An assay method to measure N1-acetylpolyamine oxidase (PAO) activity with N1,N11-didansylnorspermine (DiDNS333) as the substrate by high performance liquid chromatography (HPLC) was developed. Dansylpolyamines were synthesized, and their activity as a substrate for partially purified PAO from rat liver was evaluated. Among the dansylpolyamines, DiDNS333 was a useful substrate for the development of the PAO assay method. DiDNS333 was degraded by PAO to 1-dansylamido-3-propanal (DNS3al) and N1-dansylnorspermidine (DNS33). As DNS3al was separated into two peaks by HPLC of the assay mixture containing aminoguanidine, determination of DNS33 was used for the development of the assay method. When the assay method was applied to inhibition studies, the DNS33 peak on the chromatogram was consistently produced, and weak interference was found in the incubation with higher concentrations of natural polyamines. This result suggested that contaminating polyamines in biological samples do not interfere with this method. When the assay method was applied to cell extract from Chinese hamster ovary cell samples, the PAO activity, even at the low level in the cells, could easily be detected with as little as 10 μg of protein, which corresponds to 1×105 cells. This HPLC method is a rapid, sensitive and useful assay for the measurement of PAO activity.
Resveratrol, a natural polyphenol in grapes, is known to prevent the cardiovascular diseases and to exert the antiangiogenic effect in in vivo models with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF). We examined the effect of resveratrol on tubule formation of cultured endothelial F-2 cells. In collagen gel matrix, F-2 cells formed an extended network of tubular structures in response to VEGF or bFGF. Resveratrol dose-dependently prevented the VEGF-induced tubule formation, but failed to inhibit the angiogenic response to bFGF. We next examined whether the inhibition of nitric oxide (NO) production is linked to the antiangiogenic effect of resveratrol on VEGF-stimulated F-2 cells, because NO plays a crucial role in VEGF-induced tubular network formation. NO production was increased by VEGF, but not by bFGF, and resveratrol inhibited VEGF-stimulated NO production. NG-nitro-L-arginine methyl ester (L-NAME) potently inhibited NO production under all conditions, including VEGF stimulation, and abrogated VEGF-induced tubule formation. However, L-NAME did not inhibit bFGF-induced tubule formation. To investigate the bFGF-induced in vivo antiangiogenic effect of resveratrol, we examined the effect of resveratrol on prostaglandin E2 (PGE2) production and cyclooxygenase (COX) expression in NRK-F fibroblasts. COX-2 and its derived PGE2 are important factors for bFGF-induced in vivo angiogenesis. Resveratrol dose-dependently prevented both COX-2 induction and PGE2 production in bFGF-stimulated fibroblasts. These results suggest that resveratrol exerts the inhibitory effects on VEGF- and bFGF-induced angiogenesis through different mechanisms including inhibition of NO production in VEGF-stimulated endothelial cells and inhibition of COX-2 induction in bFGF-stimulated fibroblasts.
Hepatic iron overload has been frequently observed in the liver of patients with chronic liver diseases. In this study, the effect of hepatic fatty acid accumulation on the iron metabolism was investigated. Mice fed a choline-deficient diet developed severe steatosis associated with increased total amount of non-heme iron in the liver. Hepatic lipid contents were well correlated with the iron amount. The choline-deficient diet significantly downregulated hepcidin while increases in hemojuvelin and transferrin receptor 2 and a decrease in Tmprss6 expression were observed. Moreover, ferroportin expression was downregulated in the livers of choline-deficient mice while increases in transferrin receptor 1 and divalent metal transporter 1 and a decrease in ferritin expression were observed in accordance with increased hepatic iron content. The expression of hepcidin and ferroportin mRNA was negatively correlated to hepatic lipid concentrations. These results suggest that enhanced dietary iron intake and reduced hepatic iron efflux occur in the mice fed a choline-deficient diet. In addition, a possible link between hepatic iron and lipid metabolism is also suggested.
ABCA1, a member of the ATP-binding cassette transporter family, regulates high-density lipoprotein (HDL) metabolism and reverses cholesterol transport. Its expression is upregulated mainly by the activation of the liver X receptor (LXR), retinoid X receptor (RXR), and peroxisome proliferator-activated receptors (PPARs). To identify natural compounds that can upregulate ABCA1 expression, we developed a reporter assay using U251-MG (human glioma cell line) cells that stably express a human ABCA1 promoter-luciferase and performed a cell-based high-throughput screening of 118 natural compounds. Using this system, we identified honokiol, a compound extracted from Magnolia officinalis, as an activator of the ABCA1 promoter. We found that honokiol also increased ABCA1 mRNA and protein expression levels in a dose-dependent manner in U251-MG cells without significant cell death and also increased ABCA1, ABCG1 and apolipoprotein E (apoE) expression levels in THP-1 macrophages. PPAR antagonists did not diminish the induction of ABCA1 expression by honokiol in U251-MG cells. Cotreatment of the cells with honokiol and T0901317 (synthetic LXR ligand) further increased the ABCA1 expression level, whereas cotreatment with 9-cis retinoic acid had no additive effect compared with treatment with honokiol alone. We also found that honokiol has binding affinity to RXRβ. In this study, we identified for the first time honokiol as an upregulator of ABCA1 expression, which is mediated by the binding of honokiol to RXRβ as a ligand.
