A sensitive and reliable method for the determination of hypusine and deoxyhypusine in eIF-5A protein, an initiation factor of protein synthesis, was developed. An advantage of this method is the use of Nε-(5-aminopentyl) lysine, an analogue of deoxyhypusine, as an internal standard. The application made it possible to determine hypusine in less than a mg of protein samples from cultured HTC cells and rat organs. After acid hydrolysis of protein samples to which had been added the internal standard, the hydrolysates were fractionated by carboxymethyl cellulose column chromatography. Also, diamine fractions containing a few pmol of hypusine and deoxyhypusine were successfully analyzed by a reversed phase HPLC with a fluorescence detection of o-phthalaldehyde. The method was applied for the determination of hypusine and deoxyhypusine in drug-treated HTC cells and normal rat organs. The results from HTC cells were discussed based on the known effects of each drug on hypusine biosynthesis.
We investigated the effects of several types of aminopeptidase inhibitors on tumor cell-associated aminopeptidase activity and invasion. The aminopeptidase expressed by the human metastatic HT1080 fibrosarcoma cells was effectively suppressed by actinonin A, bestatin, leuhistin and matlystatin A, which are capable of inhibiting the purified aminopeptidase N, but not by arphamenine B specific for aminopeptidase B. The aminopeptidase N inhibitors inhibited HT1080 cells from degrading the subendothelial matrix and from invading into Matrigel in parallel with their aminopeptidase inhibitory activities. Matlystatin A, with multiple inhibitory activity against both aminopeptidase N and matrix metalloproteinases (MMP), was the most effective inhibitor of invasion. However, leuhistin and bestatin, without MMP inhibitory activity, also exhibited significant inhibition of invasion. The results suggest that aminopeptidase N plays a crucial role in the degradation and invasion of extracellular matrices by fibrosarcoma cells and that aminopeptidase inhibitors may be useful for preventing the spread of malignant tumors.
A noncollagenous high molecular weight protein (HMW) was isolated from bovine articular cartilage by CsCl density gradient centrifugation followed by DEAE-cellulose chromatography and gel filtration chromatography. The molecular weight was estimated to be 320 kDa and the reduced HMW had a molecular weight of 105 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid composition of HMW showed a high content of aspartic acid, but no hydroxylysine and hydroxyproline were detected, indicating that the protein was not a collagen. Affinity purified rabbit antibody against HMW reacted specifically with HMW, but there was no immunoreaction with cartilage-specific proteoglycan, type II collagen and type I collagen. HMW showed dose-dependent attachment to types I, II, III and V collagens. However, HMW did not bind to fibronectin, decorin and heparin. Furthermore, the binding of HMW to heat-denatured collagens was lower than native collagens. In these observation, HMW may be recognized the triple helix structure of collagen for the interaction.
It has been reported that the biological activities of several β-glucans vary due to differences in their physicochemical properties. In this study we investigated the ability of β-glucans to trigger H2O2 production and activate signaling pathway on peritoneal macrophages. The most effective β-glucan tested on H2O2 production was zymocel which was a particulate preparation from the yeast cell wall. In contrast, gel-forming β-glucans which are known as immunoenhancers did not trigger the H2O2 production by macrophages at all. To identify the related signaling pathway for the particulate β-glucans-triggered H2O2 production, several inhibitors were applied. Hydrogen peroxide production triggered with phorbol myristate acetate was inhibited by pretreatment of macrophages with H-7, a protein kinase C inhibitor. However, β-glucans-triggered H2O2 production was not affected by H-7. The results suggested that genistein-sensitive tyrosine kinase and bromophenacyl bromide-sensitive phospholipase A2 participated in the particulate β-glucans-triggered H2O2 production, although the phagocytosis of particulate β-glucans was not inhibited by either reagents. In conclusion, gel-forming (1→3)-β-D-glucans-induced activation was not sufficient to trigger H2O2 on macrophages, and pathways for particulate β-glucans-triggered H2O2 production were regulated differently from those for phagocytosis of β-glucans.
