Calprotectin, a complex of two calcium-binding proteins that belong to the S100 protein family, is abundant in the cytosolic fraction of neutrophils. A high level of calprotectin reportedly exists in extracellular fluid during various inflammatory conditions, such as rheumatoid arthritis, cystic fibrosis and abscesses. However, the exact biological role(s) of the factor is now under investigation. We recently observed that neutrophils contain a factor that shows growth-inhibitory and apoptosis-inducing activities against various cell types including tumor cells and normal fibroblasts, and we identified that factor as calprotectin. The findings suggest that calprotectin exerts a regulatory activity in inflammatory processes through its effect on the survival or growth states of cells participating in the inflammatory reaction. It is also possible that calprotectin, at a high concentration, might have a deleterious effect on fibroblasts and influence the recovery of inflammatory tissue. Therefore, the protein factor may be a new drug target to control inflammatory reactions. We found that a few of the Amaryllidaceae alkaloids effectively inhibited the growth-inhibitory and apoptosis-inducing activities of calprotectin. In this article, we focus on the biological functions of calprotectin in extracellular fluids, focusing on its apoptosis-inducing activity.

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This paper reports a sensitive and specific enzyme-linked immunosorbent assay for determination of the antiarrhythmic drug mexiletine in human serum. Anti-mexiletine antibody was obtained by immunizing rabbits with an antigen conjugated with mercaptosuccinyl bovine serum albumin using N-(ε-maleimidocaproyloxy)succinimide as a heterobifunctional coupling agent. Enzyme labeling of mexiletine with β-D-galactosidase was performed using glutaraldehyde. In this assay, the mexiletine to be quantified is chemically modified by acetic anhydride allowed to compete with a mexiletine-β-D-galactosidase conjugate for binding to a limited amount of an anti-mexiletine antibody which was used to coat the wells of a microtiter plate. Mexiletine concentrations lower than 80 ng/ml were measurable reproducibly by the enzyme-linked immunosorbent assay. This assay was specific for mexiletine and showed very slight cross-reactivity with its major metabolite, 2-hydroxymethylmexiletine (1.5%), but none with p-hydroxymexiletine. The values of serum mexiletine levels from 15 patients by this enzyme-linked immunosorbent assay were comparable with those measured by HPLC. There was a good correlation between the values determined by the two methods. The enzyme-linked immunosorbent assay should be a valuable tool in therapeutic drug monitoring and pharmacokinetic studies of mexiletine.

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Hypocholesterolemic activity of dietary polyunsaturated fatty acids is observed after relatively short-term but not long-term feedings, and their long-term feedings are suspected to accelerate aging through tissue accumulation of lipid peroxides and age pigments (lipofuscin). To define the long-term effects of fats and oils in more detail, female mice were fed a conventional basal diet supplemented with lard (Lar), high-linoleic (n-6) safflower oil (Saf), rapeseed oil (Rap), high-α-linolenic (n-3) perilla oil (Per), or a mixture of ethyl docosahexaenoate and soybean oil (DHA/Soy) from 17 weeks to 71 weeks of age. The DHA/Soy and Per groups had decreased serum cholesterol levels compared with the Lar and Saf groups, but the difference between the Lar and Saf groups was not significant. The 3-hydroxy-3-methyglutary-CoA (HMG-CoA) reductase activity in the liver was also significantly lower in the Per and DHA/Soy groups. However, no significant difference in lipofuscin contents in the brain and liver was observed among the 5 dietary groups, despite significant differences in peroxidizability indices of the dietary and/or tissue lipids. These results indicate that n-3 fatty acid-rich oils are hypocholesterolemic by suppressing hepatic HMG-CoA reductase activity compared with animal fats and high-linoleic (n-6) oil, but tissue lipofuscin contents are not affected by a long-term feeding of fats and oils with different degree of unsaturation in mice.

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The interaction of a protein-bound polysaccharide (PSK) isolated from Basidiomycetes with smooth muscle myosin components was evaluated by limited digestion, urea/glycerol gel electrophoresis, affinity chromatography and overlay assay using a peptide array. PSK was bound to the regulatory light chain (RLC) of myosin, but not to the essential light chain. The binding to PSK was definitely observed for unphosphorylated RLC, compared to phosphorylated one. From the amino acid sequence of the RLC, 490 peptides were synthesized on a cellulose membrane. Overlay assays showed that the PSK-binding on the molecule of RLC were localized in the N- and C-terminal basic regions and these sites were conserved in RLC from the human smooth muscle and nonmuscle cells.

