A new method was developed to measure the content of a Lumbricus component in a traditional Chinese medicine (TCM). An antiserum specific to Lumbricus was elicited in a rabbit following immunization with a suspension of Lumbricus fragments. A characteristic antigen protein, 70 kDa, was found in Lumbricus and was purified almost to singleness using a column chromatography series of gel filtration and DEAE-Sepharose. A selected antibody enzyme immunoassay (SAEIA) was developed using the antiserum and the purified 70 kDa protein as a solid-phase antigen. The SAEIA was specific to Lumbricus species, and showed no cross-reaction with any crude drugs other than Lumbricus. This SAEIA detected 70 kDa protein in the amount of 10 ng /ml with excellent reproducibility (coefficient of variation=3.0%) and an EC50 of 0.24 μg/ml. Using this assay, Lumbricus levels were easily determined in a Lumbricus-based TCM Kazecoll, but not in the control Kazecoll (Kakkonto) prepared without Lumbricus. The SAEIA for 70 kDa protein was simple, accurate, reproducible and may provide a general analytical method for the quality control of Lumbricus-based TCMs.
Pretreatment with streptomycin at a low concentration influenced the susceptibility to streptomycin of a strain of Escherichia coli carrying a streptomycin-resistance plasmid, pSA1700, derived from Pseudomonas aeruginosa. This phenomenon was due to a mutation that occurred at about 10-8-10-10 of frequency in a regulatory gene involved in gene expression on the chromosome of E. coli. A product encoded by the regulatory gene on the chromosome of E. coli might normally repress gene expression by binding to part of the promoter region of the streptomycin-resistance gene derived from P. aeruginosa.
Electron spin resonance(ESR) studies using 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) and sodium 3, 5-dibromo-4-nitrosobenzensulfonate (DBNBS) as a spin-trapping agent revealed the formation of both hydroxyl and carbon-centered radical-derived spin adducts in Cu2+-containing 50 mM Tris-HCl buffered solutions (pH 7.1) of D-glucosamine, D-mannosamine, and D-galactosamine, which were previously shown to have the ability to break the single-strand of plasmid pBR322 DNA in a nucleotide sequence-specific manner.HCl-free D-glucosamine has higher DNA breaking activity, and this activity is promoted more by the presence of Cu2+ than the original D-glucosamine hydrochloride, exhibits stronger radical signals in the ESR spectrum. It is suggested that D-glucosamine is unstable around neutral pH, being converted into certain intermediate(s) such as a dihydropyrazine compound, which generate(s) carbon-centered radicals, and that, besides the hydroxyl radical, the intermediate(s) is/are responsible for DNA strand breakage.
Immortalized hybrid cells were generated by the somatic fusion of the cells from the forebrain of embryonic mouse with N18TG2 neuroblastoma cells. Three monoclonal hybrid cell lines, designated NF26, NF81, and NF83 (n__-euroblastoma f__-orebrain hybrid cells), expressing an α isoform of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were isolated, and their expression was demonstrated by immunoblotting and immunocyto-chemistry using a monoclonal antibody specific to the α isoform of the enzyme. The kinase activity of the hybrid cells was 2- to 3-fold higher than that of the parent neuroblastoma line N18TG2 cells. The neuronal origin of these lines was shown by their immunoreactivity to neurofilament protein, a neuron specific marker. Lines NF26, NF81, and NF83 are the first cell lines to express the gene of the α isoform of CaM kinase II in the brain.
Physarumin, a carbohydrate-binding protein (hemagglutinin or lectin), was isolated from the plasmodium of Physarum polycephalum. Physarumin agglutinated not only several species of erythrocytes but also tumor cells such as AH109A ascites hepatoma cells, sarcoma 180 ascites cells and mouse leukemia P388 cell lines. Physarumin had tumor cell growth -inhibitory activity, and induced the apoptosis of P388 cell lines. Physarumin-induced apoptosis required binding to a 68 kDa counter-receptor on the P388 cell surface. Since the agglutinating and antiproliferative activities of physarumin were inhibited by asialofetuin and thyroglobulin, respectively, it is suggested that physarumin reacts with the galactose moiety of carbohydrate chains of physarumin receptor.
