RESUMO
Abstract Herein we evaluated the histopathological alterations and expression patterns of multixenobiotic resistence (MXR) and autophagic proteins in liver samples of fish chronically exposed to anthropogenic contaminants in a highly polluted river, and then again after they had been transferred to good quality water. Two groups were established: euthanized on the day of capture (0 h), and maintained for 30 days in a tank (30 d). The fish of 0 h presented liver with vacuolated and hypertrophic hepatocytes. Also, it was observed strong immunostaining of cathepsin-D, LC3-II and P-gp. Necrosis and apoptosis were also observed throughout the liver. Conversely, the second group (30 d) showed recovery of the liver normal histology and weak immunoreaction of the studied proteins. So, our results indicated that there was a hepatic recovery in the fish kept in good quality water, as showed by the decreased expression of cathepsin-D, LC3-II, and the MXR (P-gp). Therefore, the alterations here observed could be proposed as potential biomarkers to be tested for following the impacts of remediation or mitigation measures to environmental impacts.
Assuntos
Animais , Masculino , Feminino , Catepsina D/análise , Hepatócitos/química , Peixes , Fígado/patologia , Fígado/química , Imuno-Histoquímica , RiosRESUMO
Despite the importance of Dengue virus (DENV) infection in human health, there is not a fully effective vaccine or antiviral treatment against the infection. Since lipids such as cholesterol are required during DENV infection, its uptake and synthesis are increased in infected cells. Ezetimibe is an FDA-approved drug that reduces cholesterol uptake by inhibiting the endocytosis through Niemman-Pick C1-Like 1 (NPC1L1) receptor, expressed on the membrane of enterocytes and hepatocytes. Our results indicate that an increase in the amount of NPC1L1 occurs on the surface of Huh-7â¯cells during DENV infection, which correlates with an increase in cholesterol levels. Blockage of NPC1L1 with ezetimibe in concentrations up to 50⯵M does not reduce cell viability but diminished total cellular cholesterol, the percentage of infected cells, viral yield, viral RNA and protein synthesis without affecting DENV binding and/or entry to Huh-7â¯cells. Moreover, ezetimibe inhibited DENV replicative complex formation and lipid droplets accumulation. All these results indicate that ezetimibe is an excellent drug to inhibit DENV infection and confirm that cholesterol is a key target to inhibit viral infection.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Ezetimiba/farmacologia , Hepatócitos/efeitos dos fármacos , Proteína C1 de Niemann-Pick/antagonistas & inibidores , Receptores Virais/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Anticolesterolemiantes/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colesterol/análise , Hepatócitos/química , Hepatócitos/virologia , Humanos , RNA Viral/análise , Carga ViralRESUMO
Classical observations have shown that during the interphase the chromosomal DNA of metazoans is organized in supercoiled loops attached to a compartment known as the nuclear matrix (NM). Fragments of chromosomal DNA able to bind the isolated NM in vitro are known as matrix associated/attachment/addressed regions or MARs. No specific consensus sequence or motif has been found that may constitute a universal, defining feature of MARs. On the other hand, high-salt resistant DNA-NM interactions in situ define true DNA loop anchorage regions or LARs, that might correspond to a subset of the potential MARs but are not necessarily identical to MARs characterized in vitro, since there are several examples of MARs able to bind the NM in vitro but which are not actually bound to the NM in situ. In the present work we assayed the capacity of two LARs, as well as of shorter fragments within such LARs, for binding to the NM in vitro. Paradoxically the isolated (≈2 kb) LARs cannot bind to the NM in vitro while their shorter (≈300 pb) sub-fragments and other non-related but equally short DNA fragments, bind to the NM in a high-salt resistant fashion. Our results suggest that the ability of a given DNA fragment for binding to the NM in vitro primarily depends on the length of the fragment, suggesting that binding to the NM is modulated by the local topology of the DNA fragment in suspension that it is known to depend on the DNA length. J. Cell. Biochem. 118: 4487-4497, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
DNA/química , Hepatócitos/química , Regiões de Interação com a Matriz , Matriz Nuclear/química , Animais , DNA/metabolismo , Hepatócitos/metabolismo , Masculino , Matriz Nuclear/metabolismo , Ratos , Ratos WistarRESUMO
