RESUMO
Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca(2+) and H(+) and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.
Assuntos
Conexina 43/metabolismo , Sistema de Condução Cardíaco/metabolismo , Ventrículos do Coração/metabolismo , Líquido Intracelular/metabolismo , Isquemia Miocárdica/metabolismo , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Modelos Animais de Doenças , Eletrocardiografia , Imunofluorescência , Junções Comunicantes/metabolismo , Immunoblotting , Técnicas In Vitro , Masculino , Reperfusão Miocárdica , Fosforilação , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Myocardial ischemia causes profound changes in both active membrane currents and passive electrical properties. Because these complex changes develop and progress concomitantly, it has not been possible to elucidate the relative contributions of any one component to arrhythmogenesis induced by acute ischemia. Cx43+/- mice express 50% of the normal level of connexin43 (Cx43), the major ventricular electrical coupling protein, but are otherwise identical to wild-type (Cx43+/+) mice. Comparison of arrhythmogenesis in Cx43+/- and +/+ mice can provide insights into the role of changes in electrical coupling as an independent variable in the complex setting of acute ischemia. METHODS AND RESULTS: Acute ischemia was induced in isolated perfused mouse hearts by occlusion of the left anterior descending coronary artery. Spontaneous ventricular tachyarrhythmias (VT) occurred in more than twice as many Cx43+/- hearts than Cx43+/+ hearts. VT was induced in nearly 3 times as many Cx43+/- hearts. Multiple runs and prolonged runs of spontaneous VT were more frequent in Cx43+/- hearts. Onset of the first run of VT occurred significantly earlier in Cx43+/- hearts. Premature ventricular beats were also more frequent in Cx43+/- hearts. The size of the hypoperfused region was equivalent in both groups. CONCLUSIONS: Reduced expression of Cx43 accelerates the onset and increases the incidence, frequency, and duration of ventricular tachyarrhythmias after coronary artery occlusion. Thus diminished electrical coupling per se plays a critical role in arrhythmogenesis induced by acute ischemia.
Assuntos
Arritmias Cardíacas/etiologia , Conexina 43/metabolismo , Isquemia Miocárdica/complicações , Animais , Arritmias Cardíacas/fisiopatologia , Pressão Sanguínea , Conexina 43/deficiência , Conexina 43/genética , Eletrofisiologia , Ventrículos do Coração/fisiopatologia , Técnicas In Vitro , Camundongos , Isquemia Miocárdica/fisiopatologia , Perfusão , Taquicardia/fisiopatologiaRESUMO
UNLABELLED: Analysis of mice with genetically altered expression of cardiac connexins can provide insights into the role of individual gap junction channel proteins in cell-to-cell communication, impulse propagation, and arrhythmias. However, conflicting results have been reported regarding conduction velocity slowing in mice heterozygous for a null mutation in the gene encoding connexin43 (Cx43). METHODS: High-resolution optical mapping was used to record action potentials from 256 sites, simultaneously, on the ventricular surface of Langendorff perfused hearts from 15 heterozygous (Cx43+/-) and 8 wildtype (Cx43+/+) mice (controls). A sensitive method for measuring epicardial conduction velocity was developed to minimize confounding influences of subepicardial breakthrough and virtual electrode effects. RESULTS: Epicardial conduction velocity was significantly slower (23 to 35%, P<0.01) in Cx43+/- mice compared to wildtype. There was no change in conduction patterns or anisotropic ratio (Cx43+/- 1.54+/-0.33; Cx43+/+ 1.57+/-0.17) suggesting that Cx43 expression was reduced uniformly throughout myocardium. The magnitude of reductions in conduction velocity and Cx43 protein expression (45%) were similar in mice in which the null allele occurred in a pure C57BL/6J genetic background versus a mixed (C57BL/6J X 129) background. Action potential duration did not differ between mice of different genotypes. CONCLUSIONS: A approximately 50% reduction of Cx43 expression causes significant conduction velocity slowing in the Cx43+/- mouse heart. The apparent lack of conduction velocity changes reported in previous studies may be related to technical factors rather than variations in genetic background. High-resolution optical mapping is a powerful tool for investigating molecular determinants of propagation and arrhythmias in genetically engineered mice.