A mouse strain named ASK that was originally isolated from El (epilepsy) mice has been shown to be highly sensitive to anaphylactic shock. Here, we characterized the bases of the sensitivity of ASK mice in comparison with the parental strain, El. More than 90% of ASK mice, but not El mice that had been sensitized either actively or passively, died within 1 h following an antigen challenge. The anaphylactic death was effectively blocked by diphenhydramine. Plasma histamine levels increased by 30-50 fold in ASK after the antigen challenge, but only a 2-3-fold increase was observed in El mice. All (El×ASK) F1 mice, either male or female, showed an ASK-like phenotype, suggesting that the impaired plasma histamine response in El mice is due to some recessive mutation(s). Consistent with the plasma histamine responses, cultured mast cells derived from El bone marrow showed impaired potency to degranulate in response to surface IgE engagement, in contrast to ASK mast cells which undergo normal degranulation. Another characteristic feature of ASK mice is their sensitivity to histamine, since 75% of the mice were killed by the subcutaneous administration of 100-200 mg/kg histamine, while C3H and BALB/c mice were resistant to even 600 mg/kg histamine. Taken together, the major bases of the susceptibility to anaphylactic shock in ASK mice are thought to be the enhanced sensitivity to histamine and the recovered degranulation machinery in mast cells that is impaired in El mice.
Interactions of anandamide (N-arachidonylethanolamide), an endogenous compound for cannabinoid receptors, with the receptors for 5-hydroxytryptamine (5-HT), benzodiazepine, and γ-aminobutyric acidA (GABAA) receptors in bovine synaptic membrane were examined. Anandamide decreased the 5-HT receptor bindings at concentrations of 1-100 μM, although it did not cause any change in benzodiazepine or GABAA receptor bindings. A high concentration of anandamide, 100μM, significantly decrease both [3H]5-HT and [3H]ketanserin bindings. The present study revealed that the pharmacological activity of anandamide might be partially mediated through the 5-HT receptor.
The effect of methylcarbonylmethyl 2(S)-[4-(4-guanidino-benzoyloxy) phenyl] propionate methanesulfonate (TT-S24) on experimental pancreatitis in rats was examined in comparison with that of camostat. TT-S24 showed a preventive effect on increases in plasma amylase activity and pancreatic weight induced by cerulein injection. TT-S24 also reduced an increase in plasma amylase activity induced by taurocholate. TT-S24 effectively prevented the mortality induced by an injection of a mixture of trypsin and taurocholate. TT-S24 showed no effect on an increase in amylase activity 6 h after duodenum ligation (closed duodenal loop pancreatitis), indicating that the drug had no effect on the initiation and propagation step of closed duodenal loop pancreatitis. On the other hand, TT-S24 reduced an increase in amylase activity 6 h after release of the duodenum ligation. TT-S24 showed anti-trypsin, anti-kallikrein, anti-thrombin and anti-plasmin activities. The effect of TT-S24 on some experimental pancreatitis models was nearly equal to or somewhat more potent in most instances to that of camostat. Therefore, TT-S24 should be useful in the clinical treatment of pancreatitis.
Using an organotypic slice culture of the hippocampus, the effects of epileptic activities on synapse reorganization following axotomy were investigated. The maximal amplitude of field excitatory postsynaptic potentials that reflected the number of functional synaptic contacts were recorded 7 d after the mossy fibers or Schaffer collaterals were transected at 8 d in vitro. Fifty μM picrotoxin elicited epileptiform bursts, whose severity in the CA1 region was lower than that in the CA3 region. Synapse reformation of the mossy fibers was significantly prevented by picrotoxin, and that of Schaffer collaterals also tended to be attenuated. Ten μM bicuculline, 1 mM pentylenetetrazol or 2 mM 4-aminopyridine also induced epileptic activities in the CA3 region and significantly depressed synapse formation of the mossy fibers. Using cultures of dispersed neurons, we found that the prolonged depolarization of membrane potentials promoted neurite outgrowth. Taken together, we concluded that the preventing effects of epileptic activities on synapse reorganization following axotomy was due to the inhibition of the synaptogenesis process, not to a blockade of axon outgrowth.
