Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Biochemical Genetics (v.36, #1-2)
Evidence for Interactions Between Rat Hepatoma Cell Apoptosis and Differentiation by Eric Renault; Sophie Sarrazin; Jean Deschatrette (pp. 1-13).
Partial copper depletion of a variant rathepatoma cell line induces a transient inhibition ofgrowth and the genesis of stable, well-differentiatedrevertants. We report a burst of cell death, synchronous with the peak of reversion. The characteristicsof this cell mortality were typical of apoptosis andincluded detachment from the plastic support, chromatincondensation and fragmentation, and internucleosomal DNA degradation. Although commitment to celldeath was induced by copper deficiency, the apoptoticprocess was partially inhibited as assessed fromelectrophoretic patterns of DNA degradation.Redifferentiation was closely linked to the apoptotic deathprogram. Analysis of rescued detached cells in all threemedia (standard, Cu-, Fe-)indicated that the frequency of revertants wassignificantly higher among floating as opposed to adherent cell populations.Nevertheless, experimental copper depletion increased by104 times the revertant frequency amongadherent cells. We propose that redifferentiation of thevariant hepatoma cells (and concomitant recovery oftumorigenicity) is determined by the gene expressionpattern of programmed cell death.
Keywords: APOPTOSIS; DIFFERENTIATION; HEPATOMA; COPPER; ENDONUCLEOLYSIS
Isolation of Protease-Free Alcohol Dehydrogenase (ADH) from Drosophila simulans and Several Homozygous and Heterozygous Drosophila melanogaster Variants by Tim Smilda; Dick A. Lamme; Graziella Collu; Peter A. Jekel; Peter Reinders; Jaap J. Beintema (pp. 15-36).
The enzyme alcohol dehydrogenase (ADH) fromseveral naturally occurring ADH variants ofDrosophila melanogaster and Drosophilasimulans was isolated. Affinity chromatography withthe ligand Cibacron Blue and elution with NAD+ showed similarbehavior for D. melanogaster ADH-FF, ADH-71k,and D. simulans ADH. Introduction of a secondCibacron Blue affinity chromatography step, withgradient elution with NAD+, resulted in pure and stable enzymes. D.melanogaster ADH-SS cannot be eluted from theaffinity chromatography column at a high concentrationof NAD+ and required a pH gradient for itspurification, preceded by a wash step with a high concentration ofNAD+. Hybrid Drosophila melanogasteralcohol dehydrogenase FS has been isolated fromheterozygous flies, using affinity chromatography withfirst elution at a high concentration NAD+, directlyfollowed by affinity chromatography elution with a pHgradient. Incubation of equal amounts of pure homodimersof Drosophila melanogaster ADH-FF and ADH-SS,in the presence of 3 M urea at pH 8.6, for 30 min at roomtemperature, followed by reassociation yielded activeDrosophila melanogaster ADH-FS heterodimers. Noproteolytic degradation was found after incubation ofpurified enzyme preparations in the absence or presenceof SDS, except for some degradation of ADH-SS after verylong incubation times. The thermostabilities of D.melanogaster ADH-71k and ADH-SS were almostidentical and were higher than those of D.melanogaster ADH-FF and D. simulans ADH. Thethermostability of D. melanogaster ADH-FS waslower than those of D. melanogaster ADH-FF andADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constantswith the ligand Cibacron Blue at pH 8.6, which are twotimes higher at pH 9.5. The Ki values forD. simulans ADH are three times lower at bothpH values. D. melanogaster ADH-SS and ADH-FS havesimilar Ki values, which are lower than thosefor D. melanogaster ADH-FF at pH 8.6. But at pH9.5 the Ki value for ADH-FS is the same as atpH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters ofDrosophila melanogaster ADH-FF, ADH-SS, andADH-71k and Drosophila simulans ADH, at pH 8.6and 9.5, showed little or no variation inKm eth values. TheKm NAD values measured at pH 9.5for Drosophila alcohol dehydrogenases are alllower than those measured at pH 8.6. The rate constants(kcat) determined for all fourDrosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D.melanogaster ADH-FS showed nonlinear kinetics.
Keywords: ALCOHOL DEHYDROGENASE; DROSOPHILA; HETEROZYGOTE; PURIFICATION; HYBRID FORMATION
Modeling Studies of Conformational Changes in the Substrate-Binding Loop in Drosophila Alcohol Dehydrogenase by Tim Smilda; Peter Reinders; Jaap J. Beintema (pp. 37-49).
