Neuronal-type nicotinic acetylcholine receptors (N-nAChR) are co-localized with muscle-type (M-)nAChR in the postjunctional endplate membrane of adult skeletal muscle fibers. The postsynaptic desensitizing functions of the N-nAChR at the neuromuscular junction and at single skeletal muscle cells have been investigated using aequorin luminescence and fluorescence confocal imaging. A biphasic elevation of local intracellular Ca²⁺ is elicited by prolonged nicotinic action at the mouse muscle endplates. The contractile fast and noncontractile slow Ca²⁺ components are operated by postsynaptic M- and colocalized N-type nAChR, respectively. We have named the latter slow one RAMIC (receptor-activity modulating intracellular Ca²⁺). The N-nAChR are activated by nicotine and choline, and RAMIC are antagonized by methyllycaconitine and dihydro-β-erythroidine. Neuromuscular functions may be regulated by a dual nAChR system to maintain the normal postsynaptic excitability. Certain N-nAChR may be also endowed with the same functional role in the central nervous system.

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Changes in amount and activity of enzyme protein are critical factors in regulating intracellular metabolisms. However, since the metabolisms are proceeding in environment with complex architecture consisted of various membranes, spatial factors should be taken into consideration for the regulation. In this review, involvement of interaction between cytosolic and membrane proteins in metabolic regulation are discussed. It had been reported that hexokinase activity was found in mitochondrial fraction in spite of almost exclusive distribution of other glycolytic enzymes to soluble fraction, the tendency being marked in the brain and many types of tumor cells whereas mitochondrial hexokinase activity was quite low in the liver. Interested in such enzyme and tissue specificities, we investigated the significance and mechanism of the unique intracellular distribution of hexokinase. We found that mitochondria-bound hexokinase was more active than the cytosolic type in producing glucose 6-phosphate (G6P), probably due to the advantage in utilizing ATP produced in mitochondria. In addition, we also found that the binding stabilized hexokinase against G6P inhibition. As to the binding, it was reported that G6P released hexokinase from mitochondria while Mg²⁺ promoted the binding. In this respect, we found that polyamines promoted the binding at much lower concentration than that of Mg²⁺, and mitochondria-bound form had small hydrophobic domain at terminal region for the binding to porin on the outer membrane. Then, we found a protease which specifically cleaved the domain with little effect on catalytic activity and molecular size of the bindable form. Such a modifying protease was purified and identified as lysosomal cathepsin L. The protease activity of was high in the liver and low in the brain, suggesting that the difference in the activity was responsible for the afore-mentioned tissue specificity. On the other hand, we examined regulatory mechanism for active oxygen production in neutrophils, since the production of superoxide anion (O₂^-) by NADPH oxidase was very low at the resting state while markedly increased on phagocytosis and chemical stimulation. Since the stimulants for the activation were so various in chemical nature, we postulated mechanism to converge the stimulation to the activation. Incidentally, we found increase in phosphorylation of 46-47 K protein, irrespective of the type of stimulation. Use of inhibitors and examination on the phosphorylation condition indicated protein kinase C (PKC) as the phosphorylating enzyme. In addition, we observed the 46-47 K protein existed in cytosol at resting state, while it was translocated to cell membranes in concurrence with the phosphorylation. Similar findings were obtained in many laboratories and those proteins were named cytosolic activating factors (and then p47-phox, etc.). These proteins associate with membrane proteins to constitutes the active from of NADPH oxidase. Next, we examined mechanism to shut off the O^-₂ production, and found that the inactivation through disassembly of the constituents was attained by dephosphorylation of phosphorylated p47-phox by cytosolic protein phosphatase. Then we have also found that protein kinases other than PKC were involved in regulation of NADPH oxidase activity. Though phosphorylation of p47-phox etc. is deeply involved in the activation of NADPH oxidase, membrane perturbation, so-called priming, is required for the activation. We also reported some possible indications for the priming, and possible involvement of cytoskeletons in O^-₂ production. Apart from protein phosphorylation, it has been reported that amphiphilic acidic compounds are potent activator for NADPH oxidase. We also have examined their effects to find that these compounds also caused the assembly of the NADPH oxidase constituents. [the rest omitted]

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This review summarizes our studies on the development of new reactivities of the indole nucleus and on its application for the synthesis. These studies involve the following five main subjects : 1) The Vilsmeier-Haack reaction was applied to 1, 2, 3, 4-tetrahydrocarbazole and its N-alkyl compounds. The conditions and the mechanisms of the formation of three kinds of products obtained from the latter compound were clarified, and among the three products, 1, 9-dimethylcarbazole-3-aldehyde was found to be useful for the syntheses of olivacine and ellipticine. 2) The Fischer indole synthesis of various 2-substituted phenylhydrazones was examined in detail and it was found that the Fischer indole synthesis of 2-sulfonyloxyphenylhydrazones served a new and convenient method for the synthesis of 7-oxygenated indoles. This reaction was applied to the synthesis of eudistomidin-A. 3) The reactivities of ethyl indole-2-carboxylate for acylation and bromination were also studied, and the use of this compound as a starting material for the synthesis of 4-methoxy-β-carbolines was successfully investigated. 4) Acylation of ethyl pyrrole-2-carboxylate was concisely studied and this reaction was applied to the syntheses of benzene ring-substituted indoles and benz[f]indoles involving eupolauramine. 5) Two kinds of method for the debenzylation of N-benzylindoles were developed using either AlCl₃-benzene or methyl lithium, and they are complementary with each other.