We studied the effects of nine cytotoxic drugs on three groups of B-lymphoblastoid cell lines transformed by Epstein-Barr virus (EBV) : group 1, mortal cell lines from normal individuals; group 2, immortalized cell lines from normal individuals with strong telomerase activity; group 3, mortal cell lines from Werner's syndrome (WS) patients. Aminoglycoside antibiotics and alkylating drugs showed significantly stronger cytotoxic effects on immortalized cell lines than on mortal cell lines or the cell lines before immortalization. In contrast, topoisomerase II inhibitors showed no difference or they tended to be less cytotoxic to immortalized cell lines. Mortal cell lines from normal individuals and WS patients showed no difference in sensitivity against all the drugs examined except for the topoisomerase I inhibitor, camptothecin, which had a stronger cytotoxic effect on WS cell lines than other cell lines. We discuss the mechanisms underlying these cytotoxic effects.
The effect of scymnol on the development of lesions in a rat peripheral arterial occlusion model, involving injection of 5% lactic acid into the femoral artery, was investigated.In this model oral administration of scymnol significantly prevented edematous swelling and development of lower limb lesions, including gangrene, and also reduced changes in blood coagulation parameters, platelet aggregation and retention rate at a dose of 10 or 30 mg/kg. However, it had no effect on these clotting system functions in sham-operated rats at a dose of 10 mg/kg. The effects of scymnol were also compared with those of ticlopidine and argatroban.The findings suggest that scymnol may be clinically useful for preventing thrombotic peripheral arterial occlusive disorders. Its prophylactic action appears to be mainly due to its potent ability to protect against endothelial cell damage due to lactic acidosis.
Enzyme immunoassay (EIA) for the determination of compound K (C-K), a major metabolite of ginsenoside Rb1 (G-Rb1) from Panax ginseng root by intestinal bacterial flora, was explored. Bovine serum albumin (BSA) was coupled to the C-26 position on the unsaturated side chain of C-K. β-D-Galactosidase was introduced at the C-26 position of the saturated side chain. Antiserum, obtained by immunization of rabbits with C-K-BSA conjugate, possesed high affinity and specificity toward C-K. The EIA for C-K by the double antibody method was established in the range of 0.1-100 ng/tube.Plasma C-K after the oral administration of C-K and G-Rb1 to rats was determined by the established EIA. C-K was rapidly absorbed from the gastrointestinal tract after the administration, then slowly decreased. On the other hand, C-K appeared late and was retained for a long period of time in the plasma after the administration of G-Rb1, which itself is hardly absorbed.
Hemolysis of human erythrocytes induced by free radicals initiated from lipid-soluble 2, 2'-azobis(2, 4-dimethylvaleronitrile) (ADVN) was examined under various conditions. From the ESR spectra of the spin-labeled erythrocytes, it was found that the fluidity of the membrane did not change during the radical-induced hemolysis. The curves of the time courses of the extent of oxidation and the conformational change of band 3 proteins were hyperbolic, though the hemolysis curves were sigmoidal. In spite of the necessity of lipid peroxidation, the peroxidation did not seem to relate directly to the hemolysis. It was observed that the hemolytic holes were formed by a lateral clustering of band 3, an anion exchange protein in erythrocytes. The competitive reaction model between lipid peroxidation and the redistribution of oxidized band 3 proteins, which was previously presented for the hemolysis initiated from water-soluble, 2, 2'-azobis(amidinopropane)dihydrochloride (AAPH), could explain well the curves for the hemolysis by ADVN. Further, the rates of lipid peroxidation at various concentrations of ADVN and AAPH were calculated on the basis of the hemolysis curves, and they were compared with the experimental values estimated from the curves for the lipid peroxidation. The curves which showed a dependence of the calculated rate constant on the concentration of radical initiators were similar to those of the experimental values. These results indicate that the competitive reaction model is appropriately represents the hemolysis induced by free radicals which also originated from lipid-soluble initiators.
