A fluorimetric method using eriochrome cyanine R (ECR) was applied to determine trace amounts of albumin in bronchoalveolar lavage fluid (BALF). The ECR-albumin adduct showed intense fluorescence and the calibration curve was linear in the range of 2.5—100 μg/ml of human serum albumin (r=0.999). The sensitivity was high enough to determine trace amounts of albumin in BALF. In the present study, the results obtained by the ECR method were compared with those of an EIA, and a good linear correlation was observed between the two methods (r=0.946). The ECR method is simpler and more rapid than EIA. The concentrations of albumin in BALF samples were determined by the ECR method. The albumin level in BALF of healthy nonsmokers (n=9) was lower than that of healthy smokers (n=9) (42.9±20.7 and 48.3±15.7 μg/ml, respectively). On the other hand, the albumin level of BALF samples obtained from patients with hypersensitivity pneumonitis was increased versus in healthy subjects. The determination of albumin in BALF samples by the ECR method is useful for investigating lung diseases.

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In this study, we investigated the properties of monkey liver aldehyde oxidase directed toward the clarification of species differences. The aldehyde oxidase preparation purified from male cynomolgus monkey liver cytosol showed a major 150 kDa Coomassie brilliant blue (CBB)-stained band together with a minor 130 kDa band using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both bands were identified as being aldehyde oxidase by a database search of the MS data obtained with nano-liquid chromatography, quardrupole time of flight, mass spectrometry (nano-LC Q/TOF MS). Based on the sequence coverage, the 130 kDa protein was presumed to be deficient in 20—30 kDa mass from the N-terminus. Full male cynomolgus monkey aldehyde oxidase cDNA was cloned and sequenced with the four degenerate primers designed by considering the peptide sequences containing the amino acids specific for monkey aldehyde oxidase. The deduced amino acid sequences had 96% amino acid identity with those of human enzyme. The aldehyde oxidase expressed in Escherichia coli also exhibited two immunoreactive bands on SDS-PAGE/Western blot analysis. Further, the biphasic pattern was observed for Eadie–Hofstee plots of the (S)-enantiospecific 2-oxidation activity of RS-8359 with the expressed and cytosolic monkey liver aldehyde oxidase. The results suggested that two forms of aldehyde oxidase in monkey were the expression products by a single gene. In contrast, the similarly expressed rat aldehyde oxidase showed only one immunoreactive protein and monophasic pattern. The biphasic phenomenon could be caused by the existence of two aldehyde oxidase isoforms or two active sites in a single enzyme or some other reasons. Further studies on the problems of the biphasic pattern and species differences in aldehyde oxidase are needed.

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2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). While evidence is accumulating that the CB1 receptor plays important regulatory roles in various nervous tissues and cells, the physiological roles of the CB2 receptor, which is abundantly expressed in the immune system, are yet to be determined. In this study, we examined in detail the effect of 2-arachidonoylglycerol on the phagocytosis of opsonized zymosan by HL-60 cells that had differentiated into macrophage-like cells. We found that the addition of 2-arachidonoylglycerol augmented the phagocytosis of opsonized zymosan by the differentiated HL-60 cells. The effect was observed from 1 nM and increased with increasing concentrations of 2-arachidonoylglycerol. Treatment of the cells with SR144528 or pertussis toxin abolished the effect of 2-arachidonoylglycerol, indicating that the CB2 receptor and Gi/o are involved in the augmented phagocytosis. Phosphatidylinositol 3-kinase and extracellular signal-regulated kinase were also suggested to be involved; treatment of the cells with wortmannin or PD98059 abrogated the 2-arachidonoylglycerol-augmented phagocytosis. These results strongly suggest that 2-arachidonoylglycerol, derived from stimulated inflammatory cells, has an important role in augmenting the phagocytosis of invading microorganisms by macrophages/monocytes thereby stimulating inflammatory reactions and immune responses.

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D-Aspartate is present in the central nervous system and various endocrine organs, and modulates their neuroendocrine function. In islets of Langerhans, α and β cells contain D-aspartate. Here we show that INS-1E clonal β cells contain the highest amount of D-aspartate. Immunohistochemical analysis with specific antibodies against D-aspartate indicated that D-aspartate is co-localized with insulin. Upon the addition of K⁺, both D-aspartate and insulin are secreted from the cells in a Ca²⁺-dependent manner. A Ca²⁺ ionophore, A23187, also triggers the release of D-aspartate and insulin in the presence of Ca²⁺. Bafilomycin A₁, a specific inhibitor of V-ATPase and V-ATPase-linked secondary transport, inhibits the secretion of D-aspartate. These results support the idea that D-aspartate is present in insulin-containing secretory granules and co-secreted with insulin through exocytosis.

