Barnidipine is a 1, 4-dihydropyridine calcium antagonist. HPLC was conducted on a polybutadiene coated alumina column using an alkaline mobile phase and an electrochemical detector to determine the content of this drug in serum and plasma. A good linear relationship between barnidipine concentration and peak height was found in 5-500ng/ml with a correlation coefficient of 0.998. The detection limit was 1 ng/ml. The within-day and day-to-day variations were examined for control human serum. Relative standard deviation of within-day assay for serum spiked with 10 ng/ml barnidipine·HCl was 6.9% and the recovery was 104%. A pharmacokinetic study was made in which the time course of barnidipine in dog plasma was followed.

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T lymphocyte unresponsiveness, induced in mice by a single gastric intubation of 0.2 ml cedar pollen extract (CPE, containing 4μg protein/ml)/mouse daily for 3 to 28 consecutive days, was evaluated by the absence of a proliferative response of popliteal lymph node (PLN) T lymphocytes to CPE in vitro. T lymphocyte unresponsiveness increased with the period of gastric intubation of CPE and reached more than 80% of the control on day 28. The unresponsiveness to CPE was antigen-specific and T lymphocyte-mediated. In vitro CPE-specific T lymphocyte proliferation was significantly suppressed by intestinal intraepithelial lymphocytes (IELs) and hepatic mononuclear cells (MNCs), but not by spleen cells or PLN T lymphocytes from mice fed CPE for 28 days. The effector activity of IELs and MNCs was obviously antigen-specific. These results suggest that lymphocytes in the intestine and liver of mice fed CPE would be involved in the induction and maintenance of CPE-specific T lymphocyte unresponsiveness.

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In a previous study, we reported that one of the gel-forming (1→3)-β-D-glucans, grifolan (from Grifola frondosa, GRN), stimulated cytokine production from macrophages in vitro. However, several other gel-forming (1→3)-β-D-glucans, such as sonifilan (SPG) and SSG, did not induce cytokine production from macrophages. The ultrastructure of gel-forming (1→3)-β-D-glucans, especially the triple-and single-helix, does not affect the cytokine-inducing activity. The action on tumor necrosis factor α (TNFα) release was correlated with the molecular weight of GRN, since the highest molecular weight fraction of GRN, Mr≥450000, exhibited the strongest activity. Although, native SSG (Mr≥2000000) did not induce cytokine production, chemical modification involving debranching of the side chain glucosyl residues of SSG resulted in TNFα inducing activity. These results suggest that the branching ratio and molecular weight of (1→3)-β-D-glucans are important factors for the production of cytokines from macrophages. GRN-inducible TNFα release was reduced by co-culturing with SPG, SSG, or the soluble β-glucan, laminarin (LAM). Pretreatment alone with SPG or LAM was not sufficient for significant inhibition of GRN-inducible TNFα release. TNFα production induced with 50μg/ml of zymosan (ZyM) was also reduced by addition of SPG, but TNFα production, stimulated with a higher concentration (100μg/ml) of ZyM or with lipopolysaccharide (LPS), was not reduced significantly. The inhibitory effect of LAM on the uptake of GRN by RAW264.7 cells was not completely correlated with TNFα release. These results suggest that macrophages may incorporate β-glucans through certain (1→3)-β-D-glucan-specific mechanisms and/or other endocytosis pathways, and that the β-glucan-specific route is partially associated with cytokine production. In conclusion, TNFα release by macrophages is induced only by β-glucans with high molecular weights and lower branching ratios, and the mechanism for the recognition of β-glucans is multiple and assumed to be divided into several parts involving various cellular functions.

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To understand the roles of the 5'-flanking region of the recognition sites in binding specificity and the affinity of integration host factor (IHF), a variety of DNA fragments with a 13-bp consensus sequence, 5'-WATCAAN₄TTR-3'[Friedman, Cell, 55, 545 (1988)], but with different 5'-flanking sequences were investigated by gel retardation and methylation interference assays. It has been well-established that the putative A/T rich element distal from the 5'-end of the consensus made a significant contributions to the binding of IHF. However, many of the DNA fragments used here revealed specific binding to IHF without such an A/T element. Several bases neighboring to the 5'-end of the consensus sequence had significant effects on the binding specificity as well as its affinity, and these results indicate that the sequence-directed bendability of the flanking region plays an important role in the specific recognition by IHF.

