Mer tyrosine kinase promotes the survival of t(1;19)-positive acute lymphoblastic leukemia (ALL) in the central nervous system (CNS).

Patients with t(1;19)-positive acute lymphoblastic leukemia (ALL) are prone to central nervous system (CNS) relapses, and expression of the TAM (Tyro3, Axl, and Mer) receptor Mer is upregulated in these leukemias. We examined the functional role of Mer in the CNS in preclinical models and performed correlative studies in 64 t(1;19)-positive and 93 control pediatric ALL patients. ALL cells were analyzed in coculture with human glioma cells and normal rat astrocytes: CNS coculture caused quiescence and protection from methotrexate toxicity in Mer(high) ALL cell lines, which was antagonized by short hairpin RNA-mediated knockdown of Mer. Mer expression was upregulated, prosurvival Akt and mitogen-activated protein kinase signaling were activated, and secretion of the Mer ligand Galectin-3 was stimulated. Mer(high) t(1;19) primary cells caused CNS involvement to a larger extent in murine xenografts than in their Mer(low) counterparts. Leukemic cells from Mer(high) xenografts showed enhanced survival in coculture. Treatment of Mer(high) patient cells with the Mer-specific inhibitor UNC-569 in vivo delayed leukemia onset, reduced CNS infiltration, and prolonged survival of mice. Finally, a correlation between high Mer expression and CNS positivity upon initial diagnosis was observed in t(1;19) patients. Our data provide evidence that Mer is associated with survival in the CNS in t(1;19)-positive ALL, suggesting a role as a diagnostic marker and therapeutic target.

The role of ZAP70 kinase in acute lymphoblastic leukemia infiltration into the central nervous system.

Central nervous system infiltration and relapse are poorly understood in childhood acute lymphoblastic leukemia. We examined the role of zeta-chain-associated protein kinase 70 in preclinical models of central nervous system leukemia and performed correlative studies in patients. Zeta-chain-associated protein kinase 70 expression in acute lymphoblastic leukemia cells was modulated using short hairpin ribonucleic acid-mediated knockdown or ectopic expression. We show that zeta-chain-associated protein kinase 70 regulates CCR7/CXCR4 via activation of extracellular signal-regulated kinases. High expression of zeta-chain-associated protein kinase 70 in acute lymphoblastic leukemia cells resulted in a higher proportion of central nervous system leukemia in xenografts as compared to zeta-chain-associated protein kinase 70 low expressing counterparts. High zeta-chain-associated protein kinase 70 also enhanced the migration potential towards CCL19/CXCL12 gradients in vitro CCR7 blockade almost abrogated homing of acute lymphoblastic leukemia cells to the central nervous system in xenografts. In 130 B-cell precursor acute lymphoblastic leukemia and 117 T-cell acute lymphoblastic leukemia patients, zeta-chain-associated protein kinase 70 and CCR7/CXCR4 expression levels were significantly correlated. Zeta-chain-associated protein kinase 70 expression correlated with central nervous system disease in B-cell precursor acute lymphoblastic leukemia, and CCR7/CXCR4 correlated with central nervous system involvement in T-cell acute lymphoblastic leukemia patients. In multivariate analysis, zeta-chain-associated protein kinase 70 expression levels in the upper third and fourth quartiles were associated with central nervous system involvement in B-cell precursor acute lymphoblastic leukemia (odds ratio=7.48, 95% confidence interval, 2.06-27.17; odds ratio=6.86, 95% confidence interval, 1.86-25.26, respectively). CCR7 expression in the upper fourth quartile correlated with central nervous system positivity in T-cell acute lymphoblastic leukemia (odds ratio=11.00, 95% confidence interval, 2.00-60.62). We propose zeta-chain-associated protein kinase 70, CCR7 and CXCR4 as markers of central nervous system infiltration in acute lymphoblastic leukemia warranting prospective investigation.

EGFR signaling in the brain is necessary for olfactory learning in Drosophila larvae.

