RESUMO
OBJECTIVE: The study aimed determining whether assessment of cartilage oligomeric matrix protein (COMP) degradation products could serve as a serological disease course and therapeutic response predictor in arthritis. METHODS: We generated a panel of monoclonal antibodies against COMP fragments and developed a novel capture enzyme-linked immunosorbent assay (ELISA) for detecting COMP fragments in patients with osteoarthritis (OA) and rheumatoid arthritis (RA). This test was also used to monitor COMP fragments in surgically-induced OA, collagen-induced arthritis (CIA), and tumor necrosis factor (TNF) transgenic animal models. RESULTS: Compared with a commercial COMP ELISA kit that detected no significant difference in COMP levels between OA and control groups, a significant increase of the COMP fragments were noted in the serum of OA patients assayed by this newly established ELISA. In addition, serum COMP fragment levels were well correlated with severity in OA patients and the progression of surgically-induced OA in murine models. Furthermore, the serum levels of COMP fragments in RA patients, mice with CIA, and TNF transgenic mice were significantly higher when compared with their controls. Interestingly, treatment with TNFα inhibitors and methotrexate led to a significant decrease of serum COMP fragments in RA patients. Additionally, administration of Atsttrin [Tang, et al., Science 2011;332(6028):478] also resulted in a significant reduction in COMP fragments in arthritis mice models. CONCLUSION: A novel sandwich ELISA is capable of reproducibly measuring serum COMP fragments in both arthritic patients and rodent arthritis models. This test also provides a valuable means to utilize serum COMP fragments for monitoring the effects of interventions in arthritis.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Osteoartrite/metabolismo , Adolescente , Adulto , Animais , Antirreumáticos/farmacologia , Proteína de Matriz Oligomérica de Cartilagem , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas da Matriz Extracelular/efeitos dos fármacos , Feminino , Glicoproteínas/efeitos dos fármacos , Humanos , Masculino , Proteínas Matrilinas , Metotrexato/farmacologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/farmacologia , Líquido Sinovial/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adulto JovemRESUMO
The ability of purified extracellular matrix components to promote the initial migration of amphibian neural crest (NC) cells was quantitatively investigated in vitro. NC cells migrated avidly on fibronectin (FN), displaying progressively more extensive dispersion at increasing amounts of material incorporated in the substrate. In contrast, dispersion on laminin substrates was optimal at low protein concentrations but strongly reduced at high concentrations. NC cells were unable to migrate on substrates containing a high molecular mass chondroitin sulfate proteoglycan (ChSP). When proteolytic peptides, representing isolated functional domains of the FN molecule, were tested as potential migration substrates, the cell binding region of the molecule (105 kD) was found to be as active as the intact FN. A 31-kD heparin-binding fragment also stimulated NC cell migration, whereas NC cells dispersed to a markedly lower extent on the isolated collagen-binding domain (40 kD), or the latter domain linked to the NH2-terminal part of the FN molecule. Migration on the intact FN was partially inhibited by antibodies directed against the 105- and 31-kD fragments, respectively; dispersion was further decreased when the antibodies were used in combination. Addition of the ChSP to the culture medium dramatically perturbed NC cell migration on substrates of FN, as well as of 105- or 31-kD fragments. However, preincubation of isolated cells or substrates with ChSP followed by washing did not affect NC cell movement. The use of substrates consisting of different relative amounts of ChSP and the 105-kD peptide revealed that ChSP counteracted the motility-promoting activity of the 105-kD FN fragment in a concentration-dependent manner also when bound to the substrate. Our results indicate that NC cell migration on FN involves two separate domains of the molecule, and that ChSP can modulate the migratory behavior of NC cells moving along FN-rich pathways and may therefore influence directionally and subsequent localization of NC cells in the embryo.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/farmacologia , Matriz Extracelular/fisiologia , Crista Neural/fisiologia , Proteoglicanas/farmacologia , Ambystoma , Animais , Antígenos de Superfície , Adesão Celular , Moléculas de Adesão Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Embrião não Mamífero , Matriz Extracelular/ultraestrutura , Fibronectinas , Cinética , Crista Neural/citologiaRESUMO
Membrane microcarriers were used to determine the ability of regional extracellular matrices to direct neural crest cell differentiation in culture. Neural crest cells from the axolotl embryo responded to extracellular matrix material explanted from the subepidermal migratory pathway by dispersing and by differentiating into pigment cells. In contrast, matrix material from the presumptive site of dorsal root ganglia stimulated pronounced cell-cell association and neurotypic expression. Cell line segregation during ontogeny of the neural crest that leads to diversification into pigment cells of the skin or into elements of the peripheral nervous system appears to be controlled in part by local cell-matrix interactions.