We have recently demonstrated that TRB3, a novel stress-inducible protein, is an unstable protein regulated by the ubiquitin-proteasome system. The expression level of TRB3 protein is down-regulated by anaphase-promoting complex/cyclosome-cell division cycle division 20 homolog 1 (APC/CCdh1) through its D-box motif. Here we demonstrate that TRB3 regulates the stability of cell division cycle 25 A (Cdc25A), an essential activator of cyclin dependent kinases (CDKs). The expression level of Cdc25A protein is suppressed by over-expression of TRB3, while knockdown of TRB3 enhances the endogenous Cdc25A expression level. On the other hand, Cdc25A degradation induced by DNA damage is significantly rescued by TRB3. When serine residues in the DSG motif, which is the critical sequences for the degradation of Cdc25A induced by DNA damage, is mutated to alanine (Cdc25ADSG2X), both stimulatory and protective effects of TRB3 on the Cdc25A degradation is disappeared. TRB3 protein interacts with both wild Cdc25A and mutant Cdc25ADSG2X. Expression level of the endogenous TRB3 protein is down-regulated in a genotoxic condition. These results suggest TRB3 is a regulator for adjusting the expression level of Cdc25A both in a normal and a genotoxic conditions.
The purpose of this study was to examine the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), an inhibitor of L-type amino acid transporters, on the cell growth suppression in KB human oral cancer cells and to study the roles of cell cycle regulatory factors in the BCH-induced growth inhibition. The effect of BCH on cell growth suppression and the influence of BCH to cell cycle regulatory factors in KB cell growth inhibition were examined using cell cycle analysis, immunoblotting and immunoprecipitation. The BCH treatment induced cell cycle arrest at G1 phase in KB cells. The expression of cyclin D3 was remarkably decreased by BCH treatment. The BCH inhibited the expression of cyclin-dependent protein kinase 6 (CDK6) in a time-dependent manner. In addition, the expression of CDK inhibitor p27 was increased by BCH treatment in KB cells, but not CDK inhibitors p21 and p15. These results suggest that, in KB cells, the inhibition of LAT1 by BCH causes cell cycle arrest at G1 phase by inhibiting cyclin D3–CDK6 complex whereas increasing expression of a CDK inhibitor p27.
Antioxidant property and hematopoietic repair capacity are important characteristics of radioprotective agents. Some studies have demonstrated that 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG), a molecule isolated from the waterlily, has antioxidant, hematopoietic repair, and anti-inflammatory activities. In this study, we try to determine whether PGG extracted from a lily, Nymphaea tetragona var. angusta, has radioprotective effects on splenocytes in vitro against 60Co γ-ray irradiation with absorption doses of 2 Gy and 4 Gy. Results show that PGG treatment dramatically enhances the proliferation of splenocytes compared with irradiated but untreated controls. In addition, PGG treatment before irradiation protects the splenocytes from lethal effects of irradiation and decreases DNA damages as identified by the alkaline comet assay. PGG-treated cells also show less radiation-induced apoptosis. These cells have lower concentrations of the pro-apoptotic protein p53 and more of the anti-apoptotic protein Bcl-2. The results presented in this study suggest that PGG has a cytoprotective effect on immune cells exposed to normally damaging amount of radiation. Thus, PGG could be an effective, non-toxic radioprotective agent.
Chitooligosaccharides (COS), oligosaccharides composed of two to seven glucosamine residues, are known to exhibit various biological activities. In this study, we investigated the effects of COS in an in vivo mouse sleep deprivation-induced fatigue model in an effort to develop a functional food with anti-fatigue efficacy. Male Balb/c mice were orally administered 500 mg (kg d)−1 of COS lactate or COS HCl for 2 weeks, and severe fatigue was induced by sleep deprivation. To evaluate the extent of fatigue, the swimming time, representing the immobility time, was measured in a forced swim test. As a result, oral intake of COS lactate-manifested anti-fatigue effects could be observed by the attenuation of fatigue-induced body weight loss and shorter immobility period. In addition, COS lactate was shown to alleviate the fatigue-induced increase in cortisol and lipid peroxidation and a decrease in superoxide dismutase (SOD) activity. Of particular note, the oral administration of COS lactate increased the mitochondrial membrane potential and the mitochondrial number significantly, indicating that COS lactate may enhance mitochondrial function. In support of this, COS lactate increased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and cytochrome c (Cyt C) mRNA, indicating that it may increase mitochondrial biogenesis. These results suggest that COS lactate can be an effective anti-fatigue functional food, and this anti-fatigue effect may result from, at least in part, the enhancement of mitochondrial biogenesis and the inhibition of free radical generation.
