RESUMO
OBJECTIVE: The current study aimed to evaluate the contribution(s) of specific opioid receptor systems to the analgesic and detrimental effects of morphine, observed after spinal cord injury in prior studies. STUDY DESIGN: We used specific opioid receptor agonists to assess the effects of µ- (DAMGO), δ- (DPDPE) and κ- (GR89696) opioid receptor activation on locomotor (Basso, Beattie and Bresnahan scale, tapered beam and ladder tests) and sensory (girdle, tactile and tail-flick tests) recovery in a rodent contusion model (T12). We also tested the contribution of non-classic opioid binding using [+]- morphine. METHODS: First, a dose-response curve for analgesic efficacy was generated for each opioid agonist. Baseline locomotor and sensory reactivity was assessed 24 h after injury. Subjects were then treated with an intrathecal dose of a specific agonist and re-tested after 30 min. To evaluate the effects on recovery, subjects were treated with a single dose of an agonist and both locomotor and sensory function were monitored for 21 days. RESULTS: All agonists for the classic opioid receptors, but not the [+]- morphine enantiomer, produced antinociception at a concentration equivalent to a dose of morphine previously shown to produce strong analgesic effects (0.32 µmol). DAMGO and [+]- morphine did not affect long-term recovery. GR89696, however, significantly undermined the recovery of locomotor function at all doses tested. CONCLUSIONS: On the basis of these data, we hypothesize that the analgesic efficacy of morphine is primarily mediated by binding to the classic µ-opioid receptor. Conversely, the adverse effects of morphine may be linked to activation of the κ-opioid receptor. Ultimately, elucidating the molecular mechanisms underlying the effects of morphine is imperative to develop safe and effective pharmacological interventions in a clinical setting. SETTING: USA. SPONSORSHIP: Grant DA31197 to MA Hook and the NIDA Drug Supply Program.
Assuntos
Analgésicos Opioides/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Análise de Variância , Animais , Peso Corporal/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/uso terapêutico , D-Penicilina (2,5)-Encefalina/uso terapêutico , Locomoção/efeitos dos fármacos , Masculino , Morfina/uso terapêutico , Medição da Dor , Piperazinas/uso terapêutico , Pirrolidinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
OBJECTIVE: We report the prevalence, characteristics and clinical outcomes of women with sexually transmitted infections (STIs) in pregnancy in the Western Sydney Local Health District (WSLHD) serving a large culturally and socio-economically diverse community in New South Wales (NSW), Australia, over the last 10 years. METHODS: A retrospective cohort study of all pregnant women booked for antenatal care at three hospitals in WSLHD between September 2012 and August 2022 inclusive. Characteristics and birth outcomes associated with STIs diagnosed in pregnancy (chlamydia, gonorrhea, and syphilis) are reported using multivariable logistic regression adjusting for relevant confounders. RESULTS: During 2012-2022, there were 102 905 births and 451 women (0.44%) with an STI diagnosis during pregnancy. The number of women with a history of chlamydia prior to their current pregnancy has increased over the last 10 years (P < 0.001). STIs in pregnancy were more common in younger women aged <20 years (adjusted odds ratio [aOR] 7.30, 95% confidence interval [CI] 5.04-10.57), 20-24 years (aOR 3.12, 95% CI 2.46-3.96), and >40 years (adj OR 1.67, 95% CI 1.07-2.59), in women with body mass index >30 (aOR 1.73, 95%CI 1.37-2.19), and those who smoked (aOR 2.24, 95% CI 1.71-2.94) and consumed alcohol (aOR 3.14, 95% CI 1.88-5.23) and illicit drugs (aOR 2.10, 95% CI 1.31-3.36). STIs in pregnancy were borderline associated with stillbirth (aOR 2.19 95% CI 0.90-5.36) but did not have a significant impact on preterm birth (aOR 1.21, 95% CI 0.87-1.68), admission to neonatal intensive care unit (NICU) (aOR 1.02, 95% CI 0.77-1.34), or having a small-for-gestational-age (SGA) baby (aOR 0.97, 95% CI 0.74-1.27). CONCLUSIONS: Sociodemographic factors such as age, weight, smoking, and alcohol and drug use, were associated with the STI incidence in pregnancy. While the latter did not have an impact on preterm birth, NICU admission, and SGA in our cohort, there was a borderline association with stillbirth. Future research should identify barriers and facilitators to testing in a multicultural population and understanding the drivers of higher rates of STIs in certain population groups.
