the NEM reductase activity was recovered in the cytosolic fraction, indicating that NEM reductase in Y. lipolytica was the cytosolic enzyme. We also determined the stereochemical specificity of the reduction of N-ethylcitraconimide by NEM reductase in Y. lipolytica, showing that 4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by NEM reductase, and two hydrogen atoms from NADPH and H2O added to opposite faces of the double bond of N-ethylcitraconimide.

Candida↗

PubMed1997

The effects of unsaturated fatty acids, oxidizing agents and Michael reaction acceptors on the induction of N-ethylmaleimide reductase in Escherichia coli: possible application for drug design of chemoprotectors.

Menadione and dimethyl maleate, Michael reaction acceptors, induced N-ethylmaleimide (NEM) reductase activity in Escherichia coli strain DH5a. Linoleic acid also induced NEM reductase activity, but oleic acid, which is less susceptible to lipid peroxidation than linoleic acid, did not induce NEM reductase activity. In addition, NEM reductase activity was induced by menadione and linoleic acid also in strain DH5, Y1088 and Y1090. Linoleic acid is not a Michael reaction acceptor, but is known to produce Michael reaction acceptors such as alkenals and 4-hydroxyalkenals as a result of free-radical-initiated lipid peroxidation. Thus, our findings suggested that lipid peroxidation was involved in the induction of NEM reductase by linoleic acid. The electrophilic property of Michael reaction acceptors provides the signal for induction of phase II enzymes such as glutathione S-transferase and quinone reductase in mammals. The inducer potency of phase II enzymes has been used to design chemoprotective drugs. Therefore, the inducible nature of this enzyme will serve not only for the elucidation of its physiological function, but also for the evaluation of chemoprotective drugs.

Enzyme Induction↗

PubMed1996

Positron emission tomography study of the alterations in brain distribution of [11C]methamphetamine in methamphetamine-sensitized dog.

We newly prepared a MAP-sensitized dog by repeated MAP treatment and studied the brain distribution of [11C]MAP in the normal and the MAP-sensitized dog using PET. The maximal level of accumulation of [11C]MAP in the sensitized dog brain was 1.4 times higher than that in the control. No difference was found in the metabolism of MAP between the two conditions. The significant increase of [11C]MAP in the MAP-sensitized brain indicates that subchronic MAP administration causes some functional change in the uptake site of MAP. The pharmacokinetic change may, in part, account for behavioral sensitization.

Animals↗

PubMed1996

A case of aconitine poisoning with analysis of aconitine alkaloids by GC/SIM.

Described here is a fatal case of accidental aconitine poisoning following the ingestion of aconite, Torikabuto, mistaken for an edible grass, Momijigasa. A 61-year-old man developed symptoms of nausea, diarrhea, and discomfort of the body about 2 h after the ingestion and was taken to an emergency room. Resuscitation and antiarrhythmic drugs were ineffective, and ventricular tachycardia and fibrillation developed and lasted for 6 h. He was transferred to a coronary care unit and complete sinus rhythm was obtained on an electrocardiogram 30 h after his admission. The patient fell into a coma and died of brain edema diagnosed by CT on the 6th day. Consent for autopsy was denied by the family but was given for gas chromatography/selected ion monitoring (GC/SIM) to analyze the toxicity of aconitine alkaloids in the blood and the urine. Only a faint amount of jesaconitine was detected, while aconitine, mesaconitine and hypaconitine were not detectable in the blood 24 h after ingestion. On the other hand, aconitine and its related alkaloids such as mesaconitine, jesaconitine, and hypaconitine were clearly detected in the urine.

Aconitine↗

PubMed1996

Induction of IFN-gamma and IL-1 alpha production in macrophages stimulated with phosphopolysaccharide produced by Lactococcus lactis ssp. cremoris.

The induction of interferon (IFN) and interleukin-1 (IL-1) production in murine macrophages by a phosphopolysaccharide, produced by a dairy lactic acid bacteria, Lactococcus lactis ssp. cremoris, was investigated. When the phosphopolysaccharide was added into macrophage cultures at concentrations from 1 to 200 micrograms/ml, substantial IFN titers (6.2-79.2 IU/ml) were detected. Using the reverse transcription-polymerase chain reaction (RT-PCR), the expression of mRNA encoding IFN-gamma was verified in spleen macrophage cultures. Macrophages stimulated with the phosphopolysaccharide also produced IL-1 alpha at a concentration of 50 micrograms/ml. This study showed for the first time that phosphopolysaccharide derived from a dairy lactic acid bacterium can induce IFN-gamma and IL-1 alpha production in macrophages. These findings strongly suggest that the phosphopolysaccharide is a type of 'biological response modifier' and the fermented dairy foods containing Lactococcus lactis ssp. cremoris can be designated as a physiologically functional food.

Animals↗

PubMed1996

Analysis of the thromboxane/prostacyclin balance in human urine by gas chromatography/selected ion monitoring: abnormalities in diabetics.

We microanalyzed 2,3-dinor-6-keto-prostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha 1) and 11-dehydrothromboxane B2 (11-dehydro-TXB2, 2) in human urine. Samples containing a [2H4]-analogue as an internal standard were extracted by chromatography using Sep Pak tC18 and silica gel. The compounds were then analysed by means of the lactone ring opening reaction and dimethylisopropylsilylation. The conversion of 1 to 1-methyl ester (ME)-propylamide (PA)-9, 12, 15-dimethylisopropylsilyl (DMIPS) ether derivative and of 2 to 1-ME-6-methoxime (MO)-9, 12, 15-tris-DMIPS ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using a DB-5 column. We were able to detect 1 (222-1031 pg/mg creatinine) and 2 (18-155 pg/mg creatinine) in human urine. Furthermore, the thromboxane/prostacyclin (IX/PGI) ratio in the urine of diabetics was higher than that of healthy volunteers. This method can be used to determine the TX/PGI balance in human urine.

6-Ketoprostaglandin F1 alpha↗