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Kinds of Mois Terms modified by Mois Selected AbstractsChlamydia pneumoniae infections prevent the programmed cell death on THP-1 cell lineFEMS MICROBIOLOGY LETTERS, Issue 1 2002C.Romano Carratelli Abstract Chlamydia pneumoniae is an obligate intracellular bacterium which frequently causes airway infection in humans and has been implicated in chronic inflammatory disease and atherosclerosis. Here we show that infection with C. pneumoniae protects THP-1 cells against the apoptosis which spontaneously occurs in macrophages in the absence of an activation signal. Analysis by flow cytometry at different post-infection times revealed that 50±7% of THP-1 cells were apoptotic at 48 h after onset of the experiments, whereas C. pneumoniae -infected cultures (multiplicity of infection, MOI = 30) displayed only 18±4% of cells in apoptosis. At MOI = 20 and MOI = 10 the cells susceptible to apoptosis at 48 h were 28±5% and 35±6% respectively. Moreover, the results show that heat-inactivated bacteria do not give significant protection against apoptosis, even at higher MOI (MOI = 30), while UV-treated Chlamydia did provide a degree of protection against apoptosis. These data suggest that the anti-apoptotic effect of C. pneumoniae requires a heat-labile component released during infection, and that the effect is not lipopolysaccharide-dependent. [source] Campylobacter and IFN, interact to cause a rapid loss of epithelial barrier integrityINFLAMMATORY BOWEL DISEASES, Issue 3 2008Louisa E.N. Rees PhD Abstract Background: The intestinal epithelium is a single layer of polarized cells and is the primary barrier separating foreign antigen and underlying lymphoid tissue. IFN, alters epithelial barrier function during inflammation by disrupting tight cell junctions and facilitating the paracellular transport of luminal antigens. The aim of this work was to determine whether Campylobacter infection of cells exposed to IFN, would lead to greater disruption of cell monolayers and hence increased bacterial translocation. Methods: Monolayers were polarized on Transwell polycarbonate membranes for 14 days and then cultured in the presence or absence of 100 U/mL IFN,. Campylobacter was added to the apical side of the monolayer at an MOI of 30. Transepithelial electrical resistance (TEER) was recorded and bacteria in the basal well counted every 2 hours. Cells were stained for occludin, actin, and nuclear DNA, and cell viability determined by measurement of apoptosis. Results: In the presence of IFN,, TEER dropped significantly after 18 hours, indicating a reduction in barrier function. A further significant decrease was seen in the presence of both IFN, and Campylobacter, indicating a synergistic effect, and cellular morphology and viability were affected. Bacterial translocation across the monolayer was also significantly greater in the presence of IFN,. Conclusions: These combined effects indicate that Campylobacter infection concomitant with intestinal inflammation would result in a rapid and dramatic loss of epithelial barrier integrity, which may be a key event in the pathogenesis of Campylobacter -mediated colitis and the development of bloody diarrhea. (Inflamm Bowel Dis 2007) [source] Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2009Y.D. Niu Abstract Aims:, To evaluate host range and lytic capability of four bacteriophages (rV5, wV7, wV8 and wV11) against Escherichia coli O157:H7 (STEC O157:H7) from cattle and humans. Methods and Results:, Four hundred and twenty-two STEC O157:H7 isolates (297 bovine; 125 human) were obtained in Alberta, Canada. The four phages were serially diluted and incubated for 5 h with overnight cultures of STEC O157:H7 to estimate their multiplicity of infection (MOI). All bovine STEC O157:H7 were subjected to pulsed-field gel electrophoresis (PFGE) and phage typing (PT). Phage wV7 lysed all human and bovine isolates irrespective of PFGE genotype or PT phenotype and exhibited the lowest MOI (0·004,0·006, P < 0·0001) of all phages. Phages rV5 and wV11 exhibited a lower MOI (0·002,0·04, P < 0·0001) than did phage wV8 (25,29) and they had a narrower host range than wV7 or wV8. Phages rV5, wV11 and wV8 lysed 342 (81·0%), 321 (76·1%) and 407 (96·4%), respectively, of the 422 isolates. Susceptibility of bovine STEC O157:H7 to rV5, w11 and wV8 was influenced by PFGE genotype and/or PT phenotype. Conclusions:, Phages exhibited activity against the majority of bovine and human STEC O157:H7 isolates. PFGE genotype and/or PT phenotype of the host-target influenced their vulnerability to phage attack. Susceptibility of bovine STEC O157:H7 to phage may also differ among farms. Both lytic capability and host range should be considered in the selection of therapeutic phage for on-farm control of STEC O157:H7. Significance and Impact of the Study:, The present work indicates that a four-phage cocktail should be equally effective at mitigating STEC O157:H7 isolates both of bovine and of human origin. Given that some STEC O157:H7 exhibited resistance to some but not all phages, a phage cocktail is the logical approach to efficacious on-farm therapy. [source] Differential cytokine expression by human dendritic cells in response to different Porphyromonas