The effect of extracellular calcium on the intracellular calcium level of newborn rat skin basal cells was examined. The intracellular calcium level of basal cells was affected by the extracellular calcium concentration on culture of the cells on a collagen-coated filter, but not on a plate. The intracellular calcium ion concentration ([Ca2+]i) of cells on a collagen-coated filter with 0.05mM external CaCl2 was 15nM and increased to 20-22nM on culture with 2.0mM extracellular CaCl2. From these results we concluded that the culture matrix (e.g. culture plates, or collagen-coated filters) affects the response of the basal cells to changes in the extracellular calcium content.The intracellular calcium level of Swiss 3T3 cells cultured both on plates and collagen-coated filters were affected by the extracellular calcium concentration. Their [Ca2+]i was determined as 49-50nM in the presence of 0.05mM external CaCl2, and 54-56nM in the presence of 2.0mM CaCl2. These different responses to extracellular Ca2+ may be due to differences in the proliferative profiles of basal cells and fibroblasts.
We developed a method for the preparation of everted membrane vesicles from cells of Staphylococcus aureus. The cells were first treated with ampicillin to weaken the peptidoglycan layer, then the cells were passed through a French press cell. The resulting vesicles were roughly 0.1μm in diameter, judging from electron microscopic observations. We detected fairly high membrane-bound ATPase activity in the membrane vesicles. We observed respiratory-driven quenching of quinacrine fluorescence, which indicates that inward H+ transport took place. These results indicate that the vesicles are everted. We characterized the membrane-bound ATPase. We also detected Na+/H+ antiport, erythromycin/H+ antiport and chloramphenicol/H+ antiport activities in the membranes of S. aureus.
Isolation of several hydroxysteroid sulfotransferase (HS-ST) cDNAs from the rat liver cDNA library has demonstrated the possible expression of these HS-ST isoforms in rat liver. We devised a method to detect unequivocally ST-20 and ST-40 mRNAs by a reverse transcription-polymerase chain reaction, using specific primers. ST-40 mRNA was expressed only in liver, but ST-20 mRNA was present predominantly in the liver and slightly in extrahepatic tissues. On chromatofocusing, expressed ST-20 or ST-40 enzymes were eluted at approx.pH8.2 and 5.7-4.7 or at pH6.4-5.4, respectively. Chromatofocusing of adult female rat liver cytosols resolved HS-ST isoenzymes in a broad range of pH, and ST fractions A, B, C, D and E were eluted at approx. pH 8.2, 7.6, 7.5-6.8, 6.2 and 6.1-5.5, respectively. After PAP-agarose affinity column chromatography and SDS-polyacrylamide gel electrophoresis (PAGE), their N-terminal amino acid sequences were determined. ST isoenzymes present in fractions B and E showed identical N-terminal amino acid sequences with those of ST-21 and ST-20, respectively, whereas the ST isoenzymes present in fractions C and D had the same N-terminal amino acid sequence as those of ST-40 (and/or ST-41). The results demonstrated the presence of at least three HS-ST isoenzymes in adult female rat liver.
Effects of the administration of γ-(9H-purine-6-yl)thiomethyl L-glutamate (6-MPG), a water-soluble derivative of 6-mercaptopurine, on concomitant and sinecomitant immunity against the implanted MethA tumor were studied in BALB/c mice. In the concomitant immunity experiments, mice were intradermally inoculated with 1×105MethA cells at the right inguinal region on day 0. In sinecomitant immunity experiments, mice were similarly inoculated on day -21, and the grown tumor was excised on day -11. Both the tumor-bearing and tumor-ectomized animals were re-inoculated with 3×106MethA cells intradermally at the left inguinal region on day 10. Administration of 6-MPG (100mg/kg, i.p.) on days 3 through 7 significantly inhibited growth of the re-inoculated tumor in both series of experiments. Cyclophosphamide, adriamycin, mitomycin C and cis-diamminedichloroplatinum (II) had no significant effect on the growth of the re-inoculated tumor in the tumor-ectomized mice. Spleen cells harvested from the 6-MPG-treated tumor-ectomized mice showed a strong tumor-neutralizing activity (Winn assay).
