RESUMO
Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis are arising in the field of deuterium labelling, where compounds bearing deuterium (2H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for 2H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4-2H]-NADH). Here we report on a strategy that combines a clean reductant (H2) with a cheap source of 2H-atoms (2H2O) to generate and recycle [4-2H]-NADH. By coupling [4-2H]-NADH-recycling to an array of C=O, C=N, and C=C bond reductases, we demonstrate asymmetric deuteration across a range of organic molecules under ambient conditions with near-perfect chemo-, stereo- and isotopic selectivity. We demonstrate the synthetic utility of the system by applying it in the isolation of the heavy drug (1S,3'R)-[2',2',3'-2H3]-solifenacin fumarate on a preparative scale.
Assuntos
Biocatálise , Técnicas de Química Sintética/métodos , Deutério/química , Marcação por Isótopo/métodos , Oxirredutases/química , Óxido de Deutério/química , Estrutura Molecular , Niacinamida/química , Succinato de Solifenacina/química , EstereoisomerismoRESUMO
We demonstrate electrochemical control of the nitrogenase MoFe protein, in the absence of Fe protein or ATP, using europium(iii/ii) polyaminocarboxylate complexes as electron transfer mediators. This allows the potential dependence of proton reduction and inhibitor (CO) binding to the active site FeMo-cofactor to be established. Reduction of protons to H2 is catalyzed by the wild type MoFe protein and ß-98TyrâHis and ß-99PheâHis variants of the MoFe protein at potentials more negative than -800 mV (vs. SHE), with greater electrocatalytic proton reduction rates observed for the variants compared to the wild type protein. Electrocatalytic proton reduction is strongly attenuated by carbon monoxide (CO), and the potential-dependence of CO binding to the FeMo-cofactor is determined by in situ infrared (IR) spectroelectrochemistry. The vibrational wavenumbers for CO coordinated to the FeMo-cofactor are consistent with earlier IR studies on the MoFe protein with Fe protein/ATP as reductant showing that electrochemically generated states of the protein are closely related to states generated with the native Fe protein as electron donor.
RESUMO
We describe an approach to generating and verifying well-defined redox states in metalloprotein single crystals by combining electrochemical control with synchrotron infrared microspectroscopic imaging. For NiFe hydrogenase 1 from Escherichia coli we demonstrate fully reversible and uniform electrochemical reduction from the oxidised inactive to the fully reduced state, and temporally resolve steps during this reduction.
Assuntos
Técnicas Eletroquímicas , Hidrogenase/química , Cristalização , Escherichia coli/enzimologia , Hidrogenase/metabolismo , Oxirredução , Espectrofotometria InfravermelhoRESUMO
During the past decade, both in vitro and in vivo studies have provided new insights into the cellular and molecular mechanisms that govern angiogenesis and arteriogenesis. However, therapeutic angiogenesis clinical trials using recombinant protein or gene therapy formulations of single angiogenic growth factors have yielded at best only modest success to date. Among the second generation of angiogenic agents are therapeutic transgenes that enhance expression of two or more proangiogenic cytokines. These include synthetic constructs that mimic that activity of endogenous transcriptional regulators and other upstream, regulatory factors that have the potential to induce formation of morphologically and physiologically functional vessels. These agents are now beginning to be evaluated in clinical trials for patients with advanced ischemic cardiac and peripheral vascular disease.
Assuntos
Terapia Genética/tendências , Isquemia Miocárdica/terapia , Doenças Vasculares Periféricas/terapia , Citocinas/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Isquemia Miocárdica/fisiopatologia , Neovascularização Fisiológica , Doenças Vasculares Periféricas/fisiopatologia , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Because there is no muscle disease specific measure of quality of life (QoL), we wanted to develop and validate an individualized muscle disease specific measure of QoL for adults suitable for both clinical and research use. METHODS: A literature review exploring QoL and its measurement resulted in the development of a theoretical model of QoL. This was used alongside qualitative interviews (n = 41) and a postal survey (n = 252) to design a questionnaire. The psychometric properties, validity (n = 95), reliability (n = 40), and responsiveness (n = 25) of the scale were assessed. RESULTS: The Individualized Neuromuscular Quality of Life questionnaire (INQoL) consists of 45 questions within 10 sections. Four of these focus on the impact of key muscle disease symptoms (weakness, locking [i.e., myotonia], pain, and fatigue), five look at the impact (degree and importance of impact) muscle disease has on particular areas of life, and one section asks about the positive and negative effects of treatment. The questionnaire is structured to allow for variations in the individual characteristics that influence quality of life. Psychometric evaluation established construct validity and test-retest reliability. A preliminary assessment of responsiveness was obtained. CONCLUSIONS: The Individualized Neuromuscular Quality of Life is a validated muscle disease specific measure of quality of life developed from the experiences of patients with muscle disease and can be used for individuals or large samples.
