A new method for the selective modification of proteins which is capable to proceed under mild conditions, was developed by introducing a heterobifunctional reagent named N-(m-maleimide benzoyloxy) succinimide. Applications of the reagent in two new methods for enzyme labeling of antigens or antibodies, and for preparing drug immunogens were reviewed. Various enzyme immunoassay of drugs and hormons as well as antibodies were developed with the two immuno-reagents prepared by these methods. Also applications of these enzyme immunoassays for several studies are presented.
Kyotorphin (Try-Arg) is a neuropeptide originally isolated from bovine brains. We have demonstrated that kyotorphin-induced opioid analgesia may be caused via a release of enkephalin from the brain. Kyotorphin was found unevenly in the rat brain and concentrated in the synaptosomal fraction. Furthermore we demonstrated that kyotorphm is synthesized in the synaptosomal fraction, suggesting that kyotorphin might play a neurotransmitter/neuromodulator role in the brain.
In order to investigate the constituents of Osmanthus species, the components of the leaves of Osmanthus fortunei CARR. were examined. Six secoiridoid glycosides, four lignan glycosides, three acyl glycosides, decaffeoylacteoside, seven flavonoid glycosides and twenty-two other components were identified from the methanolic extract. Their structures of the main components were elucidated on the basis of analyses of carbon-13 nuclear magnetic resonance spectra and the other physicochemical evidence.
The reactions of 1-methyl-4-oxo-cis-and trans-2, 6-diphenylthianium tetrafluoroborate with KOH have been carried out. The s-cis (mp 93°C) and s-trans (mp 85°C) conformers of 5-(methylthio)-1, 5-diphenyl-(E)-1-penten-3-one of reaction products can be isolated by various solvents and temperature of recrystalization. The various kinds of spectra of s-cis and s-trans forms have been measured in a solution and solid state. The s-cis and s-trans forms exist as an equilibrium mixture of them in the solution and the molen state. On the other hand, the s-cis form is more stable in the solid state and s-trans form gradually rearranges to s-cis form at 80°C. Upon prolonged heating in the presence of water, s-cis and s-trans forms undergo an elimination of methylmercaptane. Moreover, Michael adduct is formerd by heating in alkali solution.
Ketalization of a few acetophenones with glycerol was performed with azeotropic removal of water. These ketals were benzoylated and cis compounds (1) were isolated by crystallization from methanol and condensed with imidazole, pyrazole and triazole. They were hydrolyzed to alcohols (3) and converted to the methanesulfonates (4). Imidazole derivatives (5) similar to ketoconazole were obtained from 4 by condensation of phenols and thiophenols. Antimicrobial test of the synthesized compounds (5d, fα), jα)) showed strong antimicrobial activities against Penicillium chrysogenum and Trichophyton rubrum. Most compounds synthesized had strong preventive effects against downy mildew and sheath blight. Triazole derivatives (5m, n) showed similar effects against not only such diseases but gray mould rot.
Anti-sialidase activity was studied in a part of a research on pharmacological actions of medicinal plants. Kakkon-to ( ?? ?? ?? ) (extracted granular 2.5 g/20 ml H2O) showed sialidase inhibition activity (64.4%). Taiso ( ?? ?? ), Mao ( ?? ?? ), Keihi ( ?? ?? ) and Shakuyaku ( ?? ?? ) which were constituents of Kakkon-to inhibited sialidase activity and Mao showed the strongest inhibition effects. The medicinal plants extracts showing the inhibition of sialidase contain various tannins. The anti-sialidase activity of tannic acid varied according to the concentration of tannic acid and galloylglucoses (1, 2, 6-tri-, 1, 2, 3, 6-tetra- and 1, 2, 3, 4, 6-pentagalloyl-β-D-glucose) found in Gobaishi ( ?? ?? ?? ) and Boshokushi ( ?? ?? ?? ) etc. Those extracts showed very strong inhibition, depending on the number of galloyl group but without relation with gallic acid and glucose. Effects of galloylglucoses on Vero cells were examined. Vero cells were impaired at the concentration of 10μg/ml. Anti-sialidase activity of flavones was examined and among flavones, kaempferol showed the strongest inhibition effects.
In out previous paper, a mixture of Ti (IV) and 4-(2-pyridylazo) resorcinol (Ti-PAR reagent) was recommended to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide. The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide. The Ti-PAR reagent was successfully applied to assays of both hydrogen peroxide and sulfite added as a food additive for bleaching by the same procedure. Aliquot amounts of sample solution were pipetted into six volumetric flasks, and graded amounts of standard hydrogen peroxide solution were pipetted into them. The absorbances of sample solutions colored by the Ti-PAR reagent were plotted as ordinate and the concentration of added hydrogen peroxide as abscissa. In the presence of hydrogen peroxide in the sample, the plots of absorbance verses concentration of added hydrogen peroxide as standard intersected the abscissa on the left-hand side of its origin, and the value of the intercept showed the concentration of hydrogen peroxide in the sample solution. On the other hand, the added hydrogen peroxide was consumed through the redox reaction with sulfite in sample. Consequently, the plots of absorbance verses concentration of hydrogen peroxide added as standard intersected the abscissa on the right-hand side of its origin, the value of the intercept of abscissa showed the concentration of sulfite in the sample solution. The present method made it possible to determine both hydrogen peroxide and sulfite in amount ranging from 0.02 to 6.5 ppm and from 0.04 to 9 ppm, respectively, by using 100 g of food sample. In both cases the coefficients of variation were less than 5%.
