We have developed a quick, highly sensitive immunoassay method for drugs by latex agglutination inhibition. An antiserum against primidone (PRM) was obtained by immunizing rabbits with PRM-bovine serum albumin conjugate. PRM-rabbit serum albumin conjugate sensitized latex was agglutinated with diluted antiserum, and the agglutination was inhibited by free PRM quantitatively. Turbidity of the agglutination suspension was measured by spectrophotometry as absorbance. Larger latex gave higher sensitivity than the smaller, because its agglutination was inhibited more intensely by free PRM. The assay values of this method were correlated well with those obtained by an enzyme immunoassay method.

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In order to obtain specific antisera to digitoxin, four new types of hapten-bovine serum albumin (BSA) conjugates were synthesized from digitoxin. The haptens were linked to the carrier protein through hemisuccinate and hemisuccinylglycine bridges at the C-3'and C-3"positions in the digitoxose chain. The antisera were prepared by immunizing rabbits with each digitoxin-BSA conjugate and the properties of the antisera were investigated by RIA with ³H-labeled digitoxin. Among these antisera, the antiserum raised against digitoxin 3'-hemisuccinate-BSA conjugate possessed high specificity for digitoxin, exhibiting only minor cross-reactions with digitoxigenin bisdigitoxoside (4.0%), dihydrodigitoxin (2.8%), digoxin (2.4%), digitoxigenin monodigitoxoside (0.23%) and digitoxigenin (<0.05%).

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To elucidate insecticidal activity of spider toxins, metal ions in venoms and in the bodies were determined by thin layer chromatography, spark source mass spectrometry, ion chromatography, inductively coupled plasma emission spectrometry and atomic absorption spectrometry. Two kinds of spiders were used, Nephila clavata and Nephila maculata. Metals from their venom glands were extracted with hydrochloric acid and the metal concentrations were almost the same in the two species. Many kinds of metals, Fe, Zn, Pb, Cu, Ca, Mg, Na, P and S were found at higher levels in the venoms at concentrations higher than in the bodies. The contents of metal ions were low in the dragonfly and the cicada which are considered to be preys. Clavamine, the main insecticidal component in N. clavata, was effective on larvae of a mosquito with Ca²⁺, Fe³⁺ or Pb²⁺, but ineffective with Mg²⁺, Zn²⁺, Fe²⁺ or Cu²⁺. It is suggested that the metal chelates play an important role in the intoxication and detoxication of the spider toxins.

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Incubation of papain (EC 3.4.22.2) with ascorbic acid (AsA) and Cu²⁺ in acetate buffer (pH 5.6) results in an irreversible loss of enzyme activity by site-specific generation of free radicals [H. Kanazawa, S. Fujimoto, A. Ohara, Biol. Pharm. Bull., 16, 11 (1993)]. In this study, the effect of some compounds, known free radical scavengers, on the relationship between the inactivation of papain by the Cu²⁺ -AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu²⁺ -AsA system, although hydrogen peroxide (H₂O₂) by itself, known to be generated during the autoxidation of AsA, did not inactivate the enzyme. The oxidation of AsA was unaffected by catalase. Both thiourea and sodium thiocyanate completely protected the enzyme from inactivation, while AsA was partially oxidized only in the initial stage. In the presence of potassium iodide, both the inactivation of the enzyme and the oxidation of AsA were characterized by a rapid initial phase followed by a stable phase where no reaction took place and, subsequently, a slower phase. Histidine partially prevented the inactivation of the enzyme and the oxidation of AsA. The present results suggest that H₂O₂ serves as a source of secondary, highly reactive species, probably hydroxyl radicals, which are responsible for the inactivation, and that the protection from inactivation by some radical scavengers, such as thiourea, sodium thiocyanate, potassium iodide, and histidine, is based on the removal of metal ions (Cu²⁺ or Cu⁺) at the specific site of inactivation.

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The effect of the trichosporin-Bs, peptide antibiotics, on the respiration of mitochondria was investigated. Trichosporin-Bs stimulated the respiratory rate of state 4 rat liver mitochondria in a dose-dependent manner. The maximum respiratory rate obtained by trichosporin-Bs was essentially the same as for 2, 4-dinitrophenol and SF6847. Trichosporin-B-VIb released oligomycin-inhibited respiration of mitochondria. This means that trichosporin-Bs are uncouplers of oxidative phosphorylation. The stimulatory effect of trichosporin-B-VIb, a component of trichosporin-Bs, on mitochondrial respiration was increased by inorganic phosphate but not by other permeant anions studied. These results suggest that the stimulation of mitochondrial respiration by trichosporin-B-VIb is mediated by the same mechanism as is operating in the case of hypelcins and alamethicins. Furthermore, the relative potencies of trichosporin-Bs on the mitochondrial respiration and their relative hydrophobicities were examined. A clear relationship was observed between the uncoupling potencies of trichosporin-Bs and their relative hydrophobicities.

