Nafamostat mesilate, an ester drug with extensive hydrolysis in vivo, exhibits species difference in the relative contribution for its hydrolysis in blood and tissues. For the rat, the main hydrolysis site may be blood and human may be tissue (mainly by liver). The paper gave in vivo evidence that human tissue may give more contribution for its hydrolysis. In the initial phase of drug administration, the drug accumulating level in tissue was low; the efflux fraction from tissue into blood can be ignorable comparing with the drug influx into tissue. Based on urine and plasma metabolite analysis, we concluded that in the initial phase almost all the drug hydrolysis in blood was excreted into urine. Then according to the initial urine metabolite analysis, we can estimate the drug hydrolysis rate in blood. The rate of drug diffusion from blood into tissues can be deduced based on the mass balance analysis of the initial blood drug. With the estimated rate constants, the drug efflux from tissues into blood was calculated according to equation: OF_T-B (efflux from tissues) = OF_B-U (blood hydrolysis fraction)+OF_B-T (influx into tissues)−D_B (hydrolysis in blood). The net flow (influent flux minus effluent flux) represented the drug hydrolysis fraction in tissue. As the result indicated, in human about 20% drug administrated was hydrolyzed in blood and nearly 80% in tissues. The relative hydrolysis fraction indicated that the main hydrolysis site in human body may locate in tissue, which was different to rats.

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We previously found that the increases in Ca²⁺ content in the lenses of three hereditary cataract model rats, UPL rat (UPLR), Shumiya cataract rat (SCR) and Ihara cataract rat (ICR), are inhibited by aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, and that the mechanisms of Ca²⁺ enhancement in these rat models differ. In this study, we compare the mechanisms for dysfunction in Ca²⁺ regulation in UPLR, SCR and ICR. Decreases in the activity of Ca²⁺-ATPase were found in the lenses of SCR and ICR concurrent with cataract development. In contrast, the Ca²⁺-ATPase activity in UPLR with opaque lenses was higher than in those with transparent lenses. On the other hand, ATP levels were markedly decreased in UPLR with opaque lenses. The expression of cytochrome c oxidase (CCO)-1 mRNA and CCO activity in UPLR lenses was found to decrease during cataract development. The nitric oxide (NO) and lipid peroxide levels were also increased in the lenses of UPLR, SCR and ICR with opaque lenses. In UPLR, excessive NO may cause damage to the mitochondrial genome, resulting in a decrease in ATP production and increase in Ca²⁺-ATPase activity. The decrease in ATP content may cause the decrease in Ca²⁺-ATPase function resulting in the elevation in lens Ca²⁺. In SCR and ICR, excessive NO may cause an enhancement of lipid peroxidation resulting in the oxidative inhibition of Ca²⁺-ATPase. The decrease in Ca²⁺-ATPase activity may cause the elevation in the level of lens Ca²⁺, thus leading to lens opacification. Our findings show that the Ca²⁺ contents in the cataractous lenses of all three model rats are increased, the mechanisms for this Ca²⁺ enhancement is different in each rat model.

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Licochalcone A, a flavonoid found in licorice root (Glycyrrhiza glabra), is known for its anti-microbial activity and its reported ability to inhibit cancer cell proliferation. In the present study, we investigated whether licochalcone A inhibits rat vascular smooth muscle cell (rVSMC) proliferation. Our data indicate that 5 μM licochalcone A inhibited platelet-derived growth factor (PDGF)-induced rVSMC proliferation, possibly through its ability to block the progression of the cell cycle from G₁ to S phase. In addition, 5 μM licochalcone A significantly inhibited the PDGF-induced expression of cyclin A, cyclin D1, CDK2, and CDK4, and the phosphorylation of Rb. Licochalcone A also reversed the decrease in p27^kip1 expression reduced by PDGF. Finally, licochalcone A inhibited the PDGF-induced activation of extracellular signal-regulated kinase (ERK)1/2. Together, these data provide the first evidence that licochalcone A can regulate rVSMC proliferation and suggest that licochalcone A inhibits the proliferation of rVSMCs by suppressing the PDGF-induced activation of the ERK1/2 pathway and Rb phosphorylation, resulting in cell cycle arrest.

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The mitochondrial ADP/ATP carrier (AAC), which has six transmembrane regions (TM) with cytosolic N- and C-termini, exchanges matrix ATP for cytosolic ADP in the mitochondrial inner membrane. Structural aspects of the bovine type 1 AAC (bAAC1) and the yeast type 2 AAC (yAAC2) are quite similar, whereas the molecular activity of yAAC2 is four times higher than that of bAAC1. To examine the relationship between the structure and the functional difference, substrate transport activities of serial chimeric proteins having N-terminal bAAC1 and C-terminal yAAC2 were estimated from growth activities of their yeast transformants on a medium containing non-fermentable glycerol. The chimera having the boundary of bAAC1 and yAAC2 between a TM and its C-terminal adjoining loop had activity, but chimera having the boundary between a TM and its N-sided loop did not. These results indicate that a set of a TM and its C-sided loop is important to the AAC function. In addition, the mutant, in which the first TM and its C-sided loop (the first matrix loop) in yAAC2 are replaced with those of bAAC1, exhibited a change in reactivity for a SH cross-linking reagent copper-o-phenanthroline, suggesting that the interaction of these regions is also involved in the structural feature of AAC. Because the mutant had similar transport activities to bAAC1, the structural property provided by the interaction between the first TM and the first matrix loop is probably involved in activity of AAC.

