Home About us Contact
|
Home About us Contact | ||
|
|
||
Vero Cells (vero + cell)
Selected AbstractsAntioxidant properties of extracts and compounds from Psoralea morisianaEUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 7-8 2005Antonella Rosa Abstract The antioxidant activity of various extracts (MeOH, petroleum ether, EtOAc) from the aerial parts of Psoralea morisiana, an endemic Sardinian plant, was evaluated during autoxidation and iron-mediated oxidation of linoleic acid at 37,°C and during cholesterol oxidation at 140,°C, in the absence of solvent. The activity of erybraedin,C, bitucarpin,A and plicatin,B, isolated from the extracts, was investigated under the same experimental conditions and compared to that of BHT and ,-tocopherol. All the extracts, erybraedin,C (major constituent of the extracts) and plicatin,B showed powerful antioxidant properties. None of the extracts and pure compounds showed any prooxidant activity. The cytotoxicity of the extracts, erybraedin,C, and plicatin,B was further evaluated in VERO cells, a line of fibroblasts derived from monkey kidney. Erybraedin,C, at non-cytotoxic concentrations, showed a strong inhibition of FeCl3 -induced oxidation in VERO cells. [source] Nuclear pore complex oxalate binding protein p62: Its expression on oxalate exposure to VERO cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2004P. Sivakamasundari Abstract Oxalate rich stones are the most common among the various stones. Oxalate binding protein plays a vital role in the transport of oxalate. Nuclear pore complex (NPC) contains a protein of molecular weight 62 kDa and it has maximum oxalate binding activity. The physiological significance of the presence of oxalate binding protein in the NPC is not well understood. In order to study its function, the expression of this protein during oxalate stress condition and the morphological changes on oxalate exposure to synchronized VERO cells have been determined. VERO cells were synchronized at different stages of cell cycle using cell cycle blockers and expression of the NPC p62 was assessed using enzyme linked immunosorbent assay (ELISA) technique with p62 antibody (MAb 414). Expression of NPC p62 was more pronounced in 1.0 mM oxalate concentration in mitotic phase than in S phase, suggesting cell cycle dependency. During oxalate exposure there is cell aggregation and complete degeneration of cell morphology occurs, which in turn lead to the expression of certain genes, including the NPC oxalate binding protein p62. Thus, oxalate induces degeneration of cells (may be due to the lipid peroxidation) and leads to the expression of NPC oxalate binding protein and the expression is of cell cycle dependent manner. © 2004 Wiley-Liss, Inc. [source] Fluorinated 1,2,4-Triazolo[1,5- a]pyrimidine-6-carboxylic Acid Derivatives as Antimycobacterial AgentsARCHIV DER PHARMAZIE, Issue 2 2009Hamdy M. Abdel-Rahman Abstract A series of fluorinated 1,2,4-triazolo[1,5- a]pyrimidine-6-carboxylic acid derivatives was designed and synthesized as fluoroquinolone analogues. The synthesized compounds were screened against Mycobacterium tuberculosis H37Rv strain at 6.25 ,g/mL concentration. Compound 4, the 7-oxo-2-(trifluoromethyl)-4,7-dihydro-1,2,4-triazolo[5,1- a]pyrimidine-6-carboxylic acid was found to be a very potent inhibitor, being able to inhibit 92% growth of M. tuberculosis H37Rv at 6.25 ,g/mL concentration. At the same time, it proofed to be nontoxic to mammalian cells (IC50 > 62.5 ,g/mL in VERO cells). [source] Differentiation Pattern of Vero Cells Cultured on Poly(L-Lactic Acid)/Poly(Hydroxybutyrate-co-Hydroxyvalerate) BlendsARTIFICIAL ORGANS, Issue 4 2004Arnaldo R. Santos Jr Abstract:, This study evaluates the effect of poly(L-lactic acid) (PLLA) and poly(hydroxybutyrate-cohydroxyvalerate) (PHBV) bioabsorbable polymers and their blends on the induction of alteration of cell growth pattern in vitro. Vero cells were cultured on PLLA, PHBV, and different blends (100/0, 60/40, 50/50, 40/60, and 0/100). The cell adhesion assay showed that the best results were obtained with the (60/40, 50/50) blends. Scanning electron microscopy showed that the cells on (100/0) and (60/40) samples grew with a round morphology preferentially in the porous