We have applied a color-developing reagent, p-nitrobenzene diazonium fluoroborate (diazo reagent) as a post-column derivatization tool for the specific determination of acetoacetate (AcAc) in high performance liquid chromatography (HPLC). A mobile phase consisting of 50 mM KH₂PO₄, 4 mM tetra-n-butylammonium phosphate (TBAP) as an ion-pair reagent and 2 v/v% methanol, pH 3.5, diazo reagent solution with 0.2% triton X-100, and alkaline solution of 1.5 mol/l NaOH were pumped using three independent pumps. Specific color development on-line was monitored at 645 nm. A calibration curve for AcAc standard solution with an injection volume of 20 μl showed a good linearity in the range 0.01—2.5 mM with a correlation coefficient of 0.999. For the determination of 3-hydroxybutyrate (3-HOBA), 3-HOBA was converted to AcAc by an enzymatic-coupling method using 3-HOBA dehydrogenase and lactate dehydrogenase. Analytical recoveries of AcAc and 3-HOBA added to serum and urine were satisfactory.

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We found that glufosinate (DL-GLUF) was distributed in the spinal fluid in glufosinate poisoning. A 50-year old Japanese man (weighing 67 kg) attempted to commit suicide by ingesting about 100 ml of BASTA (containing DL-GLUF 18.5 g; ratio of D-GLUF to L-GLUF: 1 : 1). He was transported to our hospital, where serious respiratory depression was seen 26 h after ingestion, and management with artificial ventilation was initiated. The D-GLUF concentration 1 h after ingestion was 191.1 μg/ml, almost the same as that of L-GLUF 193.5 μg/ml, but by 3 h after ingestion, these levels had sunk to 60.3 μg/ml and 52.3 μg/ml, respectively, with the concentration of L-GLUF lower than that of D-GLUF. Later, at 27 and 35 h after ingestion, the D-GLUF level was still higher than the L-GLUF level, and the total amounts of urinary excretion were 2835 mg for D-GLUF and 2298 mg for L-GLUF, each variable thus showing a difference between the enantiomers. Cerebrospinal fluid taken from the patient 27 h after poison ingestion revealed the presence of DL-GLUF on CG-MS analysis, and quantitative HPLC analysis of the enantiomers indicated that the D-GLUF concentration was 0.48 μg/ml, and the L-GLUF concentration 0.12 μg/ml. The levels in blood collected at the same time were: D-GLUF, 1.44 μg/ml, and L-GLUF, 0.35 μg/ml. Also, the cerebrospinal fluid contained about one-third of the blood levels of both DL-GLUF enantiomers. He was discharged without any sequelae after 11 d of hospitalization.

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The application of brassinolide (BL) to the lamina joint region of rice (Oryza sativa L., cv. Nipponbare) seedlings caused marked bending of laminae, and BL influenced rice root growth under intact conditions. A remarkable increase in lamina inclination at BL 1 μM and an evident increase in root elongation at BL 0.01 μM were observed compared with the control. A total of 786 and 508 proteins extracted from the lamina joint and root, respectively, were detected in images of two-dimensional polyacrylamide gel electrophoresis with isoelectric focusing or immobilized pH gradient tube gel, and 21 proteins changed by BL treatment were analyzed using a gas-phase protein sequencer and mass spectrometer. Proteins related to photosynthesis and stress tolerance were mainly found in the lamina joint and root, respectively. Advanced degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) large subunit was caused by both BL and ethylene. Calmodulin, which decreased in roots treated with BL, increased as a result of ethephon treatment. Proteins that showed homology to glutathione S-transferase (EC 2.5.1.18) increased with BL application in both the lamina joint and root. The results suggest that the physiological functions of these proteins detected using powerful proteome analysis are implicated in rice lamina inclination and/or root elongation triggered by BL.

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The actin-binding protein p57, a member of the coronin protein family, is expressed in a variety of immune cells. It has five WD repeats and a coiled-coil motif containing a leucine zipper, both of which are known to mediate protein–protein interactions. In order to identify the precise actin-binding regions in p57, and to assess the contribution of these structural motifs, we prepared various truncated p57 as fusion proteins with glutathione S-transferase (GST) and examined their actin-binding activity. A co-sedimentation assay demonstrated that p57^1—371 (C-terminal truncated p57) had the ability to bind F-actin, but p57^372—461 (a fragment containing the coiled-coil motif) did not. A segment consisting of the N-terminal 34 amino acids of p57 (p57^1—34) was found to bind to F-actin in the co-sedimentation assay. Furthermore, fluorescence microscopic observation showed that p57^1—34 was co-localized with F-actin in COS-1 cells after the transfection with the p57^1—34 construct. Deletion of ¹⁰KFRHVF¹⁵, a sequence conserved among coronin-related proteins, from p57^1—34 abolished its actin-binding activity, suggesting that this sequence with basic and hydrophobic amino acids is crucial for p57 to bind to F-actin. However, the N-terminal deletion mutant p57^63—461 retained the binding ability to F-actin. This result suggests the presence of a second actin-binding region. Further deletion analysis revealed that p57^111—204, which includes the second and third WD repeats, also exhibited weak actin-binding activity in the co-sedimentation assay. Taken together, these data strongly suggest that at least two regions within Met-1 to Asp-34 and Ile-111 to Glu-204 of p57 are responsible for its binding to the actin cytoskeleton.

