Prion protein knockout (PRNP^−/−) cattle have been developed and may be used to produce bovine material such as serum, collagen, and gelatin. However, genetically engineered animals (GE animals) must not be imported or made commercially available in Japan, because they are not authorized for food use in Japan. We used real-time polymerase chain reaction (real-time PCR) to develop method of detection for neomycin- and the puromycin-resistance genes in beef samples. Plasmids containing the neomycin-resistance gene and the puromycin-resistance gene were used as standard reference molecules. The results clearly showed that the method we developed is capable of quantitatively detecting the neomycin- and the puromycin-resistance genes in the plasmids in the presence of genomic DNA extracted from a beef sample. We also applied the method to testing of beef samples imported from the United States (U.S.). This method will make it possible to monitor beef for contamination by material from GE cattle to assure food safety.

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The naphthohydroquinone rubimaillin, which has an angular-type three cyclic skeleton and was isolated from the Chinese medical plant Rubia cordifola, was found to inhibit lipid droplet accumulation in mouse macrophages and to selectively inhibit cholesteryl ester synthesis (IC₅₀: 18 μM). The metabolism of cholesterol from lysosomes to lipid droplets was inhibited by the compound with a similar IC₅₀ (45 μM). Moreover, rubimaillin inhibited acyl-CoA:cholesterol acyltransferase (ACAT1) activity in ACAT1-expressing cells (IC₅₀: 80 μM). Thus, these data strongly suggest that rubimaillin inhibits macrophage ACAT activity in order to decrease cholesteryl ester (CE) synthesis, leading to a reduction in the number of lipid droplets. Furthermore, rubimaillin was found to inhibit the ACAT2 isozyme in ACAT2-expressing cells (IC₅₀: 22 μM). We concluded that rubimaillin is a dual inhibitor of ACAT1 and ACAT2, but is more selective for the ACAT2 isozyme.

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The gene encoding for nucleoside diphosphate kinase from Plasmodium falciparum was obtained by polymerase chain reaction (PCR) and expressed in Escherichia coli. Tracking kinases is strenuous work due to many functional and technical deficits. Tracking of Plasmodium falciparum nucleoside diphosphate kinase (PfNDK) was carried out by conventional enzyme assays combined by isothermal titration calorimetry (ITC). ITC proved an efficient tracking method with rapid, accurate, and confident target confirmation. In addition, it provides substrate affinity and full thermodynamic profile in one experiment. Magnesium ions were found to be essential for nucleoside diphosphate (NDP) kinase activity; however, the absence of Mg²⁺ did not completely interfere with the binding of nucleotides. The substrate recognition was found to depend on enthalpic forces with little entropic contributions. However, in the absence of magnesium ions the nucleotides actively bind to the enzyme driven by hydrophobic forces. The enzyme showed specific activity that was within the average of known enzymes; however, it was at least two-fold higher than that of the human enzyme.

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Based on the inherent α-galactosidase activity, squid liver contains two different α-N-acetylgalactosaminidases (α-GalNAcases): α-N-acetylgalactosaminidase I (α-GalNAcase I), which typically exhibits the α-galactosidase activity and α-N-acetylgalactosaminidase II (α-GalNAcase II), which is devoid of such activity. The molecular properties of the α-GalNAcases that may account for their enzymological differences are as yet unknown. In this study, we have characterized and compared the chemical and immunological properties of α-GalNAcase I and α-GalNAcase II. Analysis of the N-terminal sequence of the first twenty amino acids revealed the striking homology between α-GalNAcase I and α-GalNAcase II. Digestion of α-GalNAcase I and α-GalNAcase II generated the peptide maps that display similarities in peptide pattern, indicating their close relationship in structure. Polyclonal antibodies were generated in rabbits against the purified α-GalNAcase I and α-GalNAcase II for comparison of the immunological properties. Both Western blot and surface plasmon resonance (SPR) studies showed that the anti-α-GalNAcase II antibody reacted with both α-GalNAcase I and α-GalNAcase II, whereas the anti-α-GalNAcase I antibody reacted only with α-GalNAcase I, indicating the presence of common as well as unique antigenic determinants on α-GalNAcase I and α-GalNAcase II. Taken together, these results suggest that α-GalNAcase I and α-GalNAcase II are closely related with regard to structure and that their nonhomologous domains are possibly responsible for the differences in enzymatic properties.

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MinC plays an important role in regulation of the cell division site in Escherichia coli. Previous studies using sedimentation and electron microscopic methods suggested that MinC interacts with the FtsZ polymer and inhibits further FtsZ polymerization. However, it is difficult to clarify details regarding specific molecular interactions by such static analytic methods. In this study, a fluorescence resonance energy transfer (FRET) method was developed to directly observe the interaction between Cy3-labeled MinC and Cy5-labeled FtsZ in solution. FRET analysis indicated that MinC interacts with monomeric rather than polymeric FtsZ in solution. This suggests that interactions between monomeric FtsZ and MinC are important for controlling of FtsZ polymerization by MinC.