Assuntos
Complicações Infecciosas na Gravidez , Resultado da Gravidez , Infecções Sexualmente Transmissíveis , Humanos , Feminino , Gravidez , Adulto , Estudos Retrospectivos , Prevalência , Complicações Infecciosas na Gravidez/epidemiologia , New South Wales/epidemiologia , Adulto Jovem , Infecções Sexualmente Transmissíveis/epidemiologia , Resultado da Gravidez/epidemiologia , Infecções por Chlamydia/epidemiologia , Modelos Logísticos , Gonorreia/epidemiologia , Sífilis/epidemiologia , Cuidado Pré-Natal/estatística & dados numéricos , Nascimento Prematuro/epidemiologia , Adolescente , Fatores de Risco , Natimorto/epidemiologia , Recém-NascidoRESUMO
Microorganisms express a family of cell-surface adhesins that specifically recognize and bind components of the extracellular matrix. Adhesion of microorganisms to host tissues represents a critical phase in the development of many types of infections. Recent studies have focused on the mechanisms of microbial attachment at a molecular level, including the identification of ligand-binding domains in several cell-surface adhesins from Gram-positive bacteria and the construction of adhesin-deficient isogenic mutants.
Assuntos
Adesinas Bacterianas/fisiologia , Adesão Celular/fisiologia , Matriz Extracelular/fisiologia , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/etiologia , Colágeno/metabolismo , Desenho de Fármacos , Fibronectinas/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Virulência/fisiologiaRESUMO
STUDY DESIGN: This was designed as an experimental study. OBJECTIVES: Locomotor training is one of the most effective strategies currently available for facilitating recovery of function after an incomplete spinal cord injury (SCI). However, there is still controversy regarding the timing of treatment initiation for maximal recovery benefits. To address this issue, the present study compares the effects of exercise initiated in the acute and secondary phase of SCI. SETTING: Texas A&M University, College Station, TX, USA. METHODS: Rats received a moderate spinal contusion injury and began an exercise program 1 (D1-EX) or 8 days (D8-EX) later. They were individually placed into transparent exercise balls for 60 min per day, for 14 consecutive days. Control rats were placed in exercise balls that were rendered immobile. Motor and sensory recovery was assessed for 28 days after injury. RESULTS: The D1-EX rats recovered significantly more locomotor function (BBB scale) than controls and D8-EX rats. Moreover, analyses revealed that rats in the D8-EX group had significantly lower tactile reactivity thresholds compared with control and D1-EX rats, and symptoms of allodynia were not reversed by exercise. Rats in the D8-EX group also had significantly larger areas of damage across spinal sections caudal to the injury center compared with the D1-EX group. CONCLUSION: These results indicate that implementing an exercise regimen in the acute phase of SCI maximizes the potential for recovery of function.
Assuntos
Terapia por Exercício/métodos , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/reabilitação , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/diagnóstico , Traumatismos da Medula Espinal/fisiopatologia , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVE: To characterize the in vivo role epiphycan (Epn) has in cartilage development and/or maintenance. METHODS: Epn-deficient mice were generated by disrupting the Epn gene in mouse embryonic stem cells. Epn/biglycan (Bgn) double-deficient mice were produced by crossing Epn-deficient mice with Bgn-deficient mice. Whole knee joint histological sections were stained using van Gieson or Fast green/Safranin-O to analyze collagen or proteoglycan content, respectively. Microarray analysis was performed to detect gene expression changes within knee joints. RESULTS: Epn-deficient and Epn/Bgn double-deficient mice appeared normal at birth. No significant difference in body weight or femur length was detected in any animal at 1 month of age. However, 9-month Epn/Bgn double-deficient mice were significantly lighter and had shorter femurs than wild type mice, regardless of gender. Male Epn-deficient mice also had significantly shorter femurs than wild type mice at 9 months. Most of the deficient animals developed osteoarthritis (OA) with age; the onset of OA was observed earliest in Epn/Bgn double-deficient mice. Message RNA isolated from Epn/Bgn double-deficient knee joints displayed increased matrix protein expression compared with wild type mice, including other small leucine-rich proteoglycan (SLRP) members such as asporin, fibromodulin and lumican. CONCLUSION: Similar to other previously studied SLRPs, EPN plays an important role in maintaining joint integrity. However, the severity of the OA phenotype in the Epn/Bgn double-deficient mouse suggests a synergy between these two proteins. These data are the first to show a genetic interaction involving class I and class III SLRPs in vivo.