Forsythiae Fructus has been used as a herbal medicine for a fruit of Forsythia viridissima LINDLEY or Forsythia suspensa VAHL (Oleaceae). In Korea, the fruit of Forsythia viridissima is used and in China, the fruit of Forsythia suspensa is used generally. There are differences in the amount and distribution of constituents between Forsythia viridissima (FV) and Forsythia suspensa (FS). Accordingly, a discrimination of these two herbal drugs is needed. In this study, we designed FV genetic marker based on the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA that can discriminate Forsythia viridissima and Forsythia suspensa and species-specific amplification product 252 bp was confirmed. Using the real-time polymerase chain reaction (PCR) (allelic discrimination) analysis, an accurate discrimination between Forsythia viridissima and Forsythia suspensa was accomplished. Accordingly, with the use of PCR analysis based on ITS region sequence of ribosomal DNA and the real-time PCR analysis which can efficiently discriminate between Forsythia viridissima and Forsythia suspensa was developed.
Ras guanyl nucleotide-releasing protein 2 (RASGRP2) is a calcium- and diacylglycerol-responsive guanine nucleotide exchange factor. Previously, we reported that XRASGRP2, a homolog of human RASGRP2, is expressed in the vascular region of the Xenopus embryo during embryogenesis. Here, we report the regulatory mechanisms of human rasgrp2 gene expression in vascular endothelial cells. Although three alternative splicing variants, differing in the first exon of 5′-untranslated region (5′-UTR), have been found for the human rasgrp2 gene, reverse transcription-polymerase chain reaction (RT-PCR) showed that the cDNA containing the distal first exon (D1E) was expressed only in human umbilical artery endothelial cells. We analyzed the 5′-flanking region of the human rasgrp2 gene by a luciferase assay, which revealed that not only a promoter but also silencer regions were present upstream of D1E, suggesting that rasgrp2 expression is controlled by a combination of transcriptional promotion and repression. Gel super shift assay demonstrated that OCT1/POU2F1 bound to the silencer region. These findings may help furthering our understanding of vasculogenesis and/or angiogenesis in vascular endothelial cells.
The purified silkworm excretory red fluorescent protein (SE-RFP) has exhibited a potent broad spectrum antimicrobial activity. The anti-microbial assays of purified SE-RFP against several pathogenic bacterial (both Gram positive and Gram negative) and fungal strains were performed by agar cup plate method. The minimum inhibitory concentration (MIC) of SE-RFP against pathogenic bacteria and fungi was evaluated by agar dilution technique. The SE-RFP has exhibited highest activity (lowest minimum inhibitory concentration and largest zone of inhibition) against Staphylococcus aureus and Candida albicans among the tested bacteria and fungi, respectively. For the first time, we are reporting here the bioactivity of a red fluorescent protein purified from the silkworm excreta against clinically important bacteria and fungi. The bioactive SE-RFP has two absorption peaks at 280 and 603 nm and, it has exhibited fluorescence emission peaks at 334 and 619 nm upon exciting at 280 and 603 nm, respectively. The SE-RFP being an aqua-soluble, economically feasible and eco-friendly protein, it can therefore be used for the practical applications as an effective antimicrobial agent.
Numerous clinical trials have shown that calcium channel blocker (CCB) therapy improves the clinical outcome in patients with cardiovascular diseases. Since the progression of several types of cardiovascular diseases is closely associated with inflammation, alleviation of inflammation may be one potential mechanism of those beneficial effects of CCB therapy. We examined whether a new CCB (azelnidipine) could influence the inflammatory response of human peripheral blood mononuclear cells (PBMCs), which are recruited to inflammatory lesions and modulate inflammation. We investigated whether azelnidipine affected intracellular signaling and cytokine production by phytohemagglutinin (PHA)-stimulated human PBMCs in vitro. PBMCs were obtained from 10 healthy volunteers and stimulated with PHA. Then relative intracellular calcium ion concentration ([Ca2+]i) was assessed by fluorescence microscopy, and the production of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) were measured by enzyme-linked immunosorbent assay. Stimulation with PHA significantly raised [Ca2+]i and enhanced the production of MCP-1 and TNF-α by human PBMCs. Azelnidipine significantly diminished the PHA-induced rise of [Ca2+]i, and the production of MCP-1 and TNF-α. These findings indicate that azelnidipine might have an anti-inflammatory influence on human PBMCs, although the mechanisms and the difference from other CCBs still remain unclear and further exploration should be required.
