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Biomedical subjects

C Martínez-A

Publications and source records attributed to C Martínez-A.

At least 19 recordsLinked to original sources
PubMed2004

Nanomechanics of the formation of DNA self-assembled monolayers and hybridization on microcantilevers.

Biomolecular interactions over the surface of a microcantilever can produce its bending motion via changes of the surface stress, which is referred to nanomechanical response. Here, we have studied the interaction forces responsible for the bending motion during the formation of a self-assembled monolayer of thiolated 27-mer single-stranded DNA on the gold-coated side of a microcantilever and during the subsequent hybridization with the complementary nucleic acid. The immobilization of the single-stranded DNA probe gives a mean surface stress of 25 mN/m and a mean bending of 23 nm for microcantilevers with a length and thickness of about 200 microm and 0.8 microm, respectively. The hybridization with the complementary sequence could not be inferred from the nanomechanical response. The nanomechanical response was compared with data from well-established techniques such as surface plasmon resonance and radiolabeling, to determine the surface coverage and study the intermolecular forces between neighboring DNA molecules anchored to the microcantilever surface. From both techniques, an immobilization surface density of 3 x 10(12) molecules/cm(2) and a hybridization efficiency of 40% were determined. More importantly, label-free hybridization was clearly detected in the same conditions with a conventional sensor based on surface plasmon resonance. The results imply that the nanomechanical signal during the immobilization process arises mainly from the covalent attachment to the gold surface, and the interchain interactions between neighboring DNA molecules are weak, producing an undetectable surface stress. We conclude that detection of nucleic acid hybridization with nanomechanical sensors requires reference cantilevers to remove nonspecific signals, more sensitive microcantilever geometries, and immobilization chemistries specially addressed to enhance the surface stress variations.

Base Sequence↗
PubMed2003

Exacerbated inflammatory responses in transgenic mice expressing an inhibitor of apoptosis protein (OpIAP).

Members of the inhibitor of apoptosis protein family are involved not only in suppressing apoptosis, but also in signal transduction, cell division, and are associated with some types of cancers. Here we show that transgenic expression of the inhibitor of apoptosis protein OpIAP in murine T lymphocytes leads to a significant increase in T-cell receptor-induced cell activation, proliferation and cytokine production. Transgenic T lymphocytes expressing OpIAP have a lower proliferation threshold in response to T-cell receptor stimulation. Unstimulated OpIAP transgenic T lymphocytes show elevated nuclear levels of NF-kappaB transcription factor that increase after in vivo antigen peptide treatment. OpIAP transgenic animals present an exacerbated inflammatory response in an experimental contact hypersensitivity model, suggesting increased T-cell activation in vivo. These data indicate a new role for the inhibitor of apoptosis proteins in T-lymphocyte activation and proliferation.

Animals↗
PubMed2002

An endogenous hybrid mRNA encodes TWE-PRIL, a functional cell surface TWEAK-APRIL fusion protein.

TWEAK and APRIL are two recently identified tumour necrosis factor (TNF) ligand family members, implicated in angiogenesis and immune regulation, respectively. TWEAK is a transmembrane protein expressed on the cell surface, whereas APRIL acts solely as a secreted factor. In this report, using RACE, RT-PCR, cDNA library screening and an RNase protection assay, we characterize a hybrid transcript between TWEAK and APRIL mRNAs. The encoded TWE-PRIL protein is composed of TWEAK cytoplasmic and transmembrane domains fused to the APRIL C-terminal domain. TWE-PRIL mRNA is expressed and translated in human primary T cells and monocytes, and endogenous TWE-PRIL protein was detected in primary human T lymphocytes and monocytic cell lines. TWE-PRIL is membrane anchored and presents the APRIL receptor-binding domain at the cell surface. It is a biologically active ligand, as it stimulates cycling in T- and B-lymphoma cell lines. Much like membrane-bound and secreted TNF-alpha, the different cellular localizations of TWE-PRIL and APRIL suggest that they exert distinct biological roles.