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We previously reported that mevalonate pyrophosphate decarboxylase (MPD) is located in the cytosol and that MPD level in the liver is higher than in other rat tissues. In the present study, we further investigated the tissue distribution of MPD in guinea pigs by immunoblotting using anti-rat MPD antiserum. When immunoblot analysis was carried out using guinea pig brain, the antiserum reacted with 46-kDa protein as well as a substance with the same molecular weight of MPD in mice. Protein of 46-kDa detected in guinea pig liver treated with 0.1% pravastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor indicating a liver-specific effect, was increased 3-fold as compared with nontreated guinea pigs; however, 46-kDa protein in the brain treated with pravastatin was similar to that treated without pravastatin. When the subcellular distribution of MPD in the brain, liver, kidney, and testis, was examined by cell fractionation, MPD was mostly detected in the cytosol fraction of all tissues. From these data, the 46-kDa protein was identified as MPD. Next, when the tissue distribution of MPD was examined, MPD in the liver was higher than in other tissues. The relative amount of MPD in guinea pig kidney was higher than in rats and similar to in mice, as MPD in the liver of the same species was taken as 1. Furthermore, the correlation coefficient between guinea pigs and rats or mice in the tissue distribution of MPD was 0.69 or 0.72, respectively. These data indicate a relationship in tissue distribution between guinea pigs and rats or mice, although the tissue-specific regulator of MPD between species somewhat differed.

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Nitric oxide (NO) and heme oxygenase-1 (HO-1) play important roles in the regulation of stem cell proliferation and differentiation. However, it has not been examined whether human periodontal ligament (PDL) cells can differentiate into osteoblast-like cells by NO activity mediated via HO-1. The objective of this study was to determine the effect of NO on proliferation and differentiation in human PDL cells, and to identify the underlying mechanism of its actions. Primary human PDL cells were cultured with NO donor sodium nitroprusside (SNP); cell proliferation and differentiation were measured. NO production, cell viability and cell proliferation were evaluated using the Griess reagent, MTT assay and BrdU incorporation, respectively. To analyze differentiation, we measured alkaline phosphatase (ALP) activity, osteocalcin (OC), osteonectin (ON) expression, and bone sialoprotein (BSP) by Western blotting. SNP-induced NO production is associated with inducible nitric oxide synthase induction in a time and dose-dependent manner. SNP resulted in decreased cell proliferation and increased expression of osteogenic differentiation markers such as ALP, OC, ON and BSP. Maximal HO-1 was reached with 0.05 mM SNP and gradually decreased with 1.0 mM. Treatment with an HO-1 inhibitor and selective inhibitors of extracellular regulated kinase 1/2 and nuclear factor-kappaB blocked the SNP-induced growth inhibition, as well as osteoblastic differentiation. These data suggest that NO-induced osteogenic differentiation through HO-1 may be an important mediator of periodontal regeneration or bone tissue engineering.

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Bone marrow mesenchymal stem cells (MSCs) have the potential to be used in the cellular therapy of solid organs. However, tissue regeneration is limited by the death of transplanted cells. One of the main mechanisms of stem cell death in transplanted organs is through ischemia. In the present study, we sought to investigate whether a plant-derived antioxidant, berberine (BBR), could protect MSCs against MSCs apoptosis in a model of ischemia consisting of serum deprivation- and hypoxia-induced apoptosis in vitro. We also investigated the potential mechanism(s) that may mediate the action of berberine. We found that berberine significantly attenuated hypoxia-induced MSC apoptosis. Further study revealed that berberine could scavenger the reactive oxygen species (ROS), inhibit the c-jun NH(2)-terminal kinase (JNK), the loss of mitochondrial membrane potential and the release of cytochrome c (Cyt C) and caspase-3. In addition, we also showed that berberine could activate phosphoinositide-3 kinase (PI3K)/Akt and that pretreatment with PI3K/Akt inhibitors prevented berberine-induced inhibition of ROS, JNK and subsequent apoptosis, suggesting that the protective effects of berberine were PI3K/Akt-dependent. Taken together, these findings reveal that berberine protects against MSC apoptosis by preventing ROS-dependent and JNK-driven cell apoptosis in a PI3K/Akt-dependent manner. These data indicate that berberine is a promising anti-apoptotic agent for improving MSC survival during cell transplantation.

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The differentiation function and paracrine action of mesenchymal stem cells (MSCs) have been reported previously, although it is not clear which accounts for the short-term effects on post-MSC transplantation. In this study, rats with acute myocardial infarction (AMI) induced by occlusion of the left coronary artery were randomized to the MSC transplantation group and MI group. The differentiation and paracrine action of MSC were observed after 2 weeks of MI. We found that MSC transplantation improved cardiac function [left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), left ventricular +dp/dt and ejection fraction (EF)]. Only a few grafted MSC differentiated into myocardial cells or vascular endothelial cells. Conversely, a significant increase in capillary density was observed. MSC transplantation upregulated the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). These results indicate that an increase in capillary density and improved cardiac function post-MSC transplantation are closely related to the up-regulation of VEGF and bFGF.