The radical scavenging effects and protective activities against oxidative stress of Korean mistletoe (Viscum album coloratum) lectin were investigated in vitro and with a cellular system using LLC-PK1 renal epithelial cells. The Korean mistletoe lectin (KML) showed 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with an IC50 value of 42.6 μg/ml. It also exerted nitric oxide (NO), superoxide anion (O2−), and hydroxyl radical scavenging activities in concentration-dependent manners. These results suggest that KML is a promising antioxidant by scavenging free radicals. Furthermore, under the LLC-PK1 cellular model, the cells showed declines in viability and increases in lipid peroxidation through oxidative stress induced by sodium nitroprusside (SNP) and pyrogallol, generators of NO and O2−, respectively. However, KML significantly and dose-dependently inhibited cell cytotoxicity and lipid peroxidation. In addition, 3-morpholinosydnonimnie (SIN-1), a generator of peroxynitrite (ONOO−) formed by simultaneously releases of NO and O2−, caused cytotoxicity, lipid peroxidation, and NO overproduction in the LLC-PK1 cells while KML ameliorated ONOO−-induced oxidative damage. Furthermore, overexpressions of cyclooxygenase-2 and inducible NO synthase induced by SIN-1 were observed, but KML down-regulated the expression levels of both genes. KML also reduced SIN-1-induced nuclear factor kappa B expression and the phosphorylation of inhibitor kappa B alpha in LLC-PK1 cells. These results indicate that KML has protective activities against oxidative damage induced by free radicals.
Diterpene kahweol, one of the major components in coffee, has anti-cancer, anti-oxidative, and anti-inflammatory activity. In this study, we explored the molecular mechanism of the anti-inflammatory activity of kahweol. Lipopolysaccharide (LPS)-activated RAW264.7 cells were used to explore the modulatory role of kahweol on nitric oxide (NO) and prostaglandin E2 (PGE2) production and the activation of signaling proteins and transcription factors using immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Kahweol diminished both the production of NO and PGE2 and the mRNA expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2. Interestingly, this compound suppressed the phospho-signal transducers and activators of transcription (STAT)-1 and p65/nuclear factor (NF)-κB levels in the nucleus but not c-Jun and c-fos. In conjunction, the phosphorylation of Akt and Janus kinase (JAK)2 also decreased. Therefore, our data suggest that kahweol in coffee may be an anti-inflammatory modulator with NF-κB/STAT-1-targeted inhibitory properties in LPS-activated RAW264.7 cells.
Ghrelin, a 28-amino acid brain–gut peptide expressed in periphery tissues and the central nervous system, has been demonstrated to increase insulin sensitivity in adipocytes. Recent data have indicated that insulin resistance exists in the brain and is related to Alzheimer's Disease (AD). The aim of this study was to investigate whether ghrelin increased high glucose-induced hippocampal neuron insulin sensitivity, and further modulated tau phosphorylation. Hippocampal neurons were cultured in concentrations of 25 mM and 75 mM glucose. The effect of ghrelin on hippocampal neuronal insulin sensitivity was detected by [3H]-2-deoxy-D-glucose uptake. The expression of Akt, glycogen synthase kinase-3β (GSK-3β) and tau phosphorylation was determined via Western blotting. Culturation in 75 mM glucose resulted in decreased neuronal glucose uptake and an increase in the level of tau phosphorylation at Ser 199. In neurons treated with ghrelin for 1 h, neuronal glucose uptake was increased and tau hyperphosphorylation was improved. Ghrelin activated Akt and GSK-3β phosphorylation, whereas phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin eliminated ghrelin's effect on neuronal glucose uptake and tau phosphorylation. This study demonstrated that ghrelin increased insulin-stimulated neuronal glucose uptake in 25 mM or 75 mM glucose, raised insulin sensitivity, improved insulin resistance and decreased tau abnormal phosphorylation via the PI3-K/Akt-GSK pathway. Ghrelin is a potential new medicine in the treatment of AD.