Assuntos
Matriz Extracelular/fisiologia , Crista Neural/citologia , Fenótipo , Ambystoma mexicanum/embriologia , Animais , Antígenos de Superfície/análise , Adesão Celular , Moléculas de Adesão Celular , Agregação Celular , Diferenciação Celular , Células Cultivadas , Epiderme/fisiologia , Células Epiteliais , Gânglios Espinais/embriologia , Gânglios Espinais/fisiologia , Melanócitos/citologia , Neurônios/citologia , Pigmentos Biológicos/metabolismoRESUMO
The peripheral nervous system is created by a spatiotemporally co-ordinated migratory process during which the precursor cells, the neural crest (NC) cells, transverse the embryo to reach distantly located sites. Original transplantation experiments implicated the extracellular matrix (ECM) as a pivotal factor in the regulation of this process, and subsequent in vitro and in vivo studies have uncovered a number of ECM molecules potentially responsible for the NC cell-ECM interaction. Recent genetic manipulations in mice sustain the importance of certain matrix constituents, while precluding a significant role for others and, surprisingly, for all primary integrin receptors expressed by NC cells. The gradually crystallizing paradigm envisions that guidance of the disseminating NC cells, as well as the arrest at their final tissue locations, is governed by specific 'inhibitory' ECM-associated signals. This implies that homing of peripheral neurons and their supportive cells might be dictated by a delicate equilibrium between the multiple actions of stimulatory and inhibitory molecules, which is modulated further by defined responses of the dispersing cells to these ECM components during their successive phases of phenotypic diversification.
Assuntos
Movimento Celular/fisiologia , Matriz Extracelular/fisiologia , Crista Neural/citologia , Animais , Crista Neural/fisiologiaRESUMO
To determine whether subendothelial laminins (LNs) could be implicated in the extravasation of neoplastic lymphocytes, we have examined the distribution of a number of LN isoforms in human vascular structures of adult individuals and have assayed the ability of the isolated LN molecules to promote adhesion of lymphoma and leukemic cells in vitro using a novel cell adhesion assay, CAFCA, Centrifugal Assay for Fluorescence-based Cell Adhesion (E. Giacomello et al., Biotechniques, 26: 758-762, 1999; P. Spessotto et al., Methods Mol. Biol., 139: 321-343, 2000). The use of previously characterized LN chain-specific antibodies showed that the vast majority of the smaller vascular compartments, known to correspond to sites of lymphocyte transmigration, expressed the subunits involved in the structuring of 9 of the 12 LN isoforms known to date. Eight LN isoforms (i.e., LN-1, -2, -4, -5, -8, -9, -10, and -11) and four naturally occurring LN complexes were isolated from various tissues and cultured cells by combined gel filtration, ion exchange, and immunoaffinity chromatographies, and the identity/composition of the isolated LNs/LN complexes was asserted by immunochemical means and amino-acid sequencing. Notwithstanding the widespread colocalization of LN isoforms, a panel of neoplastic B- and T-cell lines and lymphocytes isolated from patients affected by chronic lymphocytic B-cell leukemia attached preferentially and with high avidity to purified LN-8, purified LN-10, and LN-10-containing protein complexes, whereas lymphocytes derived from patients diagnosed with acute lymphocytic leukemia failed to bind to these LNs. All of the tested neoplastic lymphocytes failed to adhere to the isolated LN-1, LN-4, LN-9, and LN-11 and attached moderately well to purified LN-2 and LN-5. The interaction of transformed lymphocytes with LNs was cation-dependent and interchangeably mediated by the alpha3beta1 and alpha6beta1 integrins. The degree of engagement of the two LN receptors was dependent upon their relative levels of cell surface expression, whereas, irrespective of the phenotype, lymphocytes deprived of either of these receptors were incapable of LN binding. The findings suggest that LN-8 and LN-10 may act in an independent or complementary fashion as primary components of the endothelial basement membrane favoring the interaction of extravasating neoplastic lymphocytes. Thus, our results would demonstrate that different LN isoforms may evoke diverse cellular responses in different cell types and that this divergence may be the basis for the redundancy of LN distribution in a number of vascular structures.