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The sperm proteasome has been reported to be involved in sperm penetration through the proteinaceous egg-coat during fertilization in ascidians and mammals. However, such an extracellular role for the sperm proteasome in fertilization is not known in other deuterostomes. Here, we investigated the effects of two proteasome inhibitors on fertilization of the sea urchin Anthocidaris crassispina. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization, whereas E-64-d, chymostatin or leupeptin showed no inhibition at 100 μM. MG-132 inhibited the egg-jelly-induced acrosome reaction, but not the reaction induced by the Ca²⁺ ionophore ionomycin. MG-132 and MG-115, but not E-64-d, inhibited the fertilization of dejellied eggs by acrosome-reacted sperm. Furthermore, MG-132-susceptible proteasome activity was detected in the acrosomal contents. These results suggest that the sperm proteasome plays a key role not only in the acrosome reaction, in particular, in a process before the increase in intracellular Ca²⁺ concentration but also in the sperm penetration through the vitelline coat, most probably as a lysin.

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This study was designed to examine the in vitro effects of adenosine (Ado) on hydrogen peroxide-induced endothelial dysfunction in rats. Endothelial dysfunction was induced by exposing isolated rat mesenteric arteries to hydrogen peroxide (0.5 mM) for 12 h using an organ culture system. The protective effects of adenosine were tested by exposing isolated mesenteric arteries to adenosine (3×10⁻⁷ mol/l, 10⁻⁶ mol/l, 3×10⁻⁶ mol/l)+hydrogen peroxide (0.5 mM) for 12 h. This exposure to hydrogen peroxide induced a significant concentration-dependent inhibition of endothelium-dependent relaxation (EDR). Coculture of segments of mesenteric artery with adenosine (3×10⁻⁷, 10⁻⁶, and 3×10⁻⁶ mol/l) attenuated the hydrogen peroxide-induced impairment of vasorelaxation. This impairment was accompanied by a reduction in nitrite/nitrate, nitric oxide (NO) synthase (NOS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and an increasing in malondislehyde (MDA) and lactate dehydrogenase (LDH) activities in the aorta. These results indicate that adenosine can be used to attenuate hydrogen peroxide-induced endothelial dysfunction, an effect that may be related to antioxidation, thus enhancing NO production by preventing the decrease in NOS.

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In order to evaluate the effects produced by N-(2-hydroxybenzoyl) tyramine (riparin II) isolated from the unripe fruit of Aniba riparia (NEES) MEZ (Lauraceae) on the central nervous system, different behavioral tests were performed. Riparin II (rip II) was administered orally (p.o.) and intraperitoneally (i.p.) in male mice, at doses of 25, 50 and 75 mg/kg, and tested on elevated plus maze (EPM), open field, rota rod and hole board tests. The results revealed that rip II caused considered increase of the number of head dips in hole board test and increased the number of entries and the time of permanence in the open arms in plus maze test in both routes. No significant effect was evidenced on rota rod and open field test, except an increase observed in the number of rearing. These results showed that riparin II presents anxiolytic-like effects in the plus maze and hole board tests which are not influenced by the locomotor activity as detected in the open field test.

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In the present study were studied the antinociceptives properties of monoterpene R-(+)-limonene (LM) in chemical and thermal models of nociception in mice. The R-(+)-limonene was administered, intraperitoneally (i.p.), at doses of 25 and 50 mg/kg. The results showed significant inhibition produced on chemical nociception induced by intraperitoneal acetic-acid and in the second phase of subplantar formalin test, but did not manifest a significant effect in hot-plate test. The R-(+)-limonene-induced antinociception in second phase of formalin test was insensitive to naloxone (1 mg/kg, s.c.). It was also demonstrated that R-(+)-limonene (25, 50 mg/kg) neither significantly enhanced the pentobarbital-sleeping time nor impaired the motor performance in rota-rod test, indicating that the observed antinociception is unlikely to be due to sedation or motor abnormality. In conclusion it may be suggested that the R-(+)-limonene presented antinociceptive activity and that, probably, this action can be related with peripheral analgesia, but, not with the stimulation of opioids receptors.

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Piper longum L. has been used as a crude drug for the treatment of disorders of poor peripheral blood circulation in Asia. However, the detailed mechanism of its action has not been clarified as yet. In the present study, we examined the effects of several extracts of Piper longum L. on rabbit platelet function. Thromboxane A₂ receptor agonist U46619 caused rabbit platelet aggregation, which was potently inhibited by the ethanol or butanol extract of Piper longum L. The ethanol extract inhibited U46619-induced platelet aggregation in a concentration-dependent manner, but only weakly inhibited that induced by thrombin. The maximum response to U46619 was reduced by 100% ethanol extract concentration dependently, suggesting that the inhibitory mode of U46619-induced platelet aggregation by the ethanol extract was non-competitive. The extract also inhibited U46619-induced phosphoinositide hydrolysis with a similar concentration dependency to the platelet aggregation. Furthermore, the extract inhibited binding of [³H]SQ29548 to thromboxane A₂ receptor in intact platelets in a concentration-dependent manner. These results suggest that Piper longum L. contains a constituent(s) that inhibits platelet aggregation as a non-competitive thromboxane A₂ receptor antagonist.

