We report here the transient relaxation of plasmid DNA by inhibitors of DNA gyrase in Escherichia coli. Relaxation of plasmid DNA in the presence of nalidixic acid, an inhibitor of the A subunit of DNA gyrase, lasted less than 60 min. The effect of nalidixic acid was not observed in a nalA26 mutant, a strain resistant to nalidixic acid due to a mutation in the gyrA gene. Novobiocin, whose target is the B subunit of DNA gyrase, also caused transient DNA relaxation. Resupercoiling of relaxed DNA in the presence of nalidixic acid was inhibited by pretreatment of the cells with chloramphenicol. As mutations of both the rpoH and recA genes did not affect the resupercoiling reaction, this step seems to require protein synthesis that is independent of both heat shock and SOS respones.
A base non-specific acid ribonuclease (RNase RCL2) was purified from bullfrog liver [H. Yagu et al. Biol. Pharm. Bull., 18, 219-222 (1992)]. The sequence study and comparison of the amino acid sequence of the enzyme with homologous RNases from oyster, drosophila and chicken liver suggested that the RNase RCL2 consisted of two components, large protein fraction (182 amino acid residues) and peptide 2 (20 amino acid residues) or peptide 1 (18 amino acid residues), and that both components bind with disulfide bridge. The RNase preparation was probably formed from single polypeptide protein by processing with some proteses. The amino acid sequence of RNase RCL2 showed that the RNase belongs to the RNase of RNase T2 family and its sequence most resembles chicken liver acid RNase. In RNase RCL2, the amino acid residues which sonsist of the active site and major base recognition site of RNase Rh, a typical RNase of RNase T2 family, are very well conserved except for Tyr57 (RNase Rh numbering), and part of the amino acid residues of the minor base recognition site (Phe101 and Pro92) are also conserved.
We examined the fatty acid composition in an Escherichia coli pgsA3 mutant lacking the potential to synthesize phosphatidylglycerolphosphate, a precursor of phosphatidylglycerol. The contents of C18 : 1cis-9 (oleic acid) and C18 : 1cis-11 (cis-vaccenic acid) in the total phospholipids extracted from the pgsA3 mutant growing at 37°C were higher and that of C14 : 0 was lower than the wild type cells, resulting in a higher level of unsaturation of fatty acids (ratio of unsaturated fatty acids to saturated ones) in the mutant. The higher level of the unsaturated fatty acids in the pgsA3 mutant was more obvious in cardiolipin than in phosphatidylethanolamine. On the other hand, at 28°C, at which the pgsA3 mutant shows limited cell growth, the content of unsaturated fatty acids in cardiolipin decreased in the pgsA3 mutant compared with the wild type. We consider that the pgsA3 mutant maintains cellular homeostasis by altering the level of unsaturated fatty acids in cardiolipin, and the mechanism is influenced by temperature.
In order to establish the measurement of gastric mucin secreted from cultured mucous cells, rat gastric mucin was purified from secreted mucus with Sepharose CL-4B column chromatography. Gastric mucin was measured by dot blot analysis using an enzyme-linked lectin (soybean agglutiin) assay in a good concentration-dependent manner. Surface epithelical cells were dispersed by limited digestion of a rat everted stomach and collected by density gradient centrifugation with Percoll. These cells were inoculated onto gelled collagen dishes, then cultured in a medium supplemented with 10% fetal calf serum under a 5% CO2 atomosphere in air. Changing the medium after a 2-d culture, the cells were cultured for another 3 d. During the culture, the numbers of cells each day were almost, equal, but mucin contents in the cells increased, and then dropped at day 5 after inoculation. At that time, the edge of the cell layer peelede off and the cells adhered to each other. Using 2-d cultured cells, the effects of some secretagogues on mucin secretion were investigated. Carbachol, secretin, CCK-8 and prostaglandin E2 (PGE2) strongly stimulated mucin secretion, and gastrin I weakly did. However, histamine offered no stimulation.