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Fatty acyl-Co A : sphingosine acyltransferase (ceramide synthase, EC 2. 3. 1. 24) is mainly localized in the microsomal and mitochondrial membranes. Attempts to isolate the enzyme have failed, largely because there has been little or no detection of the enzyme activity in detergent extracts. In this study, we solubilized the membrane-bound enzyme from bovine brain mitochondria with a Tris-HCI buffer containing 2% Triton X-100 and, after removal of the detergent, reconstituted it with the membrane lipid liposomes. The specific activity of the reconstituted enzyme was approx. 8 times higher than that of the solubilized enzyme. We next examined the lipid dependence of the enzyme, using various phospholipid liposomes. The ability of phospholipids to enhance the activity of solubilized ceramide synthase was specific and structure-related. The most potent stimulator was phosphatidylserine liposomes, suggesting an important role of the net negative charges. This paper also describes a highly reproducible high-performance liquid chromatographic (HPLC) procedure for the determination of ceramide synthase activity. Combination of the HPLC method with the reconstituted enzyme system appears to be suitable for elucidating the characteristics of this enzyme.

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The membrane-association of early biosynthetic form of cathepsin D has been demonstrated in hepatoma cells, and this membrane-association is not mediated by mannose 6-phosphate residues, implying that a mannose 6-phosphate receptor-independent mechanism operates in the sorting of cathepsin D. In this paper, to demonstrate whether cathepsin D is associated with the lysosomal membranes, an in vitro binding experiment was carried out employing lysosomal cathepsin D or microsomal procathepsin D isolated from rat liver. Immunoblotting analysis revealed that an intermediate form of cathepsin D was associated with the lysosomal membranes ; this lysosomal membrane-associated cathepsin D was released from the membranes by washing with Na₂CO₃ (pH 10.6) but not with solutions containing mannose 6-phosphate. This suggested that cathepsin D associates with the membranes by ionic-interaction, and that the membrane-associated cathepsin D resides as a peripheral membrane protein in the lysosomal membrane fraction. To confirm that the intermediate form of cathepsin D specifically interacts with the lysosomal integral membrane proteins, the lysosomal membrane fraction was treated with trypsin and the binding experiment was conducted. The result showed that the binding capacity of cathepsin D to the lysosomal membranes was apparently abolished and cathepsin D did not rebind to the membranes. These data suggest that the intermediate form of cathepsin D is preferentially recognized by the lysosomal membranous protein which complements the mannose 6-phosphate receptor-dependent intracellular sorting mechanism.

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Some epidemiological data have linked dietary polyphenols with lower risk of coronary heart disease. Polyphenols might impair lipoprotein oxidation which is believed to be an important step in initiating atherogenesis. The purpose of this study was to determine if grape extract known to contain polyphenolic substances can block copper-induced oxidative modification of human low density lipoprotein (LDL). LDL oxidation was monitored spectrophotometrically by measurement of change in absorbance at 234nm. Incubation of LDL (0.05mg protein/ml) with 1.66μM cupric chloride produced a lag phase of 130 min before onset of the propagation phase where polyunsaturated fatty acids undergo conversion to conjugated lipid hydroperoxides. However, in the presence of grape extract at a final concentration equal to an 8000-fold dilution, the lag phase was extended to 185 min. A 4000-fold and 2000-fold dilution of grape extract produced lag phases of 250 and 465 min, respectively. LDL oxidation was essentially blocked for at least 10h with a 1000-fold dilution of grape extract. In other experiments, incubation of LDL (0.2mg protein/ml) with 5μM cupric chloride for 1-4h increased both thiobarbituric acid-reactive substances and electrophoretic mobility of LDL on agarose gel. In addition, there was loss of immunoreactivity of LDL with a murine monoclonal antibody against human apolipoprotein B-100. However, these oxidative changes to LDL by copper were prevented when diluted grape extract was present during incubation. It is concluded that grape extract contains antioxidants in the form of polyphenols with the capacity to inhibit oxidative modification of LDL.