Three-dimensional structures of sevenshort-chain dehydrogenases/reductases show that theseenzymes share common structural features. Sequencealignment studies of Drosophila alcoholdehydrogenase (DADH), with an unknown 3D-structure, and fourshort-chain dehydrogenases/reductases with known X-raystructures suggest that DADH shares the same structuralfeatures. However, the substrate binding regions, which are located in the C-terminal region of theseenzymes, share little sequence homology, because of thewide variety of substrates used. X-ray structures ofshort-chain dehydrogenases/reductases indicate that conformational changes occur in a loop, inthe C-terminal region, upon substrate binding. Thissubstrate-binding loop is located between a strand anda helix and may contain one or two small helices. Secondary structure predictions and modelingstudies of this substrate-binding loop in DADH predictthat the two helices may also be present in this enzyme.The naturally occurring variants of Drosophila melanogaster alleloenzymes ADH-S and ADH-Fdiffer in a replacement of threonine by lysine atposition 192, which is located at a central position inthe substrate-binding loop. The positive charge oflysine may move significantly on substrate binding,resulting in a direct charge interaction withNAD+ in the enzyme-substrate complex,explaining a very strong influence of pH on the bindingof ADH-S for the NAD+ analogue Cibacron Blue. Thisindicates that the ADH S/F polymorphism has a directinfluence on the catalytic properties of the enzyme.
Keywords: DROSOPHILA; ALCOHOL DEHYDROGENASE; MODELING STUDIES; SUBSTRATE-BINDING LOOP
A Family of Drosophila Genes Encoding quaking -Related Maxi-KH Domains by Christine Fyrberg; Jodi Becker; Peter Barthmaier; James Mahaffey; Eric Fyrberg (pp. 51-64).
We recently identified a Drosophilagene, wings held out (who), that specifies a STAR(signal transduction and RNA activation) proteinexpressed within mesoderm and muscles. Genetic evidencesuggests that WHO regulates muscle development and functionin response to steroid hormone titer. who is related tothe mouse quaking gene, essential for embryogenesis andneural myelination, and gld-1, a nematode tumor suppressor gene necessary for oocytedifferentiation, both of which contain RNA binding“maxi-KH” domains presumed to link RNAmetabolism to cell signaling. To initiate a broaderstudy of Drosophila WHO related proteins we used degenerate primers encodingpeptides unique to maxi-KH domains to amplify thecorresponding genes. We recovered nine genes, allspecifying single maxi-KH domain proteins havingtripartite regions of similarity that extend over 200amino acids. One is located within the 54D chromosomesubdivision, and one within 58C, while the remainingseven are within the 58E subdivision. At least four of these STAR proteins are expressed in ageneral manner, suggesting that maxi-KH domains areemployed widely in Drosophila.
Keywords: DROSOPHILA GENETICS; K HOMOLOGY DOMAINS; STAR PROTEINS; NUCLEIC ACID-BINDING MOTIFS
Rapid Genotyping of Mice with Hemoglobinopathies and Globin Transgenes by J. Barry III Whitney; Aya Leder; Jada Lewis; Raymond A. Popp; Chris Paszty; Edward M. Rubin; W. Ronald Shehee; Tim M. Townes; Oliver Smithies (pp. 65-77).
The hematology of the laboratory mouse has beenwell characterized. Normal genetic differences at thealpha- and beta-globin gene loci serve as useful markersfor a wide variety of types of experimental studies. There are a number of naturallyoccurring or induced mutations that disrupt globinexpression and produce thalassemic phenotypes. Inaddition, much has been learned of the workings of theglobin locus control region from studies of transgenicmice, including those with mutations induced by targetedsite-specific modifications. After a new mutation ortransgene has been created, it must be maintained in living mice, and the genotypes of theoffspring must be ascertained. While it is possible todetermine genotypes by DNA analyses, such assays aretime consuming and relatively expensive. An osmoticchallenge test -- originally developed for thegenotyping of large-deletion alpha-thalassemia mutationsin mice -- has proven useful in detecting bothsevere and milder alpha- and beta-thalassemias, as wellas some transgenic genotypes in mice carrying human globin genes.Reliable genotyping can, in some cases, be completedwithin a few minutes with minimal expense.Quantification of red cell fragility for a variety ofthalassemic and transgenic mice is described here, alongwith a simplified test suitable for rapid, routinegenotyping. The osmotic challenge test is perfectlyreliable for distinguishing genotypes that causesignificantly decreased release of hemoglobin from the redcells, but it is also useful for some of the conditionsin which overall erythrocyte osmotic fragility isessentially normal.
Keywords: HEMOGLOBINOPATHY; MICE; TRANSGENIC; HEMOLYSIS; HEMOGLOBIN