The neuroprotective effects of theanine and catechins contained in green tea are discussed. Although the death of cultured rat cortical neurons was induced by the application of glutamic acid, this neuronal death was suppressed with exposure to theanine. The death of hippocampal CA1 pyramidal neurons caused by transient forebrain ischemia in the gerbil was inhibited with the ventricular preadministration of theanine. The neuronal death of the hippocampal CA3 region by kainate was also prevented by the administration of theanine. Theanine has a higher binding capacity for the AMPA/kainate receptors than for NMDA receptors, although the binding capacity in all cases is markedly less than that of glutamic acid. The results of the present study suggest that the mechanism of the neuroprotective effect of theanine is related not only to the glutamate receptor but also to other mechanisms such as the glutamate transporter, although further studies are needed. One of the onset mechanisms for arteriosclerosis, a major factor in ischemic cerebrovascular disease, is probably the oxidative alteration of low-density lipoprotein (LDL) by active oxygen species. The oxidative alterations of LDL were shown to be prevented by tea catechins. Scavenging of ^·O₂⁻ was also exhibited by tea catechins. The neuroprotective effects of theanine and catechins contained in green tea are a focus of considerable attention, and further studies are warranted.

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A simple and sensitive procedure for determination of ethanol in biological samples was established. In this procedure, ethanol in biological samples was first converted to acetaldehyde by yeast alcohol dehydrogenase. Then, acetaldehyde formed was derivatized to 2,4-dinitrophenylhydrazone, which was determined by gas chromatography with an electron-capture detection. Ethanol concentration in 1 ml of rat blood plasma can be measured as 19.5 nmol/ml with 98.0% recovery. Since this procedure enable to determine minute amount of ethanol in biological samples, the method is useful to study the metabolism of ethanol.

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The purpose of this study was to establish a simple and rapid method for selecting a nonionic surfactant to prepare an emulsion that can maintain a stable emulsification phase. As an index of the degree of emulsification, the white chromaticity of the prepared sample was measured using a color difference meter. When liquid paraffin was used as an oil, the color difference (dE(H)) was shown as a change in a V-shaped curve depending on changes in the hydrophile-lipophile balance (HLB) number of sorbitan ester, polyoxyethylene sorbitan ester, or polyethyleneglycol ester. Although there was a difference in the minimal value of dE(H) in these experiments, the dE(H) was similar to the required HLB of liquid paraffin (HLB=10). On the other hand, no relationship was found between the HLB number and color difference when polyglycerol ester was used. It seems that there was possibility of the effect by the lowering of apparent HLB with a collapse of the hydration. These results indicate that color difference could be utilized for the selection of surfactants, except for polyglycerol ester.

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We previously reported that a human analogue of pulmonary surfactant protein-C (SP-C), SP-CL16 (6—28), with 23 residues was the most active analogue in a reconstituted lipid mixture and had the shortest chain among the poly leucine analogues examined.
In this study, we verified the influence of albumin, a component of serum, on the surface activity of surfactant. Surface activity was measured using the Langmuir–Wilhelmy surface balance (WSB), pulsating bubble surfactometer (PBS), and stable microbubble test (MBT). The surface activity of synthetic lung surfactant (SLS) was only slightly influenced by albumin (0.1—10 mg/ml) as compared with that of a ternary mixture of phospholipids. The ternary mixture of phospholipids showed a decrease in surface activity due to albumin. In particular, SLS did not show interaction of surface activity with albumin in vitro (WSB, PBS, and MBT). In contrast, dipalmitoylphosphatidylcholine/phosphatidylglycerol/palmitic acid had significantly weaker surface activity in the presence of albumin. Surfactant-TA showed interaction of surface activity with albumin in the MBT. The number of stable microbubble increased in the presence of albumin at a concentration of 0.1 mg/ml.

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The effect of the 14-3-3 protein, an adaptor protein of intracellular signal pathways, on histamine release from rat peritoneal mast cells was investigated. The exogenous 14-3-3 protein from bovine brain increased the Ca²⁺-dependent histamine release from permeabilized mast cells, but only slightly affected the non-permeabilized cells. Partial amino acid sequences showed that the bovine brain 14-3-3 protein contained 14-3-3β, γ and ζ isoforms, and that these recombinant isoforms were prepared. Among them, 14-3-3ζ was an active species while the 14-3-3β and γ were inactive for histamine release from the permeabilized mast cells. Approximately 15% of the histamine release was stimulated by 14-3-3ζ at 2.5 μM, and half-maximal stimulation occurred at 1 μM. Treatment of the mast cells with wortmannin or staurosporine completely inhibited the stimulatory effect on histamine release caused by Ca²⁺ or Ca²⁺/14-3-3ζ, and genistein partially inhibited both stimulatory effects. PD 98059, however, had little effect on the histamine release. These results suggest the possibility that 14-3-3ζ is associated with signal transduction for degranulation of the mast cells.