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For the establishment of a screening system to detect inhibitors of vascular endotherial growth factor (VEGF) expression, a stable transformant of Chinese hamster ovary cells was isolated and cloned by transfection of a hypoxia-inducible factor 1 (HIF-1)-dependent VEGF promoter reporter gene. The expression of the reporter gene in the clone cells, as measured by luciferase activity, was stable. Hypoxic responses were best observed at an initial cell density of 2×10⁴/well. The maximal increase of luciferase activity was 30 fold. In the highest cell density of 8×10⁴/well (2.1×10⁵/cm²), basal activity was increased 13—15 fold compared to that at the lower cell densities, and did not respond to hypoxia. Addition of CoCl₂, which is known to mimic hypoxia, increased luciferase activity more than 10 times in normoxia. Nitric oxide donors, which are known to suppress the activation of HIF-1, inhibited expression of the VEGF promoter reporter gene under hypoxia. Histone deacetylase inhibitors, trichostatin A and sodium n-butyrate which are known to stimulate transcription of many genes enhanced its transcription in hypoxia. These results indicate that the stable transformant is a useful tool for screening of HIF-1 modifiers.

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Clonidine hydrochloride has been used for pre-anesthetic medication to provide a pre-operative sedation in pediatric surgery. The purpose of this study is to determine the plasma clonidine concentration, which gives satisfactory sedation in pediatric surgery. Sixteen pediatric patients (age: 1—11 years, weight: 9—33 kg) received either 2 or 4 μg/kg of clonidine lollipop before entering the operating room. Plasma clonidine concentrations were determined 120 min after administration of clonidine lollipop. Pre-operative sedation was evaluated by 5-point scoring systems at entering the operating room. The changes in systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were also assessed before and after administration of clonidine lollipop. The patients with satisfactory sedation had higher plasma clonidine concentration than that of the patients with unsatisfactory sedation (0.45±0.16 ng/ml vs. 0.26±0.16 ng/ml, p<0.05). The clonidine concentrations in the satisfactory group ranged from 0.28 to 0.81 ng/ml. There was no significant difference in hemodynamic parameters (SBP, DBP and HR) before and after administration of clonidine lollipop in both satisfactory and unsatisfactory sedation groups. Plasma clonidine concentration of 0.3—0.8 ng/ml would be sufficient to produce satisfactory sedation without changes in hemodynamic parameters in pediatric surgery.

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Changes in several biomarkers in bronchoalveolar lavage fluid (BALF) during an early stage of lung injury induced with oleic acid were examined in guinea pigs. In addition, a possible contribution of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase to the oxidative changes in the lung injury was investigated. An intravenous injection of oleic acid increased the levels of lipid peroxidation products, lactate dehydrogenase, and total proteins, decreased the ratio of glutathione to glutathione disulfide in the BALF, and also affected the levels of other oxidative biomarkers such as superoxide dismutase and catalase in the BALF in a dose-dependent manner. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, inhibited the oxidative changes in the BALF and the decrease in partial pressure of oxygen in artery induced with oleic acid, while allopurinol, a xanthine oxidase inhibitor, had no inhibitory effects. The results demonstrate that oxidative stress would be an important mechanism of oleic acid-induced lung injury, and indicate that the NADPH oxidase-dependent pathway contributes significantly to the generation of reactive oxygen species in oleic acid-induced lung injury.

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Three antiinflammatory saponin components were isolated from the alkaline hydrolysate of a butanol-soluble portion of Kalopanax pictus bark extract through an in vivo activity-guided fractionation procedure. The hydrolysate showed inhibition of adjuvant induced arthritis in rats. After further fractionation, the ethyl acetate fraction exhibited antiarthritic activity, which resulted in the isolation of α-hederin, α-hederin methyl ester, and kalopanaxsaponin I. All compounds showed inhibition of vascular permeability in mice, but only α-hederin methyl ester showed anticarrageenan activity in rats and antiarthritic activity in rats and mice.

