RESUMO
Programmes surveying surgical site infection (SSI) have been implemented throughout the world and are associated with a reduction in SSI rates. We report data on non-prosthetic surgery from the Italian SSI surveillance programme for the period 2009 to 2011. Participation in the programme was voluntary. We evaluated the occurrence of SSI, based on protocols from the European Centre for Disease Prevention and Control, within 30 days of surgery. Demographic data, risk factors, type of surgery and presence of SSI were recorded. The National Coordinating Centre analysed the pooled data. On 355 surgical wards 60,460 operations were recorded, with the number of surveyed intervention doubling over the study period. SSI was observed in 1,628 cases (2,6%) and 60% of SSI were diagnosed through 30-days post discharge surveillance. Operations performed in hospitals with at least two years of surveillance showed a 29% lower risk of SSI. Longer intervention duration, American Society of Anesthesiologists' (ASA) score of at least three, and pre-surgery hospital stay of at least two days were associated with increased risk of SSI, while videoscopic procedures had reduced SSI rates. Implementation of a national surveillance programme was helpful in reducing SSI rates and should be prioritised in all healthcare systems.
Assuntos
Infecção Hospitalar/epidemiologia , Tempo de Internação/estatística & dados numéricos , Vigilância da População/métodos , Avaliação de Programas e Projetos de Saúde/métodos , Infecção da Ferida Cirúrgica/epidemiologia , Adulto , Idoso , Infecção Hospitalar/prevenção & controle , Coleta de Dados/métodos , Feminino , Pesquisas sobre Atenção à Saúde , Humanos , Controle de Infecções , Itália/epidemiologia , Pessoa de Meia-Idade , Análise Multivariada , Alta do Paciente , Cuidados Pós-Operatórios , Fatores de Risco , Fatores Socioeconômicos , Infecção da Ferida Cirúrgica/classificação , Infecção da Ferida Cirúrgica/prevenção & controle , Fatores de TempoRESUMO
Large number of extracellular signals is received by plasma membrane receptors which, upon activation, transduce information into the target cell interior via trimeric G-proteins (GPCRs) and induce activation or inhibition of adenylyl cyclase enzyme activity (AC). Receptors for opioid drugs such as morphine (micro-OR, delta-OR and kappa-OR) belong to rhodopsin family of GPCRs. Our recent results indicated a specific up-regulation of AC I (8-fold) and AC II (2.5-fold) in plasma membranes (PM) isolated from rat brain cortex exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. Increase of ACI and ACII represented the specific effect as the amount of ACIII-ACIX, prototypical PM marker Na, K-ATPase and trimeric G-protein alpha and beta subunits was unchanged. The up-regulation of ACI and ACII faded away after 20 days since the last dose of morphine. Proteomic analysis of these PM indicated that the brain cortex of morphine-treated animals cannot be regarded as being adapted to this drug because significant up-regulation of proteins functionally related to oxidative stress and alteration of brain energy metabolism occurred. The number of delta-OR was increased 2-fold and their sensitivity to monovalent cations was altered. Characterization of delta-OR-G-protein coupling in model HEK293 cell line indicated high ability of lithium to support affinity of delta-OR response to agonist stimulation. Our studies of PM structure and function in context with desensitization of GPCRs action were extended by data indicating participation of cholesterol-enriched membrane domains in agonist-specific internalization of delta-OR. In HEK293 cells stably expressing delta-OR-G(i)1alpha fusion protein, depletion of PM cholesterol was associated with the decrease in affinity of G-protein response to agonist stimulation, whereas maximum response was unchanged. Hydrophobic interior of isolated PM became more "fluid", chaotically organized and accessible to water molecules. Validity of this conclusion was supported by the analysis of an immediate PM environment of cholesterol molecules in living delta-OR-G(i)1alpha-HEK293 cells by fluorescent probes 22- and 25-NBD-cholesterol. The alteration of plasma membrane structure by cholesterol depletion made the membrane more hydrated. Understanding of the positive and negative feedback regulatory loops among different OR-initiated signaling cascades (micro-, delta-, and kappa-OR) is crucial for understanding of the long-term mechanisms of drug addiction as the decrease in functional activity of micro-OR may be compensated by increase of delta-OR and/or kappa-OR signaling.