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Nickel compounds have toxic and carcinogenic effects. Several cellular targets have been identified and the toxicity is thought to be mediated by genetic and epigenetic factors. Gene expression from chromatin is regulated by posttranslational histone modifications, ATP-dependent chromatin remodeling, and the incorporation of histone variants. Nickel compounds decrease acetylation levels of all four histones and increase ubiquitylation of H2A and H2B and dimethylation of H3 lysine 9. Less attention has been focused on histone variants in nickel toxicity. Here we demonstrate that a null mutation of H2A.Z (HTZ1 in Saccharomyces cerevisiae), a variant of H2A, decreases the sensitivity to soluble nickel compounds. In addition, we show that a mutation in the acetylatable residues in Htz1p does not alter the sensitivity to nickel compounds. Furthermore, sensitivity to nickel compounds of the null mutant of SWR1 encoding the catalytic subunit of the ATP-dependent chromatin remodeling complex that specifically loads Htz1p into chromatin, was identical to that of the htz1 mutant. Taken together, these results reveal that the incorporation into chromatin, but not acetylation, of Htz1p is important to the toxicity of nickel compounds.

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Salmonellosis is a major bacterial zoonosis that causes a variety of disease syndromes, from self-limiting enteritis to fatal infection in animals and food-borne infection and typhoid fever in humans. Recently, the emergence of multidrug-resistant strains of Salmonella sp. has caused more serious problems in public health. The present study investigated the antibacterial effects of Houttuynia cordata water extract (HCWE) against murine salmonellosis. In RAW 264.7 cells, there was no detectable cytotoxic effect of HCWE at any concentration between 25 and 100 μg/ml after 8-h incubation. The antibacterial activity of HCWE was then examined in a Salmonella enterica serovar (Salmonella typhimurium), and was found to increase in a dose-dependent manner at concentrations from 25 to 100 μg/ml during 8-h incubation. HCWE also affected RAW 264.7 cells including morphologic change and bacterial uptake, but there was no significant difference in bacterial replication in RAW 264.7 cells. With HCWE alone, nitric oxide (NO) production by RAW 264.7 cells did not increase, but when RAW 264.7 cells were infected by S. typhimurium, with or without HCWE, NO production with HCWE was 2-fold higher than that without HCWE. Treatment with HCWE did not affect inducible NO synthase (iNOS) mRNA expression by RAW 264.7 cells, but when RAW 264.7 cells with HCWE were infected by S. typhimurium, iNOS mRNA expression was increased during 8-h incubation. Furthermore, HCWE showed virulence reduction effects in S. typhimurium-infected BALB/c mice. After a lethal dose of S. typhimurium, the mortality rate in the HCWE untreated group was 100% at 7 d, but the HCWE 25, 50, and 100 μg/ml groups survived until 11, 17, and 23 d, respectively. These data suggest that HCWE is stable and beneficial in the treatment of bacterial infection including intracellularly replicating pathogens and may solve antimicrobial misuse and overuse.

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Andrographis paniculata NEES is a medicinal plant that is commonly used in Asia. This work demonstrates that 25 μg/ml of ethanolic extract from A. paniculata (EEAP) and 5 μg/ml of andrographolide, a bioactive compound in EEAP, effectively inhibit the expression of Epstein–Barr virus (EBV) lytic proteins, Rta, Zta and EA-D, during the viral lytic cycle in P3HR1 cells. Transient transfection analysis revealed that the lack of expression of Rta, Zta and EA-D is caused by the inhibition of the transcription of BRLF1 and BZLF1, two EBV immediate-early genes that encode Rta and Zta, respectively. This study finds that the inhibition prevents the virus from producing mature viral particles. Meanwhile, andrographolide is not toxic to P3HR1 cells when the concentration is below 5 μg/ml, indicating that the compound is potentially useful as an anti-EBV drug.

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20(R)-ginsenoside Rg3 (20(R)-Rg3) has shown multiple pharmacological activities and been considered as one of the most promising approaches for fatigue treatment. However, 20(R)-Rg3 has a low bioavailability after oral administration in human due to the first-pass effect. Recently, nasal route has gained increasing interest as it can avoid first-pass effect for its lower enzymatic activity compared with the gastrointestinal tract and liver. In order to provide an animal experimental evidence of 20(R)-Rg3 intranasal administrated preparation, the anti-fatigue effect of 20(R)-Rg3 after intranasal administration was investigated. Two weeks after 20(R)-ginsenoside Rg3 was administrated intranasally to mice at three different doses, the anti-fatigue effect of 20(R)-Rg3 was evaluated by the weight-loaded swimming test and biochemical parameters related to fatigue, such as serum urea nitrogen (SUN), lactic dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), blood lactic acid (LA) and hepatic glycogen. The results showed that compared with the negative control group, the intermediate-dose and the high-dose groups significantly prolonged the weight-loaded swimming time (p<0.05; p<0.01), and also increased the hepatic glycogen levels (p<0.05); SUN levels were decreased considerably in three 20(R)-Rg3-treated groups (p<0.01). In addition, the low-dose group obviously decreased the content of blood LA (p<0.05). However, the levels of LDH, SOD and MDA did not show a significant change. Our results predicted a benefit of 20(R)-Rg3 as an anti-fatigue treatment by intranasal administration. The mechanism was related to the increase of the storage of hepatic glycogen, and the decrease of the accumulation of metabolite such as lactic acid and serum urea nitrogen.

