Hyaluronic acid (HA) synthesized by normal human epidermal keratinocytes cultured in a serum-free medium was monitored by a highly sensitive HPLC method, which was established by us for the simultaneous determination of HA, chondroitin sulfate (ChS) and dermatan sulfate (DS) as their unsaturated disaccharides. The major glycosaminoglycan (GAG) in the medium of the keratinocytes was HA, and the ability of the cells to synthesize HA increased relatively with an increase in cell numbers during the logarithmic phase and reached a maximum level after the cells became confluent. HA synthesis by the keratinocytes was inhibited by the addition of calcium chloride to the culture medium, and was strongly stimulated by the addition of retinoic acid (RA), respectively. It was shown that the ability of the cells to synthesize HA exists in the spinous cell stage. Furthermore, we found that HA synthesis by the cells was slightly increased by the addition of dibutyryl cyclic AMP (dbcAMP). Our results indicate that the measurement of time-course levels of HA in the culture medium is useful for the screening of active substances for proliferation and differentiation of the keratinocytes.

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We developed a novel method for measuring glycated (glc) proteins in biological samples, based on the colorimetry of 2-keto-glucose which is released from the glc protein (ketoamine) on heating with hydrazine. The ketoamine-induced coloration remained constant at room temperature (25-27°C) for 1 h. The method gave reliable precision and accuracy. However, high concentrations of serum pigments caused positive interference, suggesting that hemolytic or hyperbilirubinemic serum would give false-positive results. The concentration of glc protein in clinical serum samples measured by the present method (y) correlated well with those (fructosamine values, x) measured by the nitroblue tetrazolium-reducing method : y=1.27x-1.69 (r=0.92, n=93). The concentrations (μM, mean±S.D.) of glc protein in sera from normal and diabetic subjects were 275±37 (n=32) and 403±98 (n=32), respectively, and the concentrations (nmol/mg hair, mean±S.D.) of glc protein in back hairs from non-diabetic and diabetic rats were 3.7±0.3 (n=10) and 8.6±1.5 (n=10), respectively. Thus, the technique gave reasonable concentrations of glc proteins in humans and rats with diabetes mellitus, indicating it to be reliable and diagnostically useful.

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The effects of nerve growth factor (NGF) on PC12h cells have been studied with special reference to the relationship between neurite outgrowth and the activities of the enzymes involved in signal transduction in the early stage of NGF action. Pretreatment of cells with selective inhibitors of tyrosine kinase (ST638 and genistein) and phospholipases C (neomycin B) and A₂ (p-Bromophenacyl bromide ; BPB and indomethacin) depress neurite outgrowth following 60 min treatment with NGF (0.1 μg/ml). The inhibitor of intracellular calcium mobilization, TMB-8, also significantly depresses the NGF-induced neurite outgrowth. Arachidonic acid release, induced by NGF, from the cell lipids is reduced following previous inhibition of above mentioned enzymes. Based on these results, it is suggested that NGF requires tyrosine kinase in its early stage of action (within 60 min) in PC12h cells, phospholipases C and A₂ to release Ca²⁺ from intracellular stores and arachidonate from cellular phospholipids, in order to induce neurite outgrowth.

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The degradation of ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) was examined on cultured porcine aortic endothelial cell (EC) and rat vascular smooth muscle cell (SMC) by HPLC analysis. The degradation of ET-1 was observed on SMC and was slightly observed on EC. Membrane fractions of SMC had a strong potency for the degradation of ET-1 and retained activity even in plasma. This activity was inhibited with some of the noble inhibitors which were known as enkephalinase inhibitors. These results suggest that endothelin degradation enzyme on SMC is related to enkephalinase and that this enzyme plays a significant role in the degradation of ET-1 in vivo.

