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Cell Toxicity (cell + toxicity)
Selected AbstractsAntioxidant and hepatoprotective effects of punicalagin and punicalin on acetaminophen-induced liver damage in ratsPHYTOTHERAPY RESEARCH, Issue 3 2001Chun-Ching Lin Abstract Punicalagin and punicalin were isolated from the leaves of Terminalia catappa L., a Combretaceous plant distributed throughout tropical and subtropical beaches, which is used for the treatment of dermatitis and hepatitis. Our previous studies showed that both of these compounds exert antioxidative activity. In this study, the antihepatotoxic activity of punicalagin and punicalin on acetaminophen-induced toxicity in the rat liver was evaluated. After evaluating the changes of several biochemical functions in serum, the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased by acetaminophen administration and reduced by punicalagin and punicalin. Histological changes around the hepatic central vein and oxidative damage induced by acetaminophen were also recovered by both compounds. The data show that both punicalagin and punicalin exert antihepatotoxic activity, but treatment with larger doses enhanced liver damage. These results suggest that even if punicalagin and punicalin have antioxidant activity at small doses, treatment with larger doses will possibly induce some cell toxicities. Copyright © 2001 John Wiley & Sons, Ltd. [source] IFN-, induces apoptosis in mouse embryonic stem cells, a putative mechanism of its embryotoxicityDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2000Gang-Ming Zou It has been reported that interferon (IFN)-, should inhibit in vitro mouse embryo growth by direct cell toxicity. However, the mechanism involved has not been clearly established. In the present study, this question was addressed using the embryonic stem (ES) cell model. It was found that IFN-, induces a dose-dependent apoptosis in ES cells, as assessed by trypan-blue staining, by Annexin-V labeling and DNA analysis. Moreover, IFN-, treatment cooperates with Fas-mediated apoptosis, a phenomenon that has been recently reported. As Bcl-2 oncoprotein functions as a death repressor molecule in an evolutionarily conserved cell death pathway, its expression was analyzed by flow cytometry. It was demonstrated that Bcl-2 is expressed in ES cells. When compared to untreated ES cells, IFN-,-treated, apoptotic cells expressed a lower Bcl-2 level and a normal level of Fas, whereas surviving cells expressed a normal level of Bcl-2 but a lower Fas expression. Altogether, these data suggest that IFN-, may influence early mouse embryo development by promoting apoptosis, which may constitute a novel mechanism of IFN-, embryotoxicity. [source] Dynamics of genome evolution in facultative symbionts of aphidsENVIRONMENTAL MICROBIOLOGY, Issue 8 2010Patrick H. Degnan Summary Aphids are sap-feeding insects that host a range of bacterial endosymbionts including the obligate, nutritional mutualist Buchnera plus several bacteria that are not required for host survival. Among the latter, ,Candidatus Regiella insecticola' and ,Candidatus Hamiltonella defensa' are found in pea aphids and other hosts and have been shown to protect aphids from natural enemies. We have sequenced almost the entire genome of R. insecticola (2.07 Mbp) and compared it with the recently published genome of H. defensa (2.11 Mbp). Despite being sister species the two genomes are highly rearranged and the genomes only have ,55% of genes in common. The functions encoded by the shared genes imply that the bacteria have similar metabolic capabilities, including only two essential amino acid biosynthetic pathways and active uptake mechanisms for the remaining eight, and similar capacities for host cell toxicity and invasion (type 3 secretion systems and RTX toxins). These observations, combined with high sequence divergence of orthologues, strongly suggest an ancient divergence after establishment of a symbiotic lifestyle. The divergence in gene sets and in genome architecture implies a history of rampant recombination and gene inactivation and the ongoing integration of mobile DNA (insertion sequence elements, prophage and plasmids). [source] In vivo production of catalase containing haem analoguesFEBS JOURNAL, Issue 12 2010Myriam Brugna Haem (protohaem IX) analogues are toxic compounds and have been considered for use as antibacterial agents, but the primary mechanism behind their toxicity has not been demonstrated. Using the haem