RESUMO
Reverse transcription of HIV-1 genomic RNA requires two obligatory strand transfers. During the first strand transfer reaction, the minus strand strong-stop DNA (ss-cDNA) is transferred by hybridization of complementary sequences located at the 3' ends of the ss-cDNA and genomic template, respectively. In HIV-1, the major components of ss-cDNA transfer are the terminally redundant structured TAR elements and the nucleocapsid protein NCp7, which actively chaperones the hybridization of cTAR DNA to TAR. In the present study, we investigated the annealing kinetics of TAR with fluorescently labelled cTAR derivatives both in the absence and in the presence of NC(12-55), a peptide that contains the finger and C-terminal domains of NCp7. The annealing of TAR with cTAR involves two second-order kinetic components that are activated by at least two orders of magnitude by NC(12-55). The NC-promoted activation of cTAR-TAR annealing was correlated with its ability to destabilize the lower half of TAR stem, in order to generate the single-stranded complementary regions for nucleating the duplex structures. The two kinetics components have been assigned to two different pathways. The rapid one does not lead to extended duplex formation but is associated with a limited annealing of the terminal bases of cTAR to TAR. On the other hand, extended duplex formation follows a slower pathway that is limited kinetically by the nucleation of residues located mainly within the central double-stranded segment of both cTAR and TAR stems. An alternative mechanism involving an interaction through TAR and cTAR loops has been observed but is a minor pathway in the present conditions.
Assuntos
Sequência de Bases , Proteínas do Capsídeo/metabolismo , Produtos do Gene gag/metabolismo , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , DNA Viral , Produtos do Gene gag/genética , Humanos , Matemática , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Proteínas Virais/genética , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2'-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson-Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.
Assuntos
Desoxiadenosinas/análogos & derivados , Desoxiadenosinas/química , Corantes Fluorescentes/química , Sondas de Oligonucleotídeos/química , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Compostos Organofosforados/química , Nucleosídeos de Purina/química , Espectrometria de Fluorescência , TermodinâmicaRESUMO
PURPOSE: Postpneumonectomy empyema (PPE) is a possible complication after a pneumonectomy in patients with lung cancer. The use of intrapleural (IP) antibiotic irrigation to treat infections in the pleural space may be indicated after systemic antimicrobial therapy, and drainage of the pleural space has been insufficient. METHODS: Adult patients ≥18 years old who received IP antibiotic irrigation between 2006 and 2011 were included. Demographic data, past medical history, surgical procedure, systemic antibiotics, and culture data were collected. Additionally, the IP antibiotic administered, the dose, and how it was prepared and administered were collected. RESULTS: A total of 18 patients were evaluated in this retrospective descriptive analysis. The majority of patients underwent an extrapleural pneumonectomy (EPP; 72%). Most patients received systemic antibiotics before IP antibiotic administration (95%). Vancomycin was the most common antibiotic used for both systemic therapy (100%) and IP irrigation (94%). The median number of IP antibiotic doses received per patient was 5.5 (interquartile range [IQR] 1-9). Recurrence of PPE within 6 months of initial PPE resolution occurred in 28% of patients. Intrapleural antibiotic irrigation was well tolerated in all patients. CONCLUSION: Vancomycin is most commonly used for IP antibiotic irrigation at our institution after patients have undergone a thoracic surgery, which was most commonly an EPP.
Assuntos
Anti-Infecciosos/administração & dosagem , Empiema Pleural/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/cirurgia , Pneumonectomia/efeitos adversos , Complicações Pós-Operatórias/tratamento farmacológico , Idoso , Empiema Pleural/diagnóstico , Empiema Pleural/epidemiologia , Feminino , Humanos , Neoplasias Pulmonares/epidemiologia , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/epidemiologia , Estudos Retrospectivos , Irrigação Terapêutica/métodosRESUMO
Recovery is a process by which an individual recovers their self-esteem, dreams, self-worth, pride, choice, dignity and meaning. This paper discusses these issues, as well as offering suggestions on how clinicians and the system can help support the process. Recovery is about treating the whole person, identifying their strengths, instilling hope, and helping them to function at an optimal level by allowing them to take responsibility for their life. Also discussed is the importance of using setbacks as learning opportunities, the internal and external obstacles that people in recovery face, and the need to embrace the humanity of people in recovery.
