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OBJECTIVES: Universal test and treat (UTT) is recommended for people living with HIV (PLHIV) to reduce morbidity/mortality and minimize transmission. However, concerns exist that this strategy may lead to more crowded hospitals, longer wait times and poorer service, adversely impacting health outcomes for clients with severe disease. We assessed how UTT was related to markers of disease progression in PLHIV overall and specifically among clients with low CD4 count/high World Health Organization (WHO) stage. METHODS: The analysis was conducted using data from a stepped-wedge trial of UTT in 14 government-managed health facilities in Eswatini from 2014 to 2017. Disease progression was defined as CD4 count falling below 200 cells/µL or baseline value, > 10% weight loss, body mass index (BMI) dropping below 18.5, incident tuberculosis (TB) or HIV-related death; these outcomes also were assessed individually. We assessed multivariate Cox proportional hazard models overall and specifically among clients with CD4 count < 350 cells/µL or WHO stage 3-4 at enrolment. RESULTS: Eight hundred and seven of 3176 clients demonstrated at least one marker of disease progression over 2339 person-years of follow-up. Overall, 62.4% of clients were female; 57.2% were < 35 years old. Compared to clients not exposed to UTT, those exposed to UTT had a lower rate of disease progression overall [adjusted hazard ratio (aHR) 0.60; 95% confidence interval (CI) 0.46-0.78] and a lower rate of CD4 decline (aHR 0.40; 95% CI 0.27-0.58). When the analysis was limited to clients with CD4 count < 350 cells/µL or WHO stage 3-4, UTT was not associated with disease progression (aHR 0.92; 95% CI 0.66-1.29). CONCLUSIONS: UTT reduced HIV disease progression overall and was not detrimental for clients with more severe disease.
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Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Teste de HIV/estatística & dados numéricos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Fármacos Anti-HIV/administração & dosagem , Contagem de Linfócito CD4 , Progressão da Doença , Essuatíni/epidemiologia , Feminino , Infecções por HIV/diagnóstico , Infecções por HIV/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
OBJECTIVES: Current WHO guidelines recommend the treatment of all HIV-infected individuals with antiretroviral therapy (ART) to improve survival and quality of life, and decrease infection of others. MaxART is the first implementation trial of this strategy embedded within a government-managed health system, and assesses mortality as a secondary outcome. Because primary findings strongly supported scale-up of the 'treat all' strategy (hereafter Treat All), this analysis examines mortality as an additional indicator of its impact. METHODS: MaxART was conducted in 14 Eswatinian health clinics through a clinic-based stepped-wedge design, by transitioning clinics from then-national standard of care (SoC) to the Treat All intervention. All-cause, disease-related, and HIV-related mortality were analysed using the Cox proportional hazards model, censoring SoC participants at clinic transition. Median follow-up time among study participants was 292 days. There were 36/2034 deaths in SoC (1.77%) and 49/1371 deaths in Treat All (3.57%). RESULTS: Between September 2014 and August 2017, 3405 participants were enrolled. In SoC and Treat All interventions, respectively, the multivariable-adjusted 12-month all-cause mortality rates were 1.42% [95% confidence interval (CI): 0.66-2.17] and 1.60% (95% CI: 0.78-2.40), disease-related mortality rates were 1.02% (95% CI: 0.40-1.64) and 1.10% (95% CI: 0.46-1.73), and HIV-related mortality rates were 1.03% (95% CI: 0.40-1.65) and 0.99% (95% CI: 0.40-1.58). Treat All had no impact on all-cause [hazard ratio (HR) = 1.12, 95% CI: 0.58-2.18, P = 0.73], disease-related (HR = 1.04, 95% CI: 0.52-2.11, P = 0.90), or HIV-related mortality (HR = 0.93, 95% CI: 0.46-1.87, P = 0.83). CONCLUSION: There was no immediate benefit of the Treat All strategy on mortality, nor evidence of harm. Longer follow-up of participants is needed to establish long-term consequences.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/mortalidade , Padrão de Cuidado/organização & administração , Adulto , Essuatíni , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mortalidade , Guias de Prática Clínica como Assunto , Resultado do Tratamento , Adulto JovemRESUMO
Soft polyethylene oxide (PEO)/chitosan mixtures, reinforced with hard titanate nanotubes (TiNTs) by co-precipitation from aqueous solution, have been used to produce compact coatings by the 'drop-cast' method, using water soluble PEO polymer and stable, aqueous colloidal solutions of TiNTs. The effects of the nanotube concentration and their length on the hardness and modulus of the prepared composite have been studied using nanoindentation and nanoscratch techniques. The uniformity of TiNT dispersion within the polymer matrix has been studied using transmission electron microscopy (TEM). A remarkable increase in hardness and reduced Young's modulus of the composites, compared to pure polymer blends, has been observed at a TiNT concentration of 25 wt %. The short (up to 30 min) ultrasound treatment of aqueous solutions containing polymers and a colloidal TiNT mixture prior to drop casting has resulted in some improvements in both hardness and reduced Young's modulus of dry composite films, probably due to a better dispersion of ceramic nanotubes within the matrix. However, further (more than 1 h) treatment of the mixture with ultrasound resulted in a deterioration of the mechanical properties of the composite accompanied by a shortening of the nanotubes, as observed by the TEM.
