Down-regulation of CD46 secondary to stimulation with measles virus (MV) was investigated using CD46-positive cell lines and Japanese wild-type MV strains. The cells used were simian cell lines B95a and Vero in which MV strains have been adapted to be amplified, and Chinese hamster ovary (CHO) cell transfectants expressing human CD46 with (CHO(tail+)) or without (CHO(tail-)) the cytoplasmic tail. Of four Vero-adapted and three B95a-adapted MV strains, one Vero-adapted strain named Khono (KO), down-regulated CD46 within 60 min (early down-regulation) in all cell lines examined except Vero. No strains other than Toyoshima (TY), which induced early down-regulation only in CHO(tail+) cells, induced early down-regulation of CD46 in any combinatioon. On the other hand, conventional down-regulation of CD46 was observed 24 h post-MV inoculaiton (late down-regulation) when cell lines used were adapted to MV strains. Thus, we concluded that there are two modes of CD46 down-regulation by MV and the unique strain KO markedly induces early down-regulation. Also, the CD46 homologue of B95a, which fails to act as a MV receptor, is down-regulated concomitantly with MV replication (>24 h) in cells principally by compenent virus strains.
The overproduction of Na+/H+ antiporter NhaA in Escherichia coli caused growth retardation. Quantities and the activity of the antiporter were studied for their causative roles in terms of this retardation. We constructed a series of nhaA-expression plamids differing in their transcriptional and translational efficiencies. Low-level nhaA expression complemented the defect of an nhaA mutant and allowed the mutant to survice on a high-NaCl or high-LiCl medium. However, when the production of NhaA was strongly induced by the combination of a stronger promoter, an efficient translational initiation signal and a high copy number plamid, the growth of the cells carrying the plasmid was severely retarded. This growth retardation correlated well with the amount of NhaA protein produced from the plasmids. Surprisingly, the growth retardation caused by overproduction of NhaA was enhanced more extensively at an alkaline pH than at a neutral pH, in which the antiporter activity was stimulated. However, these retardations were also observed for mutant NhaA antiporters without the activity. These results indicated that the growth retardation was due to an overproduction of the antiporter rather than its increased antiporter activity, and also affected by a pH-dependent change in NhaA, possibly its structrual change.
The inhibitory effects of various sulfated compounds on the activitives of sialidases purified from porcine liver and human placenta were investigated. Among the sulfated compounds tested, heparin, dextran sulfate, condroitin sulfates and sulfatide significantly inhibited the 4-methylumbelliferyl-α-N-acetylneuraminic acid (4-MU-NeuAc) sialidase activities of the two enzyme preparations, but glucose 6-sulfate and glucosamine 6-surfate did not. Potassium sulfate showed an inhibitory effect only at high concentrations. When the sialidase activities were measured using natural substrates, the sialidase activities for the (α2-3) and (α2-6) sialyllactoses, and colominic acid, were markedly inhibited by heparin and sulfatide similar to 4-MU-NeuAc, although the fetuin sialidase activity was not significantly influenced by them. The sialidase activity hydrolyzing GM3 was strongly inhibited by heparin, but not by sulfatide.
We identified proteins whose amounts were altered in an Escherichia coli pgsA3 mutant lacking the potential to synthesize phosphatidylglycerolphosphate, a precursor of phosphatidylglycerol. Proteins whose amounts were increased in the mutant were protease Do, periplasmic oligopeptide-binding protein, tryptophanase, and an unidentified protein, while the decreased one was flagellin. Transformation of the mutant with a plasmid containing the wild type pgsA gene complemented the phenotype, indicating that the pgsA3 mutation is resposible for the phenotype.
