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1.
Life Sci ; 308: 120948, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36096241

RESUMO

AIMS: To assess the potential direct effects of the integrase strand-transfer inhibitors (INsTIs) dolutegravir, bictegravir, and raltegravir, drugs used as treatment for people living with human immunodeficiency virus (PLWH), on human adipose cells. MAIN METHODS: Drugs were added to the differentiation medium of human Simpson-Golabi-Behmel syndrome (SGBS) adipose cells and morphological adipogenesis was monitored for 10 days. Also, adipocytes were exposed to drugs following differentiation (day 14). The gene expression levels of selected adipogenesis markers, adipocyte metabolism markers, adipokines, and cytokines were determined by quantitative-reverse transcription polymerase-chain reaction. The release of adiponectin and leptin into the culture medium was measured using specific enzyme-linked immunosorbent assay, and release of interleukin-6 and chemokine (CC motif) ligand-2 using Multiplex assays. KEY FINDINGS: Overall morphological adipogenesis was unaltered by INsTIs. The expression of adipogenesis marker genes (peroxisome proliferator-activated receptor-Ɣ and lipoprotein lipase) was slightly reduced in dolutegravir-treated differentiating adipocytes. Bictegravir repressed gene expression and the release of pro-inflammatory cytokines in differentiating adipocytes. Dolutegravir and raltegravir increased interleukin-6 gene expression, but only dolutegravir increased interleukin-6 release. Dolutegravir repressed adiponectin expression and release in differentiating adipocytes and had a similar but milder effect on leptin. Drug treatment of mature adipocytes reduced adiponectin gene expression in response to dolutegravir. SIGNIFICANCE: The INsTIs studied do not have a significant effect on human adipose cell differentiation but exert distinct effects on gene expression and secretion of adipokines and cytokines. These findings will help understand and manage the effects of INsTI-containing treatments on body weight and metabolic dysregulation in PLWH.


Assuntos
Adipocinas , Leptina , Adipócitos/metabolismo , Adipocinas/metabolismo , Adiponectina/metabolismo , Amidas , Citocinas/metabolismo , Compostos Heterocíclicos com 3 Anéis , Humanos , Inflamação/metabolismo , Integrases/metabolismo , Integrases/farmacologia , Interleucina-6/metabolismo , Leptina/metabolismo , Ligantes , Lipase Lipoproteica , Oxazinas , Receptores Ativados por Proliferador de Peroxissomo , Piperazinas , Piridonas , Raltegravir Potássico/metabolismo , Raltegravir Potássico/farmacologia
2.
Oncogene ; 40(22): 3815-3825, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33958722

RESUMO

The integration of viral DNA into the host genome is mediated by viral integrase, resulting in the accumulation of double-strand breaks. Integrase-derived peptides (INS and INR) increase the number of integration events, leading to escalated genomic instability that induces apoptosis. CD24 is a surface protein expressed mostly in cancer cells and is very rarely found in normal cells. Here, we propose a novel targeted cancer therapeutic platform based on the lentiviral integrase, stimulated by integrase-derived peptides, that are specifically delivered to cancerous cells via CD24 antigen-antibody targeting. INS and INR were synthesized and humanized and anti-CD24 antibodies were fused to the lentivirus envelope. The activity, permeability, stability, solubility, and toxicity of these components were analyzed. Cell death was measured by fluorescent microscopy and enzymatic assays and potency were tested in vitro and in vivo. Lentivirus particles, containing non-functional DNA led to massive cell death (40-70%). Raltegravir, an antiretroviral drug, inhibited the induction of apoptosis. In vivo, single and repeated administrations of INS/INR were well tolerated without any adverse effects. Tumor development in nude mice was significantly inhibited (by 50%) as compared to the vehicle arm. In summary, a novel and generic therapeutic platform for selective cancer cell eradication with excellent efficacy and safety are presented.