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The possibility has been raised that Folium mori is clinically effective for the treatment and prevention of diabetes. In the present study, the effects of Folium mori on cell proliferation and expression of neuropeptide Y (NPY) in the dentate gyrus of rats with streptozotocin (STZ)-induced diabetes were investigated by 5-bromo-2′-deoxyuridine (BrdU) immunohistochemistry and NPY immunohistochemistry. In rats with STZ-induced diabetes, cell proliferation and NPY expression in the dentate gyrus were suppressed, and treatment with Folium mori was shown to increase new cell formation and NPY expression in the dentate gyrus in both normal rats and those with STZ-induced diabetes. In light of previous studies, this result appears to indicate that increased expression of NPY in the dentate gyrus induced by treatment with Folium mori is associated with the observed effect of Folium mori extract on cell proliferation. Based on the present results, it is suggested that Folium mori treatment may aid in the recovery from the central nervous system complications of diabetes mellitus.

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The possible role of quercetin, a naturally occurring plant flavonoid, in protecting against oxygen–glucose deprivation (OGD)-, excitotoxins-, and free radical-induced neuronal injury in mouse cortical cell cultures was investigated. Pre- and co-treatment with quercetin (100 μM) inhibited 50 min OGD-, 20 μM N-methyl-D-aspartate (NMDA)-, and 50 μM kainate-induced neurotoxicity by 36, 22, and 61%, respectively. Quercetin significantly ameliorated free radical-induced neuronal injury caused by buthionine sulfoximine, sodium nitroprusside, ZnCl₂, and FeCl₂. These results suggest that quercetin may contribute a neuroprotective action against ischemic neural injury, partially via antioxidant actions.

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To investigate the effect of serotonin depletion on the intracerebroventricularly (i.c.v.) administered MK-801-induced plasma interleukin-6 (IL-6) levels, we pretreated mice with parachlorophenylalanine (PCPA, 300 mg/kg, i.p.) 3 d before an i.c.v. injection of MK-801 (1 μg). The i.c.v. MK-801-induced rise of plasma IL-6 level was markedly enhanced in the PCPA-pretreated mice. However, serotonin depletion by PCPA pretreatment did not affect the i.c.v. MK-801-induced increase in plasma corticosterone level. These results suggest that serotonergic system is involved in the suppressive regulation of MK-801-induced plasma IL-6 level.

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In this study, we examined whether several types of non-opioid agents would inhibit the pain-related responses of melanoma-bearing mice. Orthotopic inoculation with melanoma into the hind paw induced marked tactile allodynia and mechanical hyperalgesia. A peroral injection (p.o.) of gabapentin (100—300 mg/kg) inhibited the allodynia and hyperalgesia, without effects on gross behaviors. An intraperitoneal injection (i.p.) of ketamine hydrochloride (30 mg/kg) produced partial inhibition in allodynia and hyperalgesia and prostate posture at 15 min after injection. Diclofenac sodium (10 and 30 mg/kg, i.p), mexiletine hydrochloride (20 mg/kg, i.p.), clonidine hydrochloride (0.1 mg/kg, i.p.) and suramin (100 mg/kg, i.p.) were without effects on allodynia and hyperalgesia. Subcutaneous injections of baclofen (3 mg/kg) and N^G-nitro-L-arginine methyl ester (100 mg/kg) were also without effects. Repeated administration of gabapentin (150 mg/kg, p.o.) produced constant inhibitions, suggesting no analgesic tolerance. Gabapentin may be useful for the management of cancer pain.

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We investigated the effect of diesel exhaust particles (DEPs) on normal human bronchial epithelial (NHBE) cells. Inclusion of DEPs in culture media was lethal to NHBE cells. NHBE cells are more susceptible to DEPs than other normal human lung cells, normal human pulmonary artery endothelial cells and normal human embryonic lung fibroblasts. DEP-induced cell death was mainly due to necrosis. Using the fluorescence probes diacetoxymethyl 6-carboxy-3′,6′-diacetoxy-2′,7′-dichloro-3′,6′-dideoxydihydrofluorescinate and 4,5-diaminofluorescein diacetate, it was observed that hydrogen peroxide and nitrogen monoxide, respectively, were generated within DEP-exposed NHBE cells. DEP cytotoxicity increased or decreased with an increase or decrease in the cellular level of reduced glutathione (GSH) by treatment with L-buthionine-(R,S)-sulfoximine or ethyl reduced glutathionate, respectively. In addition, DEPs themselves decreased the cellular level of GSH in a dose-dependent manner. Upon exposure of NHBE cells to high concentrations of DEPs, their cellular GSH was depleted almost throughout. Further, the following agents decreased DEP cytotoxicity: 1) antioxidants 2,2,5,7,8-pentamethylchroman-6-ol, ebselen, and N,N′-bis(salicylidene)ethylenediaminomanganese(II) dihydrate (EUK-8); 2) iron ion-chelating agents disodium bathophenanthrolinedisulfonate and desferrioxamine mesylate; 3) nitrogen monoxide synthase inhibitors N^G-nitro-L-arginine methyl ester hydrochloride and N^G-methyl-L-arginine acetate salt; and 4) an endocytosis inhibitor quinacrine. On the basis of these observations, the mechanism of DEP cytotoxicity toward NHBE cells is discussed.