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SodD, a Cu/Zn superoxide dismutase in Dictyostelium discoideum, shows 48% identity to the cytosolic Cu/Zn superoxide dismutase (SOD) of Saccharomyces cerevisiae (SOD1). The sodD gene is expressed in D. discoideum cells at late-developmental stages. However, gene expression was not detected in the sporeless mutant, indicating that sodD is a spore cell-specific gene. The D. discoideum mutant, in which sodD was disrupted, grew and formed a multicellular structure normally, therefore the gene is not essential for growth and development. The mutant spores were sensitive to UV-light compared to the wild-type spores, indicating that SodD protects spores from cellular damage caused by UV-light.

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A novel drug efflux gene (named sepA, staphylococcal efflux pump gene) is cloned from antiseptic-resistant mutants of Staphylococcus aureus into Escherichia coli. The sepA gene conferred the reduction of susceptibility to acriflavine and the acceleration of ethidium bromide efflux from the E. coli cells. The sepA (474 bp) encoded the protein that has four predicted transmembrane segments. These results indicate that sepA gene is a multidrug-resistant gene and encodes a drug efflux protein.

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Binding of Fas ligand to Fas induces apoptosis. The Fas–Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. We have previously obtained the mouse anti-Fas antibody HFE7A (m-HFE7A), which specifically induces apoptosis in inflammatory cells. In order to apply m-HFE7A for human therapy, we performed antibody humanization of m-HFE7A by grafting the mouse complementarity-determining regions (CDRs) to a human antibody. Five versions of humanized HFE7A (h-HFE7A) demonstrated the same antigen-binding affinity and same competition-binding activity against Fas as the chimeric HFE7A. Furthermore, these h-HFE7As induced the same degree of apoptosis in WR19L12a cells that express human Fas on their surface as chimeric HFE7A does. To further probe the structural basis for antibody humanization, we determined the three-dimensional structure of the h-HFE7A antigen-binding fragment (Fab) by X-ray crystallography and compared it with the crystal structure of the parent m-HFE7A Fab previously determined. The main-chain conformation in each h-HFE7A CDR is almost identical to that in m-HFE7A with root mean square (rms) deviations of 0.14—0.77 Å. However, a significant segmental shift was observed in the CDR-L1 loop. Together with the high temperature factors of the CDR-L1 residues, both the loops are flexible, suggesting that the CDR-L1 loop would undergo conformational change upon binding to the antigen. Our results indicate that the humanization of m-HFE7A succeeded in maintaining the main-chain conformation as well as the flexibility of the CDR loop.

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We previously found that multiple intraperitoneal administration of magnolol from Magnolia obovata inhibited tumor metastasis and growth in vivo, and that the anti-metastatic effect of magnolol was due to the inhibition of the tumor cell invasion. The purpose of this study was to clarify the inhibitory mechanism of magnolol on the growth of tumor cells, and we expect that magnolol may have the ability to induce apoptosis in tumor cells. In an in vitro proliferation assay, 100 μM of magnolol inhibited the proliferation of B16-BL6, THP-1, BAE and HT-1080 cells, but 30 μM of magnolol did not affected cells proliferation. In addition, 100 μM of magnolol induced apoptotic cell death within 24 h in three tumor cell lines, B16-BL6, THP-1 and HT-1080, not BAE cells, and then up-regulated the activity of caspase-3 and caspase-8. The up-regulation of caspases activity by 100 μM of magnolol was suppressed by the inhibitor of all caspases, z-VAD-fmk. These data suggest that magnolol possesses ability to inhibit tumor growth, and the ability is due to the induction of apoptosis with the activation of caspases.

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In the present study, the effect of Ginseng radix on cell proliferation in the dentate gyrus of rats with streptozotocin-induced diabetes was investigated via immunohistochemistry. Aqueous extract of Ginseng radix was shown to exert no significant effect on weight in normal rats, while it prevented weight loss in rats with streptozotocin-induced diabetes. Cell proliferation in the dentate gyrus of diabetic rats was increased by Ginseng radix treatment, but it had no effect on cell proliferation in normal rats. These results suggest that Ginseng radix may help in improve the central nervous system complications of diabetes mellitus.

