Biochemical and molecular biological approaches to two anion translocators, a Cl--translocating ATPase and sulfate permease were described for Acetabularia acetabulum, a unicellular marine alga. The primary structures of an almost complete cDNA clone of the 50 kDa subunit and a partial cDNA clone of the 54 kDa subunit of the Cl--ATPase were highly similar to the β and α subunits of the F type ATPase, respectively. A partial cDNA clone encoding the α subunit of chloroplast ATPase, and partial cDNA clones coding for the β subunits of chloroplast and mitochondrial ATPases were also obtained from A. acetabulum. The presence of a small multigene family for the F type ATPases with different ion specificities was strongly suggested for the organism. Sulfate uptake system in this organism was also studied and partial cDNA clones encoding CysA and sulfate binding proteins were obtained. ca. 1.7 kb RNA for cysA gene and 1.55 kb for sbp gene were detected by Northern analysis, respectively. A putative malK gene was also partially cloned.
The pharmacodynamics of ginsenoside-Rg1 (Rg1), -Rb1 (Rb1) and-Rb2 (Rb2) in rats were studied. In these studies, obvious differences were found in their pharmacodynamics. That is, Rg1 was easily hydrated to the same prosapogenins in both rat stomach and 0.1N HCl solution, but Rb1 and Rb2 were little decomposed (metabolised) in rat stomach and a small quantity of their hydroperoxide derivatives were found, but they were easily hydrated to their prosapogenins by 0.1 N HCl. These ginsenosides were decomposed (metabolised) to several prosapogenins by enteric bacteria and enteric enzymes. The modes of their decomposition (metabolism) were respectively different. In particular, the terminal sugar moiety at the C-20 hydroxy group was noted to affect the rates of their decomposition. The amount of Rg1 and Rb1 absorbed from the gastrointestinal tract of rat were 1.9% and 0.1% of the dose, respectively. And that of Rb2 was determined to be 3.7% of the dose by using 3H-labeled Rb2. Rg1 was excreted into the urine and bile in a 2 : 5 ratio. Rb1 and Rb2 were mainly excreted into the urine.
The acid-catalyzed reaction of N-acyl-and N-sulfonylhydroxylamines with benzene proceeded smoothly to give C-C products ; 2-and 4-hydroxybiphenyls. The reaction and the thermolysis of N-aryloxypyridinium salts involve common intermediates. The results of product analysis, the orientation of the reaction, effects of substituents on the nitrogen atom and on the phenyl ring suggested a mechanism involving a phenoxenium ion. The positive charge of the phenoxenium ion localized not on the oxygen atom but on the ortho and para carbons of the benzene ring. C-O product : diphenylethers are formed when the heterolysis of the N-O bonds is slow and the aromatic solvent has high nucleophilicity, suggesting an SN2-like reaction on the oxygen atom. The phenoxenium ions are also concerned with a rearrangement of O-arylhydroxylamine to 2-aminophenol. An ion-molecule pair involving phenoxenium ion and ammonia as an intermediate of the intramolecular ortho rearrangement.
Cytokinins which promote cell division and cell differentiation of plants are structurally classified into two goups : purine-type cytokinins and phenylurea-type cytokinins. Structural modification of the two types led a potent cytokinin, N-(2-chloro-4-pyridyl)-N'-phenylurea (forchlorfenuron). Structure-activity relationship was discussed. The mode of the two types of cytokinins was studied, and a putative receptor which is common to both types was detected using a highly-labelled synthetic cytokinin. Examples of horticultural application of the compound were illustrated.
