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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.

A rapid, specific and precise method using GC/SIM was applied to separate and quantify Aconitum alkaloids in Aconitum tubers. The TMS derivatives of each alkaloid produced a well defined peak on selected ion recording (SIR). Good linear response over the range of 100 pg - 7.5 ng was demonstrated for each alkaloid. Furthermore, we investigated the changes in the contents of Aconitum alkaloids, i.e., hypaconitine, mesaconitine, aconitine, and jesaconitine, in Aconitum tubers paying special attention to the harvesting season. Mesaconitine had the highest value in all seasons. Mesaconitine and aconitine showed the highest value in the samples harvested in May 1991 while hypaconitine had the highest value among those harvested in December 1990. As to the total amount of the alkaloids, the highest value, 4190 microg/g, and the lowest value, 1881 microg/g, were observed in the samples harvested in May and September, respectively. GC/SIM was very useful for the determination of Aconitum alkaloids in the tubers.

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.

The microdetermination of 2,3-dinor-6-ketoprostaglandin F 1α ( I ) in human urine is described. Samples to which the [ 2 H 4 ]-analogue was added as an internal standard were extracted by chromatographic sample preparation using a Bond Elut C 18 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.

[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 814GBq/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 l 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.

The purpose of the study was to evaluate the quality of Toki-shakuyaku-san extract granules (TJ-23) using chemiluminescence (CL). A linear relationship was obtained between the log value of the CL of TJ-23 and the reaction temperature. An excellent correlation (r = 0.999) was found between the slope of this curve (delta A) and the colour intensity due to the browning reaction occurring at the early stage of the Maillard reaction.

Background. Modified nucleosides such as 1-methyladenosine and pseudouridine exist as minute components of transfer ribonucleic acid (tRNA) and are excreted in the urine in large amounts in the presence of malignancy. Although use of these modified nucleosides as tumor markers has long been studied and many reports have detailed their relationship with malignant tumors and the urinary excretion of various modified nucleosides, there have been no reports on modified nucleosides in esophageal carcinoma. Methods. Monoclonal antibody patterns against 1-methyladenosine and pseudouridine were studied in esophageal carcinoma, freshly resected esophageal carcinoma tissue specimens fixed in 10% neutral formaldehyde solution, embedded in paraffin, and sectioned for immunohistochemical study. Inhibition enzyme-linked immunosorbent assay (ELISA) was used to examine urinary excretion of these modified nucleosides in patients with esophageal carcinoma. Results. Although rare in normal esophageal epithelium, these modified nucleosides were strongly stained in esophageal carcinoma cells. Most carcinoma cells exhibited a cytoplasmic pattern, although some cells at the infiltrating edge displayed a nuclear pattern. These modified nucleosides were intensely imaged in 11 of 12 cultured esophageal cell lines, the exception being one line that had a much longer doubling time. Using ELISA, urinary excretion of these modified nucleosides was found to be signifiantly higher in patients with esophageal carcinoma than in healthy subjects; such excretion correlated with carcinoma size and stage and tended to decrease after treatment. Conclusions. These findings indicate that the modified nucleosides 1-methyladenosine and pseudouridine may be useful as tumor markers for esophageal carcinoma.

Hinokitiol (I) and tropolone (II) showed characteristic cytotoxic effects in vitro on five kinds of human and murine cell lines and blastic lymphocytes from mouse splenocytes. The cytotoxic effect of I on the growth of murine and human tumor cell lines, including RL male-1, MH134, HL60, K562 and KATO-III was definite when examined by thymidine incorporation into DNA and its 50% inhibitory concentration (IC50) on all cells was 0.3-0.6 microgram/ml. Compound II also showed comparable cytotoxic effects on these cell lines, indicating a little lower activity when compared to I. Furthermore, I and II also completely suppressed the [3H]thymidine ([3H]TdR) incorporation of mitogen-induced blastic lymphocytes. The suppressive activity on mouse lymphocyte proliferative response to concanavaline-A was also found with both compounds at a low concentration of 0.32 microgram/ml. As compound I is known to be of fairly low toxicity (LD50: 453 + 24 mg/kg in mice), the antitumor and immuno-suppressive effect of hinokitiol (I) should be further investigated.