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Sanguisorbae radix (SR), the root of Sanguisorba officinalis L. (Rosaceae), has been traditionally used for its anti-inflammatory, anti-infectious and analgesic activities in Korea. Previous work has shown that SR prevents neuronal cell damage induced by Aβ (25—35) in cultured rat cortical neurons. The present study was carried out to further investigate the neuroprotective effect of SR on oxidative stress-induced toxicity in primary culture of rat cortical neurons, and on ischemia-induced brain damage in rats. SR, over a concentration range of 10—50 μg/ml, inhibited H₂O₂ (100 μM)-induced neuronal death, which was significantly inhibited by MK-801 (5 μM), an N-methyl-D-aspartate (NMDA) receptor antagonist, and verapamil (20 μM), an L-type Ca²⁺ channel blocker. Pretreatment of SR (10—50 μg/ml), MK-801 (5 μM), and verapamil (20 μM) inhibited H₂O₂-induced elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) measured by a fluorescent dye, Fluo-4 AM. SR (10—50 μg/ml) inhibited H₂O₂-induced glutamate release into medium measured by HPLC, and generation of reactive oxygen species (ROS) measured by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA). In vivo, SR prevented cerebral ischemic injury induced by 2-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. The ischemic infarct and edema were significantly reduced in rats that received SR (10, 30 mg/kg, orally), with a corresponding improvement in neurological function. Catechin isolated from SR inhibited H₂O₂-induced neuronal death in cultures. Taken together, these results suggest that SR inhibits H₂O₂-induced neuronal death by interfering with the increase of [Ca²⁺]_i, and inhibiting glutamate release and generation of ROS, and that the neuroprotective effect of SR against focal cerebral ischemic injury is due to its anti-oxidative effects. Thus SR might have therapeutic roles in neurodegenerative diseases such as stroke.

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Dantrolene, a drug used to treat malignant hyperthermia, inhibits abnormal Ca²⁺ release from the sarcoplasmic reticulum. Glutathione reductase (Glutathione: NADP⁺ oxidoreductase, EC 1.8.1.7), a member of the pyridine-nucleotide disulfide oxidoreductase family of flavoenzymes, catalyzes the reduction of glutathione disulfide (GSSG) to reduced form (GSH) in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). In the present study, the in vitro effects of dantrolene on human erythrocyte glutathione reductase were investigated. For this purpose, initially, human erythrocyte glutathione reductase was purified 2555.56 fold in a yield of 29.74% using both 2′,5′-ADP Sepharose-4B affinity gel chromatography and Sephadex G-200 gel filtration chromatography. The purity of the enzyme was controlled by sodium dodecyle sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) which showed a single band. A constant temperature (+4 °C) was maintained during the purification process. Enzyme activity was determined with the Beutler method at 340 nm by means of a spectrophotometer. Dantrolene showed remarkable in vitro inhibitory effects on the enzyme. K_i constant and 50% inhibitory concentration (IC₅₀) value for dantrolene were determined by Lineweaver–Burk graphs and plotting activity % vs. [I], respectively. K_i constant for dantrolene was found to be 0.1116±0.04 mM; IC₅₀ value was 0.0523 mM. Dantrolene displayed non-competitive inhibition.

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The effect of Laennec, a hydrolyte of human placenta, on immune-mediated liver injury was investigated in vivo and in vitro in murine. Vena caudalis administration of concanavalin A (Con A) was employed to establish an in vivo liver-injury model, and in vitro hepatotoxicity was induced by 8 h interaction between Con A pre-treated hepatocytes and Con A-stimulated autologous splenic lymphocytes. Laennec was used for pre-treatment in the two models. Laennec decreased biochemical marker activity (alanine aminotransferase, ALT; lactate dehydrogenase, LDH) in serum and recovered the activity of superoxide dismutase (SOD) and myeloperoxidase (MPO), as well as the content of malondialdehyde (MDA) and nitric oxide (NO) in liver tissue. We also found that the DNA ladder induced by Con A in vivo was attenuated by Laennec. Furthermore, the leakage of aspartate aminotransferase (AST) and LDH in the supernatant of the co-culture system was decreased by addition of Laennec. Potential protective mechanisms were elucidated by DNA fragmentation assay and intercellular adhesion molecule-1 (ICAM-1) induction/inhibition experiments. Results showed that ICAM-1, which is related to the interaction between hepatocytes and lymphocytes, was inhibited by Laennec. These findings indicated that Laennec has potent activity against immune-mediated liver injury.