Lung cancer is a leading cause of morbidity and mortality worldwide. Cyclooxygenase-2 (COX-2) expression is upregulated in lung carcinomas and is considered an attractive therapeutic target. In this study, the effect of curcumin and curcumin analogues on COX-2 expression induced by phorbol 12-myristate 13-acetate (PMA) were investigated. We found that a novel curcumin analogue (GL63) inhibited PMA-induced COX-2 mRNA and protein levels in H460 cells to a greater degree than curcumin. To understand the molecular mechanisms governing COX-2 regulation, the effect on COX-2 mRNA degradation was examined; we found that GL63 significantly decreased COX-2 mRNA stability by reducing cytoplasmic localization and protein abundance of human antigen R (HuR). The 3′-untranslated region (3′-UTR) report gene assay also showed GL63 substantially reduced the 3′-UTR green fluorescent protein values, indicating that the destabilizing effect on COX-2 mRNA may be couple with the posttranscriptional inhibition of COX-2. Taken together, our results provide evidence that the novel curcumin analogue can effectively inhibit PMA-induced COX-2 expression in H460 cells, a mechanism associated with COX-2 mRNA stability and post-transcriptional regulation.
Topiramate pharmacokinetics is influenced by individual factors such as patient age, renal function and co-treatment. The aim of this study was to develop a population pharmacokinetic model of topiramate to assist dosage adjustments in individual patients. Steady-state topiramate plasma concentrations in patients with epilepsy were determined by HPLC using fluorescent labelling. Demographic, biochemical data and dosing history including concomitant drug therapy were collected from patients' charts. Nonlinear mixed effects modelling was used to fit a one-compartment pharmacokinetic model. The influence of patient weight and gender, body surface area, age, creatinine clearance, serum transaminases, topiramate daily dose and co-treatment with carbamazepine, valproic acid, benzodiazepines, and risperidone on topiramate pharmacokinetics was evaluated. Additionally, the relationship between topiramate plasma concentration and clinical response was investigated. Volume of distribution of topiramate was 0.518 l/kg. For a typical patient oral clearance was estimated at 1.47 l/h, with interindividual variability of 39.2%. Clearance was 70% higher in patients co-treated with carbamazepine and was found to increase with patient age. Somnolence was the most frequently observed adverse event. Incidence of headache was associated with topiramate plasma concentration. Somnolence, ataxia, tremor, speech disorders and fatigue were associated with adjunctive therapy with carbamazepine, valproic acid, benzodiazepines, risperidone, and clozapine. No association of topiramate plasma concentration with frequency of seizures or patient quality of life was observed. The developed model can be used for Bayesian estimation of pharmacokinetic parameters based on sparse plasma samples and for selection of optimum dosing in routine patient care.
The aim of this study was to elucidate the efficacy of combination therapy with irinotecan and amrubicin for lung cancer and the influence of administration schedule in a xenograft mouse model and human cancer cell culture. We investigated the antitumor activity of irinotecan and amrubicin on human small cell lung cancer cell line LX-1 inoculated in mice in vivo and the cytotoxic effect of SN-38 and amrubicinol on human lung cancer cell lines A549 and PC-6 in vitro. Combined administration of irinotecan and amrubicin in divided doses inhibited tumor growth by approximately 90%, with complete recovery observed in one case. Furthermore, combined administration in divided doses induced little loss of body weight. Combination index analysis revealed that the cell growth inhibitory effect of SN-38 combined with amrubicinol was additive, regardless of schedule or cell line. The effect of combination treatment with SN-38 and amrubicinol on cell cycle was investigated. Cell cycle showed arrest at both the S and G2/M phases. The results indicate that combination therapy with irinotecan and amrubicin can be expected to yield improved outcomes, including less toxicity, especially with divided administration.
Adhesion and migration of tumor cells are crucial steps in tumor invasion and metastasis. In the present study, we investigated the effects of saponin monomer 13 of dwarf lilyturf tuber (DT-13) on metastasis of human breast cancer cells (MDA-MB-435) during hypoxia. The effects and molecular mechanisms of DT-13 on MDA-MB-435 cells metastatic phenotype in vitro and in vivo were evaluated by RNA interference; quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays. DT-13 had no significant effects on cell adhesion and migration under normoxia conditions. Under hypoxic conditions, MDA-MB-435 adhesion to vitronectin was inhibited by about 43.5% or 60.8% after exposure of the cells to DT-13 at 1 μM or 10 μM, respectively. DT-13 decreased the migratory response by hypoxia at 1 or 10 μM, and inhibition ratios were 20% and 30%, respectively. DT-13 inhibited hypoxia-induced expression of αvβ3 integrin, tissue factor (TF) and early growth response gene-1 (Egr-1) and decreased excretion of matrix metalloproteinase 9 (MMP-9) of MDA-MB-435 cells under hypoxic conditions. After Egr-1 short interference RNA (siRNA) treatment, DT-13 could still inhibit the up-regulation of TF mRNA and protein levels and its pro-coagulant activity (PCA) under hypoxia. In nude mice, DT-13 decreased extravasation of MDA-MB-435 cells in the lung after tail vein injection. Our data suggest that DT-13 inhibits MDA-MB-435 cells metastasis during hypoxia via regulation of TF, and the effect of DT-13 on TF is partly mediated by Egr-1.