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The enzyme responsible for glutathione (GSH)-independent denitration of organic nitrate esters was purified by gel chromatography, ion-exchange chromatography and affinity chromatography from rabbit hepatic cytosol. The enzyme showed a molecular mass of 175kDa and consisted of three subunits of 59kDa. The enzyme exerted its maximum activities at around pH 9, when isosorbide dinitrate (ISDN) was used as substrate. The enzyme possessed a low K_m value (10^-6M) for various organic nitrate esters. The present enzyme is likely to be involved in the denitration of organic nitrate esters in conjunction with known enzymes, GSH S-transferase (GST) and cytochrome P450.

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Diabetic KK-CA^y mice were specifically bred for high and low sensitivity to the addition of exogenous acetylcholine (ACh). The sensitivity to ACh was measured by the change in pulse rate 2 min after the administration of ACh (10mg/kg, s.c.). The two groups of mice, with high and low sensitivity to ACh, were specially selected and mated sequentially until the 12th filial generation. Although higenamine (100μg/kg, i.p.), a β₁-adrenergic agonist (a compound derived from aconite), had no effect per se, it inhibited aconitine (another compound derived from aconite extract)-induced bradycardia within 30s of administration in ACh-low sensitive mice but not in ACh-high sensitive mice. The effects of aconitine and higenamine alone did not differ between these two groups of mice. This demonstrates that the high muscarinic and high β₁-adrenergic sensitive mice may be stratified into two groups based upon an antagonistic interaction between higenamine and aconitine.

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The present study was undertaken to determine whether cardiac response to β₁-adrenergic agonists is altered in rats with chronic heart failure (CHF), and whether this alteration is related to β-adrenergic receptor down-regulation in the viable tissue of the left ventricle of these rats. For this purpose, the cardiac response to denopamine, a selective β₁-adrenergic agonist, and the change in cardiac β-adrenoceptor density were examined in rats with CHF. A non-selective β-adrenergic agonist, isoprenaline, was also examined as a comparison. Cardiac output and stroke volume indices were reduced 12 weeks after left coronary artery ligation, suggesting that CHF had developed at this time. Denopamine (2, 4 and 8μg/kg i.v.), and isoprenaline (0.01μg/kg i.v.) increased the cardiac output and stroke volume indices in sham-operated rats, whereas such increases were attenuated in the CHF rat. The cardiac β-adrenergic receptor density, measured by [³H] CGP-12177 binding assay, was reduced in homogenates and microsomal membranes in the viable tissue of the left ventricle of the CHF rat (homogenates : 29% reduction, microsomal membrane : 23% reduction). These results suggest that the cardiac responsiveness to denopamine is diminished in the CHF rat and this alteration is accounted for, in part, by a decrease in cardiac β-adrenoceptor density.

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We studied the effects of the protein synthesis inhibitor, cycloheximide (CH), on cell killing by doxorubicin (DOX) in vitro and in vivo. At the concentration of CH used (1μg/ml) the cytotoxicity of DOX was reduced in cultured P388 leukemia cells. An analysis of the DNA histogram obtained by flow cytometry showed that DOX exerts its growth-inhibitory effect by blocking the cell cycle at the G₂/M phase in P388 cells. Treatment with CH diminished this blocking effect. When CH was added to the growth medium before DOX exposure, no change in intracellular DOX accumulation was observed. Treatment with CH (15mg/kg) significantly diminished the lethality of DOX (20mg/kg) in mice and it also reduced the antitumor activity of mice with P388 leukemia. Thus, CH inhibited cell death induced by DOX in vitro and in vivo. These results suggest that CH has an antagonistic effect on the pharmacological actions of DOX in cells and mice. The cytoprotective effect of CH may be due to protein synthesis inhibition.