Survival, following the addition of a cAMP analog and high K+ to the medium, of cultured fetal septal cholinergic neurons was examined after nerve growth factor (NGF) deprivation. The number of acetylcholinesterase positive cells, which had progressively grown during 11-13 d of culture with NGF (50 ng/ml), was greatly reduced following 5 d of extended culture without NGF (55% of that with NGF). The degeneration of the cholinergic neurons was markedly reduced by addition of dibutyryl cAMP (dbcAMP, 1 mM), forskolin (10μM) or KCl (15 mM) to the medium. K252b, which blocks the survival of NGF, had no effect on the action of dbcAMP. H-8 and nifedipine inhibited the survival of dbcAMP and high KCl, respectively. These results suggest that NGF, dbcAMP and high K+ promote the survival of septal cholinergic neurons acting via the receptor tyrosine kinase, protein kinase A and a Ca2+-dependent mechanism, respectively.
Symptoms such as those in Parkinson's disease are known to be induced by the neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). We tried to quantitatively measure synaptosomal MPP+ uptake using an MPP+ selective electrode to study the correlation between MPP+ uptake and respiratory inhibition. Synaptosomal MPP+ uptake was low but could be increased by the addition of glucose as an energy substrate, or increased with an increase in the concentration of MPP+. The rate of uptake was 0.2 nmol/mg protein/min at 50μM MPP+. Tetraphenylboron (TPB-), which enhances cation permeability, increased MPP+ uptake, and the increase was proportional to the TPB- concention. When external MPP+ concentration was increased above 200μM, ATP was depleted and the uptake of MPP+ decreased, which resulted in the release of intrasynaptosomal MPP+. MPP+ uptake was also decreased by depolarization of the membrane potential in synaptosomes. MPP+ was presumed to be distributed across both the synaptosomal and inner mitochondrial membranes, and to be affected by membrane potential as a lipophilic cation. When respiration of the inner mitochondria was inhibited by increasing the intrasynaptosomal MPP+ concentration, the concentration of MPP+ in cytosol was presumed to increase by the release of MPP+ from the mitochondria, and synaptosomal MPP+ uptake would then be decreased.
The effects of sex hormones on the induction of hepatic CYP4A by clofibrate were examined in intact and gonadectomized rats of both sexes by measuring the activity of lauric acid ω-hydroxylation (LAH). Although the specific activity of LAH is the same in both sexes, only in male rats, was the activity significantly increased by administration of clofibrate. In female rats, ovariectomy or testosterone treatment resulted in an enhanced response to clofibrate, similar to that observed in male rats. In ovariectomized female rats, the effect of clofibrate was decreased by estradiol. In orchiectomized male rats, testosterone had no effect on the induction of LAH by clofibrate and estradiol suppressed induction. Changes in LAH activities co-realted well with those in CYP4A proteins and CYP4A1 mRNA. These results suggest that some factors associated with female sex hormones were involved in the suppressive regulation of CYP4A induced by clofibrate.
A cDNA clone which covers the entire coding region for the precursor of adrenodoxin was isolated from a rat adrenal cDNA library. This precursor consists of amino-terminal 64 residues of extrapeptide for transport into mitochondria and the following 124 residues of mature peptide region. The amino acid sequence of rat mature adrenodoxin showed 85-98% homology with mouse, human, chicken, porcine, bovine and sheep counterparts, whereas that of the extrapeptide showed significantly lower values.
Sulochrin oxidase, an enzyme catalyzing regio- and stereospecific phenol oxidative coupling reaction to form (+)-bisdechlorogeodin from sulochrin, was isolated from Penicillium frequentans CMI 96659. By chromatographies on DEAE-cellulose, hydroxyapatite, Phenyl-Sepharose, Mono P, Mono Q, and HPLC gel filtration columns, sulochrin oxidase was purified to apparent homogeneity. The purified enzyme had a molecular weight of 230 K as estimated by gel filtration and 110 K as estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis under denaturing conditions, suggesting that the active enzyme was a homodimer. The enzyme showed pI 4.0 and an optimum pH of 6. The enzyme activity was strongly inhibited by the copper-chelating reagent, diethyldithiocarbamate, and did not recover its full activity even after removing the inhibitor by dialysis. However, enzyme activity was fully restored by the addition of Cu2+. Thus, sulochrin oxidase is considered to be a copper protein. The enzyme showed high substrate specificity for benzophenone compounds such as sulochrin and dihydrogeodin.
