RESUMO
The aim of this study was to determine the myeloid-related protein-8 and myeloid-related protein-14 levels in the gingival crevicular fluid of smoker patients with generalized aggressive periodontitis (SAgP), smoker patients with chronic periodontitis (SCP), smoker patients with gingivitis (SG-smoker control), non-smoker patients with generalized aggressive periodontitis (AgP), non-smoker patients with chronic periodontitis (CP), and non-smoker patients with gingivitis (G-non-smoker control). The periodontal statuses of the patients were determined by periodontal clinical measurements and radiographical evaluations. The levels of myeloid-related protein-8 and myeloid-related protein-14 in the gingival crevicular fluid were assessed using enzyme-linked immuno sorbent assay. The myeloid-related protein-8 and myeloid-related protein-14 levels in the gingival crevicular fluid of patients with generalized aggressive periodontitis (non-smoker and smoker) were found to be statistically higher than patients with chronic periodontitis (non-smoker and smoker) and patients with gingivitis (non-smoker and smoker). Myeloid-related protein-8 and myeloid-related protein-14 levels of non-smokers were significantly higher than smokers in all types of periodontitis and gingivitis. The decreased myeloid-related protein-8 and myeloid-related protein-14 level could have prevented the haemostasis of calcium which plays a significant role in the migration of neutrophiles. Smoking affects myeloid-related protein-8 and myeloid-related protein-14 levels and may inhibit the antimicrobial efficiency against microorganisms. Due to these reasons smoker generalized aggressive periodontitis patients need to be treated in detail and their maintenance durations should be shortened.
Assuntos
Transportadores de Cassetes de Ligação de ATP/análise , Calgranulina B/análise , Líquido do Sulco Gengival/química , Gengivite , Periodontite , Fumar/efeitos adversos , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Índice Periodontal , Valores de Referência , Estatísticas não Paramétricas , Adulto JovemRESUMO
Abstract The aim of this study was to determine the myeloid-related protein-8 and myeloid-related protein-14 levels in the gingival crevicular fluid of smoker patients with generalized aggressive periodontitis (SAgP), smoker patients with chronic periodontitis (SCP), smoker patients with gingivitis (SG-smoker control), non-smoker patients with generalized aggressive periodontitis (AgP), non-smoker patients with chronic periodontitis (CP), and non-smoker patients with gingivitis (G-non-smoker control). The periodontal statuses of the patients were determined by periodontal clinical measurements and radiographical evaluations. The levels of myeloid-related protein-8 and myeloid-related protein-14 in the gingival crevicular fluid were assessed using enzyme-linked immuno sorbent assay. The myeloid-related protein-8 and myeloid-related protein-14 levels in the gingival crevicular fluid of patients with generalized aggressive periodontitis (non-smoker and smoker) were found to be statistically higher than patients with chronic periodontitis (non-smoker and smoker) and patients with gingivitis (non-smoker and smoker). Myeloid-related protein-8 and myeloid-related protein-14 levels of non-smokers were significantly higher than smokers in all types of periodontitis and gingivitis. The decreased myeloid-related protein-8 and myeloid-related protein-14 level could have prevented the haemostasis of calcium which plays a significant role in the migration of neutrophiles. Smoking affects myeloid-related protein-8 and myeloid-related protein-14 levels and may inhibit the antimicrobial efficiency against microorganisms. Due to these reasons smoker generalized aggressive periodontitis patients need to be treated in detail and their maintenance durations should be shortened.
Assuntos
Humanos , Masculino , Feminino , Adulto , Adulto Jovem , Periodontite , Fumar/efeitos adversos , Líquido do Sulco Gengival/química , Transportadores de Cassetes de Ligação de ATP/análise , Calgranulina B/análise , Gengivite , Valores de Referência , Ensaio de Imunoadsorção Enzimática , Índice Periodontal , Estatísticas não ParamétricasRESUMO
BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. Patients with non-alcoholic steatohepatitis (NASH) have increased plasmatic and hepatic concentrations of bile acids (BA), suggesting that they can be associated with the progression of the disease. Hepatic nuclear receptors are known to modulate genes controlling BA metabolism; thus, in this work we aimed to compare the expression of liver nuclear receptors -farnesoid X (FXR), small heterodimer partner (SHP) and liver X alpha (LXRα) receptors- and BA transporters -sodium+/taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP)- in liver biopsy samples of patients with simple steatosis (SS) and NASH. MATERIAL AND METHODS: Forty patients with biopsy-proven NALFD were enrolled between 2009 and 2012; liver biopsies were classified as SS (N = 20) or NASH (N = 20) according to the NAFLD activity score. Gene expression of nuclear FXR, LXRα, SHP, NTCP and BSEP was analyzed by real-time reverse transcription polymerase chain reaction and protein level was quantified by western blot. RESULTS: Gene expression of FXR, SHP, NTCP and BSEP was significantly up-regulated in the NASH group in comparison with SS patients (P < 0.05). In contrast, protein level for FXR, SHP and NTCP was decreased in the NASH patients vs. the SS group (P < 0.05). Gene and protein profile of LXRα did not show differences between groups. CONCLUSIONS: The results suggest that liver nuclear receptors (FXR and SHP) and BA transporters (NTCP and BSEP) are associated with the progression of NAFLD.