Methanol extracts of 36 samples of 21 Umbelliferae plants were screened for polyacetylenic compounds using the ELISA for panaxytriol, and their antiproliferative activity was checked by MTT assay using the tumor cell lines MK-1, HeLa and B16F10. The presence of antiproliferative polyacetylenes was suggested in Angelica acutiloba (fruit), Anethum graveolens (root), Bupleurum rotundifolium (fruit), Carum carvi (fruit and root), Coriandrum sativum (fruit), Cryptotaenia japonica (leaf), Glehnia littoralis (fruit), Heracleum moellendorffii (root) and Torilis japonica (fruit).Panaxynol and falcarindiol were successfully isolated from the root of Heracleum moellendorffii as antiproliferative polyacetylenes.
A new experimental device was developed to investigate respiratory diseases. The moisture and heat released from respiratory organs and the body surface of a rat were determined by means of this device as well as the rectal temperature. The high recovery of results was statistically confirmed, and the measured values at various environmental temperatures were significantly different from each other. Some standard drugs, such as ephedrine, aminophylline and chlorpromazine, were examined. Their stimulant or depressant actions were clearly observed. The results of some traditional medicines for the treatment of rhinitis and bronchial asthma from this measuring system were consistent with their clinical applications. These results suggest that this new experimental system is not only effective in the experimental understanding of cold-hot syndrome, but also contributes to the evaluation of the effects of traditional medicines.
Iontophoretic delivery of desmopressin acetate (DDAVP) was assessed for delivery efficiency and drug stability, both in vitro and in vivo. The effect of current intensity and duration of current application on the decomposition of DDAVP was investigated in vitro. It was shown that when a current of 0.1 mA was applied for 5 min, the decomposition of DDAVP was negligible. In vivo experiments under the same conditions showed that the antidiuretic response to DDAVP persisted for about 6 h. Furthermore, when this iontophoresis was repeated 3 times at intervals of 4 h, the antidiuretic response persisted for about 11 h. These results suggest that repeated short-termiontophoresis is a safe and effective technique for transdermal delivery of DDAVP.
We investigated the absorption and transport of 2', 3'-didehydro-3'-deoxythymidine (D4T) and its ester prodrugs from the nasal cavity in rats. The absorption of D4T and its acetate (C2-D4T) was rapid and almost complete, although the hemi-succinate (Suc-D4T) was absorbed rather slowly; the plasma concentrations of the prodrug, Suc-D4T, and regenerated D4T remained unchanged throughout the experimental period (180 min). Concentrations in the cerebrospinal fluid (CSF) following intravenous (i.v.) and intranasal (i.n.) administration were also measured. After i.n. administration, drug concentrations were higher in the fraction derived from the subarachnoid space located close to the nasal mucosa than those in the fractions located far from the nasal cavity. This difference was not found following the i.v. administration of the drugs. Following nasal administration, the intact Suc-D4T was found in the CSF at a concentration higher than that of D4T, although transport of the intact prodrug to the CSF was not observed following i.v. administration. These results suggest that direct transport of the drugs from the nasal cavity to the CSF significantly contributes to the higher concentrations in CSF of D4T and/or its ester prodrugs, and indicate the possible value of nasal administration for the treatment of patients with AIDS dementia.