We studied whether the peroxisomal proliferation, induction of 3-hydroxy-3-methyglutaryl-CoA reductase (HMG-CoA reductase) and activation of cholesterol synthesis by gemfibrozil shown in whole body (Hashimoto F., Ishikawa T., Hamada S. and Hayashi H., Biochemical. Pharm., 49, 1213-1221 (1995)) is also detected at a culture cell level, and we made a comparative analysis of the effects of clofibric acid. Gemfibrozil at 0.25 mM increased the activity of some peroxisomal enzymes (catalase and the cyanide-insensitive fatty acyl-CoA oxidizing system) after incubation for 72 h. However, contrary to whole body experiments, gemfibrozil decreased the activity of HMG-CoA reductase and cholesterol synthesis from [14C]acetate. At 1 mM, gemfibrozil decreased not only the activity of HMG-CoA reductase and cholesterol synthesis, but also the protein conetnt of the cells and peroxisomal enzyme activity, indicating nonspecific inhibition at this concentration. Clofibric acid (0.25 and 1 mM) increased the activity of peroxisomal enzymes, but decreased the activity of HMG-CoA reductase and cholesterol synthesis. With respect to the direct effect on HMG-CoA reductase in the cell homogenate, gemfibrozil at 0.25 mM did not affect the activity, but it clearly inhibited the activity at 2 mM and above. Clofibric acid at 2 mM hardly affected the activity, but it clearly decreased the activity at 5 mM and over. That is, gemfibrozil directly inhibited the activity more strongly than clofibric acid. The direct inhibition of the enzyme itself required higher concentrations of both agents than did inhibition at the culture cell level.These results suggest that the cytotoxicity of gemfibrozil is greater than that of clofibric acid, and that gemfibrozil, as well as clofibric acid, can induce peroxisomal enzymes in the culture cell level. In contrast to whole body results, gemfibrozil may suppress cholesterol synthesis from [14C]acetate through the inhibition of HMG-CoA reductase at the culture cell level. The decreases in the reductase activity caused by gemfibrozil and clofibric acid at the culture cell level may not be caused by the direct inhibition of the enzyme.
Nonsteroidal anti-inflammatory drugs have been shown to be potent inhibitors of mammalian 3α-hydroxy-steroid dehydrogenase. Here, we report that the drugs of the 2-arylpropionic acid class act as both activators and inhibitors for a predominant isoform of the human liver enzyme which is involved in the metabolism of steroid hormones, bile acids, drug ketones and xenobiotic aromatic hydrocarbons. Flurbiprofen, fenoprofen, ibuprofen, naproxen, ketoprofen and suprofen stimulated the activity of the human enzyme (1.5-2.4-fold) at low concentrations of less than 20-100 μM, whereas at higher concentrations they inhibited the acitivity. Comparison of the effects of the structurally related compounds with the drugs revealed that the essential structure required as the activator molecule is 2-phenylpropionic acid with a hydrophobic substituent on the aromatic ring. In addition, an R-enantiomer of ibuprofen showed higher activation (3-fold) than its S-enantiomer. Kinetic analysis with respect to NADP+ concentration indicated that R- and S-ibuprofens are nonessential activators showing binding constants of 23 and 34 μM, respectively. Neither enantiomers activated, but rather inhibited the enzyme, with Met replacing Arg-276 which has been shown to interact with the 2'-phophate of NADP+. The inhibitions of the mutant enzyme by R- and S-ibuprofens were competitive with respect to the substrate, giving Ki values of 95 and 18 μM, respectively. The results suggest that the human liver 3α-hydroxysteroid dehydrogenase isoform possesses the two distinct sites, activator and inhibitor sites, to which anti-inflammatory 2-arylpropionates stereoselectively bind.
We have investigated the anti-metastatic effect of Celosia argentea seed extracts (CAE), which have traditionally been used as a therapeutic drug for eye and hepatic diseases in China and Japan. Intraperitoneal (i.p.) administration of CAE for 7d before tumor inoculation significantly inhibited liver metastasis caused by intraportal injection of colon 26-L5 carcinoma cells in a dose-dependent manner. CAE also showed concentration dependent mitogenic activity on BALB/c whole splenocytes, whereas incubation of the non-adherent fraction of splenocytes with CAE did not induce this activity. CAE has the ability to induce interleukin (IL)-12 production from macrophages in vitro. Following i.p. administration of CAE the maximal levels of IL-12 and interferon (IFN)-γ production in serum were achieved at 2-3 and 6 h, respectively. Experiments using macrophage- or NK cell-deficient mice revealed that CAE-induced IL-12 in serum was not mediated by macrophages and that IFN-γ production was mainly dependent on natural killer (NK) cells. Since CAE was inactive when the contributions of macrophages were removed in our system, its inhibitory mechanism is likely to be mainly associated with the activation of macrophages to an anti-metastatic state rather than NK cells. CAE administration resulted in increased production of IL-2, IFN-γ and decreased production of a Th2 cytokine (IL-4) from splenocytes stimulated by PMA and A23187. Thus, the anti-metastatic effect by CAE is based on its immunomodulating properties including induction of cytokines such as IL-12, IL-2 and IFN-γ leading to a Th1 dominant immune state and activating macrophages to the tumoricidal state. This may provide a basis for the inhibition of cancer metastasis.