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Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor through which dioxins and carcinogenic polycyclic aromatic hydrocarbons cause altered gene expression and toxicity. Ten aza-polycyclic aromatic hydrocarbons (aza-PAHs), consisting of nitrogen substituted naphthalenes, phenanthrenes, chrysenes, and benzo[a]pyrenes (BaPs), were subjected to analysis of their structure-activity relationships as an AhR ligand by using a yeast AhR signaling assay, in which AhR ligand activity was evaluated as lacZ units. Most of the aza-PAHs showed similar or more potent AhR ligand activities than the corresponding parent PAHs. About a 100-fold increased in ligand activity was observed in 10-azaBaP compared with BaP. Halogen-substitution effects on AhR ligand activity in aza-polycyclic aromatics were also investigated with quinoline, benzo[f]quinoline (BfQ), benzo[h]quinoline (BhQ) and 1,7-phenanthroline (1,7-Phe). Position-specific induction of AhR ligand activity was observed in aza-tricyclic aromatic compounds, BfQ, BhQ, and 1,7-Phe, and the ratio of the ligand activities (lacZ units/μM) of monochlorinated and monobrominated aza-tricyclic aromatic compounds to those of the corresponding parent non-halogenated compounds ranged from 2.2- to 254-fold. Greatest enhancement of ligand activity was observed in 2-brominated BfQ (2-Br-BfQ), and its ligand activity was higher than that of BaP. These results suggest that even monohalogenation markedly enhances AhR ligand activity in aza-PAHs.

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We examined whether dietary intake of cattle liver-supplemented food induces reproductive effects in dams and developmental effects in embryos in the mouse model. Seven groups of 19 to 35 female mice each were given either powdered food or the food supplemented with crude liver homogenate, its lipophilic component, the defatted liver homogenate or vitamin A (retinol palmitate) during a 25-d period spanning from a week prior to mating to gestation day 18 (GD18). Fetal mortality and incidence of external abnormalities of the fetuses whose dams were given the diet supplemented with the crude liver homogenate increased dose-dependently with an increase in the supplemented amount of the crude liver homogenate. On the other hand, the defatted liver homogenate did not induce any reproductive or teratological effect. The vitamin A (VA)-supplemented food (950 IU/5 g food as VA) induced approximately the same levels of the incidence of total external abnormalities appearing at the same affected regions or organs as the foods supplemented with the 700 mg crude liver homogenate (1029 IU/5 g food as VA) and its lipophilic component (950 IU/5 g food as VA). The content of VA (as 1029 IU/5 g food) in the crude liver homogenate was found to be approximately equal to that in the lipophilic component (950 IU/5 g food as VA). Therefore, it was concluded that VA plays an important role in induction of the lethal and teratogenic effects in the fetuses whose dams were given the powdered foods supplemented with the crude liver homogenate and its lipophilic component.

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In a previous works searching for new drugs with high efficiency, we reported the in vitro and in vivo antileishmanial activity of a series of diarylheptanoid structurally related to curcumin against L. amazonensis. This work describes the in vitro antileishmanial activity of a new series of diarylheptanoids and diarylpentanoids derivatives. These drugs were assayed against Leishmania amazonensis, L. braziliensis and L. chagasi promastigotes containing a high percentage of metacyclic forms and the axenic amastigote form of L. amazonensis and using Pentamidine Isethionate as reference drug. Parasites in the log late phase culture were incubated with several concentrations of the drugs solubilized in dimethyl sulphoxide (DMSO) and then counted in a Neubauer's chamber. Controls without the drugs and with DMSO were done in parallel. The results showed that all diarylheptanoids and diarylpentanoids had a very good antileishmanial activity.

