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1.
Methods Mol Biol ; 2797: 159-175, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38570459

RESUMO

Homogenous time-resolved FRET (HTRF) assays have become one of the most popular tools for pharmaceutical drug screening efforts over the last two decades. Large Stokes shifts and long fluorescent lifetimes of lanthanide chelates lead to robust signal to noise, as well as decreased false positive rates compared to traditional assay techniques. In this chapter, we describe an HTRF protein-protein interaction (PPI) assay for the KRAS4b G-domain in the GppNHp-bound state and the RAF-1-RBD currently used for drug screens. Application of this assay contributes to the identification of lead compounds targeting the GTP-bound active state of K-RAS.


Assuntos
Descoberta de Drogas , Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Quelantes
2.
PLoS Genet ; 16(12): e1009186, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33306674

RESUMO

Cells are exposed to frequent mechanical and/or chemical stressors that can compromise the integrity of the plasma membrane and underlying cortical cytoskeleton. The molecular mechanisms driving the immediate repair response launched to restore the cell cortex and circumvent cell death are largely unknown. Using microarrays and drug-inhibition studies to assess gene expression, we find that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. We identified 253 genes whose expression is up-regulated (80) or down-regulated (173) in response to laser wounding. A subset of these genes were validated using RNAi knockdowns and exhibit aberrant actomyosin ring assembly and/or actin remodeling defects. Strikingly, we find that the canonical insulin signaling pathway controls actin dynamics through the actin regulators Girdin and Chickadee (profilin), and its disruption leads to abnormal wound repair. Our results provide new insight for understanding how cell wound repair proceeds in healthy individuals and those with diseases involving wound healing deficiencies.


Assuntos
Actinas/metabolismo , Comunicação Autócrina , Insulina/metabolismo , Transdução de Sinais , Cicatrização , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Profilinas/genética , Profilinas/metabolismo , Transcriptoma
3.
Anal Chem ; 92(7): 4971-4979, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32106676

RESUMO

We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthesized a novel γ-GTP-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fatores de Troca do Nucleotídeo Guanina/química , Guanosina Trifosfato/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética
4.
Sensors (Basel) ; 18(11)2018 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-30453509

RESUMO

A new label-free molecular probe for luminescent nucleotide detection in neutral aqueous solution is presented. Phosphate-containing molecules, such as nucleotides possess vital role in cell metabolism, energy economy, and various signaling processes. Thus, the monitoring of nucleotide concentration and nucleotide related enzymatic reactions is of high importance. Two component lanthanide complex formed from Tb(III) ion carrier and light harvesting antenna, readily distinguishes nucleotides containing different number of phosphates and enable direct detection of enzymatic reactions converting nucleotide triphosphate (NTP) to nucleotide di/monophosphate or the opposite. Developed sensor enables the detection of enzymatic activity with a low nanomolar sensitivity, as highlighted with K-Ras and apyrase enzymes in their hydrolysis assays performed in a high throughput screening compatible 384-well plate format.

5.
Sci Signal ; 11(550)2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279169

RESUMO

The Ras-extracellular signal-regulated kinase pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors to treat cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends on multifunctional autoprocessing repeats-in-toxin, a toxin that releases bacterial effector domains into host cells. One such domain is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance analysis and nucleotide exchange assays, we determined that the processing of KRAS by RRSP did not release any fragments or cause KRAS to dissociate from its bound nucleotide but instead only locally affected its structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor-mediated nucleotide exchange and prevent KRAS from binding to RAF. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance in its target.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Endopeptidases/química , Endopeptidases/genética , Células HeLa , Humanos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Conformação Proteica , Proteólise , Homologia de Sequência de Aminoácidos
6.
Mol Cell ; 64(5): 875-887, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27889448

RESUMO

Ras pathway signaling plays a critical role in cell growth control and is often upregulated in human cancer. The Raf kinases selectively interact with GTP-bound Ras and are important effectors of Ras signaling, functioning as the initiating kinases in the ERK cascade. Here, we identify a route for the phospho-inhibition of Ras/Raf/MEK/ERK pathway signaling that is mediated by the stress-activated JNK cascade. We find that key Ras pathway components, the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in response to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as rigosertib and paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and may function as a signaling checkpoint to suppress the Ras pathway during conditions of cellular stress.


Assuntos
Glicina/análogos & derivados , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Paclitaxel , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Sulfonas , Ativação Enzimática/efeitos dos fármacos , Glicina/farmacocinética , Glicina/farmacologia , Células HeLa , Humanos , Estresse Oxidativo , Paclitaxel/farmacocinética , Paclitaxel/farmacologia , Fosforilação , Sulfonas/farmacocinética , Sulfonas/farmacologia , Proteínas ras/metabolismo
7.
Sci Rep ; 5: 15916, 2015 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-26522388

RESUMO

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.