Assuntos
Antígeno CD24/biossíntese , Integrases/farmacologia , Lentivirus/enzimologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Fragmentos de Peptídeos/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Apoptose/efeitos dos fármacos , Antígeno CD24/imunologia , Linhagem Celular Tumoral , Humanos , Integrases/química , Lentivirus/genética , Lentivirus/imunologia , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/virologia , Fragmentos de Peptídeos/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Endocrinology ; 162(7)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33837405

RESUMO

Targeted oncogenesis is the process of driving tumor formation by engineering transgenic mice that express an oncogene under the control of a cell-type specific promoter. Such tumors can be adapted to cell culture, providing immortalized cell lines. To make it feasible to follow the process of tumorigenesis and increase the opportunity for generating cell lines, we developed a mouse strain that expresses SV40 T antigens in response to Cre-recombinase. Using CRISPR/Cas9 we inserted a cassette with coding sequences for SV40 T antigens and an internal ribosome entry site with green fluorescent protein cassette (IRES-GFP) into the Rosa26 locus, downstream from a stop sequence flanked by loxP sites: Rosa26LSL-SV40-GFP. These mice were mated with previously established Prop1-cre and Tshb-cre transgenic lines. Both the Rosa26LSL-SV40-GFP/+; Prop1-cre and Rosa26LSL-SV40-GFP/+; Tshb-cre mice developed fully penetrant dwarfism and large tumors by 4 weeks. Tumors from both of these mouse lines were adapted to growth in cell culture. We have established a progenitor-like cell line (PIT-P1) that expresses Sox2 and Pitx1, and a thyrotrope-like cell line (PIT-T1) that expresses Pou1f1 and Cga. These studies demonstrate the utility of the novel, Rosa26LSL-SV40-GFP mouse line for reliable targeted oncogenesis and development of unique cell lines.


Assuntos
Antígenos Transformantes de Poliomavirus/genética , Expressão Gênica/efeitos dos fármacos , Integrases/farmacologia , Neoplasias Hipofisárias/genética , Animais , Carcinogênese , Linhagem Celular Tumoral , Cruzamentos Genéticos , Técnicas de Introdução de Genes , Proteínas de Homeodomínio/genética , Hiperplasia , Camundongos , Camundongos Transgênicos , Hipófise/metabolismo , Hipófise/patologia , Tireotropina Subunidade beta/genética
4.
Bioorg Med Chem Lett ; 40: 127925, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33705909

RESUMO

Our research group has been studying the design of intracellular delivery peptides based on cationic lytic peptides. By placing negatively charged amino acids on potentially hydrophobic faces of the peptides, membrane lytic activity is attenuated on the cell surface, whereas it recovers in endosomes, enabling cytosolic delivery of proteins including antibodies. These lytic peptides generally contain multiple lysines, facilitating cell surface interaction and membrane perturbation. This study evaluated the effect of lysine-to-homoarginine substitution using HAad as a model delivery peptide. The resulting peptide had a comparable or better delivery efficacy for Cre recombinase, antibodies, and the Cas9/sgRNA complex with one-quarter of the concentration of HAad, implying that a subtle structural difference can affect delivery activity.


Assuntos
Portadores de Fármacos/química , Endossomos/metabolismo , Homoarginina/química , Membranas Intracelulares/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/farmacologia , Dextranos/química , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Fluoresceínas/química , Corantes Fluorescentes/química , Células HeLa , Humanos , Imunoglobulina G/farmacologia , Integrases/farmacologia , Lipossomos/química , Peptídeos/toxicidade , RNA Guia de Cinetoplastídeos/farmacologia , Ácidos Sulfônicos/química
5.
Sci Total Environ ; 724: 138248, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32247117

RESUMO

In this work, we investigated the impact of iron nanoparticle, including magnetite nanoparticles (Fe3O4 NPs) and nanoscale zero-valent iron (nZVI), on the anaerobic digestion (AD) performance. Moreover, the evolutions of antibiotic resistance genes (ARGs), class 1 integrons-integrase (intI1) and potential hosts of ARGs were also investigated. The optimal addition of Fe3O4 NPs and nZVI to promote methane production was 0.5 g/L and 1 g/L, which led to 22.07% and 23.02% increase in methane yield, respectively. The degradation rate of organic matter was also enhanced with the addition of Fe3O4 NPs or nZVI. The results of high-throughput sequencing showed that the reactors with iron NPs exhibited significant differences in microbial community structure, compared to the reactors with the non­iron NPs. Iron NPs have caused the relative abundance of the dominant bacteria (Proteobacteria, Firmicutes and Actinobacteria) generally decreased, while the dominant archaea (Euryarchaeota) increased in AD sludge. Quantitative PCR results revealed that iron NPs accelerated the reductions in total absolute abundance of ARGs, especially a beta-lactamase resistance encoded gene (blaOXA). Network analysis displayed that the attenuation of ARGs was mainly attributed to the decline of potential hosts (Proteobacteria, Firmicutes and Actinobacteria). Meanwhile, environmental factors (such as pH, soluble chemical oxygen demand and heavy metals) were also strongly correlated with ARGs.


