Disparate effects of tumor necrosis factor-alpha/cachectin and tumor necrosis factor-beta/lymphotoxin on hematopoietic growth factor production and neutrophil adhesion molecule expression by cultured human endothelial cells

Tumor necrosis factor-alpha/cachectin (TNF-alpha) and tumor necrosis factor-beta/lymphotoxin (TNF-beta) are inflammatory mediators with similar spectrums of cytotoxic activity against tumors in vitro and in vivo. We compared the effect of purified recombinant human TNF-alpha and TNF-beta on neutrophil adhesion molecule expression and hematopoietic growth factor production by cultured human umbilical vein endothelial cells. Endothelial cells acquired adhesive properties for neutrophils after a 4-hr incubation with as little as 5 U/ml TNF-alpha. TNF-alpha stimulated a dose-dependent increase in endothelial cell adhesiveness for neutrophils, with a maximal effect at 250 U/ml. In contrast, TNF-beta did not enhance endothelial-dependent neutrophil adherence until a concentration of 600 to 1200 U/ml was reached. Endothelial cells cultured for 24 hr with TNF-alpha, 10 to 1,000 U/ml, released hematopoietic colony-stimulating activity. TNF-beta failed to augment growth factor production by endothelial cells at any concentration tested. Inhibitor assays showed that the absence of detectable colony-stimulating activity was not due to direct inhibition of colony growth by TNF-beta or to release of hematopoietic inhibitors by the TNF-beta-stimulated endothelial cells. Purified natural TNF-beta was similar to recombinant TNF-beta in its effect on neutrophil adhesion molecule expression and growth factor production by endothelial cells. These results indicate that the two immunomodulatory proteins TNF-alpha and TNF-beta differ in their effects on a common target tissue. TNF-beta, which retains tumoricidal properties, shows fewer proinflammatory activities on cultured endothelial cells than TNF-alpha in vitro.     

AMG531 stimulates megakaryopoiesis in vitro by binding to Mpl

Thrombopoietin (TPO) plays a pivotal role in megakaryopoiesis. TPO initiates its biological effects by binding to its receptor Mpl. A recombinant protein consisting of a carrier Fc domain linked to multiple Mpl-binding domains was constructed, and is called AMG531. To define the biological activity of AMG531, we examined the ability of AMG531 to support CFU-Meg growth and to promote megakaryocyte maturation in vitro. AMG531 stimulates CFU-Meg growth in a dose-dependent manner, and acts in concert with erythropoietin, stem cell factor, interleukin-3, and interleukin-6 to enhance CFU-Meg growth, similar to parallel experiments with TPO. AMG531-stimulated serum-free liquid cultures support the development of mature polyploid megakaryocytes with a predominant DNA content of 32 N and 64 N, identical to that of parallel TPO-stimulated cultures. Competitive binding experiments show that AMG531 effectively competes with 125I-TPO for binding to BaF3-Mpl cells or normal platelets. Treatment of BaF3-Mpl cells with AMG531 or with TPO resulted in rapid tyrosine phosphorylation of Mpl, JAK2, and STAT5. These results indicate that AMG531 is a potent stimulant of megakarypoiesis in vitro, and provide support for its further characterization in vivo.     

Discovery of 5-(arenethynyl) hetero-monocyclic derivatives as potent inhibitors of BCR-ABL including the T315I gatekeeper mutant

Ponatinib (AP24534) was previously identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML. In this study, we explored a novel series of five and six membered monocycles as alternate hinge-binding templates to replace the 6,5-fused imidazopyridazine core of ponatinib. Like ponatinib, these monocycles are tethered to pendant toluanilides via an ethynyl linker. Several compounds in this series displayed excellent in vitro potency against both native BCR-ABL and the T315I mutant. Notably, a subset of inhibitors exhibited desirable PK and were orally active in a mouse model of T315I-driven CML.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

Tyrosine 462 of the membrane-proximal F'-G' loop of murine Mpl is not essential for high-affinity binding of thrombopoietin

The ligand binding site of Mpl, the thrombopoietin (Tpo) receptor, has not been determined. Tyr(462)of murine Mpl corresponds to Tyr(421)of the common beta chain of the human IL-3, IL-5 and GM-CSF receptors. Tyr(421)has been identified as essential for high-affinity ligand binding. To determine whether Tyr(462)is similarly required for Tpo binding, wild-type murine Mpl (Mpl-WT) or mutant receptors containing an alanine (Y462A) or lysine (Y462K) in place of Tyr(462)were expressed in BaF3 cells. In proliferation studies, the Y462A mutation had no effect on Tpo-induced growth. In contrast, the Y462K mutation led to an attenuated proliferative response to Tpo. In single-point binding studies, both Mpl-WT and Y462A cells were able to bind [(125)I]Tpo in a specific manner. In contrast, there was a marked reduction in binding of [(125)I]Tpo by Y462K cells. Mpl-WT cells bound Tpo with a K(d)of approximately 330 pM, while Y462A cells bound Tpo with a K(d)of approximately 268 pM. The binding affinity of Y462K cells was below that quantifiable by Scatchard analysis. This study suggests that unlike the corresponding Tyr(421)of the common human beta chain, Tyr(462)of murine Mpl is not required for high-affinity ligand binding, although it may be located in proximity to the ligand binding site.     

Identification of genes required for de novo DNA methylation in Arabidopsis

De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.Key words: DNA methylation, Arabidopsis, de novo, genetic screen, whole-genome sequencing     

Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor.

We have cloned a partial cDNA encoding murine stem cell factor (SCF) and show that the gene is syntenic with the Sl locus on mouse chromosome 10. Using retroviral vectors to immortalize fetal liver stromal cell lines from mice harboring lethal mutations at the Sl locus (Sl/Sl), we have shown that SCF genomic sequences are deleted in these lines. Furthermore, two other mutations at Sl, Sld and Sl12H, are associated with deletions or alterations of SCF genomic sequences. In vivo administration of SCF can reverse the macrocytic anemia and locally repair the mast cell deficiency of Sl/Sld mice. We have also provided biological and physical evidence that SCF is a ligand for the c-kit receptor.