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This report presents a demonstration of ceramidase activity in the nuclear membrane or envelope of mammalian livers. The products of ceramidase reaction were identified by means of TLC for released fatty acid and HPLC for sphingosine. The ceramidase activity was maximum over a broad neutral to alkaline region ranging from pH 7.0 to 8.8. This activity was inhibited by N-oleoylethanolamine known as a specific inhibitor for ceramidase and by anandamide to a similar extent. The enzymatic study suggests that the nuclear ceramidase has different properties from other ceramidase reported previously. As sphingomyelinase, one of enzymes involved in the sphingomyelin cycle, are known to be present in the nuclear membrane, it is now evident that at least two enzymes involved in the sphingomyelin cycle are present in the nuclear membrane.

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Genistein, a soybean-derived isoflavone, has been shown to suppress osteoclastic bone resorption. To clarify the mechanisms underlying this action, we investigated the effects of genistein on the differentiation, cytoskeleton and function in mice osteoclasts in vitro and bone metabolism in ovariectomized rats. Study design: Primary OCs were isolated from 3 week-old mice and induced by 1,25(OH)₂D₃. Then OCs were exposed to genistein at various concentration of 0 M, 10⁻⁹ M, 10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M, and 10⁻⁵ M. The number of TRAP+ cells were counted as well as the surface area of bone resorption on bone slice. F-actin change was observed by Confocal. In vivo, forty 12 week-old female SD rats were randomly assigned to four groups: (1) sham operated (Sham); (2) (OVX); (3) ovariectomized and treated with estradiol (OVX-E); (4) ovariectomized and received genistein (OVX-G). After 12 weeks, BMD, body weight, serum level of alkaline phosphatase (ALP), acid phosphatase (ACP), osteocalcin (OC), IL-1β, TNFα, IL-6 and calcitonin (CT) were evaluated. Femur were sectioned. In addition, the serum estradiol, the weight of uteri and histological behavior were also examined to indicate the side effect of genistein to the uteri. Results: In vitro, the number of TRAP+ cells decreased depending on the concentration of genistein as well as the area of bone resorption. F-actin became disorder under Confocal. In vivo, after treated with genistein, BMD and the serum level of ALP, ACP, osteocalcin increased significantly, while the serum level of IL-1β and TNFα decreased. Especially, the increase of ALP and osteocalcin of OVX-G was higher than that of OVX-E. Histologically, the pachy-trabecula were observed as well as the more mineral deposition lines. Additionally, the uterus weight index and the serum estradiol in OVX-G rats were lower significantly than those of OVX-E. The epithelia of uteri gland in OVX-G appeared cubic while those of OVX-E became squamous. Conclusions: Genistein can prevent bone resorption diseases by the promotion of bone formation and the prevention of bone resorption with slight side effect.

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The effects of lipid hydroperoxide degradation products, such as 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), on bovine brain synaptosomal ATPase activities and their membrane lipid organization were examined. When the synaptosomes were treated with HNE, this resulted in the decrease of Na⁺-K⁺-ATPase activity with the loss of sulfhydryl (SH) groups in the membrane proteins. In contrast, MDA treatment of the synaptosomes did not induce an appreciable decrease in the ATPase activity or a loss of SH groups. The decreases in ATPase activity and SH content by treatment with HNE were also observed, as a Na⁺-K⁺-ATPase preparation was used in place of the synaptosomes. On the other hand, HNE had very little effect on synaptosomal Ca²⁺- and Mg²⁺-ATPase activities. The results of the kinetic analysis of the Na⁺-K⁺-ATPase activity indicated that the decrease in the activity by HNE-modification is due to a decreased affinity for the substrate. ATP completely protected the ATPase from the HNE attack. Modification of the synaptosomes with HNE caused a decrease in the membrane lipid fluidity near the lipid/water interface, not the lipid layer interior. In addition, it was found that there is a good relationship between the lipid fluidity and the Na⁺-K⁺-ATPase activity under the presence of various concentrations of HNE, suggesting that the lipid dynamics are closely related to HNE-induced inhibition of the ATPase activity. On the other hand, MDA did not induce change in the membrane lipid fluidity. HNE and MDA are mainly incorporated into the lipid and protein fractions in the synaptosomal membranes, respectively. Based on these results, we proposed a possible mechanism of HNE-induced inhibition of synaptosomal Na⁺-K⁺-ATPase activity associated with alterations in the membrane lipid organization.

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Megakaryoblastoma (Dami cells) cultured in a serum-free medium containing albumin, proliferated for three days but died on the fourth day. This cell death was not observed when human plasma was added, suggesting that human plasma contains a cell-death inhibitory factor. In order to identify this factor, we purified it from human plasma. N-terminal amino acid sequence analysis revealed that this factor is a mixture of C-terminal fragments of selenoprotein P, a major selenocysteine-containing protein in plasma. The specific activity (unit per pmol of selenium) of selenoprotein P fragments protein was 15-fold and 1900-fold higher than that of the full-length SeP and sodium selenite, respectively.