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In order to identify the membrane-bound peptidase that is responsible for the degradation of endothelin (ET), an endothelin-1 (ET-1) degradation enzyme was solubilized from membrane fractions of porcine kidney with 1% Triton X-100, and subsequently purified by column chromatographies, i. e., diethylamino-Sepharose ion exchange, gel permeation, Con A Sepharose and hydroxyapatite chromatography. On DEAE-Toyopearl ion exchange column chromatography, the ET degradation enzyme and aminopeptidase were separated, but ET degradation enkephalinase activities were not separable. In order to separate ET degradation enzyme and enkephalinase, the active fractions were loaded on each of the column chromatographies : sephacryl S-200, Con A Sepharose or hydroxyapatite. The ET degradation activities were co-migrated with enkephalinase activities on all of the three chromatographies. In addition, the ET degradation activities were inhibited by thiorphan, phosphoramidon and EDTA, which are known to inhibit enkephalinase. These results suggest that ET degradation activity in the membrane fractions of the kidney is related to enkephalinase and may be involved in the degradation of ET-1 in vivo.

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The human thromboxane A₂ receptor (TXA₂-R)-coding gene was introduced into Chinese hamster ovary cells and a cell line (TCHO-25) stably expressing TXA₂-R, at a level of 3×10⁵/cell, was obtained. An anti-asthmatic agent AA-2414 [(±)-7-(3, 5, 6-trimethyl-1, 4-benzoquinon-2-yl)-7-phenylheptanoic acid] competitively inhibited the specific binding of a TXA₂ mimic ([³H] U-46619) to the TCHO-25 cells, with an IC₅₀ of 6.0×10^-8M, indicating that the drug is an antagonist of human TXA₂-R. The TCHO-25 cells offer a tool for the screening and characterization of human TXA₂-R antagonists.

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The fruits of Prunus mume SIEB. et ZACC. (Japanese name, ume) have been used as a traditional drug and health food. In order to study the active components of P. mume, the polysaccharide fractions were extracted with cold water, hot water and aqueous sodium hydroxide from the kernels of P. mume. We found that some of the polysaccharide fractions exhibited various types of biological activities such as mitogenesis, activation of the alternative pathway of complement and activation of clot formation in human plasma. A polysaccharide, P-1, obtained from the cold 0.5 M NaOH extract was purified by ion-exchange chromatography and gel-filtration. P-1 contained 62.0% neutral sugar as glucose and 38.4% uronic acid (as galacturonic acid), and was free from protein. The neutral sugars of P-1 were arabinose, xylose, rhamnose and galactose in a molar ratio of 9.4 : 3.4 : 1.1 : 1.0, following analysis by gas-liquid chromatography. In addition, galacturonic acid was identified by thin-layer chromatography. The molecular weight of P-1 was found to be more than 2000000 by gel-filtration on Toyopearl HW 65F. P-1 showed mitogenic activity towards spleen cells of both C3H/HeN and C3H/HeJ, suggesting that it was free from bacterial endotoxic lipopolysaccharides.

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The effect of fatty acids on 8-hydroxydeoxyguanosine (8-OH-dG) formation in calf thymus DNA treated with bleomycin (BLM)-Fe (II) in vitro was studied. The formation of 8-OH-dG was greatly reduced in the presence of stearic acid (18 : 0) or oleic acid (18 : 1), however, it increased to the control level with an increase in the number of unsaturated bonds of fatty acids (18 : 2, 18 : 3). This increase in 8-OH-dG formation may have resulted from the indirect oxidation of DNA by the peroxidized unsaturated fatty acids formed by BLM-Fe (II). The inhibitory effect of saturated fatty acids on 8-OH-dG formation was observed with those possessing more than 12 methylene chains. Stearic acid also inhibited peroxidization of unsaturated fatty acids (18 : 2, 18 : 3) treated with BLM-Fe (II), however, it had no effect on ribose degradation of DNA treated with BLM-Fe (II). These results suggest that different active oxygen species are probably involved in DNA degradation and in formation of 8-OH-dG in DNA and of peroxidized products in fatty acids.