Assuntos
Integrons , Nanopartículas , Anaerobiose , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Integrases/farmacologia , Ferro/farmacologia , Esgotos
6.
Hypertension ; 55(2): 277-83, 6p following 283, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20008675

RESUMO

The circumventricular organs (CVOs) lack a well-formed blood-brain barrier and produce superoxide in response to angiotensin II and other hypertensive stimuli. This increase in central superoxide has been implicated in the regulation of blood pressure. The extracellular superoxide dismutase (SOD3) is highly expressed in cells associated with CVOs and particularly with tanycytes lining this region. To understand the role of SOD3 in the CVOs in blood pressure regulation, we performed intracerebroventricular injection an adenovirus encoding Cre-recombinase (5x10(8) particles per milliliter) in mice with loxP sites flanking the SOD3 coding region (SOD3(loxp/loxp) mice). An adenovirus encoding red-fluorescent protein was injected as a control. Deletion of CVO SOD3 increased baseline blood pressure modestly and markedly augmented the hypertensive response to low-dose angiotensin II (140 ng/kg per day), whereas intracerebroventricular injection of adenovirus encoding red-fluorescent protein had minimal effects on these parameters. Adenovirus encoding Cre-recombinase-treated mice exhibited increased sympathetic modulation of heart rate and blood pressure variability, increased vascular superoxide production, and T-cell activation as characterized by increased circulating CD69(+)/CD3(+) cells. Deletion of CVO SOD3 also markedly increased vascular T-cell and leukocyte infiltration caused by angiotensin II. We conclude that SOD3 in the CVO plays a critical role in the regulation of blood pressure, and its loss promotes T-cell activation and vascular inflammation, in part by modulating sympathetic outflow. These findings provide insight into how central signals produce vascular inflammation in response to hypertensive stimuli, such as angiotensin II.


Assuntos
Angiotensina II/metabolismo , Sistema Nervoso Central/enzimologia , Hipertensão/metabolismo , Integrases/farmacologia , Superóxido Dismutase/metabolismo , Adenoviridae , Animais , Determinação da Pressão Arterial , Sistema Nervoso Central/efeitos dos fármacos , Modelos Animais de Doenças , Frequência Cardíaca/fisiologia , Hipertensão/fisiopatologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mediadores da Inflamação/análise , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , RNA Mensageiro/análise , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismo
7.
Microbiol Immunol ; 49(6): 559-70, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15965304

RESUMO

The site-specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication-deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive-expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell-growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre-expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SR alpha promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60-fold range of effective MOIs ("effective range") sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre-induced cell toxicity can be avoided by pre-examining the "effective range" using the purpose cell lines before starting experiments utilizing the experiment of Cre-expressing rAd.


Assuntos
Adenoviridae/genética , Integrases/genética , Proteínas Virais/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação Viral da Expressão Gênica , Vetores Genéticos/genética , Células HeLa , Humanos , Integrases/metabolismo , Integrases/farmacologia , Regiões Promotoras Genéticas , Proteínas Recombinantes/farmacologia , Linfócitos T , Ativação Transcricional , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
Hepatology ; 37(6): 1375-84, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12774017