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Labeling with stable isotopes, typically deuterium (D), is powerful tool for studying the functional structure of biomolecules by NMR. Biosynthesis of certain deuterated proteins in microorganisms cultured in deuterium oxide (D₂O) is an attractive strategy. However, the growth of almost all microorganisms is inhibited at high concentrations of D₂O. We isolated a mutant of yeast that grows well in D₂O. The expression of Hsp70 was enhanced in the mutant. The increased expression also endowed the yeast with cold-resistance. The mutant might be useful for biosynthesis of D-labeled biomolecules.

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To gain insight into the catalytic function of aromatase and its substrate specificity, we studied reversible inhibition of 16α-hydroxyandrostenedione (16α-OHAD) aromatization in human placental microsomes by several suicide substrates of androstenedione (AD) aromatization, including 4-hydroxyAD (1), 6-oxoAD (2) and its 19-hydroxy analogue 3, androst-5-ene-4,7,17-trione (4), and 10β-acetoxyandrost-5-en-7,17-dione (5) that, in contrast, do not cause a suicide inactivation of 16α-OHAD aromatization. All inhibitors examined blocked 16α-OHAD aromatization in a competitive manner with apparent K_i values ranging from 0.50 to 980 nM. The relative K_i values between inhibitors 1—5 obtained in the 16α-OHAD aromatization experiments were markedly different from those obtained in the AD aromatization experiments. The results predict that all inhibitors examined bind to the 16α-OHAD binding site in a manner that does not cause suicide inactivation of 16α-OHAD aromatization. These findings would be useful for understanding the active (binding) site structure as well as the catalytic function of aromatase.

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Protective effects of quinic acids from Aster scaber on tetrahydropapaveroline (THP)-induced cell toxicity were evaluated in rat C6 glioma cells. Among 4 quinic acid derivatives tested, (−) 4,5-dicaffeoyl quinic acid (QA3) exhibited the highest protective effect against THP-induced cell toxicity. C6 cells treated with THP exhibited the decrease in the survival rate and activities of glutathione peroxidase and catalase, but increased the level of malondialdehyde and superoxide dismutase activity. Staining C6 cells with propidium iodide and Hoechst 33342 revealed that 10 μM of THP treatment caused to necrotic and apoptotic cell death. However, preincubation of cells with QA3 prior to THP exposure recovered the cell survival rate and activities of antioxidant enzymes to control level. Taken together, the results indicate that QA3 might be a potential agent for treating or preventing diseases with oxidative stress.

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We examined the effects of KD3-671 (2-propyl-8-oxo-1-[(2′-(H-tetrazole-5-yl)biphenyl-4-yl)methyl]-4,5,6,7-tetrahydrocycloheptimidazole), an angiotensin II type1 receptor antagonist, on an experimental rat model of mesangioproliferative glomerulonephritis, anti-Thy-1 nephritis. Anti-Thy-1 nephritis was induced by intravenous injection of 300 μg/kg of anti-Thy-1.1 monoclonal antibody into rats. KD3-671 (3, 10, 30 mg/kg per day) or enalapril (30 mg/kg per day), an angiotensin II converting enzyme inhibitor, was given p.o. once daily from the day before the antibody injection (the 1st day) to the 15th day after. KD3-671 significantly inhibited an increase in the number of total and proliferating cell nuclear antigen-positive cells and the deposition of α-smooth muscle actin and fibronectin in the glomeruli of nephritic rats, but enalapril (30 mg/kg per day) suppressed only the number of total cells and the deposition of α-smooth muscle actin in the glomeruli. Moreover, to elucidate the effect of KD3-671 on matrix deposition in the glomeruli, we measured the production of fibronectin in isolated glomeruli obtained from anti-Thy-1 nephritic rats. The glomeruli in anti-Thy-1 nephritic rats produced more fibronectin than that in control rats. KD3-671 (10⁻⁸, 10⁻⁷, 10⁻⁶ M) dose-dependently attenuated fibronectin production in isolated nephritic glomeruli. These findings suggest that KD3-671 may be an effective agent for the treatment of mesangioproliferative glomerulonephritis.