gingivalis capsular serotypesJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 10 2009Rolando Vernal Abstract Aim: Capsular polysaccharides play an important role in the virulence of Gram-positive and Gram-negative bacteria. In Porphyromonas gingivalis, six serotypes have been described based on capsular antigenicity and its pathogenicity has been correlated both in vitro and in animal models. This study aimed to investigate the differential response of human dendritic cells (DCs) when stimulated with different P. gingivalis capsular serotypes. Materials and Methods: Using different multiplicity of infection (MOI) of the encapsulated strains K1,K6 and the non-encapsulated K, strain of P. gingivalis, the mRNA expression levels for interleukin (IL)-1,, IL-2, IL-5, IL-6, IL-10, IL-12, IL-13, interferon (IFN)- ,, tumour necrosis factor (TNF)- ,, and TNF- , in stimulated DCs were quantified by real-time reverse transcription-polymerase chain reaction. Results: All P. gingivalis capsular serotypes induced a T-helper type 1 (Th1) pattern of cytokine expression. K1- and K2-stimulated DCs expressed higher levels of IL-1,, IL-6, IL-12p35, IL-12p40, and IFN- , and at lower MOI than DCs stimulated with the other strains. Conclusions: These results demonstrate a differential potential of P. gingivalis capsular serotypes to induce DC responses and a higher capacity of strains K1 W83 and K2 HG184 than other K serotypes to trigger cytokine expression. [source] Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell lineJOURNAL OF FISH DISEASES, Issue 3 2005J-R Hong Abstract In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 °C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 °C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 °C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 °C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 °C) compared with the non-permissive temperature of 28 °C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis. [source] All CVB serotypes and clinical isolates induce irreversible cytopathic effects in primary cardiomyocytesJOURNAL OF MEDICAL VIROLOGY, Issue 2 2005Jeonghyun Ahn Abstract Coxsackievirus B3 (CVB3) has been identified as a major causative agent of acute and chronic myocarditis, but the involvement of other CVB serotypes in myocarditis has not been investigated. To dissect the pathological properties of different CVB serotypes toward primary cardiomyocytes, we tested their effects on primary cardiomyocyte cultures from neonatal rats. Morphological abnormalities were examined by both light and fluorescence microscopy after Hoechst 33342 staining, and loss of cell viability was estimated by MTT assay. All six CVB serotypes showed a similar degree of severe toxicity toward primary cardiomyocytes. CVB clinical isolates had cytopathic effects (CPEs) similar to those of their respective CVB reference strains. Within 1,2 days of infection with multiplicities of infection MOI 50, the cells began to experience morphological changes including cell shrinkage, rounding-up, and slight nuclear condensation. The irreversible loss of cell viability was readily observed within 3,5 days following virus infection. These results suggest that all six CVB serotypes induce direct, irreversible toxicity towards cardiomyocytes, which eventually leads to the death of infected cells. These findings indicate that the variations in CVB serotype are not the limiting factor determining the susceptibility of cardiomyocytes to CVB infection. J. Med. Virol. 75:290,294, 2005. © 2004 Wiley-Liss, Inc. [source] Late human cytomegalovirus (HCMV) proteins inhibit differentiation of human neural precursor cells into astrocytesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2007Jenny Odeberg Abstract Human cytomegalovirus (HCMV) is the most common cause of congenital infections in developed countries, with an incidence varying between 0.5,2.2%. Such infection may be the consequence of either a primary infection or reactivation of a latent infection in the mother and the outcome may vary from asymptomatic to severe brain disorders. Moreover, infants that are asymptomatic at the time of birth may still develop neurologic sequelae at a later age. Our hypothesis is that infection of stem cells of the central nervous system by HCMV alters the proliferation, differentiation or migration of these cells, and thereby gives rise to the brain abnormalities observed. We show that infection of human neural precursor cells (NPCs) with the laboratory strain Towne or the clinical isolate TB40 of HCMV suppresses the differentiation of these cells into astrocytes even at an multiplicity of infection (MOI) as low as 0.1 (by 33% and 67%, respectively). This inhibition required active viral replication and the expression of late HCMV proteins. Infection as late as 24 hr after the onset of differentiation, but not after 72 hr, also prevented the maturation of infected cultures. Furthermore, in cultures infected with TB40 (at an MOI of 1), approximately 54% of the cells were apoptotic and cell proliferation was significantly attenuated. Clearly, HCMV can reduce the capacity of NPCs to differentiate into astrocytes and this effect may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection. © 2006 