Assuntos
Analgésicos Opioides/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Lipídeos de Membrana/metabolismo , Receptores Opioides/metabolismo , Animais , Células HEK293 , Humanos , Fluidez de Membrana/fisiologia , Ratos , Transdução de Sinais/fisiologia , Relação Estrutura-AtividadeRESUMO
During CNS development neurons undergo directional migration to achieve their adult localizations. To study neuronal migration, we used a model cell line of immortalized murine neurons (gonadotropin-releasing hormone expressing neurons; GN11), enriched with caveolins and caveolae invaginations that show in vitro chemotaxis upon serum exposure. Cholesterol depletion with methyl-beta-cyclodextrin induced the loss of caveolae and the inhibition of chemotaxis, thus suggesting that GN11 migration depends upon the structural integrity of caveolae. Polarization of proteins and organelles is a hallmark of cell migration. Accordingly, GN11 cells transmigrating through filter pores exhibited a polarized distribution of caveolin-1 isoform (cav-1) in the leading processes. In contrast, during two-dimensional migration cav-1 and caveolae polarized at the trailing edge. As caveolae are enriched with signaling molecules, we suggest that asymmetrical localization of caveolae may spatially orient GN11 neurons to incoming migratory signals thereby transducing them into directional migration.
Assuntos
Cavéolas/metabolismo , Caveolinas/metabolismo , Movimento Celular/fisiologia , Neurônios/fisiologia , Animais , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Colesterol/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Indóis , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Sais de Tetrazólio , Tiazóis , beta-Ciclodextrinas/farmacologiaRESUMO
Caveolins are the main structural proteins of glycolipid/cholesterol-rich plasmalemmal invaginations, termed caveolae. In addition, caveolin-1 isoform takes part in membrane remodelling as it binds and transports newly synthesized cholesterol from endoplasmic reticulum to the plasma membrane. Caveolin-1 is expressed in many cell types, including hippocampal neurons, where an abundant SNAP25-caveolin-1 complex is detected after induction of persistent synaptic potentiation. To ascertain whether caveolin-1 influences neuronal voltage-gated Ca2+ channel basal activity, we stably expressed caveolin-1 into transfected neuroblastoma x glioma NG108-15 hybrid cells [cav1(+) clone] that lack endogenous caveolins but express N-type Ca2+ channels upon cAMP-induced neuronal differentiation. Whole-cell patch-clamp recordings of cav1(+) cells demonstrated that N-type current density was reduced in size by approximately 70% without any significant change in the time course of activation and inactivation and voltage dependence. Moreover, the cav1(+) clone exhibited a significantly increased proportion of membrane cholesterol compared to wild-type NG108-15 cells. To gain insight into the mechanism underlying caveolin-1 lowering of N-current density, and more precisely to test whether this was indirectly caused by caveolin-1-induced enhancement of membrane cholesterol, we compared single N-type channel activities in cav1(+) clone and wild-type NG108-15 cells enriched with cholesterol after exposure to a methyl-beta-cyclodextrin-cholesterol complex. A lower Ca2+ channel activity was recorded from cell-attached patches of both cell types, thus supporting the view that the increased proportion of membrane cholesterol is ultimately responsible for the effect. This is due to a reduction in the probability of channel opening caused by a significant decrease of channel mean open time and by an increase of the frequency of null sweeps.