Alterations in brain distribution of 11C-MAP, 14C-MAP, and 123I-IMP in MAP- and cocaine-sensitized animals were examined to investigate the mechanism involved in increased dopaminergic transmission and behavioral sensitization. First, a significant increase in 11C-MAP radioactivity in the striatum and hypothalamus was found in the mice pretreated with MAP for 7 days. Secondly, in MAP-sensitized rats, marked increases in 14C-MAP radioactivity were found in the striatum and limbic forebrain, respectively (370% and 650%). These findings may propose a new hypothesis that subchronic MAP administration may result in a long-term change in the presynaptic cell membrane at the nerve terminal which may in turn cause an increase in both MAP and DA uptake accompanied by an increased release of DA at the synaptic cleft.

Quenching effects of anthraquinones on the extra-weak chemiluminescence (CL) derived from lipid peroxidation in rat brain homogenates were investigated. Such anthraquinone derivatives as emodin, rhein, and alizarin quenched the CL, while anthraquinone did not quench the CL. A linear relationship between CL-quenching activity and inhibitory rate of malondialdehyde production of various compounds was demonstrated. This technique is useful for the screening method of antioxidants.

A simple and sensitive semi-micro high-performance liquid chromatography (HPLC) was established for determining the serum levels of glycyrrhizin (GL) in humans. Butyl p-hydroxybenzoate was used as the internal standard and serum was deproteinized by methanol. The samples were separated on a Capcell Pak C18 UG120 column (150 x 1.5 mm i.d.; particle size, 5 microm). The detection limit of GL in serum was 100 ng/ml, which enables determination of serum levels of GL after administration of a therapeutic dose. The time-course study suggested that the elimination rate of GL differed between subjects for the same administered dose, although the sample was too small to allow a meaningful comment. In clinical practice, GL is used for its antiviral and anti-inflammatory effects. Excessive administration of GL can induce pseudoaldosteronism; however the optimal GL concentration in serum remains to be determined. The determination method reported here is expected to aid in the safe and efficient use of the drug in clinical practice.

The subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase [EC 5.3.3.8] and the inducing effect of clofibrate, a peroxisomal proliferator, on the enzyme activity were examined in rat liver. From the results of spectrophotometric investigation of the fractions, which were prepared by sucrose discontinuous gradient centrifugation from the light mitochondrial fraction, the isomerase activity was found in the fractions enriched in mitochondria and those enriched in peroxisomes of the control and the clofibrate treated rat livers. The anti-isomerase antibody reacted with both the mitochondrial isomerase and the peroxisomal isomerase, revealing a single band with an apparent molecular weight of 30,000. However, the isomerase was induced by clofibrate administration mainly in the mitochondrial fraction. These results suggest that delta 3,delta 2-enoyl-CoA isomerase is located in the mitochondria and the peroxisomes of the normal rat liver, and that the isomerase in the mitochondria is induced by clofibrate administration.

A new, highly selective procedure for the determination of 11-dehydrothromboxane B2 (11-dehydro-TXB2) in human urine is described. Following the addition of [19,19,20,20-2H4]11-dehydro-TXB2 as an internal standard, samples were extracted with an affinity column of anti-11-dehydro-TXB2. Conversion of the immunoextracted 11-dehydro-TXB2 into its 1-methyl ester-11-n-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative was followed by gas chromatography-selected-ion monitoring. The mass spectrum of the 11-dehydro-TXB2 derivative was dominated by the base peak ion of [M-C3H7]+ at m/z 698, which accounted for more than 10% of the total ion current. A typical result showed that the immunoaffinity purification procedures provided an extremely clean alternative to more conventional methods of chromatographic fractionation, and that interfering substances from the urine matrix were almost entirely eliminated during the microanalysis.

