Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Segregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under non-selective conditions this segregational instability results in a heterogeneous population of cells, where the non-productive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in <it>Escherichia coli </it>LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNγ) gene placed under the control of different regulatory elements (promoter and ribosome-binding sites) were used as a model.</p> <p>Results</p> <p>Bacterial growth and product formation kinetics of transformed <it>E. coli </it>LE392 cells cultivated continuously were described by a structured kinetic model proposed by Lee et al. (1985). The obtained results demonstrated that both transcription and translation efficiency strongly affected plasmid segregation. The segregation of plasmid having a deleted promoter did not exceed 5% after 190 h of cultivation. The observed high plasmid stability was not related with an increase in the plasmid copy-number. A reverse correlation between the yield of recombinant protein (as modulated by using different ribosome binding sites) and segregational plasmid stability (determined by the above model) was also observed.</p> <p>Conclusions</p> <p>Switching-off transcription of the hIFNγ gene has a stabilising effect on ColE1-like plasmids against segregation, which is not associated with an increase in the plasmid copy-number. The increased constitutive gene expression has a negative effect on segregational plasmid stability. A kinetic model proposed by Lee et al. (1985) was appropriate for description of <it>E. coli </it>cell growth and recombinant product formation in chemostat cultivations.</p

Stem cells in liver regeneration and therapy

The liver has adapted to the inflow of ingested toxins by the evolutionary development of unique regenerative properties and responds to injury or tissue loss by the rapid division of mature cells. Proliferation of the parenchymal cells, i.e. hepatocytes and epithelial cells of the bile duct, is regulated by numerous cytokine/growth-factor-mediated pathways and is synchronised with extracellular matrix degradation and restoration of the vasculature. Resident hepatic stem/progenitor cells have also been identified in small numbers in normal liver and implicated in liver tissue repair. Their putative role in the physiology, pathophysiology and therapy of the liver, however, is not yet precisely known. Hepatic stem/progenitor cells also known as “oval cells” in rodents have been implicated in liver tissue repair, at a time when the capacity for hepatocyte and bile duct replication is exhausted or experimentally inhibited (facultative stem/progenitor cell pool). Although much more has to be learned about the role of stem/progenitor cells in the physiology and pathophysiology of the liver, experimental analysis of the therapeutic value of these cells has been initiated. Transplantation of hepatic stem/progenitor cells or in vivo pharmacological activation of the pool of hepatic stem cells may provide novel modalities for the therapy of liver diseases. In addition, extrahepatic stem cells (e.g. bone marrow cells) are being investigated for their contribution to liver regeneration. Hepatic progenitor cells derived from embryonic stem cells are included in this review, which also discusses future perspectives of stem cell-based therapies for liver diseases

Observations of the plume generated by the December 2005 oil depot explosions and prolonged fire at Buncefield (Hertfordshire, UK) and associated atmospheric changes

The explosions and subsequent fire at the Buncefield oil depot in December 2005 afforded a rare opportunity to study the atmospheric consequences of a major oil fire at close range, using ground-based remote-sensing instruments. Near-source measurements (less than 10km) suggest that plume particles were approximately 50% black carbon (BC) with refractive index 1.73-0.42i, effective radius (Reff) 0.45-0.85μm and mass loading approximately 2000μgm-3. About 50km downwind, particles were approximately 60-75% BC with refractive index between 1.80-0.52i and 1.89-0.69i, R eff∼1.0μm and mass loadings 320-780μgm-3. Number distributions were almost all monomodal with peak at r<0.1μm. Near-source UV spectroscopy revealed elevated trace gas concentrations of SO2 (70ppbv), NO2 (140ppbv), HONO (20ppbv), HCHO (160ppbv) and CS 2 (40ppbv). Our measurements are consistent with others of the Buncefield plume, and with studies of the 1991 Kuwaiti oil-fire plumes; differences from the latter reflecting in part contrasts in combustion efficiency and source composition (refined fuels versus crude oils) leading to important potential differences in atmospheric impacts. Other measurements made as the plume passed overhead approximately 50km downwind showed a reduced solar flux reaching the surface, but little effect on the atmospheric potential gradient (electric field). The wind speed data from the day of the explosion hint at a possible explosion signature. © 2007 The Royal Society