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The objective of this study was to clarify the relationship between angiotensin II and the pathogenesis of diabetic cardiomyopathy by observing the effects of related drugs on diabetic cardiomyopathy in rats. Captopril and Valsartan, an automatic biochemical analyzer, and radioimmunoassay technology were used to an experimental rat model of diabetic cardiomyopathy to dynamically measure the levels of creatine kinase-MB, lactate dehydrogenase-1 in the serum, and angiotensin I and II in the plasma, and to observe changes in the myocardial ultrastructure. The content of angiotensin I and II was increased and the renin–angiotensin system was in a hyperfunctional state in experimental rats with myocardial damage. Angiotensin-converting enzyme inhibitors and angiotensin II receptor 1 antagonists improved the myocardial structure and cardiac function. It is concluded that hyperfunction of the renin–angiotensin system is involved in the pathogenesis of diabetic cardiomyopathy, with an increase in angiotensin being a key factor. Preventing the increase in angiotensin II or the action of angiotensin II on its receptor can prevent the occurrence and development of diabetic cardiomyopathy.

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This article was retracted. Please see the retraction notice.

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It has been well documented that matrine, a tetracyclo-quinolizindine alkaloid, possessed a positive inotropic effect. However, the underlying mechanisms at the cellular and ion channel levels have not been completely clarified. Therefore, the present study was designed to identify the cellular target and the mechanisms of inotropic effect of matrine. Guinea pig papillary muscles were used to study the contractile force of the heart and ventricular myocytes were used to study L-type calcium channel (I_Ca-L) and intracellular calcium concentration ([Ca²⁺]_i). In electrically driven papillary muscles, matrine enhanced the contractile force in a dose-dependent manner and the positive inotropic effect was not inhibited by α- and β-adrenergic receptor antagonists. In ventricular myocytes, matrine also increased I_Ca-L in a dose-dependent manner and shifted the inactivation curve toward right. Matrine markedly enhanced the KCl-induced elevations of [Ca²⁺]_i. In a conclusion, I_Ca-L might be a main target of matrine. Matrine enhanced [Ca²⁺]_i by stimulating I_Ca-L and exerted positive inotropic effects on electrically driven guinea pig papillary muscles.

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In order to identify the active anti-inflammatory ingredient(s) in Cirsium chanroenicum (Compositae), its methanol extract and several solvent fractions were prepared; the methanol extract and the ethylacetate fraction inhibited cyclooxygenase-2 (COX-2)-mediated prostaglandin E₂ (PGE₂) and 5-lipoxygenase (5-LOX)-mediated leukotriene (LT) production in lipopolysaccharide-treated RAW 264.7 cells and A23187-treated rat basophilic leukemia (RBL-1) cells, respectively. Further bioactivity-guided fractionation of the ethylacetate fraction using column chromatography led to the isolation of pectolinarigenin (5,7-dihydroxy-4′,6-dimethoxyflavone), along with pectolinarin [pectolinarigenin 7-rhamnosyl-(1→6)-glucoside]. Pectolinarigenin strongly inhibited COX-2-mediated PGE₂ and 5-LOX-mediated LT production at >1 μM, indicating that it is a dual inhibitor of COX-2/5-LOX. However, pectolinarigenin did not affect COX-2 expression or nuclear transcription factor (NF-κB) activation. In addition, in vivo studies demonstrated that oral administration of these two compounds at 20—100 mg/kg resulted in similar inhibitory activities against several animal models of inflammation/allergy: arachidonic acid-induced mouse ear edema, carrageenan-induced mouse paw edema and passive cutaneous anaphylaxis. All of these results suggest that pectolinarigenin and pectolinarin possess anti-inflammatory activity and that they may inhibit eicosanoid formation in inflammatory lesions. These activities certainly contribute to the anti-inflammatory mechanism of C. chanroenicum.

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This study investigated the changes in the mRNA levels of the ATP binding cassette (ABC) transporters multidrug resistance 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and multidrug resistance-associated protein 2 (MRP2) following exposure to the prototypical microsomal enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) in primary cultures of cryopreserved human and cynomolgus monkey hepatocytes. Analysis was performed by real-time reverse transcription-polymerase chain reaction using primers and TaqMan probes. First, the time course of the mRNA expression of these transporters in primary cultures of human and cynomolgus monkey hepatocytes was examined in detail. The ratio of MDR1 and MRP2 mRNA to β-actin mRNA in both human and cynomolgus monkey hepatocytes remained constant from 48 to 72 h and from 24 to 72 h of culture, respectively. Second, the hepatocytes were exposed to the inducers and the changes in the levels of the transporter mRNAs were examined. Rif increased MDR1 and MRP1 mRNA levels in both human and cynomolgus monkey hepatocytes, while Ome slightly increased MDR1 and MRP1 mRNA levels in cynomolgus monkey hepatocytes. Rif and Ome increased MRP2 mRNA levels in both human and cynomolgus monkey hepatocytes. In contrast, Dex tended to decrease the mRNA levels of MDR1, MRP1, and MRP2 in both human and cynomolgus monkey hepatocytes. Cynomolgus monkey hepatocytes appeared to be more responsive than human hepatocytes to the inducers. These results indicate that primary cultures of cynomolgus monkey hepatocytes are as useful as primary cultures of human hepatocytes for evaluating the induction of MDR1, MRP1, and MRP2 mRNAs in preclinical studies.

