Biomedical subjects

M Mizugaki

Publications and source records attributed to M Mizugaki.

At least 91 records · Page 5Linked to original sources

PubMed1996

Enzyme-linked immunosorbent assay of neocarzinostatin chromophore (NCS-chr) by use of a monoclonal antibody against NCS-chr analog.

A monoclonal antibody against NCS-chr was prepared and characterized. Because of the instability of NCS-chr, chemically synthesized stable analog compound, termed PS, was used as a hapten of immunogen. The obtained antibody, termed APS, reacted with NCS-chr, but neither with NCS, NCS-polystyrene-maleic acid conjugate (SMANCS), nor UV-irradiated NCS-chr. Epitope analysis using the compounds that have a structure similar to PS showed that APS recognized the total structure, particularly cyclopenten moiety, of PS. These results suggest that APS recognizes the enediyne structure of NCS-chr. Next, the inhibition enzyme-linked immunosorbent assay (ELISA) for determination of NCS-chr was established. The standard curve showed that the microgram order of NCS-chr were accurately measurable by the established ELISA. Furthermore, it was revealed that the established ELISA was more sensitive than the antibiotic activity determination, termed Bio-assay. The established ELISA will be useful as a quantitative method of NCS-chr.

Antibiotics, Antineoplastic↗

PubMed1996

Direct determination of serum glycyrrhetic acid by a monoclonal antibody-based inhibition ELISA using ibuprofen for releasing serum albumin-bound glycyrrhetic acid.

We found that ibuprofen (IBU) had a potential for releasing serum albumin-bound glycyrrhetic acid (GA). Based on this observation, IBU was used to pretreat samples for the determination of serum GA levels by an inhibition ELISA. This method, termed IBU method was evaluated by the recovery of GA from human serum albumin (HSA) or normal human serum (NHS) that contained the exogeneously added GA (37-1000 ng/ml). The mean recovery of GA from HSA and NHS samples treated with IBU were 104.7 and 105.2%, respectively, whereas those without IBU pretreatment were 2.8 and 10.7%, respectively. Comparison of IBU method and chloroform extraction method revealed that the GA content of serum samples pretreated by each method were almost the same. These results suggest that IBU method is useful as a serum processing procedure for the determination of serum GA levels by an inhibition ELISA.

Antibodies, Monoclonal↗

PubMed1996

Preparation of a monoclonal antibody specific for 5-methyl-2'-deoxycytidine and its application for the detection of DNA methylation levels in human peripheral blood cells.

A monoclonal antibody specific for a modified nucleoside, 5-methyl-2'-deoxycytidine (m5dCyd), was prepared using 5-methylcytidine (m5Cyd)-keyhole limpet haemocyanin (KLH) conjugate, and was characterized. Termed FMC9, the antibody reacts with m5dCyd and slightly with m5Cyd and 5-methylcytosine (m5Cyt) but not with other nucleosides tested in this investigation. FMC-9 was used in an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of m5dCyd levels. Sensitivity was in the picomole range. Methylation levels in peripheral blood cells of healthy donors were determined by inhibition ELISA. The percentage of m5dCyd in peripheral blood cells of 10 healthy donors was 5.08 +/- 0.50%. These results suggest that the inhibition ELISA using FMC9 is useful to monitor m5dCyd levels in the peripheral blood cells.

Adult↗

PubMed1996

Subcellular distribution of rat liver NADPH-2,4-dienoyl-CoA reductase.

The subcellular distribution of 2,4-dienoyl-CoA reductase (EC. 1.3.1.34, DCR) in rat liver was studied biochemically and immunochemically after the induction of clofibrate. DCR activity was mainly detected in the mitochondrial fraction by sucrose density gradient centrifugation in the livers of both normal and clofibrate-treated rats. It was also shown that the polyclonal antibody against purified DCR detected the enzyme in the mitochondrial fraction. However, the antibodies, which were affinity purified using the purified mitochondrial DCR or were epitope-selected using the fusion-polypeptide expressed from the mitochondrial cDNA clone (lambda gt11-RDR181), were cross-reacted with the peroxisomal DCR. These results suggest that peroxisomal DCR is immunochemically indistinguishable from mitochondrial DCR.