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We classified hepatic lesions spontaneously developed by Long-Evans with a cinnamon-like coat color (LEC) rats into the following four stages : Normal liver, acute hepatitis, chronic hepatitis, and hepatoma, by biochemical tests of the sera, and anatomical and histopathological examination of the livers. Hepatocyte growth factor (HGF) activity in the sera of LEC rats which developed acute hepatitis, chronic hepatitis, and hepatoma was higher than that of normal LEC rats. In paticular, HGF activity in the sera of the LEC rats with acute hepatitis was about 70-fold that of normal LEC rats. However, primary cultured hepatocytes of LEC rats with hepatic lesions were hardly proliferated by stimulation with EGF and insulin in vitro or with increased HGF in vivo. These results suggest that the hepatocytes of LEC rats with hepatic lesions disorder the signal transduction of growth factors.

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To clarify the role of cholinergic neurons in the amygdala on learning and memory, scopolamine was injected topically into the bilateral amygdala of mice, and the ability to perform two types of passive avoidance tasks (step-through and step-down) was investigated. On the first day mice performed the learning trial and on the second day their retention was tested. Scopolamine (0.5 or 1μg/site) was injected bilaterally into the amygdala 30 min before or immediately after the learning trial or 30 min before the testing trial. Scopolamine impaired the performance ability of the mice dose-dependently only when it was injected 30 min before the learning trial. The results coincided well with the effect of scopolamine injected intraperitoneally. Taken together, these results suggest that the site of action for scopolamine to cause anterograde amnesia is the amygdala, and that cholinergic neurons projecting to the amygdala play an important role in memory acquisition in the two passive avoidance tasks.

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Charcoal transport, as an indicator of the degree of peristalsis, and water content in the large intestine after the intracaecal administration of barbaloin, were measured simultaneously in the same rat. Charcoal transport was significantly accelerated at both 3.5 and 6.5 h after the administration of barbaloin. At 6.5h, diarrhea instead of normal faeces was observed. Moreover, at 1 h before the acceleration of charcoal transport, a marked increase in the relative water content of the large intestine was observed. It appears that the increase in water content of the large intestine induced by barbaloin precedes the stimulation of peristalsis, attended by diarrhea. Therefore, it is suggested that the increase in water content is a more important factor than the stimulation of peristalsis in the diarrhea induced by barbaloin.

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New substituted 1, 8-naphthyridin-2 (1H)-ones have been found to exhibit highly selective phosphodiesterase (PDE) IV inhibition. These compounds inhibited polymorphonuclear leukocyte activation induced by N-formylmethionyl-leucyl-phenylalanine and caused relaxation of isolated guinea pig trachea precontracted by histamine or leukotriene D₄. In anesthetized guinea pigs, presensitized with the antigen, these compounds also alleviated airway constriction induced by the antigen. Since these compounds differ in their chemical structure compared with theophylline and other PDE IV inhibitors so far reported and some of them have been shown to be well tolerated in acute toxicity studies, they will provide a new tool for investigating the possible relationship between PDE IV inhibition and anti-asthmatic activity.

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The effect of 3, 9-bis (N, N-dimethylcarbamoyloxy)-5H-benzofuro [3, 2-c] quinoline-6-one (designated as KCA-098) on the bone mineral metabolism of chick embryonic bone was examined. KCA-098 dose-dependently inhibited bone resorption of cultured chick embryonic femora and calvariae. It increased the length, dry weight, and calcium and phosphorus contents of 9-d-old chick embryonic femurs cultivated for 6 d, indicating that it stimulated bone formation. These results show that KCA-098 has the unique effects of inhibiting bone resorption and stimulating bone formation of chick embryo. In addition, in an in vivo experiment, oral administration of KCA-098 (3.0 mg/kg/d) for 16 weeks led to an increase in calcium and phosphorus content as well as an increase in the amount of force required to break the femur from ovariectomized rats, suggesting that it may be useful for the treatment of bone diseases.