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The lithium ion (Li⁺) shows toxicity against Escherichia coli cells when present in a high concentration in the environment. Since Li⁺ is extruded from cells via a Na⁺ (Li⁺)/H⁺ antiporter, this antiporter must be involved in the detoxification of Li⁺. Two Na⁺ (Li⁺)/H⁺ antiporters (NhaA system and NhaB system) are known to be present in E. coli. We investigated the properties of the antiporters and the participation of these systems in the detoxification of Li⁺ using mutants lacking one of the antiporters, or lacking both of them. Although the affinity for Li⁺ of the two systems was almost the same, the V_max value for Li⁺ transport of the NhaA system was about 12 times larger than that of the NhaB system. Wild type cells were unable to grow in the presence of 0.7 M LiCl. Although a wild type cell and a mutant lacking the NhaB system grew in the presence of 0.6M LiCl, a mutant lacking the NhaA system did not. This second mutant grew in the presence of 0.1 to 0.2 M LiCl. A mutant lacking both the NhaA and NhaB systems could not grow in the presence of 30 mM LiCl.

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The effects of platelet-derived growth factor (PDGF) on the start and rate of DNA synthesis were investigated with or without (R)-trichostatin-A (TS-A) in primary cultured and synchronized smooth muscle cells (SMC) of rat aorta. After the SMC at the G₀ phase were precultured with 10 and 100 ng/ml PDGF for 3 h and washed out, they were then stimulated with 3% fetal bovine serum (FBS). FBS-stimulated DNA synthesis was determined every 3 h for 24-30 h. The SMC pre-cultured with PDGF started DNA synthesis at an earlier time, dependent on the concentrations, suggesting an acceleration of competence. After the SMC were precultured with PDGF (30 ng/ml) plus TS-A (0.1, 0.3, 1.0 and 3.0 μg/ml) for 3 h and washed out, the SMC were then stimulated by 1% FBS. TS-A delayed the starting time of DNA synthesis in a concentration-dependent manner. During the prolonged culture with PDGF (1, 3, 10 and 100 ng/ml) and FBS (3%), the rate of DNA synthesis was more rapid than in cells pretreated with PDGF alone, suggesting an acceleration of progression. These results suggest that 1) PDGF stimulates DNA synthesis dually during the competence and progression phases, and 2) PDGF-induced competence is inhibited by TS-A in primary cultured SMC.

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The present study reveals that piperine, a pungent alkaloid present in various Piper species, is cytotoxic to cultured brain neurons. Exposure to piperine (12.5-100 μM) for 72 h caused a concentration-dependent reduction in the survival of primary cultured neurons from various regions of the embryonic rat brain under high density cell culture conditions. There were relative regional differences in the susceptibility to cytotoxic effects of piperine in which septum and hippocampus showed higher vulnerability among the eight regions. The primary cultures of septal and hippocampal neurons under low density cell culture condition were performed to evaluate the contribution of non-neuronal cells. The concentration-response profiles in both high and low density cell culture conditions were comparable (septum : EC₅₀=43 and 27μM, hippocampus : EC₅₀=50 and 44μM, under high and low density cell culture conditions, respectively) suggesting a minor role of non-neuronal cells on cytotoxicity of piperine to developing neurons.

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An animal model having both hypertension and reduced renal function was produced by intraperitoneal injection of puromycin aminonucleoside (PAN) in spontaneously hypertensive rat (SHR). Using this model, two different dihydropyridine Ca blockers, CS-905 and nicardipine, were compared with regard to the relationship between hypotensive effects and changes in renal function in a conscious state. A single oral administration of CS-905 or nicardipine at doses of 3 or 10 mg/kg produced a dose-dependent decrease in blood pressure and an increase in heart rate. Glomerular filtration rate (GFR) was decreased only at 10 mg/kg. However, there was a substantial difference between the two drugs with respect to the relationship between blood pressure and GFR. The decrease of GFR by nicardipine was observed when blood pressure was at the lowest level, while GFR decreased by CS-905 returned to the initial level when blood pressure reached a nadir. Percent decrease of GFR by CS-905 was significantly less than that by nicardipine although both agents produced almost the same degree of peak hypotension. These results suggest the decrease in GFR by Ca blockers depends not only on the degree of hypotension but other factors as well, such as the rate of blood pressure lowering. Despite the hypotension, both agents produced a marked natriuresis. Since the natriuresis was not accompanied by an increase in GFR, it was assumed that the natriuretic effect of Ca blockers stemmed from their tubular effects rather than glomerular ones.