Animals↗

PubMed1996

[Colouration of toki-shakuyaku-san extract granules and the production of high-molecular compounds under heat-stress storages].

We previously suggested that the colouration (browning) of Toki-shakuyaku-san extract granules (TJ-23) was related to the Maillard reaction under heat-stress storage. In this study, we investigate the production of browning high-molecular compounds and discuss the relationships among the respective contents of these compounds, amino acids and reducing sugars and the colouration (delta E*(ab)) of TJ-23 under heat-stress conditions. As a result of the investigation, it was found that peaks corresponding to the high-molecular compounds were separated from the heat-stressed samples of TJ-23 at each molecular weight of about 5,000, 1,0000, 230,000 and 3,000,000, and especially the latter two peaks increased in areas up to about 11 times and 4 times at 60 degrees C from 10 through 90d respectively. At the same time, it was confirmed that the content of amino acids and reducing sugars decreased with an increase in storage temperature at 10d. Furthermore, it has become apparent that the content of high molecular compounds was correlated with colour differences (delta E*(ab)) of heat-stressed TJ-23, and then those compounds were related to the decreases of glucose and amino acids. From these results, we confirmed that the browning of Kampo medicines extract granules might proceed partially by Maillard reaction under higher temperature and/or longer time conditions.

Amino Acids↗

PubMed1996

Effects of clofibrate on flow cytometric populations in rat liver.

A flow cytometric study was performed on the liver of normal and clofibrate-treated rats. The flow cytometric patterns of these fractions showed three distinct populations (R1, R2 and R3). The R3 region was remarkably increased in the clofibrate-treated nuclear fraction, and was applied to sucrose linear density gradient centrifugation. Mt1, Mt2, Mt3 and Mt4 fractions were isolated and reacted with rhodamine-123. An essential enzyme for beta-oxidation, delta 3, delta 2-enoyl-CoA isomerase, was mainly expressed in Mt1 of the control-nuclear fraction, but not in Mt1, Mt2 and Mt4 in the clofibrate-treated nuclear fraction. These results suggest that clofibrate affects the flow cytometrical population of the mitochondria and changes the expression level of beta-oxidation enzyme(s) of the mitochondria in rat liver.

Animals↗

PubMed1996

[Alterations in brain distribution of methamphetamine in methamphetamine-sensitized animals].

[11C]Methamphetamine ([11C]MAP) was synthesized by an automated on-line [11C]methylation system for a position emission tomography (PET) study. The author newly prepared a MAP-sensitized dog by repeated treatment of MAP, and studied the brain distribution of [11C]MAP in normal and MAP.sensitized dogs. 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. A significant increase in [11C]MAP uptake into the sensitized dog brain was prevented by haloperidol and cocaine pretreatments. These results suggest that changes in the pharmacokinetic profile of MAP in the brain affect the development or expression of MAP-induced behavioral sensitization.

Animals↗

PubMed1995

Immunochemical detection of urinary 5-methyl-2'-deoxycytidine as a potential biologic marker for leukemia.

A monoclonal antibody against 5-methylcytidine was prepared and characterized. This antibody, termed AMC, was reactive with compounds that had 5-methylcytosine structure (i.e. 5-methyl-2'-deoxycytidine, 5-methylcytidine and 5-methylcytosine). AMC had the highest reactivity to 5-methyl-2'-deoxycytidine among reactive compounds and had no or very slight cross-reactivity to cytidine-related compounds and any other compounds. Analysis of immunoreactive materials in urine revealed that 5-methyl-2'-deoxycytidine rather than 5-methylcytidine was, contrary to our expectation, the major component. Then the inhibition ELISA system using AMC was established and urinary levels of 5-methyl-2'-deoxycytidine in healthy individuals and cancer patients were determined. The mean excretion levels of healthy individuals was 0.90 +/- 0.43 nmol/mumol creatinine and the cut-off value was set at the mean + 2 S.D. of healthy individuals (1.76 nmol/mumol creatinine). Among various types of cancer tested, elevated levels of 5-methyl-2'-deoxycytidine were detected in leukemia patients. From these results, urinary 5-methyl-2'-deoxycytidine might be applicable as a biologic marker for leukemia.

