Werner syndrome lymphoblastoid cells are sensitive to camptothecin-induced apoptosis in S-phase

Werner Syndrome (WRN) is an autosomal recessive disorder showing an endogenous mutator phenotype in combination with an elevated risk of predominantly mesenchymal cancer. The gene mutated in WRN patients codes for 3’→5’ DNA helicase and 3’→5’ exonuclease activities. We have found similar S-phase arrest in both WRN and control cells after treatment with the DNA-topoisomerase-I-trapping drug camptothecin; this may be responsible for the drug-exposure-related growth inhibition seen in both cell types. A clearer phenotypic difference between WRN and control immortalized B-cell lines (LCLs) is obtained by examining cell death. The mechanism of camptothecin-induced cell death in WRN-deficient LCLs appears to be through apoptosis, a phenotype that strongly differentiates WRN-deficient from wild-type LCLs. We hypothesize that, in cells deficient for WRN function, a topoisomerase-I-DNA intermediate persists. Conflict with DNA replication may lead to apoptosis, increased mutation rates, and cancer in WRN. Received: 23 September 1998 / Accepted: 26 October 1998     

Scl binds to primed enhancers in mesoderm to regulate hematopoietic and cardiac fate divergence

Scl/Tal1 confers hemogenic competence and prevents ectopic cardiomyogenesis in embryonic endothelium by unknown mechanisms. We discovered that Scl binds to hematopoietic and cardiac enhancers that become epigenetically primed in multipotent cardiovascular mesoderm, to regulate the divergence of hematopoietic and cardiac lineages. Scl does not act as a pioneer factor but rather exploits a pre‐established epigenetic landscape. As the blood lineage emerges, Scl binding and active epigenetic modifications are sustained in hematopoietic enhancers, whereas cardiac enhancers are decommissioned by removal of active epigenetic marks. Our data suggest that, rather than recruiting corepressors to enhancers, Scl prevents ectopic cardiogenesis by occupying enhancers that cardiac factors, such as Gata4 and Hand1, use for gene activation. Although hematopoietic Gata factors bind with Scl to both activated and repressed genes, they are dispensable for cardiac repression, but necessary for activating genes that enable hematopoietic stem/progenitor cell development. These results suggest that a unique subset of enhancers in lineage‐specific genes that are accessible for regulators of opposing fates during the time of the fate decision provide a platform where the divergence of mutually exclusive fates is orchestrated.     

Functional role of the Werner syndrome RecQ helicase in human fibroblasts

Werner syndrome is an autosomal recessive human genetic instability and cancer predisposition syndrome that also has features of premature aging. We focused on two questions related to Werner syndrome protein (WRN) function in human fibroblasts: Do WRN‐deficient fibroblasts have a consistent cellular phenotype? What role does WRN play in the recovery from replication arrest? We identified consistent cell proliferation and DNA damage sensitivity defects in both primary and SV40‐transformed fibroblasts from different Werner syndrome patients, and showed that these defects could be revealed by acute depletion of WRN protein. Mechanistic analysis of the role of WRN in recovery from replication arrest indicated that WRN acts to repair damage resulting from replication arrest, rather than to prevent the disruption or breakage of stalled replication forks. These results identify readily quantified cell phenotypes that result from WRN loss in human fibroblasts; delineate the impact of cell transformation on the expression of these phenotypes; and define a mechanistic role for WRN in the recovery from replication arrest.     

Tissue- and development-specific distributions of cytoskeletal elements in growing cells of the maize root apex

ABSTRACT

Indirect immunofluorescence performed using sections of actively growing maize root apices fixed and then embedded in low-melting-point Steedman's wax has proved efficient in revealing the arrangements and reorganizations of motility-related cytoskeletal elements which are associated with root cell development and tissue differentiation. This powerful, yet relatively simple, technique shows that specific rearrangements of both microtubular (MT) and actin microfilament (MF) arrays occur in cells as they leave the meristem and traverse the transitional region interpolated between meristem and elongation region. Cytoskeletal and growth analyses have identified the transition zone as critical for both cell and root development; it is in this zone that cell growth is channelled, by the cytoskeleton, into a strictly polarized mode which enables root tips to extend rapidly through the soil in search of water and nutrients. An integrated cytoskeletal network is crucial for both the cytomorphogenesis of individual cells and the overall morphogenesis of the plant body. The latter process can be viewed as a reflection of the tight control which cytoskeletal networks exert not only over cell division planes in the cells within meristematic apices but also over the orientation of cell growth in the meristem and elsewhere. Endoplasmic MTs interconnecting the plasma membrane with the nucleus are suggested to be involved in cell division control; they may also act as a two-way cytoskeletal communication channel for signals passing to and fro between the extracellular environment and the genome. Moreover, the dynamism of endoplasmic MTs exerts direct effects on chromatin structure and the accompanying nuclear architecture and hence can help exert a cellular level of control over cell growth and cell cycle progression. Because the inherent dynamic instability of MTs depends on the concentration of tubulin dimers within the cytoplasm, we propose that when asymmetric cell division occurs, it will result in two daughter cells which differ in the turnover rates of their MTs. This phenomenon could be responsible for different cell fates of daughter plant cells produced by such cell divisions.     