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Since the bromide preparations useful in the treatment of intractable infantile epilepsy show a tendency to accumulate in the body, they may cause chronic toxicosis. To prevent this, determination of the bromide ion concentration in the serum is essential. After establishing a simple and rapid technique using energy-dispersive X-ray spectroscopy for the analysis of the serum total bromide level, we applied this technique in a clinically diagnosed epilepsy patient. The standard curve for total bromide showed linearity (r=0.999) in the range of 10—2000 μg/ml, and the lowest detection limit was 5 μg/ml. The mean recovery rate of bromide added to reference serum to yield a concentration of 50 μg/ml was 93.5% (n=5, coefficient of variation=9.1%). Analysis took only 20 min. On analysis of the serum of a 10-year-old girl whose treatment was initiated with orally administered potassium bromide 1.0 g/kg, a good correlation was found between the total bromide level obtained with energy-dispersive X-ray spectroscopic analysis and the level of bromide ions determined by ion-exchange HPLC. The determination of serum total bromide by rapid energy-dispersive X-ray spectroscopic analysis is a useful method of monitoring to prevent bromide poisoning.

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Five iridoid glycosides, including the two new compounds scropolioside-D₂ (1) and harpagoside-B (2), were isolated from the aerial parts of Scrophularia deserti DEL (Scrophulariaceae). Their structures were elucidated on the basis of spectral data to be 6-O-[2″,4″-di-O-acetyl-3″-O-trans-cinnamoyl)-α-L-rhamnopyranosyl]-8α-hydroxymethyl-1α,5β,6α,7α,9β-pentahydro-7(8)-epoxy-2-oxaind-3-ene-1-O-β-D-glucopyranoside-6′-O-acetate (1) and 5-O-β-hydroxy-8-O-β-trans-cinnamoyl-8α-methyl-1,6,7,9-tetrahydro-2-oxaind-3-ene-1-O-β-D-glucopyranoside (2), respectively. In addition, three more iridoid glycosides, scropolioside-D (3), koelzioside (4), and 8-O-acetyl-harpagide (5), were also isolated and characterized from this source. The biological activity and the structure activity relationship of the compounds were also studied, and scropolioside-D (3) and harpagoside-B (2) were found to possess significant antidiabetic and antiinflammatory activity, respectively.

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Nitric oxide (NO) has been implemented in various pathological processes. In the present study, 47 highly-oxygenated isopimarane-type and novel carbon framework staminane-type diterpenes isolated from Orthosiphon stamineus of Indonesia, Okinawa, Myanmar and Vietnam were evaluated for their inhibitory activity in NO production by lipopolysaccharide (LPS)-activated macrophage-like J774.1 cells. All the isolated diterpenes showed concentration-dependent inhibition of NO production in LPS-activated macrophage-like J774.1 cells, and based on the results, their structure–activity relationshis were established.

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A series of novel 2-benzylamino-3-substituted quinazolin-4(3H)-ones have been synthesized by treating 3-amino-2-benzylamino quinazolin-4(3H)-one, with different aldehydes and ketones. The starting material 3-amino-2-benzylamino quinazolin-4(3H)-one was synthesized by nucleophilic substitution of thiomethyl group of 3-amino-2-methylthio quinazolin-4(3H)-one by benzylamine. The title compounds were investigated for analgesic and anti-inflammatory activities. All the test compounds exhibited significant analgesic activity, whereas the compound III is equipotent with diclofenac sodium. The compounds I, II and III showed more potent anti-inflammatory activity than diclofenac sodium.

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We studied the effects of apigenin and 2,4,5-trimethoxycinnamic acid (TMCA) on the behavioral despair test (forced swimming test), and the central noradrenergic, dopaminergic and serotonergic activities in mice. Apigenin at intraperitoneal doses of 12.5 and 25 mg/kg significantly decreased the duration of immobility in the forced swimming test in mice. At 100 mg/kg, the duration of immobility was returned to the control level in the test. On the other hand, TMCA treatment (25—200 mg/kg, i.p.) failed to significantly alter the duration of immobility. Based on the behavioral data, we examined changes in the monoamine turnover in mice having been subjected to forced swimming for 40 min. The monoamine turnover was measured in seven brain regions. Forced swimming exposure induced a significant decrease in dihydroxyphenylacetic acid (DOPAC)/dopamine (DA) in the striatum and amygdala and in 5-hydroxyindoleacetic acid (5-HIAA)/5-hydroxytriptamine (5-HT) in the hypothalamus, and a significant increase in DOPAC/DA in the thalamus and hypothalamus and in 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG)/norepinephrine (NE) in the amygdala, frontal cortex, hypothalamus, and midbrain. Apigenin (25 mg/kg) treatment produced attenuation of forced swim test-induced decrease of DA turnover in the amygdala and increase of DA turnover in the hypothalamus. Furthermore, intraperitoneal administration of haloperidol (0.2 mg/kg), a dopamine D₂ antagonist, blocked the apigenin (25 mg/kg)-induced decrease in immobility in the forced swimming test. These behavioral and biochemical results indicate the antidepressant properties of apigenin, which may be mediated by the dopaminergic mechanisms in the mouse brain.