Assuntos
Transtornos Mentais/reabilitação , Recuperação de Função Fisiológica , Apoio Social , Comportamento de Escolha , Humanos , Serviços de Saúde Mental , Autoimagem , Responsabilidade SocialRESUMO
The human NPY Y(1) receptor undergoes fast agonist-induced internalization via clathrin-coated pits then recycles back to the cell membrane. In an attempt to identify the molecular determinants involved in this process, we studied several C-terminal truncation mutants tagged with EFGP. In the absence of agonist, Y(1) receptors lacking the last 32 C-terminal amino acids (Y(1)Δ32) are constitutively internalized, unlike full-length Y(1) receptors. At steady state, internalized Y(1)Δ32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y(1)Δ32 receptors by hypertonic sucrose or by co-expression of Rab5aS34N, a dominant negative form of the small GTPase Rab5a or depletion of all three isoforms of Rab5 indicates the involvement of clathrin-coated pits. In contrast, a truncated receptor lacking the last 42 C-terminal amino acids (Y(1)Δ42) does not constitutively internalize, consistent with the possibility that there is a molecular determinant responsible for constitutive internalization located in the last 10 amino acids of Y(1)Δ32 receptors. We show that the agonist-independent internalization of Y(1)Δ32 receptors involves a tyrosine-based motif YXXΦ. The potential role of this motif in the behaviour of full-length Y(1) receptors has also been explored. Our results indicate that a C-terminal tyrosine-based motif is critical for the constitutive internalization of truncated Y(1)Δ32 receptors. We suggest that this motif is masked in full-length Y(1) receptors which do not constitutively internalize in the absence of agonist.
Assuntos
Receptores de Neuropeptídeo Y/metabolismo , Tirosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Clatrina/química , Clatrina/metabolismo , Células HEK293 , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neuropeptídeo Y/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Neuropeptídeo Y/agonistas , Receptores de Neuropeptídeo Y/genética , Transdução de Sinais , Transferrina/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state.
Assuntos
Compostos de Boro/metabolismo , Pirenzepina/análogos & derivados , Receptor Muscarínico M1/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Algoritmos , Ligação Competitiva , Compostos de Boro/química , Linhagem Celular , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Ligantes , Modelos Químicos , Pirenzepina/química , Pirenzepina/metabolismo , Multimerização Proteica , Transporte Proteico , Receptor Muscarínico M1/química , Receptor Muscarínico M1/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Fatores de TempoRESUMO
Activated human neuropeptide Y Y(1) receptors rapidly desensitize and internalize through clathrin-coated pits and recycle from early and recycling endosomes, unlike Y(2) receptors that neither internalize nor desensitize. To identify motifs implicated in Y(1) receptor desensitization and trafficking, mutants with varying C-terminal truncations or a substituted Y(2) C-terminus were constructed. Point mutations of key putative residues were made in a C-terminal conserved motif [phi-H-(S/T)-(E/D)-V-(S/T)-X-T] that we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for beta-arrestin activation and adaptor protein (AP-2) complex recruitment. Inhibitory GTP-binding protein-dependent signaling of Y(1) receptors to adenylyl cyclase and desensitization was unaffected by C-terminal truncations or mutations, while C-terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C-terminus abolished both internalization and beta-arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation-independent recruitment of beta-arrestins affected neither desensitization, internalization or recycling kinetics of activated Y(1) receptors nor beta-arrestin activation. Interestingly, combining Pro145 substitution by His and C-terminal substitutions significantly attenuates Y(1) desensitization. In the Y(2) receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist-mediated desensitization, beta-arrestin activation, internalization and recycling. Overall, our results indicate that beta-arrestin-mediated desensitization and internalization of Y(1) and Y(2) receptors are differentially regulated by the C-terminal motif and the i2 loop consensus motif.
Assuntos
Receptores de Neuropeptídeo Y/química , Receptores de Neuropeptídeo Y/metabolismo , Adenilil Ciclases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Arrestinas/metabolismo , Transporte Biológico Ativo , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores de Neuropeptídeo Y/agonistas , Receptores de Neuropeptídeo Y/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , beta-ArrestinasRESUMO
BACKGROUND: An in vitro study was conducted to determine whether haptoglobin phenotypes differed in their protective effect against oxidative stress induced by extracellular hemoglobin on red blood cells. METHODS: Conjugated dienes and thiobarbituric acid-reactive substances (TBARS) were determined in human red blood cell membranes in the presence of hemoglobin and various concentrations of each type of purified haptoglobin. In addition, the release of K+ and lactate dehydrogenase from red blood cells was measured. RESULTS: A protective effect of haptoglobin was observed in terms of results obtained for the four parameters examined, with significant differences (p<0.001) between the three haptoglobin types; type 1-1 was the most active and type 2-2 the least active. A proportion of oxidative damage was not sensitive to haptoglobin, but to desferrioxamine (an iron chelator), indicating the participation of two actors, hemoglobin and free iron, in the oxidative stress of membrane lipids. CONCLUSIONS: The antioxidant role of haptoglobin and the phenotype dependence were confirmed for preventing possible oxidative damage induced by free hemoglobin and iron release during its catabolism.