Deletion of the Src homology 3 domain and C-terminal proline-rich sequences in Bcr-Abl prevents Abl interactor 2 degradation and spontaneous cell migration and impairs leukemogenesis

The hematopoietic cells from patients with Bcr-Abl-positive chronic myelogenous leukemia exhibit multiple abnormalities of cytoskeletal function. The molecular events leading to these abnormalities are not fully understood. Previously we showed that Bcr-Abl elicits ubiquitin-dependent degradation of Abl interactor proteins. Because recent studies have suggested a role of Abl interactor proteins in the pathway that regulates cytoskeletal function, we investigated whether mutations in Bcr-Abl that interfere with the signaling to Abl interactor proteins affect its leukemogenic activity. We report here that the Src homology 3 domain and C-terminal proline-rich sequences of Bcr-Abl are required for its binding to Abl interactor 2 as well as for the induction of Abl interactor 2 degradation. Although the deletion of these regions did not affect the ability of the mutant Bcr-Abl to transform hematopoietic cells to growth factor independence, it abrogated its ability to stimulate spontaneous cell migration on fibronectin-coated surfaces. Furthermore, the mutant Bcr-Abl, defective in binding to Abl interactor 2 and inducing its degradation, failed to induce chronic myelogenous leukemia-like disease in mouse. These results are consistent with a role of Abl interactor proteins in the regulation of cytoskeletal function as well as in the pathogenesis of Bcr-Abl-induced leukemogenesis.     

Insights into alpha-K toxin specificity for K+ channels revealed through mutations in noxiustoxin

Noxiustoxin (NxTX) displays an extraordinary ability to discriminate between large conductance, calcium-activated potassium (maxi-K) channels and voltage-gated potassium (Kv1.3) channels. To identify features that contribute to this specificity, we constructed several NxTX mutants and examined their effects on whole cell current through Kv1.3 channels and on current through single maxi-K channels. Recombinant NxTX and the site-specific mutants (P10S, S14W, A25R, A25Delta) all inhibited Kv1.3 channels with Kd values of 6, 30, 0.6, 112, and 166 nM, respectively. In contrast, these same NxTX mutants had no effect on maxi-K channel activity with estimated Kd values exceeding 1 mM. To examine the role of the alpha-carbon backbone in binding specificity, we constructed four NxTX chimeras, which altered the backbone length and the alpha/beta turn. For each of these chimeras, six amino acids comprising the alpha/beta turn in iberiotoxin (IbTX) replaced the corresponding seven amino acids in NxTX (NxTX-YGSSAGA21-27-FGVDRG21-26). The chimeras differed in length of N- and C-terminal residues and in critical contact residues. In contrast to NxTX and its site-directed mutants, all of these chimeras inhibited single maxi-K channels. Under low ionic strength conditions, Kd values ranged from 0.4 to 6 microM, association rate constant values from 3 x 10(7) to 3 x 10(8) M(-1) x s(-1), and time constants for block from 5 to 20 ms. The rapid blocked times suggest that key microscopic interactions at the toxin-maxi-K channel interface may be absent. Under physiologic external ionic strength conditions, these chimera inhibited Kv1.3 channels with Kd values from 30 to 10 000 nM. These results suggest that the extraordinary specificity of NxTX for Kv1.3 over maxi-K channels is controlled, in part, by the toxin alpha-carbon backbone. These differences in the alpha-carbon backbone are likely to reflect fundamental structural differences in the external vestibules of these two channels.     

Mechanical properties of alveolar epithelial cells in culture.