Assuntos
Canais de Cálcio Tipo N/fisiologia , Colesterol/metabolismo , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Animais , Linhagem Celular , Colesterol/genética , Cinética , Camundongos , Proteínas Recombinantes/metabolismoRESUMO
This review describes the advances in our understanding of the role of G-protein coupled receptor (GPCR) localisation in membrane microdomains known as lipid rafts and caveolae. The growing interest in these specialised regions is due to the recognition that they are involved in the regulation of a number of cell functions, including the fine-tuning of various signalling molecules. As a number of GPCRs have been found to be enriched in lipid rafts and/or caveolae by means of different experimental approaches, we first discuss the pitfalls and uncertainties related to the use of these different procedures. We then analyse the addressing signals that drive and/or stabilise GPCRs in lipid rafts and caveolae, and explore the role of rafts/caveolae in regulating GPCR trafficking, particularly in receptor exo- and endocytosis. Finally, we review the growing evidence that lipid rafts and caveolae participate in the regulation of GPCR signalling by affecting both signalling selectivity and coupling efficacy.
Assuntos
Cavéolas/metabolismo , Microdomínios da Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Acilação , Animais , Ácidos Graxos/metabolismo , Humanos , Transporte Proteico , Receptores Acoplados a Proteínas G/genéticaRESUMO
1. Caveolins are integral proteins of glycolipid/cholesterol-rich plasmalemmal caveolae domains, where, they may function as a plasma membrane scaffold onto which many classes of signalling molecules, including receptors and heterotrimeric G proteins, can assemble. To ascertain whether caveolins influence G protein-mediated signal transduction, we stably expressed caveolin-1 and -3 isoforms in the neuroblastoma x glioma NG108-15 hybrid cell line, lacking endogenous caveolins. Subsequently, using whole-cell voltage clamp methods, we examined whether the modulation of N-type voltage-gated Ca2+ channels by G(o) protein-coupled, delta-type opioid receptors might be affected by recombinant caveolin expression. 2. In transfected NG108-15 cells, caveolins localized at the plasma membrane and, upon subcellular fractionation on sucrose density gradients, they co-localized in Triton-resistant, low buoyancy fractions, with endogenous G(o) protein alpha-subunits. 3. The voltage-dependent inhibition of omega-conotoxin GVIA-sensitive Ba2+ currents following either activation of delta-opioid receptors by the agonist [o-pen2,o-pen5]-enkephalin (DPDPE), or direct stimulation of G proteins with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) was significantly attenuated in caveolin-expressing cells. The kinetics of Ca2+ channel inhibition were also modified by caveolins. 4. Overall, these results suggest that caveolins may negatively affect G protein-dependent regulation of voltage-gated N-type Ca2+ channels, presumably by causing a reduction of the available pool of activated G proteins.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Caveolinas/genética , Caveolinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neurônios/fisiologia , Analgésicos Opioides/farmacologia , Animais , Caveolina 1 , Caveolina 3 , Eletrofisiologia , D-Penicilina (2,5)-Encefalina/farmacologia , Expressão Gênica/fisiologia , Glioma , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Células Híbridas , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neuroblastoma , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
alpha-Subunits of heterotrimeric G(i)-like proteins (alpha(i), alpha(o) and alpha(z)) associate with the cytoplasmic leaflet of the plasma membrane by means of N-terminally linked myristic acid and palmitic acid. An additional role for palmitate has been recently suggested by the observation that fusion with the palmitoylated N-terminus of alpha(i1) relocalizes cytosolic green-fluorescent-protein reporter to low buoyancy, Triton-insoluble membrane domains (TIFF; Triton-insoluble floating fraction), enriched with caveolin-1 [Galbiati, Volonté, Meani, Milligan, Lublin, Lisanti and Parenti (1999) J. Biol. Chem 274, 5843-5850]. Here we show that, upon transient expression in transfected COS-7 cells, myristoylated and palmitoylated alpha(o) (alpha(o)wt, where wt is wild-type) is exclusively found in TIFF, from where non-palmitoylated alpha(o)wt and alpha(o)C3S (Cys(3)-->Ser) mutant are excluded. Moreover, alpha(o) fused to N-terminally truncated human vasopressin V2 receptor (V2TR-alpha(o)), lacking myristate and palmitate, still localizes at the plasma membrane by means of first transmembrane helix of V2R, but is excluded from TIFF. Likewise, alpha(o)C3S does not partition into TIFF, even when its membrane avidity is enhanced by co-expression of betagamma-subunits. Thus membrane association, in the absence of added palmitate, is not sufficient to confer partitioning of alpha(o) within TIFF, suggesting that palmitoylation is a signal for membrane compartmentalization of dually acylated alpha-subunits.
