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Biomedical subjects

J M Frade

Publications and source records attributed to J M Frade.

At least 19 recordsLinked to original sources
PubMed2005

Lengthening of G2/mitosis in cortical precursors from mice lacking beta-amyloid precursor protein.

The beta-amyloid precursor protein (APP) is expressed within the nervous system, even at the earliest stages of embryonic development when cell growth and proliferation is particularly important. In order to study the function of APP at these early developmental stages, we have studied the development of the cerebral cortex in both wild type and App-/- mutant mice. Here, we demonstrate that APP mRNA is expressed in cortical precursor cells and that APP protein is concentrated within their apical domains during interphase. However, during mitosis, APP re-localizes to the peripheral space surrounding the metaphase plate. In APP-deficient cortical precursors, the duration of mitosis is increased and a higher proportion of cortical precursor cells contained nuclei in late G2. We conclude that during cortical development APP plays a role in controlling cell cycle progression, particularly affecting G2 and mitosis. These observations may have important implications for our understanding of how APP influences the progression of Alzheimer's disease, since degenerating cortical neurons have been shown to up-regulate cell cycle markers and re-enter the mitotic cycle before dying.

Amyloid beta-Protein Precursor↗
PubMed2002

Control of the cell cycle by neurotrophins: lessons from the p75 neurotrophin receptor.

Although traditionally little attention has been paid to the interplay between neurotrophins and the cell cycle, a number of recent findings suggest an important role for these growth factors in the regulation of this aspect of the cellular physiology. In this article, we review the evidence from a number of studies that neurotrophins can influence cell cycle progression or mitotic cycle arrest both in the nervous system as well as in other cell types. The contrary response of different cells to neurotrophins in terms of cell cycle regulation derives in part from the fact that these factors use two different receptor types to transmit their signals: members of the Trk family and the p75 neurotrophin receptor (p75NTR). With this in mind, we outline the current state of our knowledge regarding the molecular basis underlying the control of cell cycle progression by neurotrophins. We focus our interest on the receptors that transduce these signals and, in particular, the striking finding that p75NTR interacts with proteins that can promote mitotic cycle arrest. Finally, we discuss the mechanisms of cell death mediated by p75NTR in the context of cell cycle regulation.

Animals↗
PubMed2001

Functional expression of chemokine receptor 2 by normal human eosinophils.

BACKGROUND: Within the granulocytes, the CC chemokines preferentially activate basophils and eosinophils on binding to chemokine receptors (CCRs). In vivo administration of neutralizing anti-monocyte chemoattractant protein 1 (MCP-1) antibodies can block accumulation of eosinophils in the lungs of antigen-challenged animals. OBJECTIVE: We studied a panel of chemokines for chemotactic activity in normal human eosinophils from healthy donors with a special focus on MCP-1, identified the respective receptor required for the biological response of eosinophils, and investigated mediators used for signal transduction. METHODS: Cells were enriched by magnetic cell sorting. Receptor expression in eosinophils was shown by RT-PCR and fluorescence-activated cell sorting. The biological response was tested in chemotaxis and calcium mobilization assays. RESULTS: Eosinophils have detectable mRNA for CCR2, and the receptor protein is expressed on cell surfaces. MCP-1 induces chemotaxis and calcium mobilization in eosinophils. The chemotactic activity of MCP-1 revealed a double-peaked dose-response curve; one of the peaks is abolished by addition of a blocking antibody to CCR2, but it is insensitive to blocking of CCR1 or CCR3. Specific enzyme inhibitors ruled out signaling characteristics of CCR2 in eosinophils. CONCLUSION: Normal human eosinophils express functional CCR2 on cell surfaces.

Calcium Signaling↗
PubMed2000

Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones.

During their early postmitotic life, a proportion of the nascent retinal ganglion cells (RGCs) are induced to die as a result of the interaction of nerve growth factor (NGF) with the neurotrophin receptor p75. To analyse the mechanisms by which NGF promotes apoptosis, an in vitro culture system consisting of dissociated E5 retinal cells was established. In this system, NGF-induced apoptosis was only observed in the presence of insulin and neurotrophin-3, conditions that favour the birth of RGCs and other neurones expressing the glycoprotein G4. The pro-apoptotic effect of NGF on the G4-positive neurones was evident after 10 hours in vitro and was preceded by a significant upregulation of cyclin B2, but not cyclin D1, and the presence of mitotic nuclei in these cells. Brain-derived neurotrophic factor prevented both the increase of cyclin B2 expression in the G4-positive neurones and the NGF-induced cell death. Finally, pharmacologically blocking cell-cycle progression using the cyclin-dependent kinase inhibitor roscovitine prevented NGF-induced cell death in a dose-dependent manner. These results strongly suggest that the apoptotic signalling initiated by NGF requires a driving stimulus manifested by the neuronal birth and is preceded by the unscheduled re-entry of postmitotic neurones into the cell cycle.