RESUMO

The fetal liver, the major site of hematopoiesis during embryonic development, acquires additional functions near birth. Among the important liver functions is the response to xenobiotic exposure due to expression of several cytochromes P450 (CYP) and drug efflux transporters. Expression of these genes is regulated by nuclear receptors such as the pregnane X receptor (PXR). In this study, regulation of xenobiotic responses during fetal liver development was analyzed using a fetal hepatocyte primary culture system derived from embryonic day 15 (E15) livers. Hepatocyte nuclear factor (HNF) 4alpha regulates the expression of many genes preferentially in the liver. Expression of several xenobiotic response genes as well as HNF4alpha was increased in fetal hepatocytes stimulated by the hepatic maturation factors oncostatin M (OSM) and Matrigel. To determine the contribution of HNF4alpha to xenobiotic responses in the fetal liver, fetal hepatocytes containing floxed HNF4alpha alleles were cultured and the HNF4alpha gene was inactivated by infection with an adenovirus containing the Cre gene. Expression of CYP3A11 and PXR was suppressed by inactivation of HNF4alpha. An HNF4alpha binding site was characterized in the PXR promoter and found to be required for activation of the PXR promoter in fetal hepatocytes. In conclusion, HNF4alpha is the key transcription factor regulating responses to xenobiotics through activation of the PXR gene during fetal liver development.


Assuntos
Proteínas de Ligação a DNA , Fígado/embriologia , Fosfoproteínas/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Sítios de Ligação/genética , Células Cultivadas , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Técnicas de Transferência de Genes , Fator 4 Nuclear de Hepatócito , Hepatócitos/metabolismo , Integrases/farmacologia , Ligantes , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/antagonistas & inibidores , Receptor de Pregnano X , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteínas Virais/farmacologia
9.
Jpn J Cancer Res ; 93(10): 1154-63, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12417046

RESUMO

To enhance the efficacy of suicide gene therapy for prostate cancer under androgen deprivation, we designed a promoter system that consists of the prostate-specific membrane antigen (PSMA) promoter / enhancer (PEPM) and Cre-loxP DNA recombination system. We constructed two kinds of plasmids. One plasmid contains a Cre recombinase (Cre) under the control of PEPM and the other expresses CMV-lox-luciferase / herpes simplex virus thymidine kinase (TK). In PSMA-positive LNCaP cells, the promoter activity of the PEPM-Cre plus CMV-lox-luciferase demonstrated 800-fold greater activity compared with that of the PSMA promoter alone. However, no enhancement of the promoter activity was observed in the PSMA-negative cells. Furthermore, in contrast to prostate specific antigen promoter / enhancer (PP), the promoter activity of PEPM did not decrease when the LNCaP cells were cultured in charcoal-stripped fetal bovine serum (CFBS). In an in vitro gene therapy model with LNCaP cells, the cell growth inhibition in the presence of ganciclovir (GCV) was more evident in the cells transfected with the PEPM-Cre plus CMV-lox-TK than in the cells with the PP-TK, and the difference in efficacy between the two plasmids was more remarkable when the cells were maintained in CFBS medium. The therapeutic effect of PEPM-Cre plus CMV-lox-TK was also observed in xenografted LNCaP cells on nude mice when the plasmids were directly injected into tumors and GCV was administered intraperitoneally. These findings indicate that the combination of the PSMA promoter / enhancer and the Cre-loxP system can enhance the PSMA promoter activity even under androgen ablation conditions and can exert its anti-tumor effect both in vitro and in vivo.


Assuntos
Antígenos de Superfície , Carboxipeptidases/genética , Elementos Facilitadores Genéticos , Terapia Genética/métodos , Integrases/farmacologia , Regiões Promotoras Genéticas , Neoplasias da Próstata/terapia , Proteínas Virais/farmacologia , Animais , Ganciclovir/uso terapêutico , Glutamato Carboxipeptidase II , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Timidina Quinase/genética , Transfecção , Transplante Heterólogo , Células Tumorais Cultivadas
10.
Blood ; 98(7): 2248-55, 2001 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-11568013