Assuntos
Caveolinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Polietilenoglicóis/química , Compostos de Sulfidrila/metabolismo , Tensoativos/química , Acilação , Animais , Sequência de Bases , Células COS , Caveolina 1 , Primers do DNA , Ácido Palmítico/metabolismo , Ligação Proteica , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
After incubation of intact living cultured rat cerebellar granule cells at 37 degrees C with a new GM1 ganglioside analog, carrying a diazirine group and labeled with (125)I in the ceramide moiety, followed by photoactivation, a relatively small number of radiolabeled proteins were detected in a membrane-enriched fraction. A protein of about 55 kDa with a pI of about 5 carried a large portion of the radioactivity even if incubation and cross-linking were performed at 4 degrees C and in the presence of inhibitors of endocytosis, suggesting that it is cross-linked at the plasma membrane. Immunoprecipitation and Western blotting experiments showed the positivity of this protein for tubulin. Trypsin treatment of intact cells ruled out the involvement of a plasma membrane surface tubulin. Release of radioactivity from cross-linked tubulin after KOH treatment (but not hydroxylamine treatment) suggested that the photoactivated ganglioside reacts with an ester-linked fatty acid anchor of tubulin. Low buoyancy, detergent-resistant membrane fractions, isolated from cells after incubation with the GM1 analogue and photoactivation, proved their enrichment in endogenous and radioactive GM1 ganglioside, sphingomyelin, cholesterol, signal transduction proteins, and tubulin. It is noteworthy that radioactive tubulin was also detected in this fraction, indicating the presence of tubulin molecules carrying a fatty acid anchor in detergent-resistant, ganglioside-enriched domains of the plasma membrane. Parallel experiments carried out with a phosphatidylcholine analogue, also carrying a diazirine group and labeled with (125)I in the fatty acid moiety, showed the specificity of tubulin interaction with GM1. Taken together, these results indicate that some tubulin molecules are associated with a lipid anchor to detergent-resistant glycolipid-enriched domains of the plasma membrane. This novel feature of membrane domains can provide a key for a better understanding of their biological role.
Assuntos
Membrana Celular/metabolismo , Cerebelo/metabolismo , Gangliosídeo G(M1)/metabolismo , Glicolipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Neurônios/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Membrana Celular/química , Células Cultivadas , Cerebelo/citologia , Cromatografia em Camada Fina , Reagentes de Ligações Cruzadas , Detergentes , Gangliosídeo G(M1)/química , Glicolipídeos/química , Lipídeos de Membrana/química , Lipídeos de Membrana/isolamento & purificação , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Tripsina , Tubulina (Proteína)/química , Tubulina (Proteína)/isolamento & purificaçãoRESUMO
Nef protein of HIV/SIV lentiviruses affects G-protein-mediated signaling, and physically associates to Lck, a myristoylated and palmitoylated Src-like tyrosine kinase. To assess whether Nef interacts with alpha-subunits of heterotrimeric G proteins (Galpha), carrying the same lipidation motif as Lck, we transiently expressed Nef and G(o)alpha (wild-type or nonpalmitoylated C3S mutant), individually or in combination, in transfected COS-7 cells. Recombinant Nef was mostly recovered in particulate fractions, and a Nef-Green Fluorescent Protein chimera was localized at the plasmalemma by in vivo fluorescence imaging. Moreover, Nef and C3S were entirely solubilized by cold Triton X-100, and excluded from low buoyant density sucrose gradient fractions, containing caveolin-1, whereas wild-type G(o)alpha was partially resistant to Triton extraction, and colocalized with caveolin-1. After coexpression, Nef recruited soluble C3S to membranes, and the two proteins were coimmunoprecipitated by G(o)alpha and Nef antisera. We conclude that Nef interacts with nonpalmitoylated G(o)alpha, presumably outside caveolin-rich microdomains of the plasma membrane.