Molecular properties of fibrin-based matrices for promotion of angiogenesis in vitro.

The molecular properties of fibrin-based matrices, such as fibrillar structure and covalent modifications with adhesion domains, influence the angiogenic behavior of human umbilical vein endothelial cells (HUVECs) in vitro. The fibrillar structure of fibrin-based matrices was influenced by pH but not by covalent incorporation of exogenous adhesion domains. Native fibrin-based matrices polymerized at pH 10 formed organized and longitudinally oriented fibrin fibrils, which provided a good angiogenic substrate for endothelial cells. Furthermore, upon covalent incorporation of the model ligand L1Ig6, which binds to the integrin most prominently expressed on the surface of angiogenic endothelial cells, alpha(v)beta3, these matrices became angiogenesis-promoting when polymerized at physiological pH. The amount of incorporation of L1Ig6 into the matrices depended on the fibrinogen concentration on all three fibrin chains. Soluble forms of L1Ig6 diffused rapidly out of the matrix. Most important, L1Ig6-modified matrices were very specific in inducing the angiogenic phenotype of HUVECs, whereas control cells did not differentiate on these matrices. Our results indicate that artificial extracellular matrices can influence cell behavior in two ways. One way is based on the three-dimensional fibril structure of the matrix molecules themselves, and the other is due to providing specific binding sites for direct cell-matrix interactions that lead to the activation of second-messenger cascades and thus promoting angiogenic differentiation.

Mutagenesis and selection of PDZ domains that bind new protein targets.

PDZ domains are a recently characterized protein-recognition module. In most cases, PDZ domains bind to the C-terminal end of target proteins and are thought thereby to link these target proteins into functional signaling networks. We report the isolation of artificial PDZ domains selected via a mutagenesis screen in vivo, each recognizing a different C-terminal peptide. We demonstrate that the PDZ domains isolated can bind selectively to their target peptides in vitro and in vivo. Two of the target peptides chosen are the C-terminal ends of two cellular transmembrane proteins with which no known PDZ domains have been reported to interact. By targeting these artificial PDZ domains to the nucleus, interacting target peptides were efficiently transported to the same subcellular localization. One of the isolated PDZ domains was tested and shown to be efficiently directed to the plasma membrane when cotransfected with the full-length transmembrane protein in mammalian cells. Thus, artificial PDZ domains can be engineered and used to target intracellular proteins to different subcellular compartments.

The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain.

The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

Autofluorescence of living cells.

We have investigated the autofluorescence of viable mammalian cells (DU-145 and V79) with a confocal laser scanning microscope equipped with a UV laser. Our aim was to investigate the autofluorescence dependence on different treatments in mitochondria and lysosomes by using different reagents and to improve the confocal laser scanning microscope image quality by deconvolution. The following conclusions were drawn from the results: (1) not all of the autofluorescence comes from mitochondria; (2) one can significantly affect the signal which comes from the mitochondria; (3) the other organelles involved are probably lysosomes; (4) it is harder to affect the autofluorescence signal from the lysosomes than that from the mitochondria, and (5) deconvoluted autofluorescence images provide better information than undeconvoluted ones.

The 85-kilodalton phosphoprotein (pp85) of human herpesvirus 7 is encoded by open reading frame U14 and localizes to a tegument substructure in virion particles.