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The proliferation of vascular smooth muscle cells (VSMCs) induced by injury to the intima of arteries is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis. Uncaria rhynchophylla is traditional Chinese herb that has been applied to the treatment of convulsive disorders, such as epilepsy, in China. In the present study, we examined whether corynoxeine exerts inhibitory effects on platelet-derived growth factor (PDGF)-BB-induced rat aortic VSMC proliferation and the possible mechanism of such effects. Pre-treatment of VSMCs with corynoxeine (5—50 μM) for 24 h resulted in significant decreases in cell number without any cytotoxicity; the inhibition percentages were 25.0±12.5, 63.0±27.5 and 88.0±12.5% at 5, 20 and 50 μM, respectively. Also, corynoxeine significantly inhibited the 50 ng/ml PDGF-BB-induced DNA synthesis of VSMCs in a concentration-dependent manner without any cytotoxicity; the inhibitions were 32.8±11.0, 51.8±8.0 and 76.9±7.4% at concentrations of 5, 20 and 50 μM, respectively. Pre-incubation of VSMCs with corynoxeine significantly inhibited PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, whereas corynoxeine had no effects on mitogen-activated protein kinase (MAPK/ERK)-activating kinase 1 and 2 (MEK1/2), Akt, or phospholipase C (PLC)γ1 activation or on PDGF receptor beta (PDGF-Rβ) phosphorylation. These results suggest that corynoxeine is a potent ERK1/2 inhibitor of key PDGF-BB-induced VSMC proliferation and may be useful in the prevention and treatment of vascular diseases and restenosis after angioplasty.

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Objective: It was studied to determine if nicorandil can improve frequent urination in rats with partial bladder outlet obstruction (BOO) without changing the blood pressure. Materials and methods: Voiding behavior was observed 6 to 8 d after obstruction in female rats with BOO that loaded 30 ml/kg of water. A drug was administered orally. Changes in systemic blood pressure and heart rate were studied in conscious BOO rats using the tail cuff method. Results: The voiding frequency was increased and the average voided volume was decreased in BOO rats compared with normal rats. Nicorandil (1 mg/kg), cromakalim (0.1 mg/kg) and isosorbide dinitrate (ISDN; 1000 mg/kg) decreased voiding frequency significantly in BOO rats. Nicorandil also increased the average voided volume significantly. Although cromakalim and ISDN at doses effective at decreasing voiding frequency caused blood pressure to drop, nicorandil at an effective dose did not affect blood pressure and heart rate. Conclusion: Nicorandil improved frequent urination without changing the blood pressure. These results suggested that a hybrid of a K_ATP channel opener and nitric oxide donor, nicorandil was bladder-selective compared with vasculature in BOO rats.

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The present study was conducted to determine whether cutaneous itch involves mu-opioid receptors in either of the spinal cord or lower brainstem or in both regions in mice. An intraplantar injection of serotonin hydrochloride (100 nmol/site) induced biting, an itch-related behavior. The behavior was inhibited by subcutaneous (0.3—1 mg/kg) and intracisternal (1—10 nmol/site), but not intrathecal (1—10 nmol/site), injections of naloxone hydrochloride. An intradermal injection of serotonin (100 nmol/site) to the rostral back induced scratching, an itch-related behavior, which was inhibited by subcutaneous (1 mg/kg) and intracisternal (10 nmol/site) injections of naloxone. These results suggest that mu-opioid receptor in the lower brainstem, but not spinal cord, is a site of central pruritogenic action of opioids and is involved in the facilitatory regulation of itch signaling.

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The lymphocyte immunosuppressant-sensitivity test (LIST) has been reported extensively as being able to estimate the pharmacological efficacy of immunosuppressive drugs in individual patients. This study measured the IC₅₀ values for 6 drugs, cyclosporine, tacrolimus, methylprednisolone, 6-mercaptopurine, mycophenolic acid, and mizoribine, against mitogen-induced proliferation of peripheral-blood lymphocytes in 29 renal transplant recipients. We also examined relationship between the IC₅₀ values and 4 factors; age, duration of dialysis, percentage of lymphocytes in total white blood cells, and blastogenesis stimulation index by mitogen. There were no significant correlations between the IC₅₀ values and these factors, except that the tacrolimus IC₅₀ value was correlated weakly with the stimulation index (p<0.05, r=−0.429). It was concluded that the pharmacological efficacy of immunosuppressive drugs cannot be inferred from the patient characteristics or their laboratory data. LIST is an effective method to elucidate the pharmacodynamic properties of immunosuppressive agents in individual renal transplant recipients without being influenced by the recipient clinical data.

