We found and previously reported a new mammalian DNA polymerase inhibitor from a sea alga, Gigartina tenella, (Ohta K., et al., Chem. Pham. Bull., 46, 684-686, 1998). It was a new sulfolipid compound that belonged in the class of sulfoquinovosyldiacylglycerol. The biochemical properties have been investigated here. The compound, temporarily designated KM043, potently inhibited the activities of mammalian DNA polymerase α(pol. α) and DNA polymerase β(pol. β) and terminal deoxynucleotidyl transferase (TdT), and moderately, human immunodeficiency virus reverse transcriptase (HIV-RT). KM043 dose-dependently inhibited their activities, and each of their IC50 values was 0.25 μM for pol. a, 0.38 μM for TdT, 3.6 μM for pol. β, or 11.2 μM for HIV-RT, and almost complete inhibition of each was achieved at 1.0 to 2.0 μM for pol. αand TdT, 7.5 μM for pol. β and about 30 μM for HIV-RT. However, the compound did not influence the activities of prokaryotic DNA polymerases such as E. coli DNA polymerase I, and DNA metabolic enzymes like DNase 1. Inhibition of pol. α or β by KM043 was non-competitive with both the DNA template and the substrate deoxythymidine 5'-triphosphate (dTTP). KM043 was weakly cytotoxic to cultured HeLa-S3 cells, and the IC50 value was 80 μM. KM043 could synergistically enhance the cytocidal effect of an anti-cancer chemotherapy agent, bleomuycin. In the presence of 50 μM KM043, the effect ratio of (bleomycin plus KM043) /(bleomysin only) decreased from 0.76 to 0.22.
We noticed that an intraperitoneal injection of Freund's incomplete adjuvant (FIA) into mice could stimulate the induction of a writhing reaction. The FIA emulsion-induced writhing reaction was found to be remarkably inhibited by preadministration of oral indomethacin, a non-steroidal anti-inflammatory and analgesic drug. The induction of the writhing reaction was also inhibited by intravenous preadministration of sodium ascorbate (SAs) in saline. In the experiments where SAs was added to FIA, t was demonstrated that SAs had dual activity of suppression and enhancement. At lower concentrations SAs functioned as a suppressor of the writhing reaction, while at concentrations higher than about 1 mg/50 μl/mouse it acted as an enhancer of the reaction. Furthermore, this writhing reaction induced by FIA+Sas emulsion was also inhibited by preadministraion of SAs itself as well as indomethacin. These results suggested that the mechanism of the erithing reaction induced by FIA was concerned with the production of prostaglandins (PGs), and SAs might be involved in regulation of the writhing reaction.In this paper, we propose a mouse writhing model induced by FIA or FIA+SAs emulsion as a novel pain model useful for assessment of analgesic and anti-inflammatory agents.
The molecular mechanism of light signal perception was analyzed using stem sections of etiolated rice (Oryza sativa L.) seedings irradiated with red light from a fluorescent lamp. The membrane and cytosol fractions were labeled by 40 nM [γ-32P]ATP for 10s at 0°C and proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Phosphorylation of three proteins with molecular weights of 16, 17 and 18 kDa in the rice increased with the intensity of red light irradiation (50 μmol/m2/s) for 16 min. Most of the phosphorylation activity was present in the cytosol fraction. The three proteins cross-reacted with the anti-mucleoside diphosphate (NDP) kinase antibody. Phosphorylation of these proteins was correlated with changes in the activity of NDP kinase. These proteins phosphorylated histone III-S, a substurate for measuring the protein kinase activity. By phospho-amino acid analysis, phosphoserine was found present in the phosphorylated proteins. These rapidly phosphorylated proteins would thus appear to have the features of NDP kinase.