We investigated the effects of hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) on the expression of α2- and β2-adrenergic responses in primary cultures of adult rat hepatocytes. HGF (1 and 5ng/ml) rapidly stimulated the expression of β2-adrenergic responses and significantly increased α2-adrenergic responses with a maximal response at 21h. The stimulatory effects of HGF were reduced dependent on the initial plating density and were completely blocked by cycloheximide (5μM). On the other hand, PDGF (10ng/ml) significantly increased the β2-adrenergic response, but only slightly increased the α2-adrenergic responses. Expression of the α2- and β2-adrenergic responses by PDGF was independent of the initial plating density. The expression of these responses was blocked by cycloheximide (5μM). Northern blot analysis of the hepatocyte mRNA showed increased expression induced by the HGF and PDGF of the both α2- and β2-adrenergic receptor mRNA, and this expression was inhibited by actinomycin D (5ng/ml). These results indicate that the expression of α2- and β2-adrenergic responses produced by these two growth factors is regulated differently by the cell density of the primary cultures. The results suggest that the expression of α2- and β2-adrenergic responses is mediated through increased synthesis of these receptor proteins.
Effects of ethanol, acetoin and 2, 3-butanediol on the central nervous system (CNS) were investigated by using the analysis of EEG (electroencephalogram) spectral powers recorded at the frontal cortex in rats. High doses of ethanol were required for exhibiting an increase of EEG spectral powers in the delta (0-4Hz) and theta (4-8Hz) waves when it was given either orally or intravenously. On the other hand, when ethanol was injected intracerebroventricularly, the drug caused a potent increasing effect of EEG spectral powers. Both acetoin and 2, 3-butanediol were found to increase in EEG spectral powers by oral and intravenous administrations at relatively low doses. In addition, acetoin and 2, 3-butanediol were more effective than ethanol in increasing EEG spectral powers in the delta and theta bands after intracerebroventricular administration. From these findings it can be concluded that both acetoin and 2, 3-butanediol have a potent CNS depressant effect.
The effects of oral treatment with sodium malate, an active ingredient of Juzen-taiho-to, on the nephrotoxicity, bone marrow toxicity, hepatotoxicity and gastrointestinal toxicity caused by i.p. administration of 9 doses of 3.0mg/kg/d cisplatin (CDDP) (on days 3, 4, 5, 6, 7, 8, 10, 11 and 12) were examined in ddY mice inoculated with sarcoma 180 (S-180) cells on day 1 of the study. The CDDP-induced increases in blood urea nitrogen, serum creatinine, serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminases and relative stomach weight and the decreases in food intake and body weight were inhibited nearly to the control levels without reducing the antitumor activity of CDDP against S-180 by the oral treatment with sodium malate of 12 doses of more than the equimolar amount of CDDP (on days 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14 and 15). However, the CDDP-induced decreases in white blood cell and platelet counts and relative spleen and thymus weight could not be inhibited completely by combination with sodium malate, even at a dose of twice the equimolar amount of CDDP. The sodium malate-induced reduction of CDDP-induced nephrotoxicity and hepatotoxicity was observed after oral administration, as well as with i.p., s.c. and i.v. administration, and the effect was almost the same for each route of administration. Sodium malate also reduced the toxicity induced by high doses of CDDP (4.5, 6.0, 7.5, 9.0 and 12.0mg/kg/d) at doses of twice the equimolar amount of CDDP. Sodium malate at a dose of 10.68mg/kg/d (twice as high as carboplatin, CBDCA) did not reduce the nephrotoxicity, bone marrow toxicity, hepatotoxicity and gastrointestinal toxicity caused by i.p. administration of 9 doses of 15.0mg/kg/d CBDCA on days 3, 4, 5, 6, 7, 8, 10, 11 and 12 in ddY mice inoculated with sarcoma 180 (S-180) cells on day 1 of the study. From this study, it was suggested that sodium malate could become a useful agent for the reduction of CDDP-induced toxicity, particularly nephrotoxicity and hepatotoxicity.