Amino Acid Sequence↗
PubMed2001

Forkhead transcription factors contribute to execution of the mitotic programme in mammals.

Cell cycle progression is a process that is tightly controlled by internal and external signals. Environmental cues, such as those provided by growth factors, activate early signals that promote cell cycle entry. Cells that have progressed past the restriction point become independent of growth factors, and cell cycle progression is then controlled endogenously. The phosphatidylinositol 3OH kinase (PI(3)K)/protein kinase B (PKB) pathway must be activated in G1 to inactivate forkhead transcription factors (FKH-TFs) and allow cell cycle entry. Here we show that subsequent attenuation of the PI(3)K/PKB pathway is required to allow transcriptional activation of FKH-TF in G2. FKH-TF activity in G2 controls mammalian cell cycle termination, as interference with FKH transcriptional activation by disrupting PI(3)K/PKB downregulation, or by expressing a transcriptionally inactive FKH mutant, induces cell accumulation in G2/M, defective cytokinesis, and delayed transition from M to G1 of the cell cycle. We demonstrate that FKH-TFs regulate expression of mitotic genes such as cyclin B and polo-like kinase (Plk). Our results support the important role of forkhead in the control of mammalian cell cycle completion, and suggest that efficient execution of the mitotic programme depends on downregulation of PI(3)K/PKB and consequent induction of FKH transcriptional activity.

3T3 Cells↗
PubMed2001

Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization.

Redistribution of specialized molecules in migrating cells develops asymmetry between two opposite cell poles, the leading edge and the uropod. We show that acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of ganglioside GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively. This segregation to each cell pole parallels the specific redistribution of membrane proteins associated to each raft subfraction. Our data suggest that raft partitioning is a major determinant for protein redistribution in polarized T cells, as ectopic expression of raft-associated proteins results in their asymmetric redistribution, whereas non-raft-partitioned mutants of these proteins are distributed homogeneously in the polarized cell membrane. Both acquisition of a migratory phenotype and SDF-1alpha-induced chemotaxis are cholesterol depletion-sensitive. Finally, GM3 and GM1 raft redistribution requires an intact actin cytoskeleton, but is insensitive to microtubule disruption. We propose that membrane protein segregation not only between raft and nonraft domains but also between distinct raft subdomains may be an organizational principle that mediates redistribution of specialized molecules needed for T cell migration.

Actins↗
PubMed2001

Transplanted long-term cultured pre-BI cells expressing calpastatin are resistant to B cell receptor-induced apoptosis.

Long-term cultured pre-B cells are able to differentiate into immunoglobulin (Ig)M-positive B cells (IgM(+) cells) when transplanted into severe combined immunodeficient (SCID) mice. Based on previous studies, here we report the development of a reconstitution assay in nonobese diabetic/SCID (NOD/SCID) mice using pre-B cells, which allows us to study the role of calpains (calcium-activated endopeptidases) during B cell development as well as in B cell clonal deletion. Using this model, we show that calpastatin (the natural inhibitor of calpains) inhibits B cell receptor-induced apoptosis in IgM(+) cells derived from transplanted mice. We thus hypothesize an important function for calpain in sculpting the B cell repertoire.

Animals↗
PubMed2001

Chemokine receptor homo- or heterodimerization activates distinct signaling pathways.

Chemokine receptors of both the CC and CXC families have been demonstrated to undergo a ligand-mediated homodimerization process required for Ca2+ flux and chemotaxis. We show that, in the chemokine response, heterodimerization is also permitted between given receptor pairs, specifically between CCR2 and CCR5. This has functional consequences, as the CCR2 and CCR5 ligands monocyte chemotactic protein-1 (MCP-1) and RANTES (regulated upon activation, normal T cell-expressed and secreted) cooperate to trigger calcium responses at concentrations 10- to 100-fold lower than the threshold for either chemokine alone. Heterodimerization results in recruitment of each receptor-associated signaling complex, but also recruits dissimilar signaling path ways such as G(q/11) association, and delays activation of phosphatidyl inositol 3-kinase. The consequences are a pertussis toxin-resistant Ca2+ flux and trig gering of cell adhesion rather than chemotaxis. These results show the effect of heterodimer formation on increasing the sensitivity and dynamic range of the chemokine response, and may aid in understanding the dynamics of leukocytes at limiting chemokine concentrations in vivo.