Assuntos
Lipídeos/fisiologia , Prenilação de Proteína/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Biofísica/métodos , Membrana Celular/metabolismo , Células Cultivadas , Guanosina Trifosfato/metabolismo , Humanos , Insetos/metabolismo , Metilação , Ligação Proteica/fisiologia , Quinases raf/metabolismo
8.
Curr Biol ; 24(2): 144-155, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24388847

RESUMO

BACKGROUND: Cells heal disruptions in their plasma membrane using a sophisticated, efficient, and conserved response involving the formation of a membrane plug and assembly of an actomyosin ring. Here we describe how Rho family GTPases modulate the cytoskeleton machinery during single cell wound repair in the genetically amenable Drosophila embryo model. RESULTS: We find that Rho, Rac, and Cdc42 rapidly accumulate around the wound and segregate into dynamic, partially overlapping zones. Genetic and pharmacological assays show that each GTPase makes specific contributions to the repair process. Rho1 is necessary for myosin II activation, leading to its association with actin. Rho1, along with Cdc42, is necessary for actin filament formation and subsequent actomyosin ring stabilization. Rac is necessary for actin mobilization toward the wound. These GTPase contributions are subject to crosstalk among the GTPases themselves and with the cytoskeleton. We find Rho1 GTPase uses several downstream effectors, including Diaphanous, Rok, and Pkn, simultaneously to mediate its functions. CONCLUSIONS: Our results reveal that the three Rho GTPases are necessary to control and coordinate actin and myosin dynamics during single-cell wound repair in the Drosophila embryo. Wounding triggers the formation of arrays of Rho GTPases that act as signaling centers that modulate the cytoskeleton. In turn, coordinated crosstalk among the Rho GTPases themselves, as well as with the cytoskeleton, is required for assembly/disassembly and translocation of the actomyosin ring. The cell wound repair response is an example of how specific pathways can be activated locally in response to the cell's needs.


Assuntos
Citoesqueleto/enzimologia , Drosophila melanogaster/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Dactinomicina/química , Drosophila melanogaster/citologia , Ativação Enzimática
9.
J Cell Sci ; 125(Pt 24): 5984-97, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23038780

RESUMO

The repair of injured tissue must occur rapidly to prevent microbial invasion and maintain tissue integrity. Epithelial tissues in particular, which serve as a barrier against the external environment, must repair efficiently in order to restore their primary function. Here we analyze the effect of different parameters on the epithelial wound repair process in the late stage Drosophila embryo using in vivo wound assays, expression of cytoskeleton and membrane markers, and mutant analysis. We define four distinct phases in the repair process, expansion, coalescence, contraction and closure, and describe the molecular dynamics of each phase. Specifically, we find that myosin, E-cadherin, Echinoid, the plasma membrane, microtubules and the Cdc42 small GTPase respond dynamically during wound repair. We demonstrate that perturbations of each of these components result in specific impairments to the wound healing process. Our results show that embryonic epithelial wound repair is mediated by two simultaneously acting mechanisms: crawling driven by cellular protrusions and actomyosin ring contraction along the leading edge of the wound.


Assuntos
Actomiosina/metabolismo , Drosophila/metabolismo , Animais , Drosophila/citologia , Drosophila/embriologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo
10.
Cell Mol Life Sci ; 69(15): 2469-83, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22349211

RESUMO

Wound repair on the cellular and multicellular levels is essential to the survival of complex organisms. In order to avoid further damage, prevent infection, and restore normal function, cells and tissues must rapidly seal and remodel the wounded area. The cytoskeleton is an important component of wound repair in that it is needed for actomyosin contraction, recruitment of repair machineries, and cell migration. Recent use of model systems and high-resolution microscopy has provided new insight into molecular aspects of the cytoskeletal response during wound repair. Here we discuss the role of the cytoskeleton in single-cell, embryonic, and adult repair, as well as the striking resemblance of these processes to normal developmental events and many diseases.


Assuntos
Citoesqueleto/fisiologia , Cicatrização/fisiologia , Actomiosina/fisiologia , Animais , Caderinas/fisiologia , Sinalização do Cálcio/fisiologia , Membrana Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Embrião de Mamíferos/fisiopatologia , Embrião não Mamífero/lesões , Embrião não Mamífero/fisiopatologia , Humanos , Modelos Biológicos , Morfogênese/fisiologia , Lesões Pré-Natais/fisiopatologia
11.
Dev Dyn ; 241(3): 608-26, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22275148

RESUMO

BACKGROUND: Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. RESULTS: We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. CONCLUSIONS: All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics.


Assuntos
Proteínas de Drosophila/biossíntese , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/biossíntese , Proteínas de Transporte Vesicular/biossíntese , Proteína da Síndrome de Wiskott-Aldrich/biossíntese , Síndrome de Wiskott-Aldrich/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Proteínas de Drosophila/análise , Proteínas de Drosophila/imunologia , Desenvolvimento Embrionário , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/imunologia , Oogênese , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/imunologia , Proteína da Síndrome de Wiskott-Aldrich/análise , Proteína da Síndrome de Wiskott-Aldrich/imunologia
12.
Bioarchitecture ; 1(3): 114-121, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21922041

RESUMO

Cell wounding is a common event in the life of many cell types, and the capacity of the cell to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. Cell wounding is most frequent in tissues exposed to high levels of stress. Survival of such plasma membrane disruptions requires rapid resealing to prevent the loss of cytosolic components, to block Ca(2+) influx and to avoid cell death. In addition to patching the torn membrane, plasma membrane and cortical cytoskeleton remodeling are required to restore cell function. Although a general understanding of the cell wound repair process is in place, the underlying mechanisms of each step of this response are not yet known. We have developed a model to study single cell wound repair using the early Drosophila embryo. Our system combines genetics and live imaging tools, allowing us to dissect in vivo the dynamics of the single cell wound response. We have shown that cell wound repair in Drosophila requires the coordinated activities of plasma membrane and cytoskeleton components. Furthermore, we identified an unexpected role for E-cadherin as a link between the contractile actomyosin ring and the newly formed plasma membrane plug.