Effects of a traditional Chinese medicine, Shimotsu-to (a combined prescription of cnidium rhizome, peony root, angelica root and rehmannia root), and its included crude fractions were investigated on an adjuvant-induced chronic inflammation model of mice. The aqueous extract (30, 100 and 300 mg/kg, i.p.) of Shimotsu-to reduced the carmine content, granuloma weight, inflammation cell count and pouch fluid weight in the inflammation model, respectively. The extract of Shimotsu-to without cnidium at the same doses did not produce significant changes in these four inflammatory parameters. The same doses of extracts of Shimotsu-to without peony, and without angelica, weakly reduced these parameters, except for pouch fluid weight. The extract (30, 100 and 300 mg/kg) of cnidium significantly reduced these four parameters. The same doses of peony extract reduced carmine content, granuloma weight and pouch fluid weight, but less than those of the cnidium extract. The extract of cnidium and peony at the same doses reduced in an additive manner these inflammatory parameters in their combination. These results demonstrated that the Shimotsu-to extract reduced angiogenesis, granuloma formation, inflammatory cell migration and pouch fluid exudation in the adjuvant-induced chronic inflammation model. Cnidium represented the main ingredient for producing the anti-chronic inflammatory effects of Shimotsu-to extract. Cnidium and peony exhibited additive anti-inflammatory effects in combination.
We investigated the effects of cimetidine, omeprazole and atropine, antisecretory anti-ulcer agents, on the healing of gastric and duodenal ulcers simultaneously induced in the same rats. Furthermore, we examined the effect of histamine, an acid secretory agent, on the healing of both ulcers. When the effects of test drugs were assessed on the 15th day after local application of acetic acid, repeated oral administration of cimetidine (50 and 100 mg/kg twice daily) or omeprazole (25 and 50 mg/kg once daily) markedly accelerated the healing of both gastric and duodenal ulcers. Atropine (10 mg/kg twice daily, p.o.) showed a healing effect on duodenal ulcers only. The repeated subcutaneous administration of histamine (30 mg/kg 3 times daily) apparently delayed the healing of duodenal ulcers but not gastric ulcers. In conclusion, this experimental chronic ulcer model in rats is useful for directly comparing the effects of anti-ulcer drugs on the healing of gastric and duodenal ulcers. In addition, the increase in acid secretion appears to have a greater influence on the delay of ulcer healing in the duodenum than in the stomach.
The effects of 19-oxygenated lanosterol derivatives on lanosterol 14α-demethylase (P-45014DM) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were studied. 3β-Hydroxylanost-9 (11)-en-19-oic acid (6) was found to be an effective inhibitor (IC50 : 0.5 μM) of P-45014DM in the reconstituted system using purified pig liver P-45014DM and was most active in inhibiting HMG-CoA reductase activity (IC50 : 1.0μM) in mouse L cells. In [2-14C] acetate incorporation studies using mouse L cells, 6 was found to reduce the incorporation of acetate into C27-sterol (desmosterol) with a concomitant increase in radiolabeled C30-sterols. These results demonstrate that 6 is a dual-action inhibitor of cholesterol biosynthesis.
The anti-fatigue effect of 50% ethanol extract ([M]) from the dried whole body of Agkistrodon blomhoffii blomhoffii BOIE. was investigated using an acute weight-loaded forced swimming (AWLFS) test by monitoring swimming times, blood biochemical parameters, thiobarbiturate-reactive substances (TBARS) as an index of lipid peroxide and antioxidative enzyme activities in blood and tissue. [M] (500 mg/kg/d), given orally for three successive days, significantly prolonged swimming times. It also inhibited the elevation of TBARS in plasma, liver, brain, kidney and soleus, and inhibited the lowering of catalase activity in erythrocyte, liver and soleus. However, it had no inhibitory effect on the elevation of creatine-kinase activity, free fatty acid and lactic acid levels or on the decrease in glucose level in serum. Also, it decreased the plasma TBARS level and increased the superoxide dismutase activity of plasma and erythrocytes in normal rats. From these results, it can be considered that [M] has an anti-fatigue effect.