The effect of IS-741 (N-[(2-ethylsulfonylamino)-5-trifluoromethyl-3-pyridyl] cyclohexanecarboxamide monohydrate) on a model for pancreatitis has been previously reported. Recent patho-histological observations of remedial tests using rats found that the IS-741 administered group showed a low degree of tissue infiltration by inflammatory cells (polymorphonuclear leukocytes). We therefore examined cell adhesion, which is the first step in tissue infiltration by activated neutrophils, and investigated the effect of IS-741 on cell adhesion between human umbilical vein endothelial cells (HUVEC) and human promyelo-leukemia cell line (HL-60) cells during lipopolysaccharide stimulation in vitro. IS-741 significantly inhibited the adhesion of HL-60 cells to HUVEC. Further investigation of IS-741 on individual cells revealed that IS-741 mainly affected HL-60 cells. Investigation of the inhibitory effect of IS-741 at the molecular level (targeting adhesion molecules also revealed that IS-741 had no effect on the appearance of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1) on HUVEC, which supports the theory that IS-741 is mainly effective on HL-60 cells, even at the molecular level. However, the inhibition of adhesion was noticed in experiments in which an anti-ICAM-1 or anti-VCAM-1 antibody was added to the adhesion test system. Therefore, IS-741 is likely to affect adhesion molecules which belong to the β1 or β2 integrin family.
Metallothioneins (MTs) occur thoroughout the animal kingdom and they are induced in vivo by metals, hormones, cytotoxic agents, and some kind of stress. It is well known that various stresses such as starvation and immobilization can induce MT synthesis in animal tissues, but the influence of dietary restriction is unknown. The MT levels in the liver increased by food-deprivation and then decreased by refeeding, and a long period of starvation down-alters hepatic MT levels. When the stress is intensified, the induced quantity of hepatic MT is reduced. It became clear that hepatic MT concentrations are controled within a two fold limit when stressed by dietary restriction. MT was also induced in rat liver at recovery stage following an exhaustive running exercise, and thionein was synthesized first and then zinc bound to the protein. The half-life of hepatic MT induced by exercise (which is a nonmetallic inducer) was estimated at 5.2 h. Preinduced MT markedly suppressed exercise-induced lipid peroxidation in rat liver.
This study examines the possible involvement of endogenous platelet-derived growth factor (PDGF) in the angiotensin II-induced growth of rat aortic smooth muscle cells. In quiescent confluent cells, anti-PDGF-AB neutralizing antibody inhibited angiotensin II-induced DNA synthesis and protein synthesis. PDGF-AA, -AB, and -BB produced concentration-dependent increases in DNA synthesis and protein synthesis. Genistein did not inhibit PDGF-AB-induced [3H]thymidine incorporation and [3H]leucine incorporation. PDGF-AB stimulated mitogen-activated protein (MAP) kinases, and PDGF-induced MAP kinase activation was inhibited by genistein. Angiotensin II induced PDGF-A chain messenger RNA expression, and genistein inhibited angiotensin-induced PDGF gene expression. These findings suggest that endogenous PDGF is, at least in part, involved in angiotensin II-induced cell growth in rat vascular smooth muscle cells. It appears that genistein inhibits angiotensin II-induced DNA synthesis partly by inhibiting PDGF-A gene expression.
NK-104 is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase with a very potent lipid-lowering effect. Biotransformation profiles of NK-104 in bile from rat, rabbit and dog given an intravenous infusion of NK-104 were investigated. Structural assignment was made by liquid chromatography (LC)-mass spectrometry (MS)-MS and proton NMR analyses. The predominant component was intact NK-104 in all the animals. At least eight other metabolites were present in rat, four in rabbit, and 10 in dog. These bile metabolites were purified and isolated by preparative HPLC. Biotransformation pathways elucidated for NK-104 were as follows : (a) lactonization ; (b) β-oxidation of the side-chain; (c) hydroxylation of the quinoline ring; (d) conjugation with β-glucuronic acid and taurine. β-oxidative degradation of the side-chain in the case of other HMG-CoA reductase inhibitors is necessary for epimerization of the hydroxy group which has an R-configuration. However, M-16, glucuronide of the ketolactone derivative, was obtained as a key metabolite suggesting another β-oxidation pathway for the side-chain.