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α₁-Acid glycoprotein (AGP) is an acute phase protein. Whereas the expression of AGP in an inflammatory state is enhanced by inflammatory cytokines including interleukin-1, 6 (IL-1 and IL-6), and tumor necrosis factor-α (TNF-α), the biological significance of AGP remains unclear. In the current study, the anti-inflammatory effect of AGP on the acute inflammatory state was examined in vivo and in vitro. AGP suppressed carrageenan-, dextran- and kaolin-induced paw edema and vascular permeability in rat. These results suggest that both initial inflammatory mediators (serotonin and histamine) and later inflammatory mediators (prostaglandin and bradykinin) are involved in the anti-inflammatory effects of AGP. In fact, prostaglandin E₂ (PGE₂) generation in plasma was significantly inhibited by AGP. Moreover, AGP inhibited the migration of neutrophils treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) through membrane filter. In addition, AGP significantly suppressed superoxide generation from neutrophils that has been treated with fMLP or phorbol 12-myristate 13-acetate. These results imply that the anti-inflammatory effect of AGP may involve the inhibition of neutrophils migration. The data obtained in this study support a scenario in which an increase in AGP concentration in pathological conditions suppresses inflammation reactions induced by autacoids and neutrophils activities and that AGP plays an important role in the maintenance in the body.

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The CXC chemokine receptor CXCR2 has been implicated in the pathogenesis of several chronic diseases including atherosclerosis. To enable animal studies towards understanding the role of human CXCR2 (hCXCR2) in disease development, we previously generated hCXCR2 knockin (hCXCR2(+/+)) mice. We have demonstrated that the phenotype and the acute immune response of the hCXCR2(+/+) mice was identical to that of wild-type mice, indicating that hCXCR2 indeed takes over the function of endogenous mouse CXCR2 (mCXCR2). In the present paper, we extend these findings by studying whether hCXCR2 functionally replaces the role of mCXCR2 in a chronic disease model for atherosclerosis. We first defined which of two well-described atherosclerosis models (ApoE(−/−) or LDLR(−/−) mice) is most suited for this purpose. When expression of mCXCR2 and that of its ligands in atherosclerotic lesions were compared in these mice, increased expression levels were observed only in LDLR(−/−) mice. Further, cultured atherosclerotic aortas from LDLR(−/−) mice did secrete significantly higher levels of CXCR2 ligands compared to aortas from healthy controls. Since these results support the role of CXCR2 in the atherogenesis in the LDLR(−/−) mice, double mutant hCXCR2(+/+)/LDLR(−/−) mice were generated and diet-induced atherosclerosis in these mice was compared to that in LDLR(−/−) mice. Upon an atherogenic diet, the hCXCR2(+/+)/LDLR(−/−) mice developed plaque lesions in a similar manner to those in LDLR(−/−) mice, indicating successful functional replacement of mCXCR2 by hCXCR2 in this disease model. We conclude that hCXCR2(+/+)/LDLR(−/−) mice present an attractive model to study the role of hCXCR2 in atherosclerosis development and for future testing of novel pharmaceuticals designed to antagonize hCXCR2.

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α₁-Adrenoceptor antagonists are clinically useful for the improvement of urinary obstruction due to benign prostatic hyperplasia (BPH), and their therapeutic effects are mediated through the blockade of prostatic α₁-adrenoceptors. The present study was undertaken to predict the magnitude and duration of α₁-adrenoceptor occupancy in the human prostate after oral α₁-adrenoceptor antagonists. Prostatic α₁-adrenoceptor-binding parameters of silodosin were estimated by measuring specific [³H]prazosin binding in rat prostate after oral administration of this drug. The plasma concentration of silodosin after oral administration in rats and healthy volunteers was measured using a high-performance liquid chromatographic method. The α₁-adrenoceptor-binding affinities (K_i) of silodosin, tamsulosin, and terazosin in the human prostate and plasma concentrations of tamsulosin and terazosin were obtained from the literature. Using the α₁-adrenoceptor binding parameters of silodosin in rat prostate, α₁-adrenoceptor occupancy in the human prostate was estimated to be around 60—70% at 1—6 h after oral administration of silodosin at doses of 3.0, 8.1, and 16.1 μmol. Thereafter, the receptor occupancy was periodically decreased, to 24% (8.1 μmol) and 54% (16.1 μmol) 24 h later. A similar magnitude and time course of α₁-adrenoceptor occupancy by silodosin in the human prostate were estimated using α₁-adrenoceptor-binding affinities (K_i) in the human prostate. Despite about two orders of differences in the plasma unbound concentrations after clinically effective oral dosages of silodosin, tamsulosin, and terazosin, there was a comparable magnitude of prostatic α₁-adrenoceptor occupancy by these drugs. In conclusion, the prediction of α₁-adrenoceptor occupancy in the human prostate by α₁-adrenoceptor antagonists may provide the rationale for the optimum dosage regimen of these drugs in the therapy of BPH.