protein catalase in the Gram-positive bacterium Enterococcus faecalis as an experimental system, we show that a variety of haem analogues can be taken up by bacterial cells and incorporated into haem-dependent enzymes. The resulting cofactor-substituted proteins are dysfunctional, generally resulting in arrested cell growth or death. This largely explains the cell toxicity of haem analogues. In contrast to many other organisms, E. faecalis does not depend on haem for growth, and therefore resists the toxicity of many haem analogues. We have exploited this feature to establish a bacterial in vivo system for the production of cofactor-substituted haem protein variants. As a pilot study, we produced, isolated and analysed novel catalase variants in which the iron atom of the haem prosthetic group is replaced by other metals, i.e. cobalt, gallium, tin, and zinc, and also variants containing meso-protoheme IX, ruthenium meso-protoporphyrin IX and (metal-free) protoporphyrin IX. Engineered haem proteins of this type are of potential use within basic research and the biotechnical industry. Structured digital abstract ,,MINT-7722358, MINT-7722368: katA (uniprotkb:Q834P5) and katA (uniprotkb:Q834P5) physically interact (MI:0915) by copurification (MI:0025) [source] Self-sterilizing catheters with titanium dioxide photocatalyst thin films for clean intermittent catheterization: Basis and study of clinical useINTERNATIONAL JOURNAL OF UROLOGY, Issue 5 2007Yuki Sekiguchi Objective: Clean intermittent catheterization (CIC) requires a large number of disposable catheters or a large amount of water and disinfectant. We made titanium dioxide (TiO2)-coated catheters for CIC using technology we have developed previously, and examined the photocatalytic antibacterial effect of this catheter using only light energy and the safety of this type of catheter for practical clinical use. Methods: TiO2 -coated catheters were filled with bacterial cell suspensions and illuminated with a 15-W black-light lamp for testing antibacterial potency. Next, we soaked control toxic materials (zinc diethyldithiocarbamate) and the tips of TiO2 -coated catheters in M05 medium, and evaluated cell toxicity from the numbers of V79 colonies in these dilutions. Then, bodyweight curves and histological tissue changes were observed over a period of time in mouse-transplanted TiO2 -coated catheters and control catheters. Finally, we investigated the use of these TiO2 -coated catheters in 18 patients by questionnaire and bacterial culture of TiO2 -coated catheters and control catheters. Results: The survival rate of Escherichia coli in the liquid inside the TiO2 catheter decreased to a negligible level within 60 min under ultraviolet (UV)-A illumination. The survival rate of Staphylococcus aureus, Pseudomonas aeruginosa and Serratia marcescens also decreased to a negligible level within 60 min. V79 cells showed no cytotoxicity of this catheter, and there was no difference in bodyweight or foreign body reaction between mouse-transplanted TiO2 -coated catheters and control catheters. In a preliminary clinical analysis of 18 patients who voluntarily used this catheter, the rate of positive bacterial culture of the tips of TiO2 -coated catheters was 20% versus 60% for conventional catheters after 4 weeks of use. Conclusion: TiO2 -coated silicone catheters were easily sterilized under certain light sources and were shown to be safe in an experiment using cultured cells and in animal experiments. Sterilizing catheters with TiO2 photocatalyst thin films are expected to be used clinically for clean intermittent catheterization after proper modification based on this study. [source] The Dose-Response Effects of Ethanol on the Human Fetal Osteoblastic Cell LineJOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2001A. Maran Abstract Alcohol is a risk factor for the development of osteoporosis, especially in men. Chronic alcohol abuse decreases bone mass, which contributes to the increased incidence of fractures. To better understand the mechanism of action of ethanol on bone metabolism, we have studied the dose-response effects of ethanol on conditionally immortalized human fetal osteoblasts (hFOB) in culture. Ethanol treatment had no significant effects on osteoblast number after 1 day or 7 days. Ethanol treatment did not reduce type I collagen protein levels at either time point at any dose but slightly reduced alkaline phosphatase activity after 7 days. The messenger RNA (mRNA) levels for alkaline