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Three Kampo medicines, Boiogito (BOT), Bofutsushosan (BTS) and Orengedokuto (OGT), used for obese patients were investigated for their effects on adipogenesis in cultured rat white adipocytes. Administration of the three extracts suppressed adipogenesis in concentration-dependent manners (1—100 μg/ml) without any cytotoxicity. Changes in mRNA expression levels were analyzed using a Rat 230 2.0 Affymetrix GeneChip® microarray system. DNA microarray analysis (total probe set: 31099) using cDNAs prepared from adipocytes revealed that BOT, BTS and OGT increased the expression of 133—150 genes and decreased the expression of 42—110 genes by ≥2-fold. We identified 329 downregulated genes and 189 upregulated genes among a total set of 514 probes (overlap: 4). Overall, genes related to cellular movement, cell death, cell growth/differentiation and immune responses were the most downregulated, while those related to lipid metabolism and cell signaling were the most upregulated. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted to confirm the microarray results. Analysis of the clustering profiles of the microarray results revealed that BOT and BTS changed the expression levels of similar genes mainly involved in small molecule biochemistry and cell differentiation, while OGT altered 10 genes related to lipid metabolism, in contrast to the effects of BOT and BTS. We also measured mRNA expression levels of seven selected genes highly contributing to the lipid metabolism by using semiquantitative RT-PCR assay, that were acetyl-Coenzyme A carboxylase alpha (ACACA), AE binding protein 1 (AEBP1), patatin-like phospholipase domain containing 8 (PNPLA8), secretoglobin (SCGB1A1), adrenergic (ADRB3), adiponectin (ADIPOQ), monoglyceride lipase (MGLL). Beta-actin (ACTB) gene was used as an endogenous internal standard. The present findings indicate that these three herbal extracts have the potential to prevent adipogenesis in rat white adipocytes through different mechanisms via modulation of gene expression levels.

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The present study was performed to evaluate the cardiovascular effects of ethanolic extract from the root bark of Ulmus macrocarpa (RBUM) in rats. The effects of RBUM on the vascular response of isolated rat aorta and the blood pressure of spontaneously hypertensive rats (SHRs) were evaluated. In addition, its antioxidant activity in H9c2 cells was investigated. In the free radical scavenging assay using 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH), RBUM exhibited significant scavenging activity with an EC₅₀ value of 14.3 μg/ml. RBUM also induced resistance to hydrogen peroxide-mediated oxidative insult in H9c2 myocardial cells. In isolated rat aortic preparations, RBUM exhibited potent vascular relaxant effect with an EC₅₀ value of 1.9 μg/ml. This relaxation was significantly inhibited by denudation of the endothelial layer, pretreatment with N^G-nitro-L-arginine methyl ester (10 μM), raising extracellular K⁺ (45 mM), and pretreatment with tetraethylammonium (10 mM). In an antihypertensive study with SHRs, long-term administration with RBUM (100 mg/kg) for 42 d decreased systolic blood pressure (approximately 20 mmHg). In SHRs after 42 d of treatment, RBUM recovered aortic relaxation to acetylcholine and sodium nitroprusside, and attenuated lipid peroxidation in liver of SHRs. These results suggest that chronic treatment with RBUM exerts antihypertensive effects in SHRs, and its direct vasorelaxant and antioxidant properties may contribute to reduce elevated blood pressure.

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Oxidation of low density lipoprotein (LDL) is strongly implicated as a key process in the onset of atherosclerosis. In this study, nine alkylated (C₁₀–C₅) flavonoids from Sophora flavescens were examined for their inhibitory effects on copper-induced LDL oxidation. Of the flavonoids tested, sophoraflavanone G (1), kurarinone (2), kurarinol (3), norkurarinol (4), and kuraridin (9) inhibited the generation of thiobarbituric acid reactive substances (TBARS) with IC₅₀s of 7.9, 14.5, 22.0, 26.9, and 17.5 μM, respectively. The most potent inhibitor, compound 1, also demonstrated significant activities in complementary in vitro investigations, such as lag time (130 min at 5 μM), relative electrophoretic mobility (REM) of ox-LDL (80% inhibition at 20 μM), and fragmentation of apoB-100 (inhibition of 71% at 20 μM). Analysis of the structures of these compounds reveals that a resorcinol moiety in the B-ring is strongly correlated with protection of LDL-oxidation.

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Royal jelly (RJ) is known to contain excellent nutrition and a variety of biological activities. The present study was designed to investigate the effects of RJ on insulin resistance (hyperinsulinemia) in fructose-drinking rats (FDR; insulin resistance animal model). Male Wistar rats (6 weeks old) received 15% fructose solution in drinking water for 8 weeks. FDR showed significant increases in plasma levels of insulin and triglyceride, Homeostasis Model Assessment ratio (HOMA-R, an index of insulin resistance), and systolic blood pressure, but not blood glucose levels, when compared with control rats. RJ (100, 300 mg/kg, p.o.) treatment for 8 weeks significantly decreased the plasma levels of insulin and triglyceride, HOMA-R, without affecting blood glucose or total cholesterol levels and tended to lower systolic blood pressure. In isolated and perfused mesenteric vascular beds of FDR, RJ treatment resulted in a significant reduction in sympathetic nerve-mediated vasoconstrictor response to periarterial nerve stimulation (PNS) and tended to increase the calcitonin gene-related peptide (CGRP) nerve-mediated vasodilator response to PNS, compared with those in untreated FDR. However, RJ treatment did not significantly affect norepinephrine-induced vasoconstriction or CGRP-induced vasodilation. These results suggest that RJ could be an effective functional food to prevent insulin resistance associated with the development of hypertension.