Calcium Signaling↗
PubMed2001

Expression of CCR9 beta-chemokine receptor is modulated in thymocyte differentiation and is selectively maintained in CD8(+) T cells from secondary lymphoid organs.

Chemokines appear to have an important role in the seeding of lymphoid progenitors in the thymus, the regulation of the coordinated movements of the maturing T cells within this organ, and the egress of the resulting naive T cells to secondary lymphoid organs. CCR9, the specific receptor for the beta-chemokine TECK/CCL25, is selectively expressed in thymus, lymph node, and spleen. Using a specific anti-CCR9 polyclonal antibody, K629, and a semiquantitative reverse transcriptase-polymerase chain reaction procedure, a detailed study of CCR9 expression in the thymus and secondary lymphoid organs was performed. The results show that CD4(+)CD8(+) double-positive thymocytes have the highest CCR9 expression in thymus. Single-positive CD8(+) thymocytes continue to express this receptor after abandoning the thymus as mature naive T cells, as suggested by the existence of a CD8(+)CD69(low)CD62L(high) CCR9(+) cell subset. Consistent with this, CD8(+) lymphocytes from lymph nodes, spleen, and Peyer patches express a functional CCR9, as its expression correlates with migration in response to CCL25. Conversely, CD4(+) thymocytes lose CCR9 before abandoning the thymus, and CD4(+) T cells from secondary lymphoid organs also lack CCR9 expression. Analysis of CCR9 expression in thymocytes from mice of different ages showed that CCR9 levels are affected by age, as this receptor is more abundant, and its response to CCL25 is more potent in newborn animals. Collectively, these results suggest that CCR9 has a role in thymocyte development throughout murine life, with clear differences between the CD4(+) and CD8(+) lineages.

Aging↗
PubMed2001

The transient expression of C-C chemokine receptor 8 in thymus identifies a thymocyte subset committed to become CD4+ single-positive T cells.

Developing T cells journey through the different thymic microenvironments while receiving signals that eventually will allow some of them to become mature naive T cells exported to the periphery. This maturation can be visualized by the phenotype of the developing cells. CCR8 is a ss-chemokine receptor preferentially expressed in the thymus. We have developed 8F4, an anti-mouse CCR8 mAb that is able to neutralize the ligand-induced activation of CCR8, and used it to characterize the CCR8 protein expression in the different thymocyte subsets. Taking into account the intrathymic lineage relationships, our data showed that CCR8 expression in thymus followed two transient waves along T cell maturation. The first one took place in CD4(-) CD8(-) double-negative thymocytes, which showed a low CCR8 expression, and the second wave occurred after TCR activation by the Ag-dependent positive selection in CD4(+) CD8(+) double-positive cells. From that maturation stage, CCR8 expression gradually increased as the CD4(+) cell differentiation proceeded, reaching a maximum at the CD4(+) CD8(-) single-positive stage. These CD4(+) cells expressing CCR8 were also CD69(high) CD62L(low) thymocytes, suggesting that they still needed to undergo some differentiation step before becoming functionally competent naive T cells ready to be exported from the thymus. Interestingly, no significant amounts of CCR8 protein were detectable in CD4(-) CD8(+) thymocytes. Our data showing a clear regulation of the CCR8 protein in thymus suggest a relevant role for CCR8 in this lymphoid organ, and identify CCR8 as a possible marker of thymocyte subsets recently committed to the CD4(+) lineage.

Animals↗
PubMed2001

Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection.

We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity-determining region 1 (CDR1) of three clonally-related heavy chains from the human monovalent antigen-binding fragments Fab S19, S8 and S20 on gp120 binding and HIV-1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg(30) and Asp(31) somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg(30) and Asp(31) are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120-driven process, supporting a role for affinity maturation and intraclonal evolution of HIV-1 neutralizing antibodies.