Assuntos
Avaliação da Deficiência , Doenças Neuromusculares/psicologia , Qualidade de Vida/psicologia , Inquéritos e Questionários/normas , Atividades Cotidianas/psicologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Imagem Corporal , Feminino , Humanos , Entrevista Psicológica , Masculino , Transtornos Mentais/diagnóstico , Transtornos Mentais/epidemiologia , Pessoa de Meia-Idade , Doenças Neuromusculares/reabilitação , Valor Preditivo dos Testes , Psicometria/métodos , Reprodutibilidade dos TestesRESUMO
Adenovirus-mediated gene transfer of Fas ligand (FasL) inhibits neointimal formation in balloon-injured rat carotid arteries. Vascular smooth muscle (VSM) cells coexpressing murine FasL and p35, a baculovirus gene that inhibits caspase activity, are not susceptible to FasL-mediated apoptosis in vitro but are capable of inducing apoptosis of VSM cells that do not express p35. We reasoned that coexpression of p35 in FasL-transduced VSM cells in vivo would promote their survival, enhance FasL-induced apoptosis of adjacent VSM cells, and thereby facilitate a greater inhibition of neointimal formation. In balloon-injured rabbit femoral arteries, either Ad2/FasL/p35 or Ad2/FasL was infused into the injured site and withdrawn 20 min later. Both vectors induced a dose-dependent reduction (p < 0.05) of the neointima-to-media ratio when assessed 14 days later. However, Ad2/FasL/p35 exhibited a significantly greater inhibition of neointimal formation than Ad2/FasL. In a more clinically relevant model of restenosis, rabbit iliac arteries were injured with an angioplasty catheter under fluoroscopic guidance. Adenoviral vectors were delivered locally to the injured site over a period of 2 min, using a porous infusion balloon catheter. Twenty-eight days after gene transfer angiographic and histologic assessments indicated a significant (p < 0.05) inhibition of iliac artery lumen stenosis and neointimal formation by Ad2/FasL/p35 (5 x 10(11) particles per artery). The extent of inhibition was comparable to that achieved with Ad2/TK, an adenoviral vector encoding thymidine kinase (5 x 10(11) particles per artery) and coadministration of ganciclovir for 7 days. These data suggest that coexpression of p35 in FasL-transduced VSM cells is more potent at inhibiting neointimal formation and as such represents an improved gene therapy approach for restenosis.
Assuntos
Apoptose , Reestenose Coronária/prevenção & controle , Inibidores de Cisteína Proteinase , Artéria Femoral/lesões , Artéria Ilíaca/lesões , Glicoproteínas de Membrana/genética , Proteínas Virais/genética , Adenovírus Humanos , Animais , Oclusão com Balão , Proteína Ligante Fas , Artéria Femoral/patologia , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Artéria Ilíaca/patologia , Proteínas Inibidoras de Apoptose , Masculino , Coelhos , Timidina Quinase/genética , Túnica Íntima/patologiaRESUMO
BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that regulates expression of genes involved in O(2) homeostasis, including vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis. We sought to exploit this native adaptive response to hypoxia as a treatment for chronic ischemia. METHODS AND RESULTS: A hybrid protein consisting of DNA-binding and dimerization domains from the HIF-1alpha subunit and the transactivation domain from herpes simplex virus VP16 protein was constructed to create a strong, constitutive transcriptional activator. After transfection into HeLa, C6, and Hep3B cells, this chimeric transcription factor was shown to activate expression of the endogenous VEGF gene, as well as several other HIF-1 target genes in vitro. The bioactivity of HIF-1alpha/VP16 hybrid gene transfer in vivo was examined in a rabbit model of hindlimb ischemia. Administration of HIF-1alpha/VP16 was associated with significant improvements in calf blood pressure ratio, angiographic score, resting and maximal regional blood flow, and capillary density (all P:<0.01). CONCLUSIONS: The HIF-1alpha/VP16 hybrid transcription factor is able to promote significant improvement in perfusion of an ischemic limb. These results confirm the feasibility of a novel approach for therapeutic angiogenesis in which neovascularization may be achieved indirectly by use of a transcriptional regulatory strategy.
Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/terapia , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/uso terapêutico , Vacinas de DNA/administração & dosagem , Angiografia , Animais , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Circulação Colateral/efeitos dos fármacos , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/uso terapêutico , Fatores de Crescimento Endotelial/metabolismo , Eritropoetina/biossíntese , Estudos de Viabilidade , Terapia Genética/métodos , Hematócrito , Proteína Vmw65 do Vírus do Herpes Simples/genética , Membro Posterior/diagnóstico por imagem , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Injeções Intramusculares , Linfocinas/metabolismo , Masculino , Neovascularização Fisiológica/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas Nucleares/uso terapêutico , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/uso terapêutico , Ativação Transcricional/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
BACKGROUND: Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Traditionally AAV has been purified from cell lysates using CsCl gradients; this approach however is not likely to be useful in large-scale manufacturing. Moreover gradient-purified AAV vectors tend to be contaminated with significant levels of cellular and adenoviral proteins and nucleic acid. To address the issue of purification we have developed a process scale method for the rapid and efficient purification of recombinant AAV (rAAV) from crude cellular lysates. METHODS: The preferred method for the purification of rAAVbetagal includes treatment of virally infected cell lysates with both trypsin and nuclease followed by ion exchange chromatography using ceramic hydroxyapatite and DEAE-Sepharose in combination with cellufine sulphate affinity chromatography. RESULTS: Purification of rAAV particles from crude cellular lysates co-infected with adenovirus was achieved using column chromatography exclusively. Column-purified rAAV was shown to be greater than 90% pure, free of any detectable contaminating adenovirus, biologically active, and capable of directing efficient gene transfer to the lungs of both cotton rats and mice. CONCLUSIONS: This study demonstrates the feasibility of using column chromatography alone for the isolation of highly purified rAAV vector. The methods described here are advancements in procedures to purify rAAV and are adaptable for commercial production of clinical-grade rAAV vector.
Assuntos
Cromatografia/métodos , Dependovirus/isolamento & purificação , Animais , Linhagem Celular , Cromatografia por Troca Iônica , DNA Recombinante/isolamento & purificação , Dependovirus/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Humanos , Pulmão/metabolismo , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sigmodontinae , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The prevalence of tarsal coalition is probably 1% or less. The two sites most commonly affected are the calcaneonavicular joint and the middle facet of the talocalcaneal joint. Diagnosis should be suspected in the preteen or teenage patient with insidious or sudden onset of pain in the midfoot to hindfoot associated with a lack of motion in the subtalar joint. Initial treatment with immobilization or an orthosis may relieve symptoms, but most patients will have persistent symptoms that warrant surgical correction. Long-term results indicate that excision of the coalition is moderately successful in relieving symptoms in the calcaneonavicular bar. Long-term success with excision of subtalar bars is less clear, although early relief of symptoms is usually possible.
Assuntos
Pé Chato , Deformidades Congênitas do Pé , Ossos do Tarso/anormalidades , Adolescente , Criança , Pré-Escolar , Feminino , Pé Chato/etiologia , Pé Chato/cirurgia , Deformidades Congênitas do Pé/diagnóstico por imagem , Deformidades Congênitas do Pé/cirurgia , Humanos , Lactente , Recém-Nascido , Masculino , RadiografiaRESUMO
BACKGROUND: The medical literature has described the prevalence of latex allergy in the spina bifida population and its implications for surgical intervention. We report three cases of severe and unexpected intra-operative anaphylaxis secondary to latex exposure in non-spina bifida patients. METHODS: A retrospective review of case notes identified three non-spina bifida patients who suffered intra-operative anaphylaxis due to latex allergy. Personal and telephone interview and patient chart review was performed to detail a past history of multiple latex exposure, atopy, the anaphylaxis event and the postoperative outcome. RESULTS: Three non-spina bifida patients are described. One suffered a cardiopulmonary arrest, the remaining two patients had severe vascular hypotension and airway resistance that was only relieved after administration of vasoconstrictors and bronchodilators. Postoperatively, all three tested strongly positive to latex allergen testing. Each patient had a history of multiple surgical latex exposure and specific allergies or allergic-type symptoms pre-operatively. CONCLUSION: We believe that the predictors of a severe allergic reaction to latex with surgical exposure in non-spina bifida patients may be similar to those predictors known in the spina bifida population. Identification of such at-risk patients will reduce the risk of significant intra-operative morbidity and possible mortality by the introduction of a latex-free operating environment.