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We evaluated the effectiveness of bakumijiogan (BJG), an herbal formula in traditional Chinese medicine used to treat atopic dermatitis (AD), using a NC/Jic mouse model of AD. AD symptoms were induced by repeated injections of Dermatophagoides farinae antigen (Df-antigen) into the ear auricle at 2- to 3-d intervals for 16 d. Ear thickness dramatically increased up to 16 d after the first injection of Df-antigen. Daily oral administration of BJG from 7 d before to 16 d after the first injection significantly reduced ear swelling. Serum concentrations of total immunoglobulin (Ig)E and Df-antigen-specific IgG₁ were augmented when assayed 17 d after the first injection of Df-antigen, and these increases were slightly suppressed by BJG administration. Serum interferon (IFN)-γ and lesional IFN-γ mRNA levels were significantly higher, whereas lesional IL-1α and tumor necrosis factor-α mRNA levels were lower in BJG-treated mice than those in control mice. These results suggest that BJG suppressed AD-like symptoms by correcting the Th1/Th2 imbalance skewed toward Th2. Evaluation of herbal constituents in BJG revealed that the combination of two herbal ingredients, ophiopogon tuber and schisandra fruit, mainly contributed to the effects of BJG.

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Platycodin D (PD) isolated from Platycodi Radix has been reported to have anti-inflammatory and anti-tumor activities. In this study, we have investigated anti-inflammatory activities of prosapogenin D (PrsD) and prosapogenin D methyl ester (PrsDMe) of PD. The results indicated that PrsDMe concentration-dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production, however, PrsD did not inhibit NO production in LPS-induced macrophages. Furthermore, PrsDMe inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) without appreciable cytotoxic effects. In the transfectant RAW 264.7 cells, PrsDMe was observed to reduce the level of nuclear factor-κB (NF-κB) activity. PrsDMe also inhibited the degradation of an inhibitory protein called inhibitor κB (IκB). Therefore, it was suggested that PrsDMe inhibited the expression of LPS-induced iNOS and COX-2 genes by suppressing NF-κB activation at the transcriptional level. Also, PrsDMe showed carrageenan-induced acute anti-inflammatory activity and the adjuvant-induced anti-arthritic activity in mice. In conclusion, we suggest that these compounds exert an anti-inflammatory effect through the regulation of the NF-κB pathway. The different activities of PD, PrsD and PrsDMe are based on the structure of the sugar substituent or methyl group at the C₂₈-carboxyl position.

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A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding α-subunit of heterotrimeric guanosine 5′-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein α-subunits from various biological sources suggested that, unlike in animal cells, classification of Gα-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Gα-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

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An ethanol extract of rhubarb rhizome exhibited marked glucose transport activity in differentiated L6 rat myotubes. Activity-guided fractionation resulted in the isolation of two anthraquinones, chrysophanol-8-O-β-D-glucopyranoside (1) and chrysophanol (2). The anti-diabetic effect was examined by glucose transport activity, glucose transporter 4 (Glut4) expression in myotubes, and the level of insulin receptor (IR) tyrosine phosphorylation as influenced by tyrosine phosphatase 1B, each of which is a major target of diabetes treatment. Chrysophanol-8-O-β-D-glucopyranoside up to 25 μM dose-dependently activated glucose transport in insulin-stimulated myotubes. Increased tyrosine phosphorylation of IR due to tyrosine phosphatase 1B inhibitory activity with an IC₅₀ value of 18.34±0.29 μM and unchanged Glut4 mRNA levels was observed following chrysophanol-8-O-β-D-glucopyranoside treatment. Chrysophanol up to 100 μM exerted mild glucose transport activity and elevated the tyrosine phosphorylation of IR via tyrosine phosphatase 1B inhibition (IC₅₀=79.86±0.12 μM); Glut4 mRNA expression was also significantly increased by 100 μM. The ED₅₀ values of the two compounds were 59.38±0.66 and 79.69±0.03 μM, respectively. Therefore, these two anthraquinones from rhubarb rhizome, chrysophanol-8-O-β-D-glucopyranoside and chrysophanol, have mild cytotoxicity and anti-diabetic properties and could play metabolic roles in the insulin-stimulated glucose transport pathway.

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Probiotics are live microorganisms which have health-promoting attributes. These bacteria must overcome biological barriers, including acid in the stomach and bile in the intestine to exert beneficial effects. The encapsulation consists in a provision of an outer layer to protect the core material from damage. Microencapsulating in calcium alginate, nowadays, is being used to bacteria immobilization owing to its easy handling, nontoxic nature, and low cost. The aims of this study were to improve the microencapsulating method for probiotic bacteria and to investigate whether the material used as coating, afford an increase on strain survival under simulated gastrointestinal conditions. Lactic acid bacteria used in this work were isolated from feces of young and healthy pigs and they were selected because of their probiotic properties. Our results showed that the optimal encapsulation process was achieved using 1 : 1 (v/v) 20% non fat milk cell suspension mixed with 1.8% sodium alginate solution. Alginate capsules hardening was carried out using 0.1 m calcium chloride solution for 30 min. This microencapsulating technique could protect the probiotic bacteria against gastric environment, allowing viable cells get to the intestinal tract. So it could be a useful way to deliver these beneficial bacteria to host.