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Ferrous ferric chloride (FFC^®) is a distinct form of aqueous iron composed of a complex of ferrous chloride and ferric chloride that participates in both oxidation and reduction reactions. The author's previous study showed that the supplementation of culture medium with FFC stimulated the proliferation and differentiation of keratinocytes and melanocytes in newborn mice. FFC also stimulated the proliferation of cultured human keratinocytes, fibroblasts, and melanocytes. However, it is not known whether FFC stimulates the proliferation and differentiation of mammalian skin cells as well as hair growth in vivo. To answer this question, FFC-containing skin lotions (FFC Super Essence Plain^® and Moisture^® Type, Akatsuka Co.) were painted on the dorsal skin of newborn C57BL/10JHir (B10) mice and tested for their proliferation- and differentiation-stimulating effects on keratinocytes, fibroblasts, and melanocytes as well as for their stimulating effects on the hair growth. This treatment stimulated the proliferation and differentiation of epidermal keratinocytes, dermal fibroblasts, and epidermal and dermal melanocytes in the skin as well as hair growth. From 2 to 3 weeks after birth B10 mice generally lose their hairs except those on the head at the telogen stage of the hair growth cycle due to the expression of the alopecia. The treatment with FFC lotions markedly inhibited the alopecia hair-loss. These results suggest that FFC can stimulate the proliferation and differentiation of keratinocytes, fibroblasts, and melanocytes in the skin as well as the hair growth, and, in addition, can inhibit the alopecia hair-loss.

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Cyclosporin A (CsA) has been used as an immunosuppressive drug to prevent organ transplant rejection and to treat autoimmune diseases. CsA has a proliferative effect on human gingival fibroblasts (HGF) in vitro. However, the molecular mechanisms underlying CsA-induced proliferation in HGF remain to be elucidated. This study was aimed to investigate the CsA responsive proteins in HGF using systematic proteomic approach. Cell viability was determined by MTT assay and reactive oxygen species (ROS) was measured by fluorescent spectrometer. Proteins profiled by two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and electrospray ionization quadrupole time-of-flight mass spectrometry (EIQ-TOF MS). To confirm the expression changes of proteins by proteomics analysis, Western blot was performed using specific antibody. CsA increased the cell viability of HGF in a dose- and time-dependent manner. Significantly, seventeen proteins were overexpressed in the CsA-treated HGF, whereas three proteins were found to be expressed less than the untreated cells. The identified proteins were mainly related with cell proliferation, metabolism, and oxidation. The overexpression of peroxiredoxin 1 (Prx 1) confirmed by Western blotting and reduction of cytosolic reactive oxygen species (ROS) levels in the CsA-treated HGF demonstrated that Prx 1 may play a crucial role in the HGF proliferation induced by CsA. Upregulation of Galectin 3 in CsA-treated HGF indicated that it is related to CsA-induced proliferation. These proteomic analysis data will provide an efficient approach in understanding the mechanisms of HGF proliferation by CsA.

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Impaired heart function is the main reason for increased mortality of diabetes mellitus. Development of drugs with cardioprotective effects against diabetic myocardiopathy would benefit patients with diabetes. In this study, we tested the cardioprotective effects of Daming capsule (DMC), a traditional Chinese formula, on heart function in streptozocin (STZ)-induced diabetic rats with high fat-diet (HFD). DMC 100 mg/kg/d markedly decreased fasting blood glucose (FBG) and total cholesterol (TC), but did not affect triglycerides (TG) in diabetic rats at 30 d. The decreased heart rate (HR) and prolonged QT and PR interval induced by diabetes mellitus were significantly reversed by DMC (p<0.05). The mechanism may involve that DMC attenuated L-type calcium channel α_1c subunit increasing and Kv4.2 decreasing at both mRNA and protein level in diabetic rats. Additionally, DMC could obviously ameliorate the impaired heart function of diabetic rats by decreasing elevated left ventricular end-diastolic pressure (LVEDP) and increasing the attenuated maximum change velocity of left ventricular pressure in the isovolumic contraction or relaxation period (±dp/dt_max). Transmission electron microscopy (TEM) results showed that myocardium injury was attenuated by DMC (100 mg/kg/d) in STZ-induced diabetic rats with HFD. In conclusion, DMC could recover the prolonged QT interval and PR interval and elevated diastolic and systolic function of diabetic heart. This protective effect may partially be mediated through affecting the mRNA and protein expression of Kv4.2 and α_1c as well as preventing cardiomyocyte morphological remodeling.

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Salidroside is a major constituent of Rhodiola rosea L. that elicits beneficial effects for ischemic cardiovascular diseases. The aim of this study was to investigate the protective effects of salidroside on endothelial cells apoptosis induced by the hypoxia mimicking agent, cobalt chloride. After challenge with cobalt chloride for 24 h, loss of cell viability and excessive apoptotic cell death were observed in EA.hy926 endothelial cells, and the level of intracellular reactive oxygen species (ROS) increased concentration-dependently. However, the endothelial cell apoptosis and excessive ROS generation were attenuated markedly by salidroside pretreatment. In addition, salidroside inhibited activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) induced by cobalt chloride, decreased expression of Bax and rescued the balance of pro- and anti-apoptotic proteins. These findings suggest that salidroside protects endothelial cells from cobalt chloride-induced apoptosis as an antioxidant and by regulating Bcl-2 family. Salidroside may represent a novel therapeutic agent for the treatment and prevention of hypoxia and oxidative stress-related diseases.

