A method for the quantitative determination of human serum albumin in plasma and urine by post-column high-performance liquid chromatography with fluorescence detection is presented. Human serum albumin in plasma and urine is separated by gel-permeation column chromatography, which is followed by fluorescence detection. The detection uses the fluorescence enhancement observed when 8-anilino-1-naphthalenesulfonic acid binds to human serum albumin. The method is rapid to perform and is highly sensitive (detection limit is 2.5 ng/injection volume at a signal-to-noise ratio of three).
A specific enzyme immunoassay (EIA) method has been developed for 17α-estradiol 17-N-acetylglucosaminide as a model compound instead of 15α-hydroxyestrogen 15-N-acetylglucosaminides. Two new haptens, 3-(ω-carboxyalkyl) ether derivatives of 17α-estradiol 17-N-acetylglucosaminide, were synthesized and conjugated with bovine serum albumin (BSA). the EIa system was newly established using specific antiserum elicited against 3-(1-carboxypropyl) ether of 17α-estradiol 17-N-acetylglucosaminide (17NAG CPE)-BSA conjugate and β-galactosidase-labeled 17NAG CPE as a labeled antigen.An appropriate dose-response curve of EIA for 17α-estradiol 17-N-acetylglucosaminide was obtained in the range of 20-1000 pg/tube. The specificity of EIA proved to be satisfactory in terms of cross-reactivities to related compunds including 15α-N-acetylglucosaminides. The proposed method will be applicable to the preparation of antisera for use in EIA of 15α-hydroxyestrogen 15-N-acetylglucosaminides.
In immunoblotting analysis using rabbit antibody to bovine adrenodoxin, the total proteins of the bovine adrenal cortex gave two bands, suggesting the presence of two forms of adrenodoxin in vivo : full-length and carboxy-terminal deleted adrenodoxins. To examine the effect of the carboxy-terminal deletion of adrenodoxin on its activity, cDNAs for Arg115stop mutant adrenodoxin and for Asp113stop mutant adrendoxin were constructed. The wild type [Ad(2-128)] and carboxy-terminal deleted [Ad(2-114) and Ad(2-112)] recombinant adrenodoxins expressed in Escherichia coli were purified to give a single band on SDS-PAGE. They showed an A414/A276 value of 0.92. In an NADPH-cytochrome c reduction assay, the Km values for cytochrome c in the reconstituted system with Ad(2-128), Ad(2-114) and Ad(2-112) were 39, 235 and 618 nM, respectively. The Vmax values were 638, 700 and 898 mol/min/mol flavin, respectively. In an NADPH-acetylated cytochrome c reduction assay, the maximum activity of Ad(2-128) was obtained at 50 mM NaCl, while the maximum activities of Ad(2-114) and Ad(2-112) were obtained at 100 mM NaCl; the latter values were 4-times higher than that of Ad(2-128). In the presence of 100 mM NaCl, the Km values for acetylated cytochrome c in the system reconstituted with Ad(2-128), Ad(2-114) and Ad(2-112) were 220, 33 and 22 μM, respectively. The Vmax values were 352, 305 and 382 mol/min/mol flavin, respectively. These results indicate that the effects of the carboxy-terminal deletion of adrendoxin on NADPH-cytochrome c and acetylated cytochrome c reductions are different; the carboxy-terminal region (residues 113-128) of adrendoxin largely contributes to the binding with cytochrome c but disturbs the binding with acetylated cytochrome c.
The antitumor activity of cytostatic 5'-deoxy-5-fluorouridine (5'-dFUrd) depends on its being converted to 5-fluorouracil (5-FUra) by the enzyme thymidine phosphorylase (dThdPase, EC 220.127.116.11). We prepared mouse anti-human dThdPase monoclonal antibodies to serve as tools for clinical studies with this drug. Partially purified dThdPase obtained from HCT116 human colon cancer cells grown in athymic mice was used as an antigen for the immunization of mice. Six hydridomas were cloned which produced anti-human dThdPase antibodies, as detected by Western blot analysis with human dThdPase. With these antibodies, we developed an ELISA method sensitive enough to measure dThdPase levels, even in tumor tissue samples weighing as little as 10 mg. In addition, one monoclonal antibody was suitable for immunologically staining the enzyme in tumor tissues. Thus, these anti-human dThdPase monoclonal antibodies could be used to measure levels of the enzyme in tumor cells, which is essential for the activation of 5'-dFUrd.