The ability of three cardiac glycosides, ouabain, digitonin and digitoxin, to induce emesis and their mechanism(s) of action were investigated n Suncus murinus. The intraperitoneal injection of ouabain but not digitonin nor digitoxin caused vomiting in a dose-dependent manner. However, the administration of ouabain into the cerebroventricle did not cause emesis. Ouabain-induced emesis was partly prevented by surgical abdominal vagotomy. Pretreatment with tropisetron, a selective 5-HT3 (5-hydroxytriptamine) receptor antagonist, did not affect the emetic response evoked by ouabain. These results suggest that ouabain exerts emetic effects via peripheral mechanism(s), but 5-HT3 receptors are not involved in the pathway.
In order to understand the mechanism involved in the aromatase inactivation by androst-5-ene, 4, 7, 17-trione (4), a suicide substrate of aromatase, 5β, 6β-epoxyandrosta-4, 7, 17, 19-tetraone (6) was synthesized as a candidate for a reactive electrophile involved in irreversible binding to the active site of aromatase upon treatment of 19-oxo-5-ene steroid 5 with hydrogen peroxide in the presence of NaHCO3. The epoxide 6 was a competitive inhibitor of human placental aromatase (K1=34 μM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (K1=36 μM, a rate constant for inactivation (kinact)=0.027 min-1). NADPH stimulated the inactivation rate, but the substrate androst-4-ene-3, 17-dione blocked the inactivation. A nucleophile, L-cysteine, did not cause a significant change in the inactivation. When both the epoxide 6 and its 19-methyl analog 7 were subjected separately to a reaction with N-acetyl-L-cycteine in the presence of NaHCO3, the 19-oxo compound 6 disappeared from the reaction mixture more rapidly (t/1/2=6.0 min) than the 19-methyl analog 7 (t1/2=16 min). On the basis of these results, it is suggested that the 5β, 6β-epoxy-19-oxo steroid 6 may be the reactive electrophile that alkylates a nucleophilic residue of the amino acid of the active site.
To establish a method of evaluating propolis, the effects of the ethanol and water extracts from various collecting of propolis from different countries and plant sources on hyaluronidase activity were investigated along with their absorption spectra and specific absorbance (E1%1 cm value). The relations between the hyaluronidase inhibitory activities of these extracts and their E1%1 cm values were also examined, and the following was found : 1) the enzyme inhibitory activities of the ethanol extracts were more potent than those of the water extracts; 2) the enzyme inhibitory activities of the ethanol extracts from Araucaria angusrtifolia (BERT). O. KTZE were low compared with those of other ethanol extracts; 3) the enzyme inhibitory activities of all the ethanol extracts correlated excellently with their E1%1 cm values, but in the water extracts, they decreased with increase in E1%1 cm values; 4) the water extracts of Chinese propolis from Hebei, Jiangsu, Sichuan and Zhejiang Provinces inhibited weakly compared with that from Brazillian and other Chinese propolis; 5) the shapes of absorption bands of the propolis extracts and the E1%1 cm values were approximately dependent on the place or the plant source from which propolis was collected.These experimental results indicatea that, for the exact evaluation of propolis, the enzymatic method, measuring the hyaluronidase inhibitory activity, was superior to the physicochemical method.
Total DNAs were prepared from the leaves of Atractylodes lancea DE CANADOLLE, A. chinensis KOIDZUMI, A. lancea var. simplicifolia KITAMURA, A. japonica KOIDZUMI ex KITAMURA and A. ovata DE CANDOLLE. The DNAs were subjected to random amplified polymorphic DNA (RAPD) analysis. Some primers showed the definitive polymorphic DNA patterns in A. lancea, A. japonica and A. ovata. The RAPD of A. lancea var. simplicifolia and one of A. chinensis gave similar patterns to those of A. lancea, but one of the other A. chinensis gave a similar pattern to A. japonica. Futhermore, quantitative analysis of atractylon, hinesol, β-eudesmol and atractylodin in the rhizomes was done using gas chromatography. Though atractylon was detected not only in A. japonica and A. ovata but also in some of A. lancea, their RAPD profiles revealed the presence of intraspecific variation with A. lancea.