Animals↗
PubMed1999

The zinc finger protein NRIF interacts with the neurotrophin receptor p75(NTR) and participates in programmed cell death.

NRIF (neurotrophin receptor interacting factor) is a ubiquitously expressed zinc finger protein of the Krüppel family which interacts with the neurotrophin receptor p75(NTR). The interaction was first detected in yeast and then biochemically confirmed using recombinant GST-NRIF fusions and p75(NTR) expressed by eukaryotic cells. Transgenic mice carrying a deletion in the exon encoding the p75(NTR)-binding domain of NRIF display a phenotype which is strongly dependent upon genetic background. While at the F(2 )generation there is only limited (20%) embryonic lethality, in a congenic BL6 strain nrif(-/-) mice cannot survive beyond E12, but are viable and healthy to adulthood in the Sv129 background. The involvement of NRIF in p75(NTR)/NGF-mediated developmental cell death was examined in the mouse embryonic neural retina. Disruption of the nrif gene leads to a reduction in cell death which is quantitatively indistinguishable from that observed in p75(NTR)(-/-) and ngf(-/-) mice. These results indicate that NRIF is an intracellular p75(NTR)-binding protein transducing cell death signals during development.

Amino Acid Sequence↗
PubMed1999

Signaling through CD43 induces natural killer cell activation, chemokine release, and PYK-2 activation.

Natural killer (NK) cell activation is the result of a balance between positive and negative signals triggered by specific membrane receptors. We report here the activation of NK cells induced through the transmembrane glycoprotein CD43 (leukosialin, sialophorin). Engagement of CD43 by specific antibodies stimulated the secretion of the chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, which was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, signaling through CD43 increased the cytotoxic activity of NK cells and stimulated an increase in the tyrosine kinase activity in antiphosphotyrosine immune complexes of NK cell lysates. PYK-2 was identified among the tyrosine kinase proteins that become activated. Hence, PYK-2 activation was observed after 20 minutes of CD43 stimulation, reached a maximum after 45 to 60 minutes, and decreased to almost basal levels after 120 minutes of treatment. Together, these results demonstrate the role of CD43 as an activation molecule able to transduce positive activation signals in NK cells, including the regulation of chemokine synthesis, killing activity, and tyrosine kinase activation.

Antigens, CD↗
PubMed1999

Genetic evidence for cell death mediated by nerve growth factor and the neurotrophin receptor p75 in the developing mouse retina and spinal cord.

The role of nerve growth factor (NGF) and of the neurotrophin receptor p75 (p75(NTR)) in programmed cell death was investigated in the retina and the spinal cord of mouse embryos. Large numbers of cells express p75(NTR) in and along the developing optic nerve and in the mantle zone of the spinal cord. In embryos carrying deletions in the ngf or the p75(NTR) gene, cell death was reduced in the retina and in the spinal cord. Increased numbers of Islet-1-immunoreactive cells were detected in the dorsal spinal cord, and the mantle zone was enlarged in both mutants. These results indicate that NGF/p75(NTR)-dependent mechanisms are used to remove cells when axonal tracts elongate in developing neuroepithelia.

Animals↗
PubMed1998

Roles of chemokines and receptor polarization in NK-target cell interactions.

We report that the ability of NK cells to produce chemokines is increased in NK-target cell conjugates. The chemokines produced play a critical role in the polarization and recruitment of NK cells as well as in the NK effector-target cell conjugate formation. Chemokines induce the formation of two specialized regions in the NK cell: the advancing front or leading edge, where chemokine receptors CCR2 and CCR5 cluster, which might guide the cells toward the chemotactic source, and the uropod, where adhesion molecules ICAM-1 and -3 are redistributed. NK cell polarity was intrinsically involved in conjugate formation. The redistribution of both adhesion receptors and CCR was preserved during the formation of NK-target cell conjugates. Time-lapse videomicroscopy studies of the formation of effector-target conjugates showed that morphologic poles are also functionally distinct; while the binding to target cells was preferentially mediated through the leading edge, the uropod was found at the rear of migrating NK cells and recruited additional NK cells to the vicinity of K562 target cells. Inhibition of cell polarization and adhesion receptor redistribution blocked the formation of NK-K562 cell conjugates and the cytotoxic activity of NK cells. We discuss the implication of NK-cell polarization in the development of cytotoxic responses.