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A study was carried out to evaluate the potential pharmacokinetic interaction between digoxin and acenocoumarol. The binding of digoxin to rabbit cardiac tissue homogenates was assessed in vitro, using the equilibrium dialysis technique. An increase in the first-order constant (p<0.05) and a reduction in the partition coefficient in the equilibrium situation (p<0.001) of digoxin were observed when the cardiac homogenates were previously treated with acenocoumarol. In the in vivo study, the kinetics of digoxin administered in single and multiple dosage regimens were compared in control rabbits and in rabbits treated simultaneously with acenocoumarol. Kinetic analysis of the results was performed using Non-linear Mixed Effects Modeling (NONMEM). In the presence of acenocoumarol, the population distribution volume (Vd) of digoxin was increased by 40—60%, no differences being found as regards the elimination clearance. Also, joint administration of both drugs led to a reduction in digoxin concentrations in the heart (p<0.01) at the end of the dosage regimen. Both sets of results point to the hypothesis of a hitherto unreported possible pharmacokinetic interaction between the two drugs affecting the distribution process. This interaction could lead to lower plasma digoxin levels, in view of the increased Vd, and a possible reduction in the therapeutic effect, owing to the decrease in affinity and in concentration in heart tissue.

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We previously reported that fluvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, a strong lipid lowering drug, exerted an anti-atherosclerotic effect at doses insufficient to lower serum lipids in cholesterol fed rabbits. The evidence demonstrated that the superoxide anions from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a critical role in several steps in the development of atherosclerosis. This study was designed to determine the effects of HMG-CoA reductase inhibitors on the production of the superoxide anions of NADPH oxidase in isolated rat peritoneal neutrophils. Fluvastatin (1—10 μM) decreased phorbol 12-myristate 13-acetate (PMA, 10 nM)-dependent reactive oxygen species (ROS) generation in a concentration-dependent manner. It also (10 μM) decreased PMA-dependent O₂ consumption of the rat neutrophils. These effects were reversed by the addition of mevalonate, a metabolite in the HMG-CoA reductase pathway. Treatment with pravastatin did not show any significant changes. Fluvastatin (10 μM) decreased ROS, such as hydroxyl radicals and superoxide anions generated by the Fenton reaction, and by the xanthine–xanthine oxidase system. Rats were treated with either fluvastatin (5 mg/kg per day, p.o.) or pravastatin (5 mg/kg per day, p.o.) for 1 week. Treatment with fluvastatin decreased the PMA-dependent ROS generation. The fluvastatin induced effect on the PMA-dependent ROS generation was reversed by the combined administration with 40 mg/kg mevalonate per day. The antioxidative effect of fluvastatin was thought to have caused not only the scavenging action of the radicals but also to have inhibited ROS generation by inhibiting the NADPH oxidase activity. This antioxidative potential of fluvastatin via the inhibition of NADPH oxidase activity may be profitable in preventing atherosclerosis.

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To examine whether oral administration of proteoglycan derived from Phellinus linteus, which is known as the medicinal mushroom, can prevent or treat collagen-induced arthritis (CIA) in mice as experimental model of autoimmune disease. CIA was induced by intradermal injection of type II collagen (CII) emulsified with complete freund's adjuvant (CFA) into the base of the tail (on day 7) followed by a booster injection on day 21 into the footfad. To examine the ability of proteoglycan to effect the inhibition of CIA, doses of proteoglycan were orally administered on day 0 (pre-administration) or day 28 (post-administration) at two groups. The inhibition of CIA by oral administration of proteoglycan was associated with decrease in anti-CII IgG and IgG2a antibodies (Abs) as well as varying kinds of cytokines including IL-12, TNF-α, and IFN-γ. The results showed that administration of proteoglycan was followed by decrease of CIA of the mice in pre- and post-administration groups. Our findings suggest that immunomodulating proteoglycan isolated from P. linteus may be crucially involved in the prevention and treatment of autoimmune joint inflammation such as rheumatoid arthritis, although no definite role of anti-CII Abs in the human disease has been established.

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The cardiac toxicity of trastuzumab was studied in chick embryos. Fertilized eggs of White Leghorns were incubated and investigated. Trastuzumab 5 mg/egg (low dose) or 15 mg/egg (high dose) was injected into the air sac of a fertilized egg on the 16th day of incubation. Electrocardiograms (ECGs) were recorded 0 to 60 min after the injection. After low dosing of trastuzumab, the heart rate was not different compared with the control. However, the heart rate was significantly decreased by high dosing of trastuzumab. In addition, arrhythmia was produced by high dosing of trastuzumab. These findings indicate that trastuzumab has a marked dose- and time-dependent influence on the heart rate in chick embryos.

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Unsei-in, a traditional medicine, is prescribed against pruritic cutaneous diseases, but the mechanisms of antipruritic action are still unknown. In the present study, we examined the antipruritic effects of Unsei-in in mice. Single administration of Unsei-in did not inhibit substance P-induced itch-associated response (scratching) in mice. However, repeated treatment with Unsei-in for 7 d significantly inhibited substance P-induced scratching. The same repeated treatment with Unsei-in suppressed the expression of NK₁ tachykinin receptors in the skin. These results suggest that Unsei-in inhibits substance P-associated itching and that the inhibition is at least partly due to the suppression of the expression of NK₁ tachykinin receptors in the skin.