RESUMO
Typically, pure niobium oxide coatings are deposited on metallic substrates, such as commercially pure Ti, Ti6Al4 V alloys, stainless steels, niobium, TiNb alloy, and Mg alloys using techniques such as sputter deposition, sol-gel deposition, anodizing, and wet plasma electrolytic oxidation. The relative advantages and limitations of these coating techniques are considered, with particular emphasis on biomedical applications. The properties of a wide range of pure and modified niobium oxide coatings are illustrated, including their thickness, morphology, microstructure, elemental composition, phase composition, surface roughness and hardness. The corrosion resistance, tribological characteristics and cell viability/proliferation of the coatings are illustrated using data from electrochemical, wear resistance and biological cell culture measurements. Critical R&D needs for the development of improved future niobium oxide coatings, in the laboratory and in practice, are highlighted.
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A combination of genetic and biochemical analysis is contributing to an understanding of the regulation and role of calcium-independent cell adhesion molecules. Recent progress ranges from the analysis of the control of promoter expression by homeobox genes to detailed analysis of the sites of homophilic and heterophilic interactions via mutagenesis strategies.
Assuntos
Cálcio/fisiologia , Moléculas de Adesão Celular/biossíntese , Regulação da Expressão Gênica , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Moléculas de Adesão Celular/fisiologia , Drosophila/genética , Drosophila/fisiologia , Humanos , Processamento Pós-Transcricional do RNA , Transcrição GênicaRESUMO
Cell adhesion molecules (CAMs) are multifunctional proteins and are involved in a number of important regulatory processes in the brain, including cell growth, migration and regeneration. Recent studies using model in vitro systems have identified additional binding interactions in which CAMs, particularly those of the Ig superfamily, can participate. Signal transduction pathways are activated following CAM action in the process of neurite outgrowth. Key components in these pathways, such as kinases and phosphatases, are being identified. Receptor phosphatases themselves contain protein motifs characteristic of CAMs and may themselves be involved in adhesion-mediated cell recognition events.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Regeneração Nervosa/fisiologia , Transdução de Sinais/fisiologia , Animais , Axônios/fisiologia , Previsões , Humanos , Proteínas da Mielina/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Sistema Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neuritos/fisiologia , Proteínas Tirosina Fosfatases/metabolismoRESUMO
This study investigates the removal of Pb(II) using polymer matrix membranes, cellulose acetate/vinyl triethoxysilane modified graphene oxide and gum Arabic (GuA) membranes. These complexation-NF membranes were successfully synthesized via dissolution casting method for better transport phenomenon. The varied concentrations of GuA were induced in the polymer matrix membrane. The prepared membranes M-GuA2-M-GuA10 were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, atomic force microscope and bio-fouling studies. Thermal stability of the membranes was determined by thermogravimetric analysis under nitrogen atmosphere. Dead end nanofiltration was carried out to study the perm- selectivity of all the membranes under varied pressure and concentration of Pb(NO3)2. The complexation-NF membrane performances were significantly improved after the addition of GuA in the polymer matrix membrane system. M-GuA8 membrane showed optimum result of permeation flux 8.6 l m-2 h-1. Rejection of Pb(II) ions was observed to be around 97.6% at pH 9 for all the membranes due to electrostatic interaction between CA and Gum Arabic. Moreover, with the passage of time, the rate of adsorption was also increased up to 15.7 mg g-1 until steady state was attained. Gum Arabic modified CA membranes can open up new possibilities in enhancing the permeability, hydrophilicity and anti-fouling properties.