Wiley-Liss, Inc. [source] Crosslinking polymerization leading to interpenetrating polymer network formation.JOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 21 2003Abstract As part of our continuing studies concerned with the elucidation of the crosslinking polymerization mechanism leading to interpenetrating polymer network (IPN) formation, in which IPNs consist of both polymethacrylates and polyurethane (PU) networks, this article explores the polyaddition crosslinking reactions of multifunctional poly(methyl methacrylate- co -2-methacryloyloxyethyl isocyanate) [poly(MMA- co -MOI)] [MMA/MOI = 90/10] with various diols leading to PU network formation. Thus, the equimolar polyaddition crosslinking reactions of poly(MMA- co -MOI) with ethylene glycol (EG), 1,6-hexane diol, and 1,10-decane diol (DD) were carried out in N -methyl pyrrolidone at a 0.25 mol/L isocyanate group concentration at 80 °C. The second-order rate constants decreased from EG to DD. The deviation of the actual gel point from the theoretical one was smaller from EG to DD. The intrinsic viscosity of resulting prepolymer demonstrated almost no variation with progressing polymerization for the EG system, whereas it gradually increased with conversion for the DD system. Close to the gel point conversion it increased rather drastically for both systems. The swelling ratio of resulting gel was higher from EG to DD. These are discussed mechanistically in terms of the significant occurrence of intramolecular cyclization and intramolecular crosslinking reactions leading to shrinkage of the molecular size. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3243,3248, 2003 [source] Crosslinking polymerization leading to interpenetrating polymer network formation.JOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 4 2003Abstract At the start of our research program concerned with the elucidation of the crosslinking polymerization mechanism leading to interpenetrating polymer network (IPN) formation, in which IPNs consist of both polymethacrylates and polyurethane (PU) networks, this article deals with the polyaddition crosslinking reaction leading to PU network formation. Therefore, 2-methacryloyloxyethyl isocyanate (MOI) was radically copolymerized with methyl methacrylate (MMA) in the presence of CBr4 as a chain-transfer agent. The resulting poly(MMA- co -MOI)s, having pendant isocyanate (NCO) groups as novel multifunctional polyisocyanates, were used for polyaddition crosslinking reactions with ethylene glycol as a typical diol. The second-order rate constants depended on both the functionality of poly(MMA- co -MOI) and the NCO group concentration. The actual gel points were compared with the theoretical ones calculated according to Macosko's equation; the deviation of the actual gel point from the theoretical value became more remarkable for a greater functionality of poly(MMA- co -MOI) and at a lower NCO group concentration or at a lower poly(MMA- co -MOI) concentration. These are discussed mechanistically, with consideration given to the significance of intramolecular cyclization and intramolecular crosslinking reactions leading to the shrinkage of the molecular size of the prepolymer, along with the data of the intrinsic viscosities of resulting prepolymers and the swelling ratios of resulting gels. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 606,615, 2003 [source] Cytokine induction by respiratory syncytial virus and adenovirus in bronchial epithelial cellsPEDIATRIC PULMONOLOGY, Issue 3 2007Jong-Seo Yoon MD Abstract In order to broaden our knowledge of the primary immune responses to respiratory syncytial virus (RSV) and adenovirus infections, we compared the concentrations of interleukin (IL)-6, IL-8, and regulated on activation, normal T cell expressed and secreted (RANTES) produced in vitro during RSV and adenovirus infections of bronchial epithelial cells. We infected BEAS-2B cells,a human bronchial epithelial cell line,with RSV, adenovirus serotype 3, or serotype 7 and measured the concentrations of IL-6, IL-8, and RANTES in the cell culture supernatants. When the multiplicity of infection (MOI) was 1, RSV induced the production of markedly higher concentrations of IL-6, IL-8, and RANTES than the adenovirus. When the MOI of the adenovirus was increased to 100, the production of IL-6 and IL-8 increased. However, the amounts produced were still lower than those produced by RSV with the MOI of 1. There was no statistically significant increase in the production of RANTES in spite of the MOI of the adenovirus was increased to 100. Adenovirus serotype 7 induced the production of considerably more IL-6 and IL-8 than serotype 3 in the MOI of 100. However, neither adenovirus serotype triggered an increase in the production of RANTES in spite of the MOI of 100. This demonstrates that RSV could have a superior capacity to stimulate the production of IL-6, IL-8, and RANTES in the bronchial epithelial cells. This study may help to explain the differences in the clinical outcomes of RSV and adenovirus infections. Pediatr Pulmonol. 