Dasatinib Inhibits CXCR4 Signaling in Chronic Lymphocytic Leukaemia Cells and Impairs Migration Towards CXCL12

Chemokines and their ligands play a critical role in enabling chronic lymphocytic leukaemia (CLL) cells access to protective microenvironmental niches within tissues, ultimately resulting in chemoresistance and relapse: disruption of these signaling pathways has become a novel therapeutic approach in CLL. The tyrosine kinase inhibitor dasatinib inhibits migration of several cell lines from solid-organ tumours, but effects on CLL cells have not been reported. We studied the effect of clinically achievable concentrations of dasatinib on signaling induced by the chemokine CXCL12 through its'' receptor CXCR4, which is highly expressed on CLL cells. Dasatinib pre-treatment inhibited Akt and ERK phosphorylation in CLL cells upon stimulation with CXCL12. Dasatinib also significantly diminished the rapid increase in actin polymerisation observed in CLL cells following CXCL12 stimulation. Moreover, the drug significantly inhibited chemotaxis in a transwell assay, and reduced the percentage of cells able to migrate beneath a CXCL12-expressing murine stromal cell line. Dasatinib also abrogated the anti-apoptotic effect of prolonged CXCL12 stimulation on cultured CLL cells. These data suggest that dasatinib, akin to other small molecule kinase inhibitors targeting the B-cell receptor signaling pathway, may redistribute CLL cells from protective tissue niches to the peripheral blood, and support the investigation of dasatinib in combination strategies.     

Intermittent illumination increases biophotolytic hydrogen yield by Anabaena cylindrica.

Intermittent illumination increased H2 and C2H4 yields per unit of light from growing cells and from nitrogren-starved cells by 1.7- and 1.35-fold, respectively, as compared with continuous illumination.     

Mammalian carbamyl phosphate synthetase (CPS). DNA sequence and evolution of the CPS domain of the Syrian hamster multifunctional protein CAD

Glutamine-dependent carbamoyl-phosphate synthetase (EC 6.3.5.5) catalyzes the first step in de novo pyrimidine biosynthesis. The mammalian enzyme is part of a 240-kDa multifunctional protein which also has the second (aspartate carbamoyltransferase, EC 2.1.3.2), and third (dihydroorotase, EC 3.5.2.3) activities of the pathway. Shigesada et al. (Shigesada, K., Stark, G.R., Maley, J.A., and Davidson, J.N. (1985) Mol. Cell Biol. 175, 1-7) produced a truncated cDNA clone from a Syrian hamster cell line that contained most of the coding region for this protein. We have completed sequencing this clone, known as pCAD142. The cDNA insert contained all of the coding region for the glutaminase (GLN) and carbamyl phosphate synthetase (CPS) domains but lacked a short amino-terminal segment. By comparing the primary structure of the mammalian chimera to monofunctional proteins we have identified the borders of the functional domains. The GLN domain is 21 kDa, close to the size of the functionally similar polypeptide products of the Escherichia coli pabA and hisH genes. The domain has the three regions of homology common to trpG-type glutamine amidotransferases, as well as a fourth region specific to the carbamyl phosphate synthetases. The CPSase domain is similar to other reported CPSases in size (120 kDa), primary structure (37-67% amino acid identity), and homology between its amino and carboxyl halves. Analysis of the nucleotide and amino acid sequence identities among the various carbamyl phosphate synthetases suggests that the gene fusion which joined the GLN and CPS domains was an early event in the evolution of eukaryotic organisms and that the Saccharomyces cerevisiae enzyme consisting of separate subunits arose by defusion from an ancestral multifunctional protein.     

Identification and partial characterization of a latent ATP,Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol

Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase F_A-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M _r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF Phenylmethanesulphonyl Fluoride - TLCK L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride - TPCK L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone     

Activation of pro-urokinase by plasmin: non-Michaelian kinetics indicates a mechanism of negative cooperativity

Enzyme kinetic plots relating the initial rate of activation of pro-urokinase to urokinase by plasmin, according to the concentration of substrate, were smooth downward curves and indicated that an apparent decrease in binding affinity occurred with increase in the concentration of pro-urokinase. Such nonlinear plots were obtained with plasmin 1 and also plasmin 2. Over sections of each curve it was possible to estimate apparent kinetic constants. At the uppermost concentrations of substrate tested, these were Km 2.9 microM and kcat 35.5 min-1 for plasmin 1, and at the lowermost concentrations, Km 9.5 nM and kcat 2.0 min-1. Linear plots were obtained when the single proteolytic cleavage was made by K5-plasmin or undegraded plasmin in the presence of 1.0 mM 6-aminohexanoic acid (6-AHa). Constants were estimated for catalysis of this reaction by K5 plasmin to be Km 6.0 microM and kcat 38 min-1 (r = 0.987). The catalytic efficiency of plasmin, at the lowermost concentrations of pro-urokinase tested, was therefore 33-fold higher than that of K5-plasmin. Plotting of data for the cleavage of pro-urokinase by plasmin 1 (in the absence of 6-AHa) according to the model of Hill, gave a slope of 0.5 at the lowermost concentrations of pro-urokinase increasing to 1.0 at higher concentrations (greater than 0.3 microM); such a profile is characteristic of negative cooperativity. The rates of formation of plasmin and urokinase in a mixture containing a low concentration of plasminogen and pro-urokinase were measured and compared to those predicted by a computer program designed to calculate theoretical rates using available kinetic data. The observed rates of generation of both plasmin and urokinase coincided to those predicted from the negative cooperativity model. The mechanism of the negative cooperativity may reside in a conformational change induced by binding of pro-urokinase to the kringle structure of plasmin. This property may be of significance in controlling the fibrinolytic properties of the urokinase-type plasminogen activator system.     

Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.     

lacZ gene fusions and insertion mutagenesis in the TL-region of Agrobacterium rhizogenes Ri plasmid

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.     

Tripartite sequences within and 3'' to the sea urchin H2A histone gene display properties associated with a transcriptional termination process.

We have defined a DNA sequence that behaves as an RNA polymerase II termination signal by using the human HeLa cell transient expression system. Surprisingly, this sequence is tripartite, including part of the coding region of the sea urchin H2A histone gene together with two separate sequences in the 3' flanking region of the gene. We demonstrate that this signal functions both in its normal gene environment and also when placed within the human alpha-globin gene. However, we have failed to detect a discrete 3' terminus. Rather, our data indicate the presence of an extremely heterogeneous series of nonpolyadenylated RNAs. These heterogeneous nonpolyadenylated RNAs are stable when transcribed from the intact histone gene but are highly unstable within the human alpha-globin gene. This provides evidence for the role of poly(A) in the stability of mRNA.