Secretome analysis can be described as a subset of proteomics studies consisting in the analysis of the molecules secreted by cells or tissues. Dengue virus (DENV) infection can lead to a broad spectrum of clinical manifestations, with the severe forms of the disease characterized by hemostasis abnormalities and liver injury. The hepatocytes are a relevant site of viral replication and a major source of plasma proteins. Until now, we had limited information on the small molecules secreted by hepatic cells after infection by DENV. In the present study, we analysed a fraction of the secretome of mock- and DENV-infected hepatic cells (HepG2 cells) containing molecules with <10kDa, using different proteomic approaches. We identified 175 proteins, with 57 detected only in the samples from mock-infected cells, 59 only in samples from DENV-infected cells, and 59 in both conditions. Most of the peptides identified were derived from proteins larger than 10kDa, suggesting a proteolytic processing of the secreted molecules. Using in silico analysis, we predicted consistent differences between the proteolytic processing occurring in mock and DENV-infected samples, raising, for the first time, the hypothesis that differential proteolysis of secreted molecules would be involved in the pathogenesis of dengue. BIOLOGICAL SIGNIFICANCE: Since the liver, one of the targets of DENV infection, is responsible for producing molecules involved in distinct biological processes, the identification of proteins and peptides secreted by hepatocytes after infection would help to a better understanding of the physiopathology of dengue. Proteomic analyses of molecules with <10kDa secreted by HepG2 cells after infection with DENV revealed differential proteolytic processing as an effect of DENV infection.
Assuntos
Vírus da Dengue , Fígado/metabolismo , Proteólise , Proteômica/métodos , Dengue/metabolismo , Células Hep G2 , Hepatócitos/química , Hepatócitos/virologia , Humanos , Fígado/virologiaRESUMO
Several cellular molecules and components, specifically, cholesterol and lipid rafts have been described as necessary elements for dengue virus entry and signaling in several human cells. Thus, changes in lipid rafts formation and cholesterol levels were evaluated. Here we report that the amount of total cholesterol and lipid rafts formation increase early after infection of Huh-7 cells. This augment correlates with an increase in the amount of low density lipoprotein receptor (LDLr) on the surface of infected cells and also with a lower phosphorylation level of the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). None of the changes were observed in Huh 7 cells infected with VSV used as a control. These results suggest that dengue virus infection increases intracellular cholesterol levels at early times post infection by triggering the modulation of LDL particles uptake and the increase in the enzymatic activity of HMG-CoA reductase.
Assuntos
LDL-Colesterol/metabolismo , Vírus da Dengue/fisiologia , Interações Hospedeiro-Patógeno , Hidroximetilglutaril-CoA Redutases/metabolismo , Microdomínios da Membrana/metabolismo , Replicação Viral , Linhagem Celular , Hepatócitos/química , Hepatócitos/virologia , HumanosRESUMO
An inexpensive modular perfused chamber (MPC) designed for low- and normal-temperature live-cell imaging is presented. The device consists of four lathed pieces of stainless steel assembled as a cylindrical open chamber that can hold either round or square glass coverslips. The chamber is connected to a thermal-bath operating with recirculation. For image acquisition at 4°C, cooled air is blown toward the coverslip surface to prevent condensation. Principal advantages of this device are thermal stability in the sample environment, rapid response to changes in temperature set point, and easy sample insertion. The device enables the study of dynamic processes in cells governed by large temperature differences such as those imposed by hypothermic preservation of cells (0-4°C) followed by rewarming to normothermia (37°C). The capabilities of the MPC were demonstrated by monitoring the internalization of fluorescent quantum dots (QDs) in rat hepatocytes after hypothermic storage and during rewarming with an inverted microscope.
Assuntos
Técnicas de Cultura de Células/instrumentação , Temperatura Baixa , Técnicas Citológicas/instrumentação , Microscopia/instrumentação , Animais , Desenho de Equipamento , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Hepatócitos/química , Hepatócitos/metabolismo , Microscopia/métodos , Pontos Quânticos , Ratos , Ratos WistarRESUMO
We described herein the discovery of 1-(2-(benzo[d] [1,3]dioxol-6-yl)ethyl)-4-(2-methoxyphenyl) piperazine (LASSBio-772), as a novel potent and selective alpha 1A/1D adrenoceptor (AR) antagonist selected after screening of functionalized N-phenylpiperazine derivatives in phenylephrine-induced vasoconstriction of rabbit aorta rings. The affinity of LASSBio-772 for alpha 1A and alpha 1B AR subtypes was determined through displacement of [(3)H]prazosin binding. We obtained Ki values of 0.14 nM for the alpha 1A-AR, similar to that displayed by tamsulosin (K(i) = 0.13 nM) and 5.55 nM for the alpha 1B-AR, representing a 40-fold higher affinity for alpha 1A-AR. LASSBio-772 also presented high affinity (K(B) = 0.025 nM) for the alpha 1D-AR subtype in the functional rat aorta assay, showing to be equipotent to tamsulosin (K(B) = 0.017 nM).