2007; 42:277,282. © 2007 Wiley-Liss, Inc. [source] Effects of K doping and annealing on the grain size and giant dielectric constant of KxTi0.02Ni0.98,xO ceramicsPHYSICA STATUS SOLIDI (A) APPLICATIONS AND MATERIALS SCIENCE, Issue 5 2007Pradip Kumar Jana Abstract Monovalent ion (MOI) doping effect on the grain size and giant dielectric constant (, , , 104) of lead free environment friendly NiO-based KxTi0.02Ni0.98,xO (KTNO, x = 0.04, 0.25, and 0.30) type ceramics have been investigated. Increasing K concentration (MOI) enhances the grain size along with the increase of , , values. The dielectric constant of KTNO also increases with increase of the sintering time and temperature (within the range of our measurement) due to enhancement of crystalline size. The giant , , value and the leakage current in KTNO show weak field dependent behavior. Estimated temperature coefficient of permittivity (TCP) of KTNO is very low (less than 6%). All these properties meet the requirements of the ceramics used in X7R EIA specification. High , , in KTNO is attributed to the boundary layer mechanism. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Infection of replication-deficient adenoviral vector enhances interleukin-8 production in small airway epithelial cells more than in large airway epithelial cellsRESPIROLOGY, Issue 4 2001YUZO KODAMA Objective: In clinical trials or experiments of gene therapy, airway administration of an adenoviral-based vector (E1A-deleted) elicits a dose-dependent inflammatory response with limitation in the duration of transgene expression. The purpose of this study was to evaluate the possibility that the adenoviral-based vector directly enhances IL-8 production independent of adenoviral E1A in normal human airway epithelial cells and to examine the different responses between primary human bronchial epithelial cells (HBE) and primary human small airway epithelial cells (HSAE) in production of IL-8 following exposure to an adenovirus vector. Methodology: Interleukin (IL)-8 levels were evaluated in the culture medium from HBE and HSAE treated with increasing doses of E1A-deleted adenoviral vector contained the Escherichia coli LacZ reporter gene (AdCMVLacZ). To clarify the mechanism of enhancing IL-8 production in airway epithelial cells by infection with adenovirus vector, ,v,5 agonistic antibody as an analogue of adenoviral capsid and adenoviral capsid vector denatured by exposure to ultraviolet (UV) light were used in the present study. Results: Inoculation of HBE with AdCMVLacZ at a multiplicity of infection (MOI) of between 1 and 200 resulted in a dose-dependent expression of LacZ, and maximal expression was observed at a MOI of 100. In contrast, inoculation of HSAE with AdCMVLacZ resulted in maximum expression of LacZ at a MOI of 10. Interleukin-8 levels in culture media from the same experiments revealed significantly greater production of IL-8 in HSAE inoculated with AdCMVLacZ at a MOI of 50, compared to HBE under the same conditions. The capsid-denatured adenoviral vector did not enhance IL-8 production, and ,v,5 agonistic antibody induced IL-8 enhancement. Conclusion: These results suggest that the adenoviral vector directly induces the expression of airway epithelial inflammatory cytokines in the pathogenesis of inflammation and that small airway cells have a greater affinity for adenovirus than other airway epithelial cells. [source] Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID miceTHE JOURNAL OF GENE MEDICINE, Issue 10 2004Jiro Kikuchi Abstract Background An Erratum has been published for this article in Journal of Gene Medicine 7(6), 2005, 836. Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). Methods CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied. Results We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 ± 0.1%) at MOI of 5 × 103 vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 ± 20.1 ng) from 106 CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5,10 × 105) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 ± 0.9 ng/mL, maximum 3.6 ± 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. Conclusions Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients. Copyright © 2004 John Wiley & Sons, Ltd. [source] A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell linesTHE JOURNAL OF GENE MEDICINE, Issue 9 2003Emmanuel Richard Abstract Background Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells. Methods We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O6 -methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency. Results EPP lymphoblastoid (LB) cell lines, K562 and cord-blood-derived CD34+ cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O6 -benzylguanine (BG)/N,N,-bis(2-chloroethyl)- N -nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus-containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation. Conclusions This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells. Copyright © 2003 John Wiley & Sons, Ltd. [source] Laminin acts via focal adhesion kinase/phosphatidylinositol-3, kinase/protein kinase B to down-regulate ,1 -adrenergic receptor signalling in cat atrial myocytesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2009Y. G. Wang We previously reported that short-term (2 h) plating of cat atrial myocytes on the extracellular matrix protein, laminin (LMN) decreases adenylate cyclase activity and ,1 -adrenergic receptor (,1 -AR) stimulation of L-type Ca2+ current (ICa,L). The