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Effects of malonylginsenoside Rb₁ (GRb_1-m) isolated from dried root of Panax ginseng C. A. MEYER (Araliaceae) on neuronal survival and neurite outgrowth were compared with those of ginsenoside Rb₁ (GRb₁) using organ culture of chick embryonic dorsal root ganglia (DRG) and cell culture of DRG neurons. In the organ culture, nerve growth factor (NGF) showed neurite outgrowth promoting effect. GRb_1-m(30μM) significantly potentiated this NGF-induced neurite outgrowth with similar potency to that of GRb₁ (30μM). Low density cell culture of DRG neurons was employed to minimize the contribution of contaminating non-neuronal cells. NGF (10 ng/ml) prolonged duration of neuronal survival. Neither GRb_1-m nor GRb₁ (1-30μM) changed the prolongation effect of NGF, nor did NGF show a significant effect on the neurite elongation and re-elongation after axotomy by laser beam irradiation. However, GRb_1-m (10μM) potentiated initial neurite elongation when co-treated with NGF. In the process of re-elongation of neurites, GRb_1-m (1, 30μM) also promoted it in the presence of NGF. These results suggest, first, that GRb_1-m potentiates NGF-induced neurite outgrowth of chick embryonic DRGs and DRG neurons, but behaves in a slightly different manner from GRb₁, and, second, that the effects of the two saponins may work primarily on neurons causing the potentiation of NGF-induced neurite outgrowth.

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The hypnotic activity of N³-phenacyluridine in 2.0μmol/mouse by intracerebroventricular (i.c.v.) injection was 20 times stronger than that of known N³-benzyluridine. In 0.5μmol/mouse, i.c.v., this compound strongly potentiated both pentobarbital- and diazepam-induced sleep as compared to N³-substituted uridines, including N³-benzyluridine. Furthermore, the compound caused motor incoordination as well as decreasing spontaneous activity in the same dose. These results indicate that among the N³-substituted uridines and related compounds previously reported, N³-phenacyluridine possesses potent depressant effects.

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We investigated the possible protective effects of benidipine (Coniel^[○!R]), a calcium antagonist, on mechanical dysfunction, metabolic damage and changes in vascular reactivity during ischemia and reperfusion in the Langendorff-perfused rat heart. The responses of perfusion pressure to U-46619, a vasoconstrictor, and acetylcholine, an endothelial-dependent vasodilator, were also determined as indices of the vascular function. Thirty min of reperfusion following 30 min of global ischemia produced contractile failure and the marked release of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK). Additionally, the ischemia and reperfusion augmented the vasoconstrictor response to U-46619, and depressed the endothelium-dependent vasodilator response to acetylcholine. These hearts were treated with 1 or 10 nM benidipine from 20 min before ischemia to the beginning of ischemia. While benidipine at 10 nM had a modest negative inotropic action, 1 nM of this drug had minimal depressant effects on the preischemic function. The depressed contractile function after the ischemia was improved, and the increased releases of LDH and CPK were significantly ameliorated by benidipine. Also, benidipine restored the augmented contractile response to U-46619 and preserved the vasodilator response to acetylcholine. These results demonstrate that pretreatment with benidipine prevents myocardial injury following ischemia and reperfusion. The cardioprotective effects of benidipine may in part be due to the protection of vascular reactivity by this drug.

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The present investigation was undertaken to compare the effects of β-alanyl-L-histidinato zinc (AHZ) and its zinc-chelating ligands on bone metabolism in tissue culture. Calvaria were removed from 3-week-old male rats and cultured for up to 72h in Dulbecco's modified eagle medium containing zinc sulfate, AHZ, di (N-acetyl-β-alanyl-L-histidinato) zinc (AAHZ), and di (histidino) zinc (HZ). The bone calcium content and alkaline phosphatase activity were significantly increased in the presence of AHZ or AAHZ (10^-8-10^-5 M). Those increases were seen at 10^-7 to 10^-5 M zinc sulfate and HZ. The bone deoxyribonucleic acid (DNA) content was significantly increased by AHZ or AAHZ with 10^-7 to 10^-5 M, while 10^-7 M zinc sulfate and HZ had no effect. Thus, AHZ and AAHZ had more potent effect than that of zinc sulfate and HZ. The effect of AHZ, AAHZ and HZ (10^-5 M) increasing bone alkaline phosphatase activity was abolished by the presence of 10^-4 M dipicolinate, a chelator of zinc. Moreover, the effect of these zinc compounds on bone metabolic indices was not seen in the presence of 10^-6 M cycloheximide. The present results suggest that the effect of AHZ and AAHZ on bone metabolism is more potent than that of zinc sulfate and HZ.