13.
J Cell Biol ; 193(3): 455-64, 2011 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-21518790

RESUMO

When single cells or tissues are injured, the wound must be repaired quickly in order to prevent cell death, loss of tissue integrity, and invasion by microorganisms. We describe Drosophila as a genetically tractable model to dissect the mechanisms of single-cell wound repair. By analyzing the expression and the effects of perturbations of actin, myosin, microtubules, E-cadherin, and the plasma membrane, we define three distinct phases in the repair process-expansion, contraction, and closure-and identify specific components required during each phase. Specifically, plasma membrane mobilization and assembly of a contractile actomyosin ring are required for this process. In addition, E-cadherin accumulates at the wound edge, and wound expansion is excessive in E-cadherin mutants, suggesting a role for E-cadherin in anchoring the actomyosin ring to the plasma membrane. Our results show that single-cell wound repair requires specific spatial and temporal cytoskeleton responses with distinct components and mechanisms required at different stages of the process.


Assuntos
Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Drosophila melanogaster/metabolismo , Actinas/metabolismo , Actomiosina/química , Alelos , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Cruzamentos Genéticos , Microtúbulos/metabolismo , Mutação , Miosinas/metabolismo , Cicatrização
14.
Gene ; 451(1-2): 15-22, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19914359

RESUMO

The control of gene expression in the human parasite Leishmania occurs mainly at the post-transcriptional level. Nevertheless, basic cell processes such as ribosome biogenesis seem to be conserved. Mature ribosomal RNAs (rRNAs) are synthesized from typical RNA polymerase I (Pol I) promoters and processed by pathways analogous to other eukaryotes. To further understand Pol I transcription control in these parasites, we have analyzed transcription termination and processing of the rDNA in Leishmania amazonensis. 3'-end S1 mapping of rRNA precursors identified three termini, one corresponding to the mature 28S rRNA and two to the rDNA intergenic spacer (IGS), termed T1 and T2, for precursors which are 185 and 576 nucleotides longer, respectively. Both T1 and T2 are associated with conserved G + C rich elements that have the potential to form hairpin structures and T-rich clusters. We found that two fragments of 423 bp and 233 bp, flanking sites T1 and T2 respectively when placed upstream of the green fluorescent protein gene (GFP), negatively affected the Pol I-driven transcription of this gene, which suggests the presence of a transcription terminator element in these regions. Deletion analysis pointed to a CCCTTTT heptamer as part of the putative terminator and suggested that the hairpins are processing signals.


Assuntos
Leishmania/genética , Processamento de Terminações 3' de RNA , RNA Ribossômico/genética , Regiões Terminadoras Genéticas , Transcrição Gênica , DNA Espaçador Ribossômico/genética , Sequências Repetidas Invertidas , Leishmania/metabolismo , RNA Polimerase I/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética
15.
Development ; 136(16): 2849-60, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19633175

RESUMO

Wiskott-Aldrich Syndrome (WAS) family proteins are Arp2/3 activators that mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. Wasp and Scar/WAVE, the two founding members of the family, are regulated by the GTPases Cdc42 and Rac, respectively. By contrast, linear actin nucleators, such as Spire and formins, are regulated by the GTPase Rho. We recently identified a third WAS family member, called Wash, with Arp2/3-mediated actin nucleation activity. We show that Drosophila Wash interacts genetically with Arp2/3, and also functions downstream of Rho1 with Spire and the formin Cappuccino to control actin and microtubule dynamics during Drosophila oogenesis. Wash bundles and crosslinks F-actin and microtubules, is regulated by Rho1, Spire and Arp2/3, and is essential for actin cytoskeleton organization in the egg chamber. Our results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism for how cells might coordinately regulate these structures.


Assuntos
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/genética , Animais , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/genética , Microtúbulos/metabolismo , Oogênese/fisiologia , Ovário/citologia , Ovário/metabolismo , Proteínas de Transporte Vesicular/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteínas rho de Ligação ao GTP/genética
16.
J Med Entomol ; 42(1): 48-56, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15691008

RESUMO

Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green fluorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of fluorescent proteins. Tagged flagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its fluorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasites.


Assuntos
Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Trypanosoma/genética , Animais , Linhagem Celular , Células Cultivadas , Vetores Genéticos , Haplorrinos , Rim , Estágios do Ciclo de Vida , Rhodnius/parasitologia , Transfecção , Trypanosoma/crescimento & desenvolvimento , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Proteína Vermelha Fluorescente
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