Quenching effects of purine and pyrimidine derivatives on the extra-weak chemiluminescence (CL) derived from the Maillard reaction of L-lysine with D-arabinose were investigated. The pyrimidine derivatives 2'-deoxy cytidine, uridine, and uracil quenched the CL. Cytidine did not quench the CL. Purine derivatives, e.g. uric acid and 1-methyl adenosine were particularly effective in quenching the CL. 5-Methyl adenine and xanthine also quenched the CL, but adenosine had no effect. A comparison of the CL-quenching abilities of compounds that have common basic structure was made; those with ribose at the 5-position were the strongest quenchers. A linear relationship between CL-quenching activity and the HOMO energy of the pi orbital for the various compounds was shown.

N-Oxidation of pyrimidines having unsymmetrically substituted 4-and 6-positions with hydrogen peroxide in acetic acid gave mixture of their 1-oxides and 3-oxides. Ring transformation of these 1-oxides and 3-oxides into isoxazoles occurred on treatment with mineral acid, and provided information on the position of the N-oxide group in the pyrimidine N-oxides. Nuclear magnetic resonance spectroscopy with a lanthanide shift reagent was also found to be useful in identifying the pyrimidine 1-and 3-oxides.

Lipase [triacylglycerol lipase, EC 3.1.1.3] has been purified to homogeneity from Rhizopus japonicus NR 400 by chromatography on hydroxylapatite, octyl-Sepharose and Sephacryl S-200. It showed a molecular weight of about 30,000 by SDS-PAGE and a specific activity of 68,900 units/mg protein. The enzyme catalyzed the hydrolysis of tricapryn and tricaprylin rapidly in comparison with other triglycerides. This lipase had an optimum pH of around 5, and albumin enhanced its activity between pH 3 and 8. The composition of fatty acids liberated from linseed oil by the lipase was similar to that in the case of pancreatic lipase. The lipase activity was not affected by the addition of 1 mM metal ions or bile salts. Stimulation of the lipase activity was observed upon addition of albumin to the reaction mixture. Immunotitration experiments were also performed with antibodies raised against the purified lipase.

N-Acetylcysteamine and coenzyme A esters of cis-2-alkenoic acids have been found to undergo isomerization to the corresponding trans-isomers during their preparation by the mixed anhydride method and also during their alkaline hydrolysis. The isomerization might proceed by interaction of the free thiol group and the cis-double bond of 2-alkenoic thiol esters. The use of pyridine as a base and three or more equivalents of the mixed anhydride to the thiol compound prevented the formation of the trans-isomer. Addition of hydrogen peroxide during alkaline hydrolysis also prevented the isomerization completely.

2,4-Dienoyl-CoA reductase has been separated from Escherichia coli grown in the presence of linoleic acid and purified to homogeneity. The enzyme has a molecular weight close to 50,000 as determined by gel filtration on Sephacryl S-200 Super-fine. The reductase was rather stable in a buffer containing citric acid and kept its full activity on heating at 55 degrees C for 10 min in the pH range of 5.5 to 6.5, but was completely inactivated on heating at 58 degrees C for 10 min. Phosphocellulose column chromatography revealed that the reductase was not involved in the multi-enzyme complex (molecular weight of 260,000) of fatty acid oxidation.

Nuclear magnetic resonance spectroscopy with a lanthanide chemical shift reagent was applied for the structural determination of cis-and trans-isomers of 3-alkenoic acids, prepared as substrates for some enzyme reactions related to fatty acid metabolism. The configuration of N-acetylcysteamine derivatives of cis-2-alkenoic acids was also determined by the same technique.