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Several antitumor anthracyclines, including those in preclinical stages, were examined for their action in reversing tumorous phenotypes of H- or K-ras 3T3 cells (NIH3T3 cells transformed by human H- or K-ras oncogene) into normal phenotypes, such as flattened cell morphology, anchorage dependent cell growth, etc. (referred to as anti-ras activity). The study elucidated relationships between the chemical structure of anthracyclines and the anti-ras activity. The human tumor cell line T24, which has a mutated H-ras gene, responded to the anthracyclines, as did K- or H-ras 3T3 cells, in respect to the phenotypic alterations. Pirarubicin was more than 4 times as active as aclarubicin in inhibiting the growth of solid tumors of K-ras 3T3 cells in nude mice, possibly reflecting a difference in anti-ras activity between the two antibiotics.

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Stereoselectivity in the oxdative metabolism of propafenone (PPF) enantiomers to 5-hydroxypropafenone was studied with untreated and inducer-treated mouse hepatic microsomes. In the untreated microsomes, Lineweaver-Burk plots of the 5-hydroxylation of R (-)- and S(+)-PPF were single lines, and the intrinsic clearance (V_max/K_m) value of R (-)-PPF was 1.3-fold higher than that of S(+)-PPF. When racemic PPF was used as a substrate, the plot was shifted to the upper region, in comparison with that estimated from the sum of the individual 5-hydroxylase activities of each enantiomer, suggesting enantiomer/enantiomer interaction. In phenobarbital-induced microsomes, the Lineweaver-Burk plot for R(-)-PPF was a single line, but that for S (+)-PPF was biphasic. When racemic PPF was used as a substrate, the plot was biphasic and was shifted to the upper region in comparison with that estimated from the sum of the individual 5-hydroxylase activities of each enantiomer. The observed value of intrinsic clearance of the PPF racemate at lower concentrations (V_max1/K_m1) was consistent with the estimated value, suggesting no interaction between R(-)- and S(+)-PPF. These findings indicate that most of the 5-hydroxylation of R(-)- and S(+)-PPF is catalyzed by common cytochrome P-450 species, but a part of S(+)-PPF 5-hydroxylation is catalyzed by another phenobarbital inducible cytochrome P-450 species, particularly at lower substrate concentrations.

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We have investigated the pharmacokinetic alteration in rats of recombinant interleukin-2 (rIL-2) by immunocomplexing with a monoclonal antibody against rIL-2. Serum rIL-2 levels after the intravenous administration of the immune complex at a dose of 100μg/rat as rIL-2 were significantly higher than those after intravenous administration of rIL-2 alone at the same dose. Pharmacokinetic analysis indicated that the distribution volume of rIL-2 decreased from 74.0 to 10.3 ml/rat, while the elimination rate of rIL-2 was little changed by immunocomplexing with the antibody. On the other hand, serum rIL-2 levels after the subcutaneous administration of the immune complex at a dose of 100μg/rat as rIL-2 were sustained longer than those after the subcutaneous administration of rIL-2 alone at the same dose, and T_max shifted from 0.83 to 3.0h by immunocomplexing with the antibody. Pharmacokinetic analysis also revealed that the mean-residence-time of rIL-2 increased from 1.98 to 6.52h, and the area-under-the curve of rIL-2 decreased slightly, from 834 to 548 ng·h/ml, by immunocomplexing with the antibody.

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A murine monoclonal antibody (MAb) was prepared by immunizing BALB/c mice with a proteoglycan fraction derived from Grifola frondosa (Maitake mushroom), followed by the hybridization of spleen cells with mouse myeloma cells. The MAb (subclass ; Ig G_2b), designated MPG2, reacted with schizophyllan (SPG), curdlan, scleroglucan, laminarin and lentinan, but not with dextran, pullulan, mannan and xylan. Immunohistochemistry (ABC-GO method) showed that MAb MPG2 reacted with lysosomal proteoglycan and (1→6)-β-branched laminaritriose taken up by rabbit peritoneal macrophages. These results suggest that this MAb may recognize mainly (1→3)-β-D-glucan, and may be useful for determining the immunological properties of Grifola frondosa-derived proteoglycan.