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Reactive oxygen species (ROS) play an important role in normal metabolic and signaling processes. Excess ROS, however, can cause severe cardiovascular damage. Thus, the present study was designed to examine effects of H₂O₂ and xanthine plus xanthin oxidase (X/XO) on the serotonin (5HT), histamine (His) and acetylcholine (ACh)-induced contractions of porcine coronary arteries. In addition, to explore the site of ROS formation and species of it, the inhibitory effects of edaravone and EDTA were also tested. 5HT- and His-induced contractions were suppressed by H₂O₂ and X/XO treatment. However, these suppressions of ACh-induced contraction by H₂O₂ treatment was relatively weak and X/XO treatment caused no suppression on ACh-induced contraction. In the presence of edaravone which is thought to be a scavenger for ·OH, significant decrease of inhibition of 5HT- and His-induced contractions was observed when coronary artery strips were treated with X/XO, but not H₂O₂. On the other hand, inhibitory effects by EDTA treatments were also observed in X/XO treatments. These results suggest that 1) ROSs produced by additions of H₂O₂ or X/XO are considered to be responsible for several physiological functions of coronary artery contractions, 2) the site of ROS produced by X/XO system, probably ·OH, was outside the cell, but the inhibitory action of H₂O₂, was inside the cell, and 3) a low susceptibility of ACh-induced contraction to H₂O₂ and X/XO may indicates the signal transduction pathway(s) of ACh-induced contraction is different from those of 5HT and His.

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Our previous publication established a model to predict that the phenyl group of the C-3 side chain of azole antifungal compounds interacts with the phenol group of Tyr118 through the formation of π–π face-to-edge interaction. To verify this prediction, wild type and three site-directed mutants of the Y118 residue of Candida albicans sterol 14α-demethylase P450 (CACYP51) were constructed and heterologously expressed in Saccharomyces cerevisiae with deletion of the CYP51 gene. With the strains obtained and microsome enzymes separated, cell susceptibility and CACYP51 activity were examined with the 5 novel azole compounds based on the molecular modeling in comparison with fluconazole. After alteration of Y118 with Y118A, Y118F, and Y118T by a single base substitution, the expression levels of CACYP51 protein were not affected. However, these mutations markedly decreased its catalytic activity respectively; the mutation changes also decreased azole susceptibility, indicating the structural importance of the Y118 residue in maintaining CACYP51 activity and in determining azole susceptibility. In addition, our synthetic compounds with the phenyl group side chain attached to C3 produced higher susceptibility against S. cerevisiae with expression of CACYP51 and exhibited more potent inhibitory effects on CACYP51 activity in comparison with fluconazole, suggesting that the phenyl group of C3 side chain substitutes is also important for selective binding to target enzymes.

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Oxidative and nitrosative stress are known to exert various adverse effects on biological systems and this seems to be one of the major contributor of nephrotoxicity induced by cyclosporine A (CsA), which is a major clinical challenge, despite its potent immunosuppressive effect. Sulphated polysaccharides of marine origin are well known for its antioxidant properties, among its other biological applications. CsA administration (25 mg/kg body weight, orally, for 21 d) showed increased level of oxidants and xanthine oxidase activity. CsA induced nitrosative stress was evident from a marked elevation in the expression of inducible nitric oxide synthase mRNA in renal tissue and a concomitant increase in plasma nitric oxide level. Augmented levels of malondialdehyde, 8-hydroxy-2-deoxyguanosine and protein carbonyl coupled with diminished protein thiols; hallmarks of lipid peroxidation, DNA damage and protein oxidation were noted in CsA administered rats. Membrane damage was further confirmed by altered ATPase activities in the renal tissue. Simultaneous treatment with sulphated polysaccharides (5 mg/kg body weight, subcutaneously) remarkably prevented the above alterations mediated by oxidative and/or nitrosative stress during CsA induction. Hence, these findings conclude that the use of an antioxidant agent like sulphated polysaccharides could be a useful tool in reducing CsA-induced nephrotoxicity.

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Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the μ-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) or its analog YRFB (Tyr-D-Arg-Phe-βAla-NH₂) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂ (Ac-RYYRIK-NH₂). All chimeric peptides were found to possess high receptor binding affinities for both μ-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH₂ connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED₅₀ of 1.34—4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED₅₀; 1.08 pmol/mouse). In contrast to their μ-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED₅₀ 5.55—100< pmol/mouse) than when administered i.t. (ED₅₀: 1.34—4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of μ-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on μ-opioid receptor/ORL1 receptor heterodimers.