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We established microdetermination methods of prostaglandin (PG) metabolites by GC-selected ion monitoring (GC-SIM) and applied them to the clinical investigations. At first the microdetermination of Δ¹⁷-6-keto-PGF_1α, a hydrolyzed metabolite of PGI₂, is described. An authentic Δ¹⁷-6-keto-PGF_1α was prepared from eicosapentaenoic acid (EPA) incubated with a homogenate from the bovine aortic intima. [¹⁸O]Δ¹⁷-6-Keto-PGF_1α was synthesized to obtain an internal standard for GC-SIM of Δ¹⁷-6-keto-PGF_1α. A good linear response over the range of 10 pg-5 ng was demonstrated. Chromatographic conditions using a MP-65HT column presented nearly baseline separation of Δ¹⁷-6-keto-PGF_1α and 6-keto-PGF_1α. Furthermore, a monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate Δ¹⁷-6-keto-PGF_1α in the human blood sera. Using the prepared immunoaffinity columns of this antibody, Δ¹⁷-6-keto-PGF_1α was clearly detected in the human blood sera by GC/MS analysis. We were able to detect Δ¹⁷-6-keto-PGF_1α of the amount ranging from 6 to 26 pg/ml in the human blood plasma. The present method can be applied to the determination of Δ¹⁷-6-keto-PGF_1α in the human urine and plasma. Diabetes mellitus induces platelet alterations such as hyperaggregation. Variations in PG production seem to be related to this phenomenon but the changes in PG levels remain unclear. So we microanalyzed the 11-dehydrothromboxane B₂ (TXB₂) and 2, 3-dinor-6-keto-PGF_1α, which were stable metabolites of TXA₂ and PGI₂, in the urine and investigated the relationship between the thromboxane/prostacyclin (TX/PGI) ratio and diabetes mellitus. The TX/PGI ratio in the urine of diabetics was higher than that of healthy volunteers. In murine, the TX/PGI ratio of STZ-induced mice was also higher than that of non-induced mice. The ratio of db/db mice also increased with the progress of diabetes mellitus. Furthermore, we investigated the relationship between the retinal vein occlusion (RVO), a thrombotic disease in which the retinal vein is blocked by blood aggregations, and the TX/PGI ratio. The TX/PGI level in patients with the RVO, who were not combine diabetes, was significantly higher than that in healthy volunteers. One of the causes of the RVO may be due to the variation of thromboxane production. This GC-SIM method can be used to determine the TX/PGI ratio in the urine.

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As a part of our studies on the characterization of bioactive saponin constituents of horse chestnut trees, a quantitative method using high performance liquid chromatography (HPLC) has been developed for four principle saponin constituents, such as escins Ia, Ib, IIa, and IIb, isolated from the seeds of European horse chestnut trees (Aesculus hippocastanum L., Hippocastanaceae). As an application of this HPLC method, we examined the contents and compositions of these escins in the seeds of Japanese horse chestnut trees (A. turbinata BLUME) and in several commercial materials named as "β-escin". Additionally, the distribution of escins in the Japanese horse chestnut trees was examined, and escins were found to be contained only in the seeds.

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The determination of sennoside A (SA) and sennoside B (SB) by capillary zone electrophoresis was developed. The separation of SA and SB was performed in 100 mM of the 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer (pH 10.0), and the migration time of SA and SB was found to be both less than 7 min. This method was applied to the analyses of seven commercial formulations containing SA and SB without previous treatment. The satistical comparison of the results obtained from both capillary electrophoresis and HPLC methods revealed an absolute correlation.

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Fluvastatin (FV) is a highly potent inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Recently, its antioxidant effect caused by inhibiting the formation of low density lipoproteins (LDL) in vitro has been reported. In this study, we reported the antioxidant effects of FV and its major metabolites in human (M-2, M-3, M-4, M-5, and M-7) on lipid peroxidation using rat liver microsomes. The extent of NADPH- induced microsomal (Ms) lipid peroxidation was determined by the thiobarbituric acid (TBA) assay. The antioxidant effect of each compound was shown as the percentage of inhibition on the formation of TBA reactive substances (TBARS) against the vehicle control. Probucol (PR), a potent antioxidant drug, was used as a reference control. The concentration of each compound in this experiment was set at 0.1 mM (final conc.). FV inhibited the formation of TBARS by 30 to 60% without depending on the used Ms concentrations (0.025-0.2 mg protein/ml). The antioxidant effects of M-2, M-3, and M-5 were comparable to that of FV at low Ms concentarations. At the highest Ms concentration, however, the antioxidant effects of these metabolites were considerably higher than that of FV. Inhibition of the formation of TBARS by M-4 or M-7 was approximately 30% of the control and independent of the used Ms concentrations. The antioxidant effect of PR was comparable to those of M-2, M-3, and M-5 in this study. Pravastatin (PV), a potent inhibitor of HMG-CoA reductase, reduced the formation of TBARS around 20% at 0.25 or 0.5 mg protein/ml of Ms concentrations. But the value of percentage of inhibition was around 5% at 0.1 or 0.2 mg protein/ml of Ms concentrations. In conclusion, the antioxidant effects of FV, M-2, M-3, and M-5 were found to be comparable to that of PR.

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