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Produtos do Gene nef/metabolismo , HIV-1/metabolismo , Animais , Sequência de Bases , Células COS , Primers do DNA , Proteínas de Ligação ao GTP/imunologia , Produtos do Gene nef/imunologia , Proteínas de Fluorescência Verde , Soros Imunes , Proteínas Luminescentes/metabolismo , Testes de Precipitina , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Evidence has accumulated indicating that LHRH might behave as an autocrine/paracrine growth inhibitory factor in some peripheral tumors. However, LHRH receptors in tumor cells have not been fully characterized, so far. The present experiments were performed to analyze: 1) the messenger RNA expression; 2) the molecular size; and 3) the signal transduction pathway of LHRH receptors in prostate cancer. For these studies, the human androgen-dependent LNCaP and androgen-independent DU 145 prostate cancer cell lines were used. 1) By RT-PCR, a complementary DNA product, which hybridized with a 32P-labeled oligonucleotide probe specific for the pituitary LHRH receptor complementary DNA, was found both in LNCaP and in DU 145 cells. 2) Western blot analysis, using a monoclonal antibody raised against the human pituitary LHRH receptor, revealed the presence of a protein band of approximately 64 kDa (corresponding to the molecular mass of the pituitary receptor) in both cell lines. 3) In LNCaP and DU 145 cells, pertussis toxin completely abrogated the antiproliferative action of a LHRH agonist (LHRH-A). Moreover, LHRH-A substantially antagonized the pertussis toxin-catalyzed ADP-ribosylation of a Galpha(i) protein. Finally, LHRH-A significantly counteracted the forskolin-induced increase of intracellular cAMP levels in both cell lines. These data demonstrate that the LHRH receptor, which is present in prostate cancer cells, independently of whether they are androgen-dependent or not, corresponds to the pituitary receptor, in terms of messenger RNA expression and protein molecular size. However, at variance with the receptor of the gonadotrophs, prostate cancer LHRH receptor seems to be coupled to the Galpha(i) protein-cAMP signal transduction pathway, rather than to the Galpha(q/11)-phospholipase C signaling system. This might be responsible for the different actions of LHRH in anterior pituitary and in prostate cancer.
Assuntos
Expressão Gênica , Neoplasias da Próstata/metabolismo , RNA Mensageiro/análise , Receptores LHRH/genética , Receptores LHRH/metabolismo , Transdução de Sinais , Adenosina Difosfato Ribose/metabolismo , Animais , Western Blotting , AMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Gosserrelina/farmacologia , Humanos , Masculino , Peso Molecular , Toxina Pertussis , Hipófise/química , Ratos , Ratos Sprague-Dawley , Receptores LHRH/química , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Three immune-enzymatic tests for urinary HIV antibodies were examined in order to assess their sensibility, specificity and delta value. The highest sensibility was noticed for the Seradyn test (98.8%), followed by the Wellcozyme test (98.1%) and finally the SUDS rapid test (56.8%). The resultant specificity was 98.5% for the Seradyn test, 91.3% for the Wellcozyme test and 97.3% for the SUDS test. The measurement of delta value showed a higher capability of discrimination for Seradyn test, that could be considered the most reliable for epidemiological purposes.
Assuntos
Anticorpos Anti-HIV/isolamento & purificação , Anticorpos Anti-HIV/urina , Técnicas Imunoenzimáticas , Adulto , Feminino , Humanos , Masculino , Sensibilidade e EspecificidadeRESUMO
Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules.