Assuntos
Conexina 43/genética , Miocárdio/metabolismo , Potenciais de Ação , Análise de Variância , Animais , Conexina 43/metabolismo , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia de Fluorescência , Miocárdio/química , Perfusão , Ramos Subendocárdicos/patologia , Gravação em VídeoRESUMO
We tested the hypothesis that cardiac allograft dysfunction in acute cardiac rejection may be related, in part, to diminished expression of connexin43, a gap junction channel protein that facilitates intercellular communication and coordinates electrical and mechanical cardiac function. We measured connexin43 levels using quantitative confocal immunofluorescence microscopy of endocardial biopsies from heart transplant recipients with histologic evidence of either no rejection or acute cellular rejection. Expression of connexin43 diminished significantly during acute cellular rejection and returned to baseline levels following resolution of rejection. Reversible down-regulation of connexin43 may contribute to ventricular dysfunction in allograft rejection.
Assuntos
Conexina 43/metabolismo , Regulação para Baixo , Rejeição de Enxerto/metabolismo , Transplante de Coração/efeitos adversos , Miocárdio/metabolismo , Doença Aguda , Adulto , Biomarcadores , Endocárdio/metabolismo , Endocárdio/patologia , Rejeição de Enxerto/patologia , Transplante de Coração/patologia , Humanos , Microscopia de Fluorescência , Índice de Gravidade de DoençaRESUMO
Close to 20% of the patients infected with the AIDS virus develops neurological deficit; eventhough HIV does not invade neurons. Consistently with the neurological deficit, HIV(+) subjects show abnormalities in brainstem auditory and visual evoked potentials (BSAEP and VEP) and in sleep patterns. The HIV-derived glycoprotein 120 has been postulated as a neurotoxic; therefore, it may be playing a crucial role in the generation of BSAEP and VEP, as well as in sleep disturbances. To study the role of the virus-derived proteins on the development of these electrophysiological signals' alterations, we have used the feline immunodeficiency virus (FIV)-derived gp120 and evaluated the changes in these electrophysiological signals. We employed 15 adult male Sprague-Dawley rats (250-350 g), chronically implanted for evoked potential and sleep recordings. Results showed that the i.c.v. administration of FIVgp120 (5 ng/10 microliter) produces changes in the latency of both cortical auditory evoked potentials (CAEPs) and VEPs and a decrease in both REM sleep and SWS. These data support the notion that FIVgp120 is neurotoxic to the central nervous system of cats and rats and that this protein suffices to cause electrophysiological alterations. In addition, it suggests that a similar effect may be occurring in humans as a result of HIVgp120's neurotoxic effects.
Assuntos
Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Potenciais Evocados Visuais/efeitos dos fármacos , Vírus da Imunodeficiência Felina , Proteínas do Envelope Viral/farmacologia , Análise de Variância , Animais , Gatos , Masculino , Ratos , Ratos Sprague-Dawley , Sono/efeitos dos fármacos , Vigília/efeitos dos fármacosRESUMO
This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI. The enzyme from P. aeruginosa PA40 was purified to > 70% homogeneity. The Km of amikacin for this enzyme was 1.4 microM, the Vmax/Km ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed.