The effect of human placental extract (HPE) on liver regenaration in rats was investigated. After intravenous administration of HPE to α-naphthylisothiocyanate (ANIT)-intoxicated rats, the labeling index in hepatocytes was significantly increased to a level 16.5 times higher than that of the control. A 1/500 dilution of HPE directly stimulated DNA synthesis of the hepatocytes in primary culture. HPE heated at 121°C did not stimulate the labeling index in vivo or hepatocyte DNA synthesis in primary culture, suggesting that HPE contains heatunstable but potent mitogens for hepatocytes. HPE contains hepatocyte growth factor (HGF), but the mitogenic effect of HPE cannot be explained by the effect exerted by HGF alone, since both the labeling index in vivo and hepatocellular DNA synthesis in vitro stimulated by HPE were much higher than those stimulated by HGF alone when the applied doses of HGF were set to be almost the same level between each case. When HPE was fractionated on a heparin-sepharose column, the mitogenic effect of HPE was found to be located mainly in the heparin-bound fraction. Hepatocyte DNA synthesis induced by this fraction was enhanced cooperatively by the heparin-unbound fraction, suggesting that there are some modulators in the heparin-unbound fraction which enhance the proliferative activity of the heparin-bound fraction by a synergetic mechanism. Both HPE and heated HPE completely recovered the biochemical marker activity for liver function (glutamic-pyruvic transaminase, GPT;alkaline phosphatase, ALP; lactate dehydrogenase, LAP; γ-glutamyltransferase, γ-GTP activities and the bilirubin concentration) almost to the control level in the serum of ANIT-intoxicated rats, indicating that HPE also contains a heat-stable fraction which repairs liver function.
TA-993, a new 1, 5-benzothiazepine derivative having a l-cis configuation, has a selective increasing action on limb blood flow, in addition to an antiplatelet aggregating action. The cardiovascular action of TA-993 is quite different from diltiazem, which is a well-known 1, 5-benzothiazepine derivative having a d-cis configuration.Therefore, we compared the cardiovascular actions of d-cis and l-cis isomers of TA-993 with those of diltiazem. l-cis-Diltiazem, as well as TA-993, progressively increased femoral, brachial and common carotid blood flow with little change in arterial pressure or vertebral blood flow. However, the peak response to l-cis-diltiazem (20 min after the administration) was observed earlier than that to TA-993 (60 min after the administration). On the other hand, d-cis-TA-993, as well as diltiazem, caused transient hypotension, tachycardia and increases in vertebral, brachial, femoral and common carotid blood flow. Furthermore, their peak effects were observed immediately after the administration. Potency ratios of the vasorelaxing effects of TA-993, l-cis-diltiazem and d-cis-TA-993 to diltiazem in the isolated and K+-contracted canine femoral artery were 0.096, 0.032 and 1.209, respectively. pA2 values for TA-993 and diltiazem against Ca2+-induced contractions in the isolated and K+-depolarized canine saphenous artery were 5.50±0.11 and 7.12±0.18, respectively. These results indicate that TA-993 shares a common profile with l-cis-diltiazem, and suggest that 1, 5-benzothiazepine derivatives of a l-cis configuration are a different class of drug from that of the d-cis configuration.