The effect of gel-forming (1→3)-β-D-glucan on the immunological activities of murine kupffer cells was examined. A branched type gel-forming (1→3)-β-D-glucan, GRN, was administered intravenously to mice. GRN associating to kupffer cells was detected by an immunohistochemical technique using anti-GRN antibody. A kinetic study of the activation of kupffer cells revealed that GRN could induce the enhanced production of cytokines and nitric oxide on 4 to 7 d after the administration. The activities are further augmented by adding GRN in the culture. The cytostatic activity of kupffer cells against murine lymphoma, EL-4, was also augmented by a time course similar to nitric oxide production. The cytostatic activity was reduced by adding an inhibitor of nitric oxide synthase, implying that the cytostatic activity of kupffer cells to EL-4 was dependent on nitric oxide. The administration of GRN increased the expression of CD11b, known as a β-glucan receptor, on kupffer cells at day 7. The above data suggest that GRN could activate murine kupffer cells to enhance the production of cytokines and nitric oxide, and that the activation required 4 or 7 d, at least, after the administration with GRN.
Fe(II)-tetrakis-N, N, N', N'(2-pyridylmethyl) ethylenediamine (Fe-TPEN) catalyzes the dismutation of superoxide, and blocks the toxic effect of paraquat on Escherichia coli growth and survival. We examined antioxidative effects of Fe-TPEN on lipid peroxidation and t-butyl hydroperoxide induced cell damage. Fe-TPEN inhibited the FeSo4/H2O2 induced lipid peroxidation in the rat liver homogenates with an IC50 value of 30.2 μM, and protected Ac2F cell damage by t-butyl hydroperoxide in a dose-dependent manner (EC50 value is 2.6 μM). Also, hepatoprotective effect of Fe-TPEN (5 mg/kg, i.p.) was investigated using CCl4 induced liver injury in rats. This complex inhibited the elevation of serum alanine aminotransferase (AST) and aspartate aminotransferase (ALT) levels in CCl4 induced liver injuries, and improved submassive necrosis and fatty degeneration of the hepatocytes. Fe-TPEN also prevented the loss of total and nonprotein SH contents, glutathione peroxidase and glutathione-S-transferase activity in cytosol of rat liver. Although the exact mechanism of action is not clear, antioxidative properties as well as attenuation of hepatocellular defense systems by Fe-TPEN seem to be important on its potent hepatoprotective effect in CCl4-intoxicated rat.
To gain insight into the aromatization sequence of androst-4-ene-3, 6, 17-trione (1), a suicide substrate of aromatase, the aromatization of its 19-hydroxy and 19-oxo analogs 2 and 3 with human placental microsomes, was studied using GC-MS. Steroids 2 and 3 were separately incubated with the microsomes in the presence of NADPH in air. The GC-MS analysis of the trimethylsilyl derivative of the aromatization product indicated that both the 19-oxygenated steroids 2 and 3 were aromatized to yield 6-oxoestrogens, 6-oxoestrone (4) and 6-oxoestradiol (5), in each experiment. The aromatization rates of substrates 2 and 3 were 605±48 and 1794±75 pmol/mg protein/10 min, respectively. These relatively higher rates, compared to that of the parent steroid 1 (73.2±6.6 pmol/mg protein/10 min), indicates that the suicide substrate 1 is aromatized through the 19-oxygenated intermediates 2 and 3.
A series of arylthiolated 2, 3-dimethoxy-1, 4-benzoquinones was synthesized and tested for the effect on the respiratory system and the lipid peroxidation in bovine heart mitochondria (BHM). These quinones showed intense inhibitory activities on the respiratory system in BHM. Their inhibitory activity in the succinate oxidase system was greater than that in the NADH oxidase system. No difference between the difference spectra, with and without these quinones, of the reduced minus oxidized forms of cytochromes (cyt.) suggested that these quinones inhibit at the site after cyt. a+a3 in the respiratory chain. Moreover, these quinones were as efficient as exogenous ubiquinone-10 (UQ-10) for the inhibition of lipid peroxidation. 5- And 5, 6-di-arylthio groups on the quinone ring were found to be favorable for inhibition of the respiratory system and lipid peroxidation. Our results suggest that arylthiolated 2, 3-dimethoxy-1, 4-benzoquinones act as antioxidants by increasing the amount of endogenous reduced UQ-10 in BHM.