Polyclonal immunoglobulin (Ig) productions in human peripheral blood lymphocyte cultures activated with staphylococcal phage lysate (SPL) were studied. 1) In mitogenic activity, optimal dose of SPL (25μl/ml, 25μg/ml protein concentration) and pokeweed mitogen (PWM, 10μg/ml) gave the maximum 3H-thymidine uptake on the fifth day in lymphocyte cultures. 2) Maximal Ig production in lymphocyte cultures with SPL (25-50μl/ml) was observed on the seventh day for each Ig class IgG, IgM, and IgA. However, IgF production of lymphocyte stimulated with SPL was less than IgG production with PWM. 3) Ig production with SPL was augmented by the addition of T cell to B cell culture, and when the ratio of T cell to B cell was 1 : 1, maximum Ig production was observed. T cell and B cell alone, on the other hand, did not secret Ig. 4) Ig production in lymphocyte cultures with SPL was enhanced in the presence of 1×105 macrophages, and no enhancement was observed in the absence of macrophages. 5) SPL-induced culture supernatant of lymphocytes also enhanced Ig production at a concentration of 25%. AS noted above, SPL have both mitogenic activity and enhancement effect of Ig production, and these effects augmented in the presence of T cell and macrophages. Moreover, the presence of factors for Ig production in supernatant of SPL-stimulated lymphocyte cultures were suggested.
The crystallinity of cyclodextrins has been evaluated by using powder X-ray diffraction techniques. α-Cyclodextrin·6H2O and β-cyclodextrin·12H2O were transformed into dehydrate forms by heating at 110°C for 3 h in vacuo. When the cyclodextrin hydrate or dehydrate was ground by vibrational mill, it converted from crystalline state to amorphous state. Crystallinities of intact and ground cyclodextrins were calculated by Ruland's method. In Ruland's method, as the integral upper limit affects significantly the evaluation of crystallinity, we determined the crystallinity by using continuous regions of the integral upper limit. The crystallinities and disorder parameters were determined as 83.4%, 3.2A2, for α-cyclodextrin·6H2O, 87.6%, 4.3A2, for β-cyclodextrin·12H2O and 51.5%, 4.3A2 for β-cyclodextrin dehydrate, respectively. The crystallinities of these three crystals decreased to about 8% by 3 min grinding.
Recrystallization behavior of nifedipine from the supersaturated solution containing various polymers was mainly investigated to clarify the mechanism of the supersaturation phenomena from the solid dispersions of nifedipine with enteric coating agents in JP X 2nd fluid (pH 6.8). The recrystallization of nifedipine was remarkably inhibited in the medium containing non-ionic water soluble cellulose polymers or anionic cellulose polymers (enteric coating agents). However, the inhibitory effect on the recrystallization of nifedipine was little using the other type of polymer, such as polyvinylpyrrolidone or methacrylic acidmethacrylic acid methyl ester copolymer. Microscopic observations of the shape of recrystallized nifedipine suggested that a physico chemical interaction between polymer and nifedipine, such as an adsorption of polymers on the surface of nifedipine nuclei, was a main factor affecting the crystal growth of nifedipine.
A prompt, simple method for determining the protein-unbound and total salicylic acid concentrations in human, beagle and mouse blood samples by means of the measurement of the fluorescence from salicylic acid was studied. Both protein-unbound and total salicylic acid concentrations were accurately determined with a ca. 50μl of sample, serum or plasma : Protein-unbound salicylic acid was separated from protein-bound salicylic acid by the use of a ultrafiltration membrane (Centriflow[○!R] CF25) ; 15μl of the filtrate or untreated blood sample was added to Tris-HCl buffer ; and the fluorescence intensity of salicylic acid measured at 300 nm (excitation) and 400 nm (emission). It was found that the metabolites of salicylic acid affect hardly the measurement of salicylic acid with this method.
Ethanol precipitate (HW-50) from hot water extract of lyophylized antarctic krill, Euphausia superba, was found to have gastric secretion inhibitory activity. Chemical analysis revealed that HW-50 contains a large amount of nucleic acids. By use of Sodium dodecylsulfate-phenol method, the nucleic acids were extracted from HW-50. The crude NA-fraction thus obtained contained 67.5% deoxyribonucleic acid, 29.5%, ribonucleic acid and 2.0% protein. The intraperitoneal administration of the NA-fraction caused a signifi. cant inhibition of gastric juice secretion in pylorus-ligated rats at a dose of 10 mg/kg (i.p.). The fraction was digested either with DNase or RNase, followed by the measurement of the biological activity. As a result, it was demonstrated that DNase-treated NA-fractions maintained gastric secretion inhibitory activity.
The plasma concentrations of seven barbiturates from in vitro parameters were predicted in the present study. Elimination rate constant (λ1) and distribution volume of free drug (λ1) well correlated to the partition coefficient (P) but also the ratio of protein bound fraction to unbound fraction of drugs (fb/fu). In these relations, the regression lines between log λ1 and log P, log Vf and log P, log λ1 and log fb/fu, and log Vf and log fb/fu were obtained. On the bases of these regression lines, the plasma concentrations of seven barbiturates were predicted. In both relations (i.e., for log P and log fb/fu), the predicted plasma concentrations well correlated to the observed plasma concentrations. The correlation obtained by the regressions to log fb/fu was slightly better than the case obtained by the regressions to log P. This may be due to the fact that fb/fu is a physiological parameter in contrast to P which is a physicochemical parameter.