Our previous study indicated that picrotoxin competes with phenobarbital (PB) for the binding to liver microsomes, although the target of biding and the physiological role were not determined (unpublished observation). To seek information on the target site of PB, reflecting the induction of hepatic enzymes, we examined here the effect of picrotoxin on PB-mediated induction of hepatic cytochrome P450 and UDP-glucuronosyltransferase in vivo in rats. The induction of the CYP2B1/2 was estimated by immunoblot analysis and by measuring the activity of testosterone 16β-hydroxylation. Intraperitoneal injection of picrotoxin alone slightly increased CYP2B1/2 protein. An experiment on co-treatment of picrotoxin and PB showed that picrotoxin enhanced rather than antagonized the inducing effect of PB. The results suggest two possibilities : that 1) picrotoxin increases the CYP2B subfamily by binding to the same site as PB; or 2) the site in microsomes for the competition between PB and picrotoxin does not reflect the induction of P450.
Vasoactive effects of cimicifugic acids A-E, fukinolic acid and fukiic acid isolated from Cimicifuga plants were investigated using rat aortic strips. Cimicifugic acid D and fukinolic acid at 3×10-4M caused a sustained, slowly developing relaxation of aortic strips precontracted with norepinephrine (NE) in preparations with or without endothelium. Cimicifugic acid C inversely caused a weak contraction. Cimicifugic acids A, B and E and fukiic acid showed no vasoactivity at 3×10-4 M. Cimicifugic acids A-E and fukinolic acid are esters between cinnamic acids and the hydroxyl group of benzyltartaric acids. For the manifestation of vasoactivity in the rat aorta, it is concluded that in the cinnamic acid moiethy, a caffeoyl group might be necessary for the relaxaion activity, and the p-coumaroyl group causes contraction.Concentration response curves for the Ca2+-induced contracture of depolarized aortic strips with isotonic high K+ were not affected by cimicifugic acid D or fukinolic acid. The Ca2+-induced contraction of aortic strips, preincubated with 10-6 M NE in the prsence of 10-6 M nicardipine and 0.01 mM EGTA in Ca2+-free solution, were inhibited by cimicifugic acid D and fukinolic acid. These results indicated that the inhibition by cimicifugic acid D and fukinolic acid of the NE-induced contraction of rat aorta are attributable to the suppression of Ca2+ influx from the extracellular space enhanced by NE.
It is commonly accepted that bronchial asthma or rhinitis accompanies disorders in body fluids and body temperature. The effects of ephedrine and the traditional antiasthmatics "Makyo-kanseki-to" and "Goko-to" were therefore studied on such constitutional predispositions as insensible perspiration and body temperature. Ephedrine markedly increased body temperature and exhibited a strong increased action on respiratory insensible perspiration, whereas Makyo-kanseki-to and Goko-to not only prevented the elevation of body temperature, but also increased respiratory insensible perspiration following the reduction of non-evaporative heat loss from the body surface. Thus, the diagnostic criteria of these two medicines used to treat hot-type asthma or dry cough were experimentally determined. The results also suggest that there is a great possibility that the administration of antiasthmatics may elicit side effects or make diseases worse unless their actions on constitutional predispositions are taken into account, such as body temperature and body fluids.
Paeoniflorin (1) and its derivatives having in common a cage-like pinane skeleton with hemiketal-acetal system, were evaluated for their effects on memory impairment induced by scopolamine in mice using a step-down type passive avoidance task. In the test session, 1 and its derivatives were intraperitoneally (i.p.) administered at doses of 0.002, 0.01, 0.02 and 0.2 mmol/kg, and 30 min later (15 min before the experiment), scopolamine (1 mg/kg, i.p.) was given. These compounds showed dose-dependent attenuation in a dose range of 0.002-0.02 mmol/kg and also enhancement of scopolamine-induced decrease in step-down latency. The effects of these compounds, except that of 2', 3', 4', 5'-O-tetraacetyl-3-O-methylpaeoniflorin (8), followed a bell-shaped dose response profile. 8-Debenzoyl-6-deglucosyl-3-O-methylpaeoniflorin (6) showed no significant increase in the step-down latency at all tested doses. Maximum step-down latency was obtained by 3-O-methylpaeniflorin (3) and 2', 3, 3'4', 5'-penta-O-methylpaeoniflorin (7) (the minimal effective dose was 0.002 mmol/kg). Relative to 3, debenzoylation, as in 8-debenzyl-3-O-methylpaeoniflorin (4), slightly increased the latency, while deglucosylation, as in 6-deglucosyl-3-O-methylpaeoniflorin (5), significantly reduced the prolongation of latency. Removal of both glucose and benzoyl moieties resulted in the loss of activity as seen in 6. These results revealed that, in addition to the cage-like pinane skeleton, the benzoyl and the glucosyl moieties are important structural elements of the paeoniflorin skeleton as its effects on scopolamine-induced amnesia.