Characteristics of the secondary structure of neocarzinostatin apoprotein (apo-NCS) were examined by various means. Gaussian analysis of the Fourier-transform infrared (FT-IR) curve and curve-fitting of the circular dichroism (CD) spectrum for apo-NCS revealed that this peptide was abundant in beta-structures. In the presence of sodium dodecyl sulfate (SDS), the CD bands of NCS originating from phenylalanyl, tyrosyl, and cystinyl residues decreased, indicating a conformational change around the chromophore (CNS-chr). On the other hand, apo-NCS, in the SDS system, showed no change of these bands. We showed that the major parts of the protein moiety consist of beta-structures by measurements of the FT-IR and CD spectra of apo-NCS and a prediction of the secondary structure based on the amino acid sequence of the peptide. It seems that properties of the protein may be important to the hydrophobic interaction between NCS-chr and apo-NCS.

The potassium permanganate oxidation of 3-methylthio-5, 6-diphenyl-as-triazine in the presence of a phase-transfer catalyst afforded 3-methylsulfonyl-5, 6-diphenyl-as-triazine. The 3-sulfonyl-as-triazine readily reacted not only with active methylene compounds but also with methyl or methylene ketones under basic conditions, and 5, 6-diphenyl-as-triazines containing a functionalized carbon substituent at the 3-position were obtained. Similarly, 2-substituted 4, 6-dimethylpyrimidines were synthesized by the nucleophilic substitution of 4, 6-dimethyl-2-phenylsulfonylpyrimidine with various ketones.

NADPH-dependent trans-2-enoyl-CoA reductase was purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was estimated to be 80,000 by gel filtration and that of the subunit was estimated to be 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was most active toward trans-2-hexenoyl-CoA and trans-2-octenoyl-CoA but was less active toward longer chain substrates, whereas the Km values decreased progressively with increasing carbon chain length of substrates. The reductase appears to have a functional thiol group. The enzyme activity was rapidly decreased by p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid), and slowly by N-ethylmaleimide. The enzyme was protected from inhibition by these SH-reagents by the addition of NADPH. The enzyme was also inhibited by saturated acyl-CoA esters.

The structure of the chromophore of an antitumor antibiotic neocarzinostatin has been elucidated as a bicycro[7,3,0]dodecadiyne system having naphthalenecarboxylic acid, aminosugar and ethylene carbonate units.

The 1, 3-dipolar cycloaddition of 4-benzyloxy-6-methylpyrimidine 1-oxide (Ia) with dimethyl acetylenedicarboxylate (DMAD) gave dimethyl α-oxo-α'-(4-benzyloxy-6-methyl-2-pyrimidinyl) succinate (IIa), which was readily hydrolyzed to methyl 4-benzyloxy-6-methyl-2-pyrimidineacetate (IIIa). A similar reaction of 4-methoxy-(Ib) and 4-ethoxy-6-methylpyrimidine 1-oxide (Ic) gave corresponding methyl 2-pyrimidineacetates (IIIb, c). On the other hand, 6-methyl-4-piperidinopyrimidine 1-oxide (Id) reacted with DMAD to give a betaine derivative, 1-(6-methyl-4-piperidino-1-pyrimidinio)-1, 2-bis (methoxycarbonyl) ethenyl-2-oxide (IV).

2,4-Dienoyl-CoA reductase has been detected in crude extracts of E. coli. The reductase was shown to be induced many fold when the cells were grown in the presence of linoleic or oleic acid. The activity profile of the reductase on gel filtration was different from those of other enoyl reductases. These results suggest that there are two pathways even in E. coli for the degradation of cis-4-decenoyl-CoA, which is an intermediate in the beta-oxidation of linoleic acid, as recently proposed in rat liver.