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As an attempt to search for bioactive natural products exerting antiinflammatory activity, we have evaluated the effects of the methanol extract from the aerial parts of Saururus chinensis (LOUR.) BAILL (Saururaceae) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) release by the macrophage cell line RAW 264.7. Our data indicate that this extract is a potent inhibitor of NO production and it also significantly decreased PGE₂ release. Consistent with these observations, the protein and mRNA expression level of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 was inhibited by MeOH extracts of the aerial part of S. chinensis (SCM) in a dose-dependent manner. Furthermore, SCM inhibited the LPS-induced DNA binding activity of nuclear factor-κB (NF-κB), which was associated with decreased p65 protein levels in the nucleus. These results suggest that SCM inhibits LPS-induced iNOS and COX-2 expression by blocking NF-κB activation.

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The MeOH and water extracts of the Netherlands propolis were tested for their inhibitory activity toward nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage-like J774.1 cells. Both of the extract possessed significant NO inhibitory activity with IC₅₀ values of 23.8 and 51.5 μg/ml, respectively. Then 13 phenolic compounds obtained from the MeOH extract showing stronger NO inhibition were examined on their NO inhibitory activities. Caffeic acid phenethyl ester (CAPE) analogues, i.e., benzyl caffeate, CAPE and cinnamyl caffeate, possessed most potent NO inhibitory activities with IC₅₀ values of 13.8, 7.64 and 9.53 μM, respectively, which were two- to four-fold stronger than the positive control N^G-monomethyl-L-arginine (L-NMMA; IC₅₀, 32.9 μM). Further study on the synthetic analogues of CAPE revealed that both of 3-phenylpropyl caffeate (18; IC₅₀, 7.34 μM) and 4-phenylbutyl caffeate (19; IC₅₀, 6.77 μM) possessed stronger NO inhibitory activity than CAPE (10) and that elongation of alkyl side chain of alcoholic parts of caffeic acid esters enhance the NO inhibitory activity. In addition, it was found that CAPE analogues having longer carbon chain (>C₅) in alcoholic part showed toxic effects toward J774.1 cells. This NO inhibitory effect may directly correlate with antiinflammatory properties of the Netherlands propolis.

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Polyethylene glycol (PEG) has been coupled to many cationic polymers such as polyethylenimine (PEI) to improve the stability and transfection efficiency. We prepared PEG-grafted PEI with different lengths and amounts of PEG and used these graft copolymers as nonviral gene vectors. We measured the complex size and zeta-potential of polymer–DNA complexes in the presence of salt to estimate the stability of polymer–DNA complexes. We also investigated the cytotoxicity and transfection efficiency in C3 cells. In the case of graft copolymers, the stability of polymer–DNA complexes and transfection efficiency were affected by the graft length and amount of PEG side chain. PEG side chains stabilize the polymer–DNA complexes in the presence of salt, but the longer PEG side chains also interrupt the gene delivery in the cells due to the more efficient steric hindrance by longer PEG side chains, and therefore the transfection efficiency is decreased. Short PEG side chains with molecular weight of 350 kDa stabilized the polymer–DNA complexes without decreased transfection efficiency.

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Bromocriptine (BRC) has been mainly used for the inhibition of lactation, treatment of menstrual disorders, Parkinson disease, breast tumours, infertility and brain tumours as a dopamine agonist in clinics. But current BRC formulations have some side effects and bioavailability problems because of hepatic first pass effect. Transdermal application could be an alternative route to overcome all these problem and penetration properties of BRC has not been studied yet. Therefore, it was aimed to investigate the effectiveness of transdermal formulation of BRC which is applicable to the skin. For this purpose, a number of BRC gel formulations (Carbopol-934 (C-934), chitosan (CH) and Gantrez-SP215 (G-SP215) were developed and the effectiveness and bioavailability of the formulations were compared in rabbits. Commercial BRC tablets (Parlodel^®) were also given to rabbits orally and plasma levels were compared. The effects of two different penetration enhancers, sodium taurocholate (ST) and ethoxydiglycol-Transcutol^® (TR) on the BRC penetration were also investigated. The skin samples from the dorsal part of the rabbit were removed after CH gel application and investigated under electron microscope to understand the effects of the gel on the penetration and the possible penetration mechanisms through skin were also discussed. In conclusion, CH gel formulation was found to be the best formulation and comparable blood BRC concentrations were obtained when applied to the rabbit skin. Higher blood levels were obtained with the use of CH. The main penetration process was found to be through transcellular route but some other mechanisms were also found to be incorporated, after microscopic investigation. CH gel was found to be a useful carrier for BRC administration through dermal route and the penetration enhancing effect and the mechanism of CH gel were first established in this study. It was concluded that transdermal delivery of BRC may be a very promising alternative route to the oral route for the treatment.