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The L-type amino acid transporter 1 (LAT1, SLC7A5) is an Na⁺-independent neutral amino acid transporter the expression of which is located in retinal endothelial cells. Due to its broad substrate selectivity, LAT1 has been proposed to mediate the transport of amino acid-related drugs across the blood-tissue barriers. Here, we have investigated the transport screening of amino acid-mustards using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) which expresses LAT1. We synthesized 5 amino acid-mustards: tyrosine-mustard, phenylglycine-mustard, alanine-mustard, ornithine-mustard, and lysine-mustard. LAT1-mediated [³H]L-phenylalanine (Phe) uptake by TR-iBRB2 cells was inhibited in a competitive manner by tyrosine-mustard and phenylglycine-mustard as well as melphalan (phenylalanine-mustard). Phenylglycine-mustard was able to induce the efflux of [³H]Phe preloaded into the TR-iBRB2 cells expressing LAT1 through the obligatory exchange mechanism, although tyrosine-mustard, alanine-mustard, ornithine-mustard, lysine-mustard, and melphalan did not induce any significant efflux. These findings suggest that phenylglycine-mustard is a better substrate for LAT1 than melphalan and other amino acid-mustards.

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To investigate the possible effects of different beverages in the gastrointestinal tract on the sulfation of estrogen, which is a major hormone and prototype substrate for the human sulfotransferases (SULT), we analyzed the effects of these substances upon the sulfate conjugation of 17β-estradiol (E₂) in the human colon carcinoma cell line Caco-2. Sulfoconjugation activity toward E₂ was measured by incubating 20 nM E₂ with Caco-2 cells in the presence (5% (v/v)) of each beverage. Among the 35 beverages analyzed, four (aronia, blueberry, coffee, and peppermint) exhibited strong inhibitory effects on E₂ sulfation within Caco-2 cells (IC₅₀ values ranging from 1.9 to 4.4% (v/v)). These active beverages also strongly inhibited the cytosolic estrogen SULT activity of Caco-2 cells in vitro (IC₅₀ values ranging from 0.18 to 0.3% (v/v)). These inhibitory activities were extractable with ethyl acetate but not hexane or n-butanol, indicating that the molecules responsible are moderately lipophilic. Coffee was found to be the most potent inhibitor but the major constituents of this beverage, caffeic acid, caffeine, and chlorogenic acid, did not show any effects on estrogen SULT activity. Kinetic analyses further indicated that the mode of inhibition by coffee is competitive. A possible relationship between the inhibition of estrogen SULT activity by coffee in the gastrointestinal tract and the reported reduction of colon cancer incidence in women who consume coffee is discussed.

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ATP-binding cassette, sub-family C, number 2 (ABCC2) is involved in the biliary excretion of irinotecan and its metabolites, SN-38 and SN-38 glucuronide. Effects of the ABCC2 genotype on the pharmacokinetics (PK) of irinotecan and the metabolites were examined in Japanese patients with metastatic colorectal cancer receiving irinotecan plus infusional 5-fluorouracil/leucovorin (FOLFIRI). ABCC2 genotypes (−1549G>A, −1023G>A, −1019A>G, −24C>T, 1249G>A and 3972C>T) and haplotypes were analyzed for 67 patients with cancer. PK was also examined in a subset of 31 patients receiving FOLFIRI. Relationship between the ABCC2 genotypes or diplotypes and area under the time–concentration curve (AUC) of irinotecan and the metabolites normalized by irinotecan dose was analyzed. The lower AUC of irinotecan was seen in patients with A/A or G/A genotypes at 1249 of the ABCC2 gene than others (p=0.011, Mann–Whitney U teat). AUC of SN-38 in patients with A/A or G/A genotypes at −1023 was significantly lower than that in others (p=0.018). The haplotype I included −1023A (GAACGC) was the most frequent one with the allele frequency of 0.366. The AUC of SN-38 observed in patients with diplotypes harboring at least one haplotype I was lower than that observed in others (p=0.023). The haplotype IV consisted of 1249 (GGACAC) and was the fourth most frequent one with the allele frequency of 0.127. Patients with diplotypes carrying at least one haplotype IV showed lower AUC of irinotecan than others (p=0.011). Thus, ABCC2 genotype is one of the predictors of the variability of irinotecan PK in Japanese patients with metastatic colorectal cancer receiving FOLFIRI.

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We previously showed that 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC), a component of diesel exhaust particles and a degradation product of the insecticide fenitrothion, has reproductive toxicity in adult male and immature female Japanese quail (Coturnix japonica). Here we investigated effects of PNMC on the reproductive toxicity of mature female Japanese quail. The experiment consists of 3 periods of pretreatment, treatment, and post-treatment for 5 d each. The birds were reared, bred naturally for 1 week, and after 5 d of pretreatment, then injected intramuscularly with PNMC at doses 1, 10, or 100 mg/kg body weight daily for 5 d. Body weight, egg weight, and hatchability did not differ among the observation periods. However, at all doses of PNMC, the egg-laying rate showed a modest decrease during the treatment period, with recovery during the post-treatment period. Plasma concentrations of luteinizing hormone (LH) and estrodiol-17β, were significantly decreased (p<0.05), and plasma concentrations of progesterone significantly increased (p<0.05) in birds treated with 10 and 100 mg/kg PNMC. These results suggest that PNMC have acute toxicity, and inhibited LH secretion, disturbing egg-laying in mature female quail. Our findings indicate that PNMC induces endocrine malfunction at the central level and subsequently disrupts reproductive processes in mature female quails.

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