Mechanisms of the stimulatory release of lipoprotein lipase (LPL) activity from isolated rat fat pads by sodium orthovanadate (vanadate) were studied through a cAMP-dependent process. A potent inhibitor of insulin receptor tyrosine kinase, quercetin, inhibited the vanadate-increasing effect on the LPL activity in fat pads, but did not inhibit the vanadate-stimulated release of LPL activity from the fat pads. Proparanolol and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) decrased the vanadate-stimulated release in a dose-dependent manner. Isoproterenol and dibutyryl cAMP (Bt2cAMP) stimulated the release of LPL activity from fat pads. Vanadate, as well as isoproterenol, rapidly increased the cAMP content in fat pads, and this increase was almost completely inhibited by propranolol. Vanadate increased the cAMP-dependent protein kinase (PKA) activity ratios calculated from the measurement in the presence or absence of cAMP or PKA inhibitor.There results suggest that the vanadate-stimulated release of LPL activity is associated with a process involving a rapid increase in the cAMP content accompanied by the activation of PKA.
A series of eighteen substituted pyrazoles, bis- and tris-azolyl-methanes or ethanes was investigated for their interaction with the zinc enzyme carbonic anhydrase (CA). Several types of activities were detected, generally as CA activators, but CA inhibitory properties were also discovered for the very sterically-demanding derivatives of these series. Kinetic determinations by a stopped-flow technique for carbon dioxide hydration reaction allowed the determination of Michaelis-Menten constants, which are identical in the absence or in the presence of these modulators, proving a noncompetitive mechanism of activation-inhibition. MNDO calculations were used with moderate success to explain the biological results.
Effects of optical isomers of ephedrine (EPH) and methylephedrine (MEP) on the spontaneous beating rate of isolated right atrium of normal and reserpinized rat were investigated to assess direct and indirect actions on β1-adrenoceptors. l-EPH (3×10-7-3×10-5M) and d-EPH (10-6-10-4M) markedly increased beating rate of rat right atrium. l-MEP (10-5-3×10-4M) showed slight increase in heart rate. The potency of positive chronotropic effect is l-EPH>d-EPH»l-MEP. l-EPH was about 3 times as potent as d-EPH. In addition, d-MEP (3×10-5-3×10-4M) caused a decrease in heart rate. Positive chronotropic effects of EPH isomers and l-MEP were attenuated by pretreatment with atenolol (a selective β1-adrenoceptor antagonist) or reserpine treatment (8 mg/kg, s.c.). In reserpinized atria, the maximal increase by d-EPH was quite small, and l-MEP decreased heart rate. On the other hand, d-MEP, at 3×10-4M, did not show antagonist activity against the positive chronotropic effect of isoprenaline (10-10-10-5M). These results suggest that l-EPH, d-EPH and l-MEP have β1-adrenoceptor agonist activity, while d-MEP is suggested to have only low or no affinity for β1-adrenoceptors. The relatively weak activity of l-MEP is believed to be mainly mediated by released noradrenaline. It is also suggested that d-EPH has very potent noradrenaline-releasing activity.
The stereoselectivity in 4-hydroxylation of bunitrolol (BTL), a β-adrenoreceptor blocking agent, was examined in liver microsomes from monkeys (marmosets and Japanese monkeys) and compared with the results of human liver microsomes. The formation of (+)-4-OH-BTL from (+)-BTL was significantly higher than that of (-)-4-OH-BTL from (-)-BTL in the liver microsomal fractions from the two kinds of monkeys. The 4-OH-BTL-forming activity from recemic BTL was significantly lower than that from enantiomeric BTL, indicating a possible metabolic interaction between BTL enantiomers. The in vitro profiles observed in the monkeys were very similar to those in humans, but the stereoselectivity in BTL metabolism [(+)-BTL>(-)-BTL] in the primates was found to be reverse to that in rats [S. Narimatus et al., Anal. Biochem., 222, 256-261 (1994)]. The 4-OH-BTL-forming activity from BTL enantiomers was significantly suppressed by quinidine and quinine, while the former was more potent than the latter, and also by α-naphthoflavone. Furthermore, the activity was also suppressed by antisera against rat cytochromes P450-2D2 and -1A2 in concentration-dependent manners. However, kinetics showed that enantiomeric BTL 4-hydroxylation was monophasic in liver microsomes from marmosets of both genders and from male Japanese monkeys. These results suggest that cytochrome P450-2D and -1A enzymes with similar Km values are involved in BTL 4-hydroxylation in monkey liver microsomes.