Assuntos
Transportadores de Cassetes de Ligação de ATP/análise , Fígado/química , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores Nucleares Órfãos/análise , Receptores Citoplasmáticos e Nucleares/análise , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Biópsia , Western Blotting , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Fígado/patologia , Receptores X do Fígado , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Hepatopatia Gordurosa não Alcoólica/genética , Receptores Nucleares Órfãos/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de Doença , Membro 3 da Família 12 de Carreador de Soluto/análise , Membro 3 da Família 12 de Carreador de Soluto/genética , Regulação para CimaRESUMO
The Pst system is a high-affinity inorganic phosphate transporter found in many bacterial species. Streptococcus mutans, the etiological agent of tooth decay, carries a single copy of the pst operon composed of six cistrons (pstS, pstC1, pstC, pstB, smu.1134 and phoU). Here, we show that deletion of pstS, encoding the phosphate-binding protein, reduces phosphate uptake and impairs cell growth, which can be restored upon enrichment of the medium with high concentrations of inorganic phosphate. The relevance of Pst for growth was also demonstrated in the wild-type strain treated with an anti-PstS antibody. Nevertheless, a reduced ability to bind to saliva-coated surfaces was observed, along with the reduction of extracellular polysaccharide production, although no difference on pH acidification was observed between mutant and wild-type strains. Taken together, the present data indicate that the S. mutans Pst system participates in phosphate uptake, cell growth and expression of virulence-associated traits.
Assuntos
Aderência Bacteriana/fisiologia , Proteínas de Transporte de Fosfato/fisiologia , Streptococcus mutans/fisiologia , Transportadores de Cassetes de Ligação de ATP/análise , Adenosina Trifosfatases/análise , Proteínas de Bactérias/análise , Película Dentária/metabolismo , Técnicas de Inativação de Genes , Inativação Gênica , Genes Bacterianos/genética , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/análise , Mutação/genética , Óperon/genética , Proteínas de Transporte de Fosfato/genética , Proteínas de Ligação a Fosfato/análise , Fosfatos/análise , Polissacarídeos Bacterianos/metabolismo , Análise de Sequência de DNA , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Fatores de Transcrição/análise , Virulência/genéticaRESUMO
We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen species (ROS) generation in macrophages induced by advanced glycated albumin (AGE-albumin) and its relationship with cell cholesterol homeostasis, emphasizing the expression of the ATP binding cassette transporter A-1 (ABCA-1). AGE-albumin was made by incubating fatty acid-free albumin with 10 mM glycolaldehyde. ROS production and ABCA-1 protein level were determined by flow cytometry in J774 macrophages treated along time with control (C) or AGE-albumin alone or in the presence of aminoguanidine or benfotiamine. Mitochondrial function was evaluated by oxygraphy. Compared to C-albumin, AGE-albumin increased ROS production in macrophages, which was ascribed to the activities of NADPH oxidase and of the mitochondrial system. Mitochondrial respiratory chain activity was reduced in cells incubated with AGE-albumin. ROS generation along time was associated with the reduction in macrophage ABCA-1 protein level. Aminoguanidine prevented ROS elevation and restored the ABCA-1 content in macrophages; on the other hand, benfotiamine that promoted a lesser reduction in ROS generation was not able to restore ABCA-1 levels. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Macrófagos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Albumina Sérica/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Animais , Antioxidantes/farmacologia , Células Cultivadas , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Guanidinas/farmacologia , Humanos , Macrófagos/metabolismo , Camundongos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Albumina Sérica/antagonistas & inibidores , Albumina Sérica Humana , Tiamina/análogos & derivados , Tiamina/farmacologiaRESUMO