Barnidipine, (3'S)-1-benzyl-3-pyrrolidinyl methyl (4S)-2, 6-dimethyl-4-(m-nitrophenyl)-1, 4-dihydropyridine-3, 5-dicarboxylate, is a dihydropyridine calcium antagonist with asymmetric carbons at the dihydropyridine C-4 and the pyrrolidine C-3' positions. In this study, the vasodilatory activity of barnidipine and its 3 optical isomers were compared in vitro and in vivo to assess the steric effects of these asymmetric carbons. All these enantiomers produced concentration-dependent relaxation on KCl (40 mM)-induced contractions in isolated guinea pig aorta with a potency order of barnidipine>(3'R, 4R)≅(3'R, 4S)>(3'S, 4R). The potency ratio between barnidipine and the (3'S, 4R) enantiomer was 118. All enantiomers increased coronary blood flow after intra-arterial administration to aneshetized coronary-perfused dogs. The potency order almost agreed with that obtained in vitro, although the potency ratio between barnidipine and the (3'S, 4R) enantiomer was only 15. These 4 enantiomers showed stereoselectivity for time couse shanges as well. The onset and disappearance of blood flow increase after intracoronary administration of barnidipine were slower than those of other enantiomers. The duration for barnidipine was longer than those for other dihydropyridine calcium antagonists such as nifedipine or nitrendipine. The present study suggests stereoselectivity for the C-4 dihydropyridine and to a lesser degree for the C-3' of pyrrolidine in an ester moiety. The steric effects of these carbons were observed not only in the potency of vasodilatory activity but also in its duration.
Prolyl endopeptidase (PEP, EC 220.127.116.11) is an enzyme which plays a role in the metabolism of proline-containing neuropeptides, e.g., vasopressin, substance P and thyrotropin-releasing hormone (TRH), which have been suggested to be involved in learning and memory processes. In our systematic screening for PEP inhibitors from traditional Chinese medicines, we found that MeOH extract from the underground part of Rhodiola sacra S. H. Fu shows significant inhibitory activity against PEP from Flavobacterium meningosepticum. Examination of the constituents of the extract resulted in the isolation of nineteen known compounds, identified as hydroquinone (1), 4-hydroxybenzoic acid (2), caffeic acid (3), 4-hydroxycinnamic acid (4), suberic acid (5), protocatechuic acid (6), gallic acid (7), (-)-epigallocatechin 3-O-gallate (8), 2-phenylethyl β-D-glucopyranoside (9), 3-O-galloylepigallocatechin-(4β→8)-epigallocatechin 3-O-gallate (10), 2-phenylethyl α-L-grabinopyranosyl-(1→6)-β-D-glucopyranoside (11), sacranoside A (12), β-D-glucopyranosyl 4-hydroxybenzoate (13), rhodiocyanoside A (14), rhodiooctanoside (15), sarmentosin (16), heterodendrin (17), arbutin (18) and 4-O-(β-D-glucopyranosyl)-gallic acid (19). Among these, 1, 2, 5, 8-10, 13, 16, 18 and 19 have been isolated for the first time from R. sacra, among which 5, 9, 10, 13, 16, 18 and 19 have been isolated from Rhodiola plants for the first time. On the PEP inhibition, seven compounds (6-8, 10, 12, 18, 19) showed inhibition with an IC50 of 27.8, 487, 1.47, 0.437, 348, 391 and 215 μM, respectively. The kinetic study of these inhibitors indicated that they are noncompetitive inhibitors, except for 6 which is a competitive inhibitor.
To prove the relationship between the fluctuation in serum β-glucuronidase level and hepatotoxicity, an inhibitor of β-glucuronidase from G. lucidum was isolated and its hepatoprotective activity was investigated. The ether fraction of G. lucidum, which had potent β-glucuronidase-inhibitory activity, protected against CCl4-induced liver injury. From this ether fraction, ganoderenic acid A, was isolated as the potent inhibitor of β-glucuronidase. It had a potent hepatoprotective effect against CCl4-induced liver injury. These results suggest that the β-glucuronidase seems to be closely related to liver injury, which could be prevented by β-glucuronidase inhibitors.