The activation of mitogen-activated protein kinases (MAPKs) is critically involved in inflammatory events through mediation of the production of various inflammatory cytokines. The Tpl2 (tumor progression locus 2)-MEK (MAPK/ERK kinase)-ERK (extracellular signal-regulated kinase) signaling pathway plays an essential role in the production of tumor necrosis factor α (TNFα) in macrophages stimulated with lipopolysaccharide (LPS). Here, we studied the molecular mechanisms of Tpl2-mediated TNFα production using a potent Tpl2 kinase inhibitor, 1,7-naphtyridine-3-carbonitrile, and LPS-stimulated RAW264.7 cells. This inhibitor was effective in suppressing the in vitro Tpl2 kinase activity, and caused a significant reduction in TNFα production via specific suppression of the phosphorylation of MEK and ERK but not that of p38 and c-Jun N-terminal kinase (JNK). A p38 inhibitor, SB203580, also inhibited the TNFα production dose-dependently. Although the TNFα mRNA level was not altered by either inhibitor, the Tpl2 inhibitor increased the nuclear TNFα mRNA level, while decreasing that in the cytoplasm. Tip-associated protein (TAP), a key molecule in the nucleocytoplasmic transport of TNFα mRNA, was up-regulated by LPS, but this increase was impaired by the Tpl2 inhibitor. In all cases, SB203580 was without effect in the presence of LPS. These results suggest that the LPS-induced TNFα production via the Tpl2-MEK-ERK signaling pathway is regulated by changing the TAP level at the nucleocytoplasmic transport level. These results improve understanding of TNFα regulatory mechanisms and might provide a new therapeutic strategy against inflammatory diseases.
We established a highly sensitive method for evaluating P-glycoprotein activity in Caco-2 cells. Using time-lapse confocal laser-scanning microscopy, we measured the change in fluorescence of residual rhodamine 123 in Caco-2 cells. Horizontal fluorescence images of rhodamine 123 in the apical and central parts of these cells were captured for 90 min. A continuous and significant decrease in the fluorescent intensity of rhodamine 123 in the apical part of the Caco-2 cells occurred during the measurement period, while the decrease in the central part was mild. The decrease in rhodamine 123 intensity in the apical part of Caco-2 cells were abolished in the presence of 10 μM digoxin, but the decrease in the central part was not. The decrease in total rhodamine 123 over whole cells was no significant influence of digoxin was observed. This residual rhodamine 123 assay for evaluating P-glycoprotein in the apical part of Caco-2 cells imaged by confocal laser scanning microscopy is more sensitive than conventional methods and will be a valuable screening tool for studying both the inhibition and induction of P-glycoprotein activity and expression. This method may also be useful for predicting P-glycoprotein-mediated alterations in the intestinal absorption of drugs.
Arctiin and its aglucone, arctigenin from the fruits of Arctium lappa L. showed potent in vitro antiviral activities against influenza A virus (A/NWS/33, H1N1) (IFV). Based on the data from time-of-addition experiments and on release tests of progeny viruses, arctigenin was assumed to interfere with early event(s) of viral replication after viral penetration into cells, and to suppress the release of progeny viruses from the host cells. Arctiin was orally effective against either IFV-inoculated normal or 5-fluorouracil (5-FU)-treated mice, being less effective as compared with oseltamivir. Noticeably, arctiin produced a larger amount of virus-specific antibody than those of control and oseltamivir in sera collected from 5-FU-treated mice. Furthermore, oral treatment of 5-FU-treated mice with arctiin did not induce any resistant viruses, although the same treatment with oseltamivir induced resistant viruses at a 50% frequency. When the combination of arctiin and oseltamivir was administered to normal mice infected with IFV, the virus yields in both bronchoalveolar lavage fluids and lungs were significantly reduced relative to those in the mice treated with arctiin or oseltamivir alone. Thus, monotherapy of arctiin or combined therapy of arctiin with oseltamivir would be another treatment option for influenza.
Corticosteroids are routinely used in patients with pulmonary fibrosis, yet they have several adverse effects. To improve this situation, we used an animal model of pulmonary injury and early fibrosis and investigated whether the combination of an intrapulmonary inhalation device with antedrug budesonide (BUD) administered to the lung had greater efficacy and fewer systemic adverse effects compared to long-acting dexamethasone (DEX). BUD or DEX was administrated either intrapulmonary or intravenously to bleomycin-treated rats. Anti-inflammatory and antifibrotic effects were evaluated according to inflammatory cell count, total protein concentration and soluble collagen concentration in bronchoalveolar lavage fluid. The systemic immunosuppressive effects were also assessed by measuring body, spleen and thymus weight. BUD and DEX were compared with respect to their pharmacokinetic profiles in plasma and lung. Intrapulmonary treatment of BUD attenuates various inflammatory and early fibrotic indices with minimal systemic adverse effects compared with DEX. The area under the curve (AUC) of BUD by intrapulmonary spray was 6.6-fold higher than the AUC of DEX in the lung. This study suggests that antedrug BUD by intrapulmonary treatment has local anti-inflammatory and antifibrotic effects with minimal systemic adverse effects.