Assuntos
Articulação do Joelho/química , Osteoartrite do Joelho/fisiopatologia , Proteoglicanas/análise , Proteoglicanas/deficiência , Animais , Southern Blotting , Peso Corporal , Colágeno/análise , Fêmur/anatomia & histologia , Imuno-Histoquímica , Articulação do Joelho/patologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Osteoartrite do Joelho/genética , Fenótipo , Reação em Cadeia da Polimerase , Proteoglicanas/genética , RNA Mensageiro/análiseRESUMO
Endothelial 1 (E1) is identified as an endothelial cell secreted factor that stimulates collagen gel contraction by fibroblasts. This identification is based on (a) co-localization of stimulatory activity in endothelial cell conditioned media with synthetic E1 in reversed phase analysis; (b) removal of the activity from conditioned media with antiserum directed against E1; and (c) the activity of synthetic E1. Treatment of endothelial cell conditioned media with immobilized anti-E1 antibodies removed 59% of the activity from the pool suggesting that E1 is the major contraction promoter in endothelial cell conditioned medium. The mechanism of action of E1 is shown to be different from serum in that E1-promoted contraction is dependent upon the synthesis of an unknown effector protein. Synthetic E1 is shown to be a potent promoter of gel contraction with half-maximal activity occurring at 32 pM. Two other endothelins, E2 and VIC, are slightly less active than E1. A fourth endothelin species, E3, is substantially less active. A comparison of E1 with other contraction promoting peptides revealed that E1 and platelet-derived growth factor are essentially equal in specific activity, whereas TGF beta is approximately 50-fold more potent.
Assuntos
Endotelinas/fisiologia , Endotélio/fisiologia , Células Cultivadas , Colágeno , Meios de Cultura , Endotelinas/biossíntese , Endotelinas/metabolismo , Matriz Extracelular/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Recém-Nascido , Cinética , Fenômenos Fisiológicos da PeleRESUMO
We examined the mechanisms of cell attachment to fibronectin-coated substrates. Inhibition of cell attachment was obtained by species-specific antifibronectin antibodies, which presumably recognize a distinct antigenic structure in the protein located at, or in the immediate vicinity of, the cell-binding site. The inhibiting antibodies could be adsorbed on a column of Sepharose substituted with plasma fibronectin. The initial phase of cell attachment was also inhibited by addition of soluble fibronectin to the incubation medium in a reaction that exhibited specificity and concentration dependence. These data suggest that cell-binding sites are available in an active form on the surface of soluble fibronectin. However, the inhibitory effect of fibronectin was greatly enhanced by adding the protein together with heparin, heparan sulfate, collagen, or a fibronectin-binding collagen peptide (CB-7), which is consistent with an "activation" of fibronectin on binding to these matrix components. A similar activation of fibronectin was obtained by cleaving the protein with trypsin. We discuss these findings in relation to conformational rearrangements in the fibronectin molecule. Data is presented supporting a mechanism of cell attachment to fibronectin involving multiple weak interactions between cellular receptors and substrate molecules, although some steps in the attachment process appear to disobey the requirements for such a mechanism.