AMP deaminase from sheep brain was purified to homogeneity on SDS-PAGE and its general properties were investigated. The native enzyme has a molecular weight of approximately 350,000 as estimated by gel filtration and it is composed of four identical subunits with a molecular weight of 85,000 each. The purified enzyme had a specific activity of 500 units/mg protein and shows a sigmoid-shaped AMP saturation curve in the presence of 100 mM KCl. This deaminase is strongly activated by ATP and inhibited by GTP. It slightly catalyzes the hydrolysis of adenosine monosulfate (AMS), dAMP, and adenosine phosphoramidate (APA). These catalytic properties resemble those of AMP deaminase from human liver.

A gas chromatography/selected ion monitoring (GC/SIM) method for the simultaneous determination of aconitum alkaloids such as aconitine, mesaconitine and hypaconitine has been developed. Aconitum alkaloids were converted to their TMS ether derivatives by treatment with N, O-bis (trimethylsilyl) trifluoroacetamide in pyridine at room temperature. The gas chromatograms of reaction products showed in each case a well-resolved doublet consisting of a major (>70%) and a minor components, when analyzed using a 5%-phenylmethylsilicone cross-linked fused silica capillary column. These alkaloid derivatives gave simplified mass spectra with a base peak at m/z 698 for aconitine, m/z 684 for mesaconitine and m/z 596 for hypaconitine. When GC/SIM of aconitine was carried out under monitoring of the ion at m/z 698, detection limit was 10 picograms with signal-to-noise ratio of greater than 10. Linearity was obtained over a range from at least 10 to 1000pg. Similar results were obtained with another alkaloids. The use of this method for the measurement of aconitum alkaloids in biological fluids is described.

Immunocytochemical localization of Δ3, Δ2-enoyl-CoA isomerase (isomerase) was investigated in rat liver. Livers of di-(2-ethylhexyl)phthalate) (DEHP)-treated or untreated rats were perfusion-fixed and embedded in Epon or Lowicryl K4M. By light microscopy, reaction deposits for the enzyme were present in the cytoplasmic granules of hepatocytes and interlobular bile duct epithelium. Weak staining was noted in sinus-lining cells. After administration of DEHP, the granular staining of the hepatocytes was markedly enhanced, whereas the staining reaction of the sinus-lining cells decreased. The isomerase staining pattern was quite similar to that of long-chain acyl-CoA dehydrogenase (a mitochondrial marker), but different from that of catalase (a peroxisomal marker). Under electron microscopy, gold particles for isomerase were seen to be confined mainly to mitochondria of the hepatocytes, the bile duct epithelial cells and sinus-lining cells. Peroxisomes were weakly labeled. After DEHP administration, the peroxisomes were markedly induced, but the mitochondria were not. Quantitative analysis showed that the induction of the peroxisomal isomerase was only 2-fold whereas the mitochondrial isomerase was enhanced about 5-fold, 40 times as high as the peroxisomal enzyme. The results show that the mitochondria are the main intracellular site for isomerase and the peroxisomes a minor site. The mitochondrial isomerase of the rat liver is markedly induced by peroxisome proliferators, DEHP and clofibrate.

Localization of 2,4-dienoyl-CoA reductase (DCR) in rat liver was studied using immunoenzyme and immunogold techniques. The animals were fed on a laboratory diet with or without 2% di-(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for two weeks. For light microscopy (LM), semithin Epon sections were stained by immunoenzyme technique after removal of the epoxy resin. For electron microscopy (EM), ultrathin Lowicryl K4M sections were stained by the protein A-gold technique. By LM, in untreated rats reaction deposits showing the antigenic sites for DCR were present in the cytoplasmic granules. Hepatocytes, epithelial cells of interlobular bile duct, and sinus-lining cells contained these granules. After administration of DEHP, the cytoplasmic granules stained similarly. The staining intensity of the heaptocytes increased markedly, but that of the other cells decreased. The sinus-lining cells became mostly negative. By EM, gold particles indicating the antigenic sites for DCR were present in both the mitochondria and peroxisomes of hepatocytes of untreated rats. In the other cells, the gold label was confined to the mitochondria. After administration of DEHP, labelling intensity of the hepatocyte mitochondria increased markedly, but that of the peroxisomes conversely decreased. Quantitative analysis of labelling density showed that the mitochondrial DCR increased to about three times that in the untreated rat, but the peroxisomal DCR decreased to 1/6. The results show that in the rat liver, DCR exists in both, mitochondria and peroxisomes. DEHP can induce mitochondrial DCR, but not peroxisomal DCR.