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The effect of carboxamidemethylated Fc fragment (CM-Fc) from human serum immunoglobulin G on mixed lymphocyte reaction (MLR) was studied. CM-Fc enhanced the MLR response when added at the start of cultivation, but suppressed the response when added 48 h after onset of cultivation. The Fc fragment had no effect. CM-Fc elevated the levels of interleukin-1 and tumor necrosis factor when added at the start of splenocyte cultivation, and somewhat did so when added 48 h after onset of cultivation. These elevations caused the induction of prostaglandin E₂ in MLR culture supernatant. The suppression of MLR by CM-Fc was abrogated by the addition of indomethacin. We considered that CM-Fc regulated the immune response being affected to the function of macrophages.

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We examined the inhibitory effect of somatostatin on pepsinogen secretion using isolated rat gastric chief cells. Secretin and forskolin significantly increased not only pepsinogen secretion from chief cells but also cellular cAMP accumulation in a dose-dependent fashion. Somatostatin significantly inhibited secretin- and forskolin-induced pepsinogen secretion and secretin-induced cellular cAMP accumulation. However, forskolin-induced cellular cAMP accumulation was not inhibited by somatostatin. The inhibitory effect of somatostatin on secretin-induced pepsinogen secretion was abolished by pretreatment with pertussis toxin, but inhibition of forskolin-, carbachol- and cholecystokinin octapeptide-induced pepsinogen secretion was not. These results suggest that somatostatin inhibits pepsinogen secretion in two ways, one is closely related to the pertussis toxin-sensitive G-protein and the other is not determined.

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The present study was undertaken to examine whether the oxidative effect of organic hydroperoxide on cultured rat hepatocytes is enhanced by eicosapentaenoic acid (EPA). The cells were pretreated with 0.8 mM EPA which was complexed to bovine serum albumin (EPA-BSA) for 4 h, and then challenged to cumene hydroperoxide (CMHP). By the addition of CMHP to the cell culture, lipid peroxidation estimated as production of malondialdehyde (MDA) was provoked to a much greater extent in the EPA-loaded hepatocytes than in the non-loaded cells. The CMHP treatment also resulted in more severe cell injury in association with the decrease in intracellular levels of glutathione and protein thiols in the EPA-loaded cells as compared with results in the non-loaded cells. The addition of antioxidants such as N, N'-diphenyl-p-phenylenediamine (DPPD), promethazine and γ-tocopherol to the culture medium prevented the CMHP-induced oxidative injury effectively for the EPA-loaded cells but had little effect for the non-loaded cells. The potency of other unsaturated fatty acids with different numbers of double bonds in enhancing the CMHP-induced lipid peroxidation and injury was also examined. The deleterious effects of CMHP were little affected by unsaturated fatty acids with one or two double bonds but were markedly intensified by those with more than 4 double bonds. These data showed that the supplementation with highly unsaturated fatty acids makes the cells susceptible to the drug-induced oxidative stress.

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The number of alkalotolerant intestinal bacteria was 1% of the total flora in humans and 0.8% of those in rats. The β-glucosidase and β-glucuronidase activity of these intestinal bacteria was induced by elevating the pH of the medium, but the growth was not changed. The enzyme activity in a medium of pH 7 was 5- to 10-fold higher than that in a medium of pH 6. Isolated bacteria from human and rat feces were cultured in a pH 5 general anaerobic medium (GAM) broth to reach a stationary phase, then the pH of the media was changed from 5 to 8. Both β-glucosidase and β-glucuronidase were increased 9.2-12.1-fold. The activity of these enzymes was also increased 2-16-fold by adding substrates (p-nitrophenyl-β-D-glucuropyranoside or p-nitrophenyl-β-D-glucuronide). β-Glucuronidase (s) was inhibited by saccharic acid 1, 4-lactone or D-glucuronic acid. However, when lactulose was added to the medium, and then intestinal microflora were inoculated in the medium, the productivity of these enzymes dramatically decreased. We thus contend that the induction of the β-glucosidase and β-glucuronidase of intestinal bacteria by a high pH can cause colorectal cancer.