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Internal transcribed spacer (ITS) regions of nuclear ribosomal RNA gene were amplified from 23 plant- and herbarium specimens belonging to eight Plantago species (P. asiatica, P. depressa, P. major, P. erosa, P. hostifolia, P. camtschatica, P. virginica and P. lanceolata). Sequence comparison indicated that these Plantago species could be identified based on the sequence type of the ITS locus. Sequence analysis of the ITS regions amplified from the crude drug Plantago Herb obtained in the markets indicated that all the drugs from Japan were derived from P. asiatica whereas the samples obtained in China were originated from various Plantago species including P. asiatica, P. depressa, P. major and P. erosa.

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Constituent properties of licorices derived from Glycyrrhiza uralensis, G. glabra, and G. inflata are revealed by comparing 117 of licorice identified using four genetic markers; internal tracscribed spacer (ITS) on nuclear ribosomal DNA, rbcL gene, matK gene, and trnH–trnK1 intergenic region on chloroplast DNA. Regarding six main constituents of licorice; glycyrrhizin, liquiritin, liquiritin apioside, isoliquiritin, isoliquiritin apioside, and liquiritigenin, the constituent property of G. glabra resembles to that of G. inflata. On the other hand, the constituent property of G. uralensis is not similar to that of G. glabra or G. inflata and is characterized by a wide content variation of the six constituents compared to those of G. glabra and/or G. inflata. The mean contents of liquiritin, isoliquiritin, or liquilitigenin in G. uralensis are significantly higher than those of G. glabra or G. inflata. Therefore, the licorice species should be selected depending on these constituent properties for the traditional Chinese medicines or the Japanese Kampo medicines. Additionally, glycycoumarin, glabridin, and licochalcone A were reconfirmed as the species-specific typical constituents of G. uralensis, G. glabra, and G. inflata respectively. Therefore, it is resulted that the determination of the three species-specific constituents may be useful for the species identification of licorice. However, since 6% of licorice examined and hybrids were exceptions to the rule, their genetic information is necessary for the accurate species identification of licorice.

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The present study was performed to evaluate the antihypertensive effects of Morus bombycis KOIDZUMI (MK) in spontaneously hypertensive rats (SHRs). In addition, the effects on vascular responses and cardiac functions were also investigated. In isolated rat aortic preparations, the 100% ethanol extract of MK exhibited a potent vascular relaxant effect with IC₅₀ value of 3.9 μg/ml, and this vasorelaxant effect was completely abolished by pretreatment of the aortic tissues with N^G-nitro-L-arginine methyl ester or the denudation of endothelial layer. In isolated rat hearts, the MK extract significantly reduced cardiac functions such as left ventricular developed pressure and heart rate. In an antihypertensive study in SHRs, long-term administration with MK extracts (10, 30, 100 mg/kg) for 42 d dose-dependently decreased systolic blood pressure (approximately 20 mmHg). In SHRs, MK extract enhanced the aortic relaxation to acetylcholine and sodium nitroprusside after 42 d of treatment. In addition, lipid peroxidation and DNA damage in liver of SHRs were also attenuated by long-term treatment with MK extract. These results suggest that chronic treatment with MK extract exerts an antihypertensive effect in SHRs, and its direct vasorelaxant, negative inotropic actions, and anti-oxidant properties may contribute to reduce the elevated blood pressure.

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In Thailand, there are four Mitragyna species; M. speciosa, M. hirsuta, M. diversifolia, and M. rotundifolia. One, M. speciosa, is a narcotic plant and has medicinal importance for its opium-like effect. Since the use of M. speciosa has been forbidden in Thailand, the leaves of M. diversifolia or others are frequently used as substitutes but are not considered as effective. Therefore, accurate authentication of M. speciosa is essential for both medicinal and forensic purposes. The nucleotide sequences of internal transcribed spacers (ITS) and the 5.8S coding region of nuclear ribosomal DNA (rDNA) of the Mitragyna species were analyzed. The whole length of ITS1-5.8S-ITS2 region was 608 bp in M. speciosa, 607 bp in the other species. Nineteen sites of nucleotide substitutions and 3 sites of 1-bp indels were observed, and M. speciosa showed specific sequence differed from the others. Based on the ITS sequences, a distinctive site recognized by a restriction enzyme XmaI in M. speciosa was found and then PCR-restriction fragment length polymorphism (RFLP) analysis was established to differentiate M. speciosa from the others. By the method, a 409-bp PCR fragment of ITS1-5.8S (partial) rDNA region from M. speciosa was cleaved into two fragments of 119 bp and 290 bp while the other species remained undigested. This method provides an effective and accurate identification of M. speciosa.