RESUMO

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have blood cells deficient in glycosyl phosphatidylinositol (GPI)-linked proteins owing to a somatic mutation in the X-linked PIGA gene. To target Piga recombination to the erythroid/megakaryocytic lineage in mice, the Cre/loxP system was used, and Cre was expressed under the transcriptional regulatory sequences of GATA-1. Breeding of GATA1-cre (G) transgenic mice with mice carrying a floxed Piga (L) allele was associated with high embryonic lethality. However, double-transgenic (GL) mice that escaped early recombination looked healthy and were observed for 16 months. Flow cytometric analysis of peripheral blood cells showed that GL mice had up to 100% of red cells deficient in GPI-linked proteins. The loss of GPI-linked proteins on the cell surface occurred late in erythroid differentiation, causing a proportion of red cells to express low residual levels of GPI-linked proteins. Red cells with residual expression of GPI-linked proteins showed an intermediate sensitivity toward complement and thus resemble PNH type II cells in patients with PNH. Recombination of the floxed Piga allele was also detected in cultured megakaryocytes, mast cells, and eosinophils, but not in neutrophils, lymphocytes, or nonhematopoietic tissues. In summary, GATA1-Cre causes high-efficiency Piga gene inactivation in a GATA-1-specific pattern. For the first time, mice were generated that have almost 100% of red cells deficient in GPI-linked proteins. These animals will be valuable to further investigate the consequences of GPI-anchor deficiency on erythroid/megakaryocytic cells.


Assuntos
Proteínas de Ligação a DNA/farmacologia , Eritropoese/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Glicosilfosfatidilinositóis/deficiência , Integrases/farmacologia , Proteínas de Membrana/genética , Fatores de Transcrição/farmacologia , Proteínas Virais/farmacologia , Animais , Células da Medula Óssea/metabolismo , Linhagem da Célula , Proteínas de Ligação a DNA/fisiologia , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Hemoglobinúria Paroxística/patologia , Integrases/fisiologia , Fígado/citologia , Fígado/embriologia , Fígado/metabolismo , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos , Camundongos Transgênicos , Recombinação Genética , Fatores de Transcrição/fisiologia , Proteínas Virais/fisiologia
11.
J Biol Chem ; 276(36): 34213-20, 2001 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-11441016

RESUMO

Retroviral integrase (IN) recognizes linear viral DNA ends and introduces nicks adjacent to a highly conserved CA dinucleotide usually located two base pairs from the 3'-ends of viral DNA (the "processing" reaction). In a second step, the same IN active site catalyzes the insertion of these ends into host DNA (the "joining" reaction). Both DNA sequence and DNA structure contribute to specific recognition of viral DNA ends by IN. Here we used potassium permanganate modification to show that the avian sarcoma virus IN catalytic domain is able to distort viral DNA ends in vitro. This distortion activity is consistent with both unpairing and unstacking of the three terminal base pairs, including the processing site adjacent to the conserved CA. Furthermore, the introduction of mismatch mutations that destabilize the viral DNA ends were found to stimulate the IN processing reaction as well as IN-mediated distortion. End-distortion activity was also observed with mutant or heterologous DNA substrates. However, further analyses showed that using Mn(2+) as a cofactor, processing site specificity of these substrates was also maintained. Our results support a model whereby unpairing and unstacking of the terminal base pairs is a required step in the processing reaction. Furthermore, these results are consistent with our previous observations indicating that unpairing of target DNA promotes the joining reaction.


Assuntos
Vírus do Sarcoma Aviário/enzimologia , DNA/química , DNA/metabolismo , Integrases/química , Sequência de Bases , Catálise , Domínio Catalítico , DNA/efeitos dos fármacos , Escherichia coli/metabolismo , Vetores Genéticos , Integrases/farmacologia , Manganês/química , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Permanganato de Potássio/farmacologia , Ligação Proteica , Especificidade por Substrato , Fatores de Tempo
12.
J Virol ; 74(23): 11191-200, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11070016

RESUMO

Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Retroviridae/genética , Integração Viral , Sequência de Bases , DNA Ligase Dependente de ATP , DNA Ligases/farmacologia , Proteína Quinase Ativada por DNA , Integrases/farmacologia , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/fisiologia , DNA Polimerase Dirigida por RNA/farmacologia
13.
Nat Cell Biol ; 2(3): 148-55, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10707085

