ABSTRACT
Abstract Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T.cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.
Resumo O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T.cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.
Subject(s)
Humans , Animals , Trypanosoma cruzi/genetics , Xeroderma Pigmentosum , DNA Damage/genetics , Computational Biology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA Repair/geneticsABSTRACT
Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T. cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.
O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T. cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.
Subject(s)
Animals , Crosses, Genetic , DNA Damage , Gene Expression , Trypanosoma cruzi/geneticsABSTRACT
Abstract Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T.cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.
Resumo O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T.cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.
ABSTRACT
Rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) are inflammatory diseases with different bone remodeling patterns. Fibroblast-like synoviocytes (FLS) are cells involved in the transition from an acute and reparable phase to a chronic and persistent stage in these diseases. The distinction of joint phenotypes involves inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-17, and IL-22 directly or through key signaling pathways such as Wnt. To evaluate the role of FLS as the source of Wnt antagonists (sFRP3/FRZB and Dkk1) in the synovia, levels of TNF- α, IL-17, IL-22, Dkk1, and sFRP3 were measured by ELISA directly in the synovial fluid of patients with RA, PsA, or AS. Dkk1 and sFRP3 were also measured in the FLS culture supernatants after different inflammatory stimulus. sFRP3 and Dkk1 are constitutively expressed by FLS. IL-22 and sFRP3 were positively correlated (r=0.76; P<0.01) in synovial fluid. The stimulation of FLS with IL-22, but not TNF-alpha and IL-17, increased the production of sFRP3. No stimulus altered the basal expression of Dkk1. These results showed, for the first time, the ability of IL-22 to increase the expression of sFRP3/FRZB by human FLS in both in vitro and ex vivo models. This finding linked IL-22 to local inhibition of Wnt signaling and possibly to blockade of osteogenesis. Furthermore, FLS presented as a source of this inhibitor in synovial fluid, assigning to this cell a bone injury mechanism.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Interleukins/metabolism , Synoviocytes , Synovial Membrane , Cells, Cultured , Tumor Necrosis Factor-alpha , FibroblastsABSTRACT
Chagas' disease, caused by the protozoan Trypanosoma cruzi, is a major cause of cardiovascular disability in countries where it is endemic. Damage to the heart microvasculature has been proposed to be an important factor in the pathogenesis of heart dysfunction. Endothelin-1 (ET-1) is a potent vasoconstrictor and exerts its effects via specific ET A and ET B receptors. A few studies have suggested a role for ET-1 and its receptors in the pathogenesis of Chagas' disease. We investigated the effects of treatment with bosentan, an ET A/ET B receptor antagonist, on the course of T. cruzi infection (Y strain) in C57Bl/6 mice. Treatment with bosentan (100 mg kg-1 day-1) was given per os starting day 0 after infection until sacrifice. Bosentan significantly increased myocardial inflammation, with no effects on parasitemia. Although the total number of nests was similar, a lower number of intact amastigote nests was found in the heart of bosentan-treated animals. Bosentan failed to affect the infection-associated increase in the cardiac levels of the cytokines IFN-g and TNF-a and the chemokines CCL2/MCP-1, CCL3/MIP-1a and CCL5/RANTES. In vitro, pre-incubation with ET-1 (0.1 æM) 4 h before infection enhanced the uptake of the parasites by peritoneal macrophages, and this effect was abrogated when macrophages were pre-treated with bosentan (1 æM) 15 min before incubation with ET-1. However, ET-1 did not alter killing of intracellular parasites after 48 h of in vitro infection. Our data suggest that bosentan-treated mice have a delay in controlling parasitism which is compensated for exacerbated inflammation. Infection is eventually controlled in these animals and lethality is unchanged, demonstrating that ET-1 plays a minor role in the protection against acute murine T. cruzi infection.