A monoclonal antibody specific for a modified nucleoside, 1-methyladenosine, was prepared and characterized. This antibody, termed AMA-2, reacts with 1-methyladenosine and 1-methyladenine but not with other nucleosides, particularly methylated adenosines other than 1-methyladenosine and methylated guanosines, tested in this investigation. In our experiments, AMA-2 was used in an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of the levels of 1-methyladenosine in urine. Sensitivity was in the picomole range and accuracy was nearly equal to that of the high-performance liquid chromatography (HPLC) assay system. Urinary levels of 1-methyladenosine in healthy donors and patients with various advanced cancers were determined by the inhibition ELISA. The amount of 1-methyladenosine in urine of 33 healthy donors was 1.91 +/- 0.66 nmol/mumol creatinine. In 54% (51/94) of patients, urinary 1-methyladenosine was elevated above the mean plus 2 standard deviations for the healthy donors (3.23 nmol/mumol creatinine). In patients with leukemia, esophageal cancer, stomach cancer, colon cancer, and bladder cancer, urinary levels of 1-methyladenosine were significantly elevated. In patients with leukemia, urinary 1-methyladenosine levels changed almost in parallel with the change in the clinical response during chemotherapy. These results suggest that urinary 1-methyladenosine might be useful in monitoring the effectiveness of therapy.

Many tricyclic antidepressant drugs such as imipramine hydrochloride showed high extraweak chemiluminescence (CL) with similar emission spectra. Iminodibenzyl and N-methylimino-dibenzyl, compounds saturated at the 10-11 bond, generated high CL, whereas iminostibene, a compound unsaturated at the 10-11 bond, did not. Mechanisms of CL produced by autoxidation of imipramine hydrochloride were investigated. The existence of the imipramine radical and the imipramine peroxy radical during the autoxidation were supported by an electron spin resonance study. A possible pathway is proposed for CL generation from imipramine hydrochloride by autoxidation.

A series of 5-amino-3-phenylisoxazole derivatives was synthesized and evaluated as part of a search for new types of centrally acting muscle relaxants. The derivtives with an alkylaminopropyl moiety exhibited muscle relaxant activity comparable to that of tolperisone (1), but this activity was accompanied by strong general central nervous system (CNS) depressant and slight anticonvulsant activities.On the other hand, the derivative with a propanamide moiety exhibited a favorable pharmacological profile for a selective muscle relaxant with little CNS depressant action.

A simple and rapid method for the determination of paraquat in the serum was developed by a modification of the method originally reported by Knepil. Serum samples were treated with a mixture of chloroform-ethanol and ammonium sulfate and centrifuged to obtain a clear aqueous paraquat solution. Alkaline dithionite solution was added to the supernatant and the absorbance at 396 nm was recorded as A. A small amount of acetic acid was added to the same cuvette to distinguish the coloured paraquat radical and the absorbance at 396 nm resulted from the serum constituents was again measured as B. From the difference between A and B, the concentration of paraquat was determined in the range of 0.1-4 μg/ml serum, with the recovery of about 90%. This method posesses the advantages of 1) no need of special instrument and 2) short time for the determination. By using this method, paraquat concentration in the serum was monitored succesively during 7 d in a poisoning case.