Assuntos
Caveolinas , Proteínas de Ligação ao GTP/metabolismo , Genes Reporter , Proteínas Luminescentes/genética , Proteínas de Membrana/metabolismo , Acilação , Animais , Células COS , Caveolina 1 , Linhagem Celular , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Fluorescência Verde , Humanos , Microscopia de Fluorescência , Ácido Palmítico/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Caveolae are cholesterol/sphingolipid-rich microdomains of the plasma membrane that have been implicated in signal transduction and vesicular trafficking. Caveolins are a family of caveolae-associated integral membrane proteins. Caveolin-1 and -2 show the widest range of expression, whereas caveolin-3 expression is restricted to muscle cell types. It has been previously reported that little or no caveolin mRNA species are detectable in the brain by Northern blot analyses or in neuroblastoma cell lines. However, it remains unknown whether caveolins are expressed within neuronal cells. Here we demonstrate the expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion (DRG) neurons by using mono-specific antibody probes. In PC12 cells, caveolin-1 expression is up-regulated on day 4 of nerve growth factor (NGF) treatment, whereas caveolin-2 expression is transiently up-regulated early in the differentiation program and then rapidly down-regulated. Interestingly, caveolin-2 is up-regulated in response to the mechanical injury of differentiated PC12 cells; up-regulation of caveolin-2 under these conditions is strictly dependent on continued treatment with NGF. Robust expression of caveolin-1 and -2 is also observed along the entire cell surface of DRG neurons, including high levels on growth cones. These findings demonstrate that neuronal cells express caveolins.
Assuntos
Caveolinas , Gânglios Espinais/lesões , Gânglios Espinais/metabolismo , Proteínas de Membrana/genética , Animais , Sequência de Bases , Caveolina 1 , Caveolina 2 , Diferenciação Celular , Primers do DNA/genética , Proteína GAP-43/genética , Gânglios Espinais/citologia , Fatores de Crescimento Neural/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Regulação para Cima/efeitos dos fármacosRESUMO
Pertussis toxin-resistant (C351G) and also palmitoylation-negative (C3S/C351G), myristoylation-negative (G2A/C351G) and combined acylation-negative (G2A/C3S/C351G) forms of the G-protein Gi1 alpha were expressed in COS-7 cells along with the porcine alpha 2A-adrenoceptor. G2A/C3S/C351G Gi1 alpha and G2A/C351G Gi1 alpha were largely cytosolic and failed to interact with the agonist-occupied alpha 2A-adrenoceptor in membrane preparations. In contrast, C351G Gi1 alpha was almost entirely particulate and the alpha 2-adrenoceptor agonist UK14304 caused a marked stimulation of its GTPase activity and binding of [35S]GTP gamma S which was not prevented by pertussis toxin treatment of the cells. C3S/C351G Gi1 alpha was present in both the particulate and cytosolic fractions but the GTPase activity of the membrane bound fraction was only slightly activated by the alpha 2A-adrenoceptor. Coexpression of C3S/C351G Gi1 alpha and the alpha 2A-adrenoceptor along with beta 1 and gamma 2 subunits increased the P2 membrane complement of the alpha subunit and increased substantially the ratio of membrane bound to cytosolic protein. However, this also failed to allow marked stimulation of high-affinity GTPase activity by the alpha 2A-adrenoceptor despite the increased proportion of G-protein in the P2 membrane fraction. Despite the low fractional activation of C3S/C351G Gi1 alpha by the alpha 2A-adrenoceptor compared to C351G Gi1 alpha, the palmitoylation-resistant G-protein caused a marked reduction in pertussis toxin-resistant, agonist (UK14304)-mediated stimulation of adenylyl cyclase activity. UK14304 caused the same degree of effect on adenylyl cyclase activity in pertussis toxin-treated cells following transfection of the same amounts of C351G Gi1 alpha and C3S/C351G Gi1 alpha, as both appear to act to sequester beta gamma subunits. By contrast, neither G2A/C351G Gi1 alpha nor G2A/C3S/C351G Gi1 alpha resulted in effective regulation of adenylyl cyclase activity.
