A new analytical method for metallothionein (MT) developed in our laboratory was reviewed along with its application to characterize some properties of MT and to analyze chemical forms of cadmium in animal tissues on exposure to cadmium. The new analytical method consists of a high performance liquid chromatograph (HPLC) equipped with a gel permeation column for separation of proteins and a flame atomic absorption spectrophotometer (AAS) for detection of metals. MT was analyzed at iso-MT level on HPLC-AAS by elution with alkaline buffer solution. As an application, changes in the properties of MT on HPLC-AAS by replacement in vitro with copper were corelated with copper-rich MT and capacity of MT synthesis in rat kidney on exposure to cadmium.

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Reaction of p-methoxybenzyl 7β-phenylacetamido-3-methyl-2-cephem-4α-carboxylate (3) with ethoxycarbonyl nitrene was carried out. Reaction of 3 with ethoxycarbonyl nitrene afforded an adduct (5) possessing aziridine moiety as a main product and 2-ethoxycarbonylamino-3-cephem (4) as a by-product. Treatment of 5 with CF₃COOH gave a cepham (7 or 7') possessing lactone moiety by opening aziridine ring. 7 or 7'was also obtained by treatment of 5 with pyridine-NaOH followed by HCl.

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Reaction of p-methoxybenzyl 7β-phenylacetamido-3-methyl-2-cephem-4α-carboxylate (1) with NaN₃ and ICl were carried out. In CH₃CN or AcOEt, two iodoazides (2a, 2b), in CH₃NO₂ or CCl₄, the diazide (3) and in dioxane or CH₂Cl₂, the chloroazide (4) were obtained. Treatment of 2a, 2b and 4 with DABCO gave 2-azido-3-cephem. The same treatment of 3 gave 2-azido-2-cephem. By replacement of 7β-amido group or ester group, the same results were obtained. As a simple model of olefins, reaction of benzo [b] thiophene with NaN₃ and ICl gave diazide, from which HN₃ was eliminated by tert-BuOK. Configuration of 2-azidocephams and cephems were discussed.

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Thermolysis of 2-azido-3-cephem (1) and 2-azido-2-cephem (3) were carried out. Compound 1 gave β-lactam possessing seven-membered ring (2) by ring enlargement and 3 gave cyanopenam (4) by ring constriction via an azirine (5). Condensation of compound 1 and acetylenes gave 2-triazole derivatives of 3-cephems (6 and 7). Treatment of compound 3 with Ph₃P and CS₂ gave 2-isothiocyanato-2-cephem (9) which were converted to isothiocyanate derivatives (10 and 11). Antimicrobial activities were examined.

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The formation of precipitates by solutions of tannins and alkaloids at pH 5.4, 20°C was examined. Used tannins were tannic acid JP, geraniin, punicalin and chebulinic acid, and alkaloids were berberine, cinchonine, strychnine, papaverine, quinine, nicotine, atropine, caffeine and theobromine. The concentration of tannins was 0-3.0%, and that of alkaloids was 0-0.05%. The experiments with tannic acid JP and 0.01% solutions of berberine, cinchonine, papaverine, quinine and strychnine showed that the precipitate which once increased with an increase in tannin concentration, stopped increasing or quikly decreased when the concentration of tannin increased over 1.5%. Geraniin and chebulinic acid showed similar behavior. However, the experiments with punicalin and the three alkaloids showed an almost linear increase of the precipitates due to the increase of tannin concentration. The experiments with various concentrations of caffeine, nicotine, theobromine and tannic acid JP showed that the amounts of precipitates which once increased with an increase in tannin, quickly decreased upon the further increase of the tannin concentration, and finally no precipitate or almost no precipitate occurred when the concentration of tannin reached 3.0%. A marked increase of the molar ratio of tannin to berberine in the precipitate formed by tannic acid JP and berberine was observed upon the increase of the concentration of tannic acid JP. The increase of this ratio over 2 accounts for the solubilization of precipitates. The paper elecrophoresis of precipitates and solutions showed differences of the extent of dissociation of complexes due to differences of the ratio of alkaloids to tannic acid JP.