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Overcoming MDR (multidrug resistance) phenomena is a crucial aspect of cancer chemotherapy research. Artemisinin and its derivatives have been found to inhibit the proliferation of cancer cells in the μM range. They poorly inhibited the function of P-glycoprotein and did not inhibit the function of MRP1-protein. The concentrations required to inhibit by 50% the function of P-glycoprotein are 110±5 μM. Artemisinin, artesunate and dihydroartemisinin efficiently decreased the mitochondrial membrane potential, leading to a decrease in intracellular ATP in all cell lines tested: by 30 to 50% at 5 μM. Artemisinin, artesunate and dihydroartemisinin increased cytotoxicity of pirarubicin and doxorubicin in P-glycoprotein-overexpressing K562/adr, and in MRP1-overexpressing GLC4/adr, with the δ_0.5 ranging from 200 to 860 nM, but not in their corresponding drug-sensitive cell lines. In this range of concentrations these compounds did not decrease the function of P-glycoprotein, suggesting a mechanism by which the drugs did not reverse MDR phenomenon at the P-glycoprotein level but at the mitochondrial level.

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The pharmacological characteristics of Ryokan-kyomi-shinge-nin-to (RKS), a traditional oriental herbal (Kampo) medicine which has been used for the treatment of allergic asthma and rhinitis, were investigated. The number of sneezes by actively sensitized mice after a topical antigen challenge was significantly reduced by pretreatment with RKS (300 and 1000 mg/kg, p.o.). Although RKS did not inhibit the antigen-induced histamine release from rat peritoneal exudate cells (PEC), it significantly inhibited an increase in vascular permeability induced by histamine and serotonin. These results suggest that RKS has antiallergic activity in animals, and the functional antagonism of a histamine response may be one of the mechanisms of its effect. In addition, RKS prevented histamine hypersensitivity in actively sensitized mice. Because RKS did not affect sleeping time induced by pentobarbital in mice and did not inhibit gastric emptying in rats, the drug appears to be useful for treating allergic patients suffering from classical antihistamines side effects such as stomach discomfort or relative drowsiness.

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To gain insight into the nature of the substrate binding site and the catalytic function of aromatase, we studied the inhibition of androstenedione aromatization by 4β,5β-epoxy-16α-hydroxyandrostenedione (4) and its 19-hydroxy and 19-oxo derivatives, 5 and 6, as well as the biochemical aromatization of these steroids in human placental microsomes. The 19-methyl and 19-oxo compounds, 4 and 6, were weak competitive inhibitors of aromatase, with apparent K_i values of 246 μM and 270 μM, respectively, whereas the 19-hydroxy compound 5 inhibited aromatase in a non-competitive manner with the K_i of 135 μM. The 19-methyl compound 4 inactivated aromatase in a time-dependent manner with k_inact of 0.213 min⁻¹ in the presence of NADPH in air, but the other two did not cause it. The conversion of the three epoxides into estrogen, as well as 19-oxygenation of 19-methyl steroid 4 with human placental microsomes in the presence of NADPH in air, were not detected by gas chromatography-mass spectrometry. The present results are consistent with the two binding sites theory in the active site of aromatase.

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Activating the noradrenergic and cholinergic systems is known to enhance attentional processes, while stimulating dopaminergic, serotonergic, and GABAergic systems suppresses them. The objective of the present study was to investigate the pharmacological characterization in the attentional processes of a two-lever choice reaction time (CRT) task using different centrally acting drugs. We designed seven parameters in this task: the correct response (CR) rate; error response rate; nonresponse (NR) rate; differential reinforcement of other behavior (DRO) responses; number of incorrect lever pressings during both the intertrial interval and DRO periods; the mean CRT of CR; and activity during 30 trials. The compounds produced different profiles at each dose. 1) Facilitative and disruptive effects on attentional processes occurred with changes in CRT alone. Scopolamine (0.1 mg/kg) and prazosin (0.3—1 mg/kg) prolonged the CRT, whereas methamphetamine (0.3 mg/kg) shortened the CRT. 2) Attentional deficits occurred with abnormal behavior showing premature response or perseverative behavior. Scopolamine (0.2—1 mg/kg), methamphetamine (3 mg/kg), Δ⁹-tetrahydrocannabinol (10 mg/kg), and MK-801 (0.1—0.3 mg/kg) produced a marked increase in the number of total lever pressings. 3) Motor function deficits rather than attentional deficits occurred. 8-OH DPAT (1 mg/kg) and muscimol (1 mg/kg) produced a decrease in CR and an increase in NR with a marked decrease in activity and prolonged the CRT. Activating noradrenergic α₁ receptors was found to enhance the attentional processes, while blocking muscarinic receptors, α₁ receptors, and NMDA receptors, and stimulating cannabinoid receptors and the dopaminergic systems impaired the attentional processes in the two-lever CRT task.