phosphatase, type I collagen, and osteonectin were unaltered by 24 h of ethanol treatment but a high dose (200 mM) reduced mRNA levels for the two bone matrix proteins after 7 days. Ethanol treatment led to dose-dependent increases in transforming growth factor ,1 (TGF-,1) mRNA levels and decreases in TGF-,2 mRNA levels. The concentration of ethanol in the medium decreased with time because of evaporation but there was little degradation caused by metabolism. These results, which show that cultured osteoblasts are less sensitive than osteoblasts in vivo, suggest that the pronounced inhibitory effects of ethanol on bone formation are not caused by direct cell toxicity. [source] 17,-estradiol prevents cytotoxicity from hydrophobic bile acids in HepG2 and WRL-68 cell culturesJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 5 2006Matteo Ricchi Abstract Background:, Epidemiological and clinical studies suggest the possibility that estrogens might have a cytoprotective effect on the liver. The aim of the present study was to test the hypothesis that 17,-estradiol (E2) prevents hepatocellular damage induced by deoxycholic acid (DCA), a hydrophobic bile acid. Methods:, HepG2 cells were exposed for 24 h to DCA (350 µmol/L). Cell viability, aspartate aminotransferase and lactate dehydrogenase activity and apoptosis were measured as indices of cell toxicity. The effect of DCA was compared to that observed using either a hydrophilic bile acid, ursodeoxycholic acid (UDCA; 100 µmol/L), or E2 at different concentrations (1 nmol/L, 10 nmol/L, 50 nmol/L and 50 µmol/L) or mixtures of E2/DCA or UDCA/DCA. The same experiments were performed using WRL-68 cells that, at variance with HepG2, express a higher level of nuclear estrogen receptor. Results:, High concentrations of E2 and UDCA prevented DCA-induced decrease in cell viability, increase in enzyme activity and apoptosis evaluated both by 4,,6-diamidino-2-phenylindole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labeling (TUNEL) assays. In addition, DCA-related apoptosis, assessed by caspase activity, was also prevented by E2 (P < 0.01) in physiological (1,10 nmol/L) doses. The cytoprotective effects of E2 and UDCA was also observed in the WRL-68 cell line. Conclusions:, 17,-Estradiol prevents DCA-induced cell damage in HepG2 and WRL-68 cell lines to an extent comparable to UDCA. The hypothesis that the protective effect of E2 may be mediated by a mechanism that is nuclear estrogen receptor independent, deserves further verification. [source] Changes in endoplasmic reticulum stress proteins and aldolase A in cells exposed to dopamineJOURNAL OF NEUROCHEMISTRY, Issue 1 2008April A. Dukes Abstract In Parkinson's disease, oxidative stress is implicated in protein misfolding and aggregation, which may activate the unfolded protein response by the endoplasmic reticulum (ER). Dopamine (DA) can initiate oxidative stress via H2O2 formation by DA metabolism and by oxidation into DA quinone. We have previously shown that DA quinone induces oxidative protein modification, mitochondrial dysfunction in vitro, and dopaminergic cell toxicity in vivo and in vitro. In this study, we used cysteine- and lysine-reactive fluorescent dyes with 2D difference in-gel electrophoresis, mass spectrometry, and peptide mass fingerprint analysis to identify proteins in PC12 cell mitochondrial-enriched fractions that were altered in abundance following DA exposure (150 ,M, 16 h). Quantitative changes in proteins labeled with fluorescent dyes indicated increases in a subset of proteins after DA exposure: calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94 and Orp150 (149,260%), and decreased levels of aldolase A (39,42%). Changes in levels of several proteins detected by 2D difference in-gel electrophoresis were confirmed by western blot. Using this unbiased proteomics approach, our findings demonstrated that in PC12 cells, DA exposure leads to a cellular response indicative of ER stress prior to the onset of cell death, providing a potential link between DA and the unfolded protein response in the pathogenesis of Parkinson's disease. [source] PGH2 -derived levuglandin adducts increase the neurotoxicity of amyloid ,1,42JOURNAL OF NEUROCHEMISTRY, Issue 4 2006Olivier Boutaud Abstract The body of evidence indicating that oligomers of amyloid ,1,42 (A,1,42) produce toxicity to neurons, together with our demonstration that prostaglandin H2 (PGH2) oligomerizes amyloid ,1,42, led to the examination of the neurotoxicity of amyloid ,1,42 treated with PGH2. The neurotoxic effects of A,1,42 incubated with PGH2 was examined in primary cultures of cerebral neurons of mice, monitoring the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an indicator of cell toxicity. Whereas A,1,42 itself, incubated for 24 h, has little or no effect on MTT reduction, A,1,42 24 h after exposure to PGH2 produced a marked inhibition of MTT reduction, comparable with the inhibition resulting from A,1,42 that has been oligomerized by incubation for 6 days. Similar results were obtained when A,1,42 was incubated with levuglandin E2 (LGE2), a reactive aldehyde formed by spontaneous rearrangement of PGH2. The oligomers formed from reaction of A,1,42 with LGE2 exhibit immunochemical similarity with amyloid-derived diffusible ligands (ADDLs), as determined by analysis of the products of reaction of A,1,42 with LGE2 using western blotting with an antibody that is selective for ADDLs. [source] Ethanol Increases the Neurotoxic Effect of Tumor Necrosis Factor- , in Cultured Rat AstrocytesALCOHOLISM, Issue 1 2000William J. DeVito Background: The central nervous system is particularly sensitive to the cytotoxic effect of ethanol. In vivo and in vitro studies indicate that ethanol decreases cell proliferation in a number of cells types, including neurons and glial cells in the central nervous system. The cellular mechanisms involved in ethanol-induced cell toxicity, however, are unclear. In this study, we examined the effect of ethanol on tumor necrosis factor- , (TNF,)-induced cell death in a homogeneous population of cultured rat astrocytes. Methods: Flow cytometric and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) dye reduction analyses were performed on cultured rat astrocytes to determine the effect of alcohol on TNF, -induced cell death. Results: Flow cytometric analysis revealed that, in quiescent astrocytes, high concentrations of ethanol were required to increase DNA fragmentation and decrease cell viability. Preexposure of astrocytes to low concentrations of ethanol (10 to 50 mM), however, increased the sensitivity of astrocytes to TNF, with low TNF, concentrations (25 to 50 ng/ml) resulting in increased DNA fragmentation. Furthermore, MTT dye reduction analysis revealed that exposure of astrocytes to 5 mM ethanol was sufficient to increase the susceptibility of astrocytes to the cytotoxic effect of ethanol. In a number of cell types, TNF, receptor binding results in the activation of specific signal transduction cascades, including the hydrolysis of sphingomyclin to ceramide. We show that preexposure of astrocytes to a low concentration of ethanol increased the sensitivity of astrocytes to sphingomyelinase, and C2 -ceramide resulting in increased DNA fragmentation and decreased cell viability. More importantly, astrocytes prepared from rats exposed to ethanol prenatally showed increased susceptibility to TNF, -induced cell death. Conclusions: These studies suggest that ethanol increases the susceptibility of astrocytes to TNF, -induced cell death by shifting the balance of sphingolipid metabolism in favor of a pathway that increases the susceptibility of astrocytes to the cytotoxic effect of TNF,. [source] Antimalarial compounds from Kniphofia foliosa rootsPHYTOTHERAPY RESEARCH, Issue 6 2005Abraham Abebe Wube Abstract During the course of screening Ethiopian medicinal plants for their antimalarial properties, it was found that the dichloromethane extract of the roots of Kniphofia foliosa Hochst. (Asphodelaceae), which have long been used in the traditional medicine of Ethiopia for the treatment of abdominal cramps and wound healing, displayed strong in vitro antiplasmodial activity against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum with an ED50 value of 3.8 µg/mL and weak cytotoxic activity against KB cells with an ED50 value of 35.2 µg/mL. Five compounds were isolated from the roots and evaluated for their invitro antimalarial activity. Among the compounds tested, 10-(chrysophanol-7,-yl)-10-(,)-hydroxychrysopanol-9-anthrone and chryslandicin, showed a high inhibition of the growth of the malaria parasite, P. falciparum with ED50 values of 0.260 and 0.537 µg/mL, respectively, while the naphthalene derivative, 2-acetyl-1-hydroxy-8-methoxy-3-methylnaphthalene, exhibited a less significant antimalarial activity with an ED50 value of 15.4 µg/mL. To compare the effect on the parasite with toxicity to mammalian cells, the cytotoxic activities of the isolated compounds against