Abstract. Mammalian telomeres are composed of long arrays of TTAGGG repeats complexed with the TrAGGG repeat binding factor, TRF. Biochemical and ultrastructural data presented here show that the telomeric DNA and TRF colocalize in individual, condensed structures in the nuclear matrix. Telomeric TrAGGG repeats were found to carry an array of nuclear matrix attachment sites occurring at a frequency of at least one per kb. The nuclear matrix association of the telomeric arrays extended over large domains of up to 20-30 kb, encompassing the entire length of most mammalian telomeres. TRF protein and telomeric DNA cofractionated in nuclear matrix preparations and colocalized in discrete, condensed sites throughout the nuclear volume. FISH analysis indicated that TRF is an integral component of the telomeric complex and that the presence of TRF on telomeric DNA correlates with the compact configuration of telomeres and their association with the nuclear matrix. Biochemical fractionation of TRF and telomeric DNA did not reveal an interaction with the nuclear lamina. Furthermore, ultrastructural analysis indicated that the mammalian telomeric complex occupied sites throughout the nuclear volume, arguing against a role for the nuclear envelope in telomere function during interphase. These results are consistent with the view that mammalian telomeres form nuclear matrix-associated, TRF-containing higher order complexes at dispersed sites throughout the nuclear volume.

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Coagulase-negative staphylococci have emerged as important pathogens in infections associated with intravascular devices. Microbial adherence to biomaterial surfaces is a crucial step in the pathogenesis of these infections. Staphylococcal surface proteins (herein referred to as SSP-1 and SSP-2) are involved in the attachment of Staphylococcus epidermidis 354 to polystyrene. In the present study we show that the adhesin protrudes from the cell surface as a fimbria-like polymer. Furthermore, in vitro proteolytic cleavage of SSP-1 produces an SSP-2-like protein which coincides with a loss of adhesive function. SSP-1 expression is downregulated in a phenotypical variant of S. epidermidis 354 whereas SSP-2 expression is not. These results could suggest that proteolytic cleavage is a key to the regulation of the adhesive state of S. epidermidis in vivo.Over the last 2 decades coagulase-negative staphylococci (CoNS), particularly strains of Staphylococcus epidermidis, have emerged as important pathogens in association with foreign bodies, e.g., intravascular devices. Especially in immunocompromised patients, such as cancer patients and premature neonates, catheter-related sepsis due to CoNS may be a lifethreatening complication (1, 2, 5, 12, 13). For many microorganisms, adhesion to host surfaces is a crucial step in the pathogenic process and a prerequisite for colonization of these surfaces (3). It has been demonstrated that CoNS adhere avidly to a variety of biomaterials such as Teflon and polystyrene (6,8,16,19). The initial phase of attachment was found to be correlated with surface hydrophobicity. Both hydrophobicity and adherence were reduced by proteases, such as pepsin, suggesting that hydrophobic cell surface proteins may play an important role in the initial attachment to biomaterials (8,16). In a previous study (19), we identified a proteinaceous antigen of S. epidermidis 354 that apparently is involved in adherence to polystyrene. Evidence was obtained that this antigen, herein referred to as staphylococcal surface proteins (SSP-1 and SSP-2), is present on fimbria-like appendages protruding from the bacterial cell surface. A monoclonal antibody specific for SSP-1 and SSP-2 was shown to be specific and as effective as proteases in preventing this staphylococcal strain from adhering to polystyrene (19). This strain thus provides a model system to study the adherence of CoNS to intravascular devices. In the present study we present data concerning the ultrastructural organization of the staphylococcal fimbria-like appendages, the structural and functional relationship between the antigenically related SSP-1 and SSP-2 proteins, and aspects of adhesion regulation in vivo. For cryo-ultramicrotomy and immunogold-labeling purposes, staphylococci, grown on blood agar plates, were inoculated in nutrient broth (Difco) and cultured overnight at 37ЊC. After being washed with phosphate-buffered saline (PBS), the bacteria were resuspended and fixed for 1 h in PBS containing 2% paraformaldehyde and 0.25% glutaraldehyde. ...

Epidermal growth factor (EGF) is a single polypeptide of 53 amino acid residues which is involved in the regulation of cell proliferation. Egf exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of the kinase and subsequently receptor autophosphorylation. The autophosphorylation is essential for the interaction of the receptor with its substrates. These bind to the receptor by the so-called SH2 domains. The signal transduction pathways activated by EGF include the phosphatidylinositol pathway, leading to activation of protein kinase C and to increase in the intracellular Ca2+ concentration, and to the ras pathway leading to MAP kinase activation. Recently the cytoplasm has been implicated as playing an important role in EGF induced signal transduction. The EGF receptor has been demonstrated to be an actin-binding protein. In addition EGF causes a rapid actin depolymerisation and the formation of membrane ruffles. In particular these membrane ruffles have been shown to act as the first site of signal transduction after EGF binding, and thus may be considered as signal transduction structures. Finally evidence has been presented suggesting a positive role for EGF and/or the receptor in the nucleus.

Background information. Treatment of cells with UVC radiation leads to the formation of DNA cross-links which, if not repaired, can lead to apoptosis. γ-H2AX and cleaved caspase 3 are proteins formed during UVC-induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and γ-H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy).Results. Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells γ-H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of γ-H2AX a round, electron-dense nuclear structure was found, which was hitherto not identified in UV-stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy-loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV-induced nuclear structure contains a high amount of RNA.Conclusions. Because the UV-induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well-known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV-induced apoptotic pathway.