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Effects of FR194738 ((E)-N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[2-methyl-2-(3-thienylmethoxy)propyloxy]benzylamine hydrochloride), a squalene epoxidase inhibitor, on lipid metabolism were compared with those of pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in hamsters. Drugs were given for 10 d either as a diet mixture or as a bolus oral gavage, and similar results were obtained with each type of administration. FR194738 (0.01—0.32% as a diet mixture; 10—100 mg/kg as an oral gavage) dose-dependently decreased serum total cholesterol, non high density lipoprotein (HDL) cholesterol, HDL cholesterol and triglyceride levels, and the changes in serum parameters were similar. Pravastatin (0.01—0.32% as a diet mixture; 1—100 mg/kg as an oral gavage) increased serum cholesterol levels, and dose-dependently decreased serum triglyceride levels. Although oral gavage of FR194738 at 32 mg/kg and pravastatin at 3.2 and 10 mg/kg increased hepatic HMG-CoA reductase activity, the degree of the changes was far greater with the latter than the former drug. FR194738 slightly increased hepatic cholesterol content at 32 mg/kg, whereas pravastatin dose-dependently increased hepatic cholesterol content until it leveled off at 32 and 100 mg/kg. It is concluded that inhibition of squalene epoxidase and HMG-CoA reductase triggers both hypercholesterolemic (hepatic cholesterol synthesis) and hypocholesterolemic (hepatic cholesterol uptake) mechanisms. FR194738 appears to induce a greater enhancement of the latter rather than the former, whereas pravastatin has a greater effect on the former.

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Antisense oligodeoxynucleotides (asODN) are novel therapeutic agents designed to alter RNA metabolism, ultimately resulting in decreased production of disease-associated gene products. To investigate internalisation of liposomally delivered asODN in NG108-15 cells, a hybrid cell line of mouse neuroblastoma and rat glioma, and assure that uptake of marker corresponds to that of antisense, we compared the cellular uptake of fluorescently labelled marker (fluorescein isothiocyanate (FITC)–dextran) and antisense oligonucleotide (FITC–asODN), entrapped either in conventional soy phosphatidylcholine (SPC) liposomes or pH-sensitive liposomes (composed of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate in a molar ratio of 3 : 2). Both SPC and pH-sensitive liposomes were prepared by a modified freeze-thawing method. Entrapment efficiencies (about 20% of the original material) did not depend on the liposome compositions and fluorescent material used. Fluorescence activated cell sorting (FACS) analysis was used to quantify the association of fluorescent material with the NG108-15 cells, whereas confocal microscopy gave insight on the location of cell associated-fluorescence. Conventional liposomes failed to deliver fluorescent material into the cells, but in contrast, pH-sensitive liposomes significantly improved the uptake of both FITC–dextran and FITC–asODN, with the uptake of liposomal FITC–dextran being greater than the uptake of liposomal FITC–asODN. These results suggest that pH-sensitive liposomes can be applied as a carrier system in the delivery of genetic material into the cells.

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We previously reported that pressure loading of the vascular wall can activate mitogen-activated protein kinases (MAPKs), enzymes believed to be involved in the pathway for cell proliferation, partly via the vascular angiotensin system in isolated perfused rat aorta. In this study, we examined whether cyclic stretching of vascular smooth muscle cells (VSMC) also produces activation of p42 and p44 MAPKs in cultured rat VSMC and whether stretch-induced MAPK activation is mediated via angiotensin and endothelin systems in VSMC. Cyclic stretching of VSMC produced an elongation-dependent and frequency-dependent increase in p42 and p44 MAPK activity. The stretch-induced p42 and p44 MAPK activation was inhibited by the angiotensin receptor antagonist losartan and by the angiotensin-converting enzyme inhibitor, captopril. The MAPK activation was also inhibited by the endothelin receptor antagonist cyclo(D-α-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl) (BQ123) and by the endothelin-converting enzyme inhibitor phosphoramidon. Replacement of medium with culture medium of stretched cells caused MAPK activation, which was inhibited by losartan and BQ123. The results of the present study suggest that cyclic stretching of VSMC can activate p42 and p44 MAPKs and that the MAPK activation is mediated via angiotensin and endothelin systems in VSMC.

