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Low Multiplicity (low + multiplicity)
Selected AbstractsVirus-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] A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretionMOLECULAR MICROBIOLOGY, Issue 6 2004Lian-Yong Gao Summary Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871,Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host. [source] Determination of all misorientations of tetragonal lattices with low multiplicity; connection with Mallard's rule of twinningACTA CRYSTALLOGRAPHICA SECTION A, Issue 4 2003Hans Grimmer Two congruent lattices are considered, which are misoriented in such a way that they have a fraction 1, of symmetry translations in common. Whereas for cubic lattices body or face centring does not affect the `multiplicity' or `twin index' ,, this is not generally true for tetragonal lattices. Consider a fixed misorientation and let ,P and ,I be the multiplicities for tP and tI lattices with the same axial ratio ca. Grimmer [Mater. Sci. Forum (1993), 126,128, 269,272] has given an explicit formula for ,P (depending on the misorientation and the axial ratio) and showed that ,I = ,P2, ,P or 2,P. Here stronger results on the occurrence of the three possibilities are presented. Lists of all axial ratios ca of tP and tI lattices admitting misorientations with ,, 5 are given. For each of these misorientations, the twin mirror planes and their normals are listed, so that a synopsis of all possible twin laws of tetragonal crystals by reticular merohedry with ,, 5 is obtained. It is shown that the two twin laws observed in ,-Sn can be described by reticular pseudomerohedry with ,I,=,2 and obliquity ,,=,2.6134°. [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] 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] | ||||||||||