Signaling via the epidermal growth factor receptor (EGFR) pathway has emerged as one of the key mechanisms in the development of the central nervous system in Drosophila melanogaster. By contrast, little is known about the functions of EGFR signaling in the differentiated larval brain. Here, promoter-reporter lines of EGFR and its most prominent activating ligands, Spitz, Keren, and Vein, were used to identify the brain structures relevant for the EGFR pathway. Unexpectedly, promoter activity of all these pathway components was found in the mushroom bodies, which are known to be a higher brain center required for olfactory learning. We investigated the role of the EGFR pathway in this process by using different mutant larvae with reduced pan-neuronal EGFR signaling and those with reduced EGFR signaling in mushroom bodies only. Expression of a dominant-negative form of EGFR as well as silencing of the ligands via RNA interference was applied and resulted in significantly impaired olfactory learning performances. General defects in the ability to taste or smell as well as impaired EGFR signaling during embryonic development could be excluded as major reasons for this learning phenotype. In addition, targeted expression of a constitutively active form of the ligand Spitz also led to a significantly reduced learning ability. Thus, very low levels as well as very high levels of EGFR signaling are deleterious for olfactory learning and memory formation. We hypothesize that EGFR signaling in a certain range maintains a homeostatic situation in the mushroom bodies that is necessary for proper learning and memory.

Influence of disulfide bonds in human beta defensin-3 on its strain specific activity against Gram-negative bacteria.

Antimicrobial peptides (AMPs) play an important role in the host defense against various microbes. One of the most efficient human AMPs is the human beta defensin-3 (hBD-3) which is produced by, e.g. keratinocytes and lung epithelial cells. However, the structure-function relationship for AMPs and in particular for defensins with their typical three disulfide bonds is still poorly understood. In this study the importance of the three disulfide bonds for the activity of the AMPs is investigated with biological assays and with biophysical experiments utilizing different membrane reconstitution systems. The activities of natural hBD-3, hBD-3-c (cyclic variant with one disulfide bond), and hBD-3-l (linear variant without disulfide bonds) and fragments thereof were tested against specific Gram-negative bacteria. Furthermore, hemolytic and cytotoxic activities were analyzed as well as the potency to neutralize immune cell stimulation of lipopolysaccharide (LPS). Experiments using reconstituted lipid matrices composed of phospholipids or LPS purified from the respective Gram-negative bacteria, showed that the membrane activity of all three hBD-3 peptides is decisive for their capability to kill bacteria and to neutralize LPS. In most of the test systems the linear hBD-3-l showed the highest activity. It was also the only peptide significantly active against polymyxin B-resistant Proteus mirabilis R45. However, the stability of hBD-3 against protease activity decreases with decreasing number of disulfide bonds. This study demonstrates that the refining of AMP structures can generate more active compounds against certain strains.

An exceptional salt-tolerant antimicrobial peptide derived from a novel gene family of haemocytes of the marine invertebrate Ciona intestinalis.

A novel gene family coding for putative antimicrobial peptides was identified in the EST (expressed sequence tag) database of the sea squirt Ciona intestinalis, and one of these genes was molecularly cloned from the Northern European Ciona subspecies. In situ hybridization and immunocytochemical analysis revealed that the natural peptide is synthesized and stored in a distinct haemocyte type, the univacuolar non-refractile granulocytes. By semiquantitative RT-PCR (reverse transcription-PCR) analysis, it was shown that the expression of the gene is markedly up-regulated in haemocytes after immune challenge. To evaluate the antimicrobial potency of the putative defence protein, we synthesized a peptide corresponding to its cationic core region. The peptide was highly effective against Gram-negative and Gram-positive bacteria including several human and marine pathogens as well as the yeast Candida albicans. Notably, the antibacterial activity of the peptide was retained at salt concentrations of up to 450 mM NaCl. Using two different methods we demonstrated that the peptide kills Gram-negative and Gram-positive bacteria by permeabilizing their cytoplasmic membranes. CD spectroscopy revealed that, in the presence of liposomes composed of negatively charged phospholipids, the peptide undergoes a conformational change and adopts an alpha-helical structure. Moreover, the peptide was virtually non-cytolytic for mammalian erythrocytes. Hence, the designed salt-tolerant antimicrobial peptide may represent a valuable template for the development of novel antibiotics.

A reverse search for antimicrobial peptides in Ciona intestinalis: identification of a gene family expressed in hemocytes and evaluation of activity.