Assuntos
Cisteína/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Receptores Adrenérgicos alfa 2/fisiologia , Serina/genética , Toxina Adenilato Ciclase , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Células COS , Chlorocebus aethiops , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Mutagênese Sítio-Dirigida , Ácido Palmítico/metabolismo , Toxina Pertussis , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Wild-type and palmitoylation defective mutants of the murine G protein G11alpha were transfected into HEK293 cells. Wild-type G11alpha was membrane associated, Cys9Ser Cys10Ser G11alpha was present in the soluble fraction whilst both Cys9Ser G11alpha and Cys10Ser G11alpha were distributed between the fractions. Expression of the rat TRH receptor resulted in agonist stimulation of inositol phosphate accumulation. The degree of stimulation produced by TRH following co-transfection of the palmitoylation-resistant forms of G11alpha compared to the wild-type protein correlated with the amount of membrane-associated G protein.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Humanos , Camundongos , Mutação , Ácidos Palmíticos/química , Ácidos Palmíticos/metabolismo , Ratos , Receptores do Hormônio Liberador da Tireotropina/efeitos dos fármacos , Receptores do Hormônio Liberador da Tireotropina/genética , Hormônio Liberador de Tireotropina/farmacologia , TransfecçãoRESUMO
Many signaling molecules contain the consensus protein sequence Met-Gly at their N-termini that specifies N-myristoylation. Additionally, some of these proteins contain a cysteine at position-3 (Met-Gly-Cys) that can undergo palmitoylation. As many acylated proteins [G-protein subunits (alpha and beta gamma); c-Src and Src-family tyrosine kinases; H-Ras and Ras-related GTPases; endothelial nitric oxide synthase] are known to be targeted to caveolae membranes, it has been suggested that acylation is required or greatly facilitates this targeting event. However, it remains unclear whether myristoylation of Src-family kinases is necessary or sufficient for caveolar targeting. Our current study aims at clarifying the role of myristoylation in caveolar targeting using well-characterized acylation mutants of two model proteins, namely Gi1 alpha and c-Src. Here, we have used: i) detergent-free subcellular fractionation and ii) acylation mutants of Gi1 alpha and c-Src to systematically evaluate the relative contribution of myristoylation and palmitoylation to their caveolar targeting. Myristoylation (G2A) and palmitoytation (C3S) mutants of Gi1 alpha were poorly targeted to caveolae-enriched membrane fractions, while approximately 35% of total wild-type Gi1 alpha co-fractionated with caveolin, a caveolar marker protein. Similarly, a myristoylation minus mutant of c-Src was quantitatively excluded from caveolae. In contrast to a previous study, we conclude that myristoylation of Gi1 alpha and c-Src proteins is required for their correct caveolar targeting. However, the caveolar targeting of Gi1 alpha is dramatically augmented approximately 4-fold by palmitoylation. Our current studies are directly supported by the earlier in vivo observation that N-terminal myristoylation of v-Src is required for v-Src to phosphorylate caveolin on tyrosine residues in intact cells.
Assuntos
Caveolinas , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Tirosina Quinases/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Células COS , Proteína Tirosina Quinase CSK , Caveolina 1 , Fracionamento Celular , Sequência Consenso , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Ácidos Mirísticos/metabolismo , Ácidos Palmíticos/metabolismo , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Transfecção , Quinases da Família srcRESUMO
The present study evaluates the influence of cholera toxin and its B-subunit on thermic responses to morphine in the rats. The holotoxin (1 microg/rat) and the B-subunit (5 microg) were administered ICV and three days later rats were challenged ICV with morphine and tested for changes of body temperature. Cholera toxin, but not its B-subunit, modified the time course of the hyperthermic response induced by a low dose of morphine (2.5 microg), converted the hypothermia due to a higher dose of morphine (18 microg) to a consistent hyperthermia and only partially reduced the greater hypothermia induced by 36 microg of morphine. Cholera toxin-induced modifications of thermic responses to morphine were paralleled with a decreased Gs(alpha) immunoreactivity and a reduced ability for the toxin to catalyse the "in vitro" ADP-ribosylation of Gs(alpha) in hypothalamic membranes. In contrast, at the same time when morphine-induced effects on body temperature were assessed, no changes in pertussis toxin-mediated ADP-ribosylation of Gi(alpha)/Go(alpha), or basal adenylate cyclase activity, or binding of mu-opioid receptor selective ligand [3H]-DAMGO were observed in hypothalamic areas from rats treated with cholera toxin. These findings suggest that adaptative events secondary to prolonged activation of Gs(alpha) play a role in the modifications of thermic responses to morphine induced by CTX.