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In our previous study, Corni Fructus (Cornus officinalis SIEB. et ZUCC.), a component crude drug of the Chinese prescription Hachimi-jio-gan, was reported to reduce glucotoxicities, up-regulate renal function, and consequently ameliorate glycation-associated renal damage as well as Hachimi-jio-gan. Based upon these facts, we prepared Corni Fructus fractions and evaluated which fraction contained the effective components against diabetes, using one iridoid glycoside and three polyphenol fractions, which were expected to possess stronger activities than Corni Fructus, administered orally at a dose of 20 mg/kg body weight/d for 10 d, respectively. As a result, iridoid glycosides and low molecular weight polyphenol fractions could reduce the pathogenesis of diabetic renal damage, each having different mechanisms, i.e., iridoid glycosides successfully decreased the hyperglycemic state and affected renal advanced glycation end-product (AGE) accumulation, such as Nε-(carboxyethyl)lysine and Nε-(carboxymethyl)lysine, while low molecular weight polyphenol fractions could reduce renal lipid peroxidation, the receptor for AGE, and inducible nitric oxide synthase. Overall, these data suggest that iridoid glycosides and low molecular weight polyphenols purified from Corni Fructus improve metabolic parameters associated with the development of diabetic renal damage. The main active components of these fractions are discussed.

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Embryotoxic and teratogenic effects of curcumin on the development of zebrafish embryo were investi-gated in this study. The LD₅₀ values of curcumin (24-h incubation) were estimated at 7.5 μM and 5 μM for embryos and larvae, respectively. The developmental defects caused by curcumin treatments include bent or hook-like tails, spinal column curving, edema in pericardial sac, retarded yolk sac resorption, and shorter body length. In curcumin-treated larvae, fluorescence signals of curcumin were found in edamae sac and some skin cells. Together, these results indicate that zebrafish are suitable model organisms to study the toxic effects of curcumin.

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A new lignan, sylvestrin (1), was isolated from the MeOH-soluble fraction of the roots of Anthriscus sylvestris HOFFM. (Umbelliferae), along with six lignans (2—7), three coumarins (8—10), and a polyacetylene (11). The structure of sylvestrin was determined to be 2-(3′,4′,5′-trimethoxybenzylidene)-3-(3″,4″-methylendioxybenzyl)-γ-butyrolactone (1) by spectroscopic means, including 2D-NMR. The eleven compounds were assessed for their abilities to activate a caspase-3 in human promyeloid leukemic HL-60 cells. The intracellular caspase-3 activity of (−)-deoxypodophyllotoxin (3), angeloyl podophyllotoxin (5), deoxypicropodophyllin (6), picropodophyllotoxin (7), and falcarindiol (11) increased approximately 4.6, 3.6, 3.7, 3.9, and 3.9-fold, at 0.001, 1, 1, 1, and 20 μM, respectively, over that of the untreated control. In addition, compounds 3, 5, 6, and 7 showed apoptosis-inducing activities that were measured by DNA fragmentation in HL-60 cells.

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Propiverine hydrochloride, oxybutynin hydrochloride and terodiline hydrochloride have both anticholinergic and antispasmodic effects, and are used for the management of urinary frequency and incontinence. The average standard therapeutic doses of these drugs differ greatly. We retrospectively analyzed their pharmacological effects with consideration given to muscarinic acetylcholine receptor binding affinities, anticholinergic activities, and inhibitory effects on KCl-induced contraction. Muscarinic acetylcholine receptor occupancies and the inhibitory ratios of the drugs for both acetylcholine-induced and KCl-induced contraction in a steady state after oral administration of standard doses were calculated based on pharmacokinetics and the receptor occupancy theory. The average muscarinic acetylcholine receptor occupancy and inhibitory ratio of acetylcholine-induced contraction were estimated to be 12.6±1.06% and 3.27±0.74%, respectively, with no significant differences found between the drugs for those parameters. A significant linear relationship was found between muscarinic acetylcholine receptor occupancy and the maximum ratio of increase in bladder urinary capacity. On the other hand, the inhibitory ratios of KCl-induced contraction varied from 0.01 to 0.48%. The present results suggest that muscarinic acetylcholine receptor occupancy is a principal determinant of the therapeutic effect of a drug used for treatment of urinary disturbance.

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Effects of Labrasol and other pharmaceutical excipients on the intestinal transport and absorption of rhodamine123, a P-glycoprotein substrate (P-gp) were examined. Intestinal transport and absorption studies were examined by an in vitro diffusion chamber method and an in situ closed loop method. We evaluated the intestinal membrane damage produced by Labrasol by measuring the release of protein and alkaline phosphatase (ALP). Labrasol (0.075—0.1% (v/v)) increased the absorptive transport of rhodamine123 and decreased its secretory transport in the in vitro transport studies. However, Labrasol did not change the transport of Lucifer yellow, a non-P-gp substrate, suggesting that the effect of Labrasol on the transport of drugs was specific for rhodamine123. We observed almost no intestinal membrane damage in the presence of Labrasol. These findings suggest that the increase in the absorptive transport of rhodamine123 in the presence of Labrasol may not be due to its intestinal membrane damage. In the in situ absorption studies, we found that Labrasol (0.1% (v/v)) significantly enhanced the intestinal absorption of rhodamine123 in rats, although the absorption enhancing effect of Labrasol was much less than that of verapamil. These findings suggest that low concentrations of Labrasol might inhibit the function of P-gp in the intestine, thereby increasing intestinal absorption and bioavailability of P-gp substrates including rhodamine123. However, we may also consider the contribution to the enhanced intestinal absorption of rhodamine123 via a passive transport in addition to the inhibitory action of Labrasol for the function of P-gp in the intestine.