Assuntos
Adesão Celular , Matriz Extracelular/fisiologia , Fibronectinas/fisiologia , Animais , Células Cultivadas , Colágeno/fisiologia , Fibronectinas/imunologia , Laminina/fisiologia , Fígado , Ratos , Receptores de Superfície Celular/fisiologia , TemperaturaRESUMO
We examined the effects of thrombospondin (TSP) in the substrate adhesion of bovine aortic endothelial cells. The protein was tested both as a substrate for cell adhesion and as a modulator of the later stages of the cell adhesive process. TSP substrates supported the attachment of some BAE cells, but not cell spreading or the formation of focal adhesion plaques. In contrast, cells seeded on fibrinogen or fibronectin substrates were able to complete the adhesive process, as indicated by the formation of focal adhesion plaques. Incubation of cells in suspension with soluble TSP before or at the time of seeding onto fibronectin substrates resulted in an inhibition of focal adhesion formation. Furthermore, the addition of TSP to fully adherent cells in situ or prespread on fibronectin substrates caused a reduction in the number of cells, which were positive for focal adhesions, although there was no significant effect on cell spreading. In a dose-dependent manner, TSP reduced the number of cells with adhesion plaques to approximately 60% of control levels. The distribution of remaining adhesion plaques in TSP-treated cells was also altered: plaques were primarily limited to the periphery of cells and were not present in the central cell body, as in control cells treated with BSA. The observed effects were specific for TSP and were not observed with platelet factor 4, beta-thromboglobulin, or fibronectin. The TSP-mediated loss of adhesion plaques was neutralized by the addition of heparin, fucoidan, other heparin-binding proteins, and by a monoclonal antibody to the heparin binding domain of TSP, but not by antibodies to the core or carboxy-terminal regions of TSP. The interaction of the heparin-binding domain of TSP with cell-associated heparan sulfate appears to be an important mechanistic component for this activity of TSP. These data indicate that TSP may have a role in destabilizing cell adhesion through prevention of focal adhesion formation and by loss of preformed focal adhesions.
Assuntos
Endotélio Vascular/citologia , Glicoproteínas de Membrana/fisiologia , Animais , Plaquetas/fisiologia , Bovinos , Adesão Celular , Células Cultivadas , Endotélio Vascular/fisiologia , Fibrinogênio/fisiologia , Fibronectinas/fisiologia , Heparitina Sulfato/metabolismo , Cinética , Ligação Proteica , TrombospondinasRESUMO
A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl-Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.
Assuntos
Glicosaminoglicanos/análise , Heparitina Sulfato/análise , Neoplasias Hepáticas Experimentais/análise , Proteoglicanas/análise , Animais , Linhagem Celular , Transformação Celular Neoplásica/patologia , Fibronectinas/metabolismo , Heparitina Sulfato/biossíntese , Microssomos/metabolismo , Proteoglicanas/biossíntese , RatosRESUMO
Bovine aortic endothelial cells (BAEC), grown in vitro, are shown to synthesize and secrete factor(s) that stimulate fibroblasts to contract collagen matrices. The amount of contraction-promoting activity in the conditioned media is dependent on conditioning time and the number of cells in the culture. Production of the contraction-promoting activity continues at a high stable level for at least 5 d in serum-free medium but is abolished when the cells are exposed to an inhibitor of protein synthesis. The mechanism of action of the contraction factor(s) derived from endothelial cells was compared with that of unidentified serum factors. The endothelial cell-secreted factor(s) depends on active protein synthesis by the target cell but does not need to be present during the contraction process. The serum factors on the other hand promote collagen contraction in the absence of de novo protein synthesis but need to be continuously present. Preliminary biochemical characterization of the contraction-promoting factors produced by endothelial cells revealed properties similar to those of previously identified growth factors. However, the BAEC-secreted factor was found to be distinct from a previously identified contraction-promoting transforming growth factor beta.