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Coated fine granules with water-insoluble film composed primarily of ethylcellulose, containing 20% of sparfloxacin (SPFX) and various amounts of low-substituted hydroxypropylcellulose (L-HPC) (0-52%) in the cores and which masked the bitter taste of SPFX, were orally administered to fasting rats to determine the effect of L-HPC on bioavailability. The release of SPFX in water from four kinds of these coated fine granules containing 0, 25, 40 and 52% of L-HPC showed the pseudo first order kinetics, followed by the second phase, with refractive points between 0.25 and 0.5 h. The rate constant (K₁) up to 0.25 h increased with an increase in of the amount of L-HPC in the core, and the rate constant (K₂) in subsequent release (the second phase) was lower than K₁ in each fine granule. Areas under plasma concentration-time curves (AUC) of SPFX and the peak plasma SPFX levels (C_max) after oral administration of coated fine granules lacking L-HPC to fasting rats were suppressed to one-eighth and one-ninth, respectively, of those obtained from the core granules that rapidly released SPFX. However, AUC and C_max from the coated fine granules increased linearly with an increase in the amount of L-HPC in the cores, and nearly equaled those from the core fine granules when the content of L-HPC was 52%. These results confirmed that the addition of L-HPC to the cores increases not only the dissolution rate but also the bioavailability of SPFX.

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Phenytoin sodium was microencapsulated with ethylcellulose (EC) by a coacervation-phase separation method from ethyl acetate solution to develop a prolonged release dosage form of phenytoin. Release of phenytoin from the microcapsules (phenytoin sodium/EC) was evaluated by the JP dissolution test in JP disintegration media No. 1 and No. 2. The release rates of phenytoin from phenytoin sodium powders were extremely rapid in both media, however, the release rates from the microcapsules were much more retarded. Following the oral administration of microcapsules to rabbits, prolonged plasma concentrations of phenytoin were obtained, while microcapsules orally administered to human subjects showed prolonged urinary excretion of phenytoin metabolites.

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1, 4-Piperazinediethanesulfonic acid (PIPES), a zwitterion buffer, was titrated with KOH to measure its pH and osmolality. Both pH and osmolality changes showed three phases, and two transition points coincided with pH 5.1 and 10.2. The second phase is important in biological reactions, and from the present results, the pK₁ is 3.3 while pK₂ is 6.85 at 20°C. The slope of the osmolality curve was 2 mOsm per 1 mM KOH addition in the first and third phases, while it was 1 mOsm per 1 mM KOH in the second phase. The first phase indicates that ^±PIPES^± changes to ^-PIPES^± by the addition of KOH. In the second phase, additional KOH produces ^-PIPES^-. PIPES acts as a divalent ion, at least, in the physiological pH range of around 7.0. Concentrations of ^-PIPES^± and ^-PIPES^- around the physiological pH range are calculated as follows. [chemical formula], [chemical formula] The ionic strength of PIPES is calculated as follows. [chemical formula]

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A sensitive and specific double-antibody enzyme-linked immunoassay (EIA) for detecting a motilin-like immunoreactive substance (M-IS) in human plasma has been developed. In competitive reactions, the motilin antibody was incubated with a plasma sample (or motilin standard) and β-D-galactosidase-linked synthetic motilin. Free and antibody-bound enzymes were separated using an anti-rabbit IgG-coated immunoplate. Enzyme activity on the plate was determined by fluorophotometric analysis. This immunoassay allows the detection of 20 to 200 fmol/ml (54 to 540 pg/ml) of motilin. The mean level of M-IS detected in human plasma was 53.9±25.6 pg/ml.

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Silymarin, a commercial crude drug used as a hepatoprotective, was found to inhibit 53% of β-glucuronidase activity at a final concentration of 0.8 mg/ml. Of three compounds A, silybin and C, which were isolated from silymarin, A and silybin potently inhibited the enzyme activity, followed by C. β-Glucuronidases of intestinal bacteria, HGU-1 and HGU-2, and E. coli HB101 were noncompetitively inhibited by silybin. β-Glucuronidase of the feces of a healthy human and of a human with colon cancer were also inhibited by silybin, silymarin and saccharic acid 1, 4-lactone at 0.03-0.15 mg/ml. Silymarin and silybin protected the increase in enzyme activity in the serum of the rats treated with CCl₄.