The hypoglycemic and hypolipidemic effect of docosahexaenoic acid (DHA; C22 : 6ω-3) ethyl ester was examined in KK-Ay mice and neonatal streptozotocin-induced diabetic (NSZ) which are respectively obese and lean animal models of non-insulin-dependent diabetes mellitus (NIDDM), and in ddY normal mice. Single administration of DNA (500 mg/kg body weight) to KK-Ay mice significantly reduced (p<0.05) the blood glucose levels (BG) (p<0.05) and plasma free fatty acid levels (FFA) (p<0.05) at 10 h after oral administration when compared with control group. DHA (500 mg/kg body weight)-treated NSZ and normal mice, however, showed no change in these parameters. In addition, repeated administration of DHA (100 mg/kg) to KK-Ay mice significantly suppressed the increment of BG (p<0.05) and plasma triglyceride levels (TG) (p<0.01), and significantly decreased FFA (p<0.05) at 30 d compared with control group. DHA also significantly decreased the blood glucose at 60 and these 120 min on insulin tolerance test (ITT). From findings, it seems likely that DHA exhibits its hypoglycemic effects by increasing insulin sensitivity. It is concluded that DHA would be useful for treatment of obese type NIDDM with insulin resistance.
Methanol and aqueous extracts (TMe-ext and TAq-ext) from dried rhizomes of Alisma orientale have been screened for activity in experimental models of type I-IV allergies. In the type III allergic model, TMe-ext at oral doses of 50, 200 mg/kg showed an inhibitory effect on the direct passive Arthus reaction (DPAR) in rats, while TAq-ext did not. Four triterpenes (alisol A, alisol B, alisol A monoacetate and alisol B monoacetate) and two sesquiterpenes (alismol and alismoxide) isolated from TMe-ext also exhibited this inhibitory effect. In a type I allergic model, TMe-ext inhibited 48-h homologoes passive cutaneous anaphylaxis (PCS) in rats. In a typ II allergic model, it was found that TMe-ext inhibits reversed cutaneous anaphylaxis (RCA) in rats. Furthermore, in a type IV allergic model, TMe-ext has an inhibitory effect on the induction phase in picryl chloride-induced contact dermatitis (PC-CD) in mice. These results indicate that Alismatis Rhizoma not only inhibits antibody-mediated allergic reactions but also influences cell reactions and should be recognized as a material for the treatment of allergic reactions, and the anti-type III allergic components are partially attributable to the terpenes mentioned above.
Plasma concentrations of paeoniflorin (PF) and its major metabolite, paeonimetabolin I (PM-I), were estimated after oral administration of PF to rats at doses of 0.5 and 5 mg/kg. The maximal plasm concentrations (Cmax) of PF were 9.9 amd 20.3, and those of PM-I were 16.5 and 101.7 ng/ml at each dose, respectively. The times to Cmax (tmax) of PF were 11.6 and 13.3, and those of PM-I were 60 and 80 min, respectively. The AUC0-180 of PM-I were 1873 and 12358, and those of PF were 300 and 1174 ng min/ml, respectively.On the other hand, after intravenous administration of PM-I to rats at doses of 0.2 and 2 mg/kg (equal in molar ratio to 0.5 and 5 mg/kg PF), the plasma concentration of PM-I decreased rapidly and the plasma concentration-time curve profile of it fitted well with the two-compartment model at each dose, with terminal half lives (t1/2) of 90.9 and 90.6 min. The Vdss values were 0.91 and 3.79 l/kg, the Cltot values were 8.7 and 39.9 ml/min kg, and the AUC0-180 values were 5614.1 and 13176.0 ng min/ml, at each dose, respectively. The significant increase in Vdss and CLtot with increasing doses suggested dose-dependent pharmacokinetics.When PM-I was given orally at the same doses, the following parameters were shown : Cmax of 102.2 and 285 ng/ml at tmax 6.2 and 7.5 min and AUCs of 4145.6 and 14182.1 ng min/ml, at each dose. The bioavilability (F) values were 0.8 and 1.07, respectively.These findings indicated that the high percentage of PM-I transformed by intestinal bacteria was rapidly absorbed from the gastrointestinal tract, and a significantly high concentration of PM-I, rather than PF, was present in the plasma after oral administration of PF.
