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Low Cell Density (low + cell_density)
Selected AbstractsIs Helicobacter pylori a True Microaerophile?HELICOBACTER, Issue 4 2006Stephanie Bury-Moné Abstract Background:, There is no general consensus about the specific oxygen and carbon dioxide requirements of the human pathogen Helicobacter pylori. This bacterium is considered a microaerophile and consequently, it is grown under atmospheres at oxygen tensions 5,19% and carbon dioxide tensions 5,10%, both for clinical and basic and applied research purposes. The current study compared the growth of H. pylori in vitro, under various gas atmospheres, and determined some specific changes in the physiology of bacteria grown under different oxygen partial pressures. Methods:, Measurements of bacterial growth under various conditions were carried out employing classical solid and liquid culture techniques. Enzymatic activities were measured using spectrophotometric assays. Results:,H. pylori and all the other Helicobacter spp. tested had an absolute requirement for elevated carbon dioxide concentrations in the growth atmosphere. In contrast with other Helicobacter spp., H. pylori can tolerate elevated oxygen tensions when grown at high bacterial concentrations. Under 5% CO2, the bacterium showed similar growth in liquid cultures under oxygen tensions from microaerobic (< 5%) to fully aerobic (21%) at cell densities higher than 5 × 105 cfu/ml for media supplemented with horse serum and 5 × 107 cfu/ml for media supplemented with ,-cyclodextrin. Evidence that changes occurred in the physiology of H. pylori was obtained by comparing the activities of ferredoxin:NADH (nicotinamide adenine dinucleotide) oxidoreductases of bacteria grown under microaerobic and aerobic atmospheres. Conclusions:,H. pylori is a capnophile able to grow equally well in vitro under microaerobic or aerobic conditions at high bacterial concentrations, and behaved like oxygen-sensitive microaerophiles at low cell densities. Some characteristics of H. pylori cells grown in vitro under microaerobic conditions appeared to mimic better the physiology of organisms grown in their natural niche in the human stomach. [source] Study of Protein Splicing and Intein-Mediated Peptide Bond Cleavage under High-Cell-Density ConditionsBIOTECHNOLOGY PROGRESS, Issue 3 2003Shamik Sharma Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins. [source] Undirected motility of filamentous cyanobacteria produces reticulate matsGEOBIOLOGY, Issue 3 2010R. N. SHEPARD The roles of biology in the morphogenesis of microbial mats and stromatolites remain enigmatic due to the vast array of physical and chemical influences on morphology. However, certain microbial behaviors produce complex morphological features that can be directly attributed to motility patterns. Specifically, laboratory experiments with a strain of the cyanobacteria Pseudanabaena demonstrate that distinctive morphologies arise from the undirected gliding and colliding of filaments. When filamentous cells collide, they align and clump, producing intersecting ridges surrounding areas with low cell density, i.e. reticulate structures. Cell motility is essential for the development of reticulates and associated structures: filaments organize into reticulates faster than cell division and growth, and conditions that inhibit motility also inhibit reticulate formation. Cell density of the inoculum affects the frequency of cell,cell collisions, and thus the time required for biofilm organization into reticulate structures. This also affects the specific geometry of the reticulates. These patterns are propagated into larger structures as cyanobacterial cell numbers increase and cells remain motile. Thus, cell motility is important for templating and maintaining the morphology of these microbial communities, demonstrating a direct link between a microbial behavior and a community morphology. Reticulate geometries have been identified in natural microbial mats as well as in the fossil record, and these structures can be attributed to the motility of filamentous bacteria. [source] Non-antagonistic relationship between mitogenic factors and cAMP in adult Schwann cell re-differentiationGLIA, Issue 9 2009Paula V. Monje Abstract The expression of myelination-associated genes (MGs) can be induced by cyclic adenosine monophosphate (cAMP) elevation in isolated Schwann cells (SCs). To further understand the effect of known SC mitogens in the regulation of SC differentiation, we studied the response of SCs isolated from adult nerves to combined cAMP, growth factors, including neuregulin, and serum. In adult SCs, the induction of MGs by cAMP coincided with the loss of genes expressed in non-myelin-forming SCs and with a change in cell morphology from a bipolar to an expanded epithelial-like shape. Prolonged treatment with high doses of cAMP-stimulating agents, as well as low cell density, was required for the induction of SC differentiation. Stimulation with serum, neuregulin alone, or other growth factors including PDGF, IGF and FGF, increased SC proliferation but did not induce the expression of MGs or the associated morphological change. Most importantly, when these factors were administered in combination