Biomedical subjects
D Vaux
Publications and source records attributed to D Vaux.
The survivin-like C. elegans BIR-1 protein acts with the Aurora-like kinase AIR-2 to affect chromosomes and the spindle midzone.
Baculoviral IAP repeat proteins (BIRPs) may affect cell death, cell division, and tumorigenesis. The C. elegans BIRP BIR-1 was localized to chromosomes and to the spindle midzone. Embryos and fertilized oocytes lacking BIR-1 had defects in chromosome behavior, spindle midzone formation, and cytokinesis. We observed indistinguishable defects in fertilized oocytes and embryos lacking the Aurora-like kinase AIR-2. AIR-2 was not present on chromosomes in the absence of BIR-1. Histone H3 phosphorylation and HCP-1 staining, which marks kinetochores, were reduced in the absence of either BIR-1 or AIR-2. We propose that BIR-1 localizes AIR-2 to chromosomes and perhaps to the spindle midzone, where AIR-2 phosphorylates proteins that affect chromosome behavior and spindle midzone organization. The human BIRP survivin, which is upregulated in tumors, could partially substitute for BIR-1 in C. elegans. Deregulation of bir-1 promotes changes in ploidy, suggesting that similar deregulation of mammalian BIRPs may contribute to tumorigenesis.
Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate.
G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.
Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10.
Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-gamma (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-gamma decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-gamma stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity.
Direct physical interaction between the Caenorhabditis elegans 'death proteins' CED-3 and CED-4.
The two genes CED-4 and CED-3 (the nematode homologue of interleukin-1beta converting enzyme, ICE) of Caenorhabditis elegans are implicated in the control of cell death, but the mechanism by which this occurs is unknown. Here we provide evidence that CED-3 and CED-4 both contain sequences with homology to a domain present in RAIDD and the prodomain of certain ICE-like proteases (caspases). This domain is known to establish an interaction between RAIDD and these caspases. Similarly, CED-4 was found to interact with CED-3. Thus, the activity of the death protease CED-3 appears to be controlled by CED-4 through a direct physical interaction.
Interphase nuclei of many mammalian cell types contain deep, dynamic, tubular membrane-bound invaginations of the nuclear envelope.
The nuclear envelope consists of a double-membraned extension of the rough endoplasmic reticulum. In this report we describe long, dynamic tubular channels, derived from the nuclear envelope, that extend deep into the nucleoplasm. These channels show cell-type specific morphologies ranging from single short stubs to multiple, complex, branched structures. Some channels transect the nucleus entirely, opening at two separate points on the nuclear surface, while others terminate at or close to nucleoli. These channels are distinct from other topological features of the nuclear envelope, such as lobes or folds. The channel wall consists of two membranes continuous with the nuclear envelope, studded with features indistinguishable from nuclear pore complexes, and decorated on the nucleoplasmic surface with lamins. The enclosed core is continuous with the cytoplasm, and the lumenal space between the membranes contains soluble ER-resident proteins (protein disulphide isomerase and glucose-6-phosphatase). Nuclear channels are also found in live cells labeled with the lipophilic dye DiOC6. Time-lapse imaging of DiOC6-labeled cells shows that the channels undergo changes in morphology and spatial distribution within the interphase nucleus on a timescale of minutes. The presence of a cytoplasmic core and nuclear pore complexes in the channel walls suggests a possible role for these structures in nucleo-cytoplasmic transport. The clear association of a subset of these structures with nucleoli would also be consistent with such a transport role.
Lambda clone B22 contains a 7676 bp genomic fragment of Saccharomyces cerevisiae chromosome VII spanning the VAM7-SPM2 intergenic region and containing three novel transcribed open reading frames.