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The effects of catecholamines on the competence and progression phases in the proliferation of vascular smooth muscle cells (SMCs) in mouse and rat were investigated in primary cultures. α, β-Adrenergic agonists such as epinephrine and norepinephrine, and the α-adrenergic agonist, phenylephrine, stimulated proliferation of primary cultured SMCs, whereas the α₂-adrenergic agonist, clonidine, and β-adrenergic agonist, isoproterenol, did not. The stimulating effect of epinephrine was maximal at 0.54μM and was then decreased at higher concentrations. The α-adrenergic antagonist, phentolamine, and α₁-adrenergic antagonist, prazosin, inhibited epinephrine-induced SMC proliferation, while the α₂-adrenergic antagonist, yohimbine, and β-adrenergic antagonist, propranolol, did not. In primary cultured and synchronized SMCs at the G₀ phase, norepinephrine accelerated the rate of SMC proliferation, but did not change the starting time of DNA synthesis and proliferation. These results show that catecholamines activate the progression phase in primary cultured aortic SMCs α₁-adrenergic receptors.

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Carboxamide-methylated light chain (G₁L) from human serum IgG inhibited the secretion of tumor necrosis factor (TNF-α), one of the inflammatory cytokines, from adherent splenocytes and thioglycolate-induced peritoneal macrophages. The inhibition of TNF-α secretion by G₁L was associated with disappearance of tyrosine phosphorylation on about 40kDa protein when thioglycolate-induced peritoneal macrophages were stimulated with lipopolysaccharide (LPS). It is possible that this G₁L anti-inflammatory activity occurs through the blockage of the phosphorylation of about 40kDa protein.

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A water extract of licorice root inhibits granuloma angiogenesis in adjuvant-induced chronic inflammation (Phytother. Res., 5, 195, 1991). The present study has investigated the effects of licorice-derived compounds on granuloma angiogenesis. Isoliquiritin (0.31-3.1mg/kg), a licorice-derived flavonoid, inhibited the carmine content of granuloma tissue 50-fold greater than licorice extract. Glycyrrhizin (20-80mg/kg), a licorice-derived saponin, inhibited carmine content with a weak potency. The licorice extract (0.01-1mg/ml) also inhibited tube formation from vascular endothelial cells in a concentration-dependent manner. From the chemical structure-activities of used licorice-derived flavonoids (0.1-100μM), their potencies for anti-tube formation were in the order isoliquiritigenin>isoliquiritin>liquiritigenin»isoliquiritin-apioside. Glycyrrhizin (0.1-100μM) and glycyrrhetinic acid (0.1-10μM) increased tube formation. A glycyrrhizin (82μg/ml)-induced increase in tube formation was inhibited by isoliquiritin. The combined effect of a mixture of 82μg/ml glycyrrhizin and 4.2μg/ml isoliquiritin, a similar concentration ratio to their yield ratio in the licorice extract, corresponded to the effect of 100μg/ml extract. In conclusion, the anti-angiogenic effect of licorice extract depended on the anti-tube formation effect of isoliquiritin.

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Nattokinase is a new fibrinolytic enzyme which cleaves directly cross-linked fibrin in vitro. In this study, we investigated the thrombolytic effect of nattokinase on a thrombus in the common carotid artery of rat in which the endothelial cells of the vessel wall were injured by acetic acid. When a section of occluded vessel was stained for CD61 antigen by immunofluorescence utilizing a monoclonal antibody, the antigen was localized around the surface of the occluded blood vessels. This result suggests that the occlusive thrombosis was caused by platelet aggregation. In addition, thrombolysis with urokinase (UK ; 50000IU/kg, i.v.) or tissue plasminogen activator (tPA ; 13300IU/kg, i.v.) in our model was observed to restore the blood flow over a 60 min monitoring period. The results indicate that our chemically induced model is useful for screening and evaluating a thrombolytic agent. We evaluated the thrombolytic activity of nattokinase using this model and compared it with fibrino (geno) lytic enzyme, plasmin or elastase. On a molar basis, the recovery of the arterial blood flow with nattokinase, plasmin and elastase were 62.0±5.3%, 15.8±0.7% and 0%, respectively. The results indicate that the thrombolytic activity of nattokinase is stronger than that of plasmin or elastase in vivo.