Subject(s)
Animals , Male , Mice , Chagas Cardiomyopathy/metabolism , Endothelin-1/physiology , Parasitemia/metabolism , Receptors, Endothelin/antagonists & inhibitors , Sulfonamides/pharmacology , Trypanosoma cruzi/physiology , Acute Disease , Chagas Cardiomyopathy/parasitology , Chagas Cardiomyopathy/pathology , Cytokines/analysis , Disease Models, Animal , Parasitemia/immunology , Trypanosoma cruzi/isolation & purificationABSTRACT
Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.
Subject(s)
Sequence Analysis, DNA , DNA Polymerase I/metabolism , Escherichia coli/enzymology , Fluorescein/metabolism , DNA Primers/metabolism , Automation , Hydrogen-Ion ConcentrationABSTRACT
The bacteria Escherichia coli has been widely employed in studies of eukaryotic DNA repair genes. Several eukaryotic genes have been cloned by functional complementation of mutant lineages of E. coli. We examined the similarities and differences among bacterial and eukaryotic DNA repair systems. Based on these data, we examined tools used for gene cloning and functional studies of DNA repair in eukaryotes, using this bacterial system as a model
Subject(s)
Animals , Eukaryotic Cells , Escherichia coli/genetics , DNA Repair , Base Sequence , Cloning, Molecular , DNA Damage , Escherichia coli/enzymology , Genes, Bacterial , Models, GeneticABSTRACT
Injecao de cercarias de Schistosoma mansoni na cavidade peritoneal de camundongos normais induz adesao celular a estas lavras. Esta aderencia diminui acentuadamente quando as lavras infectantes se transformam em esquistossomulos. Este procedimento foi usado para detectar diferencas entre esquitossomulos obtidos in vivo e in vitro. A reinoculacao de esquistossomulos obtidos in vivo na cavidade peritoneal de camundongos nao acarreta adesao celular. Por outro lado, celulas aderentes foram encontradas em esquistossomulos obtidos in vitro (4 e 24 horas, respectivamente). Nossos dados referentes a esquistossomulos obtidos in vitro indicam que mais de 24 horas sao necessarias para a completa remocao de moleculas envolvidas no fenomeno de adesao celular
Subject(s)
Mice , Animals , Male , Peritoneal Cavity/parasitology , Schistosoma mansoni/physiology , Cell AdhesionABSTRACT
1. The activity of choline acetyltransferase (CAT) measured by a radilabeling method was significantly reduced in the heart, submandibular gland and esophagus of rats 20 days after inoculation with Trypanosoma cruzi (Y strain). 2. Normal enzyme activity was recovered in all these organs 100 days after inoculation. 3. In the transcerse colon, no change, 30% reduction and normal activity were found at days 20, 100 and 430 of infection, respectively. 4. The data indicate recovery of parasympathetic function in experimental Chagas' disease. Axonal regrowth and sprouting are discussed as possible candidates for the recovery mechanisms
Subject(s)
Rats , Animals , Male , Female , Chagas Disease/enzymology , Choline O-Acetyltransferase/metabolism , Esophagus/enzymology , Submandibular Gland/enzymology , Myocardium/enzymology , Parasympathetic Nervous System/physiopathology , Colon/enzymology , Nerve Regeneration , Neuronal Plasticity , Neurons/physiology , Rats, Inbred StrainsABSTRACT
O conteudo de noradrenalina do hipotalamo e do tronco encefalico de ratos controle e inoculados con a cepa Y (300.000 tripomastigotas, i.p.) de Trypanosoma cruzi foi medido pela tecnica fluorimetrica de Anton e Sayre. Os animais foram sacrificados 20 e 32 dias depois da inoculacao. Para avaliacao do grau de desnervacao simpatica do coracao dos animais infectados, a auricula direita foi observada com microscopio de fluorescencia apos tratamento histoquimico pela tecnica do acido glioxilico.O conteudo de noradrenalina do hipolatamo e do tronco encefalico dos animais infectados nao diferiu do medido nos animais controle Contudo, um quase completo desaparecimento das fibras adrenergicas foi observado no coracao dos animais chagasicos, sugerindo que o mecanismo envolvido na lesao discrimina neuronios adrenergicos centrais e perifericos