We have studied the effect of dietary supplementation with 25 mg (0.0025% of the total diet) of a lipophilic fraction (LF) from Panax ginseng on rat platelet aggregation induced by collagen or thrombin, and on blood coagulation.When platelets prepared from 15% corn oil plus LF-administered rats (COLF) were stimulated by thrombin (0.1 units/ml) and collagen (100 μg/ml), the cGMP level was significantly increased as compared with those from 15% corn oil only-administered rats (CO). The levels of cAMP in COLF were decreased by thrombin, but was increased by collagen. Furthermore, the levels of both cGMP and cAMP were also increased by the exogenous addition of LF to thrombin- and collagen-stimulated platelets. These results mean that LF increases cGMP directly and cAMP indirectly, and thus inhibits thrombin- or collagen-induced rat platelet aggregation.Both the thrombin time (TT) and activated partial thromboplastin time (APTT) were prolonged more in citrated platelet-poor plasma from COLF than in that from CO. The level of lipids such as triglyceride, total cholesterol, high density liporotein-cholesterol and low density lipoprotein-cholesterol was decreased in serum from COLF more than in that of CO.Thus, these results suggest that dietary LF regulates the levels of cGMP and cAMP, and prolongs the time interval (TT, APTT) between the conversion of fibrinogen to fibrin. Accordingly, our data demonstrate that dietary LF has an antithrombotic effect in vivo.
We investigated whether peripherally administered aconitine increases spontaneous acetylcholine (ACh) release from the frontal cerebral cortex in freely moving rats using in vivo microdialysis, as it relates to aconitine-induced bradycardia estimated by a tail-artery cuff technique unilateral anterior hypothalamus (AH)-lesion mice. Intraperitoneally administered aconitine significantly increased cortical A Ch release within 15 min at 10 and 30 μg/kg. The increasing effect diappeared 30 min after the administration of aconitine. Aconitite-induced ACh release was not inhibited by intracerebroventricularly preadministered atropine (1 and 3 μg/rat). Atropine (1 μg/mouse) preadministered into the contralateral intact AH in mice did not affect aconitine (30 μg/kg, i.p.)-induced bradycardia. These results indicate that the cortical ACh release caused by peripherally administered aconitine dose not occur through activation of the central muscarinic receptor, and thus its ACh release may not be concerned with the occurrence of bradycardia.
A series of nitrogen-containing terpene alcohol derivatives were tested for their immunosuppressive effects in vitro, and KYKC-407 (erythro-1-(1-imidazolyl)-3, 7, 11-trimethyl-dodecane-2(S), 3(R)-diol) was found to be the most effective in suppressing the induction of cytotoxic T cell (CTL) activity. CTL induction in the coculture of C3H spleen cells with mitomycin C-treated BALB/c spleen cells was suppressed by more than 90% when 4 μM KYKC-407 was added to the culture. Under these conditions, lymphocyte proliferation was also suppressed to a similar extent by this compound. Flow cytometric analysis revealed that KYKC-407 strongly inhibited the proliferation of CD8+ T cell subset, but showed less suppressive effects on CD4+ T cells. In contrast to cyclosporin A, KYKC-407 at 1-4 μM did not inhibit the proliferative response to concanavalin A. Further, KYKC-407 suppressed CTL induction even in the presence of exogeneous IL-2, thus suggesting that the compound dose not exert its effects through inhibiting IL-2 production.