The influence of the absorption phase (the time of maximum blood concentration ; Tmax) on the pharmacokinetics of cyclosporine (CYA) in the steady state after oral administration was studied in 26 renal transplant recipients. The patients were divided into three absorption phase groups with the Tmax times as follows, group A (n=26) ; 0h⪈Tmax<3h, group B (n=6) ; 3h⪈Tmax<6h and group C (n=11) ; 6h⪈Tmax<12h. CYA (dose ; 1.6-15.0 mg/kg/d) was administered orally to all 26 patients (10-52 years, 33.1-74.2 kg) every 12h. The blood specimens used in this study were collected just before administration (0h) in the morning and at intervals of 1, 2, 3, 6, 8 and 12h after administration. The whole blood CYA levels were measured by high-performance liquid chromatography (HPLC) or by fluorescence polarization immunoassay (FPIA) based on a specific monoclonal antibody. There were no significant differences between the three groups in terms of the dosage (mg/kg/d) and area-under-the-curve (AUC). The trough levels (0, 12h) correlated well to the AUC only in group A (r=0.842-0.907, p<0.001). The morning trough levels (0h) were significantly higher than the night trough levels (12h) in groups A and B (p<0.05-0.01). On the other hand, there was an excellent correlation between the maximal blood concentration (Cmax) and the AUC of CYA in all three groups, although the Cmax of group C was significantly less than that of group A (p<0.05). Moreover, it was impossible to estimate the Tmax of each individual patient prior to the collection of blood specimens because the Tmax values after oral dosing of CYA varied greatly from patient to patient. This suggests that the monitoring of the AUC values is necessary for obtaining the most suitable therapeutic drug monitoring data of CYA with the patients who absorb slowly.
A sensitive method was investigated for the determination of binding iodine in thyroglobulin (TG). The method was based on the solid-phase preconcentration of iodide (I-) in the absorbing solution prepared by the oxygen flask combustion of TG. The optimum procedures were discussed for the preconcentration. The absorbing solution was passed through a cation-exchange column (AG-50), followed by an anion-exchange column (Bond Elut PSA). Iodide concentrated on the Bond Elut PSA was eluted with ammonia water and treated with the AG-50. An aliquot of the concentrated sample was subjected to reversed-phase HPLC with a UV detector for the determination of I-. The method was applied successfully to the determination of binding iodine in trace amounts of TG.
Recently, methotrexate (MTX) low dose therapy (5-10 mg/m2) has been used for the treatment of patients with rheumatoid arthritis. Hence a practical and sensitive high-performance liquid chromatographic method for the determination of MTX and its main metabolite 7-hydroxymethotrexate (7-OH-MTX) in human serum has been studied. After deproteinization with perchloric acid followed by the addition of pH 5.0 acetate buffer, the serum sample was purified by solid-phase extraction on a Sep-Pak C18 cartridge. The analyte was chromatographed on a reversed-phase Inertsil ODS-2 column using a phosphate buffer-acetonitrile at pH 3.0 system as the mobile phase, and the effluent from the column was monitored at 303 nm. Gradient elution was employed to increase the sensitivity for 7-OH-MTX. A good linear relationship between peak height and concentration was found for the two compounds in the range 2.5 to 100 ng/ml of the human serum, and the detection limits were about 1 ng/ml for the two compounds. The day-to-day coefficients of variation assay were 2.3% (20 ng/ml) and 4.4% (100 ng/ml) for MTX and 4.6% (20 ng/ml) for 7-OH-MTX. The present method was successfully applied to the analysis of the serum after a single oral administration of MTX 2.5 mg tablet to male dogs. MTX was rapidly absorbed, reached to the maximal level at about 1.4h and thereafter decreased monoexponentially with a half-life of about 1.4h. A metabolite, 7-OH-MTX was not detected in the serum up to 24h post dose.
We described an amidolytic method for determining the anticoagulant activity of commercially available low molecular-weight heparin (LMWH) with the use of factor Xa (FXa) and thrombin (FIIa), and a chromogenic peptidyl substrate, S-2222 or S-2238. The procedures were based on the photometric determination of the inactivation of FXa and FIIa after incubation with LMWH in the presence of antithrombin III (AT III). At first, the optimal condition of FXa and FIIa activities with respect to pH, temperature, and the amounts of AT III and LMWH-International Standards (LMWH-IS) were determined. And then, the anticoagulant activities of various LMWHs were determined under the established conditions. In the comparative study with LMWHs, significant differences were found in anti FXa and anti FIIa activities, and their ratios. The anti FIIa activity was reconfirmed to decrease with decreasing molecular weight of LMWHs. On the other hand, the anti Xa activity was, however, less dependent on the molecular weight of LMWHs.