In search of antimicrobial peptides in the tunicate Ciona intestinalis, we used the recently completed genome project and the substantial number of expressed sequence tag (EST) data available as a screening matrix. By this means, we identified a putative gene family that exhibits several structural features typical of antimicrobial peptides. We designed and synthesized a peptide corresponding to the core region of a member of this peptide family, which is predicted to adopt an amphipathic alpha-helical structure. The synthetic peptide exerted potent antimicrobial activity against a variety of bacteria and against the yeast Candida albicans but was not cytolytic for mammalian erythrocytes. Moreover, by employing a non-cell-permeable fluorescent dye it became evident that the peptide kills bacteria by permeabilizing their cytoplasmic membranes. Using the synthetic peptide as an antigen, we generated specific antibodies and localized the natural parent molecule to a compartment of a distinct hemocyte type, the univacuolar refractile granulocytes. As C. intestinalis apparently does not possess gene products that resemble well-known antimicrobial peptides of tunicates and of other animals, the aforementioned peptide family may represent a potent armamentarium of the hemocytes to combat microbial infection in sea squirts.

The antimicrobial peptide Ci-MAM-A24 is highly active against multidrug-resistant and anaerobic bacteria pathogenic for humans.

Ci-MAM-A24, a synthetic antimicrobial peptide derived from a peptide precursor from immune cells of the marine invertebrate Ciona intestinalis, has been shown to be potently active against representatives of Gram-positive and Gram-negative bacteria by permeabilising their cytoplasmic membrane. In the present study, the activity of Ci-MAM-A24 against different bacterial pathogens frequently causing therapeutic problems was tested. In particular, the killing capacity of Ci-MAM-A24 against clinically important anaerobic bacteria as well as multiresistant aerobic strains such as meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producers and multiple-resistant Pseudomonas aeruginosa strains was monitored. Virtually all strains proved to be highly susceptible to Ci-MAM-A24 at low concentrations [minimum bactericidal concentration (MBC)<10 microg/mL].

Significance of the cyclic structure and of arginine residues for the antibacterial activity of arenicin-1 and its interaction with phospholipid and lipopolysaccharide model membranes.

Arenicin-1 (Ar-1) is a beta-sheeted antimicrobial peptide from the marine lugworm Arenicola marina. To elucidate the significance of its unique 18-residue cyclic structure and of six cationic arginines for its biological activity and its interaction with biomembranes, we synthesized one linear peptide in which the two cysteines were exchanged for serines (C/S-Ar-1) and a cyclic peptide in which all arginines were replaced by lysines (R/K-Ar-1). We addressed antibacterial and hemolytic activities, the impact of the peptides on bacterial morphology, and their binding to, intercalation into, and permeabilization of model membranes composed of phospholipids or lipopolysaccharide (LPS). In accordance with high salt concentration in sea water, the antibacterial activity of Ar-1 was almost insensitive to high NaCl concentrations. In contrast, the linear derivative lost activity under these conditions against polymyxin B-resistant Proteus mirabilis. Ar-1 intercalated into phospholipid and LPS membranes and formed heterogeneous and short-lived lesions. However, when the peptide was present in both membrane leaflets, it formed defined pores. This characteristic was not observed for the linear derivative C/S-Ar-1. Apparently, the disulfide bond provides conforma-tional stability, which has an impact on salt tolerance, prevents fast degradation by trypsin, and is a prerequisite for the formation of structurally defined pores.

A Dickkopf- 3-related gene is expressed in differentiating nematocytes in the basal metazoan Hydra.

In vertebrate development the Dickkopf protein family carries out multiple functions and is represented by at least four different genes with distinct biological activities. In invertebrates such as Drosophila and Caenorhabditis, Dickkopf genes have so far not been identified. Here we describe the identification and characterization of a Dickkopf gene with a deduced amino acid sequence closely related to that of chicken Dkk-3 in the basal metazoan Hydra. HyDkk-3 appears to be the only Dickkopf gene in Hydra. The gene is expressed in the gastric region in nematocytes at a late differentiation stage. In silico searches of EST and genome databases indicated the absence of Dkk genes from the protostomes Drosophila and Caenorhabditis, whereas within the deuterostomes, a Dkk-3 gene could be identified in the genome of the urochordate Ciona intestinalis. The results indicate that at an early stage of evolution of multicellularity Dickkopf proteins have already played important roles as developmental signals. They also suggest that vertebrate Dkk-1, 2 and 4 may have originated from a common ancestor gene of Dkk-3.