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This paper reports the oxidation of Remazol black B dye by employing iron ions catalyst based gas diffusion cathodes, (GDCs). A GDC was synthesized by using a layer of carbon black and iron ions catalyst for oxygen reduction to hydrogen peroxide. The results demonstrated around 97% decolorization of Remazol black-B dye for 50 min by iron ions catalyst based GDC. The degradation study was performed under electrogenerated hydrogen peroxide at a constant voltage of - 0.6 V vs Hg/HgSO4 in which the rate of degradation was correlated with hydrogen peroxide production. Overall, the GDC's found to be effective method to degrade the dyes via electro-Fenton.
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We have used a transfection based approach to analyze the role of neural cell adhesion molecule (NCAM) in myogenesis at the stage of myoblast fusion to form multinucleate myotubes. Stable cell lines of myogenic C2 cells were isolated that express the transmembrane 140- or 180-kD NCAM isoforms or the glycosylphosphatidylinositol (GPI) linked isoforms of 120 or 125 kD. We found that expression of the 140-kD transmembrane isoform led to a potent enhancement of myoblast fusion. The 125-kD GPI-linked NCAM also enhanced the rate of fusion but less so when a direct comparison of cell surface levels of the 140-kD transmembrane form was carried out. While the 180-kD transmembrane NCAM isoform was effective in promoting C2 cell fusion similar to the 140-kD isoform, the 120-kD isoform did not have an effect on fusion parameters. It is possible that these alterations in cell fusion are associated with cis NCAM interactions in the plane of the membrane. While all of the transfected human NCAMs (the transmembrane 140- and 180-kD isoforms and the 125- and 120-kD GPI isoforms) could be clustered in the plane of the plasma membrane by species-specific antibodies there was a concomitant clustering of the endogenous mouse NCAM protein in all cases except with the 120-kD human isoform. These studies show that different isoforms of NCAM can undergo specific interactions in the plasma membrane which are likely to be important in fusion. While the transmembrane and the 125-kD GPI-anchored NCAMs are capable of enhancing fusion the 120-kD GPI NCAM is not. Thus it is likely that interactions associated with NCAM intracellular domains and also the muscle specific domain (MSD) region in the extracellular domain of the GPI-linked 125-kD NCAM are important. In particular this is the first role ascribed to the O-linked carbohydrate containing MSD region which is specifically expressed in skeletal muscle.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Músculos/citologia , Animais , Adesão Celular , Moléculas de Adesão Celular Neuronais/química , Diferenciação Celular , Fusão Celular , Linhagem Celular , Membrana Celular/ultraestrutura , Glicosilfosfatidilinositóis , Técnicas Imunológicas , Camundongos , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
The addition of nerve growth factor (NGF) to PC12 cells induces an approximate doubling in the cell surface expression of the Thy-1 glycoprotein and the neural cell adhesion molecule (N-CAM) after 24 h of culture. Although both responses are measured at the same time point, their sensitivity to NGF differed with half-maximal induction of Thy-1 apparent at NGF concentrations (approximately 0.1 ng/ml NGF) that had little effect on N-CAM expression. Phorbol ester derivatives capable of activating Ca2+/phospholipid-dependent protein kinase (protein kinase C) and the calcium ionophore A23187 were found to mimic the NGF induction of Thy-1, but not N-CAM. Similar results were observed when a synthetic diacylglycerol was added to PC12 cell cultures. Increased expression of Thy-1 consequent to phorbol ester, calcium ionophore, or NGF treatment was associated with an increase in the expression of the mRNA species that encodes Thy-1. Increased expression of Thy-1 consequent to all three treatments was also reduced by treatment with the transcription inhibitor cordycepin. Treatment of PC12 cells with high concentrations of phorbol esters was found to inhibit the NGF induction of Thy-1, but not N-CAM. Whereas the above results are consistent with activation of protein kinase C underlying the NGF induction of Thy-1, the same data are not consistent with this pathway being important in the N-CAM response.