Animals↗

PubMed1995

Microdetermination of the prostaglandin B1 in human plasma by gas chromatography/selected ion monitoring using [18O]prostaglandin B1 as an internal standard.

We devised a simple and effective purification for the microdetermination of prostaglandin B1 (PGB1), a metabolite of PGE1. [18O]PGB1 was synthesized by the repeated base-catalyzed hydrolysis of methyl ester derivatives in H(2)18O, to obtain an internal standard for the gas chromatography/selected ion monitoring (GC/SIM) of PGB1. The methyl ester-methoxime-dimethylisopropylsilyl ether derivative was prepared, then GC/SIM was carried out by monitoring the ion at m/z 448.3 for PGB1 and that at m/z 452.3 for internal standard. A good linear response over the range of 10 pg to 100 ng was demonstrated. We detected PGB1 to a level of about 40 pg/mL in human plasma. This method can be used to determine PGB1 in biological samples.

Adult↗

PubMed1995

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

[11C]Methamphetamine ([11C]MAP) was synthesized by an automated on-line [11C]methylation system for positron emission tomography (PET) study. We newly produced a MAP sensitized dog by repeated MAP treatment and studied the brain distribution of [11C]MAP in the normal and the MAP sensitized dog. 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 uptake site of MAP.

Animals↗

PubMed1995

[Serum 1-methyladenosine and pseudouridine as tumor markers in tumor-bearing mice].

It is known that in cancer patients elevated levels of modified nucleosides originated from RNA are excreted in the urine. Modified nucleosides in the serum are thought to be more useful than those in the urine as tumor markers because they are not influenced by other factors. However the determination of these nucleosides is difficult because of their low amounts. To examine the efficacy of the modified nucleosides in the serum as tumor markers, ascites and solid tumor mouse models were prepared, and the amounts of 1-methyladenosine and pseudouridine in the serum were determined. Along with the growth of ascites tumor, the amounts of 1-methyladenosine and pseudouridine in the serum increased. The modified nucleosides in the serum in a solid tumor model also increased. This is the first report on the variation in the amount of 1-methyladenosine in the serum of tumor models, and the results suggest the usefulness of measuring the amounts of 1-methyladenosine and pseudouridine in the serum as tumor markers.

Adenosine↗

PubMed1995

Comparison of serum and urinary levels of modified nucleoside, 1-methyladenosine, in cancer patients using a monoclonal antibody-based inhibition ELISA.

To evaluate the clinical usefulness of serum 1-methyladenosine, several modifications have been made in our previously established inhibition ELISA system. Horseradish peroxidase (HRP) labeled anti-mouse IgG and 3,3',5,5'-tetramethylbenzidine (TMBZ) were used as a secondary antibody and a substrate, respectively. The second blocking was done just before the addition of the secondary antibody. The standard curve of the modified ELISA system showed good linearity between 1 and 1,000 ng/ml, and the detection limit was 50 pg/well. Using the ultrafiltrated-serum samples, serum 1-methyladenosine levels in healthy individuals and cancer patients were determined. The mean level of 1-methyladenosine in 31 healthy individuals was 28.3 +/- 7.9 ng/ml, and cut off value was set at 44.1 ng/ml (Mean+ 2SD). In cancer patients, elevated levels of serum 1-methyladenosine above the cut off value were detected in 4 out of 25 cases tested, though 11 cases had elevated urinary 1-methyladenosine levels above the cut off value (3.23 nmol/mumol creatinine). Since 1-methyladenosine has no interaction with serum proteins and its molecular weight is quite low, it might be rapidly excreted into the urine.

Adenosine↗

PubMed1995

Identification of 11-dehydro-2,3-dinorthromboxane B3 in human urine based on the mass spectral properties of 11-dehydrothromboxane B3 and related compounds.