areas. The (50/50) blends had cells in the porous and smooth areas in a similar way. The (40/60) blends showed spreading cells on the smooth areas. The (0/100) sample, which had no pores, had spreading cells interconnected by filaments. Histological sections showed a confluent cell monolayer and the immunocytochemistry showed that the cells produced collagen IV and fibronectin on all substrates. Thus, we conclude that PLLA/PHBV blends were efficient in maintaining cell growth and producing an extracellular ,matrix on them. [source] Evaluation of a reversed passive latex agglutination test for the detection of Verocytotoxin (VT) expressed by strains of VT-producing Escherichia coliLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2001H. Chart Aims: To compare an experimental Reversed-Passive Latex Agglutination (RPLA) with Vero cells for the detection of Verocytotoxin expressed by VT-producing strains of Escherichia coli (VTEC). Methods and Results: The RPLA was used alongside a Vero cell tissue culture assay for the detection of VT in bacterial culture supernatant fluids and patients' faecal extracts. Conclusions: The RPLA was comparable with the Vero cell assay, although slightly less sensitive. Significance and Impact of the Study: The RPLA test proved to be a simple, rapid and convenient method of detecting VT in bacterial culture supernatant fluids and in the faeces of patients infected with VTEC. [source] Kinetic characterization of vero cell metabolism in a serum-free batch culture processBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Emma Petiot Abstract A global kinetic study of the central metabolism of Vero cells cultivated in a serum-free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum-free medium which is a valuable help for the implementation of this cell line in serum-free industrial production processes. Biotechnol. Bioeng. 2010;107: 143,153. © 2010 Wiley Periodicals, Inc. [source] Inhibition of Escherichia coli heat-labile enterotoxin by neoglycoprotein and anti-lectin antibodies which mimic GM1 receptorFEMS MICROBIOLOGY LETTERS, Issue 1 2002Caroline A Menezes Abstract Escherichia coli producing heat-labile enterotoxin is responsible for numerous cases of diarrhea worldwide, leading to considerable morbidity and mortality. The B subunits of this toxin are responsible for the binding to the receptor, the complex ganglioside GM1 which has galactose as its terminal sugar. In this study we showed that analogs of galactose (gal) and N -acetylgalactosamine (GalNAc) interfere with the binding of heat-labile toxin to GM1. Antibodies to lectins which mimic sugar structures and neoglycoprotein were employed. These compounds were able to inhibit heat-labile toxin activity efficiently in Vero cells: 37 ,g of IgG-enriched fraction from an antiserum inhibited up to 70% of this activity, and 50% of the binding of heat-labile toxin to GM1. Neoglycoprotein was more efficient than antibodies, since 2.5 ,g of this ligand completely abolished the activity of heat-labile toxin on Vero cells. These data suggest that these molecules could be developed for prophylaxis and diagnosis of diarrhea caused by heat-labile toxin. [source] Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9JOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2003A. Cherif Abstract Aims: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers. Methods and Results:Bacillus thuringiensis ssp. entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi. Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase. Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121°C. Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization. After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12·4 kDa was isolated. The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells. Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B. thuringiensis HD9 was not toxic against Vero cells. Conclusions: Entomocin 9 is a novel heat-stable, bacteriocin produced by B. thuringiensis HD9. The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments. Significance and Impact of the Study: New finding on entomocin 9 would make B. thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry. [source] Functional analysis of polyomavirus BK non-coding control region quasispecies from kidney transplant recipientsJOURNAL OF MEDICAL VIROLOGY, Issue 11 2009Gunn-Hege Olsen Abstract Replication of the human polyomavirus BK (BKV) in renal tubular epithelial cells causes viruria and BKV-nephropathy in kidney transplant recipients. Following prolonged high-level BKV replication, rearrangement of the archetype non-coding control region (NCCR) leads to a mixture of BKV variants. The aim of this study was to compare potential functional differences of 12 rearranged (rr)-NCCR variants with the archetype (ww)-NCCR (WWT) found in allograft biopsies or urine from three kidney transplant recipients including two with BKV-nephropathy. Twelve different rr-NCCRs and one archetype ww-NCCR were inserted between the early and late protein coding region of BKV(Dunlop) to make recombinant BKV genomes for transfection into Vero cells. Immunoblotting, immunofluorescence staining, and quantitative PCR demonstrated that viral protein expression and extracellular BKV loads of 10 rr-NCCR variants were similar or higher than observed for the ww-NCCR BKV. Two rr-NCCR variants (RH-2 and RH-19) were non-functional. The functional rr-NCCRs produced infectious progeny successfully infecting primary renal proximal tubular epithelial cells. The number of infected cells and extracellular BKV loads corresponded to the activity seen in Vero cells. Three rr-NCCR variants (RH-1, RH-10, RH-13) only gave rise to a few infected cells similar to ww-NCCR, whereas seven variants had intermediate activity (RH-5, RH-6, RH-8, RH-9, RH-11) or high replication activity (RH-7 and RH-18) with several hundred infected cells per well. The results indicate that both functional and non-functional BKV rr-NCCR variants arise during BKV replication in kidney transplant recipients and that most functional rr-NCCR variants confer a higher replication capacity than archetype ww-NCCR. J. Med. Virol. 81:1959,1967, 2009. © 2009 Wiley-Liss, Inc. [source] Suppression of generation and replication of acyclovir-resistant herpes simplex virus by a sensitive virusJOURNAL OF MEDICAL VIROLOGY, Issue 1 2004Tomoko Okuda Abstract The role of acyclovir-sensitive herpes simplex virus (HSV) was analyzed in the process of its replacement by a resistant virus in vitro and in vivo in the aspect of acyclovir therapy. The mode of replacement of acyclovir-sensitive HSV with acyclovir-resistant HSV was examined by the passages of acyclovir-sensitive wild type HSV in Vero cells under acyclovir-treatment. The development of resistance was monitored more adequately by counting the number of acyclovir-resistant viruses in 10,000 plaque forming units than by the conventional susceptibility assay. The resistance increased with the proportion of thymidine kinase-deficient (TK,) viruses, when the susceptibilities of acyclovir-treated HSV population to 5,-iodo-2,deoxyuridine and phosphonoacetic acid were examined. The increased resistance was due to the increased proportion of acyclovir-resistant virus but not intermediately resistant virus. Infection with mixtures of TK, and acyclovir-sensitive strains rendered TK, sensitive to acyclovir, and virus yields were reduced to the levels of acyclovir-sensitive virus in Vero cells. Their yield reduction depended on the proportion of acyclovir-sensitive viruses and induction of TK activity. This reduction in virus yields of the mixture of TK, and acyclovir-sensitive strains was confirmed by acyclovir treatment in the skin of mice with cutaneous infection. Acyclovir treatment combined with superinfection of acyclovir-sensitive virus delayed the development of herpetic skin lesions due to acyclovir-resistant virus and reduced virus yields in the infected skin. Acyclovir-sensitive virus plays an important role in suppressing the generation and replication of acyclovir-resistant virus during acyclovir therapy. J. Med. Virol. 