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A series of methylenedioxybenzene compounds were synthesized and found to have hepatoprotective effects in chemical-induced hepatotoxicity models. The purpose of the present study was to investigate the anti-fibrotic effects of a synthetic methylenedioxybenzene compound, CW209292, using the dimethylnitrosamine (DMN)-induced chronic liver injury model in rats. Liver injuries were induced in Sprague Dawley rats by injection of DMN (intraperitoneally, 10 μl/kg) 3 times per week for 4 weeks. The rats were treated with CW209292 (per os, 25 or 75 mg/kg/d) for 4 weeks. Treatment of rats with DMN for 4 weeks resulted in significant decreases in serum albumin levels, whereas concomitant treatment with CW209292 prevented these decreases. CW209292 treatment also shortened prothrombin time prolonged by DMN, providing evidence that the agent was active in preserving liver function against DMN insult. DMN treatment caused marked increases in plasma bilirubin, aspartate aminotransferase (AST), alanine transaminase (ALT), and hyaluronic acid levels; CW209292 treatment reversed these increases. CW209292 also significantly reduced hepatic hydroxyproline content as well as hepatic fibrosis and inflammation in histological examination. Additionally, immunochemically detectable hepatic collagen type IV and α-smooth muscle actin levels were decreased by CW209292 treatment. Proliferation of hepatic stellate cells isolated from DMN-treated rats was inhibited by CW209292. Furthermore, tumor growth factor (TGF)-β1 mRNA expression was increased in DMN-treated rats, whereas CW209292 treatment prevented these increases. These results suggest that CW209292 exhibits anti-fibrotic effects in Sprague Dawley rats with DMN-induced hepatic fibrosis by blocking the mRNA expression of TGF-β1 and subsequent inhibition of the proliferation of hepatic stellate cells.

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Recruitment of specific leukocyte subpopulations at the site of inflammation requires a series of cell adhesion molecules (CAMs)-mediated interactions. The major CAMs, viz., intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin are expressed on endothelium in response to various cytokines. Caffeic acid (CA), a natural phenolic compound from herbs and other sources, has been shown to prevent cardiovascular diseases. We investigated the effect of CA on the expression of CAMs by human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor (TNF-α). Adhesion of monocytes to CA-treated HUVECs was evaluated by co-culture experiments using 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF-AM) labeling of U937 cells. The expression of adhesion and chemoattractant molecules was evaluated by Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. CA significantly inhibited the TNF-α-induced increase in U937 monocyte adhesion to HUVECs as well as decreased the protein and mRNA expression levels of CAMs on HUVECs. CA also inhibited the mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8). The involvement of nuclear factor (NF)-κB in the transcriptional control of CAMs protein was assessed by degradation of inhibitory (I)κB and nuclear translocation of NF-κB using Western blotting and immunofluorescence staining. CA attenuated TNF-α-induced IκB degradation and NF-κB translocation from cytosol to the nucleus. In conclusion, TNF-α-induced NF-κB-DNA complex formation was inhibited by CA. CA reduced TNF-α-induced endothelial adhesiveness to HUVECs by inhibiting transcription factor activation, and CAMs expression suggesting its potential role in atherosclerosis diseases.

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The present study was undertaken to elucidate pathophysiological and pharmacological alterations in the right ventricle in monocrotaline-administered (MCT) rats. Examination of tissue weights of the MCT and age-matched control (CON) rats indicated the right ventricular (RV) hypertrophy until 8 weeks after a single subcutaneous administration of 60 mg/kg MCT. Apparent fibrosis in the right ventricle of the MCT rat at the 6th week (6w-MCT) was observed. Echocardiographic measurement of the cardiac and hemodynamic parameters of the MCT rat showed decreases in cardiac output and stroke volume indices at the 6th and 8th weeks. The RV Tei index, which increase represents aggravation of RV function, was augmented at the 4th to 8th week. The results suggest the genesis of cardiac and RV failure until 6 weeks after MCT administration. Injection of dobutamine (300 ng) or colforsin daropate (1 μg) into the perfused right ventricle isolated from CON rat at the 6th week resulted in a marked increase in cardiac double product, whereas injection of either agent into the right ventricle from the 6w-MCT rat elicited a small increase in the double product, followed by a sustained decrease in the developed tension. Infusion of acetylcholine (1 μg) into the RV muscle of the 6w-MCT rat resulted in prolongation of the periods for cardiac arrest and for bradycardia of the right ventricle. The results suggest that MCT administration causes the RV hypertrophy and eventually leads to the RV failure, accompanied by abnormal inotropic and chronotropic actions of the RV muscle.