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Human, rabbit and rat aortic smooth muscle cells (hSMC, rSMC and A10 cells, respectively) were cultured with cadmium chloride and compared with human Chang liver cells to characterize the response of vascular smooth muscle cells to the metal. It was revealed that all tested vascular smooth muscle cells were markedly more sensitive to cadmium cytotoxicity than Chang liver cells. Cadmium accumulated more markedly in vascular smooth muscle cells than in Chang liver cells. After exposure to cadmium, metallothionein was induced in a concentration-dependent manner in Chang liver cells, it was constitutively high in hSMC, sensitively induced in rSMC and was constitutively low and induced within narrow limits in A10 cells. The intracellular content of reduced glutathione was greater and significantly enhanced by cadmium only in A10 cells. The present data suggest that vascular smooth muscle cells are, in general, sensitive to cadmium cytotoxicity without any species-related differences, mainly due to a higher accumulation of the metal within cells.

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Ten known glycosidic compounds, betulalbuside A (1), 8-hydroxylinaloyl, 3-O-β-D-glucopyranoside (2) (monoterpen glycosides), ipolamiide (3) (iridoid glycoside), acteoside (verbascoside) (4), leucosceptoside A (5), martynoside (6), forsythoside B (7), phlinoside B (8), phlinoside C (9), and teucrioside (10) (phenylpropanoid glycosides) were isolated from methanolic extracts of Phlomis armeniaca and Scutellaria salviifolia (Labiatae). Structure elucidations were carried out using ¹H-, ¹³C-NMR and FAB-MS spectra, as well as chemical evidence. The cytotoxic and cytostatic activities of isolated compounds were investigated by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) method. Among the glycosides obtained here, caffeic acid-containing phenylpropanoid (or phenethyl alcohol, or phenylethanoid) glycosides were found to show activity against several kinds of cancer cells. However, they didn't affect the growth and viability of primary-cultured rat hepatocytes. Study of the structure-activity relationship indicated that ortho-dihydroxy aromatic systems of phenylpropanoid glycosides are necessary for their cytotoxic and cytostatic activities.

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The stereochemical characteristics of the hydrolysis of several ester-type prodrugs of propranolol, O-acetyl, O-propionyl, O-butyryl, O-pivaloyl and succinyl esters, were studied in phosphate buffer (pH 7.4), rat plasma and rat tissue homogenates. In phosphate buffer, no differences were observed in the hydrolysis rate between the esters of (R)-and (S)-propranolol. The effects of the ester moieties on the hydrolysis rate in phosphate buffer were in the following order : acetate>propionate>butyrate>succinate>pivalate. In rat plasma and tissue homogenates, the hydrolysis of the esters was accelerated, and stereoselective hydrolysis was observed. In plasma, the hydrolysis of the (R)-isomer was faster than that of the (S)-isomer except for the succinate ester. On the other hand, in the liver and intestine homogenates, the (S)-isomer was hydrolyzed faster than the (R)-isomer except for the succinate and pivalate esters in the liver homogenate. Also, the ratio of the hydrolysis rates (S/R) changed with the type of prodrug. As the length of the alkyl chain of the ester increased, the S/R ratio approached unity in liver and intestine homogenates but stayed almost constant in plasma. For the pivalate ester, stereoselective hydrolysis was observed in plasma and intestine homogenate but not in liver homogenate. The stereoselective hydrolysis of the succinate ester was observed only in intestine homogenate, but the S/R ratio was almost 1 in plasma, liver and intestine homogenates. No interconversion between (R)- and (S)-isomer was observed during the hydrolysis of any of the ester prodrugs. These results indicate that hydrolysis of ester-type prodrugs of propranolol occurs stereoselectively in rat tissues, and that its selectivity is dependent on the kind of tissue and prodrug.