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Bacampicillin was developed as a prodrug to improve the intestinal absorption of its metabolite ampicillin. This study was undertaken to characterize bacampicillin transport in Caco-2 cells. The uptake of bacampicillin in Caco-2 cells was significantly greater than those of ampicillin and pivampicillin. An Eadie–Hofstee plot obtained from 5-min uptake of 0.2—5 mM bacampicillin was linear, indicating the presence of a saturable transport system for bacampicillin with K_m and V_max of 3.6 mM and 23.9 nmol/mg protein/min, respectively. Hydrophilic organic cations such as choline, cimetidine, guanidine, nicotinamide, 1-methylnicotiamide, and tetraethylammonium failed to modulate bacampicillin uptake in Caco-2 cells whereas diphenhydramine, procainamide, and thiamine significantly depressed it. Moreover, when thiamine was preloaded in Caco-2 cells, bacampicillin uptake was significantly increased, indicating that this cationic vitamin was capable of trans-stimulating bacampicillin transport across the apical membrane of Caco-2 cells. However, trans-stimulated bacampicillin uptake was not observed in the presence of diphenhydramine. Bacampicillin uptake increased with elevation of the medium pH, and the known modulators of thiamine transport such as amiloride and oxythiamine significantly inhibited bacampicillin uptake. Thiamine also significantly decreased the apical-to-basolateral transport of bacampicillin across Caco-2 cell monolayers. However, thiamine did not exert any modulating effect on pivampicillin uptake and its apical-to-basolateral permeation in Caco-2 cells. These results suggest that bacampicillin is transported in Caco-2 cells, sharing a carrier-mediated system with thiamine.

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The effect of permeation enhancers on the percutaneous absorption of pentazocine (PTZ) was investigated in excised hairless mice using Franz diffusion cells in vitro. The enhancing effect on the percutaneous absorption of PTZ from the isopropyl myristate (IPM) solution system was improved with glyceryl monocaprylate (GEFA-C₈), which is a kind of glycerol ester of fatty acid (GEFA). The flux of PTZ through the skin was ca. 4 times higher compared with IPM alone, while a less enhancing effect of glyceryl dicaprylate (GEFA-DiC₈) and glyceryl tricaprylate (GEFA-TriC₈) on the skin permeation of PTZ was found. Moreover, maximum enhancement of PTZ flux was observed with glyceryl monocaproate (GEFA-C₆) among various alkyl chains (C₂—C₁₈) of monoglycerides. These results indicated that the IPM solution system combination with GEFA may be used to develop a transdermal formulation with improved skin permeation of PTZ.

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Breviscapine, is the total flavonoid components (the content of scutellarin >85%) extracted from the dried whole plant of Erigeron breviscapus (VANT.) HAND.-MAZZ, and its preparations are generally used in the clinic for the treatment of cerebral and cardio-vascular diseases in China. In this paper, the metabolites of breviscapine in the urine of rats after oral administration were investigated. The ten metabolites were isolated by open-column chromatography and preparative high-performance liquid chromatography, and their structures were elucidated by MS, NMR spectroscopy including ¹H-NMR, ¹³C-NMR, and NOESY (nuclear Overhauser enhancement spectroscopy), enzymatic hydrolysis and chemical evidence. The ten metabolites were identified as scutellarein-6,7-di-O-β-D-glucuronide (M-1), scutellarein (M-2), 6-O-methyl-scutellarin (M-3), 6-O-methyl-scutellarein (M-4), scutellarein-6-O-β-D-glucuronide (M-5), scutellarein-5-O-β-D-glucuronide (M-6), scutellarin (M-7), scutellarein-7-O-sulfate (M-8), apigenin-5-O-β-D-glucuronide (M-9), and apigenin-4′-O-β-D-glucuronide (M-10) respectively. The results of this study indicated that the metabolites of brevisvapine were excreted in rats urine as glucuronidated, sulfated or methylated forms, as well as the aglycone of scutellarin-scutellarein after oral administration, and the metabolic pathways were also proposed.