11-Dehydro-2,3-dinorthromboxane B3 (an enzymatic metabolite of TXB3) was identified. Urine from a healthy male adult who had received daily administration of 1.8 g (0.6 g three times) of eicosapentaenoic acid (EPA) ethyl ester was collected. A urine sample was extracted with an affinity column of anti-11-dehydro-TXB2 monoclonal antibody. Conversion of 11-dehydro-2,3-dinor-TXB3 in the urine extract into the methyl ester (ME)-11-n-propylamide (PA)-9, 12, 15-dimethylisopropylsilyl (DMIPS) ether derivative was followed by gas chromatography with selected-ion monitoring (GC/SIM) (resolution: 8000) and GC/mass spectrometry (MS). High-resolution SIM result revealed that a major component corresponded to the [M - C3H7]+ (m/z 668.4198) ion, which may be specific for the structural integrity by analogy with the corresponding 11-dehydro-TXB3 derivative. Conventional GC/MS analysis confirmed that the peak of the interest was the title compound. The mass spectrum of the 11-dehydro-2,3-dinor-TXB3 ME-PA-DMIPS ether derivative was dominated by the ions of [M - CH3]+ (m/z 696) and [M - C3H7]+ (m/z 668, base peak) and ions containing a protected 11-carboxylic acid moiety together with the unidentified additional ions which were considered to derive from urinary endogenous substances. However, the quality was sufficient for use of mass spectral identification of 11-dehydro-2,3-dinor-TXB3, the beta-oxidation product of 11-dehydro-TXB3. Characteristic fragment ions of [M - C5H9-(dimethylisopropylsilanol)n]+ (m/z 524 and 406) yielding information about the position of the incorporated double bond were prominent, the same as the corresponding ions found in the mass spectrum of the 11-dehydro-TXB3 derivative. Consequently, the fragmentation products were closely related to those of the 11-dehydro-TXB3 derivative, except for an obvious shift produced by the lack of C-2/C-3 hydrocarbon units. The results demonstrate the endogenous formation of TXA3 in humans after administration of EPA ethyl ester and excretion of 11-dehydro-2,3-dinor-TXB3 into urine as one of the enzymatic metabolites of TXB3, together with the previously reported 11-dehydro-TXB3.

Adult↗

PubMed1995

Microdetermination of 11-dehydrothromboxane B3 in human urine by gas chromatography--selected-ion monitoring using (18O2) analogue as an internal standard.

The microdetermination of 11-dehydrothromboxane B3 (11-dehydro-TXB3) in human urine is described. We prepared [18O2]11-dehydrothromboxane B3 for use as an internal standard. Samples containing an [18O2] analogue were extracted chromatographically using Sep Pak tC18 and a silica gel column. Conversion of the extracted 11-dehydro-TXB3 to 1-methyl ester-11-n-propylamide-9,12,15-dimethylisopropylsilyl 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 an MP-65HT column. Human urine was monitored using the ions of m/z 696.4511 (11-dehydro-TXB3), m/z 700.4597 (18O2]1l-dehydro-TXB3), m/z 698.4668 (11-dehydro-TXB3) and m/z 702.4918 ([2H4]11-dehydro-TXB2). 11-Dehydro-TXB3 and [18O2]11-dehydro-TXB3 appeared at the same retention time and were eluted 15 s after 11-dehydro-TXB2 on this capillary column. This difference in retention time was due to the double bond in the omega3 position in 11-dehydro-TXB3. A good linear response over the range of 10 pg to 10 ng/tube was demonstrated. We detected 11-dehydro-TXB3 in the range from 1.29 to 7.64 pg/mg creatinine in the human urine. In the healthy volunteers, the 11-dehydro-TXB3 level was less than 1% of that of the 11-dehydro-TXB2, but 11-dehydro-TXB3 was increased by dietary supplementation of eicosapentaenoic acid. This method can be applied to the determination of 11-dehydro-TXB3 in human urine.