Assuntos
Linfócitos B/patologia , Endotélio Vascular/metabolismo , Laminina/metabolismo , Linfócitos T/patologia , Animais , Anticorpos/farmacologia , Anticorpos Monoclonais , Linfócitos B/metabolismo , Membrana Basal/metabolismo , Bovinos , Adesão Celular/fisiologia , Endotélio Vascular/citologia , Humanos , Integrinas/antagonistas & inibidores , Integrinas/imunologia , Integrinas/metabolismo , Laminina/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia de Células T/patologia , Linfoma de Células T/patologia , Linfócitos T/metabolismoRESUMO
Once specified to become neural crest (NC), cells occupying the dorsal portion of the neural tube disrupt their cadherin-mediated cell-cell contacts, acquire motile properties, and embark upon an extensive migration through the embryo to reach their ultimate phenotype-specific sites. The understanding of how this movement is regulated is still rather fragmentary due to the complexity of the cellular and molecular interactions involved. An additional intricate aspect of the regulation of NC cell movement is that the timings, modes and patterns of NC cell migration are intimately associated with the concomitant phenotypic diversification that cells undergo during their migratory phase and the fact that these changes modulate the way that moving cells interact with their microenvironment. To date, two interplaying mechanisms appear central for the guidance of the migrating NC cells through the embryo: one involves secreted signalling molecules acting through their cognate protein kinase/phosphatase-type receptors and the other is contributed by the multivalent interactions of the cells with their surrounding extracellular matrix (ECM). The latter ones seem fundamental in light of the central morphogenetic role played by the intracellular signals transduced through the cytoskeleton upon integrin ligation, and the convergence of these signalling cascades with those triggered by cadherins, survival/growth factor receptors, gap junctional communications, and stretch-activated calcium channels. The elucidation of the importance of the ECM during NC cell movement is presently favoured by the augmenting knowledge about the macromolecular structure of the specific ECM assembled during NC development and the functional assaying of its individual constituents via molecular and genetic manipulations. Collectively, these data propose that NC cell migration may be governed by time- and space-dependent alterations in the expression of inhibitory ECM components; the relative ratio of permissive versus non-permissive ECM components; and the supramolecular assembly of permissive ECM components. Six multidomain ECM constituents encoded by a corresponding number of genes appear to date the master ECM molecules in the control of NC cell movement. These are fibronectin, laminin isoforms 1 and 8, aggrecan, and PG-M/version isoforms V0 and V1. This review revisits a number of original observations in amphibian and avian embryos and discusses them in light of more recent experimental data to explain how the interaction of moving NC cells with these ECM components may be coordinated to guide cells toward their final sites during the process of organogenesis.