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Sulfotransferase purified from Klebsiella K-36, a rat intestinal bacterium, stoichiometrically catalyzed the transfer of a sulfate group of phenylsulfate esters to phenolic compounds. One of the reaction products, p-nitrophenol (PNP), non-competitively inhibited the enzyme as to p-nitrophenylsulfate (PNS), a donor substrate, but competitively inhibited the enzyme as to an acceptor substrate, α-naphthol. The other reaction product, α-naphthol-O-sulfate, non-competitively inhibited the enzyme with regard to both these substrates. These kinetic data suggest that the sulfotransferase reaction proceeds according to an ordered bi bi reaction mechanism. The natural phenolic substances. gallic acid, quercetin, tannic acid, and serotonin were good substrates of K-36 sulfotransferase.

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Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the proliferation and differentiation of bone marrow cells of normal mice with a concomitant loss of myeloperoxidase activity. The decrease in the enzyme activity was due to a reduced level in the bone marrow cells. These findings indicate that myeloperoxidase is not merely a marker, but may also play a role in the differentiation of stem cells into granulocytes.

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Electrophysiologic and hemodynamic effects of SD-3212, a new antiarrhythmic drug, were examined and compared with those of flecainide, a class Ic antiarrhythmic drug, in a canine myocardial infarction model. The doses of SD-3212 were 1 mg/kg bolus injection followed by 0.1 mg/kg/min infusion and 3 mg/kg bolus injection followed by 0.3 mg/kg/min infusion, while those of flecainide were 0.3 mg/kg bolus injection followed by 0.03 mg/kg/min infusion and 1 mg/kg bolus injection followed by 0.1 mg/kg/min infusion. SD-3212 at the high dose prolonged the effective refractory period and QT interval, and depressed ventricular delayed conduction. Flecainide at the high dose also prolonged the effective refractory period and depressed ventricular delayed conduction. Flecainide reduced a maximal rate of rise of left ventricular pressure at the high dose, while SD-3212 did not significantly change it. Neither of the drugs significantly affected mean arterial blood pressure or cardiac output. Thus, SD-3212 may produce antiarrhythmic effects with less cardiac depressant effect than flecainide.

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The electronic properties of 3- and 4-substituted benzoic acids were determined by the Pariser-Parr-Pople (PPP) molecular orbital (MO) method and were then related to glycine conjugation using the mitochondria fraction of rat liver. With the exception of nitro- and cyano-substituted compounds, the conjugation activity increased linearly with the decrease in the energy (E_lumo) of the lowest unoccupied molecular orbital (LUMO) and the energy (E_homo) of the highest occupied molecular orbital (HOMO). The differences shown by the nitro- and cyano-substituted compounds have related to the low values of the atomic orbital coefficient (C_{1, c}) of the carboxyl carbon of LUMO and the negativity of the net charge on the substituent atom. These parameters are discussed in terms of the reactivity of the carboxyl carbon and the interaction between the compounds and enzymes. Regression analysis of the combination of these electronic parameters showed that there was a significant correlation. As far as the effect of the alkyl substituent was concerned, steric hindrance effect was noted in the case of methoxy and dimethylamino groups.

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The reaction of malondialdehyde (MDA) with α-N-acetyl-histidine (Ac-His) in the presence of alkanal was investigated. MDA alone did not react with Ac-His but when alkanal was present, a reaction took place. A similar result was obtained for the reaction of MDA with imidazole in the presence of alkanal. In addition, the structure of the product obtained following the reaction of MDA with imidazole in the presence of acetaldehyde was investigated. The structure of this product was identified as a 1 : 1 : 1 adduct of MDA, acetaldehyde and imidazole (2).

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A clearance study of Cd was carried out in rats that had received an intravenous bolus of CdCl₂ or Cd-saturated metallothionein-II (Cd-MT-II) at 0.3 mg Cd/kg body weight. Urinary concentrations of Cd, and those corrected by the urine-plasma inulin ratio, were higher, by 2 or 3 orders of magnitude, for Cd-MT-II. than for CdCl₂. Fractional clearance of Cd was 0.0006 to 0.0029 for CdCl₂, and 0.130 to 0.487 for Cd-MT-II. These findings denoted that the glomerular filtration of Cd was greater in the Cd-MT-II administration than in CdCl₂ administration. This difference in the glomerular filtration of Cd gave a clear explanation for the greater nephrotoxic potential of Cd-MT-II. Also, our data revealed that when Cd was administered as Cd-MT-II, the concentration of Cd was 20 to 70 times higher at the end-point of the renal tubules than at its starting-point. This and the histopathological reports so far strongly suggest that the distally located tubules are highly resistant to intraluminal Cd.

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