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Inhibitory effects of various phenolic esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, amidinopiperidine-4-alkanoic acids or trans-4-amidinocyclohexane-4-alkanoic acids on trypsin, thrombin, plasmin and pancreatic kallikrein were examined. Their inhibitory effects were strongly affected by the acid portion and phenolic group constituting the esters. The effects of the acidic portion and phenolic group on the inhibitory effect varied with each protease ; they were most effective on thrombin and plasmin and least effective on kallikrein. The inhibitory effect of these esters on trypsin was affected mainly by acid portion.

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Previous studies have showed that halothane has depressed ventricular activation in a canine myocardial infarction model. It is also well known that class I antiarryhthmic drugs depress ventricular activation in the infarcted myocardium. In the present study, we examined whether some electrophysiologic interactions between halothane and two class I antiarrhythmic drugs, lidocaine and procainamide, occur in a canine myocardial infarction model. Halothane, lidocaine and procainamide prolonged the activation time in the infarcted zone, and the combination of halothane and either lidocaine or procainamide markedly prolonged the activation time or blocked the delayed activation in the infarcted zone. Although the mechanism of the interaction is not clear, the present results suggest a synergistic interaction between halothane and class I antiarrhythmic drugs. Therefore, care should be taken that doses of class I antiarrhythmic drugs during halothane anesthesia will not be an overdose.

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We investigated the effect of cadmium on the synthesis of glycosaminoglycans (GAGs) in confluent cultures of vascular endothelial cells derived from bovine aorta. Cadmium markedly increased the incorporation of [³H] glucosamine into GAGs in both the cell layer and the conditioned medium but decreased [³⁵S] sulfate in the cell layer. This suggested that cadmium induced the GAG synthesis by endothelial cells accompanied by a reduction of the sulfation of cell-associated GAGs. Of the tested heavy metals, the [³⁵S] sulfate incorporation in the cell layer was significantly decreased by lead ; zinc slightly but significantly increased the [³⁵S] sulfate incorporation ; manganese and copper failed to change the [³H] glucosamine and [³⁵S] sulfate incorporation ; and the incorporation of [³H] glucosamine was increased only by cadmium. On the other hand, vascular smooth-muscle cells and fibroblastic Balb/3T3 cells responded to cadmium in a way similar to vascular endothelial cells, while fibroblastic IMR-90 cells, Chang liver cells and epithelial LLC-PK1 cells altered the [³H] glucosamine and [³⁵S] sulfate incorporation after exposure to cadmium in different manners. The alteration of GAGs induced by cadmium may be involved in the pathogenesis of the metal.

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The inhibitory effect of Corydalis tuber (Corydalis turtschaninovii BESSER forma yanhusuo Y. H. CHOU et C. C. HSU) was tested on crude rat lens aldose reductase, an enzyme involved in the complications of diabetes. The methanolic extract (CM-ext) inhibited aldose reductase, while the aqueous extract (CA-ext) was ineffective. Only dehydrocorydaline, of the seven alkaloidal components isolated from CM-ext, inhibited aldose reductase. It is suggested that the inhibitory effect of CM-ext on aldose reductase may be partially attributed to dehydrocorydaline.

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Karounidiol [D : C-Friedo-oleana-7, 9 (11)-diene-3α, 29-diol] and 7-oxodihydrokarounidiol [7-oxo-D : C-friedo-olean-8-ene-3α, 29-diol], isolated from the seeds of Trichosanthes kirilowii, were examined for the effect on 12-O-tetradecanoylphorbol-13-acetate (TPA, 1μg/ear)-induced inflammation, following application of this tumorpromoting agent, to the ears of mice. Both compounds inhibited the inflammatory activity induced by TPA and the 50% inhibitory dose for TPA-induced inflammation was 0.3 and 0.4 mg/ear, respectively. Furthermore, at 2 μmol/mouse, karounidiol markedly suppressed the promoting effect of TPA (1 μg/mouse) on skin tumor formation in mice following initiation with 7, 12-dimethylbenz [a] anthracene (50 μg/mouse).

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