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1/síntese química , Aorta/efeitos dos fármacos , Benzodioxóis/síntese química , Membrana Celular/efeitos dos fármacos , Piperazinas/síntese química , Receptores Adrenérgicos alfa 1/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Benzodioxóis/farmacologia , Membrana Celular/metabolismo , Hepatócitos/química , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Fenilefrina/farmacologia , Piperazinas/farmacologia , Prazosina/metabolismo , Prazosina/farmacologia , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Coelhos , Ratos , Receptores Adrenérgicos alfa 1/química , Sulfonamidas/farmacologia , Tansulosina , Técnicas de Cultura de Tecidos , Trítio , Vasoconstrição/efeitos dos fármacosRESUMO
The aim of this study was to evaluate the effect of different protein sources with different amino-acid profiles on liver cell development in "Wistar" rats submitted to a food restriction and recovery model. The food restriction model was based on a 50% ingestion restriction for the rats fed with the control diet (21 days) and "ad libitum" recovery (a period of 21 days). The protein sources used in this study were: Yeast autolysate (YA), whey protein concentrate from bovine milk (WPC), a mixture containing the YA and WPC in the proportion of 64:36 (protein base), commercial casein (CC) which was used as the experimental treatment (EC) and control treatment (CP). The following parameters were evaluated in this study: the amino-acid profile of the protein sources, the development of liver cells (liver weight and growth rates - RNA, DNA, protein), weight and number of hepatocytes in the whole organ. The results showed that the treatment with (YA) was the most affected by the food restriction, showing an incomplete (leucine-deficient) amino-acid profile, a lower development of liver cells, lower growth of the liver due to a lower growth by cellular hyperplasia (number of cells), lower capacity of cell division and DNA synthesis. However, it showed a higher ability for RNA synthesis, thus indicating that growth in the restricted phase was mainly due to increase in the size of hepatocytes (cell hypertrophy). During the repletion period, all food treatments showed normal liver development, i.e. cell growth and organ hyperplasia and hypertrophy.
El objetivo de este estudio fue evaluar el efecto que diversas fuentes de proteínas con diferentes perfiles de aminoácidos ejercían en el desarrollo de las células hepáticas en ratas "Wistar" sometidas a restricción y reposición de la ingestión de alimentos. El modelo de restricción alimentar consistía en disminuir 50% del consumo de los animales control (período de 21 días) y la recuperación con ingestión "ad libitum" (período de 21 días). Las fuentes de proteínas utilizadas en este estudio fueron: autolisado de levadura (ATL); concentrado proteico de suero de leche bovino (CPL); mezcla de CPL y ATL, en la proporción de 64:36 (base proteica), caseína comercial (CC), que fue utilizada como tratamiento experimental (CE) y como estándar (CP). Fueron evaluados el perfil de aminoácidos de las proteínas, el desarrollo de la célula hepática (peso del hígado y las tasas de crecimiento: ARN, ADN y proteína total), el peso y número de hepatocitos en el órgano. Los resultados mostraron que el grupo tratado con (ATL) fue el más afectado por el proceso de restricción de alimentos, la proteína presenta un perfil incompleto de aminoácidos(deficiente en leucina). Había menor desarrollo de las células del hígado, menor crecimiento del hígado debido a un menor crecimiento por hiperplasia celular (número de células), menor capacidad de división celular y de síntesis de ADN, sin embargo, mostraron una mayor capacidad para sintetizar ARN indicando que el crecimiento en la fase de restricción se debió principalmente al aumento en el tamaño de los hepatocitos (hipertrofia celular). Durante la fase de recuperación alimentar de todos los tratamientos hubo un desarrollo hepático normal, o sea crecimiento de las células y del órgano por hiperplasia e hipertrofia.