Assuntos
Amicacina/farmacologia , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Fosfotransferases/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Amicacina/metabolismo , Antibacterianos/metabolismo , Sequência de Bases , Southern Blotting , Primers do DNA , Cinética , Testes de Sensibilidade Microbiana , Fosfotransferases/isolamento & purificação , Reação em Cadeia da Polimerase , Pseudomonas aeruginosa/enzimologia , Especificidade por SubstratoRESUMO
We have analyzed the ability of three molecular clones of feline immunodeficiency virus (FIV) and an ex vivo variant to infect nine distinct specific-pathogen-free feline cell lines in tissue culture. The purpose of these studies was to elucidate mechanisms by which host cells regulate the level of virus infection and expression and to assess host cell cytokine responses to virus infection. Cells used for the analyzes included four IL-2-dependent continuous T-cell lines (104-C1, 104-C7, MCH5-4 and DB FeTs) which arose from long-term passage, followed by limiting dilution cloning of peripheral blood mononuclear cells (PBMCs); two IL-2-independent T-cell lines (104-C1DL and MCH5-4DL) which originated from two of the IL-2-dependent lines, 104-C1 and MCH5-4; respectively; Crandell feline kidney cells (CrFK); G355-5 brain-derived glial cells; and the T-cell lymphoma line, 3201. Cells were infected with FIV-PPR, FIV-34TF10, FIV 34TF10orf2rep, and a variant arising from FIV-PPR during ex vivo passage on 104-C1DL cells, termed FIV-PPRglial. Infection of the IL-2-dependent T-cell line, 104-C1, by FIV-PPR resulted in the specific and distinct upregulation of cytokine expression. In particular, these cells doubled their expression of the pleiotropic cytokines, interleukin-4 and interleukin-12 after FIV infection. Interferon-gamma production also increased after infection with FIV whereas, TNFalpha expression remained constant. Also, a marked upregulation of MHC class II expression was noted post infection of MCH5-4 and 104-C1 cells with FIV-PPR. Similar results were obtained after infection with FIV-34TF10orf2rep, indicating that the upregulation of cytokine expression is not an isolate-specific phenomenon. Changes in cytokine and class II expression are similar to various reports for the in vivo cytokine alterations in FIV, SIV and HIV infections. The ex vivo infection of these cell lines offers amanipulable system to examine the mechanism(s) by which lentiviruses alter cytokine expression.
Assuntos
Citocinas/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Interleucina-2/metabolismo , Rim/virologia , Linfoma de Células T/virologia , Neuroglia/virologia , Linfócitos T/virologia , Animais , Sequência de Bases , Western Blotting/veterinária , Gatos , Linhagem Celular , DNA/análise , Primers do DNA/química , Eletroforese em Gel de Poliacrilamida/veterinária , Antígenos de Histocompatibilidade Classe II/metabolismo , Rim/metabolismo , Linfoma de Células T/metabolismo , Dados de Sequência Molecular , Neuroglia/metabolismo , Reação em Cadeia da Polimerase/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Linfócitos T/metabolismo , Regulação para CimaRESUMO
A cytopathic variant of feline immunodeficiency virus (FIV) strain PPR emerged after passage of wild-type virus on an interleukin-2-independent cell line. The virus, termed FIV-PPRglial, displayed a phenotype markedly different from the parental virus, including the ability to productively infect previously refractory cell lines, induction of large syncytia, and accelerated kinetic properties. A chimeric molecular clone, FIV-PPRchim42, containing the FIV-PPRglial envelope within the backbone of FIV-PPR, exhibited all the characteristics of the FIV-PPRglial phenotype, demonstrating that the viral envelope was responsible for the acquired traits. Subsequent molecular characterization revealed that the FIV-PPRglial envelope contained five amino acid substitutions relative to wild-type FIV-PPR. Mutagenic analyses further demonstrated that the acquired phenotype was minimally attributable to a combination of three mutations, specifically, a glutamine-to-proline change within the second constant domain of the surface protein (SU); a threonine-to-proline change within the V4 loop, also in the SU; and a premature stop codon in the cytoplasmic tail of the transmembrane protein. All three changes were required to produce the FIV-PPRglial phenotype. Cotransfection studies with mutant viruses in combination with each other and with FIV-PPR indicated that the truncated cytoplasmic tail was responsible for the induction of syncytium formation. Receptor usage analyses were pursued, and distinctions were observed between FIV-PPR and FIV-PPRglial. In vitro infections with FIV-PPR, FIV-PPRglial, and FIV-34TF10 on two adherent cell lines were ablated in the presence of SDF1alpha, the natural ligand for CXCR4. In contrast, viral infection of T cells was not limited to CXCR4 usage, and inhibition studies indicate the potential involvement of a CC chemokine receptor.