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The repeated administration of methamphetamine (MAP) causes behavioral sensitization in animals. We previously reported that the high accumulation of MAP was observed in the MAP-sensitized animal brain, which suggested that this phenomenon is an important factor in the development or expression of behavioral sensitization. The purpose of the present study is to elucidate the MAP distribution in the MAP-sensitized rat using gas chromatography/mass spectrometry (GC/MS). As a result, the MAP distribution in the heart at 10 min when showing a high accumulation of MAP in the MAP-sensitized rat brain was significantly higher than that of the control rat, whereas no significant differences in the liver, kidney, abdominal muscle, femoral muscle and blood were observed. In the brain and heart, there was no different distribution at 1 min, reflecting only the influx process from blood to brain and heart. On the contrary, there was the significant difference at 10 min, reflecting both the influx and efflux process, suggesting that the efflux process of MAP from brain or heart to blood may be slow due to MAP sensitization. In conclusion, it was clear that the brain and heart specific alteration of the MAP distribution occurred in the MAP sensitization. It was considered that the high accumulation of MAP in the MAP-sensitized rat brain may be related to the expression of behavioral sensitization and that the delayed efflux of MAP in the MAP-sensitized rat heart may be connected with the cardiac toxicity.

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The cytochrome P-450 3A (CYP3A) enzyme family is responsible for most of the drug metabolism in the human liver. In this study, we demonstrated the inductive effects of phenobarbital, rifampicin, carbamazepine, phenytoin, prednisolone, ciclosporin and clotrimazole on CYP3A4, CYP3A5 and CYP3A7 mRNA expression, and established the relationship between the expression of human glucocorticoid receptor α (hGR) mRNA and the induction of CYP3A4 mRNA in cultured HepG2 cells by reverse transcription polymerase chain reaction (RT-PCR). Treatment with prednisolone, rifampicin and carbamazepine rapidly induced the level of CYP3A4 mRNA expression by 3- to 6-fold. However, phenytoin and phenobarbital gradually induced CYP3A4 mRNA level by 3 to 4-fold. The induction of CYP3A4 mRNA expression by clotrimazole and ciclosporin was negligible. Treatment with phenytoin, rifampicin, carbamazepine and ciclosporin induced approximately 2-fold increases in the expression of CYP3A5 mRNA, although prednisolone, phenytoin and clotrimazole had no effect. Treatment with rifampicin, phenytoin, clotrimazole and ciclosporin resulted in approximately a 2-fold induction of the CYP3A7 mRNA level. Treatment with rifampicin and ciclosporin induced the expression of hGRα mRNA significantly in comparison with controls, although the induction of hGRα mRNA following treatment with other drugs was negligible. In cluster analysis, the induced level of CYP3A4, CYP3A5, CYP3A7 and hGRα mRNA by these drugs could be classified into four major clusters. This suggested that each cluster might be associated with different mechanism(s) of induction by these drugs. Furthermore, we studied the associations between the expression of hGRα mRNA and the induced level of CYP3A4 mRNA by prednisolone and ciclosporin. Treatment with both prednisolone and ciclosporin showed synergistic effects on induction of CYP3A4 mRNA and, following treatment with both drugs, the expression level of CYP3A4 mRNA was 2-fold greater compared with prednisolone alone after the fifth day. Positive correlations were observed between the levels of hGRα mRNA expression and those of CYP3A4 mRNA. This observation shows that the regulation of CYP3A4 gene expression was hGRα-dependent and that ciclosporin may function as a regulator of expression via hGRα.

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The objective of this study was to propose a suitable electrode disposition and shape for iontophoretic drug delivery systems in consideration of a reduction in skin barrier function and a distribution of current density. The reduction in barrier function was evaluated with our proposed method, which measured the resistance in the short term. The distribution was estimated using an electromagnetic waves analysis program. Using rectangular electrodes, effects of distance between electrodes and boundary length of the electrode on the barrier function were examined. A distance of 2 mm decreased the barrier function effectively. The barrier function was reduced with increasing electrode boundary length. Furthermore, a surrounded square type electrode was more effective in the reduction of barrier function than a paired square type. With respect to the surrounded type electrodes, both square and circle types decreased the barrier function. However, percutaneous absorption using the circle type electrode was greater than with the square type. These phenomena are attributed to not only the electrode boundary length but also the homogeneous distribution of current density. Therefore, the surrounded circle type electrode was suitable for iontophoretic transdermal drug delivery systems.

