Actin-binding proteins in the Arabidopsis genome database: properties of functionally distinct plant actin-depolymerizing factors/cofilins

The plant actin cytoskeleton is a highly dynamic, fibrous structure essential in many cellular processes including cell division and cytoplasmic streaming. This structure is stimulus responsive, being affected by internal stimuli, by biotic and abiotic stresses mediated in signal transduction pathways by actin-binding proteins. The completion of the Arabidopsis genome sequence has allowed a comparative identification of many actin-binding proteins. However, not all are conserved in plants, which possibly reflects the differences in the processes involved in morphogenesis between plant and other cells. Here we have searched for the Arabidopsis equivalents of 67 animal/fungal actin-binding proteins and show that 36 are not conserved in plants. One protein that is conserved across phylogeny is actin-depolymerizing factor or cofilin and we describe our work on the activity of vegetative tissue and pollen-specific isoforms of this protein in plant cells, concluding that they are functionally distinct.

Insulin-like growth factor binding protein-3 mediates vascular repair by enhancing nitric oxide generation

Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury.

The Arabidopsis RNA-binding protein FCA requires a lysine-specific demethylase 1 homolog to downregulate FLC.

A repressor of the transition to flowering in Arabidopsis is the MADS box protein FLOWERING LOCUS C (FLC). FCA, an RNA-binding protein, and FY, a homolog of the yeast RNA 3' processing factor Pfs2p, downregulate FLC expression and therefore promote flowering. FCA/FY physically interact and alter polyadenylation/3' processing to negatively autoregulate FCA. Here, we show that FCA requires FLOWERING LOCUS D (FLD), a homolog of the human lysine-specific demethylase 1 (LSD1) for FLC downregulation. FCA also partially depends on DICER-LIKE 3, involved in chromatin silencing. fca mutations increased levels of unspliced sense FLC transcript, altered processing of antisense FLC transcripts, and increased H3K4 dimethylation in the central region of FLC. These data support a close association of FCA and FLD in mediating H3K4 demethylation and thus transcriptional silencing of FLC and reveal roles for antisense RNA processing and DCL3 function in this regulation.

Calcium binding proteins in the liver fluke, Fasciola hepatica

The common liver fluke, Fasciola hepatica, is a parasite of mammals. In the western world its effects are largely felt on agriculture where infection of cows, sheep and other farm animals is estimated to cause millions of dollars ofif financial losses. In the developing world, the problem is even more serious with an estimated 7 million infected people and many millions more at risk of infection. Calcium signalling is of key importance in all eukaryotic species and recent discoveries of novel types of calcium binding proteins in liver flukes (and related trematodes) suggest that there may be calcium signalling processes which are unique to this group of organisms. If so, these pathways may provide potential targets for the design of novel anthelmintic drugs. Here, we review three main groups of F. hepatica calcium binding proteins: the FH8 family, the calmodulin family (FhCaM1, FhCaM2 and FhCaM3) and the EF-hand/dynein light chain family (FH22, FhCaBP3, FhCaBP4). Considerable information has been gathered on the sequences, predicted structures and biochemical properties of these molecules. The challenge now is to understand their functions in the organism.

Misonidazole binding in murine liver tissue: a marker for cellular hypoxia in vivo

The hepatic microcirculation is believed to cause variable cellular oxygenation within the organ. In this study a marker of cellular hypoxia was used to demonstrate liver oxygen tension gradients in vivo. Covalent binding of misonidazole adducts to cellular macromolecules is enhanced by hypoxia. Autoradiographs of liver from mice treated with radiolabeled misonidazole demonstrated enhanced binding of adducts within hepatocytes surrounding hepatic veins. Livers from both hypoxic and normal mice had characteristic autoradiographic grain patterns reflecting regional oxygen tension variation in vivo. Differential binding of misonidazole adducts formed in hypoxic cells could have an application in studies of liver physiology and biochemistry.

Fasciola hepatica:development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.

Identification of a high-risk haplotype for the dystrobrevin binding protein 1 (DTNBP1) gene in the Irish study of high-density schizophrenia families

