The stability of foreign protein production in genetically modified plant cells

Summary The stability of foreign protein production in genetically engineered plant cells was studied. A cultured tobacco cell line was transformed with a chimeric molecule carrying a bacterial gene, ß-glucuronidase (GUS), under plant regulatory sequences. The specific GUS activity was monitored for 294 days with ten independently transformed cell lines either in the presence or the absence of selectable antibiotics. Specific GUS activity was stably maintained in five lines. About a two-to four-fold increase in the GUS activity was observed from three cell lines. The remaining two cell lines lost the activity within the first 70 to 210 days. The presence of antibiotics did not significantly alter the stability of the foreign protein production in all cell lines examined.     

Cultivation of plant cells in a stirred vessel: effect of impeller design

Suspension cultures of Nicotiana tabacum were grown in a batch fermentor using different agitation systems. The effects of the impeller type, size, and agitation speed on the productivity of cell mass and secondary metabolites (phenolics) have been investigated. The use of a large, flat-bladed impeller (diameter 7.6 cm; width 14.0 cm) improved culture growth significantly over systems using a regular, flat-bladed impeller (diameter 5.6 cm; width 1.5 cm). An impeller of the same dimensions as the 14.0-cm-wide, large, flat-bladed impeller with sail cloth blades yielded a higher maximum growth rate in the exponential phase but resulted in a longer lag phase. Overall (intracellular and extracellular) phenolics concentration showed a direct relationship to culture growth rate whereas extracellular concentrations were a function of agitation conditions. Power consumption and flow pattern studies were also completed to further characterize the different impellers tested.     

Expression of bacterial beta-galactosidase in animal cells

A recombinant plasmid containing the gene for bacterial β-galactosidase, situated close to the simian virus 40 early promoter, has been constructed. Transfection of CHO, L, and COS-1 cells with this plasmid led to the expression and appearance of the enzyme. Using this system, we have developed a series of promoter cloning vehicles capable of accepting promoter signals for animal genes.     

Galium procurrens,a new diploid relic species of theG. sylvaticum-group from the Balkan Peninsula

Galium procurrens is described as a new diploid relic species from Montenegro/N. Albania and SW. Bulgaria. It is related to the tetraploidG. laevigatum and other diploid and polyploid taxa of theG. sylvaticum-group inhabiting European deciduous forests.     

Hyperosmolarity enhanced susceptibility to renal tubular fibrosis by modulating catabolism of type I transforming growth factor‐β receptors

Hyperosmolarity plays an essential role in the pathogenesis of diabetic tubular fibrosis. However, the mechanism of the involvement of hyperosmolarity remains unclear. In this study, mannitol was used to evaluate the effects of hyperosmolarity on a renal distal tubule cell line (MDCK). We investigated transforming growth factor‐β receptors and their downstream fibrogenic signal proteins. We show that hyperosmolarity significantly enhances the susceptibility to exogenous transforming growth factor (TGF)‐β1, as mannitol (27.5 mM) significantly enhanced the TGF‐β1‐induced increase in fibronectin levels compared with control experiments (5.5 mM). Specifically, hyperosmolarity induced tyrosine phosphorylation on TGF‐β RII at 336 residues in a time (0–24 h) and dose (5.5–38.5 mM) dependent manner. In addition, hyperosmolarity increased the level of TGF‐β RI in a dose‐ and time‐course dependent manner. These observations may be closely related to decreased catabolism of TGF‐β RI. Hyperosmolarity significantly downregulated the expression of an inhibitory Smad (Smad7), decreased the level of Smurf 1, and reduced ubiquitination of TGF‐β RI. In addition, through the use of cycloheximide and the proteasome inhibitor MG132, we showed that hyperosmolarity significantly increased the half‐life and inhibited the protein level of TGF‐β RI by polyubiquitination and proteasomal degradation. Taken together, our data suggest that hyperosmolarity enhances cellular susceptibility to renal tubular fibrosis by activating the Smad7 pathway and increasing the stability of type I TGF‐β receptors by retarding proteasomal degradation of TGF‐β RI. This study clarifies the mechanism underlying hyperosmotic‐induced renal fibrosis in renal distal tubule cells. J. Cell. Biochem. 109: 663–671, 2010. © 2010 Wiley‐Liss, Inc.     

BTB/TAZ protein MdBT2 integrates multiple hormonal and environmental signals to regulate anthocyanin biosynthesis in apple

BT2 is a BTB/TAZ domain protein with key roles in multiple stress responses and the plant development of Arabidopsis (Figueroa et al. 2005; Ren et al. 2007; Mandadi et al. 2009). Recent studies have demonstrated that apple MdBT2 functions as a negative regulator in diverse hormonal and environmental signal‐induced anthocyanin biosynthesis, suggesting that MdBT2 integrates stress signals and anthocyanin biosynthesis.