A novel harmony search-K means hybrid algorithm for clustering gene expression data

Recent progress in bioinformatics research has led to the accumulation of huge quantities of biological data at various data sources. The DNA microarray technology makes it possible to simultaneously analyze large number of genes across different samples. Clustering of microarray data can reveal the hidden gene expression patterns from large quantities of expression data that in turn offers tremendous possibilities in functional genomics, comparative genomics, disease diagnosis and drug development. The k- ¬means clustering algorithm is widely used for many practical applications. But the original k-¬means algorithm has several drawbacks. It is computationally expensive and generates locally optimal solutions based on the random choice of the initial centroids. Several methods have been proposed in the literature for improving the performance of the k-¬means algorithm. A meta-heuristic optimization algorithm named harmony search helps find out near-global optimal solutions by searching the entire solution space. Low clustering accuracy of the existing algorithms limits their use in many crucial applications of life sciences. In this paper we propose a novel Harmony Search-K means Hybrid (HSKH) algorithm for clustering the gene expression data. Experimental results show that the proposed algorithm produces clusters with better accuracy in comparison with the existing algorithms.     

The biogeochemistry of basic cations in two forest catchments with contrasting lithology in the Czech Republic

The biogeochemistry of Ca, Mg, K, and Nawere investigated in two forested catchments in theCzech Republic, one underlain by leucogranite, theother by serpentinite. High weathering rates at theserpentinite site at Pluhv Bor resultedin Mg²⁺ as the dominant cation on the soilexchange complex and in drainage water. Other basiccations (Ca²⁺, K⁺, Na⁺) showedrelatively low concentrations and outflow instreamwater. The catchment exhibited high basesaturation in mineral soils (>70%), and nearneutral soil and stream pH, despite elevated inputsof acidic deposition. Slow growth of Norway spruceat Pluhv Bor may be caused by K deficiency, Mgoversupply and/or Ni toxicity. In contrast, thegranitic site at Lysina showed low concentrations ofbasic cations on the soil exchange complex and instreamwater. Soil and drainage water at Lysina werehighly impacted by acidic deposition. Soil pH wasextremely acidic (<4.5) throughout the soilprofile, and the base saturation of the mineral soilwas very low (<5%). Supplies of basic cationsfrom atmospheric deposition and soil processes wereless than inputs of SO^2- ₄ on anequivalence basis, resulting in low pH and highconcentrations of total Al in drainage water. Needle yellowing in Norway spruce was possibly theresult of Mg deficiency at Lysina. Because of theirextremely different lithologies, these catchmentsserve as valuable end-members of ecosystemsensitivity to elevated levels of acidicdeposition.     

The lactosylceramide binding specificity of Helicobacter pylori

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.      

Delineation of the epitope recognized by an antibody specific for N- glycolylneuraminic acid-containing gangliosides

P3 is a mouse monoclonal antibody (mAb) that binds to several NeuGc- containing gangliosides. It also reacts with antigens expressed in human breast tumors (Vazquez et al. (1995) Hybridoma , 14, 551-556). In this work, the binding specificity of P3 has been characterized in more detail using a panel of glycolipids that included several disialylated gangliosides and several chemical derivatives of NeuGc-GM3. The carboxyl group and the nitrogen function of sialic acid were found to play important roles in the antibody binding, whereas the glycerol tail appears to be nonrelevant. Molecular modeling was used to analyze the binding data, including the finding that P3 selectively recognizes the internal NeuGc in GD3. For this purpose, conformational studies of GD3 were performed using molecular dynamics. It was concluded that sialic acid binds the P3 antibody through its upper face (the one on which the carboxyl group is exposed) and the C4-C5 side of the sugar ring, whereas none or very little contact between the galactose residue and the protein is evident. Conformational analysis of GD3 revealed that, despite the large flexibility of the NeuGcalpha8NeuGc linkage, the P3 binding epitope on the external sialic acid is not well exposed for any of the possible conformations this linkage can adopt, whereas the internal sialic acid presents the epitope in a proper way for several of these conformations. As a final result, a coherent picture of the epitope that fits the wide binding data was obtained.