Conjugates of mitomycin C (MMC) with estradiol benzoate and estradiol via glutaric acid, abbreviated to EB-glu-MMC and E-glu-MMC, respectively, as previously reported, were examined concerning their pharmacokinetic behaviors and antitumor effects against two kinds of general and popular tumors, P388 leukemia and Sarcoma 180. EB-glu-MMC and E-glu-MMC were dissolved in propylene glycol. Their solution was administered intrapertioneally to rats and mice in order to examine their plasma concentration-time profiles and antitumor characteristics, respectively. After the administraiton of EB-glu-MMC, EB-glu-MMC was detected slightly in blood only in the initial stage, while E-glu-MMC and MMC were observed there for a prolonged period. In the administration of E-glu-MMC, a similar phenomenon was observed but the drug retention effect seemed lower than that in EB-gul-MMC. In the antitumor test against P388 leukemia, E-glu-MMC exhibited a better effect than EB-glu-MMC; however, neither conjugate surpassed the effect of MMC. The toxic side effect was improved in each conjugate. As to the growth inhibition against Sarcoma 180, EB-glu-MMC and E-glu-MMC produced good effect and improved the toxic side effect. Especially, in the administration of EB-glu-MMC at the dose of 30 mg eq MMC/kg, a decrease in tumor volume was observed in the latter stage. EB-glu-MMC and E-glu-MMC were demonstarted to produce prolonged retention, to enter the systemic circulation to a fair extent, and to exhibit a good effect against the general solid tumor, Sarcoma 180, in vivo.
In this study, we developed a new hollow-type suppository containing elcatonin ((Asu1, 7)-eel calcitonin, ECT), a synthetic derivative of eel calcitonin, which produces hypocalcemia, as a pharmaceutical preparation for self administration, to be used instead of parenteral injections for patients with osteoporosis. The absorption of ECT from the rectal mucous membrane was evaluated by observation of the decrease in serum calcium (Ca) concentrations following rectal administration in rabbits. ECT was efficiently absorbed from the rectum and effectively decreased serum Ca concentrations. The data of the area under the percent decrease in serum Ca concentration (ΔCa%)-time curve (ΔCa%-AUC), assumed to be an index of the pharmacodynamics (pharmacological effect) of ECT, indicated that similar hypocalcemic effects were obtained following rectal and intravenous administrations of ECT. In regard to the effect of coadministration of other compounds on rectal absorption of ECT, no significant difference in the ΔCa%-AUC between rectal ECT administration with or without nafamostat mesilate (a protease inhibitor) was observed. However, the coadministration of ECT with cytochalasin B or monensin (endocytosis inhibitors) significantly decreased the ΔCa%-AUC, indicating that rectal ECT absorption is probably inhibited by endocytosis inhibitors by endocytosis inhibitors. On the other hand, it was found that sodium decanoate, a mediumchain fatty acid (sodium salt), significantly enhanced the rectal absorption of ECT. We conclude that this ECT hollow-type suppository offers promise as a new method for the administartion of ECT.