present study sought to determine whether LMN-mediated down-regulation of ,1 signalling is due to down-regulation of adenylate cyclase and to gain insight into the signalling mechanisms responsible. ,1 -AR stimulation was achieved by 0.01 ,m isoproterenol (isoprenaline) plus 0.1 ,m ICI 118551, a selective ,2 -AR antagonist. Atrial myocytes were plated for at least 2 h on uncoated cover-slips (,LMN) or cover-slips coated with LMN (+LMN). As previously reported, ,1 -AR stimulation of ICa,L was significantly smaller in +LMN compared to ,LMN atrial myocytes. In ,LMN myocytes, 10 ,m LY294002 (LY), a specific inhibitor of PI-(3)K, had no effect on ,1 -AR stimulation of ICa,L. In +LMN myocytes, however, LY significantly increased ,1 -AR stimulation of ICa,L. Western blots revealed that compared with ,LMN myocytes, +LMN myocytes showed a significant increase in Akt phosphorylation at Ser-473, which was prevented by LY. In another approach, +LMN myocytes were infected (multiplicity of infection (MOI), 100; 24 h) with replication-defective adenoviruses (Adv) expressing dominant-negative inhibitors of focal adhesion kinase (FAK) (Adv-FRNK or Adv-Y397F-FAK) or Akt (Adv-dnAkt). Compared with control cells infected with Adv-,-galactosidase, cells infected with Adv-FRNK, Adv-Y397F-FAK or Adv-dnAkt each exhibited a significantly greater ,1 -AR stimulation of ICa,L. In ,LMN myocytes LY had no effect on forskolin (FSK)-stimulated ICa,L. However, in +LMN myocytes LY significantly increased FSK-stimulated ICa,L. Similar results were obtained in +LMN atrial myocytes infected with Adv-FRNK. We conclude that LMN binding to ,1 -integrin receptors acts via FAK/PI-(3)K/Akt to inhibit adenylate cyclase activity and thereby down-regulates ,1 -AR-mediated stimulation of ICa,L. These findings provide new insight into the cellular mechanisms by which the extracellular matrix can modulate atrial ,-AR signalling. [source] Determination of the baculovirus transducing titer in mammalian cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Zun-Ren Chan Abstract Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4,7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments. © 2005 Wiley Periodicals, Inc. [source] Virus-like particle production at low multiplicities of infection with the baculovirus insect cell systemBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Luis Maranga Abstract The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus -like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained. Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 245,253, 2003. [source] Using the rate of respiration to monitor events in the infection of Escherichia coli cultures by bacteriophage T4BIOTECHNOLOGY PROGRESS, Issue 3 2010Dominic Sauvageau Abstract The growing interest in applications of bacteriophages creates a need for improvements in the production processes. Continuous monitoring of the phage production is an essential aspect of any control strategy and, at present, there is no completely satisfactory option. The approach presented here uses IR-spectrometry to continuously measure the rate of respiration (CO2 released) of Escherichia coli infected by phage T4 at various multiplicities of infection (MOI). Within the trends in these data, or in other aspects of the rate of respiration, it was possible to reliably and reproducibly identify five features that reflected specific events in the infection process. These included two events in the host cell apparent growth rate and events in the magnitude of the host cell density, in the measurement of OD600 or in the specific rate of respiration. All of these correlations were within 95% confidence showing that they are suitable for the monitoring and control of E. coli populations infected by phage T4. This method is reliable, cheap, and can be operated in-line and in real time. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-insect cell expression systemBIOTECHNOLOGY PROGRESS, Issue 4 2009Sirika Pillay Abstract A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase process efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni ProÔ insect cell line, at a cell density of 1 × 106 cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Processing of Infectious Bursal Disease Virus (IBDV) Polyprotein and Self-Assembly of IBDV-Like Particles in Hi-5 CellsBIOTECHNOLOGY PROGRESS, Issue 3 2006Meng-Shiou Lee The capsid of infectious bursal disease virus (IBDV), with a size of 60,65 nm, is formed by an initial processing of polyprotein (pVP2-VP4-VP3) by VP4, subsequent assemblage of pVP2 and VP3, and the maturation of VP2. In Sf9 cells, the processing of polyprotein expressed was restrained in the stage of VP2 maturation, leading to a limited production of capsid, i.e., IBDV-like particles (VLPs). In the present study, another insect cell line, High-Five (Hi-5) cells, was demonstrated to efficiently produce VLPs. Meanwhile, in this system, polyprotein was processed to pVP2 and VP3 protein and pVP2 was further processed to the matured form of VP2. Consequently, Hi-5 cells are better in terms of polyprotein processing and formation of VLPs than Sf9. In addition to the processing of pVP2, VP3 was also degraded. With insufficient intact VP3 protein present for the formation of