A eficácia das estatinas em reduzir o risco de eventos coronarianos não é completamente explicada por seus efeitos em diminuir colesterol de lipoproteína de baixa densidade (LDL-C). Um dos seus efeitos adicionais pode ser decorrente da modificação na concentração de lipoproteína de alta densidade (HDL), reconhecida como ateroprotetora, principalmente por seu papel no transporte reverso do colesterol (TRC). Os transportadores de membrana do tipo ATP-binding cassette, ABCA1 e ABCG1, e o scavenger receptor BI (SRBI) são proteínas importantes envolvidas no TRC e seus genes são regulados por vários fatores de transcrição, entre eles os liver-x-receptors (LXRs). Com a finalidade de avaliarmos os efeitos dos hipolipemiantes sobre expressão dos transportadores ABC e do receptor SRBI, a expressão de RNAm do ABCA1, ABCG1, SCARB1, NR1H3 (LXRα) e NR1H2 (LRXβ) foi avaliada por PCR em tempo real em células das linhagens HepG2 (origem hepática) e Caco-2 (origem intestinal) tratadas com atorvastatina ou sinvastatina (10 µM) e/ou ezetimiba (até 5 µM) por até 24 horas. Além disso, a expressão desses genes também foi avaliada em células mononucleares do sangue periférico (CMSP) de 50 pacientes normolipidêmicos (NL) e 71 hipercolesterolêmicos (HC) tratados com atorvastatina (10mg/dia/4semanas, n=48) ou sinvastatina e/ou ezetimiba (10mg/dia/4 ou 8 semanas, n=23). A possível associação entre os polimorfismos ABCA1 C-14T e R219K e a expressão de RNAm em CMSP também foi avaliada por PCR-RFLP. O SCARB1 foi o gene mais expresso nas células HepG2 e Caco-2, seguido por NR1H2, NR1H3, ABCG1 e ABCA1 em HepG2 ou por ABCA1 e ABCG1 em Caco-2. O tratamento com estatinas (1 ou 10 µM) ou ezetimiba (5 µM), por 12 ou 24 horas, aumentou a expressão de RNAm do ABCG1, mas não de ABCA1 e SCARB1, em células HepG2. Ainda nesta linhagem, o aumento na transcrição dos genes NR1H2 e NR1H3 foi observado somente com a maior concentração de atorvastatina (10 µM) e, ao contrário, o tratamento com ezetimiba...
The efficacy of statins in reducing the risk of coronary events is not completely explained by their effects in decreasing cholesterol low-density lipoprotein (LDL-C). One of their additional effects may result from the change in concentration of high-density lipoprotein (HDL), recognized as atheroprotective, mainly for the role in reverse cholesterol transport (RCT). The membrane transporters, as ATP-binding cassette, ABCA1 and ABCG1, and scavenger receptor BI (SRBI) are important proteins involved in the RCT and their genes are regulated by various transcription factors, including the liver-X-receptors (LXRs) . In order to evaluate the effects of lipid lowering on expression of ABC transporters and SRBI receptor, the mRNA expression of ABCA1, ABCG1, SCARB1, NR1H3 (LXRα) and NR1H2 (LRXβ) was assessed by real time PCR in HepG2 (hepatic origin) and Caco-2 (intestinal origin) cells treated with atorvastatin or simvastatin (10 µM) and/or ezetimibe (up to 5 µM) for 24 hours. Furthermore, the expression of these genes was evaluated in peripheral blood mononuclear cells (PBMC) of 50 normolipidemic (NL) and 71 hypercholesterolemic (HC) patients treated with atorvastatin (10mg/d/4 weeks, n = 48) or simvastatin and/or ezetimibe (10mg/d/4 or 8 weeks, n = 23). The possible association between ABCA1 C-14T and R219K polymorphisms and mRNA expression in PBMC was also evaluated by PCR-RFLP. SCARB1 was the most expressed in HepG2 and Caco-2 cells, followed by NR1H2, NR1H3, ABCG1 and ABCA1 in HepG2 or by ABCG1 and ABCA1 in Caco-2. The treatment with statins (1 or 10 µM) or ezetimibe (5 µM) for 12 or 24 hours, increased mRNA expression of ABCG1 but not ABCA1 and SCARB1 in HepG2 cells. Moreover, in HepG2 cells, atorvastatin also upregulated NR1H2 and NR1H3 only at 10.0 µM, meanwhile ezetimibe downregulated NR1H2 but did not change NR1H3 expression. In Caco-2 cells, atorvastatin or simvastatin treatment for 12 or 24 hours reduced the amount of ABCA1 transcript and did not ...