The effect of three positional isomers, o-, m- and p-acetylaminophenyl sulfate (AOAPS, AMAPS and APAPS (acetaminophen sulfate), respectively), on the pharmacokinetics of acetaminophen (APAP) was investigated in rats. All of the intravenously administered positional isomers were rapidly eliminated from plasma, and approximately 80% of the dose was excreted in an unchanged form in the urine within 4 h, while biliary excretions represented a small percent of the doses. Following the intravenous bolus injection of APAP, plasma elimination of APAP was accelerated and the distribution volume of APAP was increased under a steady state concentration (about 10 μg APAP eq/ml) of AOAPS or APAPS, but not AMAPS, as compared with saline infusion. Total body clearances of APAP were increased from 18.3 ml/min/kg for the control to 23.9 and 26.9 ml/min/kg for AOAPS and APAPS coadministration, respectively. AOAPS and APAPS competitively displaced the serum protein binding of APAP, while AMAPS had little effect. The distribution volume of unbound APAP was anomalously increased by APAPS, while it was not affected by AOAPS or AMAPS. Tissue-to-plasma concentration ratios of APAP were significantly increased by APAPS in the liver, kidney and brain, while they were only slightly increased by AOAPS. It was suggested that APAPS has nt only the displacing activity of serum protein binding but also other specific effectiveness on the distribution of APAP.
For ex vivvo gene therapy, superoxide dismutase (SOD) must be secreted into the extracellular space and delivered to damaged cells. Recombinant DNA technique can be used to produce a secretory protein that is fused to a non-secretory protein and a signal peptide of another secretory protein gene. We constructed a secretable SOD eukaryotic expression vector which expressed human SOD cDNA by fusing it to the signal peptide DNA sequence of the human interleukin-2 (IL-2) gene. The ILSOD cDNA constructed by PCR-based gene expression was ligated into the multicloning site of the pRc/CMV plasmic (pRc/CMV-ILSOD). Rat lung epithelial like cells (L2 cells) were transfected with pRc/CMV-ILSOD by lipofection. The extracellular SOD activity of ILSOD-L2 cells (transfected cells with pRc/CMV-ILSOD) was 3 times as high as that of host cells.We used the xanthin (X)/ xanthin oxidase (XO) system to produce superoxide anions at the extracellular space. We initially investigated the direct cytotoxicity of superoxide anions upon cells. Host and ILSOD-L2 cells were killed by using X/XO, although the sensitivity of the ILSOD-L2 cells to X/XO induced cytotoxicity was significantly decreased compared with that of host cells. The production of lipid peroxidated substances in the host in the presence of X/XO increased to about twice the control (absence of X/XO) level. However, that of ILSOD-L2 cells did not change in the presence of X/XO. Therefore, ILSOD-L2 cells were resistant to X/XO induced lipid peroxidation. These findings indicated that ILSOD gene transfection protected against direct oxidant stress by X/XO.We then investigated the effect of extracellular SOD secreted from ILSOD-L2 cells on extracellular superoxide anion induced cytotoxicity in normal cells. The conditioned media of host cells had no significant effect upon X/XO induced cytotoxicity. However, the conditioned media of ILSOD-L2 cells protected against X/XO induced cytotoxicity. Furthermore, the conditioned medium of ILSOD-L2 cells was more effective than that of host cells against the production of lipid peroxidated substances by normal cells under conditions of oxidative stress. These results indicated that non-secretable protein could be delivered to target cells by means of DNA engineering. This strategy could thus provide an ex vivo means of applying gene therapy using non-secretable proteins.
