Effects of constitutively active GTPases on fibroblast behavior

The GTP-binding proteins RhoA, Cdc42 and Rac1 regulate the organization and turnover of the cytoskeleton and cell-matrix adhesions, structures bridging cells to their support, and translating forces, external or generated within the cell. To investigate the specific requirements of Rho GTPases for biomechanical activities of clonal cell populations, we compared side-by-side stable lines of human fibroblasts expressing constitutively active (CA) RhoA, Cdc42 or Rac1. There was no marked effect of any CA GTPase on cell adhesion to different extracellular matrix proteins. Cell spreading was CA Rho GTPase specific and independent of the extracellular matrix proteins allowing adhesion. Mechanical properties were dramatically restricted by CA RhoA on bi- and in tri-dimensional surroundings, were boosted by CA Rac1 on bi-dimensional surroundings only, and were not or marginally affected by CA Cdc42. In conclusion, the action of Rho GTPases appears to depend on the task cells are performing. Received 12 September 2005; received after revision 5 October 2005; accepted 1 November 2005     

IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.     

Similarities between metallothionein and low molecular weight testicular cadmium-binding protein

The incorporation of 35S-cysteine and 3H-glutamic acid was studied in mouse hepatic and renal metallothionein and in testicular cadmium-binding protein of similar molecular weight. Preferential incorporation of 35S-cysteine over 3H-glutamic acid was observed not only in hepatic and renal metallothionein, but also in testicular cadmium-binding protein. When the antigenic reactivity of these proteins was compared, all three proteins reacted with the metallothionein antibody. These similarities suggest that the low molecular weight testicular cadmium-binding protein is apparently metallothionein.     

Torus shaped bands at the 2R telomere region and at the region 68 of the salivary gland chromosomes ofDrosophila auraria

Summary The telomere of the 2R arm of the salivary gland chromosomes ofD. auraria exhibits a definite toroidal structure in routine squashed preparations, stained either by propionic orcein-carmine or by fluorescent dyes. There is evidence that a band (or bands) of region 68 (possibly homologous to that ofD. melanogaster) of the 3L chromosome arm also exhibits a toroidal structure. These toroids are associated with heterochromatin, but it is not certain that they are themselves heterochromatic.Acknowledgments. This work was supported by a grant from Volkswagenwerk-Stiftung to C.D.K. The outstanding technical assistance of Ms G. Karamanlidis is acknowledged.     

Novel and sensitive noncompetitive enzyme immunoassay for kassinin in rat plasma

A novel and sensitive noncompetitive enzyme immunoassay for kassinin is described. Kassinin was biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate. The biotinylated kassinin was trapped on anti-kassinin IgG-coated polystyrene balls and, after washing to eliminate other biotinylated substances, was eluted with HCl. The biotinylated kassinin eluted was reacted with anti-kassinin Fab'-peroxidase conjugate and trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. The detection limit of kassinin was 0.13 pg (0.1 fmol)/tube or 0.065 microgram/l of rat plasma, which was 750-fold or 15-fold lower than that for competitive radioimmunoassay.