Fluorescence thiol reagents and various substrates for proteases have been designed and synthesized. These functional organic molecules have been applied to the studies of biological macromolecular systems.
Complex formations of 12 p-hydroxybenzoic acid esters (alkylparabens) with α- and β-cyclodextrins (α-CyD, β-CyD) were studied in water and in solid state by solubility method, IR spectroscopy, thermal analyses, and X-ray diffractometry. Solid complexes for alkylparabens with α- and β-CyDs were obtained and their molar ratios were found to be 1 : 2 (alkylparaben/α-CyD) and 1 : 1 (alkylparaben/β-CyD), respectively, except methylparaben-α-CyD complex (1 : 1). The dissolution rate of the complex in water was significantly higher than that of alkylparaben itself, depending upon the magnitude of the stability of the complex. Alkylparaben molecule included within CyD cavity appeared to be inactive and antimicrobial activity was directly related to the concentration of free alkylparaben in aqueous solution. Upon addition of CyDs, a significant increase in inhibitory-zone diameter was observed by cup-plate method, suggesting improved solubility and diffusibility of the preservatives by inclusion complexation.
Class of antibodies formed in mice by the oral, rectal and intraperitoneal (i.p.) administration with egg albumin (EA) was investigated. IgM antibody to EA was formed in the following cases ; everyday oral administration of 3.0 mg of EA, everyday rectal administration of 0.4 mg of antigen and i.p. injection at intervals of 3 days of 1.0 mg of antigen, and detected at 8th, 7th and 6th days, respectively, after the first immunization. IgG antibody appeared on the next day of IgM antibody detection. Thus IgM and IgG antibodies were produced even by the rectal administration of antigen, and it is suggested that antigen is absorbed from the digestive system as a molecule (macromolecule) enough to show immunogenicity. The oral and rectal administration showed similar effect as an administration route with regard to an increase in the blood IgA level and non-induction of IgE antibody to EA. IgE antibody was induced by the i.p. injection with not only native EA but also EA which was partially digested with pepsin and trypsin in vitro, and IgE antibody titer to native EA was higher at 10μg than 100μg of the dose of native antigen. These findings suggest that the non-induction of IgE antibody by orally administration of EA did not depend upon that protein becomes smaller molecules by proteases.
The metabolism of γ-oryzanol was investigated in rabbit after oral administration of γ-oryzanol-14C (triterpenyl ester of ferulic acid-3-14C) (40 mg/kg). The radioactivity excreted in the urine for 48 hr was 6.4% of the administered dose and unchanged γ-oryzanol was not detected in the urine, the in situ absorption experiment suggested that γ-oryzanol was not metabolized in the intestinal tract and absorded as an intact form. Only acid-extracted fraction of the urine contained the radioactivity, and ferulic acid, vanillic acid, acetovanillone, hippuric acid and vanilloylglycine (glycine conjugate of vanillic acid) were identified as the major urinary metabolites of γ-oryzanol. On the basis of these data, metabolic pathway of γ-oryzanol in rabbit was postulated.
Estriol antisera were raised and studied for the use in radioimmunoassay (RIA) of urinary estrogen as an aid of diagnosis of feto-placental function. Antisera to estriol-related six haptens, i.e., estriol 16-glucuronide (E3-16-G), estriol 16-hemisuccinate (E3-16-succ), estriol 17-hemisuccinate (E3-17-succ), estriol 16, 17-dihemisuccinate (E3-16, 17-succ), estriol 3-carboxymethylether (E3-3-CME) and estriol 3-glucuronide (E3-3-G), were obtained by immunizing rabbits with the respective haptenbovine serum albumin (BSA) conjugate. Antiserum to E3-16-G-BSA reacted significantly with E3-16-G, C-17 conjugated estrogens and free estrogens. But it did not react with C-3 conjugated estrogens and other steroids. Similar properties were observed with antisera to E3-16-succ-BSA, E3-17-succ-BSA and E3-16, 17-succ-BSA, but their reactivities with C-16 or C-17 conjugated estrogens were slightly lower than that of anti-E3-16-G-BSA serum. Among antisera which were raised against estriol linked to BSA via hemisuccinate varying in linking posirion, anti-E3-16-succ-BSA serum highly reacted with E3-16-G, while anti-E3-17-succ-BSA did with E3-17-G. Anti-E3-16, 17-succ-BSA serum reacted with C-16 and C-17 conjugated estrogens to nearly the same extent. Antisera to both E3-3-CME-BSA and E3-3-G-BSA reacted with estrogens conjugated at C-3 position, but failed to react with C-16 or C-17 conjugated estrogens. The latter antiserum reacted more specifically with E3 and E3 3-G than the former. Estrogen values in pregnancy urine samples estimated by the RIA by use of anti-E3-16-G serum were not influenced by other components in urine, and showed a good correlation of y=0.81x-1.28, r=0.983 with those determined by the currently available colorimetric method with E3-kit.