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The subacute toxicity and toxicokinetics of a new fluoroquinolone antibiotic, DW-224a, were evaluated after single (on the 1st day) and 4-week (on the 28th day) oral administration of the drug at doses of 0 (to serve as a control), 10, 30, and 90 mg/kg/d, to male and female dogs (n=3 for male and female dogs for each dose). During the test period, clinical signs, mortality, body weight, food consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weight and histopathology were examined. The 4-week repeated oral dose of DW-224a resulted in vomiting, salivation, increased serum cholesterol level, and atrophy of thymus and testes. The target organ was determined to be the thymus and testes. The absolute toxic dose of DW-224a was 30 mg/kg and the level at which no adverse effects were observed was 10 mg/kg for both sexes. There were no significant gender differences in the pharmacokinetic parameters of DW-224a for each dose after both single and 4-week oral administration. The pharmacokinetic parameters of DW-224a were dose independent after a single oral administration; the time to reach the peak plasma concentration (T_max) and the dose-normalized area under the plasma concentration–time curve from time zero to 24 h in plasma (AUC_{0—24 h}) were not significantly different among the three doses. The accumulation of DW-224a after 4-week oral administration was not notable at the toxic dose of 90 mg/kg/d. For example, after 4-week administration, the dose-normalized AUC_{0—24 h} value at 90 mg/kg/d (7.69, 7.05 μg h/ml) was not significantly greater than that at 10 mg/kg/d. After 4-week oral administration, the dose-normalized C_max and AUC_{0—24 h} at 90 mg/kg/d were not significantly higher and greater, respectively, than those after a single oral administration.

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Linoleic acid hydroperoxide (LOOH) has been reported to cause an increase in the content of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), a typical oxidation product of DNA bases, in cultured cells due to coexisting iron(III) ion. We examined whether coumarins are able to suppress the formation of 8-oxodG in the DNA of human diploid fibroblasts, TIG-7 cells, treated with LOOH and iron(III) ion. Cotreatment of TIG-7 cells with esculetin (6,7-dihydroxycoumarin) significantly suppressed the increase in 8-oxodG content induced by LOOH and iron(III) ion. Pretreatment of cells with esculetin for 24 h was also effective in protecting cellular DNA against oxidative damage induced by subsequent treatment with LOOH and iron(III) ion. Pretreatment with esculin, the 6-glucoside of esculetin, was effective, but to a lesser extent. Furthermore, the free radical-scavenging activities of coumarins and hydroxycinnamic acids were examined by measuring the inhibition of spin-adduct formation of hydroxyl radicals with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Compounds bearing an ortho-catechol moiety, such as esculetin, fraxetin, and caffeic acid, significantly reduced the ESR signal intensities of the DMPO-OH spin adduct. These results indicate that esculetin is effective in protecting cells against DNA damage induced by oxidative stress and that the presence of an ortho-catechol moiety is important for antioxidant activities against reactive oxygen species.

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Effect of N⁶-benzyladenine (BA) on tanshinone formation in callus cultures of Salvia miltiorrhiza was examined in an attempt to increase the productivity of the medicinal compound, cryptotanshinone. Primary callus was induced by culturing leaf explants on Murashige and Skoog's (MS) basal medium supplemented with 1.0 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) in darkness. The callus proliferated further on MS basal medium containing 1.0 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ BA and was analyzed for cryptotanshinone by high performance liquid chromatography (HPLC). The HPLC results indicated that it contained small amounts of cryptotanshinone (0.26±0.05 mg/g dry wt). Omission of 2,4-D from the medium resulted in a marked increase in the content of cryptotanshinone in callus. The HPLC analysis revealed that the content of cryptotanshinone in the callus cultured on the MS basal medium supplemented with 0.1, 0.2, 0.5, 1.0, and 2.0 mg l⁻¹ of BA was significantly higher than the marketed crude drug (processed underground parts of S. miltiorrhiza). Maximum yield of cryptotanshinone (4.59±0.09 mg/g dry wt) was observed in the callus cultured on MS basal medium supplemented with 0.2 mg l⁻¹ BA for 60 d. Cryptotanshinone was isolated from callus through silica gel column chromatography followed by preparative TLC and characterized based on NMR and mass spectral data.