Effects of oral administration of cryomilled powder of the soft-shelled turtle (SST powder) on forced-exercise performance and stress-induced reduction in sexual and learning behavior were studied in mice. In the fatigue-preventive experiment, the SST powder was administered orally to mice which were forced to climb a descending rope for 60 min a day. The treatment significantly improved the daily performance of the mice. In the stress recovery experiment, male mice were hanged from the tail and forced to swim using their fore paws for 30 to 60 min everyday. Stress significantly reduced sexual behavior and memory retrieval (step down test). Daily oral administration of the SST powder significantly attenuated this decline in sexual behavior. However, the effects on memory retrieval were not statistically significant. These results suggest that administration of SST powder attenuates fatigue and accelerates recovery from stress in mice.
The protective effects of N-benzoyl amino acids (NAAs) and piperacillin (PIP), anionic transport inhibitors, against the nephrotoxicity of cisplatin were examined in rats. Male Wistar rats were injected i.p. with 6 mg/kg of cisplatin combined with i.p. NAAs or PIP. Rats were sacrificed on day 5 after cisplatin injection to weigh the kidney and liver, and to determine blood urea nitrogen (BUN) and serum creatinine (serum Cr) levels. Treatments with NAAs were an effective means of protection against cisplatin-induced nephrotoxicity. The combination of cisplatin with NAAs containing a short and straight chain significantly suppressed (p<0.05) the changes in body, kidney and liver weights, BUN and serum Cr. Further, betamipron (BP) at a 2000 mg/kg dose showed no apparent effect on the body, kidney and liver weights, BUN and serum Cr levels in rats. The combination of cisplatin with PIP caused a loss in body weight. The protective effects of PIP against cisplatin toxicity are inferior to those of BP when compared at 250 mg/kg doses.
In the course of our search for derivatives with potent antibacterial activity and low cytotoxicity, we have studied the relationships among the structure, antibacterial activity and cytotoxicity of thiazoloquinolone and thiazetoquinolone derivatives (the term quinolone as used in this paper includes the quinolone, 1, 8-naphthyridine and pyridopyrimidine nuclei). The antibacterial activities and cytotoxicity of these derivatives were compared with those on norfloxacin, ofloxacin, enoxacin and ciprofloxacin. The antibacterial activities of the thiazoloquinolone derivatives were more potent than those of the dihyrothiazoloquinolone derivatives, and comparable to that of ciprofloxacin. All of the thiazoloquinolone derivatives were highly cytotoxic against mammalian cells, but some of the dihyrothiazoloquinolone derivatives were less cytotoxic, being comparable in cytotoxicity to the reference drugs. The thiazetoquinolone derivatives were less cytotoxic than the thiazoloquinolone derivatives, and one of them, 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1, 3]thiazeto[3, 2-a]quinoline-3-carboxylic acid, showed the most potent antibacterial activity of all compounds tested in this study, as well as a very low cytotoxicity. The antibacterial activity and cytotoxicity of this compound were similar to that of ciprofloxacin.
To identify the active components in Mori Cortex (Chinese medicine), Mori Cortex extracts were administered orally to rats, and then blood plasma, urine, and bile were analyzed. The results showed only a few remaining metabolites of mulberroside A. Consequently, to clarify the transitional properties of mulberroside A derivatives to tissues and the in vivo abundance ratio of mulberroside A compounds, the pharmacokinetics of mulberroside A derivatives were investigated in this study. When Mori Cortex extracts were administered orally, mulberroside A was only detected in small quantities in the plasma, and its bioavailability was a bout 1%. This is due to the first pass effect, by which most mulberroside A was converted into oxyresveratrol and transported into the circulating blood, and its absorption ratio was estimated at about 50%. Oxyresvertrol was also found to be transported to tissues at high rates. As a result, when analyzing the pharmacological activity of the Mori Cortex in vitro, it is more useful to study oxyresveratarol than mulberroside A.