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β-Sitosterol is the main dietary phytosterol found in plants and has been shown to inhibit proliferation and induce apoptosis in human solid tumors such as colon and breast cancers. However, the mechanism by which β-sitosterol induces apoptosis is not completely understood in leukemic cells. This study investigated the mechanism of apoptosis induced by β-sitosterol in human leukemic U937 cells. β-Sitosterol induced cytotoxicity and apoptosis in U937 cells in a concentration dependent manner, as measured by hemocytometer counts, fluorescence microscopy, agarose gel electrophoresis, and flow cytometry analysis. The increase in apoptosis induced by β-sitosterol was associated with down-regulation of Bcl-2, degradation of poly-(ADP-ribose) polymerase (PARP) and phospholipase C (PLC)-γ1 protein, and activation of caspase-3. β-Sitosterol induced apoptosis was not associated with changes in the expression of Bcl-xL, Bax, or inhibitor of apoptosis proteins (IAPs). z-DEVD-fmk, a caspase-3 specific inhibitor, blocked caspase-3 activation and PARP degradation, and significantly attenuated β-sitosterol-induced apoptosis. This suggests that caspase-3 activation is partially essential for β-sitosterol-induced apoptosis. Bcl-2 overexpression also significantly blocked caspase-3 activation and the decrease in PARP cleavage by β-sitosterol, and effectively attenuated the apoptotic response to β-sitosterol. These results show that β-sitosterol potently induces apoptosis in U937 cells and that β-sitosterol-induced apoptosis is related to the selective activation of caspase-3 and induction of Bax/Bcl-2 ratio.

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Our in vivo assay system developed to search for allergy-preventive substances, assesses the blood flow decrease in tail vein microcirculation of mice subjected to sensitization with hen-egg white lysozyme (HEL). The blood flow decrease appears to be regulated by various factors such as nitric oxide (NO), thromboxane (TX) A₂, prostacyclin (PGI₂) and endothelin (ET)-1 together with cyclooxygenase (COX)-1, COX-2, inducible nitric oxide synthase (iNOS), and constitutive nitric oxide synthase (cNOS). In this study, we examined in detail the roles of iNOS in this assay system using an iNOS knockout (KO) mouse. We found that the blood flow decrease in the HEL-sensitized iNOS KO mice was slightly weaker than that in their wild type (WT) mice. This blood flow decrease was not affected by a selective COX-1 inhibitor, a selective COX-2 inhibitor and a PGI₂ agonist unlike the case of the WT mice. However, it was inhibited by a nonselective NOS inhibitor, a specific TXA₂ synthase inhibitor and a specific ET-1 receptor blocker as in the case of the WT mice. The present results indicate that the blood flow decrease occurs via two pathways; one is an iNOS-independent response involving TXA₂ and ET-1, and the other is an iNOS-dependent response involving COX-1, COX-2 and PGI₂. cNOS appears to play some roles in the blood flow decrease and iNOS acts as an exacerbation factor. Our method using HEL-sensitized should be useful for searching for agents that can prevent allergy via new mechanisms.

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CAWS (Candida albicans water-soluble extracellular polysaccharide fraction) is a water-soluble extracellular mannoprotein-β-glucan complex obtained from the culture supernatant following the culture of pathogenic Candida albicans in a completely synthetic medium. CAWS administered intraperitoneally induces vasculitis in mice, however, administered intravenously, it causes lethal shock. The acute lethal reaction to CAWS occurs within 1 h of intravenous administration, with the mice demonstrating anaphylactic shock-like symptoms including convulsion, diarrhea, and collapse. In this study, we analyzed the factors involved in this lethal effect. We examined physiologically active substances believed to be involved in anaphylactic shock, and found that the lethal effect of CAWS could be inhibited by blocking histamine, serotonin, and platelet activating factor (PAF) simultaneously, but by blocking only one. This finding strongly suggests that the acute lethal reaction to CAWS is a result of the simultaneous production of several physiologically active substances.

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The present study demonstrates the estrogenic effects of fluorotelomer alcohols (FTOHs). In a yeast two-hybrid assay, treatment with 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH), 1H,1H,2H,2H-perfluoro-decan-1-ol (8:2 FTOH) and 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecafluoro-1-decanol (NFDH) showed a dose-dependent interaction between the human estrogen receptor (hER) isoforms hERα or hERβ ligand-binding domain and coactivator TIF2, whereas there were no estrogenic effects of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) for these hERs. The estrogenic effects of FTOHs on hERα were higher than those on hERβ, indicating a differential responsiveness of hERs to FTOHs. The relative ranks of tested chemicals on the estrogenic effects for hERα and hERβ descended in the order of estradiol-17β>>.6:2 FTOH>NFDH>8:2 FTOH. These results suggest that certain FTOHs including 6:2 FTOH, 8:2 FTOH and NFDH interact with hER isoforms α and β in vitro. Further studies are necessary to investigate contamination levels, potential biological effects and the risks of these compounds on human health.

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