A recent report showed significant associations between several SNPs in a previously unknown EST cluster with schizophrenia. (1). The cluster was identified as the human dystrobrevin binding protein 1 gene (DTNBP1) by sequence database comparisons and homology with mouse DTNBP1. (2). However, the linkage disequilibrium (LD) among the SNPs in DTNBP1 as well as the pattern of significant SNP-schizophrenia association was complex. This raised several questions such as the number of susceptibility alleles that may be involved and the size of the region where the actual disease mutation(s) could be located. To address these questions, we performed different single-marker tests on the 12 previously studied and 2 new SNPs in DTNBP1 that were re-scored using an improved procedure, and performed a variety of haplotype analyses. The sample consisted of 268 Irish multiplex families selected for high density of schizophrenia. Results suggested a simple structure where the LD in the target region could be explained by 6 haplotypes that together accounted for 96% of haplotype diversity in the whole sample. From these six, a single high-risk haplotype was identified that showed a significant association with schizophrenia and explained the pattern of significant findings in the analyses with individual markers. This haplotype was 30 kb long, had a large effect, could be measured with two tag SNPs only, had a frequency of 6% in our sample, seemed to be of relatively recent origin in evolutionary terms, and was equally distributed over Ireland. Implications of these findings for follow-up and replication studies are discussed.

The identification of p130cas-binding proteins and their role in cellular transformation

Adaptor proteins play an important role in signal transduction by regulating the establishment and maintenance of functionally important protein complexes. A recently described member of this group of proteins is p130cas (CAS), which contains numerous sequence motifs predicted to be involved in mediating protein-protein interactions. We propose that adaptor molecules like CAS may help determine the response of a cell to a particular signal by interacting with specific subsets of cellular proteins. To test this hypothesis, we have identified potential binding partners of CAS that may play a rote in cellular transformation by the oncoproteins v-SRC and/or v-CRK. We show that individual domains of CAS associate with specific subsets of proteins in vitro, and that many of these interactions are dependent on the state of tyrosine-phosphorylation of CAS. Sequences necessary for interacting with the focal adhesion kinase pp125FAK (FAK), v-SRC and v-CRK have been mapped to distinct regions of CAS. In addition, the identification of a number of putative CAS-binding partners that are present in crk-transformed cell extracts but undetectable in normal and src-transformed cell extracts supports a model in which unique protein complexes are formed in response to different signals.

Mutations within subdomain II of the extracellular region of epidermal growth factor receptor selectively alter TGF alpha binding

The interactions of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) with the epidermal growth factor receptor (EGFR) were examined by insertion mutagenesis of the receptor. Seventeen insertions were made throughout a construct containing only the extracellular domain. This truncated receptor (sEGFR) was secreted and had a dissociation constant similar to that of the full-length solubilized receptor. Receptors with insertions within subdomain III were not secreted. Two receptors with insertions at positions 291 and 474, which border subdomain III, have significantly decreased binding to both EGF and TGF alpha relative to wild type. This confirms previous work demonstrating that subdomain III forms the primary binding site for EGF and TGF alpha. Four of the mutants within subdomain II had a decreased binding to TGF alpha relative to wild type, but had wild type binding to EGF. These results suggest that a region within subdomain II may selectively regulate the binding of TGF alpha. Two receptors which contained insertions within subdomains II and IV, approximately equidistant from the center of subdomain III, bound twofold more ligand molecules than wild type receptor, with an affinity similar to that of wild type receptor. These findings suggest that insertion at these positions allows the access of more than one ligand molecule to the binding site.

Simvastatin decreases the level of heparin-binding protein in patients with acute lung injury

Heparin-binding protein is released by neutrophils during inflammation and disrupts the integrity of the alveolar and capillary endothelial barrier implicated in the development of acute lung injury and systemic organ failure. We sought to investigate whether oral administration of simvastatin to patients with acute lung injury reduces plasma heparin-binding protein levels and improves intensive care unit outcome.

Poly[mercury(II)-di-mu-chloro-mu-pyrazine-kappa N-2 : N ']

In [HgCl2(Pyp)](n) (Pyp = pyrazine, C4H4N2), chloride-bridged HgCl4/2 strands are connected into layers by pyrazine molecules. The Hg atom is on a site of symmetry 2/m, the unique Cl atom is on a mirror plane, the unique N atom is on a twofold rotation axis, and the unique C and H atoms are in general positions.

catena-poly[mercury(II)-di-mu-chloro-mu-pyridazine-kappa N-2 : N ']

The crystal structure of [HgCl2(Pyo)](n) (Pyo = pyridazine, C4H4N2) consists of chloride-bridged strands of octahedrally coordinated mercuric centers, connected by the two neighboring N atoms of pyridazine molecules. All atoms lie in special positions:Hg with site symmetry 2/m and the others on mirror planes.

catena-poly[mercury(II)-di-mu-bromo-mu-pyridazine-kappa N-2 : N ']

The crystal structure of [HgBr2(Pyo)](n) (Pyo = pyridazine, C4H4N2) consists of strands of octahedrally coordinated mercuric centers asymmetrically bridged by bromide and connected by the two neighboring N atoms of pyridazine molecules to complete the octahedral coordination of mercury. The Hg atoms lie on inversion centers.