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Infections with influenza A viruses are still a major threat to humans and several animal species. The occurrence of highly pathogenic avian influenza viruses capable of infecting and killing humans highlights the urgency for a new and efficient strategy for the treatment of diseases caused by the virus. Andrographolide and its derivatives have been widely used for treating respiratory infections in China for decades. We have recently synthesized new andrographolide derivatives and found that some of the compounds including 14-α-lipoyl andrographolide (AL-1) have significant activity against bacterial infections with an unique mechanism of action. We report here the antiviral activity of AL-1 and other andrographolide drugs. AL-1 showed significant activity against influenza A viruses including the H5N1 avian influenza virus. The administration of AL-1 by oral gavage to mice infected with avian influenza A/Chicken/Guangdong /96 (H9N2), A/Duck/Guangdong/99 (H5N1), and human influenza A/PR/8/34 (H1N1) viruses greatly reduced the death rate, prolonged life, inhibited lung consolidation, and reduced viral titers in the lung. The most effective dosage of AL-1 in these studies ranged from 100 to 200 mg/kg/d, when administered twice daily for 7 d beginning 24 h before viral exposure. The LD₅₀ of AL-1 was 1243 mg/kg/d. AL-1 was effective against avian influenza A (H9N2 and H5N1) and human influenza A H1N1 viruses in vitro, with the 50% effective concentrations ranging from 7.2 to 15.2 μM and the selective indexes ranging from 51 to 109. Significant inhibition of viral adsorption onto red blood cells with minimum inhibitory concentrations ranging from 5.3 to 16.8 mM suggested that AL-1 was capable of directly interfering with viral hemagglutinin to block binding to cellular receptors. With potent antiviral activity and a potentially new mechanism of action, AL-1 may warrant further evaluation as a possible therapy for influenza.

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Many studies have shown that Bupleurum falcatum (BF), which is widely used in the treatment of various psychosomatic diseases in traditional Oriental medicine, is an effective therapeutic intervention for memory impairment. The purpose of this study was to examine the effect of BF on stress-induced alterations in learning and memory in rats using the Morris water maze (MWM) and elevated plus maze (EPM) behavioral tests. In addition, we examined the effects of BF treatment on the cholinergic system, as indicated by changes in neuronal choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) immunoreactivity in the hippocampus. BF (150, 300, or 600 mg/kg) was administered orally 30 min before exposure to repeated immobilization (IMO) stress (4 h/d for 14 d). The BF treatment produced a significant improvement in escape latency (time required to find the platform) in the MWM, and it also produced an anxiolytic-like effect in the EPM. Consistent with the behavioral data, BF treatment significantly attenuated the IMO stress-induced loss of cholinergic immunoreactivity in the hippocampus. These findings indicate that BF has a protective effect against repeated IMO stress-induced neuronal and cognitive impairments, and they suggest that BF may be useful in the treatment of stress-induced memory impairment.

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Cancer is one of the leading causes of death in the world. The triterpenoid compound asiatic acid derived from the tropical medicinal plant Centella asiatica displays cytotoxic activity on fibroblast cells and several other kinds of cells. The present work studies asiatic acid-mediated growth inhibition of cancer cells and the underlying mechanism. Asiatic acid markedly inhibited cancer cell proliferation. Apoptosis of SW480 human colon cancer cells was induced by asiatic acid as shown by flow cytometry, DNA fragmentation and nuclear chromatin condensation experiments. Through increasing mitochondrial membrane permeability and cytochrome c release from mitochondria into cytosol, asiatic acid induced caspase-9 activity, which further activated caspase-3 and poly(ADP-ribose) polymerase cleavage resulting in irreversible apoptotic death in the tumor cells. Taken together, these results suggest that mitochondrial death apoptosis cascade plays very important roles in asiatic acid-induced cancer apoptosis.

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Hydrogen sulfide (H₂S), as an endogenous gas signaling molecule with important biological function that has been found recently, may play a protection in ischemic reperfusion (I/R) myocardium. We investigated the cardioprotective effect of H₂S in rats model of ischemic reperfusion in vivo and a probably influence on the expression of survivin, an anti-apoptosis gene. Animals were randomly divided into 3 groups and received either vehicle, sodium hydrosulfide (NaHS) or DL-propargylglycine (PAG) respectively everyday for 1 week before surgery and the treatment continued for a further 2 d after I/R till the animals were sacrificed. We investigated the plasma H₂S concentration and blood pressure, with the electrocardiogram (ECG) together, to prove the effect of H₂S to the heart function. We also compared the heart infarct size and the expression of an anti-apoptosis gene, survivin, among groups. As the data shown, the NaHS group had great improvement in blood pressure and electrocardiogram situation. And the remarkable shrink of the infarct size and up-regulation of survivin in NaHS group comparing with the other two groups also showed the cardio protective effect of H₂S in our study.

