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.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

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.     

Physical mapping of the von Recklinghausen neurofibromatosis region on chromosome 17

The von Recklinghausen neurofibromatosis (NF1) locus has been linked to chromosome 17, and recent linkage analyses place the gene on the proximal long arm. NF1 probably resides in 17q11.2, since two unrelated NF1 patients have been identified who possess constitutional reciprocal translocations involving 17q11.2 with chromosomes 1 and 22. We have used a somatic-cell hybrid from the t(17;22) individual, along with other hybrid cell lines, to order probes around the NF1 locus. An additional probe, 17L1, has been isolated from a NotI linking library made from flow-sorted chromosome 17 material and has been mapped to a region immediately proximal to the translocation breakpoint. While neither NF1 translocation breakpoint has yet been identified by pulse-field gel analysis, an overlap between two probes, EW206 and EW207, has been detected. Furthermore, we have identified the breakpoint in a non-NF1 translocation, SP-3, on the proximal side of the NF1 locus. This breakpoint has been helpful in creating a 1,000-kb pulsed-field map, which includes the closely linked NF1 probes HHH202 and TH17.19. The combined somatic-cell hybrid and pulsed-field gel analysis we report here favors the probe order D17Z1-HHH202-TH17.19-CRYB1-17L1-NF1- (EW206, EW207, EW203, L581, L946)-(ERBB2, ERBA1). The agreement in probe ordering between linkage analysis and physical mapping is excellent, and the availability of translocation breakpoints in NF1 should now greatly assist the cloning of this locus.