In this study, the effects of a protease inhibitor, endocytosis inhibitors and an absorption-enhancing agent on the absorption of (Asu1, 7)-eel calcitonin, ECT) from the nasal mucous membrane in rabbits were examined, and the results were compared with those obtained following the rectal absorption of ECT reported in our previous paper. ECT was efficiently absorbed from the nasal mucous membrane and effectively decreased serum calcium (Ca) concentrations. The increase in the area under the precent decrease in serum Ca concentration (ΔCa%)-time curve (ΔCa%-AUC) value, assumed to be an index of the pharmacodynamics (hypocalcemic effect) of ECT, depended on the dose of ECT administered intranasally. When nafamostat mesilate, a protease inhibitor, was coadministered with ECT, the ΔCa%-AUC markedly increased. It is presumed that the influence (enzymatic barrier function) of protease on the nasal absorption of ECT is significant. However, no significant difference in the ΔCa%-AUC value was observed when an endocytosis inhibitor (cytochalasin B or monensin) was coadministered with ECT. ECT administration in combination with sodium decanoate, the sodium salt of a medium-chain fatty acid, effectively increased the ΔCa%-AUC value due to the enhancing effect of sodium decanoate on the nasal absorption of ECT. We conclude that the nasal application offers a promising approach for the administration of pharmaceutical preparations containing ECT with additives such as nafamostat mesiate and sodium decanoate.
The factors determining drug residence in skin during penetraiton across rat abdominal skin were investigated using five β-blocking agents with different lipophilicities as model drugs in vivo and in vitro. The amount of β-blocking agent in the skin at steady state correlated well with lipophilicity. The distribution of β-blocking agents to the stratum corneum and the contribution of intercellular lipids in the stratum corneum to their skin distribution were also correlated with their lipophilicity, suggesting that the stratum corneum, especially intercellular lipids in the stratum corneum, wound be responsible for the residence of β-blocking agents in the skin. Furthermore, cholesterol-3-sulfate, palmitic acid, stearic acid and oleic acid were found to interact with the β-blocking agents, which are cationized under the physiological condition, and were assumed to play an important role in the skin accumulation. On the other hand, the binding to keratinocyte was so small that keratinocyte might have little effect on the skin accumulation of the β-blocking agents. Drug transport from the stratum corneum to viable skin was suggested to be regulated by the lipophilicity of these agents. To investigate the residence of these drugs in viable skin, in vitro transport studies using stripped skin were performed. The transport rate constant across viable skin to receptor cells (k23) was inversely correlated with the lipophilicity of the drugs. The elimination rate constants from viable skin (kvs) obtained in the in vivo study were much smaller than the values of k23 obtained in the in vitro study, and they were inversely correlated with the binding to cytosol components of viable skin but not the lipophilicity. The viable skin-to-muscle concentration ratio of these drugs, obtained at the β-phase of the plasma concentration-time curve after intravenous administration, was also inversely correlated with the binding to the cytosol components of viable skin. These results suggest that kvs reflects the transport from viable skin to muscle rather than to blood circulation and that the binding of drugs to cytosol components in viable skin would be one of the important factors determining the residence in viable skin.
Two types of poly(vinyl alcohol)-gel spheres were prepared with chitosan (CS/PVA-GS) and witout chitosan (PVA-GS), and comparative studies were performed using these gel spheres (GSs). No change in particle size was observed by the addition of chitosan : nearly 45% of both particles were in the 5-10 μm range. In an in vivo gastrointestinal transit test, CS/PVA-GS prolonged the small-intestinal transit time more than PVA-GS. In an in vitro intestinal perfusion study, the mean transit time of these GSs was markedly reduced by pretreatment of the intestinal surface with a mucolytic agent, N-acetyl-L-cysteine, suggesting that the mucous layer on the intestinal surface plays an important role in controlling the transit rate of these GSs. The oral administraiton of aminophylline (theophylline) and ampicillin as model drugs incorporated in PVA-GS and CS/PVA-GS was examined in rats. While theophylline absorption from PVA-GS was not affected by the addition of chitosan, the improvement of ampicillin absorption by PVA-GS was enhanced by the chitosan combination.
We developed a method for simultaneous analysis of benzphetamine (BZ) and its metabolites, p-hydroxy-N-benzylamphetamine (pHBA), p-hydroxybenzphetamine (pHBZ), amphetamine (AP), methamphetamine and p-hydroxymethamphetamine by micellar electrokinetic chromatography (MEKC). Urine samples from 0-15 h (3h) intervals) after oral administration of BZ (10 mg) were hydrolyzed with β-glucuronidase (EC 18.104.22.168) at 37°C overnight. The treated urine was applied to a solid phase extraction column Bond Elut Certify[○!R]. After sequentially washing the column wiht water, 0.1 mol/l acetic acid and methanol, the samples were eluted with dichloromethane : isopropanol : 28% ammonium hydroxide=78.4 : 19.6 : 2.0 (v/v %). The eluate was evaporated and the residue dissolved in running buffer was analyzed by MEKC. In urine from 0-3 h, AP, pHBZ and pHBA were detected. After that, only pHBA, which is one of the major metabolites of BZ in human urine, could be detected in the urine by the present method. A method for quantitation of pHBA by MEKC is described here. The effects of acetonitrile and sodium dodecyl sulfate in the running buffer of MEKC on the separation of BZ and its metabolites are also reported.