Assuntos
Vírus da Imunodeficiência Felina/fisiologia , Linfócitos T/virologia , Sequência de Aminoácidos , Animais , Gatos , Linhagem Celular , Quimiocina CCL4 , Quimiocina CCL5/farmacologia , Efeito Citopatogênico Viral , Células Gigantes , Interleucina-2/fisiologia , Proteínas Inflamatórias de Macrófagos/farmacologia , Dados de Sequência Molecular , Mutação , Linfócitos T/efeitos dos fármacos , Transfecção , TropismoRESUMO
This review examines some of the recent advances made in the prevention and treatment of upper airway obstruction in infancy and childhood. In some instances, the advances are the result of experimental studies that corroborate or refute therapeutic notions that had been adopted prematurely. Studies performed in the past few years, for instance, have demonstrated that both systemic and local corticosteroid treatments are indeed effective in the treatment of viral croup. In contrast, other studies carried out in the same period raise doubts about the usefulness of these medications in the prevention of postextubation laryngeal edema. In other instances, the advances are the result of pioneering efforts to correct anatomical defects, usually congenital, that cause severe airway obstruction. Tracheal and laryngeal stenoses and craniofacial deformities, which only 5 years ago would have been palliated by tracheotomy, undergo now routine primary correction. Despite all these advances, upper airway obstruction remains an important source of morbidity and mortality in early childhood.
Assuntos
Obstrução das Vias Respiratórias/terapia , Cuidados Críticos/métodos , Corticosteroides/uso terapêutico , Obstrução das Vias Respiratórias/etiologia , Obstrução das Vias Respiratórias/prevenção & controle , Antibacterianos/uso terapêutico , Criança , Anormalidades Craniofaciais/complicações , Anormalidades Craniofaciais/cirurgia , Cuidados Críticos/tendências , Humanos , Lactente , Doenças do Sistema Nervoso/complicações , Doenças do Sistema Nervoso/terapia , Sistema Respiratório/lesões , Anormalidades do Sistema Respiratório/complicações , Anormalidades do Sistema Respiratório/cirurgia , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/microbiologiaRESUMO
A monoclonal antibody, MAb vpg15, inhibits feline immunodeficiency virus (FIV) infection in tissue culture. The antibody is directed to a determinant of the feline cell surface marker, CD9, implying that CD9 may serve as a viral receptor or coreceptor in this system. In cells expressing CD9, MAb vpg15 markedly delayed acute virus infection in terms of reverse transcriptase activity detected in cell culture supernatants. This effect was evident if the antibody was added before, immediately after, or 24 h after virus infection. Binding experiments showed that MAb vpg15 did not block virus binding to the cells. PCR analyses at various intervals postinfection also indicated that MAb vpg15 did not block virus uptake, reverse transcription of viral RNA, or integration into host cell DNA. Multiply spliced mRNAs were detected up to 24 h postinfection in both control and MAb vpg15-treated cells. However, viral mRNAs were markedly diminished in MAb vpg15-treated cells after this time, consistent with a failure of the FIV infection to spread in the cell culture. Treatment of chronically infected cells with MAb vpg15 also caused a sharp diminution in viral particle production, while viral mRNA levels were the same in both untreated and MAb-treated infected cells. Analyses of intracellular and extracellular levels of virus-associated antigens showed an enhanced accumulation of intracellular p24. These findings are consistent with the interpretation that MAb vpg15 acts at a posttranscriptional stage by interfering with the assembly and/or release of virus from the cell.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/fisiologia , Vírus da Imunodeficiência Felina/fisiologia , Glicoproteínas de Membrana , Receptores Virais/fisiologia , Animais , Gatos , Linhagem Celular , RNA Viral/análise , Tetraspanina 29 , Transcrição GênicaRESUMO
