Lac/Tet dual-inducible system functions in mammalian cell lines

The Escherichia coli Lac repressor (Lac system) and tetracycline responsive promoter (Tet system) systems have been used individually to regulate gene expression at the cellular as well as the organismal levels. In this study, these two systems were combined (designated Lac/Tet dual-inducible system) to regulate two inducible genes simultaneously in a single cell. The isopropyl-beta-D-thiogalactopyranoside (IPTG) and tetracycline (used for the operation of the Lac and the Tet systems) were non-cytotoxic to the cells when added together into the cells at around the optimal concentrations (IPTG: < or = 5 mM; tetracycline: < 1.5 micrograms). The rate and efficiency of induction and repression of two inducible genes regulated by the Lac/Tet dual-inducible system were similar to the results obtained when one inducible gene is regulated by one inducible system in a single cell. The Lac/Tet dual-inducible system could function in many cell lines, which was demonstrated by regulating the expression of beta-galactosidase and luciferase reporter genes in five tumor cell lines by transient transfection analysis. The feasibility of introducing a second inducible system into an already established inducible cell line was confirmed. Finally, we showed that the Lac/Tet dual-inducible system functions at translational and at functional levels in a stable cell line named 7-4-b, which contains the Ha-ras and bc1-2 inducible genes. In conclusion, this study extends the application of prokaryotic inducible systems from the regulation of a single gene to two genes and helps clarify the relationship between two genes and the effects of two genes on the cells.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Experimental pyeloureterectomy and calyceal neck transection with autotransplantation and bladder advancement

OBJECTIVE: To determine whether Dexon mesh, closely applied to the kidney, provides purchase for sutures to permit bladder/parenchymal apposition on autotransplantation and that, if this line of apposition were some distance from but surrounding renal papillae, urothelium would proliferate to cover exposed parenchyma to form a widely patent lumen; this should facilitate removal of the whole of an upper tract collecting system, retaining renal parenchyma alone. MATERIALS AND METHODS: To test this possibility and explore the practicability of the concept, nine dogs underwent bilateral nephrectomy followed by unilateral autotransplantation: the other kidney was discarded. Because the canine renal pelvis is intrarenal, the ureter was stretched maximally before passing fine scissors into the renal hilum to transect the collecting system as close to the kidney as possible in six of the nine dogs. In the remaining three dogs, partial nephrectomy was performed with division of the calyceal necks under vision. Thinned bladder wall was sutured to Dexon mesh some distance from the collecting tubules; omentum was applied to the suture line. RESULTS: Three dogs were killed prematurely at < 2 weeks because of perioperative complications. Four were killed at 2, 4, 5 and 8 weeks and two at 12 months. Dexon mesh proved to be an effective anchoring fabric, providing close apposition of bladder wall and parenchyma. There was no adhesion of the kidney to peritoneal contents. Urothelial proliferation to cover exposed parenchyma occurred early and by 12 months, a thin stroma was interposed between parenchyma and epithelium. The kidney was preserved in all but one removed electively, this dog having both cystitis and pyelonephritis at 12 months. CONCLUSIONS: This study showed that autotransplantation of a kidney after removal of its collecting system and advancement of thinned bladder wall to renal parenchyma is practicable, with regenerated urothelium bridging the deficiency by covering exposed parenchyma, to create a widely patent lumen.     

The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface

The immunity protein of colicin E7 (ImmE7) can bind specifically to the DNase-type colicin E7 and inhibit its bactericidal activity. Here we report the 1.8-angstrom crystal structure of the ImmE7 protein. This is the first x-ray structure determined in the superfamily of colicin immunity proteins. The ImmE7 protein consists of four antiparallel alpha-helices, folded in a topology similar to the architecture of a four-helix bundle structure. A region rich in acidic residues is identified. This negatively charged area has the greatest variability within the family of DNase-type immunity proteins; thus, it seems likely that this area is involved in specific binding to colicin. Based on structural, genetic, and kinetic data, we suggest that all the DNase-type immunity proteins, as well as colicins, share a "homologous-structural framework" and that specific interaction between a colicin and its cognate immunity protein relies upon how well these two proteins' charged residues match on the interaction surface, thus leading to specific immunity of the colicin.     

Retrotransposable elements R1 and R2 in the rDNA units of Drosophila mercatorum: abnormal abdomen revisited

R1 and R2 retrotransposable elements are stable components of the 28S rRNA genes of arthropods. While each retrotransposition event leads to incremental losses of rDNA unit expression, little is known about the selective consequences of these elements on the host genome. Previous reports suggested that in the abnormal abdomen (aa) phenotype of Drosophila mercatorum, high levels of rDNA insertions (R1) in conjunction with the under-replication locus (ur), enable the utilization of different ecological conditions via a population level shift to younger age. We have sequenced the R1 and R2 elements of D. mercatorum and show that the levels of R1- and R2-inserted rDNA units were inaccurately scored in the original studies of aa, leading to several misinterpretations. In particular, contrary to earlier reports, aa flies differentially underreplicate R1- and R2-inserted rDNA units, like other species of Drosophila. However, aa flies do not undergo the lower level of underreplication of their functional rDNA units (general underreplication) that is seen in wild-type strains. The lack of general underreplication is expected to confer a selective advantage and, thus, can be interpreted as an adaptation to overcome high levels of R1 and R2 insertions. These results allow us to reconcile some of the apparently contradictory effects of aa and the bobbed phenotype found in other species of Drosophila.     

Structure of poly d(AI).poly d(CT) in two different packing arrangements

X-ray diffraction analysis of poly d(AI).poly d(CT) in oriented and polycrystalline fibers has revealed the DNA structure to be a 10-fold, right-handed, antiparallel, Watson-Crick base paired double helix in two distinct packing arrangements corresponding to one and two helices, respectively, in the unit cell. The helix pitch is 32.1 A and 32.4 A in the two cases, almost 1.5 A shorter than in classical B-DNA. The resulting B'-DNA geometry, described in terms of a tetranucleotide repeat which is conformationally similar to B-DNA, has its minor groove closely shut and major groove correspondingly widened, thus striking a sharp morphological contrast to B-DNA. According to difference electron density maps, a spine of hydration along the minor groove connects both strands and provides structural stability; ordered sodium ions and water molecules are actively involved in bridging the phosphate groups of neighboring helices. The crystallographic R-values for these two allomorphs are 0.26 and 0.20, respectively, for data up to 3.0 A resolution.     

Determining the cost of a clinical intervention through the use of shadow pricing

This article reviews the scientific literature in several areas important to the delivery of palliative care: multicultural issues, education, comprehensive outcome measures and ethics. Most of the research can be classified as fundamental rather than intervention research according to the Cancer Control Framework of the National Cancer Institute of Canada. Desired outcomes of interventions are most often defined from the health care professional's perspective but need to be defined from the patient's perspective. In areas such as multicultural issues and the effect of the volunteer on the patient, there is almost no research. The complexity of studying the best way to deliver palliative care would benefit from the input of colleagues who have experience addressing these issues in other patient populations.