Effects of ephedrine, amygdalin, glycyrrhizin, gypsum and their combinations on body temperature and body fluid were studied in rats using the method developed in our previous reports. Ephedrine significantly increased respiratory evaporative water loss and heat loss in response to a marked elevation of body temperature. There was a small but significant increase in body temperature when amygdalin was orally given rats at a dose of 46.32 mg/kg. Glycyrrhizin and gypsum were unable to affect body temperature. However, gypsum was able to prevent the increased action of ephedrine on body temperature, amygdalin exhibited a preventive tendency to it, and glycyrrhizin did not affect it. The results are in good agreement with classical claims of Makyo-kanseki-to ( ?? ?? ?? ?? ?? ) and the related crude drugs in traditional medicine. Moreover, a combination of the four components reproduced the effects of Makyo-kanseki-to on body temperature and body fluid.This report ssuggests that the co-administration of ephedrine and gypsum is physiologically more desirable than ephedrine alone for dry-type asthmatic patients with a fever. Also, it experimentally supports the clinical efficacy of Makyo-kanseki-to.
The N, N-dimethylglycine esters of menahydroquinone-4 (1-mono, 1; 4-mono, 2; 1, 4-bis, 3) were established in previous reports as prodrugs that could achieve the systemic bioreductive activation-independent delivery of menahydroquinone-4 (MKH), the active form of menaquinone-4 (MK-4), in rat. The present study was undertaken to investigate if the prodrugs could undergo cleavage to parent drug (MKH) by a human tissues enzyme catalyzed hydrolytic pathway, the mechanism of the prodrugs for vitamin K-dependent carboxylation in human liver and their action in the warfarin poisoned human liver. The hydrolysis of the esters was shown to be catalyzed by esterases located in human liver but not in human plasma. The susceptibility of the esters to undergo human liver esterase hydrolysis was afected by the esterified position : 1>2>3. By using a human liver microsomal test system, the stimulation of vitamin K-dependent carboxylation with the prodrugs was determined. The prodrug could stimulate the carboxylation activity in the absence of dithiothreitol, an artificial activator of the reductive activation pathway of MK-4. The carboxylation activity of the prodrug was strongly inhibited in the presence of eserine, an esterase inhibitor. The prodrug could also stimulate the carboxylase under warfarin-poisoned conditions, where the vitamin K cycle was strongly inhibited. The results confirmed that the prodrug could generate MKH in human liver (active site), and that the resultant MKH could act as a cofactor for the carboxylase without reductive activation processes of MK-4 to MKH. Such bioreductive activation-independent vitamin K-dependent carboxylation characteristic of the prodrug leads to enhanced pharmacological efficacy in the treatment of hypoprothrombinaemia induced in patients with coumarin and cephalosporin therapies.
Two water-soluble chitosan derivatives, N-succinyl-chitosan (Suc-chi; average MW 3×105) and glycol-chitosan (Gly-chi; average MW 1.5×105), were examined concerning their biodisposition characteristics in order to evaluate their possible use as water-soluble drug carriers. Their body distribution and urinary excretion were investigated by i.v. administration of FITC-labeled Suc-chi (FTC-Suc-chi) and FITC-labeled Gly-chi (FTC-Gly-chi) to normal and Sarcoma 180 solid tumor-bearing mice. In normal mice, both polymers showed good retention in blood circulation; especially, FTC-Suc-chi exhibited a long half-life of 51 h, and its distribution to other tissues was very small. FTC-Gly-chi was distributed into the kidney to a relatively high extent. In tumor-bearing mice, FTC-Suc-shi and FTC-Gly-chi were eliminated faster from the blood circulaiton than in normal mice, that is, with half-lives of 11 and 7h, respectively. FTC-Suc-chi was less partitioned to the tumor tissue but accumulated more easily into it compared with FTC-Gly-chi. This suggested the enhanced permeability and retention (EPR) effect of Suc-chi and explained the previous result that a water-soluble Suc-chi-mitomycin C conjugate injected intravenously exhibited a good effect against Sarcoma 180 solid tumor. FTC-Gly-chi showed greater distribution to the kidney than in normal mice. Urinary excretion studies indicated the faster excretion of both polymers in tumor-bearing mice. The molecular weight of the products excreted into urine indicated that both polymers should be pretty resistant to the hydrolytic enzyme, lysozyme. Taking toxicities into account, Suc-chi is consiered to be available as a drug carrier showing long systemic retention and tumor accumulation.