The feline lentivirus, FIV, contains dUTPase (DU) as part of the enzyme cassette encoded by the viral pol gene (Elder et al., 1992, J. Virol. 66, 1791-1794). The enzyme is processed from the Pol polyprotein and is packaged into infectious virions. We report here the basic characteristics of the viral enzyme, including substrate specificity, ion requirements, and pH optimum. We also report the overexpression of DU in Escherichia coli and insertional mutagenesis of the enzyme in the context of the complete provirus or DU alone. The enzyme requires Mg2+ for full activity and competition studies employing unlabeled dNTPs indicated that DU has an absolute preference for dUTP. The pH optimum for FIV DU is pH 7.0. The limits of the protein dictate a species of M(r) 14,350, which agrees precisely with the determination by ion spray mass spectroscopy of DU isolated from virions. Cleavage sites at the junctions between DU, RT, and IN, as defined by N-terminal amino acid sequencing of each protein species, are consistent with predictions for sites of cleavage by aspartate protease. In-frame insertional mutagenesis at Tyr 75 of DU abolishes activity. Cells transfected with proviruses containing this mutation express virion-associated reverse transcriptase activity but lack DU activity. The resultant virions replicate slower than those possessing wild-type DU. Tests are currently underway to evaluate the consequences of DU mutagenesis on in vivo phenotype.
Assuntos
Vírus da Imunodeficiência Felina/enzimologia , Pirofosfatases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Gatos , Células Cultivadas , Clonagem Molecular , Análise Mutacional de DNA , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Vírus da Imunodeficiência Felina/genética , Magnésio/farmacologia , Dados de Sequência Molecular , Mutagênese Insercional , Pirofosfatases/biossíntese , Pirofosfatases/efeitos dos fármacos , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Cyanovirin-N (CV-N) is a cyanobacterial protein with potent neutralizing activity against human immunodeficiency virus (HIV). CV-N has been shown to bind HIV type 1 (HIV-1) gp120 with high affinity; moreover, it blocks the envelope glycoprotein-mediated membrane fusion reaction associated with HIV-1 entry. However, the inhibitory mechanism(s) remains unclear. In this study, we show that CV-N blocked binding of gp120 to cell-associated CD4. Consistent with this, pretreatment of gp120 with CV-N inhibited soluble CD4 (sCD4)-dependent binding of gp120 to cell-associated CCR5. To investigate possible effects of CV-N at post-CD4 binding steps, we used an assay that measures sCD4 activation of the HIV-1 envelope glycoprotein for fusion with CCR5-expressing cells. CV-N displayed equivalently potent inhibitory effects when added before or after sCD4 activation, suggesting that CV-N also has blocking action at the level of gp120 interaction with coreceptor. This effect was shown not to be due to CV-N-induced coreceptor down-modulation after the CD4 binding step. The multiple activities against the HIV-1 envelope glycoprotein prompted us to examine other enveloped viruses. CV-N potently blocked infection by feline immunodeficiency virus, which utilizes the chemokine receptor CXCR4 as an entry receptor but is CD4 independent. CV-N also inhibited fusion and/or infection by human herpesvirus 6 and measles virus but not by vaccinia virus. Thus, CV-N has broad-spectrum antiviral activity, both for multiple steps in the HIV entry mechanism and for diverse enveloped viruses. This broad specificity has implications for potential clinical utility of CV-N.