the KB cell line were evaluated and 10-(chrysophanol-7,-yl)-10-(,)-hydroxychrysopanol-9-anthrone and chryslandicin displayed very low toxicity with ED50 values of 104 and 90 µg/mL, respectively. This is the first report of the inhibition of the growth of P. falciparum by anthraquinone-anthrone dimers and establishes them as a new class of potential antimalarial compounds with very little host cell toxicity. Copyright © 2005 John Wiley & Sons, Ltd. [source] In vitro immunopotentiating properties and tumour cell toxicity induced by Lophophora williamsii (peyote) cactus methanolic extractPHYTOTHERAPY RESEARCH, Issue 9 2003M. Franco-Molina Abstract Lophophora williamsii, also known as peyote, is found primarily in dry regions from Central Mexico, including the Mexican States of Nayarit, San Luis Potosí, Zacatecas, Nuevo León, Chihuahua, Coahuila and Tamaulipas, to Texas particularly in regions along Rio Grande. Peyote extracts have been associated with stimulating the central nervous system and regulating blood pressure, sleep, hunger and thirst. However, there is no evidence of any effect of peyote on the immune system or against tumour cell growth. The present study was designed to evaluate the in vitro effects of peyote methanolic extracts on some parameters of mouse and human leukocyte immunocompetence and tumour cell growth. Peyote extract (0.18,18 µg/mL) activated nitric oxide production by murine macrophages, and stimulated up to 2.4-fold proliferation of murine thymic lymphocytes. In addition, peyote extract induced up to 1.85-, 2.29- and 1.89-fold increases in mRNA signal of IL-1, IL-6 and IL-8 by human leukocytes. Also examined were the effects of peyote extracts on murine lymphoma L5178Y-R and ,broblastoma L929, and human myeloid U937 and mammary gland MCF7 tumour cell growth using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Peyote extracts were toxic for MCF7, L5178Y-R, U937 and L929 (18 mg/mL peyote extract caused 1.3%, 8%, 45% and 60% viability respectively) cell lines. Copyright © 2003 John Wiley & Sons, Ltd. [source] Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L -lysine) block copolymer (PEG-PLL)THE JOURNAL OF GENE MEDICINE, Issue 4 2004Hiromichi Katakura Abstract Background Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L -lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. Methods A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. Results We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. Conclusions PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity. Copyright © 2004 John Wiley & Sons, Ltd. [source] Radiofrequency exposure and mammalian cell toxicity, genotoxicity, and transformationBIOELECTROMAGNETICS, Issue S6 2003Martin L. Meltz Abstract The published in vitro literature relevant to the issue of the possible induction of toxicity, genotoxicity, and transformation of mammalian cells due to radiofrequency field (RF) exposure is examined. In some instances, information about related in vivo studies is presented. The review is from the perspective of technical merit and also biological consistency, especially with regard to those publications reporting a positive effect. The weight of evidence available indicates that, for a variety of frequencies and modulations with both short and long exposure times, at exposure levels that do not (or in some instances do) heat the biological sample such that there is a measurable increase in temperature, RF exposure does not induce (a) DNA strand breaks, (b) chromosome aberrations, (c) sister chromatid exchanges (SCEs), (d) DNA repair synthesis, (e) phenotypic mutation, or (f) transformation (cancer-like changes). While there is limited experimental evidence that RF exposure induces micronuclei formation, there is abundant evidence that it does not. There is some evidence that RF exposure does not induce DNA excision repair, suggesting the absence of base damage. There is also evidence that RF exposure does not inhibit excision repair after the induction of thymine dimers by UV exposure, as well as evidence that indicates that RF is not a co-carcinogen or a tumor promoter. The article is in part a tutorial, so that the reader can consider similarities and discrepancies between reports of RF-induced effects relative to one another. Bioelectromagnetics Supplement 6:S196,S213, 2003. © 2003 Wiley-Liss, Inc. [source] Study on syntheses and anticoagulant action of heparin/rare earth nano-oxides hybrid material,BIOPOLYMERS, Issue 10 2010Kun-Jie Wang Abstract Four hybrid materials of RE2O3 -TDI-Heparin (TDI = Toluene 2,4,diisocyanate, RE = La, Eu, Nd, Sc) were prepared by the method of graft. The materials were characterized by IR, TG, and SEM, which confirmed that the heparin was grafted on the surface of TDI modified rare earth nano-oxides. The cell adhesion experiment and the anticoagulant experiment demonstrated that the materials have lower cell toxicity, better cell adhesion as well as better anticoagulant action. In addition, the clotting time of hybrid materials were shortened compared with the heparin. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 887,892, 2010. [source] Genetic analysis of G protein-coupled receptor expression in Escherichia coli: Inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptorBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Georgios Skretas Abstract The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production. Biotechnol. Bioeng. 2009;102: 357,367. © 2008 Wiley Periodicals, Inc. [source] Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injuryBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2010Jian Yao BACKGROUND AND PURPOSE Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. EXPERIMENTAL APPROACH Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. KEY RESULTS NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor ,-glycyrrhetinic acid (,-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of ,-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by ,-GA. CONCLUSION AND IMPLICATIONS Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury. [source] Development of a Method for the High-Throughput Quantification of Cellular ProteinsCHEMBIOCHEM, Issue 10 2009Paolo Paganetti Dr. Abstract Hunting for huntingtin: We describe a screening assay based on the inducible expression of the mutant huntingtin protein in cells and on its highly sensitive homogenous determination. Rapid, reproducible, and robust protein determination was achieved through the use of two donor,acceptor-labeled antibodies and time-resolved FRET. The assay was developed and validated for ultra-throughput screening of low-molecular-weight compounds modulating the expression of the mutant protein. The quantification of cellular proteins is essential for the study of many different biological processes. This study describes an assay for the detection of the intracellular mutant huntingtin, the causative agent of Huntington's disease, with a method that may be generally applicable to other cellular proteins. A small recombinant protein tag that is recognized by a pair of readily available, high-affinity monoclonal antibodies was designed. This tag was then added to an inducible fragment of the mutant huntingtin protein by genetic engineering. We show that it is possible to use time-resolved FRET to detect low intracellular levels of huntingtin by a simple lysis and detection procedure. This assay was then adapted into a homogeneous, miniaturized format suitable for screening in 1536-well plates. The use of time-resolved FRET also permits the assay to be multiplexed with a standard readout of cell toxicity, thus allowing the identification of conditions causing reduction of protein levels simply due to cytotoxicity. The screening results demonstrated that the assay is able to identify compounds that modulate the levels of huntingtin both positively and negatively and that represent valuable starting points for drug discovery programs. [source] Synthesis and Antitumor Activity of Novel Dibutyltin Carboxylates of Aminoglucosyl DerivativesCHEMICAL BIOLOGY & DRUG DESIGN, Issue 6 2009Wei Li In this study, di-n-butyltin(IV) oxide was reacted with the amino glucose analog, cis -4-[N -(1,,3,,4,,6,-tetra- O -benzoyl-2-deoxy-glucopyranosyl)imido]-4-oxo-2-butenoic acid (1a) and o -[N -(1,,3,,4,,6,-tetra- O -benzoyl-2-deoxy-glucopyranosyl) carbamoyl] benzoic acid (2a) to give the complexes bis-{cis -4-[N -(1,,3,,4,,6,-tetra- O -benzoyl-2-deoxy-glucopyranosyl)imido-4-oxo-2-butenoic acid]-di-n-butyltin} carboxylate (1) and bis-{o -[N -(1,,3,,4,,6,-tetra- O -benzoyl-2-deoxy-glucopyranosyl) carbamoyl-benzoic acid]-di-n-butyltin}carboxylate (2). These two compounds were then characterized by IR, NMR and MS. In vitro tests showed that both compounds have high cytotoxicity in four tumor cell lines (P388, HL-60, A549 and BEL-7402). Clonogenic assays demonstrated that both compounds 1 and 2 have hematopoietic cell toxicity at 10,6 m. 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