Macrophages play important roles both in immune response and in lipid metabolism and contribute to the development of atherosclerosis. To clarify the mechanism by which Shosaikoto, a Kampo medicine, shows anti-atherosclerotic action, we studied its effect on macrophage function. The production of nitric oxide (NO), prostaglandin E2 and interleukin 1 by macrophages in mice was reduced by feeding of a cholesterol-enriched diet, and the reduced production was observed 1 week after the beginning of cholesterol feeding. Furthermore, although oxidized low density lipoprotein (LDL) and lysophosphatidylcholine (LPC) reduced NO production, macrophages prepared from mice treated with Shosaikoto at a dose of 1.2 g/kg/d restored the reduced NO production by them as well as by hypercholesterolemia. When the content of LPC was measured, no difference was observed between mice fed a cholesterol-enriched diet in the presence or absence of Shosaikoto treatment, suggesting that the restorative effect of Shosaikoto is not due to the inhibition of LPC production or accumulation. Conclusively, Shosaikoto prevents the modification of macrophage function induced by atherogenic factors, which is probably lined to its displayed anti-atherosclerotic action.
The effects of a traditional Chinese medicine Sho-saiko-to (Kampo prescription) were investigated on the various metabolic disorders and antitumor activity of recombinant human tumor necrosis factor (rhTNF) administered to mice. The glycogen level in liver of rhTNF (5×104 units/mouse, i.v.)-injected mice was markedly lower at 4 h post-intoxication than that in the control, whereas the administration of rhTNF to Sho-saiko-to (500 mg/kg/d, p.o.)-pretreated mice resulted in a greater level of glycogen than that in rhTNF alone-treated mice. In mice pretreated with Sho-saiko-to, the level of fibrinogen 4 h after rhTNF injection markedly increased as compared to that in mice treated with rhTNF alone. We also estimated the NO-2 in murine macrophage cell line J774A.1 using mice serum after administration of Sho-saiko-to. Our results clearly demonstrated that J774A.1 cells stimulated with endotoxin (1 μg/ml) and rhTNF (1×104 units/ml) can effectively produce nitric oxide (NO), and ascertained the suppressive effect of Sho-saiko-to (500 mg/kg/d, p.o.)-pretreated serum on NO generation by endotoxin/TNF-activated J774A.1 cells. When the cells were incubated with endotoxin/TNF and Sho-saiko-to pretreated serum (10-100 μl), the NO level was significantly lower than that in control serum incubated with endotoxin/TNF alone. The effect of Sho-saiko-to (1 and 10 μg/ml) on in vitro cytotoxicity by rhTNF in Meth-A Sarcoma cells was observed to be in a dose dependent fashion. In addition, there was a remarkable enhancement of antitumor activity of rhTNF by Sho-saiko-to pretreatment in mice. These findings suggest that the Kampo prescription Sho-saiko-to may protect mice from severe shock syndrome by rhTNF, and that it may enhance rhTNF-induced antitumor activity.
The water extract of propolis (PWE) showed a strong hepatoprotective activity against CCl4-toxicity in rats and D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. The PWE also showed a significant hepatoprotective activity against CCl4-induced liver cell injury in cultured rat hepatocytes. The in vitro hepatoprotective activity guided fractionation and chemical analysis led to the isolation of four dicaffeoyl quinic acid derivatives from the PWE. The structure of these isolated was determined to be methyl 3, 4-di-O-caffeoyl quinate (1), 3, 4-di-O-caffeoyl quinic acid (2), methyl 4, 5-di-O-caffeoyl quinate (3), and 3, 5-di-O-caffeoyl quinic acid (4) by spectroscopic method. These compounds were more potent hepatoprotective agents than glycyrrhizin at a concentration of 10 μg/ml and 1 was the most potent among the four compounds in the cultured hepatocytes. Quinic acid (5) alone did not show hepatoprotective effects in cultured rat hepatocytes against CCl4-toxicity. On the other hand, chlorogenic acid (6) or caffeic acid alone was found to be less potent than the dicaffeoyl quinic acid derivatives.
Complete amino acid sequences of karasurin-B and karasurin-C purified from root tubers of Trichosanthes kirilowii MAXIM. var. japonica KITAMURA (Cucurbitaceae) were determined. Karasurin-B consists of 247 amino acid residues with a calculated molecular weight of 27214 Da and karasurin-C of 249 amino acid residues with a calculated molecular weight of 27401 Da. Their amino acid sequences are quite similar to that of karasurin-A, a major basic protein from the same root tubers, but the pI values are slightly different amoung them. All karasurins strongly inhibited in vitro translation in the rabbit reticulocyte system. The amino acid sequences of karasurins were compared with those of the ribosome-inactivating proteins (RIPs) from nine different species by UPGMA method, and the RIPs from Cucurbitaceae plants phylogenetically formed one cluster clearly distiguishable from those of other origin.