Assuntos
Antivirais/farmacologia , Proteínas de Bactérias , Proteínas de Transporte/farmacologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Animais , Fármacos Anti-HIV/farmacologia , Antígenos CD4/metabolismo , Gatos , Linhagem Celular , Proteína gp120 do Envelope de HIV/efeitos dos fármacos , Herpesvirus Humano 6/efeitos dos fármacos , Humanos , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Vírus do Sarampo/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Camundongos , Receptores CCR5/metabolismoRESUMO
The nonprimate lentiviruses feline immunodeficiency virus, equine infectious anemia virus, visna virus, and caprine encephalitis virus contain a gene segment in the polymerase gene that is lacking in the primate lentiviruses. A related sequence has been noted in other retroviruses, most notably the type D retroviruses. Computer searches have indicated a relatedness between this unique gene segment, termed proteaselike element and elements of both the aspartate proteinase and the dUTPase enzyme families. In this report, we show that members of both nonprimate lentiviruses and type D retroviruses possess dUTPase activity and present a formal demonstration that in feline immunodeficiency virus, the activity is encoded by the proteaselike element.
Assuntos
Produtos do Gene pol/metabolismo , Pirofosfatases/genética , Retroviridae/enzimologia , Sequência de Aminoácidos , Vírus da Imunodeficiência Felina/enzimologia , Dados de Sequência MolecularRESUMO
Feline immunodeficiency virus (FIV) encodes the enzyme deoxyuridine-triphosphatase (DU; EC 3.6.1.23) between the coding regions for reverse transcriptase and integrase in the pol gene. Here, we report the in vivo infection of cats with a DU- variant of the PPR strain of FIV and compare its growth properties and tissue distribution with those of wild-type FIV-PPR. The results reveal several important points: (i) DU- FIV is able to infect the cat, with kinetics similar to that observed with wild-type FIV; (ii) both wild-type and DU- FIV-infected specific-pathogen free cats mount a strong humoral antibody response which is able to limit the virus burden in both groups of animals; (iii) the virus burden is reduced in the DU- FIV-infected cats, particularly in tissues such as spleen and salivary gland; and (iv) the mutation frequency in DU- FIVs integrated in the DNA of primary macrophages after 9 months of infection is approximately 5-fold greater than the frequency observed in DU- FIV DNA integrated in T lymphocytes. Mutation rate with wild-type FIV remains the same in both cell types in vivo. The dominant mutations seen in macrophages with DU- FIV are G-->A base changes, consistent with an increased misincorporation of deoxyuridine into viral DNA of DU- FIVs during reverse transcription. Because this enzyme is absent from human immunodeficiency virus type 1 and other primate lentiviruses, virus replication in cell environments with low DU activity may lead to increased mutation and contribute to the rapid expansion of the viral repertoire.
Assuntos
Vírus da Imunodeficiência Felina/enzimologia , Vírus da Imunodeficiência Felina/genética , Mutação , Pirofosfatases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Capsídeo/genética , Capsídeo/imunologia , Gatos , Primers do DNA/genética , DNA Viral/genética , Síndrome de Imunodeficiência Adquirida Felina/virologia , Genes pol , Genoma Viral , Vírus da Imunodeficiência Felina/crescimento & desenvolvimento , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Distribuição TecidualRESUMO
We have characterized an apparently full-length cDNA corresponding to a rat mRNA, SE6C, previously identified by subtractive hybridization as being expressed predominantly in the striatal region of the brain. The SE6C mRNA encodes a 266 amino acid protein with significant similarity to members of the Ras-like GTP-binding protein family; thus, we have chosen the name Rhes, for Ras homolog enriched in striatum. The human homolog was found in a genomic sequence from human chromosome 22q13.1 and shares 95% identity with rat Rhes. Among the family of small G-proteins, Rhes shares 62% identity with Dexras1, a mouse dexamethasone-inducible Ras-like protein. Both Rhes and Dexras1 have substantially longer C-termini than other members of the Ras-like small G-protein family. Divergence between the C-terminal sequences of Rhes and Dexras1 suggests that, although their functions are probably similar, they have unique properties. Bacterially expressed Rhes binds GTP, suggesting that the protein indeed has GTPase functionality. Although Rhes was not induced by dexamethasone, its full expression is dependent upon thyroid hormone availability. Its accumulation is postnatal, consistent with the dependence upon thyroid hormone. It is noteworthy that most striatum-"specific" mRNAs characterized to date encode components of signal transduction cascades.