Assuntos
Anafilaxia/etiologia , Luvas Cirúrgicas , Complicações Intraoperatórias , Borracha/efeitos adversos , Anormalidades Múltiplas/cirurgia , Adolescente , Criança , Feminino , Luxação do Quadril/cirurgia , Humanos , Osteotomia , Estudos Retrospectivos , Disrafismo EspinalRESUMO
Knowledge of the mutations leading to inherited retinal degenerations provides a foundation for the development of somatic gene therapy in which potentially corrective genes are transferred to the target photoreceptor cells. Towards this end, we have evaluated the efficacy of a recombinant adeno-associated virus (AAV) vector to deliver and express the correct form of the cGMP phosphodiesterase-beta (PDE-beta) gene in the retinas of rd mice, which suffer rapid retinal degeneration due to recessive mutation in the endogenous gene. A truncated murine opsin promoter was used to drive expression of the PDE-beta cDNA. Following intraocular injection of AAV. PDE-beta, increased retinal expression of immunoreactive PDE protein was observed, including within photoreceptor cell bodies. Compared with age-matched controls, treated eyes showed increased numbers of photoreceptors and a two-fold increase in sensitivity to light as measured by in vitro electroretinography. These findings provide evidence that rescue of functional photoreceptor neurons can be achieved by somatic gene therapy.
Assuntos
Dependovirus , Técnicas de Transferência de Genes , Vetores Genéticos , Células Fotorreceptoras/fisiologia , Degeneração Retiniana/genética , Animais , Modelos Animais de Doenças , Eletrorretinografia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Células Fotorreceptoras/patologia , Degeneração Retiniana/patologia , Degeneração Retiniana/terapiaRESUMO
PURPOSE: The purpose of this report is to present a case of multifocal osteosarcoma in a patient with Fanconi anemia. PATIENTS AND METHODS: A nine year old girl with known Fanconi anemia presented with a pathologic femur fracture. Imaging studies confirmed soft tissue and bony involvement in the femur, ipsilateral tibia, and possibly contralateral femur. RESULTS: Biopsy and subsequent amputation confirmed the presence of classic osteosarcoma in the involved femur and ipsilateral tibia. The patient died seven months later of pulmonary failure secondary to metastases. CONCLUSION: Fanconi anemia is known to be associated with malignancies such as leukemias. We report a new association of Fanconi anemia with osteosarcoma.
Assuntos
Neoplasias Ósseas/complicações , Anemia de Fanconi/complicações , Osteossarcoma/complicações , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Criança , Evolução Fatal , Feminino , Humanos , Neoplasias Pulmonares/secundário , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/patologia , RadiografiaRESUMO
Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Because the gene transfer vector commonly retains only the AAV terminal repeats, propagation of recombinant AAV (rAAV) requires that the viral replication (Rep) and capsid (Cap) proteins be supplied in trans. In an effort to optimize the production of these vectors, a panel of helper plasmids was constructed to determine if expression of the rep and/or cap genes is a limiting factor for rAAV packaging. Expression of the Rep and Cap proteins was increased by replacing the endogenous AAV promoters, p5 and p40, with the Rous sarcoma virus (RSV) long terminal repeat (LTR) and the cytomegalovirus immediate-early promoter, respectively. Increased synthesis of the Cap proteins resulted in an approximately 10-fold increase in the yield of rAAV, indicating that production of capsid proteins is one limiting factor for rAAV packaging. Expression of the rep gene from the RSV LTR not only failed to increase the yield of rAAV but also prevented activation of p40 transcription with adenovirus infection, resulting in a reduced level of capsid protein synthesis.