Fundamental studies were made on hemagglutination inhibition reaction (HAIR) method with anti-estriol 16-glucuronide (E3-16-G) serum for a rapid and simplified determination of estrogen in pregnancy urine. The sheep erythrocytes fixed with formalin and treated with tannic acid were sensitized with E3-16-G-rabbit serum albumin (RSA) conjugates. An antiserum to E3-16-G-bovine serum albumin (BSA) was absorbed with both BSA and erythrocytes fixed with formalin. The degree of binding of E3-16-G with RSA and the amount of E3-16-G-RSA sensitized onto erythrocytes affected the easiness of adjusting sensitivity and the clearcut patterns of HAIR formed at the bottom of the test tubes. A HAIR reagent having sensitivity of 0.2 μg/ml (E3-16-G equivalent in E3) could be prepared by sensitizing 8% erythrocytes suspension with a 0.05 to 0.08% of E3-16-G-RSA in which 20 to 30 mol of E3-16-G being bound per 1 mol RSA, and finaly the sensitivity was adjusted by diluting an antiserum. Cross-reactivity of the HAIR method with estrogens was almost similar to that of radioimmunoassay (RIA) and the reactions were found with estrogens other than conjugated at the C-3 position i.e., E3-16-G, C-17 conjugated estrogens, free estrogens. Glucose, NaCl, serum protein, urinary protein and urine pH did not affect the results. E3-16-G added to pregnancy urine was recovered in satisfactory yield by considering the accuracy of the method. Significant correlation (y=0.78x+0.97, r=0.949) was noted between HAIR and RIA on the measured estrogen values of the same urines from pregnant women. These results indicate that our HAIR method, semi-quantitative determination of urinary estrogen, is a useful tool for feto-placental function in clinical routine.
An arginine ester hydrolase, ME-1, was isolated from the venom of Trimeresurus mucrosquamatus by gel filtration on Sephadex G-100 and ion exchange chromatographies on CM-Sephadex C-50, DEAE-Sephadex A-50, CM-Sephadex C-50 and DEAE-Sephacel. By these procedures, 36.8 mg of purified preparation was obtained from 1g of crude venom. This enzyme hydrolyzed arginine esters, such as tosyl-l-arginine methyl ester (TAME) or benzoyl-l-arginine ethyl ester (BAEE), but did not hydrolyze casein, hemoglobin, tosyl-l-lysine methyl ester (TLME), acetyl-l-tyrosine ethyl ester (ATEE), tosyl-l-argininamide (TAA) or benzoyl-l-argininamide (BAA). The esterolytic activity was inhibited by benzamidine but not by diisopropyl fluorophosphate (DFP) or trasylol. The purified preparation was homogeneous as judged by disc electrophoresis on polyacrylamide gel and isoelectric focusing. The molecular weight of ME-1 was determined to be approximately 27000, and the isoelectric point was found to be pH 5.95 by isoelectric focusing with carrier ampholyte. The esterolytic activity of the final preparation was 481.9 unit/mg. When this enzyme was injected into the skin of a rabbit, capillary premeability increased distinctly. This protein was stable to heat treatment and between pH 4 and 9. Its michaelis constant (Km) and inhibition constant (K1) values for TAME or benzamidine were found to be 4.0×10-3M and 0.292×10-3M, respectively. This protein had some carbohydrates.
N, O-diacetyl-p-aminophenol (DAPAP) formation after simultaneous administration of aspirin with phenacetin and acetaminophen was investigated in rats and rabbits. 1) When aspirin and acetaminophen were orally administered to rats, DAPAP was detected in the plasma and bile. 2) By the combined use of aspirin with phenacetin, DAPAP was detected in the plasma of rabbits and the bile of rats. 3) DAPAP was observed in the plasma and bile at the earlier stage after dosing. 4) DAPAP was hydrolyzed rapidly in Tris buffer (pH7.0) containing the serum of rats and rabbits.
The photo-stability and the structures of photo-oxidation products of aminophylline in aqueous solutions were investigated. When an aqueous solution of aminophylline was submitted to photo-oxidation in the presence of oxygen under daylight, aminophylline was decomposed to yield 1, 3-dimethylallantoin, N, N'-dimethyloxamide and ammonia as main products. It was found that photodegradation of theophylline in an aqueous aminophylline solution was closely related to that of ethylenediamine in the same solution.
The essential oil of fresh flower of Cytisus scoparius LINK was identified to contain cis-3-hexen-1-ol, 1-octen-3-ol, benzyl alcohol, phenethyl alcohol, phenol, cresols, guaiacol, eugenol, isovaleric acid, benzoic acid, palmitic acid, eight fatty acids and n-paraffins. Orobol, genistein, quercetin, kaempferol, aesculetin, p-coumaric acid and caffeic acid were isolated from the residue after steam distillation of ethereal extract.
A method for the determination of daidzin in Puerariae Radix was established by using high-performance liquid chromatography. Daidzin in Puerariae Radix was separated on the 15cm column of Nucreosil ODS with an eluent of 18% acetonitrile, and detected at 254 nm by phothometric detector. Precision of the present method was about 1% and detection limit was about several ng (S/N ratio, about 3). Daidzin in Puerariae Radix was extracted with water and the extract was injected directly into the column. Sufficient separation of daidzin and other components in Puerariae Radix water extract was obtained. The content of daidzin was calculated from the calibration curve prepared previously by use of a standard. This method was considered to be useful for the evaluation of Puerariae Radix.