Assuntos
Movimento Celular , Matriz Extracelular , Crista Neural , Animais , Embrião de Galinha , Proteínas da Matriz Extracelular , Crista Neural/citologia , Crista Neural/embriologiaRESUMO
We report the molecular cloning of the human laminin alpha3b chain variant and its mRNA expression pattern in adult human tissues when compared to the alpha3a variant. The mRNA encoding for the alpha3b variant is about 11 kb and the predicted translation product carries the complete set of domains typical for a 'full-sized' laminin alpha chain. Apart from the similar domain structure of alpha3b also the sequence of alpha3 resulted more closely related to the alpha5 than to the alpha4 chain. Quantitative analysis of the RNA expression in a broad panel of adult human tissues indicated that the alpha3b variant is more widely distributed than the alpha3a shorter variant.
Assuntos
Variação Genética , Laminina/química , Laminina/genética , Adulto , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Clonagem Molecular , Humanos , Laminina/biossíntese , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
Human synovium was analyzed for the possible expression of cartilage oligomeric matrix protein (COMP). Immunostaining with polyclonal antiserum to COMP demonstrated positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. Western blot analysis using either polyclonal or monoclonal antibodies to human COMP confirmed the presence of COMP by immunoreactive bands with the same molecular mass (approximately 110 kDa) as purified articular cartilage COMP. PCR using oligonucleotides that span human COMP exons 7-13 revealed identical amplification products from cDNA prepared from either human chondrocytes or synovium. Northern blot analysis using a biotinylated-probe to human COMP, spanning exons 12-13, also reveal an identical hybridization product to either human chondrocyte or synovium total RNA. Human synovium should be considered as a potential tissue source of COMP in any investigation of biological markers of cartilage metabolism.
Assuntos
Cartilagem Articular/química , Expressão Gênica , Glicoproteínas/genética , Membrana Sinovial/metabolismo , Sequência de Bases , Northern Blotting , Western Blotting , Proteína de Matriz Oligomérica de Cartilagem , Proteínas da Matriz Extracelular , Humanos , Proteínas Matrilinas , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Membrana Sinovial/químicaRESUMO
Matrix metalloproteinase (MMP)-19 and MMP-20 (enamelysin) are two recently discovered members of the MMP family. These enzymes are involved in the degradation of the various components of the extracellular matrix (ECM) during development, haemostasis and pathological conditions. Whereas MMP-19 mRNA is found widely expressed in body tissues, including the synovium of normal and rheumatoid arthritic patients, MMP-20 expression is restricted to the enamel organ. In this study we investigated the ability of MMP-19 and MMP-20 to cleave two of the macromolecules characterising the cartilage ECM, namely aggrecan and the cartilage oligomeric matrix protein (COMP). Both MMPs hydrolysed aggrecan efficiently at the well-described MMP cleavage site between residues Asn(341) and Phe(342), as shown by Western blotting using neo-epitope antibodies. Furthermore, the two enzymes cleaved COMP in a distinctive manner, generating a major proteolytic product of 60 kDa. Our results suggest that MMP-19 may participate in the degradation of aggrecan and COMP in arthritic disease, whereas MMP-20, due to its unique expression pattern, may primarily be involved in the turnover of these molecules during tooth development.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Metaloproteinases da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Proteoglicanas/metabolismo , Agrecanas , Sequência de Aminoácidos , Animais , Artrite Reumatoide/enzimologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Western Blotting , Cartilagem/citologia , Cartilagem/enzimologia , Cartilagem/metabolismo , Proteína de Matriz Oligomérica de Cartilagem , Domínio Catalítico , Bovinos , Eletroforese em Gel de Poliacrilamida , Matriz Extracelular/química , Matriz Extracelular/enzimologia , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/química , Glicoproteínas/química , Humanos , Lectinas Tipo C , Proteínas Matrilinas , Metaloproteinase 20 da Matriz , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz Secretadas , Metaloendopeptidases/química , Metaloendopeptidases/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteoglicanas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência/genética , Especificidade por Substrato , Suínos , Dente/citologia , Dente/enzimologia , Dente/metabolismoRESUMO