Assuntos
Endotélio Vascular/citologia , Fibroblastos/fisiologia , Animais , Fatores Biológicos/farmacologia , Bovinos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia de Afinidade , Colágeno/análise , Colágeno/metabolismo , Meios de Cultura/análise , Meios de Cultura/farmacologia , Endotélio Vascular/análise , Endotélio Vascular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Géis , Humanos , Camundongos , Neuroglia/citologia , Neuroglia/metabolismo , Ratos , Fatores de TempoRESUMO
In previous studies rat hepatocytes have been shown to adhere to substrates composed of collagen or fibronectin. In the present communication, the basement membrane protein laminin is reported to mediated the attachment and spreading of hepatocytes. The cell attachment-mediating activity of laminin was compared with that of fibronectin. The activity of fibronectin was heat sensitive, whereas laminin retained its activity after boiling. On the other hand, reduction and alkylation or periodate oxidation of the proteins affected only the cell attachment activity of laminin. Preincubation of cells with soluble fibronectin inhibited initial cell attachment to fibronectin but not to laminin substrates, and, reversely, soluble laminin selectively inhibited cell attachment to laminin. These results suggest that attachment of cells to substrates of the two proteins involves different cellular receptors recognizing distinct and nonidentical structures in the proteins.
Assuntos
Adesão Celular , Fibronectinas/fisiologia , Glicoproteínas/fisiologia , Animais , Movimento Celular , Temperatura Alta , Laminina , Fígado/citologia , Masculino , Ácido Periódico , RatosRESUMO
Human embryonic skin fibroblasts have been shown to attach and spread on laminin substrates in the absence of protein synthesis and presence of fibronectin-depleted serum and anti-fibronectin antibodies. Rates of attachment and the type of spreading are virtually identical on fibronectin and laminin-coated substrates with the development of microfilament bundles and focal adhesions. Antibodies to laminin, but not fibronectin, will prevent or reverse fibroblast adhesion to laminin, whereas antibodies to fibronectin but not laminin will give similar results on fibronectin-coated substrates. These and other results indicate that fibroblasts possess distinct receptors for laminin and fibronectin which on contact with suitable substrates promote adhesion through interaction with common intermediates. This type of adhesion is compatible with subsequent growth and extracellular matrix production.
Assuntos
Adesão Celular , Divisão Celular , Glicoproteínas/fisiologia , Anticorpos , Movimento Celular , Células Cultivadas , Citoesqueleto/ultraestrutura , Fibroblastos , Fibronectinas/metabolismo , Glicoproteínas/imunologia , Humanos , Laminina , Pró-Colágeno/metabolismo , PeleRESUMO
The distribution of cell surface heparan sulfate proteoglycans (HSPGs) was determined in rat liver by immunocytochemistry. A polyclonal antibody was raised against HSPGs purified from rat liver microsomes which specifically immunoprecipitated liver membrane HSPGs. It was shown to recognize both the heparin-releasable and membrane-intercalated form of membrane HSPGs and to recognize determinants on the core protein of these HSPGs. By immunocytochemistry membrane HSPGs were localized to hepatocytes. The distribution of HSPGs at the cell surface of the hepatocyte was restricted to the sinusoidal domain of the plasmalemma; there was little or no staining of the lateral or bile canalicular domains. Intracellularly, HSPGs were occasionally detected in cisternae of the rough endoplasmic reticulum and were regularly found in Golgi cisternae--usually distributed across the entire Golgi stack. HSPGs were also localized in some endosomes, lysosomes, and cytoplasmic vesicles of hepatocytes. We conclude that the HSPGs recognized by this antibody have a restricted distribution in rat liver: they are largely confined to the sinusoidal plasmalemmal domain and to biosynthetic and endocytic compartments of hepatocytes.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Fígado/metabolismo , Proteoglicanas/metabolismo , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteoglicanas de Heparan Sulfato , Histocitoquímica , Fígado/ultraestrutura , Microscopia Eletrônica , Organoides/metabolismo , RatosRESUMO
We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan-deficient cells did not attach to substrate composed of a heparin-binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.
