Epilepsy is one of the most frequently occurring nervous diseases. However, the fundamental cause of epilepsy is still unclear. We tried to elucidate the cellular mechanism of seizure discharge. During this research we unexpectedly found that a herbal mixture prescription shows very good effects on epileptics. Therefore, we also performed experiments on the anticonvulsant mechanism of this herbal mixture prescription, “Saikokeishito-ka-Shakuyaku” (SK). SK showed normalizing effects on intracellular calcium-related and protein-related pathological changes induced by pentylenetetrazol (PTZ) application in snail neurons and cultured neurons from the cerebral cortex of mice. In addition, SK showed marked protective effects against neuron damage induced by the cobalt focus epilepsy model, cytochalasin B and severe stress. SK also showed normalizing effects on developmental defects of cultured neurons from the cerebral cortex of an epilepsy animal model, EL mice. Moreover, SK showed complete preventive effects on the abnormal expression of one of the seizure-related (SEZ) genes, PTZ-17, induced by PTZ in Xenopus oocytes injected with PTZ-17 RNA. We also determined mouse chromosomal loci of the SEZ gene group and PTZ sensitive trait loci by linkage analysis for comparison with human synteny of epileptic families. The above-mentioned findings suggest that some herbal prescriptions will become promising drugs for the therapy against intractable nervous diseases which can not be ameliorated by pure chemical drugs in the future.
A high-performance liquid chromatography (HPLC) assay was developed for the determination of 6 new quinolones in the plasma. The plasma samples were directly introduced onto a HPLC column after filtering through a Molcut II membrane filter, which removes high molecular weight proteins. New quinolone in filtrate was separated from interfering substances and retained on a pre-column using an ODS stationary phase and then was introduced onto an analytical column with an ODS stationary phase by column switching. New quinolones were dtected by ultraviolet absorbance in the range of 269-300 nm. Determinations of new quinolones were possible over the concentration range of 50-4000 ng/ml; the limits of detection were 20 ng/ml. The recoveries of the new quinolones added to the plasma were 96.1-101.4% with a coefficient of variation of less than 5.0%. These methods were applied to drug level monitoring in the plasma of patients treated with new quinolones and in that of healthy volunteers participating in pharmacokinetic studies. In addition, these methods were applied to a drug interaction between new quinolones and metal cation (e.g.; Mg2+, Al3+ or Fe2+) containing agents. Furthermore, this method was applied to the determination of skin tissue level of ofloxacin in patients after treatment with ofloxacin. A correlation between serum levels and skin tissue levels of ofloxacin was determined for 30 patients after oral administration of ofloxacin. A good correlation was obtained and the coefficient of the correlation was 0.84.
The disintegration rates of 222Rn and its daughters in natural water were determined successfully by the use of the integral counting method with a liquid scintillation spectrometer. A significant advantage of this method is its freedom from the quenching effect. Moreover, when plural α-, and β-emitters are present, their total amounts can be determined. The simple extrapolation of integral counting curve to zero pulse-height, however, do not give the true disintegration rate for the soft β-emitters (Emax<200 keV), because the liquid scintillator (LS) has a relatively high detection threshold. Therefore, the zero detection threshold of the liquid scintillation spectrometer was determined by measuring standard 3H samples, and a modified integral counting method which extrapolates the integral counting curve to the zero detection threshold was proposed. The method has been successfully applied to various β-emitters, 222Rn samples, and coloured samples of β-emitters, giving more accurate absolute disintegration rate than the conventional integral counting method and the efficiency tracing method. In the course of the study determining 222Rn by liquid scintillation counting, we observed unexpected phenomena; the air luminescence from gaseous space above LS, and the temperature dependence of pulse-height spectra. As for the former phenomenon, we proposed a method for correcting errors due to air luminescence, a method for determining α-emitters by the air luminescence, and a rapid calibration method for 222Rn detectors. As for the latter phenomenon, we observed that the pulse-height spectra for α, and β-emitters in LS are shifted toward higher pulse-height with decreasing temperature. We found that the fluorescence intensities of the solvent of LS (toluene) is promoted at lower temperatures, and that not only toluene, but also the fluorescence intensity of a number of aromatic hydrocarbons and aliphatic hydrocarbons show the same effect as toluene. Other unexpected results are existence of metals in number of enzymes, and discrepancies between the experimental value for Kurie plot of allowed β-emitters and the value which would be expected according to Fermi’s theory, the results of which would affect the transmission probability of potential barrier for α-particles.
Fcγ Receptors (FcγR) are membrane glycoproteins that bind the Fc portion of immunoglobulin G (IgG). The cross linking of FcγR-bound IgG by multivalent antigens allows clustering of the FcγR and initiates a variety of effector mechanisms which play a key role in immune defenses against pathogens. The Fc region is composed of two identical polypeptide chains, which are related to each other by a two-fold axis. Recent elucidation of the crystal structure of human FcγRII provided two distinct views of modes of IgG-FcγR interactions, which is controversial against each other. Nuclear magnetic resonance (NMR) spectroscopy provides a unique and irreplaceable tool to solve these issues. We recently studied the interaction between the Fc fragment of mouse IgG2b and the extracellular domain of mouse FcγRII by this method. We showed that FcγRII binds to a negatively charged area of the CH2 domain, corresponding to the lower hinge region, and that the binding of FcγRII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. This conformational change may account for the 1:1 stoichiometry that we and others observed between FcγR and Fc. We therefore propose a model that explains why the interaction between IgG molecules and FcγR does not trigger cellular responses in the absence of cross linking by multivalent antigens and does not lead to spontaneous inflammatory responses that would be deleterious for the organism.
The antidiabetic effect of hot water extracts from Folium Mori was investigated in GK rat; one of the animal models of non-insulin dependent diabetic mellitus types. Folium Mori extracts (150 mg/kg) significantly reduced the blood glucose of GK rat from 203.8±29.8 to 138.5±21.2 mg/dl at 14 days after oral administration. However, in normal rats, blood glucose and insulin levels were not changed by treatment with Folium Mori. The Folium Mori also decreased blood glucose and improved glucose tolerance at 14 days after repeated administration in GK rats. The Folium Mori treatment significantly increased glucose metabolism in the glucose clamp test for GK rats. These results suggest that Folium Mori has quite unique properties such as raising insulin sensitivity and improving insulin resistance.
Mangiferin, three catechins, and two catechin dimers were isolated from the roots of Salacia reticulata (SRE), and examined their inhibitory activities against several carbohydrate metabolize enzymes (sucrase, maltase, isomaltase, α-amylase, and aldose reductase). Among them, mangiferin was found to inhibit sucrase, isomaltase, and aldose reductase from rat with IC50 values of 87, 216 and 1.4 μg/ml, respectively. The inhibitory activities of mangiferin are competitive for sucrase and isomaltase with inhibitor constant (Ki) 55 μg/ml and 70 μg/ml, respectively. In order to determine the mangiferin contents in the water extracts from the roots of S. reticulata, a quantitative analytical method by means of HPLC was developed and the mangiferin contents in SRE were determined to be in the range of 0.9-2.3% by the application of this method. A high linear correlation (r=0.934) was observed between the mangiferin contents and the sucrase inhibitory activity. In addition, this analytical procedure of mangiferin was found to be applicable for other Salacia species (S. oblonga, S. chinensis, and S. prinoides). Thus, the quantitative HPLC analysis of mangiferin was supposed to be suitable for the quality control of Salacia species and its products.