A method to prevent co-elution of steroid sulfates with proteins in serum from the pre-column in column-switching HPLC was developed. The pre-column, a polymer-coated mixed function column, was used for ionpair chromatography with 5 mM tetra-n-butylammonium (TBA) ion. As steroid sulfates, estriol 3-sulfate, dehydroepiandrosterone 3-sulfate and pregnenolone 3-sulfate were used. Human serum (25 μl) was diluted with mobile phases including 5, 100 and 500 mM TBA ion, and then injected directly into the pre-column. The peak areas of the steroid sulfates in serum samples were compared with those of the steroid standards without serum. When 25 μl of serum was diluted with mobile phase including 100 or 500 mM TBA ion, the steroid sulfates in serum were retained in the pre-column; however, the steroid sulfates from the same sample diluted with mobile phase containing 5 mM TBA ion were not retained in the pre-column. Addition of an excess amount of counter ion (TBA ion) into the serum sample made it possible to retain the steroid sulfates in the pre-column. This method was applied to column-switching HPLC for measurement of steroid sulfates in serum using a semi-microcolumn as the analytical column.
The fluorescence polarization (FP) technique was evaluated to determine molecular interaction between plant lectins and polysaccharides, yeast cells and glycopeptide after labeling the lectins with fluorescein isothiocyanate. Use of Lycoris radiata agglutinin allowed determination of the molecular interactions with large biomolecules containing mannose oligomers and polymers. Another example using a fluorescein-labeled glycopeptide also indicated that use of the FP method would allow easy observation of the molecular interactions on the quantitative base. The present technique is highly sensitive and facile because it does not require any washing procedures before measurement.
We have purified two forms of Zn2+-dependent acid phosphatase (Zn2+-APase) from bovine liver, both of which require Zn2+ to hydrolyze the substrate p-nitrophenyl phosphate in an acidic environment. The apparent molecular weights of these two forms of Zn2+-APase were estimated to be about 100000 and 62000 by gel filtration, and about 44000 and 31000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, respectively. The low-molecular weight (LMW) Zn2+-APase catalyzed the hydrolysis of myo-inositol-1-phosphate in the presence of 3 mM Mg2+ at physiological pH, but the high-molecular weight (HMW) enzyme did not. The LMW-Zn2+-APase of bovine liver was recognized by polyclonal antibodies developed against the Zn2+-APase of bovine brain, but the HMW-Zn2+-APase was not.
The effect of various metal ions on neutral phosphatase activity in the brain cytosol of rats was investigated. p-Nitrophenylphosphate was used as the substrate for the enzyme assay. Brain cytosolic phophatase activity was significantly decreased by the presence of zinc or copper in the range of 10-5 to 10-3 M in the enzyme reaction mixture, while it was significantly increased by manganse, nickel or cobalt (10-5 to 10-3 M). Calcium (10-5 to 10-3 M) did not have an appreciable effect on brain cytosolic phosphatase activity. The inhibitory effect of zinc (10-5 M) or copper (10-5 M) on phosphatse activity was seen in the presence o manganese, nickel or cobalt with 10-4 M concentration, indicating a potent inhibitory effect of these metals. Zinc or copper-decreased phosphatase activity was clearly prevented by dithiothreitol (5 mM), although the effect of each metal was further enhanced by N-ethylmaleimide (5 mM), suggesting that the metals act on the SH-groups of enzyme. Moreover, the inhibitory effect of zinc (10-5 M) was significantly enhanced by S-100A (10-6 M) or calbindin (10-7 M), although neither protein had any effect in the presence of calcium (10-5 M) or copper (10-5 M). These results suggests that zinc has an unique role in the regulation of neutral phosphatase activity in rat brain cytosol.