The steric course of the reaction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase from Escherichia coli was investigated. trans-2-[6,7,8-2H7]Octenoyl-CoA was synthesized as a substrate and incubated with partially purified trans-2-enoyl-CoA reductase in the presence of 4R- or 4S-[4-2H1]NADPH. The deuterium-labeled octenoyl-CoA was also incubated with the reductase in the presence of NADPH in 2H2O. Aliquots of octanoic acids formed were analyzed, after esterification, by gas chromatography-mass spectrometry (GC-MS) to demonstrate that the pro-4R hydrogen of NADPH was incorporated into the C-3 position of octenoyl-CoA and that hydrogen from water was introduced into the C-2 position of octenoyl-CoA. The remaining portions of octanoic acids isolated from the incubation mixtures were converted to their CoA esters by the action of acyl-CoA synthetase, and they were dehydrogenated by treatment with acyl-CoA oxidase, which had previously been shown to catalyze the anti-elimination of the pro-2R and pro-3R hydrogen atoms of acyl-CoA. The deuterium contents of the products were also analyzed by GC-MS, and the results indicated that the reduction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase occurred by an anti-addition of hydrogen via a 2-Re, 3-Re attack on the trans-double bond of the substrate.

Crude extract of Escherichia coli was found to elongate medium chain acyl-CoA primers. The reaction products were fatty acids one or two C2 units longer than the primer. Acetyl-CoA acted as the condensing unit in this reaction, while malonyl-CoA did not. The optimal pH for the reaction was 5.0 in 0.1 M citrate-phosphate buffer. NADH was the predominant electron donor for the incorporation of acetyl-CoA into fatty acids, and NADPH was one-third as effective as NADH at pH 5.0. Acyl carrier protein and cerulenin had no effect on the acetyl-CoA incorporation into the chain elongation products. Acyl-CoA compounds with medium carbon chain lengths proved to be the best as primers, and the maximum incorporation was observed with octanoyl-CoA. N-Ethylmaleimide and p-hydroxymercuribenzoate blocked the chain elongation reaction by inhibiting either condensation or 3-ketoacyl reduction.

The mass spectrum exhibited a series of ions characteristic of the expected 11-dehydrothromboxane B2 (11-dehydro-TXB2) derivative. Many of the fragmentation products could be explained in terms of simple bond fission mechanisms. A further series of fragmentations proceeded via a six-membered ring transition state or by formation of the cyclic dimethylsilylene ring system. These produced ions in the low mass region containing silicon atoms, characteristic of the protected C-11 carboxylic and moiety. The appearance of these ions may be used for the identification of 11-dehydro-TXB2 and its metabolites having a lactone ring. Six homologue and analogue derivatives were prepared for investigation of the fragmentation mechanism. The proposed fragmentation mechanisms have been supported by accurate mass measurement and mass analyzed ion kinetic energy mass spectrometry.

In the presence of a nickel phosphine complex, the coupling reaction of Grignard reagents with chloropyrimidines such as 2-chloro-4, 6-dimethyl-(III), 4-chloro-2, 6-dimethyl-(V), 4, 6-dichloro-2-methyl-(VIII), 2, 4-dichloro-6-methyl-(X), and 2, 4, 6-trichloro-pyrimidine (XIII) was studied to give the corresponding alkyl (or arkyl) substituted pyrimidines in yields ranging from 40 to 88%. Although in the cases of VIII, X, and XIII, the selectivity on the reaction due to the position of the chloride atom was not observed, this coupling was concluded to remove the defects of the Pinner reaction for the synthesis of polyalkyl (or polyaryl) pyrimidines.

In order to investigate the generality of a chain-elongation reaction observed in the metabolism of fusaric acid (5-butyl-2-pyridinecarboxylic acid) and its derivatives, various heteroaromatic (diazine, 1, 3-azole, and 1, 2-azole) carboxylic acids with a normal alkyl side-chain were synthesized.