Cationic lipid N-[3-[2-(1, 3-dioleoyloxy)propoxy-carbonyl]propyl]-N, N, N-trimethylammonium iodide (YKS-220) having a symmetrical and biodegradable structure was employed for the preparation of cationic liposomes with dioleoylphosphatidylethanolamine (DOPE). The stability, transfection activity in several cell lines and cytotoxicity of YKS-220 cationic liposomes were studied. It was found the YKS-220 cationic liposomes were very stable and their transfection activity remained even after storage at 4°C for 12 months. The transfection activity of these liposomes was assayed using CHO, COS, and HepG2 cells and found to be comparable with, or better than, that of other cationic liposomes, such as N-[1-(2, 3-dioleoyloxy)propyl]-N, N, N-trimethylammonium methylsulfate (DOTAP) liposome, N-[1-(2, 3-dioleyloxy)propyl]-N, N, N-trimethylammonium chloride (DOTMA) liposome (Lipofectin[○!R]), and 2, 3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) liposome (LipofectAMINETM). n addition, the cytotoxicity of YKS-220 cationic liposomes was far lower than that of other cationic liposomes.
The aim of the present study was to determine the effect of sulfaphenazole (SP) on the pharmacokinetics of ampiroxicam (AM) which is metabolized by cytochrome P-450 (CYP) 2C9, since SP is a potent inhibitor of CYP2C9, and so a dramatic pharmacokinetic drug interaction between both drugs is assumed after dosing. Sihgle intravenous and oral administrations of AM (5 and 7.5 mg/kg piroxicam equivalent, respectively)and SP (80 and 120 mg/kg, respectively) to rats did not significantly alter the elimination kinetics of AM and piroxicam (PX) converted from AM.. When SP was preloaded orally at 2 h before the dosing of AM, and when AM and SP were orally coadministered for 7 d, the elimination of PX from plasma was slightly retarded and the area under the plasma concentration-time curve (AUC) was increased 77 and 53%, respectively, but not significantly, compared with those after AM alone. On the other hand, a significantly devreased metabolic conversion of PX to 5'-hydroxyPX in plasma was observed by these treatments (p<0.05).In order to clarify the mechanism for the interaction, hepatic and intestinal metabolizing enzyme activities, CYP, uridine 5'-diphosphoglucuronyltransferase (UDPGT) and aryl esterase, were assayed after single and multiple oral administrations of AM or AM and SP. The enzyme activities were hardly inhibited by the treatment, indicating that the inhibition of CYP and hydrolytic enzymes by SP was approximately denied. These results suggest that SP does not significantly affect the pharmacokinetics of AM and PX in rats. However, the pharmacokinetic drug interaction between both drugs in man may not always be ignored.
The growth of Bacillus cereus was markedly inhibited by the addition of lactoferrin and was recovered by the addition of FeCl3. The growth inhibition was also reversed by the addition of erythrocytes and hemoglobin.B. cereus can use heme or heme-protein complex (hemoglobin-haptoglobin and hematin-albumin complexes) as iron sources in iron deficient conditions. Therefore, B. cereus uses these heme or heme-protein complexes to prevent the growth inhibition of lactoferrin in vivo.