A family of antigenically related proteins present in cells infected with human herpesvirus 7 (HHV-7), designated phosphoprotein 85 (pp85), comprises a complex of proteins, of which the 85-kDa species is phosphorylated. pp85 is a major determinant of human response to HHV-7 infection (L. Foà-Tomasi, E. Avitabile, L. Ke, and G. Campadelli-Fiume, J. Gen. Virol. 75:2719-2727, 1994; L. Foà-Tomasi, M. P. Fiorilli, E. Avitabile, and G. Campadelli-Fiume, J. Gen. Virol. 77:511-518, 1996; J. B. Black et al., Clin. Diagn. Lab. Immunol. 3:79-83, 1996). By immunoscreening of a cDNA library from HHV-7-infected cells with monoclonal antibody (MAb) 5E1, directed to the proteins of the pp85 complex, we mapped the gene encoding pp85 to the U14 open reading frame of the HHV-7 genome. A prokaryotically expressed fusion protein containing the U14 open reading frame reacted with MAb 5E1 in an immunoblot assay. A functional role for pp85 was defined by immunoelectron microscopy studies. Immunogold labeling of cryosections of HHV-7-infected cord blood mononuclear cells at high resolution localized the reactivity of MAb 5E1 to the outer surface of the virion tegument. This finding demonstrates that pp85, the product of the U14 gene, is a component of the HHV-7 tegument and suggests that the HHV-7 tegument is not a homogeneous structure but rather is composed of substructures, including an outermost layer containing pp85. The present findings, together with previously reported properties of MAb 5E1, including its ability to react with formalin-fixed paraffin-embedded samples, make this antibody a specific tool useful for etiopathogenetic studies of HHV-7 infection in humans and provide the basis for further development of pp85 into a specific recombinant diagnostic reagent.

A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II.

The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro . It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis - trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domain-containing splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcription.

Immunocytochemical analysis of the regeneration of myofibrils in long-term cultures of adult cardiomyocytes of the rat.

Dissociated adult rat ventricular cardiomyocytes obtained from hearts by retrograde perfusion with collagenase were investigated in long-term cultures. Myofibril regeneration, isoprotein transition of alpha- and beta-myosin heavy chain (MHC), and M-band localization of M-creatine kinase in the reconstituting heart cells were studied. Myofibril formation was demonstrated by the use of antibodies against either cardiac C-protein or myomesin as early differentiation markers. Four days after plating, small myofibrils could be identified in attached cells in a perinuclear fashion; later in culture the cells displayed various shapes and myofibril distribution. Frequently a patchy distribution of myofibrils within the extending peripheral processes could be observed. Colocalization of sarcomeres and phalloidin-stained F-actin filament bundles was demonstrated by double fluorescence staining and by the use of high intensifying video microscopy and computerized image processing. The immunofluorescence distribution of alpha- and beta-MHC isoproteins in newly isolated and cultured cardiomyocytes changed from 100% alpha-MHC and 70% beta-MHC in rod-shaped cells to about 100% beta-MHC and 70% alpha-MHC in spread out cultured cells. This shift was corroborated by a relative gradual decline in alpha-MHC at the expense of increasing amounts of beta-MHC with time in culture as assessed by sodium dodecyl sulfate gel electrophoresis of total cell homogenates. In addition, whereas rod-shaped newly isolated cardiomyocytes showed a clear M-band association of M-creatine kinase as found in adult heart tissue, adult cultivated spread out cells did not show a cross-striated pattern after incubation with antibody. Taken together, these observations suggest that adult cardiomyocytes not only undergo extensive morphological transitions in long-term cultures, but also generate new myofibrillar structures lacking M-creatine kinase and containing the beta-MHC, thus fitting the characteristics of fetal myofibrils. These results indicate a change from the adult terminally differentiated to a less differentiated state of the cardiac cells in culture.

Evidence for different receptor sites in mouse spleen cells for the Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins.

Liposomes were reconstituted from phosphatidylcholine and Sendai virus glycoproteins HN or F and their interaction with mouse spleen cells was studied. Both the HN and F liposomes were able to stimulate chemiluminescence (CL), indicating that the glycoproteins were able to interact with the cell membrane independently of each other. The induction of CL in cells which had been pretreated with liposomes by monoclonal antibodies to either HN or F demonstrated that HN and F bind to the cells independently. The presence of F liposomes on the cell surface was also confirmed by immunoelectron microscopy. Cells pretreated with HN and F liposomes revealed a different pattern of CL when challenged with intact virus or the calcium ionophore A23187 indicating that HN and F bind to different receptor sites.