Assuntos
Capsídeo/genética , Proteínas de Ligação a DNA/genética , Dependovirus/crescimento & desenvolvimento , Proteínas Virais/genética , Vírus do Sarcoma Aviário/genética , Linhagem Celular Transformada , Citomegalovirus/genética , Dependovirus/fisiologia , Regulação para Baixo , Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Plasmídeos , Montagem de Vírus/fisiologiaRESUMO
Integrase mediates integration of the retroviral genome into a host cell chromosome, an essential step in the viral life cycle. In vitro, a stable complex containing only purified human immunodeficiency virus (HIV) integrase and a model viral DNA substrate processively executes the 3'-end processing and DNA joining steps in the integration reaction. We examined the relationship of three essential components of the HIV integrase: the HHCC domain, a putative zinc-finger near the N terminus; the phylogenetically conserved "DD35E" motif, which defines the catalytic domain; and a feature recognized by its sensitivity to the alkylating agent N-ethylmaleimide (NEM). HIV integrase is a multimer, and these three components can be distributed among at least two subunits of the multimeric enzyme. The components function asymmetrically in the active multimer; the DD35E motif and NEM-sensitive site are required in trans to the HHCC region. A divalent cation-dependent interaction involving the NEM-sensitive site of one integrase subunit and the HHCC region of another subunit points to a role for these two features of integrase in multimer assembly. Deletion of the HHCC domain, or modification of integrase with NEM, impaired the assembly of a stable complex between integrase and viral DNA, suggesting that this initial step in the integration pathway requires assembly of the active integrase multimer.
Assuntos
DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/metabolismo , HIV-1/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Cloretos/farmacologia , DNA Nucleotidiltransferases/biossíntese , Ditiotreitol/farmacologia , Etilmaleimida/farmacologia , Teste de Complementação Genética , HIV-1/genética , Integrases , Cinética , Substâncias Macromoleculares , Cloreto de Magnésio/farmacologia , Compostos de Manganês/farmacologia , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/metabolismo , Mutação Puntual , Deleção de Sequência , Especificidade por Substrato , Integração ViralRESUMO
Previous studies have shown renal mesenchymal tumors (RMTs) induced in rats by a single intrarenal injection of nickel subsulfide and iron are more pleomorphic and metastatically aggressive than RMTs induced by a single ip injection of methyl(methoxymethyl)nitrosamine (DMN-OMe). While both RMT types contain high levels of K-ras activation, the specific mutational spectra within codon 12 of K-ras are quite different. Nickel subsulfide and iron-induced tumors exhibited codon 12 GGT-->GTT transversions exclusively, while DMN-OMe RMTs showed a wide array of codon 12 mutations, as well as mutations within codons 61 and 63 [K. G. Higinbotham, J. M. Rice, B. A. Diwan, K. S. Kasprzak, C. D. Reed, and A. O. Perantoni, Cancer Res., 52: 4747-4751, 1992; K. G. Higinbotham, J. M. Rice, and A. O. Perantoni, Mol. Carcinog., 5: 136-139, 1992]. In an effort to further correlate carcinogen-specific molecular events in renal tumors, we investigated the p53 tumor suppressor gene in RMTs induced by these two carcinogens for the presence of point mutations. The evolutionarily conserved portion of the coding region of the gene, including part of exon 4 through exon 10, was surveyed for point mutations utilizing single-strand conformation polymorphism and chemical cleavage of mismatches analyses. None (0 of 10) of the nickel subsulfide and iron-induced RMTs and only 1 of 10 DMN-OMe-induced tumors that were evaluated contained point mutations within this portion of the p53 gene. Direct sequencing of the one single-strand conformation polymorphism and chemical cleavage of mismatches-"positive" DMN-OMe-induced RMT revealed a GCC-->GTC (Ala-->Val) transition in codon 345 within exon 10. These results suggest that the different tumorigenic phenotypes exhibited by these two RMTs are not the result of specific mutations or patterns of mutations within the portion of the p53 gene examined and that the mutated p53 tumorigenic pathway, whereby p53 plays a major role in many human neoplasms, does not function in RMTs induced by either agent.
Assuntos
Genes p53/genética , Neoplasias Renais/genética , Mutação Puntual , Animais , Sequência de Bases , Primers do DNA , Dimetilnitrosamina/análogos & derivados , Genes p53/efeitos dos fármacos , Neoplasias Renais/induzido quimicamente , Masculino , Dados de Sequência Molecular , Níquel , Ratos , Ratos Endogâmicos F344 , Análise de Sequência de DNARESUMO
Our experience with the Sauve-Kapandji procedure for reconstruction of the rheumatoid distal radioulnar joint is reported. Twenty-one wrists in 17 patients were followed for an average of 39 months postoperatively. Average range of motion at follow-up evaluation was pronation to 78 degrees and supination to 86 degrees. X-ray films demonstrated that significant ulnarward and palmarward translocation of the carpus was prevented. The Sauve-Kapandji procedure provides a stable ulnar side support in the rheumatoid wrist with distal radioulnar degeneration.