VLPs, the excessive VP2 form subviral particles (SVPs) with a size of about 25 nm. The ratio of VLPs to SVPs is dependent on the multiplicity of infections (MOIs) used, and an optimal MOI is found for the production of both particles. VLPs were separated from SVPs with a combination of ultracentrifugation and gel-filtration chromatography, and a large number of purified particles of both were obtained. In conclusion, the insect cell lines and MOIs were optimized for the production of VLPs, and pure VLPs with morphology similar to that of the wild-type viruses can be effectively prepared. The efficient production and purification of VLPs benefits not only the development of an antiviral vaccine against IBDV but also the understanding of the structure of this avian virus that is economically important. [source] Evaluation of Production Parameters with the Vaccinia Virus Expression System Using Microcarrier Attached HeLa CellsBIOTECHNOLOGY PROGRESS, Issue 2 2005Nicole A. Bleckwenn Parameters that affect production of the recombinant reporter protein, EGFP, in the T7 promoter based VOTE vaccinia virus-HeLa cell expression system were examined. Length of infection phase, inducer concentration, and timing of its addition relative to infection were evaluated in 6-well plate monolayer cultures. One hour infection with 1.0 mM IPTG added at the time of infection provided a robust process. For larger scale experiments, anchorage-dependent HeLa cells were grown on 5 g/L Cytodex 3 microcarriers. The change to this dynamic culture environment, with cell-covered microcarriers suspended in culture medium in spinner flasks, suggested a re-examination of the multiplicity of infection (MOI) for this culture type that indicated a need for an increase in the number of virus particles per cell to 5.0, higher than that needed for complete infection in monolayer tissue flask culture. Additionally, dissolved oxygen level and temperature during the protein production phase were evaluated for their effect on EGFP expression in microcarrier spinner flask culture. Both increased dissolved oxygen, based on surface area to volume (SA/V) adjustments, and decreased temperature from 37 to 31 °C showed increases in EGFP production over the course of the production phase. The level of production achieved with this system reached approximately 17 ,g EGFP/106 infected cells. [source] Health Status of Temporary Migrants in Urban Areas in Vietnam1INTERNATIONAL MIGRATION, Issue 4 2007Liem T. Nguyen ABSTRACT The rapid economic growth after economic reform, known in Viet Nam as "Doi Moi", and the growing scope of urban migration raise specific questions for social policy, including migration and health policies. This paper compares issues of health status and its determinants as they affect temporary urban migrants versus permanent urban migrants and non-migrants. The analyses utilize multivariate logistic regression and data from the 1997 Vietnam Migration and Health Survey. The results show that temporary migrants staying in guest houses are most vulnerable to health problems. Though most of them are initially healthier, their reported health deteriorates faster than other groups of urban residents. The findings also present important implications for the current migration and health policies in Vietnam: 1) A special attention should be given to temporary migrants in guest houses; 2) Different priorities in health policy should be applied to different groups of migrants and non-migrants; 3) The current population management policy by registration system needs to be reviewed; 4) Providing clean water is one of the most important ways to improve health of temporary migrants; 5) Targeting educational investments and reducing unemployment would likely to improve overall health; 6) A higher priority on health policies targeting women would likely pay dividends, and; 7) Improving management and collaboration between government offices and interested partners is important to improving health status and reducing inequity. [source] Identifications of expressed sequence tags from Pacific threadfin (Polydactylus sexfilis) skeletal muscle cDNA libraryAQUACULTURE RESEARCH, Issue 4 2010Shizu Watanabe Abstract Pacific threadfin (Polydactylus sexfilis), locally known as Moi, is a desirable fish for aquaculture and recreational fishing. To understand the basic mechanism of muscle formation and its impacts on flesh quality, we established a cDNA library using mRNA of the skeletal muscle tissue from fingerlings. The library size was 1.1 × 108 plaque forming units mg,1 and the percentage of recombinant clones was >81%. A pilot sequencing project from 181 clones identified 129 useful expressed sequence tags (ESTs), of which 90 ESTs exhibited significant homology to known genes and 39 ESTs have low homologies to unknown genes by blast algorithm. The most abundant EST from the pilot sequence data is nikotinamide riboside kinase 2 (59 times), followed by 60S ribosomal protein L24 (12 times) and ribosomal protein L8 (5 times). Fourteen novel genes were retrieved from the sequenced clones and subjected to gene ontology annotation. Four mRNA sequences were identified as significant regulators of transcription, including Not2p, Tsc22 domain family 2, LIM domain