Antigens, CD↗
PubMed1998

Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called beta-adrenergic kinase 1 or betaARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein beta-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant betaARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines.

Arrestin↗
PubMed1998

Nerve growth factor: two receptors, multiple functions.

Nerve growth factor (NGF) was characterized over 4 decades ago, and like the other neurotrophins subsequently discovered, it is best known for its trophic role, including the prevention of programmed cell death in specific populations of neurones in the peripheral nervous system. This property can be accounted for by the activation of a tyrosine kinase receptor. NGF also regulates neuronal function, as illustrated by its role in pain and inflammation, and in synaptic plasticity. Finally, NGF recently was shown to activate the neurotrophin receptor p75 (p75NTR), a receptor with no intrinsic catalytic activity and with similarities to members of the tumor necrosis factor receptor family. During normal development, the activation of p75NTR by NGF actually kills cells in the central nervous system. One remarkable property of NGF is then that it controls cell numbers in opposite ways in the developing nervous system, a result of its unique ability to activate two different receptor types.

Animals↗
PubMed1998

The chemokine SDF-1alpha triggers a chemotactic response and induces cell polarization in human B lymphocytes.

We studied the expression and possible functional role of chemokine receptors CXCR3, CXCR4 and CCR5 in normal human B lymphocytes. B cells from both peripheral blood and tonsils expressed high levels of CXCR4 but not the other chemokine receptors tested. CXCR4 ligand, stromal cell-derived factor (SDF)-1alpha, elicited a potent chemotactic response and induced a polarized motile phenotype in B cells, resulting in redistribution of the adhesion molecule ICAM-3 to a posterior appendage of the cell, termed uropod, and of CXCR4 receptor to the leading edge of migrating B cells. Time-lapse videomicroscopy studies revealed that SDF-1alpha-treated cells recruited additional bystander B cells through the uropod. SDF-1alpha induced levels of cellular recruitment comparable to those elicited by polarization-inducing anti-ICAM-3 monoclonal antibody, in an LFA-1/ICAM-1, -3-dependent fashion. Moreover, this chemokine increased intracellular Ca2+ levels in B lymphocytes, and induced a rapid CXCR4 receptor down-regulation on the cell surface membrane. These results provide new insight into the important biological role of SDF-1alpha in physiological processes in which B cells participate, and suggest a key role for chemokines in normal B cell trafficking and recirculation.

Antigens, CD↗
PubMed1998

Microglia-derived nerve growth factor causes cell death in the developing retina.

While nerve growth factor (NGF) is best known for its trophic functions, recent experiments indicate that it can also cause cell death during development by activating the neurotrophin receptor p75. We now identify microglial cells as the source of NGF as a killing agent in the developing eye. When the retina is separated from the surrounding tissue before colonization by microglial cells, no NGF can be detected, and cell death is dramatically reduced. It is restored by the addition of microglial cells, an effect that is blocked by NGF antibodies. NGF adsorbed at the surface of beads, but not soluble NGF, mimics the killing action of microglial cells. These results indicate an active role for macrophages in neuronal death.

Animals↗
PubMed1998

Influence of the CCR2-V64I polymorphism on human immunodeficiency virus type 1 coreceptor activity and on chemokine receptor function of CCR2b, CCR3, CCR5, and CXCR4.

The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism in CCR2 (CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor activities, and their effects on the expression and receptor activities of the major HIV-1 coreceptors. CCR2b and CCR2b-V64I were expressed at similar levels, and neither molecule affected the expression or coreceptor activity of CCR3, CCR5, or CXCR4 in cotransfected cell lines. Peripheral blood mononuclear cells (PBMCs) from CCR2-V64I heterozygotes had normal levels of CCR2b and CCR5 but slightly reduced levels of CXCR4. CCR2b and CCR2b-V64I functioned equally well as HIV-1 coreceptors, and CCR2-V64I PBMCs were permissive for HIV-1 infection regardless of viral tropism. The MCP-1-induced calcium mobilization mediated by CCR2b signaling was unaffected by the polymorphism, but MCP-1 signaling mediated by either CCR2b- or CCR2-V64I-encoded receptors resulted in heterologous desensitization (i.e., limiting the signal response of other receptors) of both CCR5 and CXCR4. The heterologous desensitization of CCR5 and CXCR4 signaling by both CCR2 allele receptor types provides a mechanistic link that might help explain the in vivo effects of CCR2 gene variants on progression to AIDS as well as the reported antiviral activity of natural CCR2 ligands.