Total DNA was extracted from the leaves of seven Epimedium species grown in different places in Japan. Their genetic characterization was performed by DNA analyses of random amplified polymorphic DNA (RAPD) using 32 random primers having 10 base sequences, and by restriction fragment length polymorphism (RFLP). E. sagittatum and E. koreanum were easily distinguished by a representative amplified band pattern. It became evident that E. sagittatum had extremely different genetic composition compared to the other species. A dendrogram obtained from the similarity matrix by cluster analysis indicates that E. sagittatum can be completely isolated from the other species. Moreover, it became evident that E. grandiflorum var. higoense, E. trifoliatobinatum and E. koreanum are independent species, contrary to the previous assumption that they are subspecies or a variety. The geographical variation of E. sempervirens was confirmed by cluster analysis. E. diphyllum showed wide genetic variations, in spite of sampling from the same area.
Four cDNAs encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, were isolated from Pueraria lobata cells challenged with yeast extract elicitor using bean CHS cDNA as a probe. The longest clone contained a complete open reading frame of 1170 bp which would predict a protein of about 43 kDa. The others were not full-length clones. Using isolated cDNA as a probe, Southern blot hybridization of genomic DNA fragments revealed the presence of multiple CHS genes in the P. lobata genome. We cloned and sequenced one CHS genomic clone, gCHS14, whose 5'untranslated region showed homology with the bean CHS gene CHS15 and included the several reported sequences characteristic of stress response.
The pharmacokinetics of the two-peak plasma profiles of orally administered acetaminophen sulfate (APAPS), a major conjugated metabolite of acetaminophen (APAP), in rats was examined by a two-compartment model having two delivery routes with individual time lags : a direct delivery route of APAPS absorbed in an unchanged form and an indirect one where APAPS was absorbed as APAP after deconjugation in the lower intestine. Pharmacokinetic parameters were estimated by a non-linear least squares program, MULTI (FILT), based on the fast inverse Laplace transform algorithm. Plasma APAPS concentration profiles after oral doses were simulated satisfactorily. In a dose escalation study, the peroral availability of APAPS derived from the direct route was not changed significantly with the doses. However, that from the indirect route was decreased with the dose escalation, suggesting the contribution of a capacity-limited deconjugation of APAPS to APAP by the intestinal microflora to the non-linear pharmacokinetics of orally administered APAPS. The absorption of peroral APAPS in rats at a dose range of 30 to 120 mg APAP eq/kg could be summarized as follows : (1) approximately 25% (22 to 32%) of orally administered APAPS are absorbed from the gastrointestinal tract in an unchanged form ; (2) more than 50% (50 to 98%) are deconjugated to APAP in the lower intestinal tract ; and (3) the latter plays a significant role as an indirect and non-linear APAPS delivery system because considerable amounts of APAP thus absorbed are rapidly reconjugated to APAPS in the systemic circulation, which gives the second plasma APAPS peak.
The effect of perfusate flow rate on hepatic structure and hepatic uptake kinetics was investigated using oxacillin as a model drug and bovine serum albumin (BSA) as a reference substance in the liver perfusion system from the standpoint of a dispersion model and moment characteristics. The estimated recovery ratio (FH) of oxacillin was about 40% which was independent of the change in perfusate flow rate. The mean transit time (t^^-H) of oxacillin decreased with an increase in flow rate, while the relative variance (σ2/t^^-2H) of oxacillin was independent of the flow rate. The t^^-H of BSA decreased with an increase in the flow rate to the same extent as that of oxacillin, while σ2/t^^-2H of BSA was independent of flow rate. When the dispersion model is adopted as a model system to analyze hepatic perfusion data following the pulse input, the moment characteristics (FH, t^^-H and σ2/t^^-2H) are given in complicated equations. It is demonstrated by the present investigation that these moment equations can be extensively simplified for a drug with a medium extraction ratio (FH>50%), i.e., FH is independent of the distribution, both FH and t^^-H are independent of the dispersion process in the hepatic blood space, and both t^^-H and σ2/t^^-2H are independent of the elimination. Thus, it is shown that FH and t^^-H are exactly the indices of elimination and distribution, respectively, and σ2/t^^-2H is the index of dispersion in the blood space plus nonequilibrium in the hepatic distribution.