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Ethanol and aqueous extracts of Carica papaya has been evaluated for its anti hepatotoxic activity. The ethanol and aqueous extracts of Carica papaya showed remarkable hepatoprotective activity against CCl₄ induced hepatotoxicity. The activity was evaluated by using biochemical parameters such as serum aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase, total bilirubin and gamma glutamate transpeptidase (GGTP). The histopathological changes of liver sample was compared with respect to control.

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Natural killer (NK) cells are directly cytotoxic for tumor cells and play a primary role in regulating immune responses. We monitored levels of NK cell cytotoxic activity in cancer patients receiving D-Fraction extracted from maitake mushrooms (Grifola frondosa). Elevated levels of cytotoxic activity were maintained for one year. To elucidate the mechanisms underlying long-term activation of NK cells during treatment with D-Fraction, we examined tumor volume and levels of IFN-γ and TNF-α in MM46-bearing C3H/HeN mice to which D-Fraction was administered for 19 d. D-Fraction markedly suppressed tumor growth, corresponding with increases in TNF-α and IFN-γ released from spleen cells and a significant increase in TNF-α expressed in NK cells. This suggests that the D-Fraction activates NK cells even on the 20th day after treatment. Furthermore, D-Fraction increased macrophage-derived interleukin (IL)-12, which serves to activate NK cells. These results suggest that NK cells are not only responsible for the early effects of D-Fraction on tumor growth, but also for the long-term tumor-suppressive effects of D-Fraction through increased IL-12 released from macrophages.

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The botanical origins of Chinese and Japanese Curcuma drugs were determined to be Curcuma longa, C. phaeocaulis, the Japanese population of C. zedoaria, C. kwangsiensis, C. wenyujin, and C. aromatica based on a comparison of their 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously. Moreover, to develop a more convenient identification method, amplification-refractory mutation system (ARMS) analysis of both gene regions was performed on plants. The ARMS method for the 18S rRNA gene was established using two types of forward primers designed based on the nucleotide difference at position 234. When DNAs of four Curcuma species were used as templates, PCR amplification with either of the two primers only generated a fragment of 912 base pairs (bp). However, when DNAs of the purple-cloud type of C. kwangsiensis and C. wenyujin were used, PCR amplifications with both primers unexpectedly generated the fragment, suggesting that these two were heterozygotes. The ARMS method for the trnK gene was also established using a mixture of four types of specific reverse primers designed on the basis of base substitutions and indels among six species, and common reverse and forward primers. C. phaeocaulis or the Chinese population of C. zedoaria, the Japanese population of C. zedoaria or the purple-cloud type of C. kwangsiensis, the pubescent type of C. kwangsiensis or C. wenyujin, and C. aromatica were found to show specific fragments of 730, 185, 527 or 528, and 641 or 642 bp, respectively. All species including C. longa also showed a common fragment of 897—904 bp. Using both ARMS methods, together with information on producing areas, the identification of Curcuma plants was achieved. Moreover, the ARMS method for the trnK gene was also useful for authentication of Curcuma drugs.

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To find new anthelmintics against parasites living in host tissues, we used an in vitro assay to screen isoquinoline alkaloids for nematocidal activity on the larva of dog roundworm, Toxocara canis. To evaluate the efficacy of anthelmintics in vitro, Tsuda et al. previously introduced the concept of Relative Mobility (RM) of Toxocara larvae. After improvement of the assay system using image data processing, we generated a new index, RM₅₀, the concentration at which RM=50%. However, except for pyrantel, the RM₅₀ of most existing anthelmintics could not be calculated because of low activity. Of the isoquinoline alkaloids tested, emetine, sanguinarine, 6-methoxydihydrosanguinarine (6-MS), chelerythrine and berberine showed strong nematocidal activities. However, these compounds were highly cytotoxic; thus, the prospect of their direct application is low. We then tested the cytotoxicity (IC₅₀) of other isoquinoline alkaloids in HL60 tissue-culture cells. We continued our search for new anthelmintics by examining in detail the relationship between RM₅₀ and IC₅₀. We determined that an ideal target compound would exhibit a low RM₅₀/IC₅₀ ratio. Allocryptopine, dehydrocorydaline and papaverine were identified as potentially effective anthelmintics.