Assuntos
Analgésicos Opioides/farmacologia , Regulação da Temperatura Corporal/efeitos dos fármacos , Toxina da Cólera/farmacologia , Morfina/farmacologia , Adenosina Difosfato Ribose/metabolismo , Adenilil Ciclases/efeitos dos fármacos , Animais , Catálise , Avaliação Pré-Clínica de Medicamentos , Feminino , Proteínas de Ligação ao GTP/metabolismo , Injeções Intraventriculares , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/efeitos dos fármacosRESUMO
Prolonged exposure of rats to the synthetic cannabinoid receptor ligand, CP-55,940 (0.4 mg/kg, i.p. for 11 days), induced tolerance to analgesia, to the reduction in spontaneous locomotor activity and the incidence of splayed hind limbs. One hour after the last injection on day 11, the rats were killed and in situ hybridization was used to investigate the effect of treatment on G-protein alpha-subunit expression throughout the brain. Chronic cannabinoid exposure markedly reduced G alpha(s), G alpha(i) and G alpha(o) mRNA levels. The message for the alpha(s)-subunit was decreased in all the brain areas containing the basal autoradiographic signal; the decrease ranging from 25% in the thalamus to 45% in the mesencephalon. Also the basal G alpha(i) expression was reduced in tolerant rats showing the greatest decrease in the forebrain (63%) in the cerebellum (58%) and in the mesencephalon (38%). The reduction in G alpha(o) expression (25%) was more localized, being present only in the rostral portion of the brain (cortex, striatum and olfactory area). The alterations in alpha-subunits gene expression were not followed by any change in the amount of proteins. Our results indicate that, besides the receptor modification, alteration to the G-protein expression could be a molecular event associated with the development of cannabinoid tolerance.
Assuntos
Analgésicos/farmacologia , Encéfalo/efeitos dos fármacos , Canabinoides/farmacologia , Cicloexanóis/farmacologia , Proteínas de Ligação ao GTP/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Animais , Encéfalo/metabolismo , Mapeamento Encefálico , Hibridização In Situ , Masculino , Medição da Dor , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: Pulmonary sporotrichosis is a rare event. Sporothrix schenckii is a dimorphic fungus and develops at 37 degrees C in yeast form. Usually hyphae are not observed in tissues, although their presence has been occasionally demonstrated in biopsies. CASE: A 37-year-old man, human immunodeficiency virus-1 positive, with a CD4 cell count of 345/mm3, developed a productive cough. A sputum smear revealed the presence of a large amount of long, thin, septated micelia. The hyphae bore oval, sessile conidia. Cultures of sputum yielded numerous colonies of S schenckii. CONCLUSION: This is the first report of hyphae of S schenckii in sputum. This case emphasizes the possibilities of cytology for the diagnosis of mycotic infections. Fungi have typical morphologies, and it is possible, on the basis of microscopic evidence, to suspect the nature of the infection early and thus to direct culture procedures.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Pneumonia/microbiologia , Sporothrix/isolamento & purificação , Esporotricose/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/patologia , Adulto , Técnicas Citológicas , Humanos , Masculino , Pneumonia/diagnóstico , Esporotricose/patologiaRESUMO
The effect of albendazole was studied in 12 patients with cystic hydatid disease (CHD) of the liver. Six patients received albendazole continuously for 6 months, while 6 patients received albendazole for 6 courses of 4 weeks with a 2 week drug-free interval between cycles. The continuous therapy proved successful, with stable involution at the follow-up at 24 months, while the patients treated with discontinuous therapy showed improvement or relapse. In our experience, continuous therapy was more effective and can be considered to be a suitable alternative or percutaneous therapy in uncomplicated hydatid liver disease, as an initial treatment.