The cathartic effect of isobarbaloin, a stereoisomer of barbaloin (compound principally responsible for the cathartic activity of Aloe), was examined in male rats by oral administration. Individual differences in sensitivity in the laxative activity of isobarbaloin and barbaloin was not found. The cathartic activity (ED50) of isobarbaloin in barbaloin positive rats was 19.2 mg/kg, nearly equal to that of barbaloin (19.5 mg/kg). Also, isobarbaloin administered orally was demonstrated to decompose to aloe-emodin-9-anthrone (active metabolite of barbaloin) as well as to barbaloin. Therefore, it is considered that the mechanism underlying the cathartic effect of isobarbaloin is the same as that of barbaloin.
The interaction of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist YM90K with the competitive N-methyl-D-aspartate (NMDA) antagonist CGS19755 or with the NMDA channel blocker MK801 on the spatial memory of rats was examined by the 4-arm-baited radial maze task. When administered alone, these drugs had no effect on the spatial memory. The combination YM90K and MK801 synergistically disrupted working and reference memories, whereas the combination of YM90K and CGS19755 had no effect. These results indicate that the blockade of the AMPA recepor and NMDA channels produces a synergistic impairment of spatial memory and suggest that interaction between the AMPA and NMDA receptors plays a role in congnitive function.
We examined the antipruritic effects of various oleanolic acid glycosides from natural medicines such as Kochiae Fructus (the fruit of Kochia scoparia SCHRAD.) and Momordicae Radix (the roots of Momordica cochinchinensis SPRENG.) using a compound 48/80-induced pruritic model in mice. Oleanolic acid 3-O-monodesmosides showed an antipruritic effect, while oleanolic acid 3, 28-O-bisdesmosides and their common sapogenol oleanolic acid lacked the activity. This evidence indicated that the 3-O-glycoside moiety and the 28-carboxyl group in oleanolic acid glycosides were essential for exhibiting the antipruritic effect. Furthermore, it was found that the 3-O-glucuronides showed more potent activity than the corresponding 3-O-glucosides.
Comparisons of the biological activities of diosgenyl, methyl glycyrrhetinate or digitoxigenyl 3-O-β-L-xylopy-ranosyl-(1→6)-β-D-glucopyranoside with those of other previously tested glycosides confirmed our assumption that both the hemolytic and antifungal activities of steroid saponins are generally parallel to each other, while almost all hemolytic triterpenoid saponins and nonhemolytic ones have no antifungal activity, and that cardiac diglycosides having a (1→4) sugar linkage have stronger activities than those with a (1→6) or a (1→2) linkage. On the other hand, the case of the diosgenyl 3-O-β-L-xylopyranosyl-(1→6)-α-D-glucopyranoside didn't conform to the above assumption, but those of methyl glycyrrhetinate and digitoxigenyl did.
In order to estimate the effect of the long term administration of cyclosporine (CsA) on the shape change of erythorocytes, erythorocyte shapes which are observed with a scanning electron microscope were classfieid according to the nomenclature of Bessis for stomatocyte-echinocyte shape transformation. As a result of observing the erythrocyte shape of fifty-six patients with kidney transplantation treated with CsA, the morphological index of the erythrocytes of patients significantly increased to 0.0835±0.0085*** in comparison with 0.0004±0.0051 of those from healthy volunteers (control) (*** : p<0.001, ANOVA). Such transformations had no relation to the subject's sex or age. On the other hand, the erythrocytes of patients administered more than 100 ng/ml of CsA and posttransplanted within less than two years were transformed by CsA from the state of discocyte to echinocyte. In rats, the morphological index of erythrocytes of rats treated with 3 mg/kg/d or 5 mg/kg/d of CsA significantly increased in comparison with rats treated with saline (control). Furthermore, the erythrocytes of two patients were observed in terms of shape before the treatment with CsA. In both patients, the echinocyte type of erythrocyte increased by treatment with CsA. In vitro, the morphological index of the erythrocytes incubated with plasma containing CsA significantly increased, to 0.459±0.066*** in comparison with 0.064±0.029 of the control. It is suggested from these results that CsA treatment induces the echinocyte type of erythrocyte.
The insulin content in mouse insulinoma MIN6 cells was determined using matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF/MS). A mass spectrum of cellular insulin was obtained with a cell burst of MIN6 by hypotonic water. Signal intensities of intracellular insulin were proportional to the number of MIN6 cells. The present method was applied to the determination of intracellular insulin content of MIN6 before and after glucose stimulation.