Assuntos
Expressão Gênica , Inibidores de Hidroximetilglutaril-CoA Redutases/análise , Lipoproteínas LDL , Lipoproteínas LDL/isolamento & purificação , Lipoproteínas LDL/química , Transportadores de Cassetes de Ligação de ATP/análiseAssuntos
Transportadores de Cassetes de Ligação de ATP/análise , Antiprotozoários/farmacologia , Resistência a Medicamentos , Leishmania major/efeitos dos fármacos , Pentamidina/farmacologia , Proteínas de Protozoários/análise , Animais , Sequência de Bases , Proteínas de Fluorescência Verde , Leishmania major/química , Leishmania major/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , TransfecçãoRESUMO
Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17beta-D-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 micromol/kg i.v.) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2. DBcAMP (20 micromol/kg i.v.) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 microM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 microM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Endocitose/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Actinas/análise , Animais , Bile/fisiologia , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/química , Western Blotting , Bucladesina/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Membrana Celular/química , Colestase/metabolismo , Feminino , Imunofluorescência , Fígado/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Microscopia Confocal , Mutação , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
The identity of the sandfly vectors of Leishmania braziliensis in Valle del Cauca Department, Colombia, was originally given as Lutzomyia townsendi, but then changed to L. youngi, another member of the L. townsendi series (Verrucarum group) with isomorphic females. To identify members of this series in Valle del Cauca, we analyzed the nuclear gene elongation factor-alpha (EF-alpha) and the mitochondrial gene cytochrome b (Cyt b). DNA sequences from the L. verrucarum series (L. columbiana, L. evansi and L. ovallesi) were used as outgroups. Flies from two locations on the western cordillera of the Andes were identified as L. townsendi s.s., according to male morphology and distinctive gene lineages. In the third location, on the central cordillera of the Andes, most specimens were identified as belonging to a geographical population of L. youngi, according to male morphology, an EF-alpha lineage shared with L. youngi from the Venezuelan-type locality, and a distinctive Cyt b sub-lineage. All other specimens were identified as L. youngi with the introgressed Cyt b sequences of L. townsendi. Such interspecific introgression implies that vectorial traits and ecological associations may no longer be viewed as fixed properties of different morphospecies.
Assuntos
Insetos Vetores/genética , Leishmania braziliensis , Leishmaniose/transmissão , Mitocôndrias/genética , Psychodidae/genética , Proteínas de Schizosaccharomyces pombe , Transportadores de Cassetes de Ligação de ATP/análise , Animais , Colômbia , Grupo dos Citocromos b/análise , Grupo dos Citocromos b/genética , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/métodos , Psychodidae/classificação , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
It is widely accepted that a prolonged ouabain blockade of the Na(+),K(+)-ATPase makes cells detach from each other and from the substrate, leading to their death and that cellular resistance to ouabain is due to the presence of isoforms of Na(+),K(+)-ATPase with low affinity to this glycoside. In the present work the effect of reduced glutathione in the response of two types of renal cells to ouabain: MDCK, a ouabain-sensitive cell line and Ma104, a ouabain-resistant one, was studied. Glutathione protected MDCK cells from ouabain toxicity and inhibition of glutathione synthesis by L-buthionine-S,R-sulfoximine sensitized Ma104 cells to ouabain. As glutathione is involved with multidrug resistance (MDR) in cells expressing the multidrug resistance-related protein MRP1 and as Ma104 cells have a MDR phenotype, it was investigated whether Ma104 cells express this protein. The expression of the MRP1-mRNA in Ma104 cells was detected by reverse transcriptase-polymerase chain reaction and ribonuclease protection assay, and the protein was detected by Western blotting and immunofluorescence. Treatment of Ma104 cells with ouabain increased MRP1-mRNA expression and altered the localization of MRP1 in these cells. Our results suggest that some cells may have mechanisms to protect themselves from ouabain toxicity and that MRP1 may have a role in controlling the toxic effects of ouabain.
Assuntos
Transportadores de Cassetes de Ligação de ATP/análise , Antioxidantes/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/farmacologia , Ouabaína/toxicidade , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Western Blotting , Butionina Sulfoximina/farmacologia , Catalase/farmacologia , Linhagem Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Imunofluorescência , Glutationa/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Ouabaína/antagonistas & inibidores , Fenótipo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/farmacologiaRESUMO
Mycobacterium leprae, an obligate intracellular pathogen, can be derived only from host tissue and thus affords the opportunity to study in vivo-expressed products responsible for the particular pathogenesis of leprosy. Despite considerable progress in the characterization of the proteins and secondary gene products of M. leprae, there is little information on the nature of the proteins associated with the cell envelope. M. leprae has been fractionated into its major subcellular components, cell wall, cytoplasmic membrane, and soluble cytosol. A number of biochemical markers, including diaminopimelic acid content, monosaccharide composition, mycolic acid, and glycolipid distribution, were applied to their characterization, and two-dimensional gel electrophoresis was used to map the component proteins. A total of 391 major proteins spots were resolved, and 8 proteins were identified based on their reactivity to a panel of monoclonal antibodies and/or relative pI size. Microsequencing of six protein spots present in the cell wall fraction allowed identification of new proteins, including the protein elongation factor EF-Tu and a homolog for the Mycobacterium tuberculosis MtrA response regulator. These results, together with previous studies, contribute to the progressive knowledge of the composition of the in vivo-expressed proteins of M. leprae.