A genomic clone of 7676 bp designated B22 from Saccharomyces cerevisiae has been sequenced. The 5' end matches the previously described gene, VAM7, and the 3' end matches the previously described gene, SPM2, both of which have been assigned to the left arm of chromosome VII. The intergenic region contains three transcribed open reading frames (ORFs). The first is related to an uncharacterized ORF of Bacillus subtilis and more weakly to MesJ in Escherichia coli; this is found as a single transcript of 1.1 kb by Northern blotting. The second ORF encodes a small ras-like GTPase of 222 residues with strong homology to yeast Ypt8p and to mammalian Rab11; this is found as a single transcript of 1.1 kb by Northern blotting. The third ORF generates a transcript of 1.6 kb and encodes a protein of 382 residues including a perfect match to the consensus sequence of a C2H2 zinc finger domain; it shares a strong homology with yeast Mig1p and Cre-A from Aspergillus, Emericella and E. coli. This ORF also has a striking similarity to a putative 43 kDa zinc finger protein encoded by an ORF (YEL8) immediately downstream of YPT8, raising the possibility that a region between VAM7 and SPM2 on chromosome VII arose as a duplication of the YPT8-YEL8 region of chromosome V, followed by a translocation.
Uptake of acetylcholinesterase by neurons in the substantia nigra.
It is known that acetylcholinesterase is secreted by the dopaminergic neurons of the substantia nigra and has a subsequent action independent of cholinergic transmission. Although non-cholinergic actions of this protein have been demonstrated, the subsequent fate of acetylcholinesterase is unknown. One possibility is that acetylcholinesterase is taken up following secretion into the extracellular space. This hypothesis has been tested in vivo, in both conscious and anaesthetized guinea-pigs. Exogenous acetylcholinesterase (2-20 pM) was infused via a push-pull cannula implanted into either the substantia nigra or the surrounding extranigral regions: the amount subsequently recovered in the perfusate was then compared with control values. Only when the push-pull cannulae were implanted in the substantia nigra was there a marked decrease in the amount of acetylcholinesterase recovered; this selective retention was abolished when the perfusion medium was cooled to 4 degrees C or when the experiment was performed post mortem. Direct visualization of immunocytochemically identified nigral dopaminergic cells revealed co-localized deposits of labelled, exogenous acetylcholinesterase. Moreover, when exogenous acetylcholinesterase was boiled to prevent detection by the assay system and to eliminate any classical enzymatic action, an enhancement in perfusate levels of endogenous acetylcholinesterase was observed from nigral but not from extranigral sites, indicating that endogenous and exogenous acetylcholinesterases were in competition. These results suggest that, within the substantia nigra, secreted acetylcholinesterase may be subject to a temperature- and energy-dependent uptake mechanism.
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Identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal.
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The cytoplasmic domain of alphavirus E2 glycoprotein contains a short linear recognition signal required for viral budding.
Intracellular alphavirus nucleocapsids express a binding site for the cytoplasmic domain of the viral E2 spike glycoprotein. This binding site is recognized by the anti-idiotype monoclonal antibody, F13. The monoclonal anti-anti-idiotype antibody, raised against F13 and designated 3G10, recognizes the carboxy-terminal eight residues of the E2 cytoplasmic domain in Semliki Forest virus (SFV), identifying this as the signal for nucleocapsid interaction. F13 binding to cells infected with SFV or a second alphavirus, Sindbis virus, is inhibited by a synthetic peptide corresponding to the entire 31 residue cytoplasmic domain (E2c), and also by a synthetic peptide corresponding to the eight residue epitope recognized by 3G10. Both E2c and the eight residue peptide inhibited viral budding in microinjection experiments and when conjugated to colloidal gold are bound specifically to nucleocapsids in infected cells. These results identify a short linear signal in the E2 cytoplasmic domain required for the interaction with nucleocapsids which leads to budding of at least two alphaviruses from infected cells.
The use of antiidiotype antibodies for the characterization of protein-protein interactions.
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Identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal.
Monoclonal antibodies were raised against antibodies to distinct carboxy-terminal KDEL sequences of two soluble, resident endoplasmic reticulum proteins. These anti-idiotype reagents recognize an intrinsic membrane protein with characteristics expected of a receptor responsible for the recognition and return of resident proteins to the endoplasmic reticulum.