One strategy for improving the antitumor selectivity and toxicity profile of antitumor agents is to design drug carrier systems with suitable transport proteins. Thus, four maleimide derivatives of doxorubicin were bound to thiolated human serum albumin which differed in the stability of the chemical link between drug and spacer. In the maleimide derivatives, 3-maleimidobenzoic or 4-maleimidophenylacetic acid was bound to the 3'-amino position of doxorubicin through a benzoyl or phenylacetyl amide bond and 3-maleimidobenzoic acid hydrazide or 4-maleimidophenylacetic acid hydrazide was bound to the 13-keto position through a benzoyl hydrazone or phenylacetyl hydrazone bond. The acid-sensitive albumin conjugates prepared with the carboxylic hydrazone doxorubicin derivatives exhibited an inhibitory efficacy in the MDA-MB-468 breast cancer cell line and U937 leukemia cell line comparable with that of the free drug (using the BrdU-(5-bromo-2'-deoxyuridine)-incorporation assay and tritiated thymidine incorporation assay respectively, IC50∼0.1-1μM) whereas conjugates with the amide derivatives showed no or only marginal activity. These results demonstrate that antiproliferative activity depends on the nature of the chemical bond between doxorubicin and carrier protein. Acid-sensitive albumin conjugates are suitable candidates for further in vitro and in vivo assessment.
The effects of the plant isoflavones, daidzin and genistin, on bone loss in ovariectomized (ovx) rats fed a calcium-deficient diet were investigated. Daidzin and genistin were orally administered to ovx rats for 4 weeks. The femurs of these rats showed significantly lower density, strength (breaking forces), ash weight and calcium and phosphorus content (p<0.01) in comparison with those of sham-operated rats. These changes were largely prevented in animals receiving oral daidzin or genistin for 4 weeks at a dose of 50mg/kg/d and in animals receiving subcutaneous estrone (7.5μg/kg/d) as a positive control. Ovariectomy caused atrophy of the uterus and increased the ratio of the urinary excretion of pyridinoline and deoxypyridinoline to endogenous creatinine excretion. This was prevented by administration of daidzin or estrone, but, interestingly, not genistin. The preventive effect of daidzin treatment on bone loss in ovariectomized rats appears to be due to suppression of bone turnover.Genistin has a different mechanism of action from daidzin.
The subcellular distribution of biperiden (BP), trihexyphenidyl (TP) and (-)-quinuclidinyl benzylate (QNB) in brain, heart and lung following high dose (3.2mg/kg) i.v. administration was investigated in rats. The subcellular distribution of BP or TP used clinically conformed with that of QNB, a typical potent central muscarinic antagonist. The concentration-time courses of the brain subcellular fractions for these drugs were of two types which decreased slowly and in parallel to the plasma concentration. The subcellular distribution in the brain and heart was dependent on the protein amount of each fraction. The percent post-nuclear fraction (P2) of the total concentration in the lung was characteristically about 3-5 times larger than that in the heart. It was elucidated that the distribution in the lung differs from that in the brain and heart, with high affinity which is not dependent on the protein amount in the P2 fraction containing lysosomes. On the other hand, at a low dose (650ng/kg) of 3H-QNB, each fraction as a percentage of the total concentration in the brain increased in synaptic membrane and synaptic vesicles and decreased in nuclei and cytosol as compared with the high dose. These results show that although the tissue concentration-time courses of anticholinergic drugs appear to decrease simply in parallel to plasma concentration, the subcellular distribution exhibits a variety of patterns among various tissues.
Ocular implants containing fluorometholone (FLM) were prepared using blends of poly (DL-lactic acid) (PLA) and polyvinyl pyrrolidone (PVP). The effect of the fraction of PVP content on the release of FLM from the implant was investigated in vitro. The drug was released from the device by approximately following first order kinetics within the period of 40d. The release rate gradually increased with an increase in the PVP content. The in vivo study after implantation in the anterior chamber of rabbit eyes indicated that the PLA-PVP implant showed a good correlation between the in vitro and in vivo release of FLM. The present polymer blend implant demonstrated a constant level of FLM in the aqueous humor for one month.