An iontophoretic drug delivery system was compared with intranasal, oral and subcutaneous delivery from a standpoint of the prolongation of the antidiuretic response to desmopressin acetate (DDAVP) in diabetes insipidus rats. Iontophoretic delivery was comparable to the nasal route at a dose about five higher than the nasal route dose, and was 2 to 3 times as effective as the oral route. Effect of dose and duration of current application on the prolongation of the response to DDAVP was also investigated in order to find the effectiveness of the iontophoresis. The antidiuretic response to DDAVP delivered by iontophoresis indicated a dose-dependent prolongation and was prolonged up to about 14 h with the increase of the duration of current application; when a pulsed direct current at 0.1 mA was passed for about 1 h, the response to DDAVP was prolonged for about 9 h. DDAVP in the anodic donor steeply decreased with the application for 1 h, and the gradually decreased. We suggest that the antidiuretic response to DDAVP can be effectively controlled by regulating the absorption of DDAVP at the short-term iontophoresis eather than prolonged treatment.
In vitro selective antitumor activity was tested as a general screening parameter for biologically active substances from a wide range of species of seaweed, 1446 samples of 306 species of marine algae from Japan's coasts. The algae extracts were prepared successively first by phosphate buffered saline (PBS) and then by methanol, and then tested for in vitro selective antitumor activity against murine lymphoid leukemia L1210 cells and for low cytotoxic activity against NIH-3T3 normal cells.Strong cytotoxic activity against L1210 cells was found in 47 species of algae, also showing similar cytotoxicity to mouse NIH-3T3 normal cells. However, four species of green algae showed strong activity specifically against L1210 cells, with low cytotoxicity to normal cells. Such selective activity was conspicuous in two brown and two green algae extracts. In particular, methanol extracts from the green alga, Cladophoropsis vaucheriaeformis, exhibited high viability (86%) to normal cells, showing selective cytotoxicity to tumor cells. This alga extract was not cytocidalic, but cytostatic against L1210 cells. Furthermore, the results of a cytotoxic spectrum test with 9 cell lines including those of L1210 and NIH-3T3 demonstrated that this extract acted strongly only against leukemic cell lines L1210 and P388.
In a previous paper, we reported that an extract of silkworm faeces has a marked antiviral activity on enveloped viruses, but not on a non-enveloped virus, and we showed that it inhibits the synthesis of a viral specific gene of HVJ (Sendai virus) without affecting the viral adsorption and entry into the host cell. In this paper, we accomplished the purification of an antiviral active substance by extraction of a hydrophobic substance and thin layer chromatography. The active substance was found to be a chlorophyll-like substance with a molecular mass of about 530. This substance shows clear antiviral activity against HVJ, HSV (herpes simplex virus type-1), and HIV (humanimmunodeficiency virus type-1). Its antiviral activity was dependent on light irradiation and temperature. Furthermore, it also possesses a strong hemolytic activity under light.
^<125>I-Labeled recombinant human neuron-specific enolase (R-NSE) was inadequate for RIA as a labeled antigen.The binding activity of laveled R-NES to the antibody was markedly decreased. To supplement this defect and facilitate purification, we constructed two R-NSE derivatives, Y-NSE (one tyrosine residue was added at the N-terminal of R-NSE) and Y-NSE.H6 (six histidine residues were further added at the C-terminal of Y-NSE). The biochemical and immunochemical characteristics of these R-NSE derivatives were essentially the same to those of R-NSE. These derivatives were useful not only as standards for enzyme immunoassay (EIA), but also as labeled antigens for RIA. These results clearly indicate that the reactivity of these modified NSEs to anti-NSE antibody is almost equivalent to that of human brain γγ-enolase (B-NSE), and that even if the modified NSEs are labeled, they retain their binding affinities to antibodies in contrast to R-NSE.