Calibration↗

PubMed1994

Microdetermination of 2,3-dinor-6-ketoprostaglandin F1 alpha in human urine using gas chromatography-high-resolution selected-ion monitoring.

The microdetermination of 2,3-dinor-6-ketoprostaglandin F1 alpha (I) in human urine is described. Samples to which the [2H4]-analogue was added as an internal standard were extracted by chromatographic sample preparation using a Bond Elut C18 cartridge and a silica gel column. Conversion of the extracted I into the 1-methyl ester-6-methoxime-9,11,15-trisdimethylisopropylsilyl ether derivative was followed by gas chromatography-high-resolution selected-ion monitoring (GC-HR-SIM). Interfering substances from the urine matrix were eliminated during GC-HR-SIM analysis using a DB-1 column. A good linear response over the range 10 pg-100 ng per tube was demonstrated. Compound I could be detected in the range 26-375 pg/ml of human urine. The proposed method can be applied to the determination of I in human urine.

6-Ketoprostaglandin F1 alpha↗

PubMed1994

Expression of mRNA encoding IFN alpha in macrophages stimulated with Lactobacillus gasseri.

The ability of Lactobacillus gasseri, a dairy lactic acid bacterium, to induce interferon (IFN) was investigated in murine macrophage cultures. IFN alpha was substantially induced by some strains of L. gasseri and the titers were the highest at a concentration of 100 micrograms ml-1 of L. gasseri DSM20243T. The expression of mRNA encoding IFN alpha was detected in spleen-macrophages (SP-M phi) and Peyer's patch-adherent cells stimulated with L. gasseri DSM20243T. Actinomycin D and cycloheximide added to SP-M phi cultures showed that the mRNA was synthesized by 0.5 h, and that IFN alpha was produced within 3 to 6 h after the stimulation with L. gasseri DSM20243T. The results support the notion that dairy products containing L. gasseri can be 'physiologically functional foods'.

Animals↗

PubMed1994

Analysis of 11-dehydrothromboxane B2 in human urine improved by use of gas chromatography/high-resolution selected ion monitoring with 18O2-labelled analogue as an internal standard.

Gas chromatography/high-resolution selected ion monitoring (GC/HR-SIM) for the determination of 11-dehydrothromboxane B2 (11-dehydro-TXB2) in human urine is described. Combined use of the lactone ring opening reaction and dimethylisopropylsilylation has been adopted for the analysis of 11-dehydro-TXB2, to give a new chemically stable derivative. The feasibility of using this derivative for the analysis of 11-dehydro-TXB2 in human urine has been evaluated. Following the addition of 18O2-labelled analogue, for use as an internal standard, the samples were extracted using chromatographic separation with Bond Elut C18 and silica gel. Derivatization of the extracted 11-dehydro-TXB2 was followed by GC/SIM at resolution of 5000. This method enables selective detection of 11-dehydro-TXB2 in human urine without serious interference from other endogenous substances. In 11 volunteers, the mean urinary excretions of 11-dehydro-TXB2 was 547.6 +/- 217.6 ng per day.

Chromatography, Gas↗

PubMed1994

The distribution of [11C]cocaine in normal and cocaine-sensitization mice.

[N-11C-methyl]-cocaine ([11C]cocaine), synthesized by N-methylation of norcocaine with [11C]CH3I, was used to assist in imaging the variety of local distribution by positron emission tomography (PET). The radiochemical yield and the radiochemical purity after purification of [11C]cocaine by high performance liquid chromatography (HPLC) at a sp. act. of 814 GBq/mmol were 47-58% and > 99%, respectively. The time required for synthesis including the purification was 25-30 min from the end of [11C]CH3I trapping. The physical distribution of [11C]cocaine in organ was also investigated in mice at various time after i.v. injection. The main accumulation of radioactivity occurred in the lung, kidney and brain within 1 min after the injection. In the brain, no differences in the organ were observed except the radioactivity level in each section increased for the first 5 min, since then radioactivity decreased dramatically. Furthermore, in the behavioral sensitization model of cocaine, the peak of [11C]cocaine uptake in each brain area was shown to be 5-15 min.

Animals↗