Ten fatty acid alkyl esters isolated from Oxalis triangularis, were evaluated for the effects on melanogenesis using mouse B16 melanoma cells. Treatment of methyl linoleate, methyl linolenate, ethyl linoleate and ethyl linolenate significantly blocked forskolin-induced melanogenesis and inhibited tyrosinase activity. In addition, we found that they inhibited cAMP production, suggesting that their anti-melanogenic effect is mediated by the inhibition of cAMP production. We concluded that methyl/ethyl linoleate and linolenate isolated from Oxalis triangularis have pigment inhibition activity. These compounds may be useful as the cosmetic agent to stimulate skin whitening.
Cellular replacement therapy for diabetes mellitus has received much attention. In this study we investigated the effect of transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) induced into endoderm and early hepatic cells in streptozotocin (STZ)-induced diabetic mice. Mouse BM-MSCs were cultured in the presence of hepatocyte growth factor (HGF) and fibroblast growth factor (FGF-4) for 2 weeks and transplanted into diabetic mice. Blood glucose levels, intraperitoneal glucose tolerance test, serum insulin, body weight and islets histology were analyzed. The results demonstrated that transplantation of syngeneic induced MSCs could reverse STZ-induced diabetes in mice. The treatment of mice with hyperglycemia and islet destruction resulted in the repair of pancreatic islets. Blood glucose levels, intraperitoneal glucose tolerance test, and serum insulin were significantly recovered in induced BM-MSCs (iBM-MSCs) group. In addition, in the iBM-MSCs group the body weight and the number of islets were significantly increased compared to other groups. The results demonstrate that BM-MSCs induced into endoderm and early hepatic cells are suitable candidates for cell-based therapy of diabetes mellitus.
It has been believed that nuclear gene delivery is the most important process for gene expression, and various non-viral vectors are currently being developed with this assumption. However, some of our earlier studies revealed a surprising difference in transfection activity between viral and non-viral vectors: this difference is largely due to the result of the intranuclear disposition of DNA rather than its delivery to the nucleus (Hama S. et al. (2006), Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems. Mol. Ther., 13, 786—794). Here, we report on some direct evidence that demonstrates the importance of the release of intranuclear DNA on transfection activity. The data show that transfection activity can be substantially enhanced by integrating a multifunctional envelope-type nano device (MEND) and a biocleavable polyrotaxane (DMAE-SS-PRX) as an artificial condenser. Our integration system showed significantly higher transfection activity compared to conventional gene delivery system. Moreover, this system provides a strong support for our hypothesis that intranuclear DNA disposition plays a critical role in gene expression for non-viral vectors.
The delivery of nucleic acids to cancer cells represents a potentially useful strategy. Previously, we developed a multifunctional envelope-type nano device (MEND) for the efficient delivery of plasmid DNA. In addition, we successfully delivered short interference RNA (siRNA) into cytoplasm using a MEND which contains siRNA particles that were produced using stearyl octaarginine (STR-R8). In the present study, to achieve further gene silencing activity compared with STR-R8, various additional polycations were screened. We used protamine and 10 different polypeptides containing random sequence of basic amino acids. The ability of these polycations to form nano particles with siRNA were evaluated by measuring the size and zeta-potential of produced nano particles, and as a consequence, 6 of the polycations were selected for further evaluation. We then prepared MENDs containing the particles. The lipid composition of the MEND consisted of dioleoylphosphatidyl ethanolamine (DOPE)/phosphatidic acid (PA) (7/2). For cellular uptake and endosomal escape, the MEND was modified with PPD (polyethylene glycol (PEG)-peptide-DOPE), STR-R8 and GALA, pH-sensitive fusogenic peptide. The resulting MEND had a diameter of 120—170 nm and a zeta-potential of 15—25 mV. The MEND was transfected into HeLa cells stably expressing luciferase and the silencing activity of the polycations was compared. Most of the polycations failed to knockdown luciferase activity. However, the polypeptide containing ornithine and tryptophan (Orn/Trp) induced a higher knockdown than STR-R8. In addition, Orn/Trp induced a silencing effect at lower doses than STR-R8, as evidenced by dose-response data. In conclusion, the findings suggest that Orn/Trp is a superior polycation to STR-R8 for siRNA delivery.
