The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) in Escherichia coli. The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system. Furthermore, these two systems were ABC (ATP binding cassette) transporters consisting of four kinds of proteins : pPT104 clone encoded PotA, -B, -C, and -D proteins and pPT79 clone encoded PotF, -G, -H, and -I proteins. PotD and -F proteins were periplasmic substrate binding proteins and PotA and -G proteins membrane associated proteins having the nucleotide binding site. PotB and -C proteins, and PotH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins were similar to each other. The functions of PotA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (PotE protein) having twelve transmembrane segments, and was active in both the uptake and excretion of putrescine. The uptake was dependent on the membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine. In mouse mammary carcinoma FM3A cells, it was shown that the antizyme, which negatively regulates the amount of ornithine decarboxylase, also negatively regulates the activity of polyamine transport.
Author's work on the adaptation and protection systems against chemical substances, such as drugs, food additives, heavy metals, pesticides and environmental pollutants, in the living body during the past thirty five years was reviewed. Using the induction of heme and drug metabolizing enzymes (especially heme oxygenase) and of metallothionein in the liver of animals as a biological index, the adaptation and protection of these substances to the body were discussed. This is because that heme oxygenase is a heat shock protein which is induced by various environmental factors, and that metallothionein also has a wide variety of biological functions, and was identified as an acute phase protein in the first phase defense system against environmental stresses. The research under mRNA levels as more sensitive index will advance in the near future.
We investigated in vitro the properties of glycyrrhizin (GL), such as dissolution, absorption and resolution, using a Sho-Seiryu-To extract, a Sho-Saiko-To extract, both including a licorice root, and licorice extract. The dissolution of GL differed with the pH of the solvent. The absorption (partition coefficient) of GL decreased with an increase in pH, and increased in the presence of other active constituents, such as baicalin, baicalein, and ephedrine. In the case of the Sho-Saiko-To extract, the conversion from GL to glycyrrhetic acid (GA) by β-glucuronidase originated from E. coli occurred slowly. It was also suppressed by adding baicalin. We determined in vivo the pharmacokinetics of GA after oral administration of Kampo extracts in healthy volunteers. In each Kampo extract, the time of administration had no influence on the mean maximum blood concentration (Cmax) and the area under the blood concentration-time curve (AUC). Tmax was delayed in the case of the administration after meal (p<0.05).
Dipyridamole, a nucleoside membrane transport inhibitor, enhanced the cytotoxicity of epirubicin for mouse leukemia P388 cells by a factor of 1.8-fold and that for 30-fold doxorubicin-resistant sublines of P388 cells (P388/DOX) by a factor of 6.5-fold. This interaction was shown to be truly synergistic by DNA histogram and median effect analysis. The dipyridamole enhancement of the cytotoxicity of epirubicin was a dose-dependent effect ; it was greatest when cells were exposed to dipyridamole before treatment with epirubicin. In cell cycle experiments, 1-5μM dipyridamole increased the accumulation of G2+M phase produced by the treatment with 0.5-1μM epirubicin. Dipyridamole, however, did not appear to alter the patterns of DNA histogram in sensitive cells. These results suggest that the increase of the accumulation of G2+M phase in resistant cells is an important factor for the interaction between epirubicin and dipyridamole.
We have previously reported that dipyridamole increases the cytotoxicity of epirubicin and alters the cell cycle in doxorubicin-resistant (P388/DOX) cells, increasing the accumulation of G2/M phase by blocking the cell cycle. In cultured cells, dipyridamole increased dose-dependently the intracellular accumulation of epirubicin in the resistant cells. Simultaneous exposure of the resistant cells to epirubicin and 100μM dipyridamole resulted in a 4.2-fold increase in proportion to the control level of epirubicin after 60 min. Dipyridamole inhibited the enhanced efflux of epirubicin in doxorubicin-resistant cells. However, dipyridamole had no effect on both the influx and efflux of epirubicin in doxorubicin-sensitive cells. In mice, lethal and bone marrow toxicity induced by epirubicin were potentiated by administration of high-dose of dipyridamole. In addition, in vivo results also demonstrated that dipyridamole in combination with epirubicin produced a significant reversal of the in vivo antitumor activity of epirubicin in mice bearing P388/DOX cells. These data imply the enhancement effects of dipyridamole on the efficacy and toxicity of epirubicin.
Antimicrobial and anti-inflammatory actions of tea-leaf saponin, which was a mixture of saponin separated from leaves of Camellia sinensis var. sinensis, were investigated. Tea-leaf saponin showed relatively high antimicrobial activity against pathogenic dermal fungi and its MIC value for Microsporum audouinii was 10 μg/ml. On the other hand, tea-leaf saponin inhibited rat paw edema induced by carrageenin in a dose dependent manner. Activation of hyaluronidase, one of the enzymes involved in inflammatory reactions, was inhibited by tea-leaf saponin. It was also found that tea-leaf saponin antagonized the action of leukotrien D4, one of the chemical mediators of inflammatory reactions. Any symptom of toxic reaction was not observed when tea-leaf saponin was administered orally to mice at a dose of 2000mg/kg.
An ethanol extract of "Kijitsu" (Aurantii Fructus Immaturus, Citrus aurantium L.) collected in China was assessed for the antitumor activity using murine leukemia P388 in vivo, and the extract was found to be active by the antitumor bioassay in vivo and in vitro. The extract was separated into a petroleum ether-soluble fraction and an ethyl acetate-soluble fraction. Fractionation was carried out using an index of cell-growth inhibitory activity against mouse leukemia L1210 cells to isolate antitumor active substances or compounds. The active compounds were purified employing silica gel column chromatography and HPLC. The antitumor effect of the isolated active compounds was studied. Five compounds, auraptene, marmin, tangeretin, nobiretin and 5- [ (6', 7'-dihydroxy-3', 7'-dimethyl-2-octenyl) oxy] psoralen were isolated from Citrus aurantium L. Though they are all known compounds, 5 [ (6', 7'-dihydroxy-3', 7'-dimethyl-2-octenyl) oxy] psoralen from this plants was first isolated. These compounds showed a cell-growth inhibitory effect against L1210 and K562 in vitro.
Headspace solid phase micro extraction gas chromatography (HS-SPME-GC), which was an improved method of the usual HS-GC was applied to the analyses of components in crude drugs, such as Asiasari Radix. The crude drug (0.20g) was preheated with 1.0ml of water in a capped vial at 50°C for 25 min and the SPME fiber was injected into the vial. Then vaporized components were adsorbed and extracted with the fiber at 50°C for 5 min. The fiber was injected into the injection port of GC at 220°C for 30s. The results indicated that HS-SPME method was sensitive and rapid analysis for crude drugs under moderate conditions, and this technique described in this paper, therefore, will become a powerful method for quality control of components in crude drugs.
The synthesis of 3, 5-diphenyl-and 5-tert-buthyl-3-phenyl-1, 3, 4-oxadiazolin-2-ones by the 1, 3-dipolar addition of carbon dioxide with nitrile imine was investigated. The 1, 3-dipolar addition was carried out at 60°C for 36h in a potable reactor (autoclave) into which the required amounts of dryice (carbon dioxide), N- (α-chloroalkylidene) -N'-phenylhydrazines, triethylamine or 1, 4-diazabicyclo- [2.2.2] octane and 1, 4-dioxane were placed, with a mechanical stirrer, to give 3, 5-diphenyl-1, 3, 4-oxadiazolin-2-one in a 25% yield and 3-phenyl-5-tert-butyl-1, 3, 4-oxadiazolin-2-one in a 14% yield.