Urinary antibiotic treatments usually affect the normal urethral flora. This work was developed in order to evaluate whether Lactobacillus fermentum CRL 1058 could reduce urinary tract infections (UTI) produced by uropathogenic Escherichia coli in mice treated with antimicrobial agents. Animals were inoculated intra-urethrically with agarose beads containing lactobacilli, and were challenged with E. coli. Ampicillin (13 mg/kg/dose) was administered orally. The number of microorganisms present at different days was evaluated in the urogenital tract. Serum inflammatory and systemic immune response were also registered. The use of 5 doses of ampicillin after 3 doses of lactobacilli in agarose beads significantly affected the viability of lactic acid bacteria, while the amount of E. coli was not altered. Lactate dehidrogenase (LDH) activity and anti-E. coli antibody levels showed no statistically significant difference between the challenged and non-challenged mice. Lactobacilli reinoculation and 3 doses of ampicillin proved to be a moderately effective treatment since a smaller amount of E. coli was recovered from the organs of treated mice than from the controls. The reinforcement of lactobacilli, administered on the 9th day, produced a faster elimination of the pathogen. The ampicillin dose used allowed lactobacilli permanence in the urinary tract, and caused the elimination of the pathogen. Serum LDH values seemed to show an inflammatory immune response. No successful preventive results could be achieved. We can conclude that lactobacilli and adequately low doses of ampicillin have a positive effect on the treatment of E. coli in this UTI model.
The intestinal transport mechanism of 5-fluorouracil (5-FU) was investigated in the intestinal everted sacs and brush border membrane vesicles (BBMVs) of rats. In the everted sacs, the initial uptake of 5-FU was apparently Na+-dependent, in terms of the inhibition of the uptake by replacing the Na+ in the medium with K+, and also concentration-dependent in the presence of Na+, with a maximum transport rate of 0.74±0.24 nmol/min/cm and a Michaelis constant of 0.025±0.018 mM. Passive transport was also significant, with a membrane permeability clearance of 5.9±0.6μl/min/cm. The uptake of 5-FU was inhibited by pyrimidines (uracil and thymine) and 2, 4-dinitrophenol (DNP), a metabolic inhibitor, but not by purines (adenine and guanine), pyrimidine nucleosides (thymidine and uridine), L-alanine or D-glucose. These results suggest the involvement of carrier-mediated transport specific to pyrimidines in intestinal 5-FU transport, and this appeared to satisfy the criteria of Na+-dependent secondary active transport. However, in BBMVs, 5-FU uptake was independent of Na+, minimally dependent on concentration and not inhibited by thymine, though slightly inhibited by uracil. 5-FU uptake was also independent of pH, outward HCO-3 gradient and valinomycin-induced K+ diffusion potential. Thus, the carrier-mediated transport of 5-FU, or presumably pyrimidines, may require some other factors, which are yet to be identified, in addition to Na+. Alternatively, Na+ may not be prerequisite, and something else may be required in the absence of K+, as suggested from an additional result in everted sacs that the replacement of NaCl in the medium with mannitol failed to inhibit 5-FU uptake.
We examined the transport mechanisms of daunorubicin (DNR) and doxorubicin (ADR) in HL60 and HL60/THP cells which were the non-P-glycoprotein-mediated resistant clone of the parent HL60 cells and showed a low degree of resistance, and compared them with those of pirarubicin (THP). In both lines, it appeared that the uptakes of DNR and ADR were time-, temperature- and concentration-dependent and energy independent, and the transport of DNR consisted of saturable and nonsaturable components. They were pumped out from the cells time-, temperature- and energy-dependently. There were no differences in the accumulation amount of either DNR or ADR between HL60 and HL60/THP cells. Comparing the transport of DNR or ADR with that of THP, the uptake amounts of DNR and THP were approximately equal, and were greater than that of ADR in both types of cell. In cis-inhibition experiments, DNR inhibited the THP uptake noncompetitively in the parent and resistant cells, in contradiction of the previously reported result in which ADR showed competitive inhibition (Nagasawa, K. et al., Cancer Chemother. Pharmacol., in press). The THP accumulation appeared to be increased by preload of DNR and ADR, indicating a counter transport. Thus, DNR and ADR as well as THP might be incorporated via a common carrier-mediated transport system, but DNR uptake in part appeared to follow a nonsaturable transport, and its binding site in the carrier might differ from that of THP and ADR in both HL60 and HL60/THP cells.