Active components in grapefruit juice, which modulate a cytochrome P450 (CYP3A) activity, were investigated. CYP3A-catalyzed 6β-hydroxylation of testosterone in livers of rat and human was inhibited by the addition of an ethyl acetate-extract of grapefruit juice. Several components of grapefruit juice, including naringin, naringenin, limonin and obacunone, also showed inhibitory effects in human liver microsomes. However, the amounts of these components in grapefruit juice are too low to account for the inhibition by the ethyl acetate-extracts. Analyses with HPLC indicate the existence of inhibitory components in the extract, which are distinct from these known compounds and are specific to grapefruit juice. These results suggest that hydrophobic components other than flavonoids, probably coumarin derivatives, are responsible for the inhibitory effect of grapefruit juice.
We investigated the anti-allergic effect of tea-leaf saponin (TLS), which was a mixture of saponins separated from the leaves of Camellia sinensis var. sinensis, in guinea pigs and rats. TLS (20-100 mg/kg) dose-dependently inhibited experimentally-induced asthma, and ID50 was 61.7 mg/kg. TLS (20-100 mg/kg) dose-dependently inhibited a 48 h homologous PCA (passive cutaneous anaphylaxis) reaction, and the inhibitory effect was similar to that of tranilast. TLS (1-100 μg/ml) also inhibited the release of antigen-induced leukotriene (LT) C4 from sensitized guinea pig lung samples in a dose-dependent fashion, but did not prevent histamine release. TLS (0.01-0.5 μg/ml) inhibited histamine release from rat peritoneal mast cells induced by compound 48/80. At higher concentrations, TLS elicited histamine release. These findings suggest that TLS may be a useful protective agent against clinical allergy, and that the inhibitory effects of TLS on mediator release are in some way related to its inhibitory effect on experimental-induced asthma and PCA reaction.
Raising the flow rate of the perfusate from 10 to 20 ml/min significantly suppressed vasodilator responses to acetylcholine, but not to sodium nitroprusside, in the isolated canine mesenteric arterial bed. Both acetylcholine and sodium nitroprusside augmented guanosine 3', 5'-cyclic monophosphate (cyclic GMP) levels in the effluent from the vascular bed preparation, though cyclic GMP responses to these agents were not affected by raising the flow rate. These data suggest that the suppression, via raising the flow rate, of vasodilator responses to acetylcholine is not due to impaired function of the nitric oxide (NO)-cyclic GMP pathway in the mesenteric arterial bed.
Previous studies have shown that butylated hydroxytoluene (BHT) undergoes oxidation by cytochrome P450 to form BHT-quinone methide. BHT-quinone methide is probably responsible for BHT-induced lung damage in mice. In this study, we calculated the MO parameters for BHT analogs and the corresponding quinone methide intermediates. Except for the analogs with structures that form a highly sterically hindered quinone methide, correlations could be established between the lung toxicity in mice and electronic charges on the hydroxyl oxygen and 4-carbon atoms of BHT analogs. The same toxicity could also be correlated to the difference between the heat of formation of the quinone methide intermediates and the parent BHT analogs, and to the electronic charge on the carbonyl oxygen atom of the quinone methides. These results suggest that the metabolic activation of BHT analogs to their quinone methide intermediates is energetically dependent on the oxidation of the aromatic π-electron system, and that the toxic potency of BHT analogs is controlled by protonation of the oxygen atom of the quinone methides. These electronic features provide further evidence of the importance of the quinone methide intermediates in the mechanism of lung toxicity induced by BHT analogs.
The effect of PEG4000 solution on the in vitro release of nifedipine, a calcium entry blocker and a poorly water soluble drug was evaluated. Nifedipine tablets containing 10 mg of nifedipine, 20% of one four polymers-Eudragit L-100 and Rs, ethylcellulose and carbopol 941, 2% lubritab and an adequate quantity of Encompress to yield 300 mg weight tablets-were formulated using the direct compression method. Tablets were compressed to a hardness of 5N, and an in vitro dissolution profile was performed on the tablets in 70% ethanol and in ethanolic PEG4000 solutions.The results obtained indicated enhancement of nifedipine release from two of the polymer matrices (Eudragit L-100 and ethylcellulose) in the presence of PEG4000 and a retardant effect from carbopol 941 matrix. It is suggested that carbopols may not be suitable for use in the formation of nifedipine tablets since there are some physiological surfactants present in the gastrointestinal tract (GIT).