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The effects of conjugated linoleic acid (CLA) against anaphylaxis and allergic pruritus were investigated using a in vivo assay. Inhibitory effects of CLA were observed on the immediate (type 1) hypersensitivity reaction, with CLA significantly suppressing the decrease in blood pressure (BP) and blood flow (BF) induced by the hen egg-white lysozyme (HEL)-anaphylactic reaction in ddY mice. After oral administration, CLA showed antipruritic activity, with significant inhibition of scratching behavior induced by compound 48/80 (COM), a histamine-release agent. When painted onto the skin, CLA also inhibited COM, platelet-activating factor, and protease-induced scratching behavior, and COM-induced vasodilation of the skin. CLA offers promise as a drug for the treatment of allergic and inflammatory pruritus not only as an oral but also a topical agent. The present findings demonstrate that CLA can be effective for the prevention and treatment of allergic disease with severe pruritus.

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Dimethyl sulfoxide (DMSO) is usually used to solubilize poorly soluble drugs in permeation assays such as that using Caco2 enterocyte-like cells. The objective of this study was to evaluate the toxicity of DMSO on Caco2/TC7 cells and determinate the maximal concentration usable in permeation experiments. Caco2/TC7 cells were cultured for 21 d on 96-well plates for evaluation of toxicity. The determination of lactate dehydrogenase (LDH) release in cell supernatant and the measurement of Neutral Red (NR) uptake are used for cytotoxicity assays. DMSO solutions (0—100%) in Hank's balanced salt solution containing HEPES (25 mM), pH 7.4, were incubated with Caco-2/TC7 cells on 96 well plates. Caco2/TC7 cells were cultured on Transwell-Clear^® inserts to evaluate the influence of DMSO on the apparent permeability of the paracellular marker mannitol. DMSO 10% did not induce any significant increase in LDH release whereas a significant increase in LDH activity (ANOVA, p<0.05) occurred at a DMSO concentration of 20 to 50%. NR incorporation in viable cells was statistically reduced by 27 to 36% at DMSO concentration of 20% up to 100% (ANOVA, p>0.05). No statistical difference (p<0.05) in apparent mannitol permeability was observed between the control and 10% DMSO groups. In conclusion, at concentrations of up to 10%, DMSO did not produce any significant alteration in apical membrane permeability or on cell-to-cell tight junctional complexes.

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The multidrug transporter, MDR1-mediated interaction of digoxin with antiarrhythmic or antianginal drugs was examined in vitro by using the MDR1-overexpressing LLC-GA5-COL150 cells, which were established by transfection with human MDR1 cDNA into porcine kidney epithelial LLC-PK₁ cells. Amiodarone, its active metabolite monodesethyl-amiodarone (DEA), and quinidine markedly inhibited the basal-to-apical transport (renal secretion) of [³H]digoxin and increased the apical-to-basal transport (reabsorption), but cibenzoline and lidocaine showed slight inhibition of the transport, and disopyramide and mexiletin had no such effects. The IC₅₀ values for amiodarone, DEA and quinidine on [³H]digoxin transport in LLC-GA5-COL150 cells were 5.48 μM, 1.27 μM and 9.52 μM, respectively. These were comparable to, or only several times the achievable concentration in clinical use, suggesting that MDR1 could be responsible for the drug interaction between digoxin and amiodarone found in clinical reports and that DEA contributes the elevation of digoxin serum concentration. Similarly, dipyridamole altered the transport, but isosorbide showed only slight modification of the transport. The IC₅₀ value for dipyridamole was 40.0 μM, also only several times the achievable concentration in clinical use, indicating a risk of interaction.