Cell Line, Transformed↗
PubMed1997

Characterization of the CCR2 chemokine receptor: functional CCR2 receptor expression in B cells.

We have derived anti-human CCR2-specific mAbs by immunization with synthetic peptides corresponding to CCR2 sequences presumably involved in the interaction with its ligand(s). The characterization of these mAbs includes the ability to recognize the CCR2 receptor specifically, as well as the function based on their ability to promote Ca2+ influx or to block MCP-1-induced Ca2+ influx and chemotaxis. One mAb (MCP-1 R02) that is directed to the NH2 terminal domain of the CCR2 receptor has MCP-1 agonist activity, and two that recognize the third extracellular domain (MCP-1R04 and MCP-1 R05) have MCP-1 antagonist activity. We analyzed the presence of CCR2 in several PBL and tonsil-derived leukocyte populations and found expression of this receptor in monocytes, activated T cells, and, surprisingly, in B cells. CCR2 receptor expression in B cells was further corroborated in Southern blot using CCR2-specific probes. Moreover, both MCP-1 and the agonist mAb trigger specific B cell migration via a PTX-sensitive mechanism, indicating the presence of a functional CCR2 receptor in these cells.

Antibodies, Monoclonal↗
PubMed1997

The amino-terminal domain of the CCR2 chemokine receptor acts as coreceptor for HIV-1 infection.

The chemokines are a homologous serum protein family characterized by their ability to induce activation of integrin adhesion molecules and leukocyte migration. Chemokines interact with their receptors, which are composed of a single-chain, seven-helix, membrane-spanning protein coupled to G proteins. Two CC chemokine receptors, CCR3 and CCR5, as well as the CXCR4 chemokine receptor, have been shown necessary for infection by several HIV-1 virus isolates. We studied the effect of the chemokine monocyte chemoattractant protein 1 (MCP-1) and of a panel of MCP-1 receptor (CCR2)-specific monoclonal antibodies (mAb) on the suppression of HIV-1 replication in peripheral blood mononuclear cells. We have compelling evidence that MCP-1 has potent HIV-1 suppressive activity when HIV-1-infected peripheral blood lymphocytes are used as target cells. Furthermore, mAb specific for the MCP-1R CCR2 which recognize the third extracellular CCR2 domain inhibit all MCP-1 activity and also block MCP-1 suppressive activity. Finally, a set of mAb specific for the CCR2 amino-terminal domain, one of which mimics MCP-1 activity, has a potent suppressive effect on HIV-1 replication in M- and T-tropic HIV-1 viral isolates. We conjecture a role for CCR2 as a coreceptor for HIV-1 infection and map the HIV-1 binding site to the amino-terminal part of this receptor. This concurs with results showing that the CCR5 amino terminus is relevant in HIV-1 infection, although chimeric fusion of various extracellular domains shows that other domains are also implicated. We discuss the importance of CCR2 structure relative to its coreceptor role and the role of anti-CCR2 receptor antibodies in the prevention of HIV-1 infection.

Chemokine CCL2↗
PubMed1997

Polarization of chemokine receptors to the leading edge during lymphocyte chemotaxis.

Leukocyte migration in response to cell attractant gradients or chemotaxis is a key phenomenon both in cell movement and in the inflammatory response. Chemokines are quite likely to be the key molecules directing migration of leukocytes that involve cell polarization with generation of specialized cell compartments. The precise mechanism of leukocyte chemoattraction is not known, however. In this study, we demonstrate that the CC chemokine receptors CCR2 and CCR5, but not cytokine receptors such as interleukin (IL)-2Ralpha, IL-2Rbeta, tumor necrosis factor receptor 1, or transforming growth factor betaR, are redistributed to a pole in T cells that are migrating in response to chemokines. Immunofluorescence and confocal microscopy studies show that the chemokine receptors concentrate at the leading edge of the cell on the flattened cell-substratum contact area, induced specifically by the signals that trigger cell polarization. The redistribution of chemokine receptors is blocked by pertussis toxin and is dependent on cell adhesion through integrin receptors, which mediate cell migration. Chemokine receptor expression on the leading edge of migrating polarized lymphocytes appears to act as a sensor mechanism for the directed migration of leukocytes through a chemoattractant gradient.

Cells, Cultured↗