Assuntos
Corpo Estriado/metabolismo , DNA Complementar/genética , GTP Fosfo-Hidrolases/genética , Proteínas Monoméricas de Ligação ao GTP , Proteínas ras , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Ratos , Homologia de Sequência de Aminoácidos , Hormônios Tireóideos/fisiologiaRESUMO
The S3 and S3' subsite binding specificities of HIV and feline immunodeficiency virus proteases (FIV) proteases (PRs) have been explored by using C2-symmetric competitive inhibitors. The inhibitors evaluated contained (1S, 2R, 3R, 4S)-1,4-diamino-1, 4-dibenzyl-2,3-diol as P1 and P1' units, Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed very high potency against HIV PR in vitro, and their Ki values ranged between 1.1 and 2.6 nM. In contrast to the low restriction of P3 and P3' residues observed in HIV PR, FIV PR exhibited strong preference for small hydrophobic groups at the S3 and S3' subsites. Within this series, the most effective inhibitor against FIV PR contained Ala at P3 and P3'. Its Ki of 41 nM was 415- and 170-fold lower than those of the inhibitors without the P3 and P3' moieties or with the Phe at these positions, respectively. In addition, these compounds were tested against mutant FIV PRs, which contain amino acid substitutions corresponding to those in native HIV PR at homologous sites, and their efficacy of inhibition progressively increased up to 5-fold. The most potent FIV PR inhibitor was selected for examination of its effectiveness in tissue culture, and it was able to block nearly 100% of virus production in an acute infection at 1 microg/ml (1.1 microM) against HIV, FIV, and simian immunodeficiency virus. Furthermore, it was not toxic to cells, and even after 2 months of culture there was no sign of resistance development by virus. The findings suggest that inhibitors with small P3 residue may be efficacious against a broad range of HIV variants as well as interspecies PRs.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Desenho de Fármacos , Inibidores de Proteases/síntese química , Animais , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Gatos , Linhagem Celular , Modelos Animais de Doenças , Síndrome de Imunodeficiência Adquirida Felina/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida Felina/enzimologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , Protease de HIV/química , Protease de HIV/metabolismo , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/uso terapêutico , Humanos , Vírus da Imunodeficiência Felina/enzimologia , Cinética , Inibidores de Proteases/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/enzimologia , Vírus da Imunodeficiência Símia/enzimologia , Relação Estrutura-AtividadeRESUMO
Feline immunodeficiency virus (FIV) is a lentivirus associated with an immunodeficiency syndrome of the domestic cat. A short open reading frame (ORF2), of unknown function, is present in all FIV isolates. We have investigated the role of ORF2 in determining the cell tropism of two infectious molecular clones of FIV. FIV-PPR is able to productively infect feline peripheral blood leukocytes (PBLs) and a T lymphocyte cell line (MCH5-4), but not a feline astrocyte cell line (G355-5) or Crandell feline kidney cells (CrFK). In contrast, FIV-34TF10 is able to productively infect G355-5 and CrFK cells, but not PBLs or MCH5-4 cells. The major difference in these FIV clones is that ORF2 in FIV-PPR is capable of encoding a 79-amino-acid peptide, whereas there is a stop codon in ORF2 after 43 amino acids in FIV-34TF10. We performed site-directed mutagenesis to change the stop codon (TGA) in FIV-34TF10 to a tryptophan (TGG), the amino acid present at this location in FIV-PPR. FIV-34TF10 with ORF2 repaired (FIV-ORF2rep) productively infected PBLs, MCH5-4 cells, and primary macrophages, as well as CrFK and G355-5 cells, indicating that a protein encoded by ORF2 plays a role in determining the host cell tropism of FIV. ORF2 contains hydrophobic, acidic, and leucine-rich domains similar to those shown to be important for transactivating proteins of other lentiviruses. Coexpression of a plasmid expressing the ORF2 gene product with another construct expressing the chloramphenicol acetyl transferase (CAT) gene driven by the FIV LTR, resulted in transactivation of CAT expression in both feline and human cells.