The nasal absorption of non-glycosylated recombinant human granulocyte colony-stimulating factor produced by Esherichia coli (E. coli-rhG-CSF) was studied in rats. We investigated the effects of the basic conditions of the soding solution and the enhancing effects of various substances to find means to improve the nasal absorption of E. coli-rhG-CSF. Relative bioavaliability following the nasal administration of E. coli-rhG-CSF solution of subcutaneous administration was 3.6%. Osmotic pressure of the dosing solution had virtually no effect of the nasal absorption of E. coli-rhG-CSF. Absorption was enhanced by a decrease in pH, an increase in buffer concentration at pH 2 and an increase in drug concentration. Sodium cholate, ascorbic acid, tartaric acid, β-cyclodextrin and dimethyl-β-cyclodextrin remarkably increased the nasal absorption of E. coli-rhG-CSF. Condition adjustments to the dosing solution and the use of several enhancers were significantly concluded to increase the intranasal absorption of E. coli-rhG-CSF.
Dose-dependent gastrointestinal absorption of 5-fluorouracil (5-FU) was kinetically evaluated in rats in vivo by analyzing gastointestinal disposition after oral administration, where a linear model assuming first-order gastric emptying followed by first-order intestinal absorption was fitted to remaining fraction versus time profiles for the stomach and small intestine to estimate the rate constants of gastric emptying (kg) and intestinal absorption (ka). With an increase in dose from 1.5 nmol/rat (low dose) to 15 μmol/rat (high dose), the ka decreased from 5.95 to 0.55 min-1, suggesting the involvement of carrier-mediated transport. This study is the first to demonstrate the dose-dependent gastrointestinal absorption of 5-FU in vivo, though it has long been suggested in situ and in vivtro. Meanwhile, at both the low and high doses, the kg values, which were unaffected by dose (0.069 and 0.082 min-1, respectively, for the low and high doses), were smaller that the ka values by an order of magnitude or more and the recovery of 5-FU was negligible, compared with that of inulin (a nonabsorbable marker), in the most distal segment of ileum. These results suggest that, regardless of dose, 5-FU is highly absorbable in a gastric emptying-limited manner. Thus, well-publicized bioavailability problem (low and erratic) of 5-FU may be attributable to extensive and variable first-pass metabolism rather than poor and variable gastrointestinal absorption.
We studied the characterization of cabergoline, a new ergot alkaloid derivative and a selective dopamine D2 receptor agonist, in comparison to bromocriptine and pergolide in reserpine-treated rodents. Cabergoline (0.25-1.0 mg/kg, s.c.) improved dose-dependently the reserpine-induced akinesia that was assessed on the locomotor activity, and the efficacy lasted longer than those of bromocriptine (1.25-5.0 mg/kg, s.c.) or pergolide (0.0625-0.5 mg/kg, s.c.). Cabergoline (ED50=1.10 mg/kg, at 4 h after the administration of drugs) also reversed catalepsy, the failure to correct an externally imposed posture, and its efficacy was stronger and longer than bromocriptine (ED50=4.65 mg/kg, at 4 h). Further, reserpine-induced rigidity was improved equally by cabergoline (0.125-1.0 mg/kg, i.v.) and bromocriptine (1.0 mg/kg, i.v.). When cabergoline was administered together with 3-(3, 4-dihydroxyphenyl)-L-alanine (L-DOPA), the effects were additive. Our results indicate that the long-lasting effects of cabergoline could be beneficial for treating Parkinson's disease.
Calmodulin (CaM), a Ca2+-binding protein, is a multifunctional modulator mediating the calcium regulation of a large number of intracellular enzyme systems. Because of its highly concerved structure, it has been difficult to produce anti-CaM antibody. We describe here a simplified method for production of this antibody. BALB/c mice were immunized with a polymerized CaM, and the serum was prepared and tested for the presence of anti-CaM antibody by immunoblot analysis. The serum obtained from the immunized mice was shown to contain anti-CaM antibody. Immunoblot analysis proved that a 500-fold diluted serum was able to recognize both types of CaM with a polymerized form and monomer form. The method developed in this study may be applicable to a wide range of proteins for enhancement of antigenicity.