Two types of multiple controlled release dosage forms, hydroxypropylmethyl cellulose acetyl succinate (HPMC-AS) coated granules and double layer coated granules with HPMC-AS and ethyl cellulose (EC), were prepared for the newly developed antihistaminergic drug, TA-5707F, using a centrifugal fluidizing granulator. The in vitro dissolution rate of TA-5707F in the second fluid from the former granules could be altered by changing the grade (HPMC-AS-M, AS-H, AS-V) or coating amount. However, the plasma concentration determined in fasted dogs was not markedly affected by the HPMC-AS grades. From the granules coated with 30% (w/w) AS-V (the polymer with the highest pH required for dissolution), little TA-5707F was released into the second fluid, but a fairly high plasma concentration was detected although a constant plasma level was not maintained in the fasted dogs. The dissolution rates from the double layer coated granules were changed by the coating percent of EC. The plasma concentration of TA-5707F in dogs correlated well with the in vitro dissolution rate and was not markedly affected by food. Thus, two kinds of double layer coated granules (coated 0.75% EC and 1% EC) were administered to humans, and showed sustained-releasing plasma profiles ; that is, the plasma concentration increased gradually and reached a Cmax 5 and 8 h after administration, respectively. The amount of urinary excretion of TA-5707F and its metabolite was 70% of the dose. This value was almost equal to that of the rapid release tablet. In dogs the in vivo absorption rate was similar to the in vitro dissolution rate, but in humans it was only about half.
Immunomodulating activities of three carboxymethylated derivatives (AG-AL-CMS, AG-AL-CMI, and AM-APP-CM) of linear (1→3)-α-D-glucans from Agrocybe cylindracea and Amanita muscaria were evaluated with murine peritoneal macrophages playing an important role in tumor immunity. The ratio of macrophages in peritoneal exudate cells increased more than 50% after the administration of three carboxymethylated (1→3)-α-D-glucans. These carboxymethylated (1→3)-α-D-glucans exhibited higher potentiating activities for macrophages than carboxymethylated linear (1→3)-β-D-glucan (CMPS) in the potency of reduction of nitro blue tetrazolium, products of nitric oxide and the soluble cytotoxic factor, the amount of glucose consumption, and the activation of acid phosphatase. AG-AL-CMS, AG-AL-CMI, and AM-APP-CM were found to induce the tumor regressing factor in mouse serum, although the ability of the induction of this factor was weaker than that of CMPS. The reticuloendothelial system-potentiating activation of three carboxymethylated α-D-glucans was similar to that of the carboxymethylated β-D-glucan. AG-AL-CMS and AG-AL-CMI, but not AM-APP-CM, were suggested to possess a higher-order structure, resulting from the formation of a fluorescent complex with aniline blue.
A cDNA encoding human TFIID subunit p30β (hp30β) was isolated and sequenced. Comparison of the deduced 211 amino acid (aa) sequence with that of Drosophila melanogaster p30β (dp30β) showed 45% identity in overall length, and subunits with highly conserved C-terminal (81% identity) and less conserved N-terminal (21% identity) regions. Three acidic and basic regions were alternately placed. Potent target sites for protein kinases and a putative nuclear localization signal (NLS) were located in the N-terminal region. Interestingly, analysis of the aa sequence of p30β led us to find several interesting structural motifs/regions including 1) direct repeats, 2) a region similar to histone H4 between the two direct repeats and 3) acidic and hydrophobic aa surfaces in the potent helical region. Northern blot analysis showed ubiquitous expression of mRNA corresponding to hp30β.