Amino Acid Sequence↗
PubMed2001

Cell surface CD28 levels define four CD4+ T cell subsets: abnormal expression in rheumatoid arthritis.

CD28 is a costimulatory receptor expressed in most CD4(+) T cells. Despite the long-standing evidence for up- and downregulation of surface CD28 expression in vitro, and the key regulatory role assigned to the upregulation of CD28 counterreceptor [the CD152 (CTLA-4) molecule], in vivo CD28 induction has attracted little attention. We studied CD28 and CD152 expression and function in 33 rheumatoid arthritis (RA) patients, 20 clinically active and 13 inactive, and in 24 healthy donors. Four subsets of CD28(-), CD28(low), CD28(int), and CD28(high) peripheral blood human CD4(+) T cells were defined using three-color flow cytometry. The three CD28(+) subsets displayed a one-, two-, or threefold quantitative difference in their relative number of CD28 antibody binding sites, respectively (P < 0.01). RA patients, whether active or inactive, showed a distinct phenotype when compared to healthy donors: (i) the percentage of CD4(+)CD28(high) cells was increased twofold and the CD4(+)CD28(low) subset was reduced twofold (P < 0.01) and (ii) the CD4(+)CD28(high) cells from RA patients showed an in vivo activated phenotype, CD45RO(+)CD5(high)IL-2Ralpha(+) (P < 0.01). Active RA patients were different from inactive patients. They showed a twofold increase in mean CD28 expression (P < 0.05), whereas each of the CD28(+) subsets in the inactive RA patients showed reduced expression when compared to healthy donors. Notably, both active and inactive RA patients showed abnormal CD28 upregulation when T cells were activated in vitro with CD3 antibodies, but only inactive RA patients showed a hypoproliferative response to TCR/CD3 triggering when compared to healthy donors (P < 0.01). This defective proliferation was normalized by concurrent crosslinking with CD28 antibody. No differences were noted in the expression of CD152 or CD80, a CD28 and CD152 shared ligand. The disregulated in vivo expression of CD28 was related to the RA patients' disease activity and suggests that modulation of CD28 surface levels may be an additional mechanism to finely tune the delicate responsiveness/tolerance balance.

Arthritis, Rheumatoid↗
PubMed2001

Membrane raft microdomains in chemokine receptor function.

Cell chemotaxis requires the acquisition and maintenance of both spatial and functional asymmetry between initially equivalent cell parts. In leukocytes one becomes the leading edge and the other, the rear edge or uropod. The acquisition of this cell polarity is controlled by an array of chemoattractants, including those of the chemokine family. We propose that chemokine receptor activation in highly organized lipid raft domains is a major determinant for the correct localization of the signaling pathways leading to the cell asymmetries required for migration. The lateral organization imposed by membrane raft microdomains is discussed in the context of other chemokine receptor activities, such as its role as a human immunodeficiency virus (HIV) coreceptor.

Animals↗
PubMed2001

Chemokine receptor dimerization: two are better than one.

The chemokines participate in an exceptional range of physiological and pathological processes, including the control of lymphocyte trafficking, tumor growth, wound healing, allograft rejection, regulation of T-cell differentiation, asthma, infection with HIV and atherosclerosis. This vast array of activities is triggered by the interaction of nearly 50 different chemokines with a relatively modest number of 20 G-protein-coupled receptors. The asymmetry between the number of receptors and ligands suggests an underlying, shared control mechanism activated at a very early stage of the response. One of the first events triggered by the binding of chemokines is the homo- and hetero-dimerization of their receptors; here, we outline these events and their consequences in chemokine signaling.

Animals↗
PubMed2001

Mammalian STAG3 is a cohesin specific to sister chromatid arms in meiosis I.