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Theophylline is a mild bronchodilator and has significant extrapulmonary effects, but it may also have some anti-inflammatory properties. We investigated the immunological effects of theophylline on peripheral blood mononuclear cells (PBMC), by examining the production of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin 8 (IL-8) when PBMC were stimulated with lipopolysaccharide (LPS) or recombinant human IL-1β (rhIL-1β). At concentrations&lnE;50μg/ml, theophylline suppressed the proliferative activitiy of PBMC stimulated with phytohemagglutinin (p<0.05). IL-1β production showed 23% suppression by 10μg/ml theophylline (p<0.05), while the suppression was 26% at 25μg/ml (p<0.05), 30% at 50μg/ml (p<0.05), and 33% at 100μg/ml (p<0.05). TNF-α production was suppressed in a dose-dependent manner by theophylline, being decreased by 24% at 10μg/ml (p<0.05), by 29% at 25μg/ml (p<0.05), by 41% at 50μg/ml (p<0.01), and by 54% at 100μg/ml (p<0.01). IL-8 production, in contrast, was not affected by theophylline. rhIL-1β induced IL-8 production in a dose-dependent manner at concentrations of 1-100 units/ml, and theophylline (particularly at concentrations of 50 and 100μg/ml), increased IL-8 production in the presence of rhIL-1β. Suppression of the production of IL-1β and TNF-α by therapeutic levels of theophylline suggested that this drug might have anti-inflammatory and immunosuppressive effects.

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Two series of matrix tablets for the newly developed anti-histaminergic drug, TA-5707F, were prepared : one series with an entero-soluble polymer, hydroxypropylmethylcellulose acetyl succinate (HPMC-AS), and one series with a hydrophilic gel-forming polymer, hydroxypropylmethylcellulose (HPMC). In the JP dissolution test of HPMC-AS tablets, as the soluble-pH of HPMC-AS was increased, the dissolution of the drug in the JP XII second fluid (pH 6.8) was increasingly delayed, and the duration time of the constant plasma level in fasted dogs was increased. However, the absorption of TA-5707F in fed dogs was markedly diminished. In the JP dissolution test, the HPMC tablets showed a zero order dissolution profile, being the same in both the JP first (pH 1.2) and the JP second fluids. The H-2 tablet, containing HPMC 60SH-4000 and lactose, showed a constant plasma level in fasted dogs. However, this tablet was disintegrated by the ingestion of food. The H-3 tablet containing a lot of HPMC 60SH-4000, but not lactose, showed insufficient duration in fasted dogs, but its plasma profile was less affected by food, and the propantheline bromide pretreatment extended the constant plasma level in dogs. Thus, the H-3 tablets were considered promising for human use. Then, H-2 and H-3 tablets were administered to humans ; however, the plasma concentration profiles did not show sufficient duration time for either tablet. TA-5707F was considered to have been released slowly in human gastro-intestinal (GI) tract due to more moderate GI mobility.

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The protective effects of some neutral amino acids against hypotonic hemolysis were examined at various pHs. At pH 5.0, 7.0 and 8.0, 50% hemolysis was induced at 200, 160 and 140 mOsM, respectively, suggesting that erythrocyte membranes became more fragile to osmotic shock with decreasing pH. All amino acids tested reduced the hypotonic hemolysis at pH 5.0, but enhanced it at pH 8.0. It is therefore likely that these amino acids controlled the osmotic fragility of the cell membranes. At pH 7.0, glycine (Gly) reduced hypotonic hemolysis with increasing concentration. Phenylalanine (Phe) also reduced hypotonic hemolysis at low concentrations, but had an incrementally opposite effect at high concentrations. It was suggested that Phe interacted with erythrocyte membranes in a similar way to amphipathic drugs. Kinetic studies demonstrated that hypotonic hemolysis occurred immediately, according to osmotic shock, and that Gly and a low concentration of Phe decreased osmotic shock. Phe at a high concentration showed fast hemolysis with a short lag-time. Gly also showed fast hemolysis after the suppression of hypotonic hemolysis. Morphological observations demonstrated that these amino acids induced exvagination, exovesiculation and then invagination. It was suggested that with exvagination, the membrane expansion decreased the osmotic fragility, but the further shape change evoked membrane hole-formation.