We examined the antinociceptive effects of the crude alkaloid fractions (CAF) of nux vomica (the dried seeds of Strychnos nux-vomica L.) and the influences of various processing methods upon their antinociception in three analgesic tests in mice. In the tail-pressure test, the CAF (0.01-1 μg/kg, i.p.) of nux vomica that was unprocessed or treated with sand-, licorice-, oil- or vinegar and sand-processing showed clear antinociception. The CAF (1 μg/kg, i.p.) of vinegar-processed nux vomica showed antinociception, without effects at lower doses of 0.01 and 0.1 μg/kg and those treated with urine- or urine and sand-processing were without effects at doses of 0.01-1 μg/kg. Morphine (2 mg/kg, s.c.) showed short-lasting antinociception, without effects at a dose of 1 μg/kg. In the hot-plate test, the CAF (100 μg/kg, i.p.) of nux vomica having undergone sand-processing produced a significant antinociception, without effects at lower doses of 0.01 and 1 μg/kg. The CAF (0.01-100 μg/kg, i.p.) of nux vomica that was unprocessed or treated with oil- or vinegar and sand-processing and morphine (1 and 100 μg/kg, s.c.) were without effects. In the acetic acid-induced writhing test, the CAF (1 μg/kg, i.p.) of nux vomica that was treated with sand-processing significantly inhibited the writhing behavior, while those of nux vomica that was unprocessed or treated with oil- or vinegar and sand-processing and morphine were without effects at a dose of 1 μg/kg. The present results demonstrate the antinociceptive effects of the CAF of nux vomica and suggest that sand-processing is good for the analgesic potency of nux vomica. It is also suggested that the CAF of nux vomica has distinct antinociceptive potency, even after treatment with licorice-, oil-, vinegar and sand-processing.
Heating normal human serum produced aggregates that can be detected with a F(ab')2 anti-C3 enzyme-linked immunosorbent assay (FaC3 ELISA) for circulating immune complexes. Using this we studied the production mechanism of these aggregates. Heating serum with the inactivated complement system produced aggregates which included IgG and C3/C3 fragments. The aggregates also incorporated IgA, IgM, and albumin as well as IgG and C3/C3 fragments. These results indicate that the production of the aggregates was not due to activation of the complement system by aggregated IgG generated by heating, but mainly resulted from aggregation of serum proteins induced by heating. This study suggests that there are heating conditions under which the hemolytic activity of complement system is abolished, but aggregates are not detected with FaC3 ELISA.
Orally administered barbaloin (100 mg/kg) did not induce any diarrhea in male Wistar rats, in spite of severe diarrhea with sennoside B (40 mg/kg). Also, in gnotobiote rats mono-associated with Peptostreptococcus intermedius, a human intestinal anaerobe capable of reducing sennidins to rhein anthrone, barbaloin did not induce diarrhea ; the faecal water content (71.9%) 8 h after the administration of barbaloin was not increased, compared with that (73.9%) just before the treatment. However, severe diarrhea was induced with barbaloin in gnotobiote rats mono-associated with Eubacterium sp. strain BAR, another human intestinal anaerobe capable of transforming barbaloin to aloe-emodin anthrone ; the faecal water content was significantly increased to 85.5% 8 h after the administration, from 73.2% before the treatment. At this time, barbaloin was transformed to aloe-emodin anthrone in the feces from the gnotobiote rats mono-associated with the strain BAR, but not in feces from the conventional rats or the gnotobiote rats mono-associated with P. intermedius. These facts indicate that barbaloin is inactive as a laxative itself but is activated to aloe-emodin anthrone, a genuine purgative component, by Eubacterium sp. strain BAR.
Tuna muscle-derived angiotensin-converting enzyme inhibitory peptide, PTHIKWGD, inhibited the injury of bovine aorta endothelial cells (BAECs) induced by N-formyl-methionyl-leucyl-phenylalanine-activated polymorphonuclear leukocytes and the adhesion of a leukemia cell line, THP-1 cells, to BAECs stimulated by lipopolysaccharides. It is possible that this peptide directly affects BAECs, protecting them from activated leukocytes.