Assuntos
Vírus da Imunodeficiência Felina/crescimento & desenvolvimento , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Gatos , Células Cultivadas , DNA Viral/genética , Humanos , Vírus da Imunodeficiência Felina/química , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/isolamento & purificação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Relação Estrutura-Atividade , Ativação Transcricional , Transfecção , Proteínas Virais/químicaRESUMO
Specific pathogen-free cats were infected with the Maryland strain of FIV (FIV-MD) for the purpose of assessing the effects of FIV infection on the central nervous system (CNS). Two separate studies were performed, involving a total of 13 infected cats and six age-matched, sham-inoculated controls. All animals infected with FIV-MD seroconverted by 8 weeks post-infection and virus was recovered from peripheral blood mononuclear cells of all infected cats. All of the infected animals had lower absolute CD4+ cells counts and decreased CD4+/CD8+ ratios. Virus was recovered from the cerebrospinal fluid (CSF) of certain infected individuals, and antiviral antibody and pleocytosis were evident in the CSF of the majority of infected cats. Additionally, virus was recovered from tissue explants from the cerebellum, midbrain and brainstem of one sacrificed FIV+ cat. Specific neurological changes included anisocoria, delayed righting reflex and delayed pupillary reflex, as well as delayed visual and auditory evoked potentials, and marked alterations in sleep patterns similar to those reported for human immunodeficiency virus (HIV)-positive individuals. Histological evaluation revealed the presence of perivascular cuffing and glial nodules in FIV-infected cats. These results indicate that FIV causes an acute neurological disease that closely resembles the early neurological effects of HIV infection in humans and should serve well as an animal model for lentivirus-induced CNS disease.
Assuntos
Encefalopatias/fisiopatologia , Vírus da Imunodeficiência Felina , Infecções por Lentivirus/fisiopatologia , Doença Aguda , Animais , Gatos , Potenciais Evocados Auditivos , Potenciais Evocados Visuais , Olho/patologia , Hipocampo/patologia , Equilíbrio Postural , Reflexo Pupilar , Sono , Organismos Livres de Patógenos EspecíficosRESUMO
FIV is a lentivirus of domestic cats that causes a spectrum of diseases that is remarkably similar to the clinical syndrome produced by HIV infection in people. Both HIV and FIV has been shown to cause neurologic dysfunction. Specific Pathogen-Free (SPF) cats were placed into one of three groups: FIV-PPR infected; DU-FIV-PPR (a dUTPase mutant of the FIV-PPR clone) infected; or an age-matched control group. In both infected groups, the general clinical signs of infection included lymphadenopathy, oral ulcerations, rough hair coat, and conjuntivitis. Specific neurological changes in the FIV-PPR infected cats included hind limb paresis; delayed righting and pupillary reflexes; behavioral changes; delayed visual and auditory evoked potentials; decreased spinal and peripheral nerve conduction velocities; and marked alterations in sleep patterns. Most of these changes were also observed in the DU-FIV-PPR infected cats. However, these cats tended to have a slightly less severe disease. In this study, we have demonstrated that an infectious molecular clone of FIV closely parallels the disease course of wild type FIV-infected cats. By using a knockout gene mutant of this clone, we were able to demonstrate that the dUTPase gene is not essential for neuropathogenesis. Further use of the FIV-PPR clone should prove useful in determining the essential viral elements that are important in the neuropathogenesis of lentiviral infections.