Assuntos
Adesão Celular , Fibronectinas/metabolismo , Glicosaminoglicanos/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Linhagem Celular , Microscopia de Interferência , MutaçãoRESUMO
The distribution of heparan sulfate proteoglycans (HSPG) on cultured fibroblasts was monitored using an antiserum raised against cell surface HSPG from rat liver. After seeding, HSPG was detected by immunofluorescence first on cell surfaces and later in fibrillar deposits of an extracellular matrix. Cell surface HSPG aligned with microfilament bundles of rat embryo fibroblasts seen by phase-contrast microscopy but was diffuse on transformed rat dermal fibroblasts (16C cells) which lack obvious stress fibers. Focal adhesions isolated from either cell type and monitored by interference reflection microscopy showed a concentration of HSPG labeling with respect to the rest of the membrane. Increased labeling in these areas was also seen for fibronectin (FN) by using an antiserum that detects both plasma and cell-derived FN. Double immunofluorescent staining of fully adherent rat embryo fibroblast cells showed some co-distribution of HSPG and FN, and this was confirmed by immunoelectron microscopy, which detected HSPG at localized areas of dorsal and ventral cell membranes, overlapping cell margins, and in the extracellular matrix. During cell shape changes on rounding and spreading, HSPG and FN may not co-distribute. Double labeling for actin and either HSPG or FN showed a closer correlation of actin with HSPG than with FN. The studies are consistent with HSPG being closely involved in a transmembrane cytoskeletal-matrix interaction; the possibility that HSPG coordinates the deposition of FN and other matrix components with cytoskeletal organization is discussed.
Assuntos
Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Proteoglicanas/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Embrião de Mamíferos , Fibronectinas/metabolismo , Imunofluorescência , Técnicas de Imunoadsorção , Microscopia Eletrônica , RatosRESUMO
Cell adhesion is a process which is initiated by the attachment of cells to specific sites in adhesive matrix proteins via cell surface receptors of the integrin family. This is followed by a reorganization of cytoskeletal elements which results in cell spreading and the formation of focal adhesion plaques. We have examined the effects of a class of small galactosaminoglycan-containing proteoglycans on the various stages of cell adhesion to fibronectin-coated substrates. Our results indicate that dermatan sulfate proteoglycans (DSPGs) derived from cartilage, as well as other related small proteoglycans, inhibit the initial attachment of CHO cells and rat embryo fibroblasts to substrates composed of the 105-kD cell-binding fibronectin fragment, but do not affect cell attachment to intact fibronectin. Although this effect involves binding of DSPGs to the substrate via the protein core, the intact proteoglycan is necessary for the observed activity. Isolated core proteins are inactive. The structural composition of the galactosaminoglycan chain does not appear to be functionally significant since both chondroitin sulfate and various dermatan sulfate proteoglycans of this family inhibit cell attachment to the fibronectin fragment. Neither the percentage of cells spread nor the mean area of spread cells adhering to substrates of intact fibronectin was significantly affected by the DSPGs. However, significantly fewer cells formed focal adhesions in the presence of DSPGs as compared with untreated control cells. These results suggest that the binding of small galactosaminoglycan-containing proteoglycans to a fibronectin substrate may affect several stages in the cell adhesion process.
Assuntos
Adesão Celular/fisiologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/metabolismo , Polissacarídeos/metabolismo , Proteoglicanas/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetinae , Decorina , Proteínas da Matriz Extracelular , Fibronectinas/metabolismo , Imunofluorescência , Cinética , Ensaio Radioligante , RatosRESUMO
The pericellular matrix of human fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. The matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the major glycosaminoglycan components and fibronectin and procollagen as major polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix.