Assuntos
Antígenos de Superfície/biossíntese , Calcimicina/farmacologia , Fatores de Crescimento Neural/farmacologia , Ésteres de Forbol/farmacologia , Moléculas de Adesão Celular , Diglicerídeos/farmacologia , Humanos , Glicoproteínas de Membrana/biossíntese , Feocromocitoma , Proteína Quinase C/metabolismo , Antígenos Thy-1 , Células Tumorais CultivadasRESUMO
We present evidence that direct activation of neuronal second messenger pathways in PC12 cells by opening voltage-dependent calcium channels mimics cell adhesion molecule (CAM)-induced differentiation of these cells. PC12 cells were cultured on monolayers of control 3T3 cells or 3T3 cells expressing transfected N-cadherin in the presence of KCl or a calcium channel agonist Bay K 8644. Both potassium depolarization and agonist-induced activation of calcium channels promoted substantial neurite outgrowth from PC12 cells cultured on control 3T3 monolayers and increased neurite outgrowth from those cultured on N-cadherin-expressing 3T3 monolayers. The potassium-induced response could be inhibited by L- and N-type calcium channel antagonists and by kinase inhibitor K-252b but was unaffected by pertussis toxin. In contrast activators of protein kinase C did not stimulate neurite outgrowth, and the neurite outgrowth response induced by activation of protein kinase A was not inhibited by calcium channel antagonists or pertussis toxin. These studies support the postulate that CAM-induced neuronal differentiation involves a specific transmembrane signaling pathway and suggest that activation of this pathway after CAM binding may be more important for the neurite outgrowth response than CAM-dependent adhesion per se.
Assuntos
Moléculas de Adesão Celular/fisiologia , Neuritos , Sistemas do Segundo Mensageiro , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Células 3T3 , Animais , Caderinas/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Carbazóis/farmacologia , Divisão Celular , Ativação Enzimática , Alcaloides Indólicos , Camundongos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neuritos/ultraestrutura , Células PC12 , Toxina Pertussis , Potássio/farmacologia , Proteínas Quinases/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
We have used monolayers of parental 3T3 cells and 3T3 cells expressing one of three transfected cell adhesion molecules (CAMs) (NCAM, N-cadherin, and L1) as a culture substrate for rat cerebellar neurons. A number of tyrosine kinase inhibitors have been tested for their ability to inhibit neurite outgrowth over parental 3T3 monolayers which we show to be partly dependent on neuronal integrin receptor function, as compared with neurite outgrowth stimulated by the above three CAMs. Whereas genistein (100 microM), lavendustin A (20 microM), and tyrphostins 34 and 47 (both at 150 microM) had no effect on integrin dependent or CAM stimulated neurite outgrowth, the erbstatin analogue (10-15 micrograms/ml) and tyrphostins 23 and 25 (both at 150 microM) specifically inhibited the response stimulated by all three CAMs. CAM stimulated neurite outgrowth can be accounted for by a G-protein-dependent activation of neuronal calcium channels; experiments with agents that directly activate this pathway localized the erbstatin analogue site of action upstream of the G-protein and calcium channels, whereas tyrphostins have sites of action downstream from calcium channel activation. These data suggest that activation of an erbstatin sensitive tyrosine kinase is an important step upstream of calcium channel activation in the second messenger pathway underlying the neurite outgrowth response stimulated by a variety of CAMs, and that this kinase is not required for integrin-dependent neurite outgrowth.