The current study reports active glycosidases in the lens of ICR/f rats, which generate a hereditary cataract approximately 90 d after birth, and the variation in enzyme activity with cataract progression. Seven active glycosidases, β-D-galactosidase, α-D-glucosidase, β-D-glucosidase, β-D-glucuronidase, β-D-galactosaminidase, β-D-glucosaminidase and α-D-mannosidase, were detected in ICR/f rat lenses. Of these, β-D-glucuronidase and β-D-galactosidase showed a tendency to increase in activity with the cataract progression. Furthermore, β-D-glucosidase and α-D-mannosidase showed a transitory increase in activity at the time of cataract formation. This result suggests that several glycosidases in the lens may be involved in the hereditary cataract formation. The optimal pH and temperature of the seven active glycosidases in rat lenses were also measured in this study.
The protective effects of nine oleanene-type triterpenoidal glucuronides obtained from several fabaceous plants (Lupinus polyphyllus×arboreus Hybrid, Astragalus complanatus, Wistaria brachybotrys) on in vitro immunological liver injury in primary cultured rat hepatocytes were studied. Although all tested saponins sholwed hepatoprotective action, the levels of activity of the individual saponins differed. Structure-activity relationships for the sapogenol moiety suggested that the presence of the carbonyl group at C-22 would show a similar hepatoprotective effect to that of the hydroxyl group at C-22 while the presence of the hydroxyl group at C-30 would reduce the hepatoprotective action. This structure-activity relationship substantiated other recently obtained data. Furthermore, the β-orientation of the hydroxyl group at C-21 is more effective than the α-configuration in regard to the hepatoprotection. Furthermore, structure-activity relationships for the sugar moiety substantiated previously obtained data that the hydroxyl group at C-5" enhances the hepatoprotective action.
The method of maximum extended quasi-likelihood (MEQL) can be viewed as an estimation method in the framework of generalized linear models. The method was applied to a pharmacokinetic problem in which the pharmacokinetic model was a nonlinear function of its parameters. The behavior of the method toward the estimation of a variance function was numerically compared with those of the generalized least squares (GLS) and extended least squares methods. In general, the MEQL and GLS methods were equally better. However, the MEQL estimator often showed smaller mean squared errors for the scaling parameter than the other two estimators. Such a generally comparable but partially distinct property of the MEQL method, as compared with the GLS method, is useful to pharmacokinetic analysis.
Virus identification in clinical materials during acute phase infections could give necessary information for the treatment of infections by human immunoglobulin (hIg) or interferon (IF). But because of a lack of information, most virus infections have not been treated. We have tried to develop a real time detection system for viruses in general using an potical biosensor and a model virus, Herpes simplex virus Type 1 (HSV-1), and have proved that the HSV-1 virus propagated in Vero cells and diluted in minimum essential medium (MEM) with 10% fetal bovine serum (FBS) could be detected in high sensitivity close to 10 infectious units (50% tissue culture infective dose [TCID50] units) using purified cellular receptor molecules as the ligand because the receptor could be the most specific ligand. However, because ligands available for this system to idetify various viral infections in general are limited, we also tested this system using a purified polyclonal antibody which contained many other antigens as the ligand, and produced sensitivity comparable to that using the receptor as a ligand. In this paper, we tested the sensitivity by this system under the worst condition. That is, we used a crude homemade rabbit antiserum against measles virus with host cell debris as a ligand. It was found that less than 500 infectious (TCID50) units of virus were required for detection in a 100 μl solution, and that the efficacy of the commercially available hIg was also estimated by this system. This result suggested that this real time viral detection and titration system was applicable for the diagnosis of all viral infections even under difficult conditions without requiring any complex skills, with high enough sensitivity for clinical purposes. The efficacy of hIg preparations could also be evaluated by this system at the same time of the clinical materal sampling.