72:112,120, 2004. © 2004 Wiley-Liss, Inc. [source] Mosquito cells bind and replicate hepatitis C virusJOURNAL OF MEDICAL VIROLOGY, Issue 1 2001Raphaële Germi Abstract Several studies have demonstrated some hepatitis C virus (HCV) replication in lymphocyte and hepatocyte cell lines such as in African green monkey Vero cells. The aim of the present study was to select other cell lines able to bind and replicate HCV. Human hepatoma PLC/PRF/5 cells, human lymphoma Namalwa cells, Vero and mosquito AP61 cells were inoculated with HCV-positive plasma, washed six times and examined for the presence of the viral genome at different times post infection, using an RT-PCR method. Binding of HCV to cells was estimated by HCV RNA detection in cells 2 hr after inoculation and in the last wash of these cells. Successive virus passages in cells were carried out. All the cells studied were able to bind HCV but only AP61 and Vero cells provided evidence of replication and production of infectious virus: virus RNA was detected during 28 days post-infection in four successive virus passages. CD81 molecules, a putative HCV receptor, were detected by cytofluorometric analysis. Vero cells express CD81 molecules whereas these molecules were not detected on AP61 cells. It is suggested that other receptors are involved in HCV binding to Vero and AP61 cells. J. Med. Virol. 64:6,12, 2001. © 2001 Wiley-Liss, Inc. [source] Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cellsLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2007C.R.A. Couto Abstract Aim:, Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. Methods and Results:, A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. Conclusions:,A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. Significance and Impact of the Study:, The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health. [source] Evaluation of a reversed passive latex agglutination test for the detection of Verocytotoxin (VT) expressed by strains of VT-producing Escherichia coliLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2001H. Chart Aims: To compare an experimental Reversed-Passive Latex Agglutination (RPLA) with Vero cells for the detection of Verocytotoxin expressed by VT-producing strains of Escherichia coli (VTEC). Methods and Results: The RPLA was used alongside a Vero cell tissue culture assay for the detection of VT in bacterial culture supernatant fluids and patients' faecal extracts. Conclusions: The RPLA was comparable with the Vero cell assay, although slightly less sensitive. Significance and Impact of the Study: The RPLA test proved to be a simple, rapid and convenient method of detecting VT in bacterial culture supernatant fluids and in the faeces of patients infected with VTEC. [source] The Salmonella SpiC protein targets the mammalian Hook3 protein function to alter cellular traffickingMOLECULAR MICROBIOLOGY, Issue 6 2003Yoram Shotland Summary The Salmonella SpiC protein is secreted into the cytosol of macrophages via a unique type III secretion system that functions intracellularly to translocate proteins across the phagosomal membrane. The SpiC protein is required for survival within macrophages and inhibition of phagosome-lysosome fusion in vivo, and it is sufficient to inhibit endosome-endosome fusion in vitro. Here, we establish that SpiC targets the function of Hook3, a mammalian protein implicated in cellular trafficking. Purified GST-SpiC pulled down Hook3 from murine macrophages, and anti-Hook3 antibodies precipitated SpiC from the cytosol of Salmonella -infected macrophages. Expression of the spiC gene disrupted Golgi morphology in Vero cells and altered the distribution of lysosomes in macrophages, mimicking the phenotype of cells expressing a hook3 dominant-negative mutant. By inactivating Hook3 function, the SpiC protein may alter the lysosome network and prevent phagosome-lysosome fusion. [source] Molecular epidemiology of measles virus in JapanPEDIATRICS INTERNATIONAL, Issue 2 2004Tetsuo Nakayama AbstractBackground:,Measles virus has been classified into 22 genotypes. The present report examines the molecular epidemiology of measles virus in Japan from 1984 to 2002, and the epidemiological link between imported cases in several foreign countries and Japanese strains was elucidated from the literature. Methods:,B95a or Vero cells was used to isolate the measles virus. The measles virus genome was amplified in the N and H genes by reverse transcriptase-polymerase chain reaction and were partially sequenced. Phylogenetic analysis of a partial sequence of the N gene, from position 1230 to 1685, of the