binding factor 3 and mesenchyme homeobox 2. These results suggest that the muscle cDNA library is an useful source for identifying EST sequences of Pacific threadfin. [source] Measuring the Impact of Doi Moi on Vietnam's Gross Domestic ProductASIAN ECONOMIC JOURNAL, Issue 3 2000Le Thanh Nghiep In 1986 a wide range of policy measures, known as Doi Moi, was introduced to promote Vietnam's transition to a market economy. This paper represents the first attempt to measure the effect of Doi Moi on Vietnam's GDP. In the paper the level of GDP actually reached is compared with the level that would have been reached had the policy not been implemented, i.e. without the improvements in productivity and the increases in investment ratio that can be directly attributed to Doi Moi. Cross-time changes in GDP were depicted by a production function of capital stock, economically active labour force and technical progress. It was found that, after a time lag, Doi Moi appeared to have a significant positive effect on productivity, which by 1998 accounted for a 42% increase in GDP. [source] Government and NGO partnership in managing community-based water resources in Vietnam: a case study of Thai Long Dam ProjectBUSINESS STRATEGY AND THE ENVIRONMENT, Issue 2 2002Bach Tan Sinh Economic reform policy called ,Doi Moi' introduced by the Government of Vietnam at the end of 1980s opened new opportunities of community-based involvement in the policy and decision-making at various local levels. Innovations such as decentralization of decision-making power to lower administrative level, and recognition of the local community's role in managing their natural resources, e.g. transfer of irrigated water management right to local communities, were introduced. Significantly, this new institutional framework also facilitated greater civil society involvement in Vietnam. The Water Users Cooperative (WUC) set up through the Thai Long Dam Project mobilized local farmers to participate and manage their local resources in a sustainable manner. Through this process, the WUC was able to strengthen itself as a civil society institution that mediates between the individual and the state, as well as a forum for increasing government responsiveness and accountability. The success of the WUC of the Thai Long Project implies that the Vietnamese civil society is playing a more active role in the decision-making process. Copyright © 2002 John Wiley & Sons, Ltd. and ERP Environment [source] Corpus Meum: Disintegrating Bodies and the Ideal of IntegrityHYPATIA, Issue 3 2005DIANE PERPICH This essay shows that Jean-Luc Nancy's reconceptualization of corporeality in such texts as L'Intrus and Corpus can be an important ally to feminist theories of body. I introduce Nancy's ontology and argue that his rejection of the unified, integrated body of humanist discourses in favor of dis-integrated bodies constituted by multiple alterities and his consequent reinterpretation of body as a "being-exscribed" begin the task of thinking bodies beyond traditional dualisms and their ahistorical and rationalist frameworks. I then address three potential criticisms of Nancy's work and suggest that though there may be reasons to move cautiously in adopting the framework he provides, his work harbors resources directly beneficial to critiques of prevailing forms of gender normativity. Quel étrange moi! ,Jean-Luc Nancy, Corpus [source] The Application of Hydrogen Peroxide as a Treatment for the Ectoparasite Amyloodinium ocellatum (Brown 1931) on the Pacific Threadfin Polydactylus sexfilisJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 2 2001Dee Montgomery-Brock Ectoparasite infections can cause death or a decline in the general health of farm-raised finfish. This paper reports the findings from two studies conducted to evaluate the efficacy of hydrogen peroxide as a therapeu-tant for the control of infections of Amyloodinium sp. on cultured Pacific threadfin Polydactylus sexfilis (locally called "moi"). Threadfin with amyloodiniasis collected from a commercial farm were used in both trials. Prior to the trials, and following hydrogen peroxide treatment, the extent of infection was determined by a gill biopsy procedure. An initial trial was conducted in the laboratory to assess the response of juvenile threadfin and Amyloodinium sp. trophonts to hydrogen peroxide exposure at four dosages: 0, 75, 150, or 300 mg/L for 30 min. In a trial on a commercial farm, a hydrogen peroxide treatment at 75 mg/L for 30 min was applied to juvenile threadfin in a grow-out tank. In both trials, hydrogen peroxide was immediately flushed from the culture system with sea-water after the 30 min exposure period. In the laboratory trial, treatment with 300 mg/L hydrogen peroxide resulted in 100% mortality of the exposed group of fish. However, single treatments with hydrogen peroxide at concentrations of 75 or 150 mg/L eliminated Amyloodinium sp. trophonts without causing loss of fish. In the field trial, a single treatment with 75 mg/L hydrogen peroxide greatly reduced levels of Amyloodinium infestation, and a second treatment 6 d later reduced Amyloodinium trophonts to a nondetectable level. These findings suggest that hydrogen peroxide is a suitable chemical for the treatment of amyloodiniasis