Assuntos
Espaço Extracelular/análise , Linhagem Celular , Sulfatos de Condroitina/análise , Espaço Extracelular/ultraestrutura , Fibroblastos , Imunofluorescência , Glicoproteínas/análise , Heparitina Sulfato/análise , Humanos , Ácido Hialurônico/análise , Microscopia Eletrônica de Varredura , Pró-Colágeno/análiseRESUMO
Tenascin, together with thrombospondin and SPARC, form a family of matrix proteins that, when added to bovine aortic endothelial cells, caused a dose-dependent reduction in the number of focal adhesion-positive cells to approximately 50% of albumin-treated controls. For tenascin, a maximum response was obtained with 20-60 micrograms/ml of protein. The reduction in focal adhesions in tenascin-treated spread cells was observed 10 min after addition of the adhesion modulator, reached the maximum by 45 min, and persisted for at least 4 h in the continued presence of tenascin. This effect was fully reversible, was independent of de novo protein synthesis, and was neutralized by a polyclonal antibody to tenascin. Monoclonal antibodies to specific domains of tenascin (mAbs 81C6 and 127) were used to localize the active site to the alternatively spliced segment of tenascin. Furthermore, a recombinant protein corresponding to the alternatively spliced segment (fibronectin type III domains 6-12) was expressed in Escherichia coli and was active in causing loss of focal adhesions, whereas a recombinant form of a domain (domain 3) containing the RGD sequence had no activity. Chondroitin-6-sulfate effectively neutralized tenascin activity, whereas dermatan sulfate and chondroitin-4-sulfate were less active and heparan sulfate and heparin were essentially inactive. Studies suggest that galactosaminoglycans neutralize tenascin activity through interactions with cell surface molecules. Overall, our results demonstrate that tenascin, thrombospondin, and SPARC, acting as soluble ligands, are able to provoke the loss of focal adhesions in well-spread endothelial cells.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Endotélio Vascular/citologia , Proteínas da Matriz Extracelular/metabolismo , Splicing de RNA , Actinas/metabolismo , Animais , Anticorpos , Aorta/citologia , Bovinos , Adesão Celular , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/imunologia , Linhagem Celular , Sulfatos de Condroitina/metabolismo , Clonagem Molecular , Regulação para Baixo , Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/imunologia , Glicosaminoglicanos/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Tenascina , Vinculina/metabolismoRESUMO
A cDNA encoding a unique hyaluronan receptor has been molecularly cloned from a lambda GT11 3T3 cDNA expression library. Immunoblot analyses of cell lysates, using antibodies to peptides encoded in the cDNA, specifically react with a 58-kD protein. This protein is regulated by the mutant H-ras gene in cells containing a metallothionein promoter H-ras hybrid gene. Further, antibodies to peptide sequences encoded in the cDNA block the increase in locomotion resulting from induction of the mutant H-ras gene in this cell line. In a transblot assay, the bacterially expressed protein binds to biotinylated hyaluronan. Antibodies to peptides encoded in the cDNA react in immunoblot assays with the 58- and 52-kD proteins of a novel hyaluronan receptor complex previously implicated in cell locomotion. Furthermore, antibodies specific to the 58- and 52-kD proteins, which block ras-induced locomotion, also cross-react with the expressed, encoded protein. The gene product described here appears to be a new type of hyaluronan receptor that is involved in cell locomotion. It is named RHAMM, an acronym for receptor for hyaluronan-mediated motility.
Assuntos
Movimento Celular , Ácido Hialurônico , Receptores de Superfície Celular/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA , Escherichia coli/genética , Imunofluorescência , Regulação da Expressão Gênica , Genes ras , Receptores de Hialuronatos , Camundongos , Dados de Sequência Molecular , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Mapeamento por Restrição , Células Tumorais CultivadasRESUMO
Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. Morbidity and mortality due to infections such as sepsis, osteomyelitis, septic arthritis, and invasive endocarditis remain high despite the use of antibiotics. The emergence of antibiotic resistant super bugs mandates that alternative strategies for the prevention and treatment of S. aureus infections are developed. We investigated the ability of vaccination with a recombinant fragment of the S. aureus collagen adhesin to protect mice against sepsis-induced death. Actively immunized NMRI mice were intravenously inoculated with the S. aureus clinical isolate strain Phillips. 14 d after inoculation, mortality in the collagen adhesin-vaccinated group was only 13%, compared with 87% in the control antigen immunized group (P < 0.001). To determine if the protective effect was antibody mediated, we passively immunized naive mice with collagen adhesin-specific antibodies. Similar to the active immunization strategy, passive transfer of collagen adhesin-specific antibodies protected mice against sepsis-induced death. In vitro experiments indicated that S. aureus opsonized with sera from collagen adhesin immunized mice promoted phagocytic uptake and enhanced intracellular killing compared with bacteria opsonized with sera from control animals. These results indicate that the collagen adhesin is a viable target in the development of immunotherapeutics against S. aureus.