Assuntos
Moléculas de Adesão Celular/farmacologia , Integrinas/fisiologia , Neuritos/fisiologia , Neurônios/citologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirfostinas , Células 3T3 , Animais , Canais de Cálcio/metabolismo , Catecóis/farmacologia , Moléculas de Adesão Celular Neuronais/farmacologia , Células Cultivadas , Cerebelo , Toxina da Cólera/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Genisteína , Hidroquinonas/farmacologia , Isoflavonas/farmacologia , Complexo Antígeno L1 Leucocitário , Camundongos , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nitrilas/farmacologia , Fenóis/farmacologia , Cloreto de Potássio/farmacologia , RatosRESUMO
Binding of the neural cell adhesion molecule (NCAM) in neurons to NCAM on non-neuronal cells can stimulate axonal growth. A developmentally regulated loss of this response is associated with the insertion of 10 amino acids (called VASE) into the fourth Ig domain in up to 50% of the NCAM receptors in neurons. In the present study we have transfected PC12 cells with the major neuronal isoforms of human NCAM and tested cells expressing these isoforms for their ability to respond to NCAM in a cellular substratum. Whereas both the 140- and 180-kD isoforms of NCAM can act as functional receptors for neurite outgrowth, the presence of the VASE sequence in a minority of the receptors specifically inhibited this response. A synthetic peptide containing the VASE sequence inhibits neurite outgrowth from PC12 cells and primary neurons stimulated by NCAM. The same peptide has no effect on integrin dependent neurite outgrowth or neurite outgrowth stimulated by N-cadherin or L1. We discuss the possibility that the VASE peptide inhibits the NCAM response by preventing NCAM from binding to the FGF receptor in the plasma membrane.
Assuntos
Moléculas de Adesão Celular Neuronais/biossíntese , Neuritos/metabolismo , Neurônios/metabolismo , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Células 3T3 , Processamento Alternativo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Adesão Celular/fisiologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/fisiologia , Divisão Celular , Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/ultraestrutura , Oligopeptídeos/genética , Células PC12 , Peptídeos/genética , Ratos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , TransfecçãoRESUMO
Using a sensitive and quantitative adhesion assay, we have studied the initial stages of the intercellular adhesion of the C2 mouse myoblast line. After dissociation in low levels of trypsin in EDTA, C2 cells can rapidly reaggregate by Ca2+-independent mechanisms to form large multicellular aggregates. If cells are allowed to recover from dissociation by incubation in defined media, this adhesive system is augmented by a Ca2+-dependent mechanism with maximum recovery seen after 4 h incubation. The Ca2+-independent adhesion system is inhibited by preincubation of cell monolayers with cycloheximide before dissociation. Aggregation is also reduced after exposure to monensin, implicating a role for surface-translocated glycoproteins in this mechanism of adhesion. In coaggregation experiments using C2 myoblasts and 3T3 fibroblasts in which the Ca2+-dependent adhesion system was inactivated, no adhesive specificity between the two cell types was seen. Although synthetic peptides containing the RGD sequence are known to inhibit cell-substratum adhesion in various cell types, incubation of C2 myoblasts with the integrin-binding tetrapeptide, RGDS, greatly stimulated the Ca2+-independent aggregation of these cells while control analogs had no effect. These results show that a Ca2+-independent mechanism alone is sufficient to allow for the rapid formation of multicellular aggregates in a mouse myoblast line, and that many of the requirements and perturbants of the Ca2+-independent system of intercellular myoblast adhesion are similar to those of the Ca2+-dependent adhesion mechanisms.
Assuntos
Antígenos de Superfície/fisiologia , Cálcio/fisiologia , Adesão Celular , Músculos/citologia , Animais , Moléculas de Adesão Celular , Agregação Celular/efeitos dos fármacos , Linhagem Celular , Cicloeximida/farmacologia , Glicoproteínas/fisiologia , Monensin/farmacologia , Oligopeptídeos/farmacologia , Ratos , Tripsina/farmacologiaRESUMO
The majority of skeletal muscle fibers are generated through the process of secondary myogenesis. Cell adhesion molecules such as NCAM are thought to be intricately involved in the cell-cell interactions between developing secondary and primary myotubes. During secondary myogenesis, the expression of NCAM in skeletal muscle is under strict spatial and temporal control. To investigate the role of NCAM in the regulation of primary-secondary myotube interactions and muscle fusion in vivo, we have examined muscle development in transgenic mice expressing the 125-kD muscle-specific, glycosylphosphatidylinositol-anchored isoform of human NCAM, under the control of a human skeletal muscle alpha-actin promoter that is active from about embryonic day 15 onward. Analysis of developing muscle from transgenic animals revealed a significantly lower number of myofibers encased by basal lamina at postnatal day 1 compared with nontransgenic littermates, although the total number of developing myofibers was similar. An increase in muscle fiber size and decreased numbers of VCAM-1-positive secondary myoblasts at postnatal day 1 was also found, indicating enhanced secondary myoblast fusion in the transgenic animals. There was also a significant decrease in myofiber number but no increase in overall muscle size in adult transgenic animals; other measurements such as the number of nuclei per fiber and the size of individual muscle fibers were significantly increased, again suggesting increased secondary myoblast fusion. Thus the level of NCAM in the sarcolemma is a key regulator of cell-cell interactions occurring during secondary myogenesis in vivo and fulfills the prediction derived from transfection studies in vitro that the 125-kD NCAM isoform can enhance myoblast fusion.