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The important role of renin-angiotensin-aldosterone system blockade in the treatment of diabetes-induced cardiomyopathy and nephropathy has been clearly established. The present study examined the effect of angiotensin II type 1 receptor blocker (ARB) losartan on oxidative stress and cardio-renal function in streptozotocin (STZ)-induced diabetic rats. Losartan treatment resulted in improvement of myocardial function and suppressed cardiac and renal fibrosis compared with the diabetic group. Losartan treatment also down-regulated transforming growth factor-β1 expression and attenuated the increased expression levels of p22^phox and Nox4. Blood urea nitrogen (BUN) and urinary protein levels were increased significantly in the diabetic group. Losartan treatment significantly reduced proteinuria but not BUN level. Moreover, the elevated level of malondialdehyde in both heart and kidney were significantly reduced in the losartan-treated group compared with the diabetic group. These results provided evidence that oxidative stress plays a major role in diabetic rats induced by STZ, and treatment with the ARB might be beneficial for preventing the development and progression of diabetic disease.

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Biofilm formation plays a key role in the life cycles and subsistence of many microorganisms. The human fungal pathogen Candida albicans has a high propensity to develop biofilms and resulted resistant to traditional antifungal agents. Biofilms are composed of a mixture of cell types, including yeast, pseudohyphal and hyphal cells, and hyphae are a prominent feature of biofilms. Riccardin D is a macrocyclic bisbibenzyl isolated from the liverwort Dumortiera hirsute in our laboratory. In the present investigation, the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assay and live/dead cell staining were employed for evaluating the effects of riccardin D on C. albicans biofilms. The results demonstrated that riccardin D can interfere with the biofilm formation. To investigate whether this effect was due to the inhibition of hyphae formation, morphological observation and real-time reverse transcriptase polymerase chain reaction (RT-PCR) were employed for evaluating the effects of riccardin D on the hyphae formation and the expression of hyphae specific genes. The results showed that the hyphae formation was strongly inhibited and the mRNA expression levels of hyphae specific genes were downregulated after riccardin D treatment. We concluded that riccardin D interfered with the biofilm formation of C. albicans through downregulating the expression of hyphae specific genes and inhibiting the formation of hyphae.

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Cytochrome P450 (CYP) 3A enzymes are responsible for the metabolism of many drugs. It is useful to know CYP3A activity in individual patients undergoing drug therapy so as to predict the efficacies or adverse events. Lansoprazole is metabolized to Lansoprazole sulfone (LS) by CYP3A, while to 5-hydroxylansoprasole by CYP2C19. The aim of this study was to evaluate whether lansoprazole can be used to assess CYP 3A activity in human liver. Lansoprazole sulfoxidation activity in 14 human liver microsomes was determined as the ratio of lansoprazole/LS, measuring these parameters by high-performance liquid chromatography. Testosterone 6β-hydroxylation (T6β-OH) activity, a known marker for CYP3A activity was also measured together with lansoprazole sulfoxidation activity. Lansoprazole sulfoxidation activity was also analyzed in microsomes preincubat-ed with anti-CYP2C19 antibody. Interindividual variation was observed in lansoprazole sulfoxidation activity and T6β-OH activities of those microsomes, respectively. Lansoprazole sulfoxidation activity was significantly correlated with T6β-OH activity and CYP3A protein level. Lansoprazole sulfoxidation activity in microsomes with anti-CYP2C19 antibody was closely correlated with T6β-OH activity. In contrast, lansoprazole 5-hydroxylation activity was correlated with the CYP2C19 activity. These results suggest that metabolism of lansoprazole to LS by CYP3A occurs independently of metabolism by CYP2C19. LS can be used as a new marker of CYP3A activity.

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Fatty acid synthase (FASN) is highly expressed in breast carcinomas to support their continuous growth and proliferation, but has low expression level in normal tissues. Considerable interest has been developed in searching for novel FASN inhibitors as a therapeutic target for breast cancer. In present study, amentoflavone was isolated from Selaginella tamariscina, a traditional oriental medicine that has been used to treat cancer for many years, and was found to significantly inhibit the in vitro enzymatic activity of FASN at concentrations above 50 μM. Amentoflavone was also found to decrease fatty acid synthesis by the reduction of [³H]acetyl-CoA incorporation into lipids in FASN-overexpressed SK-BR-3 human breast cancer cells. Furthermore, this study showed that amentoflavone, at a concentration greater than 75 μM, increased the cleavage-activity of caspase-3 and poly (ADP-ribose) polymerase (PARP), and administration of pan-caspase inhibitor Z-VAD-FMK completely rescued the SK-BR-3 cells from PARP cleavages. The sequential internucleosomal DNA fragmentation in SK-BR-3 cells was observed at a concentration of 100 μM. A decrease in breast cancer cell growth was observed in SK-BR-3 cells at 12 and 24 h post treatment with 100 μM of amentoflavone, followed by a dramatic suppression after 48 h. The inhibition of cancer-growth by amentoflavone was dose-dependent, showing a slight reduction at 50 μM and significant reduction at concentrations of 75 and 100 μM. FASN-nonexpressed NIH-3T3 normal cell growth was not decreased by amentoflavone-treatment, both in time- and dose-dependent manners. These data provide evidence that amentoflavone isolated from S. tamariscina induced breast cancer apoptosis through blockade of fatty acid synthesis.

