Lack of detection of retroviral particles (HIAP-1) in the H9 T cell line co-cultured with thyrocytes of Graves' disease.

Evidence for a possible aetiopathogenetic role of endogenous and/or exogenous retroviruses (RVs) in organ- and non-organ-specific autoimmune diseases is circumstantial in both humans and animal models. Intracisternal A type particles, antigenically related to HIV, have been reported in H9 cells co-cultured with homogenates of salivary glands obtained from patients with Sjögren syndrome and with synovial fluid of patients with rheumatoid arthritis. In order to identify a possible transfer of a putative 'infective RV agent' involved in the pathogenesis of human thyroid autoimmune disease, the H9 T cell line was co-cultured not only with thyroid homogenates, but also with viable thyrocytes, both prepared from glands of patients with Graves' disease. At the end of a prolonged co-culture period (24 weeks), no RV particles could be detected by electron microscopy in the H9 cells co-cultured with both thyroid preparations. These data seem to exclude the involvement of HIAP-1 in the aetiopathogenesis of human autoimmune thyroid disease.

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of holliday junction resolution.

Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration.

Visualisation of human rad52 protein and its complexes with hRad51 and DNA.

The human Rad52 protein stimulates joint molecule formation by hRad51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRad51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRad51 invasion and nucleoprotein filament formation. When Rad52-Rad51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the Rad51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human Rad51 and Rad52 recombination/repair proteins.

Calcium-induced intercellular adhesion of keratinocytes does not involve accumulation of beta 1 integrins at cell-cell contacts and does not involve changes in the levels or phosphorylation of catenins.

On initiation of terminal differentiation human epidermal keratinocytes detach from the underlying basement membrane as a result of inactivation and subsequent loss of integrins from the cell surface. Assembly of keratinocytes into multilayered sheets requires functional E- and P-cadherin and when stratification is inhibited in low calcium medium differentiating keratinocytes continue to express functional integrins. Using immunofluorescence microscopy, we found that on addition of calcium ions to keratinocyte monolayers there was colocalisation of the beta 1 integrins and E-cadherin along the lateral membranes except for a zone close to the substratum which exclusively contained integrins. Quantitative immunoelectron microscopy showed that on induction of stable cell-cell contacts the density of beta 1 integrins was the same on the apical and lateral membranes, suggesting that the accumulation of integrins on the lateral membranes observed by immunofluorescence microscopy is due to the increased area of contact between adjacent cells and not to an increase in receptor density. There were no changes in the levels of catenins and their degree of phosphorylation after induction of cell-cell contacts. These observations provide new sights into the mechanism of calcium-dependent intercellular adhesion of keratinocytes.

Purification of human Rad51 protein by selective spermidine precipitation.

The human Rad51 protein is a structural homolog of Escherichia coli RecA. The exact role of human Rad51 within the cell is poorly understood but, like its bacterial and yeast homologs, hRad51 is believed to play a central role in homologous recombination. However, recent reports that transgenic mice lacking the RAD51 gene die early in development suggest an additional and essential function for mammalian Rad51 in cell proliferation or genome maintenance. In this paper we describe a simple and quick method for the purification of human Rad51 overproduced in E. coli. Dialysis of cell-free extracts against buffer containing low concentrations of spermidine result in the formation of hRad51 microcrystals as observed by light and electron microscopy. The crystals were easily redissolved in phosphate buffer and hRad51 was further purified to homogeneity using hydroxylapatite, affi-gel heparin and Q-sepharose chromatography. When purified by this method hRad51 is free of endo- and exonuclease activities and suitable for enzymological studies. Spermidine precipitation also provides a rapid method for the large scale purification of hRad51 suitable for physical analysis.

Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells.

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.

The AP-1 adaptor complex binds to immature secretory granules from PC12 cells, and is regulated by ADP-ribosylation factor.