Cohesins, which have been characterized in budding yeast and Xenopus, are multisubunit protein complexes involved in sister chromatid cohesion. Regulation of the interactions among different cohesin subunits and the assembly/disassembly of the cohesin complex to chromatin are key steps in chromosome segregation. We previously characterized the mammalian STAG3 protein as a component of the synaptonemal complex that is specifically expressed in germinal cells, although its function in meiosis remains unknown. Here we show that STAG3 has a role in sister chromatid arm cohesion during mammalian meiosis I. Immunofluorescence results in prophase I cells suggest that STAG3 is a component of the axial/lateral element of the synaptonemal complex. In metaphase I, STAG3 is located at the interchromatid domain and is absent from the chiasma region. In late anaphase I and the later stages of meiosis, STAG3 is not detected. STAG3 interacts with the structural maintenance chromosome proteins SMC1 and SMC3, which have been reported to be subunits of the mitotic cohesin complex. We propose that STAG3 is a sister chromatid arm cohesin that is specific to mammalian meiosis I.

Animals↗
PubMed2001

A role for chemokine receptor transactivation in growth factor signaling.

Complex cell responses require the integration of signals delivered through different pathways. We show that insulin-like growth factor (IGF)-I induces specific transactivation of the Gi-coupled chemokine receptor CCR5, triggering its tyrosine phosphorylation and Galpha recruitment. This transactivation occurs via a mechanism involving transcriptional upregulation and secretion of RANTES, the natural CCR5 ligand. CCR5 transactivation is an essential downstream signal in IGF-I-induced cell chemotaxis, as abrogation of CCR5 function with a transdominant-negative KDELccr5A32 mutant abolishes IGF-I-induced migration. The relevance of this transactivation pathway was shown in vivo, as KDELccr5A32 overexpression prevents invasion by highly metastatic tumor cells; conversely, RANTES overexpression confers built-in invasive capacity on a non-invasive tumor cell line. Our results suggest that this extracellular growth factor-chemokine network represents a general mechanism connecting tumorigenesis and inflammation.

Breast Neoplasms↗
PubMed2001

Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation.

A broad array of biological responses, including cell polarization, movement, immune and inflammatory responses, and prevention of HIV-1 infection, are triggered by the chemokines, a family of structurally related chemoattractant proteins that bind to specific seven-transmembrane receptors linked to G proteins. Here we discuss one of the early signaling pathways activated by chemokines, the JAK/STAT pathway. Through this pathway, and possibly in conjunction with other signaling pathways, the chemokines promote changes in cellular morphology, collectively known as polarization, required for chemotactic responses. The polarized cell expresses the chemokine receptors at the leading cell edge, to which they are conveyed by rafts, a cholesterol-enriched membrane fraction fundamental to the lateral organization of the plasma membrane. Finally, the mechanisms through which the chemokines promote their effect are discussed in the context of the prevention of HIV-1 infection.

Cell Movement↗
PubMed2001

CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses.

CCR6 expression in dendritic, T, and B cells suggests that this beta-chemokine receptor may regulate the migration and recruitment of antigen-presenting and immunocompetent cells during inflammatory and immunological responses. Here we demonstrate that CCR6-/- mice have underdeveloped Peyer's patches, in which the myeloid CD11b+ CD11c+ dendritic-cell subset is not present in the subepithelial dome. CCR6-/- mice also have increased numbers in T-cell subpopulations within the intestinal mucosa. In 2,4-dinitrofluorobenzene-induced contact hypersensitivity (CHS) studies, CCR6-/- mice developed more severe and more persistent inflammation than wild-type (WT) animals. Conversely, in a delayed-type hypersensitivity (DTH) model induced with allogeneic splenocytes, CCR6-/- mice developed no inflammatory response. The altered responses seen in the CHS and DTH assays suggest the existence of a defect in the activation and/or migration of the CD4(+) T-cell subsets that downregulate or elicit the inflammation response, respectively. These findings underscore the role of CCR6 in cutaneous and intestinal immunity and the utility of CCR6-/- mice as a model to study pathologies in these tissues. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Animals↗