Assuntos
Actinas/genética , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/crescimento & desenvolvimento , Moléculas de Adesão de Célula Nervosa/fisiologia , Animais , Comunicação Celular , Fusão Celular , Tamanho Celular , DNA/análise , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Glicosilfosfatidilinositóis , Humanos , Integrina alfa4beta1 , Integrinas/análise , Camundongos , Camundongos Transgênicos , Morfogênese , Fibras Musculares Esqueléticas/química , Músculo Esquelético/química , Músculo Esquelético/citologia , Moléculas de Adesão de Célula Nervosa/análise , Moléculas de Adesão de Célula Nervosa/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , Receptores de Retorno de Linfócitos/análise , Molécula 1 de Adesão de Célula Vascular/análiseRESUMO
We present evidence that the neurite out-growth stimulated by the binding of Thy-1 antibodies to PC12 cells is mediated by calcium influx through both N- and L-type calcium channels. PC12 cells cultured on a noncellular substratum in the presence of NGF, or on a cellular substratum in the absence of NGF, responded to soluble Thy-1 antibody by extending longer neurites. The response required bivalent antibody and could be blocked by removing Thy-1 from the surface of PC12 cells with phosphatidylinositol specific phospholipase C. The response could also be blocked by reducing extracellular calcium to 0.25 mM, or by antagonists of L- and N-type calcium channels. Additionally, the response could be fully inhibited by preloading PC12 cells with BAPTA/AM which buffers changes in intracellular calcium. A heterotrimeric G-protein is also implicated in the pathway as the response could be fully inhibited by pertussis toxin. These data suggest that antibody-induced clustering of Thy-1 stimulates neurite outgrowth by activating a second messenger pathway that has previously been shown to underlie cell adhesion molecule (NCAM, N-cadherin, and L1), but not integrin or NGF-dependent neurite outgrowth.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/metabolismo , Canais de Cálcio/fisiologia , Cálcio/metabolismo , Isoanticorpos/farmacologia , Glicoproteínas de Membrana/metabolismo , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/fisiologia , Neurônios/fisiologia , Células 3T3 , Animais , Canais de Cálcio/efeitos dos fármacos , Diltiazem/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Camundongos , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Toxina Pertussis , Antígenos Thy-1 , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Qualitative and quantitative changes in neural cell adhesion molecule (N-CAM) protein and mRNA forms were measured during myogenesis in G8-1 and C2 cell lines. Indirect immunofluorescence assay showed that N-CAM was constitutively expressed by myoblasts in culture and that myotubes appeared to be stained more strongly. These changes were quantified using a dot blot assay. N-CAM levels increased almost 4-fold in G8-1 cells and 15-fold in C2 cells during myogenesis. The kinetics of accumulation of N-CAM were not coordinate with other muscle markers such as creatine kinase or acetylcholine receptor levels, since N-CAM accumulated significantly ahead of these markers. Immunoblotting showed that myogenesis was not associated with changes in the extent of sialylation of N-CAM. However, distinct changes in desialo forms were observed after neuraminidase treatment. Myogenesis was accompanied by increases in 125- and 155-kD desialo forms with minor changes in 120- and 145-kD forms. Biosynthetic labeling studies showed that myoblasts specifically expressed a transmembrane isoform of 145 kD that was phosphorylated and was down-regulated in myotubes. Pulse-chase analysis of myotubes showed that the 120-kD isoform and an isoform of 145 kD that co-migrated with, but was distinct from, the 145 kD transmembrane isoform of myoblasts were precursors of the 125- and 155-kD isoforms, respectively, that accumulated in myotubes. The 125- and 155-kD isoforms in myotubes are linked to the cell membrane via phosphatidylinositol linkage and can be released by phospholipase C. Indirect immunofluorescence analysis showed that phosphatidylinositol specific phospholipase C specifically released N-CAM from the myotube membrane generating N-CAM-free myotubes, while myoblasts were unaffected by this treatment. Three N-CAM mRNA species were observed in mouse muscle cell lines. Myoblasts were characterized by their expression of 6.7- and 5.2-kb transcripts while myotubes express 5.2- and 2.9-kb transcripts. Thus, myogenesis is qualitatively associated with a down regulation of the 6.7-kb transcript and an up regulation of the 5.2- and 2.9-kb transcript.