RESUMO

The INK4A locus encodes two independent but overlapping genes, p16INK4A and p19ARF, and is frequently inactivated in human cancers. The unusual structure of this locus has lead to ambiguity regarding the biological role of each gene. Here we express, in primary mouse embryonic fibroblasts (MEFs), antisense RNA constructs directed specifically towards either p16INK4A or p19 ARF. Such constructs induce extended lifespan in primary MEFs; this lifespan extension is reversed upon subsequent elimination of the p16INK4A or p19ARF antisense constructs. In immortal derivatives of cell lines expressing antisense p16INK4A or p19ARF RNA, growth arrest induced by recovery of p16INK4A expression is bypassed by compromising the function of the retinoblastoma protein (Rb), whereas growth arrest induced by re-expression of p19ARF is overcome only by simultaneous inactivation of both the Rb and the p53 pathways. Thus, the physically overlapping p16INK4A and p19ARF genes act in partly overlapping pathways.


Assuntos
Senescência Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Fibroblastos/metabolismo , Proteínas Nucleares , Biossíntese de Proteínas , Proteínas Virais , Animais , Antígenos Virais de Tumores/biossíntese , Antígenos Virais de Tumores/farmacologia , Morte Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/antagonistas & inibidores , Inibidor p16 de Quinase Dependente de Ciclina/genética , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Integrases/biossíntese , Integrases/genética , Integrases/farmacologia , Camundongos , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/farmacologia , Proteínas E7 de Papillomavirus , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Antissenso/farmacologia , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/genética , Proteína Supressora de Tumor p14ARF , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo
15.
Cell ; 90(6): 1073-83, 1997 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-9323135

RESUMO

Gene targeting experiments have demonstrated that the expression of immunoglobulin heavy chain in the pre-B cell receptor (pBCR) and of heavy and light chains in the B cell antigen receptor (BCR) marks checkpoints in early B cell development that the cells have to pass to survive. To investigate whether the persistence of mature B cells in the peripheral immune system also depends on BCR expression, we have generated a transgenic mouse in which the BCR can be inducibly ablated through V region gene deletion. Ablation leads to rapid death of mature B lymphocytes, which is preceded by down-regulation of MHC antigens and up-regulation of CD95 (Fas) and can be delayed by constitutive bcl-2 expression.


Assuntos
Linfócitos B/química , Marcação de Genes , Células-Tronco Hematopoéticas/química , Cadeias Pesadas de Imunoglobulinas/genética , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Proteínas Virais , Animais , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Apoptose/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Feminino , Citometria de Fluxo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Integrases/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas/genética , Fenótipo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcr , Transfecção
16.
J Virol ; 71(4): 2685-92, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9060621

RESUMO

Current molecular genetic strategies to inhibit productive human immunodeficiency virus type 1 (HIV-1) replication have involved the generation of gene products which provide intracellular inhibition of essential virally encoded proteins or RNA structures. A molecular strategy to excise proviral DNA from HIV-1-infected cells and render these cells virus free would provide an attractive direct antiviral strategy, providing a mechanism to remove viral genes from infected cells. The potential of such a molecular genetic intervention was examined by using the Cre-loxP recombination system. A recombinant HIV-1 clone, designated HIV(lox), that contains loxP within a nonessential U3 region of the long terminal repeats was synthesized. The loxP motif was maintained during replication of HIV(lox) in CEM cells, as demonstrated by reverse transcriptase PCR analyses of genomic RNA isolated from virions. Two different types of HIV-1-permissive cells, CEM cells and 293 cells expressing the CD4 glycoprotein, were transformed with a Cre expression vector which was shown to encode Cre DNA binding and recombinase activities. HIV(lox) infection of CEM or CD4+ 293 cells expressing Cre resulted in a substantial reduction in virus replication compared to control cells, and evidence for the presence of the expected excision product was found. Site-specific excision of HIV-1 can therefore be achieved by using this model system with acute infection. These studies represent one step toward the development of a novel antiviral strategy for the treatment of AIDS.


Assuntos
Fármacos Anti-HIV , HIV-1/fisiologia , Integrases/metabolismo , Recombinação Genética , Proteínas Virais , Replicação Viral , Antígenos CD4 , Linhagem Celular Transformada , HIV-1/genética , Humanos , Integrases/genética , Integrases/farmacologia , Transfecção , Transformação Genética , Células Tumorais Cultivadas
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