of cultured, juvenile Pacific threadfin. [source] Influence of phage population on the phage-mediated bioluminescent adenylate kinase (AK) assay for detection of bacteriaLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2001Y. Wu Aims: The effect of phage concentration on the activity of adenylate kinase (AK) released from the cells lysed during infection was investigated in order to optimize a bioluminescent phage-mediated method for bacterial enumeration. Methods and Results: The number of bacteria lysed by phages specific to Salmonella enteritidis and E. coli was determined using a bioluminescent method for the detection of AK released. In order to optimize the assay, the effect of phage concentration and time of infection on the amount of AK released was investigated. The release of AK was greatest at a multiplicity of infection (moi) of 10,100. Conclusions: The amount of AK released from Salmonella enteritidis and E. coli G2-2 cells by specific phages, SJ2 and AT20, respectively, depended on the type of bacteria, the stage of growth, the nature of phage, moi and time. Significance and Impact of the Study: An assay is described which allows detection of E. coli and Salmonella Enteritidis within 2 h at levels of 103 cfu ml,1. [source] iNOS activity is critical for the clearance of Burkholderia mallei from infected RAW 264.7 murine macrophagesCELLULAR MICROBIOLOGY, Issue 2 2008Paul J. Brett Summary Burkholderia mallei is a facultative intracellular pathogen that can cause fatal disease in animals and humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of , 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-,, IL-6, IL-10, GM-CSF, RANTES and IFN-, when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells. [source] Survival and vitality of Gremmeniella abietina on Pinus sylvestris slash in northern SwedenFOREST PATHOLOGY, Issue 6 2006J. Witzell Summary Survival and vitality of Gremmeniella abietina on Pinus sylvestris slash was studied in northern Sweden during 2003 and 2004. Once a month between September 2003 and April 2004, two to three trees were cut down and debranched. Shoots with pycnidia were sampled at the felling date and then at every consecutive month. The percentage of germinated conidia from each shoot was calculated after 24, 48 and 72 h incubation. The vitality of G. abietina pycnidia in the slash remained high the whole period. Intact pycnidia were found on slash several months after the time of conidial sporulation, which indicates that new pycnidia may be produced on dead pine branches. Sampling of shoots from slash on 13- to 18-month-old clear-cuts showed conidial germination capacity as high as in pycnidia collected in fresh slash. Due to survival of G. abietina in slash it is recommended to postpone planting of P. sylvestris seedlings in northern boreal areas to the third vegetation period after sanitary clear-cuts. Résumé La survie et la vitalité de Gremmeniella abietina dans des rémanents de Pinus sylvestris ont étéétudiées dans le nord de la Suède pendant les années 2003 et 2004. Une fois par mois entre septembre 2003 et avril 2004, 2 ou 3 arbres ont été abattus et ébranchés. Des pousses avec pycnides ont étééchantillonnées à la date d'abattage et les mois suivants. Le pourcentage de conidies germées a été calculé pour chaque pousse après 24, 48 et 72 heures d'incubation. La vitalité des pycnides de G. abietina dans les rémanents est restée élevée tout au long de la période. Des pycnides intactes ont été trouvées dans les rémanents plusieurs mois après la période de sporulation conidienne, ce qui suggère que de nouvelles pycnides peuvent être produites sur des branches mortes de pin. Des échantillonnages de pousses dans des rémanents de coupes rases réalisées 13,18 mois plus tôt ont montré une capacité de germination des conidies aussi élevée que dans les pycnides collectées dans des rémanents fraîchement coupés. Du fait de la survie de G. abietina dans les rémanents, il est conseillé de reporter la plantation des semis de P. sylvestris dans les zones septentrionales boréales à la troisième saison de végétation après les coupes sanitaires. Zusammenfassung Das Überleben und die Vitalität von Gremmeniella abietina auf Schlagabraum von Pinus sylvestris wurde in den Jahren 2003 und 2004 untersucht. Zwischen September 2003 und April 2004 wurden in jedem Monat einmal 2,3 Bäume gefällt und entastet. Zum Zeitpunkt des Fällens und in jedem folgenden Monat wurden Triebe mit Pyknidien gesammelt. Von jedem Trieb wurde die Keimrate der Konidien nach 24, 48 und 72 Stunden Inkubation bestimmt. Während der gesamten Beobachtungsdauer blieb die Vitalität der Pyknidien im Schlagabraum hoch. Mehrere Monate nach der Sporulation wurden intakte Pyknidien gefunden, ein Hinweis darauf, dass möglicherweise neue Pyknidien auf den toten Kiefernzweigen gebildet wurden. Auf dem Schlagabraum von 13,18 Monate alten Kahlschlägen war die Keimfähigkeit der Konidien ähnlich hoch wie bei Pyknidien von frischem Schlagabraum. Aufgrund des langen Überlebens von G. abietina in Schlagabraum wird für die nördlichen borealen Gebiete empfohlen, nach phytosanitären Kahlschlägen P. sylvestris -Sämlinge erst in der dritten Vegetationsperiode zu pflanzen. [source] | ||||||||||||||||||||||||||||||||||