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Melanogenesis is a well known physiological response of human skin exposed to ultraviolet light, genetic reasons and other sources. In this study, we conducted to evaluate the effects of Radix Ginseng (RG) and Radix Trichosanthis (RT) on the melanogenesis in the B16 melanoma cells. The cells were treated for 48 h with RT at concentrations ranging from 1 to 50 μg/ml, RG at concentration of 10—1000 μg/ml, or RG at various doses (10—1000 μg/ml) with 25 μg/ml RT. Treatment with RT alone dose-dependently suppressed tyrosinase activity and melanin content compared with untreated control, and significantly inhibited cell proliferation. However, RG at various concentrations did not exhibit any significant change of them. Treatment with RT in the presence of various concentrations of RG suppressed tyrosinase activity and melanin content, similar to treatment with RT alone, but slightly increased cell proliferation. Furthermore, tyrosinase protein level was significantly decreased in treatment with 25 μg/ml RT alone and with a combination of 100 μg/ml RG. These results indicate that treatment with RG and RT significantly inhibits the melanogenesis in B16 cells, and raise the possibility that this combination may be effective in the whitening agent for the skin.

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The pancreatic lipase inhibitory activity of the rhizome of Alpinia officinarum (AO) and its antihyperlipidemic activity were measured. When the water extract of AO was fractionated stepwise with organic solvents, the ethyl acetate fraction exhibited the most potent inhibition. 3-Methylethergalangin was isolated from that fraction as an inhibitor of pancreatic lipase with an IC₅₀ value of 1.3 mg/ml (triolein as a substrate). AO and its ethyl acetate fraction significantly inhibited the serum TG level in corn oil feeding-induced triglyceridemic mice, and serum triglyceride (TG) and cholesterol in Triton WR-1339-induced hyperlipidemic mice. However, this compound and the AO ethyl acetate fraction did not show hypolipidemic activity in high cholesterol diet-induced hyperlipidemic mice. The results suggest that the hypolipidemic activity of AO and 3-methylethergalangin is due to the inhibition of pancreatic lipase.

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We examined the effects of ginseng total saponin (GTS) and several ginsenosides injected intrathecally (i.t.) or intracerebroventricularly (i.c.v.) on the antinociception induced by cold water swimming. The tail-flick response was used as an antinociceptive parameter. We found that i.t. injection of GTS time- and dose-dependently attenuated inhibition of the tail-flick response induced by cold water swimming, although GTS given i.c.v. had no significant effect on the latency of the tail-flick response induced by cold water swimming. To identify those responsible for antagonism of GTS against cold water swimming-induced antinociception, the effects of various kinds of ginsenosides (R_b1, R_b2, R_c, R_d, R_e, and R_g1) on inhibition of the tail-flick response induced by cold water swimming were examined. R_b1, R_d, R_e, and R_g1 effectively attenuated the inhibition of the tail-flick response induced by cold water swimming stress. Our results suggest that GTS injected spinally, but not supraspinally, reduces the antinociception induced by this stress, and that the responsible ginsenosides against antinociception induced by cold water swimming may be R_b1, R_d, R_e, and R_g1. Moreover, the possible involvement of the opioid system in the regulation of cold water swimming stress-induced antinociception by these four ginsenosides is discussed.

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This work describes an immunochemical approach for the quality control of Paeoniae Radix by an enzyme-linked immunosorbent assay (ELISA) based on determination of the total concentration of paeoniflorin (PF) and albiflorin (Alb), which are major bioactive constituents in Paeoniae Radix. Four hybridromas secreting monoclonal antibodies against PF and Alb were produced by fusing splenocytes from a mouse immunized by a PF-bovine serum albumin (BSA) conjugate with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A relatively higher reactivity of monoclonal antibodies (MAbs) with PF and Alb than oxypaeoniflorin (OP) and benzoylpaeoniflorin (BP) was observed, while other monoterpenes and benzoic acid did not cross-react. When PF was used as a standard, the assay can cover a measuring range of 20—600 ng/ml for PF and Alb. A series of Paeoniae Radix samples have been determined, and the results showed good agreement with that determined by traditional high-performance liquid chromatography (HPLC). The developed competitive ELISA was 100 times more sensitive than the HPLC method. Meanwhile, fifteen Chinese traditional prescriptions were determined by the competitive ELISA.