Six peptides were obtained by the digestion of carbonyl reductase purified from rabbit liver. The amino acid sequences of the six peptides were virtually identical to the corresponding regions in amino acid sequences deduced from two cloned carbonyl reductase genes (RCBR5 and RCBR6). However, there was a difference of one amino acid residue between the sequences of peptides from the purified enzyme and the corresponding region in the amino acid sequences deduced from the two cDNAs. The purified carbonyl reductase was confirmed to exhibit no reactivity towards menadione, even though the transient expression of the two cDNA for rabbit liver carbonyl reductase has been reported to cause a marked increase of menadione reductase activity in COS7 cells.The enzyme purified from rabbit liver was inactivated by thiol-specific reagents, 5, 5'-dithiobis(2-nitrobenzoic acid) and sodium tetrathionate, suggesting that menadione probably interacts with the functional cysteine residue(s), and cannot serve as a substrate of the purified enzyme. Based on these results, it is concluded that the carbonyl reductase purified from rabbit liver is not the product of cloned carbonyl reductase gene (RCBR5 or RCBR6).
Ginseng saponins and their degradation products have been screened for antagonist activity towards [3H]PAF (platelet activating factor) in washed rabbit platelet receptor binding studies. 20(S)- and Δ20-ginsenosides Rg3, protopanaxadiol-type saponins, were found to be relatively potent PAF antagonists (IC50=4.9×10-5M and 9.2×10-5M, respectively).
Acteoside, a phenylpropanoid glycoside with anti-oxidative activity, induced cell death in promyelocytic leukemia HL-60 cells with an IC50 value of 26.7μM. Analysis of extracted DNA on agarose gel electrophoresis revealed that aceteoside induced the internucleosomal breakdown of chromatin DNA characteristic of apoptosis.Apoptosis-specific DNA fragmentation was clearly detectable 4h after treatment with acteoside and was indepedent of the cell cycle phase. These data indicate that acteoside induces apoptosis in HL-60 cells.
The effects of ethanolamine on injured liver were investigated by oral administration of ethanolamine to male ddY mice 24h after carbon tetrachloride (CCl4) injection. The serum aminotransferase activities in mice with liver injury were reduced by ethanolamine treatment (10-30mg/kg body weight). Drastically increased regerative reaction of ethanolamine treated-CCl4 injured liver was also observed through an increase in 5-bromo-2'-deoxyuridine uptake. ATP concentration in liver tissue was recovered by administration of ethanolamine. These results suggest that oral administration of ethanolamine accelerates recovery from CCl4-in-duced liver injury.
In the present study we investigated irritation of the oral mucosa and the safety of gummi drugs containing acetaminophen (AAP). The oral mucosae of hamsters were macroscopically examined for any evidence of irritation after gummi drugs were inserted into the cheek pouch and left there for 1h. The cheek pouch tissue was also macroscopically and microscopically examined 24h after gummi drugs were withdrawn from the cheek pouch.As a result, no evidence of irritation was found macroscopically 1h after insertion, or macroscopically and microscopically 24h after the withdrawal of the gummi drugs or placebos as compared with negative controls (saline). Considering these results, the gummi drugs administered in the present study produced no irritation to the oral mucosa.
Strain differences of mice in the induction of DNA damage in peripheral blood cells and skin tumors were investigated using 7, 12-dimethylbenz[a]anthracene (DMBA). DMBA-induced DNA damage and skin tumorigenesis were evaluated using the single cell gel electrophoresis (SCGE) assay and 2-stage carcinogenicity study, respectively. DNA damaged cells were markedly increased in the aryl hydrocarbon hydroxylase (AHH)-inducible mice, BALB/c and C57BL/6, as compared with the AHH-noninducible mice, DBA/2, in the SCGE assay. The AHH-inducible mice were more sensitive to DMBA than the AHH-noninducible mice in the 2-stage carcinogenicity study. These results strongly suggest that the genetic capacity to metabolize PAH is associated with the mutagenecity and carcinogenecity of DMBA.