The blood–brain barrier (BBB) expresses transporters that influence both dopaminergic neuronal function and drug therapy for Parkinson's disease (PD). The purpose of the present study was to clarify changes of transporter mRNA expression at the BBB in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model of PD, in order to understand the pathophysiological role of BBB transport function in PD. At 7 d after MPTP treatment, mice showed a motor deficit and a loss of dopaminergic neurons. At the same time, L-type amino acid transporter 1 (LAT1) mRNA expression in the brain capillary fraction of the MPTP-treated mice was significantly reduced by 62.6% compared with saline-treated mice, while no significant change was observed in the expression of glucose transporter 1, creatine transporter 1, taurine transporter, organic cation transporter 2, serotonin transporter, norepinephrine transporter and dopamine transporter. LAT1 mRNA expression in whole brain was not affected at 1, 3 and 5 d after the treatment, but was reduced by 46.3% at 7 d. LAT1 mediates the transport of large neutral amino acids, including tyrosine, as well as the PD-therapeutic drug levodopa, across the BBB. Our findings indicate that decreased LAT1 expression at the BBB in PD patients may adversely affect amino acid supply from the circulating blood and levodopa distribution into the brain.
To enhance the levels of transgene expression from plasmid-based nonviral vectors, replicating plasmids containing the SV40 origin and the SV40 large T antigen gene, as a model replicating unit, were constructed. The replicating luciferase plasmid DNA produced the luciferase protein more efficiently than the non-replicating luciferase plasmid DNA, as expected. Surprisingly, the introduction of the replicating plasmid DNA containing the Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) gene was highly cytotoxic and caused cell death without nucleoside analogs. Our results confirm that transgenes on a replicating plasmid represent an excellent tool for effective protein production and suggest that efficient production of the Dm-dNK protein in tumor cells could be an attractive cancer therapy.
A Biodegradable artificial bone with inter-connective pores was prepared using a self-setting apatite/collagen composite cement as a cell scaffold for bone regenerative medicine, and investigated as to biocompatibility by X-ray computed tomography (CT) after its implantation into rats. Blocks (APN, APC and ACC) of apatite cement, apatite cement with continuous holes, and apatite/collagen composite cement with continuous holes were prepared. The APC and ACC blocks had 16 (8×2) interconnecting holes 500 μm in diameter. After the APN, APC, and ACC blocks were implanted in the back of the rats, X-ray CT images were measured every week. Before and after implantation, powder X-ray diffraction profiles of APN, APC and ACC showed diffraction patterns of hydroxyapatite with low crystallinity. Changes in the volume, inorganic content and density of the blocks in the rats were evaluated based on X-ray CT images. The volume and inorganic content of ACC decreased continuously at a constant rate. In contrast, the volume and inorganic content of APN and APC didn't show major changes. After implantation, the absorption of X-rays by ACC decreased with time. This suggested that the block was bioabsorbed significantly with time. In contrast, the absorption of APC and APN did not decrease, indicating that the blocks were not bioabsorbed.
PEGylation, the covalent attachment of polyethylene glycol (PEG) to pharmaceutical proteins, is regarded as an extremely useful procedure to generate protein drugs with intensified therapeutic properties. We examined the amino group modification of bovine lactoferrin (bLF) with linear PEG-p-nitrophenyl active esters. At pH 5.0, we specifically observed the formation of mono-PEGylated bLF in high yields. PEG-conjugation reactions advanced slowly and reached a steady state by 48 h in a buffer at pH 5.0. The hydrolysis half-lives of 5-kDa and 30-kDa PEG-p-nitrophenyl active esters at pH 5.0 were estimated to be approximately 117 and 136 h, respectively. The slow reaction and hydrolysis rates of PEG-p-nitrophenyl active esters may contribute to the formation of mono-PEGylated bLF that could not be obtained by PEGylation with linear N-hydroxysuccinimide (NHS) activated PEG.
Carboxypeptidase R (CPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is an enzyme generated by proteolytic cleavage of its zymogen (proCPR). CPR removes the C-terminal arginine from inflammatory peptides such as C3a and C5a, bradykinin, enkephalin, and the thrombin-cleaved N-terminal fragment osteopontin (cleaved N-OPN). In the mouse model of concanavalin A (Con A)-induced immune-mediated fulminating hepatitis, cleaved N-OPN is one of the important peptides that induce the production of chemokines or cytokines. In the current study using proCPR deficient mice, we showed that injection of Con A into the mouse tail vein can induce a significantly higher lethality in proCPR-deficient female but not in male mice. Furthermore, a lack of CPR activity increased serum macrophage inflammatory protein-2 (MIP-2) and high-mobility group box 1 (HMGB1) levels after Con A injection. These in vivo findings suggest that CPR helps to protect against Con A-induced hepatitis.