recent measles strains was performed in comparison with the World Health Organization reference strains. Results:,There were large outbreaks of measles in Japan in 1984, 1987,1988, 1991,1993, and 2001,2002 and each outbreak was caused by a different genotype. Genotype C1 was an indigenous strain for a long period before 1985, while D3 was isolated in 1987,1988 and D5 in 1991,1993 outbreaks. In addition, the Chicago-type D3 caused sporadic regional outbreaks from 1998 to 1999. After 2000, H1 became the dominant circulating strain. It should be noted that the Japanese strains were detected as imported cases by epidemiological linkage in several countries. Conclusion:,Among the recent circulating strain of measles virus in Japan the genotype H1 was dominant after 2000 and the Japanese strains D3, D5, and H1 were exported to several countries. It is recommended that Japan should adopt a more extensive and active vaccination strategy for measles elimination in line with other countries in the world. [source] In vitro activity of dermaseptin S1 derivatives against genital pathogensAPMIS, Issue 9 2010DIANELLA SAVOIA Savoia D, Donalisio M, Civra A, Salvadori S, Guerrini R. In vitro activity of dermaseptin S1 derivatives against genital pathogens. APMIS 2010; 118: 674,80. The aim of this study was to evaluate the biological activity of nine dermaseptin-S1 (DRS-S1) derivatives (synthesized by solid-phase methods and purified) against different pathogens causing genital infections (Trichomonas vaginalis, Herpes simplex virus, Papillomavirus). The in vitro activity on T. vaginalis was determined by counting the protozoon in a hemocytometer after vital staining with trypan blue; antiviral activity of the compounds was tested on monolayers of Vero cells for Herpes simplex virus-1 (GFP) and on 293TT cells for human papillomavirus (HPV-16) pseudovirions (GFP). The cytotoxicity of the derivatives was assessed by evaluating both the hemolytic activity and the effect on Vero and 293TT cells. The DRS-S1 longer peptides demonstrated a superior activity on T. vaginalis but also a certain cytopathic effect. The compounds with 29 amino acids exhibited activity against the two viruses tested at concentrations not toxic to cells. The results obtained show that some of the synthetic peptides assessed have inhibitory activity against the pathogens tested, indicating a potential for the development of new molecules for use as topical microbicides to prevent the sexual transmission of microorganisms. [source] Differentiation Pattern of Vero Cells Cultured on Poly(L-Lactic Acid)/Poly(Hydroxybutyrate-co-Hydroxyvalerate) BlendsARTIFICIAL ORGANS, Issue 4 2004Arnaldo R. Santos Jr Abstract:, This study evaluates the effect of poly(L-lactic acid) (PLLA) and poly(hydroxybutyrate-cohydroxyvalerate) (PHBV) bioabsorbable polymers and their blends on the induction of alteration of cell growth pattern in vitro. Vero cells were cultured on PLLA, PHBV, and different blends (100/0, 60/40, 50/50, 40/60, and 0/100). The cell adhesion assay showed that the best results were obtained with the (60/40, 50/50) blends. Scanning electron microscopy showed that the cells on (100/0) and (60/40) samples grew with a round morphology preferentially in the porous areas. The (50/50) blends had cells in the porous and smooth areas in a similar way. The (40/60) blends showed spreading cells on the smooth areas. The (0/100) sample, which had no pores, had spreading cells interconnected by filaments. Histological sections showed a confluent cell monolayer and the immunocytochemistry showed that the cells produced collagen IV and fibronectin on all substrates. Thus, we conclude that PLLA/PHBV blends were efficient in maintaining cell growth and producing an extracellular ,matrix on them. [source] Vero Cell Growth and Differentiation on Poly(l -Lactic Acid) Membranes of Different Pore DiametersARTIFICIAL ORGANS, Issue 1 2001Arnaldo R. Santos Jr. Abstract: In the last few years, the demand has increased for research on polymeric materials, which can be used as substitutes for injured tissues and organs or to improve their regeneration. In this work, we studied poly(l -lactic acid) (PLLA) membranes, a resorbable biomaterial, which were either dense or had different