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We constructed a nomogram for determining the optimal regimen of cyclosporine (CyA), based on physiological changes that occur during immunosuppressive therapy. The nomogram consists of a fixed model and a variable model. In the fixed model, the oral dose of CyA (D, mg/kg) is given by the multiple linear function of logarithmic CyA trough level (TL, ng/ml), the surrogate apparent total body clearance of CyA (CL/f_su, 1/h/kg, being equal to D/TL/12), and the erythrocyte-to-plasma distribution ratio of CyA (CyA-EP), as defined by : D=4.938×log (TL)+1.5037×CL/f_su-0.0326×CyA-EP-10.7156. In the variable model, the CL/f_su is given by the CyA-EP and the patient's intrinsic parameters (P₁, P₂), using a nonlinear equation : CL/f_su=P₁×exp (P₂×CyA-EP)/CyA-EP. An optimal CyA dose to maintain a desired trough level was calculated, and the validity of the nomogram was found satisfactory for clinical use. This offers a very concise and practical method for the therapeutic monitoring of CyA. Because the pharmacokinetics of CyA depends on physiological changes due to several disease states, and because the CyA-EP reflects the pharmacokinetics of CyA and the patient's disease state, the proposed nomogram is believed to provide an optimal dosage adjustment, taking physiological factors into consideration.

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β1 and β2 adrenoceptor ligand activity has been shown to be down-regulated in failing myocardium. It is the aim of this study to test the hypothesis that also mRNA levels are down-regulated in dilated cardiomyopathy. β1 and β2 adrenoceptor ligand activities and mRNA expressions were analyzed in left ventricular biopsies from six organ donor hearts, in papillary muscles from seven patients operated on for mitral regurgitation, and in six explanted hearts as the result of dilated cardiomyophathy. mRNA levels were determined by solution hybridization. β1 ligand activity was decreased in the cases of mitral regurgitation (p<0.01) and dilated cardiomyopathy (p<0.001). β2 ligand activity did not differ between the three groups. mRNA expression was depressed in mitral regurgitation regarding both β1 (p<0.001) and β2 (p<0.01), while no differences were observed in dilated cardiomyopathy as compared to the donor hearts. The regulation of β1 and β2 adrenoceptor ligand activity and mRNA expression appears to follow a specific pattern in dilated cardiomyopathy. The specific down-regulation of β1 ligand activity seems to occur at a posttranslational level.

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We have previously reported the inhibitory effects of diethylstilbestrol (1) and optically active indenestrol derivatives on microtubule polymerization in vitro and their disruptive effect on cytoplasmic microtubules and cytotoxicity in cultured Chinese hamster V79 cells. In the present study, the cytotoxicities of (±)-diethylstilbestrol oxide (2), (+)-, (-)-and (±)-monomethyl ethers (4) of 2, (±)-dimethyl ether (5) of 2, diethylstilbestrol pinacolone (3), E, E-dienestrol (6), Z, Z-dienestrol (7), meso-hexestrol (8), a mixture of (1R, 1'S) 4-hydroxyhexestrol and (1R, 1'S) 4'-hydroxyhexestrol (9), and the 4-hydroxy derivative (10) of diethylstilbestrol dimethyl ether were investigated in Chinese hamster V79 cells. The results indicated that the cytotoxic activity of 10 was the strongest of the compounds tested, although its activity was the almost same as that of 1. Moreover, as the activity of (-)-4 was greater than those of 2 and 1 monomethyl ethers, the effect of 4 on cytotoxic activities was elucidated. In conclusion, the present results indicate that the cytotoxic activities of hydroxylated metabolites are greater than those of each mother compound, although epoxidation of 1 leads to a product which can be broken down more readily than the parent compound.

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The effects of Panax ginseng ethanol extract and its water (WSF)-and lipid-soluble (LSF) fractions on the scopolamine-induced disruption of radial maze performance in rats were examined. Ginseng root was refluxed with ethanol, and WSF and LSF were prepared from this ethanol extract. Scopolamine (0.075-0.3mg/kg, i.p.) dose-dependently impaired the maze performance. However, the oral administration of Panax ginseng ethanol extract and WSF (2-8g dried root/kg) 90min before testing improved the maze performance disrupted by scopolamine (0.3mg/kg) in a dose-dependent manner, but LSF failed to attenuate the disruption. These data suggest that ginseng extract possesses a beneficial effect regarding spatial cognitive impairment and that the water-soluble fraction of ginseng extract mainly contributes to the effect of the ethanol extract.