Cell adhesion assays are widely used to identify novel cellular ligands, novel cell surface receptors for these ligands and to elucidate the mechanisms responsible for the underlying cellular and molecular interactions. We report here the development of a novel centrifugal assay for fluorescence-based cell adhesion (CAFCA) that offers a number of advantages over the currently available assays. CAFCA is based on two centrifugation steps: one to allow for the synchronization of the initial cell-substratum contact and one to enable both a defined removal force to be exerted onto the cells for displacement of unbound cells and determination of the relative binding strengths of adhering cells. The fluorescently tagged cells are monitored in specifically devised, disposable microplate assemblies by a two-sided fluorescence detection through the computer-interfaced SPECTRAFLUOR microplate fluorometer. The assay is rapid, accurate, reproducible and adaptable to small numbers of delicate primary cells that can ideally be labeled with the fluorochrome calcein AM (or analogous vital fluorescent dyes). Most uniquely, CAFCA provides (i) means of assessing the precise number of cells bound to a given substratum out of the total amount of cells contained within the population to be analyzed and (ii) a means of establishing the attachment strengths (i.e., dynes/cell) in a high number of samples/conditions simultaneously. CAFCA is therefore expected to make a substantial methodological and conceptual contribution to the range of available assays aimed at examining cellular interactions in vitro and promises the potential of being able to transpose automated versions of these tests for routine use in laboratories.
Assuntos
Adesão Celular , Centrifugação/métodos , Animais , Biotecnologia , Linhagem Celular , Células Cultivadas , Centrifugação/instrumentação , Meios de Cultura , Fluorescência , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , CamundongosRESUMO
A number of laminin isoforms have recently been identified and proposed to exert different functions during embryonic development. In the present study, we describe the purification and partial characterization of several isoforms isolated from chick heart and gizzard, and provide data on the molecular mechanisms underlying the interaction of avian neural crest cells with these molecules in vitro. Laminins extracted from heart and gizzard tissues were separated by gel filtration and purified to homogeneity by sequential lectin and immunoaffinity chromatography by utilizing monoclonal antibodies directed against the avian alpha 2, beta 2 and gamma 1 laminin chains. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern of the polypeptide complexes obtained and immunoblotting with polyclonal antisera allowed the identification of Laminin-2 (alpha 2 beta 1 gamma 1), Laminin-4 (alpha 2 beta 2 gamma 1), and laminins comprising the beta 1, beta 2 and gamma 1 chains associated with a shorter alpha chain which, in SDS-PAGE, co-migrate with the beta/gamma complex in the 200 kDa region. These latter laminins, which are here arbitrarily denoted Laminin-alpha x (heart tissue) and Laminin-G (gizzard tissue), are somewhat distinct in their apparent molecular weight, are differentially associated with nidogen, and appear as "T"-shaped particles similar to Laminin-6 and Laminin-7 when analyzed by transmission electron microscopy following rotary shadowing. In contrast, the avian Laminin-2 and Laminin-4 isoforms exhibit the characteristic cruciform shape described previously for their mammalian counterparts. Isolated neural crest cells differentially attached and migrated on these laminin isoforms, showing a clear preference for Laminin-G. Similarly to the EHS Laminin-1, neural crest cells recognized all avian isoforms through their alpha 1 beta 1 integrin, shown previously to be the primary laminin-binding receptor on these cells. Neural crest cell interaction with the avian laminins was dependent upon maintenance of the secondary and tertiary structure of the molecules, as shown by the marked reduction in cell attachment and migration upon disruption of the alpha-helical coiled-coil structure of their constituent chains. The results demonstrate that different laminin isoforms may be differentially involved in the regulation of neural crest cell migration and suggest that this regulation operates through interaction of the cells with a structurally conserved cell binding site recognized by the alpha 1 beta 1 integrin.