The present paper investigates the pharmacokinetics of pirarubicin (THP) in the plasma and cerebrospinal fluid (CSF) of two patients with glioma during hyperosmotic disruption of the blood-brain barrier (HODBBB) and intra-arterial combination chemotherapy.A 42-year-old Japanese man (patient A) with glioblastoma and a 21-year-old Japanese woman (patient B) with astrocytoma received a course of HODBBB and intra-arterial combination chemotherapy with THP, methotrexate, peplomycin, and vindesine. Patient A was initially administered mannitol, followed by the infusion of anticancer drugs into the right internal carotid artery. Patient B was initially administered mannitol, followed by the infusion of anticancer drugs into the right internal carotid artery and, immediately thereafter, into the right vertebral artery. Samples of blood and of CSF in the brain ventricle were obtained. THP concentration was measured by HPLC, and the pharmacokinetic parameters of this drug were estimated in plasma and CSF.In both patients, the plasma concentration of THP peaked at the end of infusion, then decreased in a bi-exponential decay pattern during the remainder of the treatment period. THP was detectable in CSF beginning 1.0 h after the initiation of infusion, then was slowly eliminated from the ventricle. The maximum CSF concentration of THP was 0.97% of plasma in patient A and 0.89% in patient B. The CSF AUC of THP was 28.4% of plasma in patient A and 13.1% in patient B.
For the transdermal delivery of tranilast (TL), a drug used for the treatment of skin diseases such as keloids and hypertrophic scars, its oily gels were prepared; its in vitro release and penetration into Yucatan micropig skin were evaluated. In the gels that consisted of hydrogenated soybean phospholipids (HSL) and octyl isononanoate (IOIN), a fatty-acid ester, the release of TL from the gels was proportional to the drug content, and the extent of TL released up to 6 h from them was approximately 70% of the amount of applied TL. On the other hand, with the gels consisting of HSL and isocetyl isostearate (ICIS), the release of TL from the gels was about half of that from IOIN gels, even at the same drug concentration. When oily gels were used, the TL skin concentration was rapidly increased compared with the level obtained with suspensions. With 0.1% IOIN gel, a high concentration of TL (ca. 160 μg/g) in the dermis was obtained and continued until at least 48 h. These results suggest that oily gels may be useful for the topical application of TL.
The urinary excretion of valproic acid (VPA) and its metabolites (3-keto VPA, 3-OH VPA, and VPA-glucuronide) in 6 epileptic patients was studied using gas chromatography-mass spectrometry. The amount of VPA and 3-OH VPA excreted in the urine was low (0.1-0.5% of the dose of VPA and 0.6-1.5% of the dose of 3-OH administered). The amount of 3-keto VPA and glucuronide (VPA-Glu) excreted was marked (5.8-26.2% and 13.1-88.7% of the dose of VPA administered, respectively). The urinary excretion of VPA and its metabolites by patients who have taken a normal amount of a VPA preparation was almost the same as that of healthy volunteers. Two epileptic patients who took a large amount of the VPA preparation showed a high excretion of VPA-Glu without an increase in their plasma VPA-Glu.
In order to obtain an insight into the retention mechanism of drugs with imidazole moiety in the connective tissue, the in vitro characteristics of the interaction between 14C-labeled imidazoles (imidazole and its 2-methyl derivative) and slices of dog aorta were studied. We found that cupro-ascorbate-catalyzed oxidative reactions for the imidazoles led to their irreversible binding with connective tissue, and that, from a study using protein modifiers, the aldehydic function intrinsic to the tissue protein was involved in the binding formation. This characteristic in vitro was observed under physiological conditions, hence it can be extrapolated to the in vivo situation and could also give a clue to the nature of the retention of imidazole-containing drugs in connective tissue.