Membrane topology of the Rickettsia prowazekii ATP/ADP translocase revealed by novel dual pho-lac reporters

Here, we report the construction and characterization of dual reporters, consisting of both an Escherichia coli alkaline phosphatase (AP) gene and an alpha-fragment of the beta-galactosidase (BG) gene, for studying membrane protein topology by the gene fusion approach. Each of the reporters, when fused to periplasmic domains of polytopic proteins, produces fusions with high AP activity and, when fused to cytoplasmic domains, produces fusions with high BG activity in E. coli strains capable of alpha-complementation. The dual nature of these reporters simplifies interpretation of data obtained with poorly expressed fusions and allows one to evaluate the reliability of topological data. Deleterious effects resulting from the cell's attempt to export the full-length BG are eliminated in this approach. We describe dual indicator plates that allow for discrimination between colonies bearing cytoplasmic fusions, periplasmic fusions, and no fusions. We have generated a set of fusions to the topologically well-studied lactose permease of E. coli and demonstrated that topological information generated by these new reporters is in good agreement with the existing model. We used this new methodology for the determination of membrane topology of the Rickettsia prowazekii ATP/ADP translocase (Tlc). Our results were in agreement with the proposed in silico topological model in which Tlc traverses the cytoplasmic membrane of E. coli 12 times with its N and C termini facing the cytoplasm.     

Marked osteoblastopenia and reduced bone formation in a model of multiple myeloma bone disease in severe combined immunodeficiency mice

Increased generation of reactive oxygen species (ROS) and low levels of antioxidants may cause morbidity in premature infants on supplemental oxygen. Glutathione (GSH)-dependent antioxidant systems protect against ROS, and regenerating GSH from GSH disulfide (GSSG) by the flavoenzyme GSH reductase (GR) is essential for the optimal function of this system. Previously, we have observed enhanced resistance to t-butyl hydroperoxide (t-BuOOH) in Chinese hamster ovary cells stably transfected with a vector (leader sequence GR [LGR]) for human GR cDNA that contained a functional synthetic mitochondrial targeting signal. The present studies were designed to investigate adenovirus-mediated gene transfer of LGR to H441 cells and resistance of such cells to t-BuOOH. Adenovirus-mediated transfection of H441 cells with LGR increased total GR activities more than 11-fold (mitochondria more than 10-fold and cytosolic more than 7-fold) and protected against t-BuOOH cytotoxicity, as indicated by lower fractional release of cellular lactate dehydrogenase (LDH) than was observed in wild-type untransfected cells (CON) or in cells transfected with a control gene (human manganese superoxide dismutase in the antisense orientation [DOS]) (*LGR 6.6 +/- 1.7; DOS 16 +/- 1.8; CON 16.6 +/- 0.7% LDH release). In addition, cells transfected with LGR retained higher GSH/GSSG ratios (*LGR 66 +/- 0.4; DOS 47 +/- 1; CON 52.6 +/- 2.3) and released less GSH + GSSG to the media in response to challenge with t-BuOOH (*LGR 0.05 +/- 0.01; DOS 0.08 +/- 0.01; CON 0.07 +/- 0.01 nmol/mg of protein) than did wild-type cells or cells transfected with a control vector, indicating an enhanced ability of the LGR cells to reduce GSSG formed in response to exposure to t-BuOOH. In conclusion, adenovirus-mediated gene transfer of LGR enhanced cellular GR activities and protected H441 cells from oxidant stresses.     

Effect of taro (Colocasia esculenta) flour addition on the functional and alveographic properties of wheat flour and dough

BACKGROUND: Taro is a plant widely produced in tropical areas for its underground corms. Taro corms are highly perishable owing to their high moisture content. In order to limit post‐harvest losses and improve the production and consumption, production and utilization of taro flour have been envisaged. In connection with this, the incorporation of taro flour into wheat‐based products has been reported to increase their keeping quality. Before consideration is given to taro flour as a substitute for wheat in bread manufacture, it is necessary to characterize the functionality of the composite flour and dough. The present study investigated the physicochemical and alveographic properties of wheat–taro composite flour. RESULTS: Three varieties of taro flour (WCN, RIN and KWN) were incorporated into wheat flour in proportions of 10%, 20% and 30% (w/w) and the water absorption capacity (WAC), retrogradation index (RI), foam capacity (FC) and other functional properties were measured. In addition, characteristics of dough rupture pressure (P), extensibility (L), elasticity index (Ie) and strength (W) of flour made from the different composites were measured using an alveograph. The results showed that WAC significantly increased from 132% (wheat flour) to 156% (30% composite flour), while RI significantly decreased from 38% to a mean value of 22%. Principal component analysis revealed that WAC and P formed a group of variables negatively correlated with a second group made of RI, FC, W, Ie and L. CONCLUSION: Incorporating taro flour up to a level of 10% had no significant effect on the functional and alveographic properties of the flours. This suggested that in order to guarantee the quality of bread made from wheat–taro composite the level of taro addition should not exceed 10%. Copyright © 2007 Society of Chemical Industry