Assuntos
Laminina/fisiologia , Crista Neural/citologia , Animais , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Galinhas , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Moela das Aves/citologia , Moela das Aves/inervação , Moela das Aves/metabolismo , Immunoblotting , Imuno-Histoquímica , Isomerismo , Laminina/isolamento & purificação , Laminina/metabolismo , Microscopia Eletrônica , Crista Neural/fisiologiaRESUMO
Cartilage oligomeric matrix protein has been implicated as an important component of endochondral ossification because of its direct effects on chondrocytes. The importance of this protein for skeletal development and growth has been recently illustrated by the identification of mutations in cartilage oligomeric protein genes in two types of inherited chondrodysplasias and osteoarthritic phenotypes: multiple epiphyseal dysplasia and pseudoachondroplasia. In the present study, we report the presence of cartilage oligomeric protein in embryonic and adult osteoblasts. A foot from a 21-week-old human fetus, subchondral bone obtained from knee replacement surgery in an adult patient, and a limb from a 19-day-postcoital mouse embryo were analyzed with immunostaining and in situ hybridization. In the human fetal foot, cartilage oligomeric protein was localized to osteoblasts of the bone collar and at the newly formed bone at the growth plate and bone diaphyses. Immunostaining was performed on the adult subchondral bone and showed positive intracellular staining for cartilage oligomeric protein of the osteoblasts lining the trabecular bone. There was no staining of the osteocytes. Immunostaining of the mouse limb showed the most intense staining for cartilage oligomeric protein in the hypertrophic chondrocytes and in the surrounding osteoblast cells of the developing bone. Cartilage oligomeric protein mRNA and protein were detected in an osteoblast cell line (MG-63), and cartilage oligomeric protein mRNA was detected from human cancellous bone RNA. These results suggest that the altered structure of cartilage oligomeric protein by the mutations seen in pseudoachondroplasia and multiple epiphyseal dysplasia may have direct effects on osteoblasts, contributing to the pathogenesis of these genetic disorders.
Assuntos
Cartilagem/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Osteoblastos/metabolismo , Animais , Cartilagem/citologia , Proteína de Matriz Oligomérica de Cartilagem , Células Cultivadas , Condrócitos/metabolismo , Primers do DNA/química , Proteínas da Matriz Extracelular/genética , Feminino , Feto , Ossos do Pé/embriologia , Ossos do Pé/metabolismo , Membro Anterior/embriologia , Membro Anterior/metabolismo , Idade Gestacional , Glicoproteínas/genética , Humanos , Hibridização In Situ , Proteínas Matrilinas , Camundongos , RNA Mensageiro/metabolismoRESUMO
Mouse cartilage oligomeric matrix protein cDNA was cloned and sequenced by a reverse transcription-polymerase chain reaction. The open reading frame encoded a product of 755 amino acids that shares a high degree of identity to and possesses all the characteristic molecular features of both rat and human cartilage oligomeric matrix protein. This suggests that cartilage oligomeric matrix protein is highly conserved during evolution. The clone was 83, 84, and 95% identical to human, bovine, and rat cartilage oligomeric matrix protein cDNA, respectively. In tissues from the adult mouse, cartilage oligomeric matrix protein was expressed not only in cartilage and tendon but in trachea, bone, skeletal muscle, eye, heart, and placenta as well, and no expression was found in other tissues. Immunohistology revealed that cartilage oligomeric matrix was deposited as early as 10 days post coitus in predifferentiated mouse embryo mesenchyme. It was detected in all cartilaginous tissues and in the skeletal muscles of the embryo at day 13. As development progressed, accumulation of cartilage oligomeric matrix protein was marked in the growth plate. At 19 days post coitus, it was prominently deposited in the hypertrophic zone of the growth plate, perichondrium, and periosteum and in the superficial layer of the articular cartilage surface but was absent in the more central areas of the epiphyseal cartilage. The restricted tissue distribution and expression of cartilage oligomeric matrix protein in developing as well as adult mouse tissues suggest the regulation of this protein at the transcriptional level. The findings reported herein are the first detailed characterization of the distribution of cartilage oligomeric matrix protein during early skeletal development of the mouse.