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The aims of this study were to examine the enhancing effects of aloe-emodin anthrone (AEA) on the colonic membrane permeability of water-soluble and poorly permeable compounds and to clarify the mechanism of the permeation-enhancing activity of AEA. The permeation-enhancing activity of AEA was estimated from changes in the permeability coefficient of 5(6)-carboxyfluorescein (CF) in rat colonic mucosa using a Ussing-type chamber. Various inhibitors were used to investigate the mechanism of action of AEA. The structural change in the membrane and the cytotoxicity of AEA in the intestinal mucosa were evaluated by measuring the electrical resistance of the membrane (R_m) and lactate dehydrogenase (LDH) activity, respectively. AEA significantly increased the permeation of CF in a dose-dependent manner. The enhanced permeability was significantly suppressed by a histamine H₁ receptor antagonist, pyrilamine, and a mast cell stabilizer, ketotifen, but not by a histamine H₂ receptor antagonist, cimetidine. The enhancing effect was also inhibited by an inhibitor of protein kinase C (PKC). Potential difference and short-circuit current values decreased, while R_m values remained constant throughout the experiment. The addition of AEA to the mucosal solution decreased R_m to 30%, but then remained constant. LDH activity with AEA was not significantly different from that of the control. In conclusion, AEA is a candidate for effective absorption enhancers without damage of the membrane and cytotoxicity. We propose that AEA stimulates mast cells within the colonic mucosa to release histamine, which probably bind to the H₁ receptor. The intracellular PKC route activated by H₁ receptor activation enhances the permeability of water-soluble and poorly permeable drugs via opening of tight junctions in rat colonic membrane.

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The pharmacokinetics of ketotifen fumarate (KF) was examined after administration in rabbits through four different routes (intravenous, intranasal, oral and rectal). The time–course of the plasma concentration of KF after intravenous administration (1 mg/kg dose) fitted a two-compartment open model. KF was rapidly absorbed and showed a high plasma concentration within 0.33 h after intranasal administration. The absolute bioavailability of KF after intranasal administration was 66%. After oral administration at a dose of 1 mg/kg, the plasma concentration of KF was below the detection limit of HPLC analysis. Even at 5 mg/kg, the value of the area under the plasma concentration–time curve (AUC) after oral administration of KF was significantly lower than that after intranasal administration of 1 mg/kg. Oral bioavailability was only 8%. The very low bioavailability of KF after oral administration might be due to the first-pass effect in the liver. We also prepared suppositories containing KF (1 mg/kg) for rectal administration in rabbits. After rectal administration, KF was rapidly absorbed and its bioavailability was 34%. These results indicated that the intranasal route appears the most effective for administering KF, and that rectal administration may be superior to oral administration in terms of bioavailability.

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The glucocorticoid receptor regulates gene expression mainly by two mechanisms; transactivation and trans-repression. A ligand with strong transrepression and weak transactivation activity is predicted to be a beneficial agent with potent anti-inflammatory activity and minor adverse effects. Recently, the profile of a synthetic steroid, RU24858, has been reported to fulfill this condition in vitro, but others have reported no dissociation between the anti-inflammatory activity and side effects in vivo. To gain further information on the profile of this compound, we evaluated its transactivation ability using a reporter gene analysis both in vitro and in vivo. In the in vitro analysis, RU24858 demonstrated only a weak transactivation activity in HeLa cells, when compared with prednisolone. In CV-1 cells, however, these two glucocorticoids exhibited equivalent transactivation activities. This behavior is independent of whether the reporter gene is regulated by mouse mammary tumor virus promoter or multiple copies of glucocorticoid response element. When the reporter plasmid was inoculated into mouse abdominal skin using a gene gun, followed by orally administration of glucocorticoids, RU24858 induced significantly higher reporter enzyme activity than prednisolone. These results suggest that the profile of RU24858 is divergent and its transactivation ability is comparable to prednisolone depending on the cell-type both in vitro and in vivo.

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The aim of this study was to evaluate the reduction in skin barrier function caused by pulsed iontophoresis by measuring resistance in the short term. Experiments under direct current (DC) and pulsed direct current (PDC) conditions were carried out using rat abdominal skin in vivo. The resistance was measured every 62.5 μs and analyzed using a two-compartment model consisting of surface and skin resistance. Moreover, the initial value and the rate constant of surface resistance were calculated with the non-linear approximation program. Using this method, effects of duty cycle, waveform, frequency and mode on the skin barrier function were examined. The barrier function decreased with increasing duty cycle. With regard to waveforms, the influence on the skin barrier function was greatest in the order of sine, rectangular, saw and triangular waveforms. A frequency of less than 100 Hz reduced the barrier function. Considering the reduction in barrier function and skin polarization, the PDC of a rectangular waveform (around 75% as duty cycle and less than 100 Hz as frequency) or sine waveform was most suitable. In addition, the difference in the amount of drug delivered by DC and by PDC was discussed.