O objetivo deste estudo foi avaliar o efeito de diferentes fontes proteicas com diferentes perfis de aminoácidos sobre o desenvolvimento celular hepático de ratos "Wistar" submetidos à restrição e recuperação alimentar. O modelo de restrição alimentar foi baseado na restrição de 50% da ingestão dos animais controle (período de 21 dias), e recuperação ad libitum (período de 21 dias). As fontes proteicas utilizadas neste estudo foram: autolisado de levedura (ATL), concentrado proteico de soro de leite bovino (CSL), mistura contendo CSL e ATL na porcentagem de 64:36 (base proteica), caseína comercial (CC), a qual foi utilizada como tratamento experimental (CE) e como tratamento padrão (CP). Avaliouse, neste estudo, o perfil de aminoácidos das fontes proteicas, o desenvolvimento celular hepático (peso do fígado e dos índices de crescimento - RNA, DNA, proteína total), peso dos hepatócitos e número de hepatócitos em todo órgão. Os resultados mostram que o tratamento com (ATL) foi o mais afetado pelo processo de restrição alimentar, apresentando um perfil de aminoácido incompleto (deficiência em leucina); apresentou menor desenvolvimento celular hepático; menor crescimento do fígado em função do menor crescimento por hiperplasia celular (número de células), menor capacidade de divisão celular e síntese de DNA. Entretanto, apresentou maior capacidade de síntese de RNA, indicando que o crescimento na fase de restrição ocorreu principalmente por aumento no tamanho dos hepatócitos (hipertrofia celular). Durante o período de restauração alimentar todos os tratamentos apresentaram desenvolvimento hepático normal, ou seja, crescimento de células e do órgão por hiperplasia e hipertrofia.
Assuntos
Animais , Masculino , Feminino , Dieta com Restrição de Proteínas , Hepatócitos/fisiologia , Hepatócitos/química , Ratos Wistar/metabolismo , Crescimento Celular , Proteínas Alimentares/análiseRESUMO
The importance of physical exercise practice in the treatment of diabetes has been reported in many studies recently, but only limited data can be found regarding its benefits on liver morphology and protein content of hepatocytes. In order to assess the changes arising from the development of type I diabetes and the benefits of a training protocol, Wistar rats were divided into four groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). The training protocol consisted of swimming for 60 min a day, 5 days/week, during 8 weeks. Liver samples were collected, processed and analyzed by histochemical and ultrastructural techniques. Biochemical tests were also conducted to examine the protein content and quantity of DNA in the liver. In morphological assessment, the presence of areas of cytoplasmic basophilia observed in control subjects was not visualized in sedentary diabetics. It was related to differences in the amount of mitochondria in the cytosol. The mitochondrial structure has not undergone relevant changes, and the number of rough endoplasmic reticulum cisterns was clearly inferior in sedentary diabetics, suggesting lower protein production. However, the biochemical analysis of protein content indicated no statistical differences between groups. The exercise, in turn, was not responsible for major changes in these characteristics. On the whole, the morphological damages arising from type I diabetes were noteworthy. Nevertheless, regular physical training was not responsible for significant improvements in some respects, making evident the need for combined application of a distinct form of treatment.
Assuntos
Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Hepatócitos/química , Fígado/patologia , Condicionamento Físico Animal , Proteínas/análise , Animais , Bioquímica , Histocitoquímica , Masculino , Microscopia Eletrônica , Ratos , Ratos WistarRESUMO
Salmonella Typhimurium harbors two Salmonella pathogenicity islands (SPIs), each encoding a type three secretion system for virulence proteins. Although there is increasing evidence of postinvasion roles for SPI-1, it has been generally accepted that SPI-1 genes are downregulated following the invasion process. Here, we analyzed the expression and translocation of SopB in vitro, in cell culture and in vivo. To this end, a sopB-FLAG-tagged strain of Salmonella Typhimurium was obtained by epitope tagging. Tagged proteins were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. SopB expression was observed in vitro under cultured conditions that mimic the intestinal niche and different intracellular environments. In agreement, bacteria isolated from infected monolayers expressed and translocated SopB for at least 24 h postinoculation. For in vivo experiments, BALB/c mice were inoculated intraperitoneally with the tagged strain of Salmonella Typhimurium. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes. Our results showed that SopB continues to be synthesized in vivo during 5 days after inoculation. Interestingly, translocation of SopB was detected in the cytosol of cells isolated from lymph nodes 1 day after infection. Altogether, these findings indicate that the expression and translocation of SopB during Salmonella infection is not constrained to the initial host-bacteria encounter in the intestinal environment as defined previously.