Assuntos
Cartilagem/embriologia , Cartilagem/fisiologia , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/genética , Animais , Sequência de Bases , Northern Blotting , Cartilagem/química , Proteína de Matriz Oligomérica de Cartilagem , Clonagem Molecular , DNA Complementar , Proteínas Matrilinas , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Synovium and cartilage from patients with osteoarthritis or rheumatoid arthritis were analyzed for expression of cartilage oligomeric matrix protein. Immunostaining of synovium with antiserum to cartilage oligomeric matrix protein demonstrated positive staining in both diseases. In osteoarthritis, there was positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. In rheumatoid arthritis, there was less intense staining within the synovial cells and marked intense staining in the deeper connective tissue. In situ hybridization performed with an antisense digoxigenin-labeled riboprobe to human cartilage oligomeric matrix protein confirmed the presence of cartilage oligomeric matrix protein mRNA in the cells of the synovial lining in both types of synovium. Quantitative polymerase chain reaction with a cartilage oligomeric matrix protein MIMIC demonstrated increased cartilage oligomeric matrix protein mRNA in rheumatoid cartilage and synovium as compared with osteoarthritic cartilage and synovium, respectively; mRNA levels in rheumatoid synovium were similar to those from osteoarthritic chondrocytes. As a result of the high expression of cartilage oligomeric matrix protein from rheumatoid synovium, inflammatory synovium should be considered as a potential tissue source of cartilage oligomeric matrix protein in any investigation of biological markers of cartilage metabolism. The upregulated expression of cartilage oligomeric matrix protein in inflammatory tissues suggests its in vivo regulation by cytokines.
Assuntos
Artrite Reumatoide/metabolismo , Cartilagem Articular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Osteoartrite/metabolismo , Membrana Sinovial/metabolismo , Proteína de Matriz Oligomérica de Cartilagem , Proteínas da Matriz Extracelular/genética , Glicoproteínas/genética , Humanos , Técnicas Imunológicas , Hibridização In Situ , Proteínas Matrilinas , RNA Mensageiro/metabolismo , Distribuição Tecidual/fisiologiaRESUMO
We describe herein a novel in vitro approach that can be used effectively to obtain valuable insights into the role of platelets, various coagulation proteins as well as proteins of the subendothelial extracellular matrix involved in the hemostatic and thrombotic processes occurring under microgravity. At difference with other experimental approaches proposed in the past our device operates in a closed system and under different shear forces, which better mimics flow conditions occurring in vessels. Furthermore our device by allowing real time monitoring of the thrombotic process and its underlying mechanisms can be regarded as a reliable system for the precise assessment of platelet function.
Assuntos
Medicina Aeroespacial/instrumentação , Coagulação Sanguínea/fisiologia , Ausência de Peso , Plaquetas/fisiologia , Testes Hematológicos/instrumentação , Hemostasia/fisiologia , Humanos , Microfluídica/instrumentação , Microfluídica/métodos , Microscopia de Fluorescência , Trombose/fisiopatologiaAssuntos
Movimento Celular/fisiologia , Animais , Adesão Celular/fisiologia , Fluorescência , HumanosAssuntos
Sistemas Computacionais , Visão Ocular/efeitos da radiação , Adulto , Exposição Ambiental , Feminino , Humanos , MasculinoAssuntos
Visão Ocular , Trabalho , Acomodação Ocular , Convergência Ocular , Humanos , Testes VisuaisRESUMO
We report the experimental realization and the characterization of polarization and momentum hyperentangled two-photon states, generated by a new parametric source of correlated photon pairs. By adoption of these states an "all-versus-nothing" test of quantum mechanics was performed. The two-photon hyperentangled states are expected to find at an increasing rate a widespread application in state engineering and quantum information.