The stability of nikkomycin Z in aqueous solution at various pH values and in the plasma of several kinds of experimental animals was studied. The degradation of nikkomycin Z in aqueous solution at pH 4 to 11.5 and in plasma was an apparent first-order reaction. The degradation rate at 37°C increased with increasing pH, from 4.0 to 7.5, and decreased with increasing pH from 7.5 to 10.2. Above pH 10.2, the degradation rate was constant. The maximal rate of nikkomycin Z degradation was observed in pH 7.5 buffer solution, in which the apparent first-order rate constant (kobs) was 8.08×10-2h-1 (t1/2=8.6 h). The degradation rate of nikkomycin Z in dog plasma at 37°C was the almost same as that in pH 7.5 buffer. The rates in rat, mouse, and rabbit plasma were much greater than that in pH 7.5 buffer; the kobs values for rat, mouse, rabbit, and dog plasma at 37°C were 1.74×10-1min-1, 3.64×10-2 min-1, 5.10×10-1 h-1, and 6.14×10-2 h-1, respectively. The degradation rate of nikkomycin Z in rat plasma was remarkedly decreased when NaF, an esterase inhibitor, was added to the plasma. The findings of faster degradation rates in rat, mouse, and rabbit plasma compared with that in pH 7.5 buffer were considered to be due to an esterase in the plasma. This notion was supported by results showing the degradation rate of nikkomycin Z in porcine liver esterase solution.
In order to develop a membrane fusible drug carrier from human erythrocytes, we attempted the reconstitution of influenza virus fusion protein hemagglutinin (HA) to an erythrocyte membrane. In this study, we succeeded in the preparation of HA-reconstituted erythrocytes (HA-erythrocytes) by the incubation of erythrocytes with influenza virus-infected CV-1 cells, and confirmed the ability of HA-erythrocytes to fuse with the cell membrane. Furthermore, by using an HA-reconstituted ghost (HA-ghost), which entrapped fluorescent-labeled ovalbumin, 25% of the protein was incorporated into cells through the fusion of the HA-ghost with the cell membrane.
We evaluated the antimicrobial sesceptibility to six strains of Escherichia coli O157 (E. coli O157) isolated from patients in Yamaguchi Prefecture between June and July, 1996. Seven antimicrobial agents that were expected to retain a high concentration in the intestine were selected. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of ciprofloxacin, polymyxin B, cefoperazone, and kanamycin for each strain were ≤6.25 μg/ml. However, the MIC of fosfomycin was 3.13-100 μg/ml, and its MBC was ≥100 μg/ml. The MIC of ampicillin and tetracycline was >100 μg/ml in some strains. In a time-kill study of E. coli O157 at a drug concentration of 12.5 μg/ml, about 104 colony forming units/ml of E. coli O157 were eradicated within 10 min by ciprofloxacin, within 30 min by polymyxin B, within 4 h by cefoperazone, and within 16 h by kanamycin. These results suggest that the new quinolones with a poor absorption rate in the intestine (such as ciprofloxacin and norfloxacin) are effective against E. coli O157. When oral administration is impossible, bile excreting cephem antibiotics (such as cefoperazone, cefriaxone, and cefotetan) may be useful.
(S)-Norreticuline and (S)-reticuline have been shown to stimulate the proliferation of cultured cells from the murine hair apparatus significantly. Furthermore, these activities were found on cultured hair cells, but not on cultured keratinocytes or fibroblasts from murine skin. In addition, (S)-norreticuline significantly stimulated mouse hair regrowth. These results suggest that (S)-norreticuline and (S)-reticuline could have specific activities on hair apparatus cells and might be useful as active compounds for accelerating hair growth.