With the use of magnetic twisting cytometry, we characterized the mechanical properties of rat type II alveolar epithelial (ATII) cells in primary culture and examined whether the cells' state of differentiation and the application of deforming stresses influence their resistance to shape change. Cells were harvested from rat lungs as previously described (Dobbs LG. Am J Physiol Lung Cell Mol Physiol 258: L134-L147, 1990) and plated at a density of 1 x 10(6) cells/cm(2) in fibronectin-coated 96 Remova wells, and their mechanical properties were measured 2-9 days later. We show 1) that ATII cells form much stronger bonds with RGD-coated beads than they do with albumin- or acetylated low-density lipoprotein-coated beads, 2) that RGD-mediated bonds seemingly "mature" during the first 60 min of bead contact, 3) that the apparent stiffness of ATII cells increases with days in culture, 4) that stiffness falls when the RGD-coated beads are intermittently oscillated at 0.3 Hz, and 5) that this fall cannot be attributed to exocytosis-related remodeling of the subcortical cytoskeleton. Although the mechanisms of force transfer between basement membrane, cytoskeleton, and plasma membrane of ATII cells remain to be resolved, such analyses undoubtedly require definition of the cell's mechanical properties. To our knowledge, the results presented here provide the first data on this topic.     

Using convex hulls to extract interaction interfaces from known structures

MOTIVATION: Protein interactions provide an important context for the understanding of function. Experimental approaches have been complemented with computational ones, such as PSIMAP, which computes domain-domain interactions for all multi-domain and multi-chain proteins in the Protein Data Bank (PDB). PSIMAP has been used to determine that superfamilies occurring in many species have many interaction partners, to show examples of convergent evolution through shared interaction partners and to uncover complexes in the interaction map. To determine an interaction, the original PSIMAP algorithm checks all residue pairs of any domain pair defined by classification systems such as SCOP. The computation takes several days for the PDB. The computation of PSIMAP has two shortcomings: first, the original PSIMAP algorithm considers only interactions of residue pairs rather than atom pairs losing information for detailed analysis of contact patterns. At the atomic level the original algorithm would take months. Second, with the superlinear growth of PDB, PSIMAP is not sustainable. RESULTS: We address these two shortcomings by developing a family of new algorithms for the computation of domain-domain interactions based on the idea of bounding shapes, which are used to prune the search space. The best of the algorithms improves on the old PSIMAP algorithm by a factor of 60 on the PDB. Additionally, the algorithms allow a distributed computation, which we carry out on a farm of 80 Linux PCs. Overall, the new algorithms reduce the computation at atomic level from months to 20 min. The combination of pruning and distribution makes the new algorithm scalable and sustainable even with the superlinear growth in PDB.     

The impact of harbour sludge disposal on benthic macrofauna communities in the Weser estuary

During an open-water disposal of about 710,000 m³ of harbour sludge in the polyhaline zone of the Weser estuary, Germany, a monitoring programme was carried out to investigate the impact on benthic invertebrates. The macrofaunal communities of four sites within the disposal area and five sites in a reference area were compared after discharge. The location and extent of the potentially affected area were inferred from a morphodynamic computer model (TIMOR 3; Zanke 1998). Disposal effects were analysed by comparing species numbers, densities, diversity and faunal similarity using multivariate methods. A loss of diversity and a decline in the abundance of several species in the disposal area were measured. The species number was reduced up to 50% and important habitat structures were absent from the disposal area. Several benthic species were affected by the disposal. The importance of species such as Mytilus edulis (Mollusca) and Lanice conchilega (Polychaeta) for the diversity of the community, and the sensitivity of these species to sediment discharge are analysed. The difficulties of separating dumping effects from natural variation in a dynamic estuarine channel system are discussed.Communicated by H.-D. Franke     

Phylogenetic relationship of Ophiostoma piliferum to other sapstain fungi based on the nuclear rRNA gene

The nuclear rRNA gene of Ophiostoma piliferum was analyzed to understand its phylogenetic relationships to other sapstain fungi. Phylograms based on nucleotide sequences of the rRNA gene showed that the relationships between O. piliferum and other Ophiostoma species varied depending on the regions of the rRNA gene analyzed. Intraspecies variation in O. piliferum was found in the internal transcribed spacer regions, and the variation was related to the geographic origin of O. piliferum strains. A useful molecular marker for differentiating O. piliferum from other sapstain Ophiostoma species was generated by the HaeIII restriction fragment length polymorphism of the 26S rRNA gene.     