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The characteristics of Glycyrrhiza plants from 12 collection sites in southeasten Kazakhstan were investigated. G. uralensis was observed at 9 of the sites from Almaty to Shu, and G. glabra was observed at 8 sites. At 4 sites near Shu, and 1 site near Almaty, G. glabra and G. uralensis grew together forming a mixed population, and intermediate-type plants between them were also observed at 3 sites. Although two nucleotide substitutions of the chloroplast rbcL gene were observed between G. uralensis and G. glabra, rbcL sequences of the intermediate-types were divided into G. uralensis-type (G-A type) and G. glabra-type (A-T type). HPLC analysis of the roots indicated that species-specific flavonoids, glabridin and glycycoumarin, were detected in the roots of G. glabra and G. uralensis, respectively, but neither flavonoid was detected in underground parts of the intermediate-types. HPLC analysis of their leaves indicated a significant difference among G. uralensis, G. glabra and the intermediate-type plants. Both G. glabra-specific and G. uralensis-specific compounds were detected in the leaves of the intermediate-type, thus suggesting that the intermediate plants are hybrids of G. glabra and G. uralensis.

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Objective of the present study was to investigate the elimination kinetics of quinaprilat and perindoprilat, the active metabolites of angiotensin-converting enzyme (ACE) inhibitors quinapril and perindopril, in hypertensive patients with renal failure under haemodialysis to evaluate the appropriate duration of off-dose of these drugs before starting of low-density lipoprotein (LDL) apheresis. The informed consent was received from 12 hypertensive patients with renal failure, who were under haemodialysis (42 to 62 years). The patients received oral administration of quinapril (10 mg) or perindopril (2 mg) once a day for four weeks. First, to evaluate the dialyzability of each metabolite, blood samples were collected before and after haemodialysis one week after the repeated doses. Second, to evaluate the elimination kinetics of quinaprilat or perindoprilat, blood samples were collected at 24, 72, 120, 192 and 240 h after the final administration. Plasma concentrations of quinaprilat and perindoprilat were measured by high-performance liquid chromatography (HPLC) and radioimmunoassay, respectively. Pharmacokinetic parameters were determined by a model-dependent method. Values of haemodialysis clearance (CL_HD) and extraction ratio (ER) were 51.5±30.2 ml/min and 0.35±0.21 for quinaprilat and 108.1±5.9 ml/min and 0.75±0.04 for perindoprilat, respectively. The terminal elimination half-lives of quinaprilat and perindoprilat were 60.7±2.1 and 79.9±14.0 h, respectively. The dialyzability of perindoprilat was much higher than that of quinaprilat probably due to low protein binding potency. The present study suggests that hypertensive patients receiving chronic therapy with quinapril or perindopril on haemodialysis should be withdrawn for at least 2 to 3 weeks before LDL apheresis.

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Efficacy of therapeutic drug monitoring (TDM) of vancomycin (VCM) was retrospectively investigated in 184 patients with methicillin-resistant Staphylococcus aureus (MRSA) infection. The incidence of nephrotoxicity was compared between the patients who received TDM practice (TDM group, n=73) and did not (non-TDM group, n=111). Creatinine clearance (CLcr) values decreased significantly after the VCM therapy in the non-TDM group (p<0.05). The patients with MRSA bacteremia or pneumonia were classified into two groups according to peak concentrations of VCM: above 25 μg/ml (Group A: n=29) and below (Group B: n=24). Mean duration of VCM therapy (14.1 d) in Group A was significantly shorter than that (27.0 d) in Group B. Mean cumulative total VCM doses (13.3 g) in Group A was significantly less than that (25.0 g) in Group B. These results indicate that monitoring peak concentration is essential to obtain better clinical effects for VCM therapy, and that the peak concentration above 25 μg/ml is more effective.

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The development of a carrier system that enables the transfer of a functional exogenous gene to non- or less frequently dividing mammalian cells is essential for increasing the available options for the treatment of various diseases. The issue of whether TFL-3, a recently developed cationic liposome, can be successfully used to achieve gene expression in primary cultured rat hepatocytes was examined. The hepatocytes were transfected for 4 h with plasmid DNA (pDNA) in TFL-3 at various time points after 4-h preculture. The transfection efficiency was determined at various times posttransfection with pDNA coding for chloramphenicol acetyltransferase (CAT), luciferase, or β-galactosidase. The amount of intranuclear pDNA present, as a consequence of the lipofection, was also quantitatively determined. Successful lipofections were observed for all pDNA tested, and the efficiencies were superior to that of commercially available LIPOFECTAMINE under our experimental conditions. The degree and rate of gene expression were dependent on incubation time prior to lipofection as well as on the density of the cells per dish, but this relationship did not hold for the amount of gene delivered to the nuclei. These results indicate that TFL-3 could be a useful vector for achieving sufficient gene expression in rat hepatocytes and suggest that the culture time prior to and following lipofection, which is related to the biological condition of the cells, may be one major factor affecting efficient gene expression in nondividing cells.