Five restriction endonucleases (ENases) and one ENase were found in a screen of 196 strains of Plesiomonas shigelloides and 147 strains of Aeromonas species. Plesiomonas and Aeromonas species are classified as Vibrionaceae, identified as food-poisoning bacteria, are closely genetically related to each other, and their ENases producing abilities have not been reported. ENases were detected at relatively low frequencies in these species as compared to those in other species, such as Salmonella species and Vibrio paraheamolyticus. All ENases were shon to be isoschizomers of already known ENases. One of the Plesimonas ENases, designated PshBI, recognizing the sequence 5'-AT/TAAT-3' should be useful, since PshBI ENase is produced at a high yield or 7000 units/g of wet cells. The specificities of other ENases are also described in this paper.
HLT, Bordetella heat-labile toxin, caused a release of radioactivity from cultured smooth muscle cells (SMC) previously labeled with [14C]arachidonic acid, and from membrane preparations from the labeled SMC. HLT, however, failed to induced release from primary SMC prepared by treatment with an enzyme solution containing collagenase and elastase. When cultured SMC were treated with the enzyme solution, their susceptibility to HLT was reduced more significantly than that of untreated cells. SMC also lost their susceptibility, after treatment with collagenase alone, but not with elastase, in a dose-dependent manner. These data suggest that collagen on the SMC surface might play an important role in HLT activity.
The oxidative metabolism of cinnarizine [(E)-1-(diphenylmethyl)-4-(3-phenyl-2-propyl)piperazine, CZ] and flunarizine [(E)-1-[bis(4-fluorophenyl)methyl]4-(3-phenyl-2-propyl)piperazine, FZ] was examined in microsomes from lymphoblastoid cells that expressed human cytochrome P450 (CYP) enzymes. Among 10 kinds of CYP enzymes examined, only CYP2D6 catalyzed p-hydroxylation of the cinnamyl phenyl ring of CZ (C-2 formation) and FZ (F-2 formation), and only CYP2B6 exhibited activity for p-hydroxylation (C-4 formation) of the diphenylmethyl group of CZ at a substrate concentration of 50 μM. On the other hand, CYP2C9 together with CYP1A1, -1A2 and/or -2A6 mediated N-desalkylation at the 1- and 4-positions of the piperazine ring of the two drugs that formed C-1 and C-3 from CZ and F-1 and F-3 from FZ, respectively, whereas CYP2C8, -2C19, -2E1 or -3A4 did not show detectable activity for these reactions under the conditions used. We then examined kinetics for the oxidative metabolism of CZ and FZ using CYP2B6 and -2D6 that have considerable activities. CYP2D6 with Km values of 2 to 4 μM had intrinsic clearance values (Vmax/Km) of 0.31 and 0.14 ml/min/nmol CYP for C-2 and F-2 formation, respectively, while CYP2B6 with a Km value of 17 μM exhibited the clearance value of 0.10 ml/min/nmol CYP for C-4 formation. These results suggest that CYP2D6 mainly mediates p-hydroxylation of the cinnamyl phenyl rings of CZ and FZ, and CYP2B6 mediates that of the diphenylmethyl group of CZ.
Bardanae Furctus (Goboshi) extract showed potent in vitro cytotoxicity and in vivo antitumor activity against human hepatoma HepG2 cells and mouse sarcoma 180 cells, respectively. In this study, the cytotoxicities of four fractions and three major components (arctiin, arctigenin, and chlorogenic acid) isolated from Goboshi extract were examined. Arctiin and arctigenin, which were contained in the ethylacetate fraction and n-butanol fraction, respectively, showed stroug cytotoxicity against HepG2 cells, but little toxicity against Chang liver cells. Chlorogenic acid isolated from the water fraction did not affect the viability of these cells. The cytotoxicity of arctigenin against Chang liver cells was markedly potentiated by treatment with a glutathione (GSH) synthesis inhibitor, L-buthionine-(S, R)-sulfoximine (BSO). On the other hand, in HepG2 cells, the cytotoxicity of arctigenin was hardly changed by BSO. The cytotoxicity of arctigenin against HepG2 cells increased in an exposure-time dependent manner. These characteristics of arctigenin were similar to those of Goboshi extract, as previously observed. We therefore conclude that the principal cytotoxic components of Goboshi extract are arctiin and its aglycone arctigenin.