Deformation-induced lipid trafficking in alveolar epithelial cells

Mechanical ventilation with a high tidal volume results in lung injury that is characterized by blebbing and breaks both between and through alveolar epithelial cells. We developed an in vitro model to simulate ventilator-induced deformation of the alveolar basement membrane and to investigate, in a direct manner, epithelial cell responses to deforming forces. Taking advantage of the novel fluorescent properties of BODIPY lipids and the fluorescent dye FM1-43, we have shown that mechanical deformation of alveolar epithelial cells results in lipid transport to the plasma membrane. Deformation-induced lipid trafficking (DILT) was a vesicular process, rapid in onset, and was associated with a large increase in cell surface area. DILT could be demonstrated in all cells; however, only a small percentage of cells developed plasma membrane breaks that were reversible and nonlethal. Therefore, DILT was not only involved in site-directed wound repair but might also have served as a cytoprotective mechanism against plasma membrane stress failure. This study suggests that DILT is a regulatory mechanism for membrane trafficking in alveolar epithelia and provides a novel biological framework within which to consider alveolar deformation injury and repair.     

Identification of a novel cytokine, ML-1, and its expression in subjects with asthma

A novel gene, designated ML-1, was identified from a human genomic DNA clone and human T cell cDNA sequences. The second exon of ML-1 gene shares significant sequence identity with the gene encoding IL-17 (IL-17). ML-1 gene expression was up-regulated in activated PBMCs, CD4(+) T cells, allergen-specific Th0, Th1, and Th2 clones, activated basophils, and mast cells. Increased expression of the ML-1 gene, but not IL-17, was seen following allergen challenge in four asthmatic subjects, suggesting its role in allergic inflammatory responses. ML-1 from transiently transfected COS-7 cells was able to induce gene expression and protein production for IL-6 and IL-8 (at 10 ng/ml of ML-1: for IL-6, 599.6 +/- 19.1 pg/ml; for IL-8, 1724.2 +/- 132.9 pg/ml; and at 100 ng/ml of ML-1: for IL-6, 1005.3 +/- 55.6 pg/ml; for IL-8, 4371.4 +/- 280.5 pg/ml; p < 0.05 for both doses vs baseline) in primary bronchial epithelial (PBE) cells. Furthermore, increased expression of ICAM-1 was found in ML-1-stimulated PBE cells (mean fluorescence intensity (MFI) = 31.42 +/- 4.39 vs baseline, MFI = 12.26 +/- 1.77, p < 0.05), a functional feature distinct from IL-17 (MFI = 11.07 +/- 1.22). This effect was not inhibited by a saturating amount of IL-17. These findings demonstrate that ML-1 is a novel cytokine with a distinct function, and suggest a different receptor for ML-1 on PBE cells.     

Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung. We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the DeltaF508 Cftr allele or an allele with a Cftr stop codon (S489X). Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 DeltaF508 Cftr mice, whereas DeltaF508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. CF mice showed significantly less ingestion of LPS-smooth P. aeruginosa by lung cells and significantly greater bacterial lung burdens 4.5 h postinfection than C57BL/6 wild-type mice. Microscopy of infected mouse and rhesus monkey tracheas clearly demonstrated ingestion of P. aeruginosa by epithelial cells in wild-type animals, mostly around injured areas of the epithelium. Desquamating cells loaded with P. aeruginosa could also be seen in these tissues. No difference was found between CF and wild-type mice challenged with an LPS-rough mucoid isolate of P. aeruginosa lacking the CFTR ligand. Thus, transgenic CF mice exhibit decreased clearance of P. aeruginosa and increased bacterial burdens in the lung, substantiating a key role for CFTR-mediated bacterial ingestion in lung clearance of P. aeruginosa.