with cAMP-stimulating agents, SC proliferation was synergistically increased without reducing the differentiating activity of cAMP. Even though the initiation of DNA synthesis and the induction of differentiation were mostly incompatible events in individual cells, SCs were able to differentiate under conditions that also supported active proliferation. Overall, the results indicate that in the absence of neurons, cAMP can trigger SC re-differentiation concurrently with, but independently of, growth factor signaling. © 2008 Wiley-Liss, Inc. [source] PPAR,1 synthesis and adipogenesis in C3H10T1/2 cells depends on S-phase progression, but does not require mitotic clonal expansionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Young C. Cho Abstract Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I), dexamethasone (D), and methylisobutylxanthine (M), administered with a fresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator-activated receptor ,1 (PPAR,1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that mitotic clonal expansion of 3T3-L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S-phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPAR,1 synthesis and lipogenesis. Different S-phase inhibitors (aphidicolin, hydroxyurea, l -mimosine, and roscovitin) each arrested cells in S-phase, under hormonal stimulation, and completely blocked PPAR,1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPAR,1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M-phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one-half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPAR, was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPAR,1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. Importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non-differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S-phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPAR, synthesis and subsequent adipocyte differentiation. © 2003 Wiley-Liss, Inc. [source] Identification of the central quorum sensing regulator of virulence in the enteric phytopathogen, Erwinia carotovora: the VirR repressorMOLECULAR MICROBIOLOGY, Issue 1 2006Tom Burr Summary In the Gram-negative phytopathogen, Erwinia carotovora ssp. atroseptica (Eca) virulence depends on the production of a N- (3-oxohexanoyl)- l -homoserine lactone (OHHL) quorum sensing (QS) signal. This work identifies the elusive ,missing link' between QS and virulence in Erwinia. We have identified and characterized a novel regulator of virulence, VirR, in Eca and show that a virR mutation completely restores virulence factor production to an Eca mutant unable to synthesize OHHL. This effect of the virR mutation translates to a restoration of virulence to wild-type levels and thus provides evidence that VirR acts to prevent the production of virulence factors at low cell density. We also show that, in Eca, transcription of virulence genes is controlled by OHHL and that this control is effected through the action of VirR. We also demonstrate that the VirR regulatory pathway is present and functional in both blackleg and soft rotting species of Erwinia. [source] Quorum sensing controls biofilm formation in Vibrio choleraeMOLECULAR MICROBIOLOGY, Issue 1 2003Brian K. Hammer Summary Multiple quorum-sensing circuits function in parallel to control virulence and biofilm formation in Vibrio cholerae. In contrast to other bacterial pathogens that induce virulence factor production and/or biofilm formation at high cell density in the presence of quorum-sensing autoinducers, V. cholerae represses these behaviours at high cell density. Consistent with this, we show here that V. cholerae strains ,locked' in the regulatory state mimicking low cell density are enhanced for biofilm production whereas mutants ,locked' in the regulatory state mimicking high cell density are incapable of producing biofilms. The quorum-sensing cascade we have identified in V. cholerae regulates the transcription of genes involved in exopolysaccharide production (EPS), and variants that produce EPS and form biofilms arise at high frequency from non-EPS, non-biofilm producing strains. Our data show that spontaneous mutation of the transcriptional regulator hapR is responsible for this effect. Several toxigenic strains of V. cholerae possess a naturally occurring frameshift mutation in hapR. Thus, the distinct environments occupied by this aquatic pathogen presumably include niches where cell-cell communication is crucial, as well as ones where loss of quorum sensing via hapR mutation confers a selective advantage. Bacterial biofilms could represent a complex habitat where such differentiation occurs. [source] Reassessing culture media and critical metabolites that affect adenovirus productionBIOTECHNOLOGY PROGRESS, Issue 1 2010Chun Fang Shen Abstract Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell-specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell-specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 × 106 cells/mL. In comparison, only 50% of reduction in the cell-specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 × 106 cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 × 106 cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | ||||||||||