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Coptidis Rhizoma and its isolated alkaloids are reported to possess a variety of activities, including neuroprotective and antioxidant effects. Thus, the anti-Alzheimer and antioxidant effects of six protoberberine alkaloids (berberine, palmatine, jateorrhizine, epiberberine, coptisine, and groenlandicine) and one aporphine alkaloid (magnoflorine) from Coptidis Rhizoma were evaluated via β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) assays, along with peroxynitrite (ONOO⁻) scavenging and total reactive oxygen species (ROS) inhibitory assays. Six protoberberine alkaloids exhibited predominant cholinesterases (ChEs) inhibitory effects with IC₅₀ values ranging between 0.44—1.07 μM for AChE and 3.32—6.84 μM for BChE; only epiberberine (K_i=10.0) and groenlandicine (K_i=21.2) exerted good, non-competitive BACE1 inhibitory activities with IC₅₀ values of 8.55 and 19.68 μM, respectively. In two antioxidant assays, jateorrhizine and groenlandicine exhibited significant ONOO⁻ scavenging activities with IC₅₀ values of 0.78 and 0.84 μM, respectively; coptisine and groenlandicine exhibited moderate total ROS inhibitory activities with IC₅₀ values of 48.93 and 51.78 μM, respectively. These results indicate that Coptidis Rhizoma alkaloids have a strong potential of inhibition and prevention of Alzheimer's disease (AD) mainly through both ChEs and β-amyloids pathways, and additionally through antioxidant capacities. In particular, groenlandicine may be a promising anti-AD agent due to its potent inhibitory activity of both ChEs and β-amyloids formation, as well as marked ONOO⁻ scavenging and good ROS inhibitory capacities. As a result, Coptidis Rhizoma and the alkaloids contained therein would clearly have beneficial uses in the development of therapeutic and preventive agents for AD and oxidative stress-related disease.

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Stemona sessilifolia, S. japonica and S. tuberosa are the three original sources of Stemonae Radix specified in the Chinese Pharmacopoeia (CP), and have been traditionally used for antitussive and insecticidal remedy. Significant variations in alkaloids composition and content, as well as different degrees of antitussive activities were found among them. In order to identify the genuine sources of Stemonae Radix accurately in genetic level, we determined the nucleotide sequences of chloroplast DNA trnL-trnF, trnH-psbA, petB-petD and trnK-rps16 regions of the species recorded in CP and S. parviflora, as well as the common counterfeits of Stemonae Radix, Asparagus species. The results revealed that the sequences of petB-petD and trnK-rps16 regions, showing relatively high substitution rate, were more informative than those of trnL-trnF and trnH-psbA regions. The sequences from all the four regions provided useful information to discriminate the three CP species from each other and from S. parviflora and the counterfeits. A phylogenetic tree reconstructed by the trnH-psbA sequences for 9 Stemona species distributed in China and Thailand showed that the three CP species belonged to the same clade, among which S. japonica and S. sessillifolia formed a sister group, showing closer relations to each other than to S. tuberosa. By contrast, S. parviflora was genetically far from the three CP species. Intra-species variations were observed in the three CP species. Especially, in S. tuberosa two types of petB-petD sequence and four types each of trnL-trnF, trnK-rps16 and trnH-psbA sequences resulted in 6 haplotypes; whereas, these differences had no relation with the different chemical types, but seemed to be consistent with geographical distribution.

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Nowadays herbal medicines of skin-whitening cosmetics are popular with women. We attempted to find the whitening activity compounds present in many herbal medicines used for this purpose and discuss their mechanisms in melanin biosynthesis. The 70% acetone extracts of 10 kinds of herbs were investigated for their mushroom tyrosinase activity inhibition. Among these 10 extracts, Chinese galls showed inhibitory activity against tyrosinase, with a 50% inhibitory concentration (IC₅₀) value of 22 μg/ml. In a B16 mouse melanoma cell cul-ture assay, Chinese galls dose-dependently inhibited melanin biosynthesis. Using ultraviolet A (UVA) or α-melanocyte-stimulating hormone (α-MSH) to stimulate B16 cells after Chinese gall treatment, the melanin biosynthesis of B16 cells was inhibited in a dose-dependent manner. The active compounds of Chinese galls were isolated by column chromatography, and the melanin biosynthesis inhibition in B16 melanoma cells was measured. Three gallotannins, 2,3,4,6-tetra-O-galloyl-D-glucopyranose, 1,2,3,6-tetra-O-galloyl-β-D-glucopyranose, and 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose, were isolated from Chinese gall extract, and their IC₅₀ values of tyrosinase inhibition activity were 54, 30, and 15 μM, respectively. By the mushroom tyrosinase inhibition kinetics assay, the three gallotannins were all determined to be non-competitive inhibitors. These results indicated that Chinese galls inhibit melanin biosynthesis, associated with hyperpigmentation and can be used as skin-whitening cosmetics for skin care.