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The effects of gomisin A, a lignan component of Schizandra fruits, on the promotion stage of hepatocarcinogenesis initiated by 3'-methyl-4-dimethylamino-azobenzene (3'-MeDAB) in male Donryu rats were investigated. When different types of tumor promotors, phenobarbital (PB) and deoxycholic acid (DCA), were administered for 5 weeks after initiation by 3'-MeDAB, preneoplastic alterations in the liver, determined by glutathione S-transferase placental form (GST-P), were markedly increased. Gomisin A significantly inhibited the increase in number and size of GST-P positive foci, regardless of the promotor. This lignan inhibited the increase in serum bile acid concentration by administration of DCA, but hardly influenced the serum bile acids in the PB-combined group. These results suggest that the inhibitory effect of gomisin A on the promotive action of DCA is based on improving bile acid metabolism, but regarding the action of PB, the effect could not be elucidated from the metabolism of bile acids.

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We investigated the optimal conditions for the application of the MTT assay to determine the viability of cell in primary cultures of rat hepatocytes. The optimal conditions were found to be : inoculation at cell density of 1.31×10⁵ cells/cm² ; concentration of 3-(4, 5-dimethylthiazoyl-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) 0.12mM ; and MTT incorporation time, 2 h or more. We also found that insulin and glucocorticoid, which are usually added to the medium to maintain cell function, had no effect on the viability of the primary cultures of rat hepatocytes.

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This paper describes the glycosidases in normal lenses and the variation in their activities during aging and with the advance of cataract. In normal human lenses, four glycosidases, α-L-fucosidase, α-D-glucosidase, β-D-glucuronidase and β-D-galactosaminidase, exhibited strong specific activities, which fell sharply between the ages of 40 and 60. In the 50-60 age group the enzyme activities fell sharply to one-tenth and below those in the lenses of the 20-30 age group. The K_m value of each enzyme also depended on age and exhibited some variability, suggesting that the substrate affinity of enzyme may be changeable. These facts show that the specific activities and K_m values of some glycosidases in normal human lenses change with aging. The activities of the four glycosidases in lenses with senile cataract were also determined as a function of advanced cataract stage. The activities of the four enzymes were weak in the lenses of the younger age groups, and were undetectable in the lenses of older groups, over 60-year of age. This shows that cataract formation may have a deleterious effect on the catalytic activity of some glycosidases and inhibit them strongly.

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1-Acetamino-3-(1-naphthyloxy)-2-propanol (AcNDP) detected in human urine was formed as a metabolite of propranolol (PL) via 1-amino-3-(1-naphthyloxy)-2-propanol (N-desisopropylpropranolol, NDP). The excreted amount of AcNDP was determined by GC-MS using an isotope dilution method. More than 40% of total AcNDP in 24h urine was detected 10h after the oral administration of PL to two volunteers, and the total amounts during 24h urine were at least 1.9-3.9% of the PL dose. As AcNDP is an intermediate metabolite of PL, its urinary amount cannot be determined exactly. Incidentally, AcNDP was chemically stable and was not formed from NDP when acetyl CoA was added to the inactivated hepatocyte system. Thus, the acetylation of NDP, an aliphatic primary amine, was confirmed to be catalyzed by N-acetyltransferase, and interestingly, the acetyl conjugation was inhibited not by sulfamethazine but by p-amino benzoic acid.

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The hybrid liposomes (90mol% DMPC/10mol% C₁₂ (EO)₈ and 90mol% DMPC/10mol% C₁₂ (EO)₁₂) have a highly inhibitory action against the growth of tumor cells. The uniform and stable structure of the hybrid liposomes was revealed on the basis of electron microscopy and dynamic light scattering measurements.

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A cDNA encoding human lanosterol synthase, the enzyme responsible for the backbone formation step in sterol biosynthesis, was cloned by extensive application of PCRs. Five degenerate oligonucleotide primers (139S, 440S, 528A, 575A and 712A) corresponding to the homologous amino acid sequences among the known 2, 3-oxidosqualene cyclase (OSC) were designed. PCR with one pair (440S and 528A) of five primers yielded a 285-bp fragment. PCRs with the primers based on the obtained fragment and the degenerate primers (139S and 712A) gave longer fragments. Finally, full nucleotide sequence of cDNA was obtained by a "rapid amplification of cDNA ends"(RACE) method.

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