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The present study was aimed at kinetically characterizing the carrier-mediated transport systems for D-glucose and taurocholate in the rat colon, compared with their respective counterparts in the small intestine. The transport of these compounds was evaluated by measuring the initial uptake into everted intestinal tissue sacs. The uptake of both D-glucose and taurocholate was highly saturable, conforming to Michaelis–Menten kinetics without an appreciable nonsaturable transport component. The Michaelis constant (K_m) was 0.43 and 0.021 mM, respectively, for D-glucose and taurocholate and the maximum transport rate (J_max) was 0.82 and 0.056 nmol/min/100 mg wet tissue weight (wtw), respectively. For both compounds, these values of K_m and J_max in the colon were one to three orders of magnitude smaller than those in the small intestine, suggesting that the transport systems in the colon have by far a higher affinity and a lower transport capacity than their counterparts in the small intestine. However, it is now evident from kinetic studies that carrier-mediated transport systems for D-glucose and taurocholate are also present in the colon. It will be interesting to explore the possibility that they could be used for oral drug delivery via the colon. Their physiological roles would also be of interest.

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General characteristics of the microclimate layer on the mucosal surface of the rat jejunum incubated in bicarbonate buffers were investigated in vitro using pH sensitive flat membrane microelectrode. Jejunal surface microclimate (JSM) pH changed from 7.30±0.03 to 5.83±0.04 when the incubation buffer pH decreased from 8.03±0.02 to 6.12±0.01. Treatment of the mucosal side with mucolytic substances L-cysteine (1% (wt/v)) and 1,4-dithio-DL-threitol (2 mM) significantly (p<0.01) increased JSM pH. Respiratory chain inhibitor, sodium azide (10 mM) also significantly (p<0.05) increased JSM pH. D-Glucose (10 mM) at the mucosal side markedly (p<0.05) decreased JSM pH, which was attenuated by Na⁺/H⁺ exchange inhibitor, amiloride (1 mM). Amiloride had no effect on JSM pH when D-glucose was not present at the mucosal side. In contrast to previous observations using bicarbonate free incubation buffers, we have demonstrated that JSM pH is not a constant value, but is dependent on pH of the incubation buffer. Additionally, Na⁺/H⁺ exchanger does not contribute to acidic properties of JSM, when there is no D-glucose in the bicarbonate incubation buffer at the mucosal side of the tissue. In conclusion, we suggest that the bicarbonate buffers which are more close to in vivo situation than bicarbonate free buffers should be preferable incubation media when examining JSM.

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We conducted a comparative study of the protective effects of chitin, chitosan, and N-acetyl chitohexaose (NACOS-6) against mice infected intravenously or intraperitoneally with Pseudomonas aeruginosa and Listeria monocytogenes. Mice pretreated with chitin, chitosan, and NACOS-6 showed resistance to intraperitoneal infections by both microbes. Only mice pretreated with chitin and chitosan showed resistance to intravenous infections by both microbes. The number, active oxygen generation, and myeloperoxidase activity of peritoneal exudate cells (PEC) in the chitin, chitosan, and NACOS-6-treated mice were greater than those of the untreated mice. Also, these PEC factors from mice pretreated with chitin and chitosan were greater than those from the NACOS-6-treated mice.

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Paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naphto(2,3c)pyran-1-one), a natural isocoumarin isolated from the capitula of Paepalanthus bromelioides (Eriocaulaceae), was assessed for its effect on the respiratory burst (zymosan-stimulated luminol-enhanced chemiluminescence and PMA-stimulated lucigenin-enhanced chemiluminescence) of polymorphonuclear neutrophils in vitro. Special attention was devoted to establishing the IC₅₀ for neutrophils. Paepalantine was able to decrease luminol and lucigenin chemiluminescence, reflecting an inhibitory effect on the respiratory burst, with an ED₅₀ of 0.44±0.05 and 0.84±0.15 μg/ml, respectively. A cell-free system was performed with paepalantine on myeloperoxidase/H₂O₂ and myeloperoxidase/H₂O₂/Cl⁻ systems. Paepalantine inhibited luminol oxidation in both systems. This inhibition was related to the interaction of paepalantine-myeloperoxidase and its scavenger effect on HOCl.

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An allegedly novel human trypsinogen cDNA clone termed trypsinogen hL was recently reported in the Biological & Pharmaceutical Bulletin (2003; 26: 361–364). This “new” trypsinogen sequence was isolated by PCR using human lung cDNAs as template and was presumed to be a new member of the human trypsinogen family. However, trypsinogen hL does not match any sequence in the human genome; whereas it is 100% identical to mouse trypsinogen 7. Thus, trypsinogen hL is not of human origin, and its cloning from a human cDNA library was clearly a result of contamination with mouse genetic material. Publication of this cloning artifact could have been avoided by a simple BLAST search of GenBank or other sequence databanks.

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