Cell viability and in vitro cytotoxicity assay methods were developed using a combination of dyes, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (WST-1), neutral red (NR) and crystal violet (CV), with HeLa cells as a bioindicator. As WST-1 produces a highly water soluble and non-cytotoxic formazan dye, it allows each assay to be carried out in one culture dish. The combined cell viability assay using WST-1, NR and CV gave an absorbance that correlated linearly with the number of cells over the range 1000 to 50000 cells/well. The combined assay was applied to the evaluation of the IC50 values for sodium dodecyl sulfate as a model toxicant, which yielded similar values to those obtained with each assay independently.
A chemiluminescence (CL) was observed immediately after the addition of luminol to thioglycollate-elicited ICR mouse peritoneal macrophages (Mφ) that had been incubated overnight with recombinant murine interferon-γ(IFN-γ) and lipopolysaccharides (LPS). The intensity of this CL was closely correlated with the cytotoxic activity of Mφ. NG-minomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthase, inhibited the induction of this CL, and L-arginine restored the L-NMMA-induced inhibition. These results suggest that NO is directly involved in the induction of CL. However, we found that immune complexes are required for the induction of CL as well as NO.
The in vivo metabolism of 1-nitropyrene, an improtant environmental pollutant, was examined in fish, focusing on nitroreduction followed by acylation. When goldfish were bathed in a solution of 1-nitropyrene or its reduction product, 1-aminopyrene, one or two metabolites were isolated from the solution, respectively. The former metabolite was identified as 1-aminopyrene and the latter two metabolites as 1-acetylaminopyrene and 1-formylaminopyrene by comparing their mass and UV spectra, and their behaviors in HPLC and TLC, with those of authentic samples.
Biodegradable microspheres containing cyclosporin A (CsA), an immunosuppressant, enabled the lymphotropic delivery of the durg over the log term by parenteral administration in mice. The CsA encapsulated in the microspheres of polylactic acid polymer (Mw, 9000) was released over 30 days in vitro. The intramuscular injection of the microspheres containing CsA in the femoral site of mice maintained the drug at a high level in the inguinal lymph nodes over 1 month, although only low blood levels of the drug were observed.
Total DNA was extracted from the fresh underground parts of three Panax separate species. The 18SrRNA regions of extracted DNA were amplified by the polymerase chain reaction (PCR) and their sequences were determined. In each species, the sequences were found to be of 1809 bease pairs (bps) but with different gene sequences. Different base substitutions were observed at nucleotide positions 497, 499, 501 and 712. The same procedure was performed on commercial samples of Ginseng Radix, Panacis Japonici Rhizoma and American Ginseng. Each sequence completely corresponded with that of each original plant, namely P. ginseng, P. japonicus and P. quinquefolius, respectively. This is the first time that 18S rRNA gene sequencing on Panax species was carried out. Prevously, Ginseng drugs have been identified mainly by their external and internal structure. Thus this method will be useful in identifying Ginseng drugs at the gene level.
We previously reported that cultured mammalian cells are effectively permeabilized by vortex-stirring the cells with a high molecular weight polyacrylic acid. The present study revealed that the efficiency of permeabilization of a non-permeant dye, Lucifer Yellow (LY), was sensitively affected by the pH of the medium. When the cells are vortex-stirred in RPMI 1640 culture medium, the pH of the medium should be adjusted with HEPES and NaOH, in stead of NaHCO3, to a pH higher than 7.6 for successful permeabilization; the higher the pH, the better the result obtained. Internalization of LY was near the background level at a pH below 7.4. When phosphate-buffered saline was substituted for RPMI 1640 medium, the optimal pH range was slightly shifted to a more acidic region. This requirement for the pH of the medium is indispensable as a supplement to the standard permeabilization procedure tentatively proposed in the preceding report : Biol. Pharm. Bull., 19, 1279-1284 (1996).