Immature secretory granules (ISGs) in endocrine and neuroendocrine cells have been shown by morphological techniques to be partially clathrin coated (Orci, L., M. Ravazzola, M. Amherdt, D. Lonvard, A. Perrelet. 1985a. Proc. Natl. Acad. Sci. USA. 82:5385-5389; Tooze, J., and S. A. Tooze. 1986. J. Cell Biol. 103:839-850). The function, and composition, of this clathrin coat has remained an enigma. Here we demonstrate using three independent techniques that immature secretory granules isolated from the rat neuroendocrine cell line PC12 have clathrin coat components associated with their membrane. To study the nature of the coat association we have developed an assay whereby the binding of the AP-1 subunit gamma-adaptin to ISGs was reconstituted by addition of rat or bovine brain cytosol. The amount of gamma-adaptin bound to the ISGs was ATP independent and was increased fourfold by the addition of GTPgammaS. The level of exogenous gamma-adaptin recruited to the ISG was similar to the level of gamma-adaptin present on the ISG after isolation. Addition of myristoylated ARF1 peptide stimulated binding. Reconstitution of the assay using AP-1 adaptor complex and recombinant ARF1 provided further evidence that ARF is involved in gamma-adaptin binding to ISGs; BFA inhibited this binding. Trypsin treatment and Trisstripping of the ISGs suggest that additional soluble and membrane-associated components are required for gamma-adaptin binding.

Extruded erythroblast nuclei are bound and phagocytosed by a novel macrophage receptor.

Resident bone marrow macrophages (RBMM) play an important role in clearance of nuclei extruded from late-stage erythroblasts (Eb). To investigate the nature of macrophage receptors involved in this process, extruded erythroblast nuclei (EEN) were purified by cultivation of erythroblasts with erythropoietin, followed by density gradient centrifugation. By electron microscopy, the majority of free nuclei had an intact plasma membrane. EEN bound avidly to RBMM in a divalent cation-independent manner at both 4 degrees C and 37 degrees C. The use of specific monoclonal antibodies (MoAbs) and inhibitors showed that this adhesive interaction was not mediated by previously characterized macrophage receptors involved in recognition of either developing hematopoietic cells or apoptotic cells. The EEN receptor was expressed on resident macrophages isolated from murine bone marrow, spleen, lymph node, and peritoneal cavity, but was completely absent from alveolar macrophages. Despite high levels of EEN binding to RBMM, few were phagocytosed even after prolonged culture. Phorbol myristate acetate (PMA) was found to stimulate phagocytosis, suggesting that this is a regulated process. These findings indicate that EEN are recognized by a novel macrophage receptor and that recognition may be triggered during the membrane remodeling that accompanies enucleation.

CD66 identifies the biliary glycoprotein (BGP) adhesion molecule: cloning, expression, and adhesion functions of the BGPc splice variant.

Within the hematopoietic lineage, the monoclonal antibody (MoAb) CD66 reacts with cells of the granulocyte lineage, but not with the majority of progenitor cells from human bone marrow. Our previous studies have shown that CD66 binds specifically to at least three carcinoembryonic antigen (CEA) superfamily members, ie, CEA itself, nonspecific cross-reacting antigen (NCA), and CGM1, but not to CGM6 (NCA-95). In this report, we show that CD66 will also identify the biliary glycoproteins (BGP). A full-length cDNA for the BGPc molecule (a cytoplasmic splice variant of BGPa) was isolated by expression cloning using the CD66 MoAbs. This protein has an identical extracellular and transmembrane sequence to BGPa with one N-terminal IgV like domain, three IgC-like extracellular domains (A1, B1, and A2), plus a transmembrane domain, but the cytoplasmic domain is spliced by 53 nucleotides. Reverse transcriptase-polymerase chain reaction experiments show that this splice variant can be detected in colonic carcinoma cell lines, in primary colonic adenocarcinomas, and in myeloid and B-cell lines to varying degrees. Quantitative analyses of BGPc RNA expression by RNase protection indicate that abundant levels occur only in the colonic, but not in the hematopoietic, cell lines tested. Studies presented here show that BGPc mediates homotypic adhesion and suggest that the cytoplasmic splicing does not alter the initial homotypic adhesion properties of BGPa.

Stacking of Golgi cisternae in Schizosaccharomyces pombe requires intact microtubules.

Fission yeast was treated with the anti-microtubule agent, thiabendazole. Cytoplasmic microtubules broke down with a half-time of less than 10 minutes followed closely by the unstacking of Golgi cisternae. The final product appeared to be single Golgi cisternae. No other organelle seemed to be affected by this treatment, which was completely reversible. The nda3 mutant strain has an altered beta-tubulin and its cytoplasmic microtubules are resistant to thiabendazole. The Golgi in this cold-sensitive mutant was unaffected by treatment at the permissive temperature but unstacked at the non-permissive temperature even in the absence of thiabendazole. Taken together these data show that disruption of the microtubular network can cause dissociation of Golgi cisternae. Newly synthesised acid phosphatase was transported and secreted to the same extent and with the same kinetics whether or not the Golgi was unstacked. The possible role of microtubules in Golgi stacking and the lack of effect on secretion are discussed.