Assuntos
Artrite Reumatoide/cirurgia , Articulação do Punho/cirurgia , Adulto , Idoso , Artrite Reumatoide/diagnóstico por imagem , Feminino , Humanos , Masculino , Métodos , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Radiografia , Rádio (Anatomia) , Estudos Retrospectivos , Ulna , Articulação do Punho/diagnóstico por imagemRESUMO
Replication of a retroviral genome depends upon integration of the viral DNA into a chromosome of the host cell. The integration reaction is mediated by integrase, a viral enzyme. Human immunodeficiency virus type 1 integrase was expressed in Escherichia coli and purified to near homogeneity. Optimum conditions for the integration and 3'-end-processing activities of integrase were characterized by using an in vitro assay with short, double-stranded oligonucleotide substrates. Mutants containing amino acid substitutions within the HHCC region, defined by phylogenetically conserved pairs of histidine and cysteine residues near the N terminus, were constructed and characterized by using three assays: 3'-end processing, integration, and the reverse of the integration reaction (or disintegration). Mutations in the conserved histidine and cysteine residues abolished both integration and processing activities. Weak activity in both assays was retained by two other mutants containing substitutions for less highly conserved amino acids in this region. All mutants retained activity in the disintegration assay, implying that the active site for DNA cleavage-ligation is not located in this domain and that the HHCC region is not the sole DNA-binding domain in the protein. However, the preferential impairment of processing and integration rather than disintegration by mutations in the HHCC region is consistent with a role for this domain in recognizing features of the viral DNA. This hypothesis is supported by the results of disintegration assays performed with altered substrates. The results support a model involving separate viral and target DNA-binding sites on integrase.
Assuntos
DNA Nucleotidiltransferases/genética , HIV-1/enzimologia , Proteínas dos Retroviridae/genética , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , DNA Nucleotidiltransferases/biossíntese , DNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , HIV-1/genética , Integrases , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oligonucleotídeos/metabolismo , Conformação Proteica , Proteínas dos Retroviridae/biossíntese , Proteínas dos Retroviridae/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Integração Viral , Dedos de ZincoRESUMO
In retroviral integration, the viral integration protein (integrase) mediates a concerted DNA cleavage-ligation reaction in which the target DNA is cleaved and the resulting 5' ends of target DNA are joined to the 3' ends of viral DNA. Through an oligonucleotide substrate that mimics the recombination intermediate formed by this initial cleavage-ligation reaction, the purified integrase of human immunodeficiency virus was shown to promote the same reaction in reverse, a process called disintegration. Analysis of a set of structurally related substrates showed that integrase could promote a range of DNA cleavage-ligation reactions. When the viral DNA component of the disintegration substrate was single-stranded, integrase could mediate a DNA splicing reaction analogous to RNA splicing.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA Viral/metabolismo , DNA/metabolismo , HIV-1/enzimologia , Sequência de Bases , DNA/química , DNA Viral/química , Esterificação , HIV-1/genética , Integrases , Dados de Sequência Molecular , Splicing de RNARESUMO
A 2.4 kb fragment of hCMV (Towne strain), containing the 5' end of the major immediate-early gene, has been cloned, sequenced, and used to construct a series of mammalian cell expression plasmids. The effects of regulatory regions present on this fragment were assessed using human glycoproteins as reporter molecules. We compared secreted levels of Factor VIII, t-PA, and HIV-1 envelope glycoproteins in cells transfected with plasmids in which intron A of the immediate-early gene was present or absent. Secretion of several glycoproteins was significantly higher when cells were transfected with intron A-containing plasmids. Mutation of three basepairs in the strong nuclear factor 1 (NF1) binding site in intron A led to reduced transient expression levels, but not to the level observed in the absence of intron A. Reduced expression from NF1 mutant plasmids was roughly correlated with reduced binding in vitro of NF1 proteins to a synthetic oligonucleotide containing the mutation. The evidence indicates that sequences in intron A positively regulate expression from the hCMV immediate-early enhancer/promoter in transformed monkey kidney cells.