It is demonstrated that 3-alkenoyl-CoA is the product of the enzymic reduction catalyzed by 2,4-dienoyl-CoA reductase with the highly purified enzyme preparation from beef liver mitochondria. Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase from beef liver was investigated. When trans-2, trans-4-decadienoyl-CoA was incubated with the reductase in the presence of 4R-[4-2H1]NADPH and H2O, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to pyrrolidine amides of fatty acids. When the substrate was incubated with the enzyme in the presence of 4S-[4-2H1]NADPH and H2O, one deuterium atom was incorporated into the product, 3-decenoate, at the C-5 position. In the case of 3-decenoate enzymatically prepared in the presence of NADPH and 2H2O, two deuterium atoms were incorporated into the product at the C-2 position. These results indicate that the reaction catalyzed by 2,4-dienoyl-CoA reductase from beef liver is a unique example of 1,4-addition of hydrogen to a 1,3-diene system conjugated with a carbonyl group of a thioester in biochemical fields. Chain length specificity of the reductase, isotope effects on reaction rates and competitive inhibitors are also discussed.

A tricyclic antidepressant, 11C-labeled imipramine was synthesized by N-methylation of desipramine with 11CH3I to assist in the imaging of the human imipramine receptor by positron emission tomography. The radiochemical yield after purification of 11C-imipramine by high performance liquid chromatography was 28-63% at a specific activity of 26-53 Ci/mmol. The time required for synthesis, including purification was 30 min from the end of 11CH3I trapping. The organ distribution of 11C-imipramine was investigated in mice at various times after i.v. injection. The main accumulation of radioactivity was in the kidney, followed by the lung and the heart. In the brain, the radioactivity levels in the hypothalamus and striatum were the highest and remained constant, differentiating them from other portions of the brain. Furthermore, the result of a binding assay with 3H-labeled imipramine suggested that the regional distribution fo 11C-imipramine in the same mouse brain correlated to that of the high affinity imipramine binding site.

Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli (E. coli) was investigated. When trans-2,cis-4-decadienoyl-CoA was incubated with 4R- or 4S-[4-2H1]NADPH in the presence of purified 2,4-dienoyl-CoA reductase, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to its pyrrolidine amide. On the other hand, when the dienoyl-CoA was incubated in the presence of NADPH and the reductase in 2H2O, two deuterium atoms were incorporated: One deuterium atom was located at the C-4 position of trans-2-decenoate, and the other at the C-5 position. The UV and shorter wavelengths of the visible spectrum of the reductase solution revealed that the reductase contained flavin as a prosthetic group. Therefore it is considered that a hydrogen atom of NADPH was first transferred to the flavin moiety of the reductase, and then the hydrogen atom was rapidly exchanged for one in the medium before its direct transfer to the substrate.

Alterations in the activities of enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase, and 2,4-dienoyl-CoA reductase in rat liver mitochondria and peroxisomes caused by clofibrate were compared in order to clarify the metabolic pathways for unsaturated fatty acids. Our results suggest that there are two pathways for fatty acids having (a) double bond(s) at the even-numbered carbon atom(s) both in mitochondria and in peroxisomes. One is the hydratase-epimerase participating pathways, and the other is the reductase participating pathway. It is considered that the former in mitochondria occurs mainly under normal conditions, and the latter becomes significant when the degradation of fatty acids is stimulated.

Two fractions containing NADPH-enoyl CoA reductase activities were isolated from crude extracts of Candida albicans. One fraction (Type-I) having larger molecular weight utilizes 5-hydroxyundec-cis-2-enoyl CoA, oct-cis-2-enoyl CoA and oct-trans-2-enoyl CoA as substrates with Km values of 2.5×10-6M, 1.1×10-6M and 5.0×10-7M, respectively, while another fraction (Type-II) having smaller molecular weight utilizes these substrates with Km values of 3.0×10-6M, 5.3×10-5M and 1.0×10-5M, respectively. This indicates that there exist comparable differences between these two in their affinities especially for the latter two substrates. Some characterizations, such as, effects of pH and of heat treatment on the activities of these reductase preparations were also investigated.