N3-Substituted derivatives of deoxyuridine (1) were synthesized and their pharmacological effects were evaluated by intracerebroventricular (i.c.v.) injection in mice. Eleven derivatives, including the methyl (2), ethyl (3), propyl (4), allyl (5), butyl (6), benzyl (7), o, m and p-xylyls (8, 9, 10), α-phenylethyl (11) and phenacyl (12) derivatives, of 1 were prepared and their pharmacological effects were evaluated by using hypnotic activity, pentobarbital-induced sleep prolongation, spontaneous activity and motor incoordination as indices of central nervous system (CNS) depressant effects. At a dose of 2.0 μmol/mouse, the values of mean sleeping time induced by 7, 8, 9 and 10 were 23, 35, 29 and 30 min, respectively. Although the alkyl (2-6) derivatives did not cause any hypnotic activity, some derivatives tested (3, 5, 6, 8-12) significantly prolonged the pentobarbital-induced sleeping time. When the CNS depressant effects of phenacyl substituted 1 were compared to that of other oxopyrimidine nucleosides, N3-phenacyluridine (13), N3-phenacylthymidine (14), N3-phenacyl-6-azauridine (15), compounds 12, 13 and 14 (1.0 μmol/mouse, i.c.v.) significantly decreased mouse spontaneous activity. Furthermore, 12-15 (1.0 μmol/mouse, i.c.v.) caused mouse motor incoordination. These results indicate that deoxyuridine derivatives have generally central depressant activity, and the benzyl and xylyl derivatives, but not alkyl derivatives, possess hypnotic activity.
Procyanidins extracted from the rhizomes of Potentilla tormentilla were fractionated according to their degree of polymerization by chromatography on Sephadex LH20[○!R]. Dimers and trimers displayed the highest anti-radical activity towards lipoperoxidation. Pentamers and hexamers possessed the most marked anti-elastase properties.
Monocytes/macrophages are known to be involved in atherogenesis, and the adherence of monocytes to the endothelium is considered an earliest characteristic of atherogenesis. Therefore, we studied the mechanism by which Shosaikoto, a Kampo medicine, shows anti-atherosclerotic action, which has been already shown in hypercholesterolemic rabbits. Hypercholesterolemia in rabbits gradually reduced the monocyte number in peripheral blood, whereas Shosaikoto treatment suppressed this decrease in circulating monocytes. Furthermore, although monocytes from hypercholesterolemic rabbits increased in adherence to endothelial cells even without lipopolysaccharide (LPS) activation, Shosaikoto treatment reduced the enhanced adherence observed in monocytes from hypercholesterolemic rabbits. These data suggested that the anti-atherosclerotic action shown by Shosaikoto resulted partly from the suppression of the enhanced adherence characteristic of hypercholesterolemia.
The inhibitory effect of 50% ethanol extracts obtained from the leaves of six Arctostaphylos plants (A. patula, A. viscida, A. canescens, A. columbiana, A. nevadensis and A. uva-ursi) (Ericaceae) was tested on melanin biosynthesis. Among them, the extracts of A. patula and A. viscida showed potent inhibition against tyrosinase. The two extracts not only inhibited the production of melanin from dopachrome by autoxidation, but exhibited SOD (superoxide dismutase)-like activity and had a moderate absorbance in the UV-B area. These results suggest that the leaves of Arctostaphylos plants, especially A. patula and A. viscida can be applied to a whitening agent for the skin.
Blood concentrations of branched chain amino acids (BCAA ; leucine, isoleucine, and valine) and glutamine (Gln) decrease markedly in sepsis. We investigated the effect of carnitine on serum concentrations of BCAA and Gln in fasted septic rats. Rats were made septic by cecal ligation and puncture. They developed extremely high blood concentrations of endotoxin, and serum concentrations of BCAA and Gln were markedly decreased 2 d after the operation. When L-carnitine was administered subcutaneously to the rats at 500 mg/kg body weight every 12 h for 2 d starting at the operation, no decrease in the serum concentrations of BCAA and Gln was observed. This indicates that the administration of carnitine can prevent the decrease of serum concentrations of BCAA and Gln in septic animals.
Four iridoidal glycosides, deacetylasperulosidic acid methyl ester (DE), scandoside methyl ester (SC), geniposide (GE) and gardenoside (GA) isolated from Gardenia jasminoides Ellis grandiflora Makino leaves, were tested hypoglycemic activity in mice. DE lowered the blood glucose level in normal mice. However, SC, GE and GA did not affect the blood glucose level in normal mice, indicating that the absolute configuration of 6 position hydroxy based (OH) should be essential for the biological activity.