Assuntos
Antígenos de Superfície/genética , RNA Mensageiro/genética , Antígenos de Superfície/isolamento & purificação , Adesão Celular , Moléculas de Adesão Celular , Linhagem Celular , Cinética , Músculos/citologia , Processamento de Proteína Pós-Traducional , Tunicamicina/farmacologia , Fosfolipases Tipo C/farmacologiaRESUMO
We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human neural cell adhesion molecule (NCAM) or chick N-cadherin as a culture substrate for PC12 cells. NCAM and N-cadherin in the monolayer directly promote neurite outgrowth from PC12 cells via a G-protein-dependent activation of neuronal calcium channels. In the present study we show that ganglioside GM1 does not directly activate this pathway in PC12 cells. However, the presence of GM1 (12.5-100 micrograms/ml) in the co-culture was associated with a potentiation of NCAM and N-cadherin-dependent neurite outgrowth. Treatment of PC12 cells with GM1 (100 micrograms/ml) for 90 min led to trypsin-stable increases in both beta-cholera toxin binding to PC12 cells and an enhanced neurite outgrowth response to N-cadherin. The ganglioside response could be fully inhibited by treatment with pertussis toxin. These data are consistent with exogenous gangliosides enhancing neuritic growth by promoting cell adhesion molecule-induced calcium influx into neurons.
Assuntos
Caderinas/fisiologia , Moléculas de Adesão Celular Neuronais/fisiologia , Gangliosídeo G(M1)/farmacologia , Neuritos/efeitos dos fármacos , Células 3T3 , Animais , Cálcio/metabolismo , Toxina da Cólera/farmacologia , Camundongos , Fatores de Crescimento Neural/farmacologia , Neuritos/fisiologia , Células PC12 , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Mouse 3T3 fibroblasts were permanently transfected with cDNAs encoding isoforms of the neural cell adhesion molecule (N-CAM) present in human skeletal muscle and brain. Parental and transfected cells were then used in a range of adhesion assays. In the absence of external shear forces, transfection with cDNAs encoding either transmembrane or glycosylphosphatidylinositol (GPI)-linked N-CAM species significantly increased the intercellular adhesiveness of 3T3 cells in suspension. Transfection of a cDNA encoding a secreted N-CAM isoform was without effect on adhesion. Cells transfected with cDNAs containing or lacking the muscle-specific domain 1 sequence, a four-exon group spliced into the muscle but not the brain GPI-linked N-CAM species, were equally adhesive in the assays used. We also demonstrate that N-CAM-mediated intercellular adhesiveness is inhibited by 0.2 mg/ml heparin; but, at higher concentrations, reduced adhesion of parental cells was also seen. Coaggregation of fluorescently labeled and unlabeled cell populations was performed and measured by comparing their distribution within aggregates with distributions that assume nonspecific (random) aggregation. These studies demonstrate that random aggregation occurs between transfected cells expressing the transmembrane and GPI-linked N-CAM species and between parental cells and those expressing the secreted N-CAM isoform. Other combinations of these populations tested exhibited partial adhesive specificity, indicating homophilic binding between surface-bound N-CAM. Thus, the approach exploited here allows for a full analysis of the requirements, characteristics, and specificities of the adhesive behavior of individual N-CAM isoforms.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Adesão Celular/fisiologia , Éxons , Músculos/metabolismo , Animais , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Clonagem Molecular , DNA/genética , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis , Heparina , Humanos , Isomerismo , Fosfatidilinositóis/metabolismo , TransfecçãoRESUMO
We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.