Lymphoid neoplasia and the control of haemopoietic differentiation.
Our broad aims are to delineate oncogenic events in lymphoid neoplasia and to search for genes that control haemopoietic differentiation. To explore lymphoid neoplasia, we have constructed transgenic mice bearing different oncogenes coupled to the immunoglobulin heavy chain enhancer (E mu), to force expression within lymphocytes. The prototype E mu-myc mice are highly prone to lymphomagenesis, generating pre-B and B cell lymphomas. In their pre-neoplastic phase, E mu-myc expression perturbs B cell development, accelerating the accumulation of pre-B cells. Lymphomagenesis requires additional oncogenic events, such as ras activation, and can be reconstructed in vitro. Transgenic mice bearing the N-myc, N-ras, v-abl and bcr-v-abl oncogenes are also prone to tumours. A striking demonstration that oncogenes can perturb lineage commitment has emerged. Introduction of the v-raf gene into cloned E mu-myc transgenic B cells frequently led to a switch in haemopoietic lineage: the cells became macrophages. Two clues to this remarkable metamorphosis are that the macrophage lines produce a myeloid growth factor and most bear marked karyotypic alterations, perhaps indicating that the balance between a few critical lineage control genes has been disturbed. To explore the hypothesis that genes encoding the DNA-binding homeo box domain participate in haemopoiesis, cDNA libraries from haemopoietic sources were screened, and several distinct homeo box cDNAs were isolated. They revealed a complex pattern of expression among haemopoietic cell lines. These genes are attractive candidates for regulators of haemopoietic differentiation.
DNA fragmentation in cytolysis.
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Macrophage subpopulations in lamina propria of normal and inflamed colon and terminal ileum.
The aim of this study was to characterise human intestinal macrophages in normal and inflamed ileum and colon. Immunoperoxidase staining with a panel of monoclonal antibodies and histochemical staining for acid phosphatase and non-specific esterase was performed. In the superficial lamina propria, normal colonic macrophages were larger and more strongly positive for acid phosphatase and non-specific esterase than those in normal terminal ileum. There were more macrophages staining with monoclonal antibody RFD1 in the superficial lamina propria of the latter. Studies in inflammatory bowel disease tissue showed the presence of macrophages staining with monoclonal antibodies RFD9 and 3G8 which were rarely present in normal tissue and represented a different pattern from that seen in infectious colitis. Studies on isolated intestinal macrophages confirmed the findings in tissue sections. Subpopulations of intestinal macrophages are likely to have different functional roles. Phenotypic changes during inflammation may be induced by mediators of inflammation or may represent a recently recruited population of cells.
Transgenic mouse models for hematopoietic tumorigenesis.
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Structure and function of Fc receptors on macrophages and lymphocytes.
Cell surface receptors for the Fc portion of immunoglobulin confer on most cells of the immune system the ability to communicate with the humoral antibody response. These Fc receptors are known to be particularly important for the function of various effector cells, such as macrophages, since they are involved in mediating a variety of activities including endocytosis, antibody-dependent cellular cytotoxicity, and triggering the release of potent inflammatory agents. Over the past few years, a considerable amount has been learned about the structure and functions of the Fc receptors expressed by murine and human cells, due to the availability of specific anti-receptor antibodies and the isolation of Fc receptor cDNA clones. In general, these receptors are transmembrane proteins whose extracellular domains contain two immunoglobulin-like regions and are thus members of the immunoglobulin gene family. Their domain structure consists of a glycosylated extracellular domain, a single membrane-spanning segment, and a relatively long cytoplasmic domain. The cytoplasmic tails exhibit a surprising degree of variation in length and amino acid sequence. This review summarizes some recent information concerning the structure and expression of the Fc receptors found on murine and human macrophages and lymphocytes. Particular attention is paid to the functional activities of these receptors, and the possible relationship between receptor function and receptor structure.