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Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are candidate cytokines which are produced by osteoblastic linage cells and promote osteoblast apoptosis, osteoclastogenesis and bone resorption. Here, we examined the effect of (+)-catechin, one of the most common grape flavonols, on osteoblastic MC3T3-E1 cells. (+)-Catechin caused a significant elevation of cell survival at 10⁻⁵ and 10⁻⁴ M and alkaline phosphatase activity at 10⁻⁵ M. Also, treatment with (+)-catechin (10⁻⁵ M) decreased bone-resorbing cytokines (TNF-α and IL-6) production and apoptosis in osteoblasts. Our data indicate that the reduction of bone-resorbing cytokines and apoptosis in osteoblasts by (+)-catechin may result in the prevention and therapy for osteoporosis and inflammatory bone diseases.

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In the present study, the effects of perilla leaf extract (PLE) and luteolin on 7,12-dimethylbenz[a]anthracene (DMBA)- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin papillomas in mice were investigated. Topical application of PLE prior to TPA treatment in DMBA-initiated mouse skin resulted in a significant reduction in tumor incidence and multiplicity. An even more potent preventive effect was observed with topical application of luteolin, which we previously identified as an antiinflammatory constituent. PLE was dissolved in drinking water at a 0.05% dose and mice ingested it ad libitum; no significant difference was observed in tumor incidence or multiplicity but there was a significant reduction in tumor volume between the PLE-treated and untreated groups. These results suggest that PLE has potent antipromotion activity and ingesting it as a daily food may provide a beneficial chemopreventive effect.

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Flavonoids have been considered the main biologically active components in propolis. However, a new variety of flavonoid-free propolis was recently found and chemically classified as type 6. Because it showed activity against oral microorganisms, this study evaluated the effects of the crude ethanolic extract of this propolis and its chemical fractions on the activity of purified glucosyltransferases (GTFs) and on the growth and adherence of mutans streptococci. The inhibitory effect of propolis extracts on GTF activities was determined either in solution or adsorbed onto saliva-coated hydroxyapatite. Streptococcus mutans Ingbritt 1600, Streptococcus sobrinus 6715, and two clinical isolates of each species were used for antibacterial assays. Susceptibilities to the test extracts were analyzed using the agar diffusion method and by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC); the effect on bacterial adherence to a glass surface was also assessed. The activity of GTFs in solution was effectively inhibited by the ethanolic extract of propolis type 6 (EEP) (>80% inhibition at 0.5 mg/ml), hexane, and chloroform fractions (60—90% inhibition at 100 μg/ml); their inhibitory effects on surface enzymes were less pronounced. The EEP, hexane, and chloroform fractions also showed significant antibacterial activity. The data showed that propolis type 6 remarkably reduced GTF activity and inhibited mutans streptococci growth and adherence; these biological activities are associated with its nonpolar components.

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The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological action of many aromatic environmental pollutants. In this study, we investigated the activation of the AhR by some vegetable constituents using the AhR-based bioassay for dioxins, i.e., the chemical activated luciferase gene expression (CALUX) assay. Ninety-five vegetable constituents, including flavonoids, tannins, saponins, and terpenes, were tested in vitro. Among them, isoflavones such as daidzein, resveratrol having a stilbene structure, and some flavonoids such as naringenin, hesperetin, and baicalein showed AhR activation.

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Matrix metalloproteinases (MMPs) are involved in invasive cell behavior, embryonic development and organ remodeling. In this report, we investigated the role of liver-derived MMP-9 in the in vivo system at liver injury. Liver injury induced MMP-9 expression in the liver 3 to 12 h after intravenous administration of anti-Fas antibody, followed by the expression of the activity and the protein detected by zymography and Western blotting, respectively, in the blood circulation. Interestingly, the MMP-9 expression was accompanied by the recruitment of hematopoietic progenitor cells from bone marrow into the circulation. The recruitment was blocked by a specific MMP-9 inhibitor, R94138, which did not affect the Fas-mediated liver injury or induced expression of MMP-9. Compulsive expression of mutant active MMP-9 in the liver also recruited the progenitor cells into the circulation. In contrast, partial hepatectomy, which treatment does not directly injure hepatocytes, did not recruit progenitor cells despite the increased expression of MMP-9 in the circulation. These results suggest that liver-derived MMP-9 induced by liver injury plays an essential role in the recruitment of hematopoietic progenitor cells from bone marrow into the blood circulation.

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We examined the in vitro effects of the benzophenone derivatives garcinol, isogarcinol, and xanthochymol on cell growth in four human leukemia cell lines. All of the compounds exhibited significant growth suppression due to apoptosis mediated by the activation of caspase-3. A loss of mitochondrial membrane potential was found in garcinol- and isogarcinol-induced apoptosis, but not in xanthochymol-induced apoptosis. The growth inhibitory effects of isogarcinol and xanthochymol were more potent than that of garcinol, which is a well- known cytotoxic benzophenone derivative.

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