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The binding of 1, 1'-ethylene-bis (1-nitrosourea) (EBNU) [carbonyl-¹⁴C], 1-ethyl-1-nitrosourea (ENU) [carbonyl-¹⁴C], ¹⁴C-potassium cyanate (KCNO), and acetic anhydride [1-¹⁴C], to rat liver chromatin was compared in vitro. The order of binding was acetic anhydride&GiEBNU>ENU>KCNO. The effects of various kinds of acylating agents (alkyl isocyanate, alkyl isothiocyanate, N-nitrosourea, KCNO, and acetic anhydride) on template activity of rat liver chromatin were compared by using exogenous bacterial DNA-dependent RNA polymerase system. The template activity was extremely increased by acetic anhydride, and slightly increased by KCNO and some alkyl isocyanates (methyl, ethyl, n-propyl, and n-butyl). The chromatin reacting with EBNU and KCNO was slightly more sensitive than the control chromatin to DNase I and Staphylococcal nuclease. We also compared the solubility of control chromatin and EBNU-and KCNO-reacted chromatin in various concentrations of magnesium chloride and calcium chloride. The solubility of carbamoylated chromatin by EBNU or KCNO was clearly higher than that of the control chromatin in the presence of magnesium or calcium ion.

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The blood concentration, urinary, fecal and biliary excretion and urinary metabolites were studied in rats after the oral administration of 2-oxo-3-[4-(1-oxo-2-isoindolinyl)-phenyl] butamide (OIB) and its ground mixture with PVP (1 : 4 w/w) as a suspension in 1% gum arabic solution. The experiments with the labelled compound revealed that a maximum blood concentration of the radioactivity was about seven times higher after the administration of the ground mixture than after that of ¹⁴C-OIB. During a 48 h period after the administration, 8.3% of the given dose was recovered from the urine in the case of ¹⁴C-OIB and 37.9% in the case of the ground mixture. After the single administration of ¹⁴C-OIB, 5.5% of the dose was excreted in the bile and 87.1% in feces for 48 hours. On the other hand, metabolites of OIB were analyzed by thin-layer chromatography and mass-spectrometry. Sixteen metabolites were detected. Unchanged compound and six metabolites, M1, M4, M7, M10, M12 and M13 were identified by comparison with the corresponding authentic samples. The other metabolites were considered to be hydroxylated on the aromatic ring in the 1-oxo-isoindolinyl moiety.

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To reduce the local irritation induced by intramuscular injection of tiamulin (TM), the cyclodextrin complexation was utilized. The inclusion complexations of TM with three cyclodextrins (α-, β-, γ-CyD) in aqueous solution and in solid phase were assessed by ultraviolet, circular dichroism, solubility method, thermal analyses and powder X-ray diffractometry. Effects of CyDs on in vitro hemolysis induced by TM were investigated as a representation of local irritation. The protective effects of CyDs against hemolysis caused by TM increased in the order of γ->β->α-CyD. The irritative effects of TM and its γ-CyD complex on the M. vastus lateralis of rabbits were studied on gross examination. TM showed a distinct discoloration of M. vastus lateralis with surrounding hemorrhagic area. The irritation of γ-CyD complex was found to be milder than that of TM itself.

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Effect of"Shoseiryu-to, "one of the Kanpo-prescriptions, on the delayed-type allergic reaction induced by picryl chloride was investigated in mice. The water extract of"Shoseiryu-to"showed an anti-allergic effect, and 50% methanolic extract of Paeoniae Radix (the root of Paeonia albiflora spp.) and Glycyrrhizae Radix (the root of Glycyrrhiza spp.), components of"Shoseiryu-to, "exhibited an anti-allergic effect. The anti-allergic activity of the extract of Glycyrrhizae Radix have been reported, so we designed experiments to clarify the active substance of Paeoniae Radix. It was revealed that, paeoniflorin, a main component of Paeoniae Radix, was one of the active substances in the extract.

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From the leaves of Rabdosia ternifolia (D. DON) HARA (Labiatae), a new diterpenoid, isodonoic acid (1) was isolated and the structure was established by spectroscopic and chemical evidence. Three known diterpenoids, isodonal (2), longikaurin E (3) and longikaurin A (4) were also isolated together with oleanolic acid and stearic acid.

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Plasma concentrations of cyclophosphamide (CPM) were studied in patients with gastric cancer before and after total gastrectomy. The plasma concentration-time curve for total CPM declined biexponentially both before and after the operation after intravenous administration of CPM. A decline in plasma levels of α-phase was accelerated by the operation. The β-phase half-life of CPM was not altered in the postsurgical state. Plasma concentration of the active metabolite (AM) was initially higher after the total gastrectomy than that before the operation. These results suggested that pharmacokinetic studies of CPM before and after the operation should be made to improve adjuvant cancer chemotherapy. Concentrations of total CPM and AM in selected tissues were also investigated.

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A flow procedure for the analysis of biological samples was developed by incorporating a highly efficient extraction-column and an on-line evaporator as an interface for switching column extraction and chromatography. Drug metabolites in the biological fluids were quantitatively extracted by aqueous liquid-liquid column distribution with eliminating tedious manual clean-up process. Extracts were switched directly to the analytical column injection by passing through the programmed course in the system. This procedure was automated by employing sequencer to provide a high sensitivity and a reproducibility.

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