Radiolabeling of proteins is a widely used approach to study their in vivo disposition patterns. However, the obtained results may largely depend on the radiolabeling method used. The purpose of the present study was to investigate the effect of the radiolabeling method on the pharmacokinetic analysis of liver targeted protein in mice. Galactosylated bovine serum albumin (Gal-BSA) was labeled with 125I or 111In, using diethylenetriaminepentaacetic dianhydride (cDTPA) or 1-(4-isothiocyanobenzyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA) as bifunctional chelating agents. The Gal-BSA was then injected in mice by a bolus intravenous injection. Samples of plasma, urine, liver, kidney, intestine and feces were collected at various time intervals and their radioactivity was measured. In none of the samples examined was there any significant difference in radioactivity distribution originating from the radiolabeling methods within 5 min after administration. After this period, 125I radioactivity in the liver started to decrease significantly faster than that of 111In, which would indicate the intracellular degradation of the protein. Consequently, the reappearance of trichloracetic acid (TCA) soluble 125I radioactivity in the plasma occurred. But whereas the hepatic uptake clearance (CLliver) of [111In]DTPA-Gal-BSA remained constant during 8 h postinjection, the CLliver of [125I]Gal-BSA at 30 min represented only one eighth of its initial values. The CLliver of [111In]SCN-Bz-EDTA-Gal-BSA resembled that of [111In]DTPA-Gal-BSA within 1 h of the ecperiment but it started to decline after this interval. The observed discrepancies most probably resulted from the formation of different radiolabeled metabolites in the hepatocytes and their different capability of crossing biological membranes. Our findings indicate that among the three methods employed, [111In]DTPA radiolabeling of Gal-BSA is the most appropriate method to study its tissue disposition.
Ginsenosides saparated by silica gel TLC were blotted to a polyvinylidene difluoride (PVDF) membrane which was treated with a NaIO4 solution followed by bovine serum albumine (BSA), resulting in a ginsenoside-BSA conjugate on a PVDF membrane. The blotted bands were stained with monoclonal antibody (MAb). The newly established Western blotting method was used for the determination of ginsenosides and their distribution in various Panax species.
A method for detecting glucuronides of glycyrrhetic acid using Western blotting was investigated. Glucuronides of glycyrrhetic acid separated by silica gel TLC were transferred to a polyvinyliden difluoride membrane. The membrane was treated with sodium periodate solution followed by bovine serum albumin, resulting in a glucuronides of glycyrrhetic acid-BSA conjugate. Individual spots were stained by monoclonal antibody against glycyrrhizin. Immunostaining of glucuronides of glycyrrhetic acid was more sensitive compared to other staining methods. The newly established immunostaining method can be expanded to the distribution of glucuronides of glycyrrhetic acid in the plant body.
Recently, we developed novel tumor necrosis factor (TNF)-α production regulators with a phthalimide skeleton derived fromthalidomide. We show here that some of these compounds are more potent inhibitors than thalidomide of angiogenesis induced by basic fibroblast growth factor in a murine angiogenesis assay.
Escherichia coli BM2506 produced macrolide 2'-phosphotransferase II [MPH (2') II]. A gene for MPH (2') II, designated mphB, is located on plasmids pTZ3721 and pTZ3723 in E. coli BM2506. In the present study, we determined the nucleotide sequence of the 6.5-kb EcoRI-PstI fragment containing mphB on pTZ3721. The DNA region of 6.5-kb EcoRI-PstI fragment contained five open reading frames (ORFs). ORF4 corresponded to mphB. Respective products deduced from ORF1, ORF2, ORF3, and ORF5 were similar to the penicillin-binding protein 4 of Streptomyces lactamduras, the repressor protein AcrR of the acrAB operon, the enzyme RdmC involved in the biosynthesis of the antibiotic aklavinone, and IS801 transposase-like protein from Pseudomonas pseudoalcakigenes, respectively. Among these genes, ORF2, ORF3, and mphB formed a gene cluster with ORF2 in the lead sequence. Our results suggest that mphB may originate from an operon related to antibiotic biosynthesis.