pore diameters (less than 45 ,m, between 180 and 250 ,m, and between 250 and 350 ,m), in relation to stimulation of cell adhesion, growth, and differentiation in vitro. We used Vero cells, a fibroblastic cell line, as the biological model of investigation. We found that cells attached slowly to all PLLA membranes studied. On the other hand, once the adhesion occurs, the cells are able to grow and differentiate on the different polymers. The cells grew to form a confluent monolayer and were capable of producing collagen Type IV and fibronectin on different PLLA membranes. This behavior indicates that cells try to create a better environment to stimulate their growth. This also indicates that Vero cells alter their differentiation pattern once they are producing extracellular matrix molecules related to epithelial differentiation. [source] Microbiological and Toxicological Effects of Perla Black Bean (Phaseolus vulgaris L.) Extracts: In Vitro and In Vivo StudiesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 2 2009Víctor Javier Lara-Díaz Three different solvents were used to obtain Perla black bean extracts. All three Perla black bean extracts were tested for antibacterial and antiparasitic activity and further analysed for intrinsic cytotoxicity (IC50). Methanol Perla black bean extract was used for acute toxicity test in rats, with the up-and-down doping method. All Perla black bean extracts inhibited bacterial growth. Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella oxytoca, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis and Listeria monocytogenes showed inhibition, while Escherichia coli and Enterobacter aerogenes did not. Acidified water and acetic acid Perla black bean extract were tested in parasites. The best IC50 was observed for Giardia lamblia, while higher concentrations were active against Entamoeba histolytica and Trichomonas vaginalis. The Vero cells toxicity levels (IC50) for methanol, acidified water and acetic acid Perla black bean extract were [mean ± S.D. (95% CI)]: 275 ± 6.2 (267.9,282.0), 390 ± 4.6 (384.8,395.2) and 209 ± 3.39 (205.6,212.4) µg/ml, respectively. In vivo acute toxicity assays did not show changes in absolute organ weights, gross and histological examinations of selected tissues or functional tests. The acetic acid and methanol Perla black bean extract proved to exhibit strong antibacterial activity and the acidified water Perla black bean extract exerted parasiticidal effects against Giardia lamblia, Entamoeba hystolitica and Trichomonas vaginalis. The three Perla black bean extracts assayed over Vero cells showed very low toxicity and the methanol Perla black bean extract in vivo did not cause toxicity. [source] Kinetic characterization of vero cell metabolism in a serum-free batch culture processBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Emma Petiot Abstract A global kinetic study of the central metabolism of Vero cells cultivated in a serum-free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum-free medium which is a valuable help for the implementation of this cell line in serum-free industrial production processes. Biotechnol. Bioeng. 2010;107: 143,153. © 2010 Wiley Periodicals, Inc. [source] Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibitionCELLULAR MICROBIOLOGY, Issue 2 2010Osvaldo Martinez Summary Cathepsins B and L contribute to Ebola virus (EBOV) entry into Vero cells and mouse embryonic fibroblasts. However, the role of cathepsins in EBOV-infection of human dendritic cells (DCs), important targets of infection in vivo, remains undefined. Here, EBOV-like particles containing a ,-lactamase,VP40 fusion reporter and Ebola virus were used to demonstrate the cathepsin dependence of EBOV entry into human monocyte-derived DCs. However, while DC infection is blocked by cathepsin B inhibitor, it is insensitive to cathepsin L inhibitor. Furthermore, DCs pre-treated for 48 h with TNF, were generally less susceptible to entry and infection by EBOV. This decrease in infection was associated with a decrease in cathepsin B activity. Thus, cathepsin L plays a minimal, if any, role in EBOV infection in human DCs. The inflammatory cytokine TNF, modulates cathepsin B activity and affects EBOV entry into and infection