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Citrus aurantium L. is commonly used as an alternative treatment for insomnia, anxiety and epilepsy. Essential oil from peel (EOP) and hydroethanolic (70% w/v) extract (HE) from leaves were obtained. Hexanic (HF), dichloromethanic (DF) and final aqueous (AF) fractions were obtained from HE by successive partitions. Swiss male mice (35—45 g) were treated orally with 0.5 or 1.0 g/kg of these preparations 30 min before the experiments for the evaluation of the sedative/hypnotic activity (sleeping time induced by sodium pentobarbital – SPB: 40 mg/kg, i.p.), anxiolytic activity (elevated plus maze – EPM) and anticonvulsant activity (induced by pentylenetetrazole – PTZ: 85 mg/kg, sc or by maximal electroshock – MES: 50 mA, 0.11 s, corneal). The results showed that EOP (0.5 g/kg) increased the latency period of tonic seizures in both convulsing experimental models. This effect was not dose-dependent. Treatment with 1.0 g/kg increased the sleeping time induced by barbiturates and the time spent in the open arms of the EPM. Specific tests indicated that the preparation, in both doses used, did not promote deficits in general activity or motor coordination. HF and DF fractions (1.0 g/kg) did not interfere in the epileptic seizures, but were able to enhance the sleeping time induced by barbiturates. The results obtained with EOP in the anxiety model, and with EOP, HF and DF in the sedation model, are in accord with the ethnopharmacological use of Citrus aurantium L., which could be useful in primary medical care, after toxicological investigation.

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We examined the serum concentrations of Δ⁵-3β-hydroxysteroids, pregnenolone (Preg), 17-hydroxypregnenolone (17-OH-Preg), dehydroepiandrosterone (DHEA), androstenediol (ADIOL) and their sulfates in 30 well controlled (Group I: HbA1c<7.0%) and 15 poorly controlled (Group II: HbA1c>7.1%) type 2 diabetic patients, and 30 normal controls. These patients were treated with diet therapy or anti-diabetic agent. The distribution of gender and age of the subjects were matched between the groups. The serum levels of sulfo-conjugated and unconjugated steroids described above were measured by GC-MS and enzyme immunoassay (EIA), respectively. The serum levels of the entire sulfo-conjugated steroid measured in this study were significantly lower in Groups I and II than in controls. On the other hand, Preg levels in both Groups I and II were significantly higher than those in controls, whereas the serum levels of the downstream unconjugated steroids were not different from controls. To investigate the effect of sulfonylurea (SU) on the serum levels of steroids, the serum concentrations of steroids between the patients who were treated with diet therapy and SU agent were compared in Group I. No significant differences were observed between both groups. These results suggest that (1) since increased Preg levels did not cause any changes in the downstream Δ⁵-3β-hydroxysteroid levels, the metabolic pathway of Δ⁴-3-ketosteroids may be accelerated in type 2 diabetes; (2) serum steroid levels were not affected by SU treatment; (3) sulfo-conjugated steroid catabolism was altered in type 2 diabetes; (4) the decreased sulfo-conjugated steroids especially ADIOLS may contribute to the alteration of sex steroid levels and onset or exacerbate infectious diseases in diabetes.

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Therapeutic effects of fosfomycin (FOF) and imipenem (IPM) were investigated in a novel model for endotoxin shock that was caused by intraperitoneal (i.p.) infection with 10⁸ colony forming units of attenuated Salmonella typhimurium. Acute lethal shock was observed in BALB/c and ddY but not in lipopolysaccharide (LPS)-nonresponder BALB/lps^d mice. Effects of FOF, but not its enantiomer, and IPM were dose- and time-dependent, since therapeutic efficacy was demonstrated in mice injected i.p. or orally at doses of more than 20 mg/kg 15 min before or 1 h after infection. Treatment with FOF 1 h postinfection (p.i.) resulted in significant decreases in bacterial numbers in spleen and liver, suggesting that the antimicrobial activity of FOF seems to closely correlate to suppression of infection-induced lethal shock. Regarding coagulation systems, FOF inhibited increase in the prothrombin time but upregulated fibrinogen concentration. Plasma levels of LPS released from bacilli were significantly higher in FOF- than IPM-treated mice and infection controls, but both antibiotics showed similar efficacy in protection.

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