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Proniosomal formulations with non-ionic surfactant were studied. The effect of hydrophilicity and hydrophobicity of one or two surfactants on drug solubility, proniosome surface structure and stability and skin permeation of haloperidol from different formulations were investigated. Haloperidol (HP) was entrapped in proniosomes with very high efficiency for all formulations. Stability studies performed at 4 °C and 25 °C for a period of 6 weeks did not reveal any significant drug leakage (p>0.05). Formulations with single surfactants were found to increase the skin permeation of HP more than formulations containing two surfactants. The number of carbons in the alkyl chain of the non-ionic surfactant influenced the in vitro permeation of HP though the epidermis and the skin permeation was increased with increase in hydrophilic–lipophilic balance (HLB) value of the surfactant. Interfacial tension and surfactant hydrophobicity appeared to be useful for elucidating mechanism of skin permeation and for comparing drug fluxes from different proniosomal formulations.

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The oligopeptide transporter PEPT1 (SLC15A1) is responsible for absorption of peptidic nutrients in the small intestine. Although the L-diastereomer of the β-lactam antibiotic cephalexin (L-cephalexin) is likely to be transported by PEPT1, there has been no direct demonstration of PEPT1-mediated L-cephalexin transport. Indeed, after the incubation with L-cephalexin, the intact form of L-cephalexin has not been identified inside vesicles/proteoliposomes prepared from brush border membrane of intestinal epithelial cells or cultured cell lines exogenously transfected with PEPT1 gene. Thus, it appears that L-cephalexin is rapidly metabolized by PEPT1 or PEPT1-associated proteins. Here, we attempted to verify whether L-cephalexin is transported by PEPT1 and whether it is hydrolyzed by PEPT1 itself, by using budded baculovirus expressing PEPT1 protein. Marked uptake of L-cephalexin in PEPT1-expressing budded baculovirus, compared with wild-type virus, indicated that L-cephalexin is a substrate for PEPT1. The uptake was found to be pH sensitive, and was strongly inhibited by the D-diastereomer of cephalexin and glycylsarcosine, but not by glycine. Thus, L-cephalexin is transported by PEPT1 itself. Upon the transport of both L- and D-cephalexin by PEPT1, dose-dependent membrane depolarization was observed; the EC₅₀ values of 0.18 and 2.9 mM, respectively, indicate that the affinity of L-cephalexin for PEPT1-mediated transport is much higher than that of the D-diastereomer. On the other hand, the L-cephalexin metabolite 7-aminodesacetoxycephalosporanic acid was not detected in PEPT1-expressing or wild-type virus at either pH 6.0 or 7.4. We conclude that L-cephalexin is transported by PEPT1 with high affinity, but is not metabolized by PEPT1 itself.

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The area under the curve (AUC) can be associated with the therapeutic or toxic effect of a drug. The limited sampling model (LSM) is an approach that is gaining popularity due to its simplicity for the estimation of AUC using 1—3 blood samples. The aim of the present simulation study was to compare the performance of LSM for various hypothetical drugs with that of the naive trapezoidal method (Trap). The 3-point (trough, peak, and downhill) sampling design following repetitive oral dosing was assumed for LSM (LSM3) and Trap (Trap3). The 2-point (trough and peak) sampling design was also assumed for LSM (LSM2) and Trap (Trap2). In addition, trough-sampling and peak-sampling designs for LSM were designated as LSM1 and LSM1′, respectively. As a result, the rank order of precision of the AUC estimation designs/methods was summarized as follows: LSM3≈Trap3≥LSM2≥Trap2≈LSM1>LSM1′. The finding suggested that LSM can not always improve the estimation performance of AUC in the 3-point sampling design, and that LSM1′ is insufficient to estimate the performance of AUC for the hypothetical drugs evaluated in the present study. Accordingly, LSM2 and LSM1 may be an efficient approach for estimating AUC following repetitive oral dosing. In addition, Trap3 and Trap2 may be promising alternatives, because Trap does not require a high investment to recruit a full-sampling model-development group.

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The aim of this research was to investigate differential gene expression of cyclin-dependent kinase inhibitors (CKIs) in white adipose tissue (WAT) and liver from high-fat fed male Wistar rats with or without vitamin C (VC) supplementation (750 mg/kg of body weight). After 56 d of experimentation, animals fed on a cafeteria diet increased significantly body weights and total body fat. Reverse transcription-polymerase chain reaction (RT-PCR) studies showed that cafeteria diet decreased p21 and p57 mRNA expression in subcutaneous WAT and increased p21 mRNA in liver. Overall, these data provide new information about the role of high fat intake on mRNA levels of several CKIs with implications in adipogenesis, cell metabolism and weight homeostasis. Interestingly, VC supplementation partially prevented diet-induced adiposity and increased p27 mRNA in liver without any changes in the other tissues and genes analyzed. Thus, hepatic mRNA changes induced by ascorbic acid indicate a possible role of these genes in diet-induced oxidative stress processes.

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