of human DCs. [source] Alternative infectious entry pathways for dengue virus serotypes into mammalian cellsCELLULAR MICROBIOLOGY, Issue 10 2009Eliana G. Acosta Summary The entry of two dengue virus (DENV) serotypes into Vero cells was analysed using biochemical inhibitors, dominant negative mutants of cellular proteins involved in endocytic pathways, fluorescence microscopy and infectivity determinations. By treatment with dansylcadaverine and chlorpromazine and overexpression of a dominant negative form of the Eps15 protein, a clathrin-mediated endocytosis for productive DENV-1 internalization into Vero cells was demonstrated whereas the infectious entry of DENV-2 in the same cell system was independent of clathrin. Treatment with the inhibitors nystatin and methyl-,-cyclodextrin, as well as transfection of Vero cells with dominant negative caveolin-1, had no effect on DENV-2 virus infection. It was also shown, by using the K44A mutant and the inhibitor dynasore, that dynamin was required for DENV-2 entry. Consequently, the infectious entry of DENV-2 into Vero cells occurs by a non-classical endocytic pathway independent of clathrin, caveolae and lipid rafts, but dependent on dynamin. By contrast, DENV-2 entry into A549 cells was clathrin-dependent, as previously reported in HeLa, C6/36 and BS-C-1 cells. Our results conclusively show, for the first time, a differential mode of infective entry for DENV-1 and DENV-2 into a common host cell, Vero cells, as well as alternative entry pathways for a given serotype, DENV-2, into different types of cells. [source] Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1CELLULAR MICROBIOLOGY, Issue 4 2008Naoko Morinaga Summary Subtilase cytotoxin (SubAB) is a AB5 type toxin produced by Shiga-toxigenic Escherichia coli, which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase-like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor-2, (eIF2,). The phosphorylation of PERK and eIF2, was maximal at 30,60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress-inducible C/EBP-homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB-treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB-induced translational inhibition and continuous prolonged proteasomal degradation. [source] Coxiella burnetii inhabits a cholesterol-rich vacuole and influences cellular cholesterol metabolismCELLULAR MICROBIOLOGY, Issue 3 2006Dale Howe Summary Coxiella burnetii directs the synthesis of a large parasitophorous vacuole (PV) required for replication. While some lysosomal characteristics of the PV have been described, the origin and composition of the PV membrane is largely undefined. Cholesterol is an essential component of mammalian cell membranes where it plays important regulatory and structural roles. Here we investigated the role of host cholesterol in biogenesis and maintenance of the C. burnetii PV in Vero cells. The C. burnetii PV membrane stained with filipin and was positive for the lipid raft protein flotillin-1, suggesting PV membranes are enriched in cholesterol and contain lipid raft microdomains. C. burnetii infection increased host cell cholesterol content by 1.75-fold with a coincident upregulation of host genes involved in cholesterol metabolism. Treatment with U18666A, lovastatin, or 25-hydroxycholesterol, pharmacological agents that inhibit cholesterol uptake and/or biosynthesis, altered PV morphology and partially inhibited C. burnetii replication. Complete inhibition of C. burnetii PV development and replication was observed when infected cells were treated with imipramine or ketoconazole, inhibitors of cholesterol uptake and biosynthesis respectively. We conclude that C. burnetii infection perturbs host cell cholesterol metabolism and that free access to host cholesterol stores is required for optimal C. burnetii replication. [source] Synthesis, Cytotoxicity and Antileishmanial Activity of Some N -(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-aminesCHEMICAL BIOLOGY & DRUG DESIGN, Issue 6 2010Elaine S. Coimbra We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3). Hydrolysis of the methyl ester adduct (5) yielded the free acid (6). The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells. [source] | |||||||||||||||||||||||||