RESUMO
This work aims to evaluate the size and lability of Cu and Zn bound to proteins in the cytosol of fish liver of Oreochromis niloticus by employing solid-phase extraction (SPE), diffusive gradients in thin films (DGT), and ultrafiltration (UF). SPE was carried out using Chelex-100. DGT containing Chelex-100 as binding agent was employed. Analyte concentrations were determined by ICP-MS. Total Cu and Zn concentrations in cytosol (1 g of fish liver in 5 ml of Tris-HCl) ranged from 39.6 to 44.3 ng ml-1 and 1498 to 2106 ng ml-1, respectively. Data from UF (10-30 kDa) suggested that Cu and Zn in cytosol were associated with â¼70% and 95%, respectively, with high-molecular-weight proteins. Cu-metallothionein was not selectively detected (although 28% of Cu was associated with low-molecular-weight proteins). However, information about the specific proteins in the cytosol will require coupling UF with organic mass spectrometry. Data from SPE showed the presence of labile Cu species of â¼17%, while the fraction of labile Zn species was >55%. However, data from DGT suggested a fraction of labile Cu species only of 7% and a labile Zn fraction of 5%. This data, as compared with previous data from literature, suggests that the DGT technique gave a more plausible estimation of the labile pool of Zn and Cu in cytosol. The combination of results from UF and DGT is capable of contributing to the knowledge about the labile and low-molecular pool of Cu and Zn.
Assuntos
Ciclídeos , Poluentes Químicos da Água , Animais , Ultrafiltração/métodos , Citosol , Zinco/análise , Fígado/química , Monitoramento Ambiental/métodos , Poluentes Químicos da Água/análiseRESUMO
Although the prevalence of autoimmune hepatitis in first-degree relatives is small, the relationship between genetic markers, especially human leucocyte antigens (HLA), and susceptibility to this disease, has been studied for over three decades. The genetic susceptibility to AIH is believed to be different in the two subtypes of the disease, AIH type 1 and AIH type 2. Type 1 AIH has anti-smooth muscle and anti-nuclear antibodies as its main markers, while those of type 2 AIH are the anti-liver/kidney microsome type 1 and anti-liver cytosol type 1 antibodies. The anti-soluble liver antigen/liver-pancreas antibodies, which, in addition to being present in both subtypes, mark an important number of patients without serological markers. Therefore, a third type of disease is questionable. The vast majority of immunogenetic studies compare the differences between the two main types and make no difference between which antibodies are present to define the subtype. This review seeks to analyze what was most important published in the AIH in this context, trying to relate the HLA alleles according to the AIH marker autoantibodies.
Assuntos
Hepatite Autoimune , Humanos , Hepatite Autoimune/genética , Predisposição Genética para Doença , Autoanticorpos , Anticorpos Antinucleares , BiomarcadoresRESUMO
This article reviews the use of the 6-acetyl-2-(dimethylamino)naphthalene (ACDAN) fluorophore to study dipolar relaxation in cells, tissues, and biomimetic systems. As the most hydrophilic member of the 6-acyl-2-(dimethylamino)naphthalene series, ACDAN markedly partitions to aqueous environments. In contrast to 6-lauroyl-2-(dimethylamino)naphthalene (LAURDAN), the hydrophobic and best-known member of the series used to explore relaxation phenomena in biological (or biomimetic) membranes, ACDAN allows mapping of spatial and temporal water dipolar relaxation in cytosolic and intra-organelle environments of the cell. This is also true for the 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) derivative which, unlike LAURDAN, partitions to both hydrophobic and aqueous environments. We will (i) summarize the mechanism which underlies the solvatochromic properties of the DAN probes, (ii) expound on the importance of water relaxation to understand the intracellular environment, (iii) discuss technical aspects of the use of ACDAN in eukaryotic cells and some specialized structures, including liquid condensates arising from processes leading to liquid immiscibility and, (iv) present some novel studies in plant cells and tissues which demonstrate the kinds of information that can be uncovered using this approach to study dipolar relaxation in living systems.
Assuntos
Corantes Fluorescentes , Água , Corantes Fluorescentes/química , Naftalenos , Água/químicaRESUMO
Superoxide dismutase (SOD) enzymes are considered the first line of Defence against reactive oxygen species. Among the intracellular isoforms of SOD, Cu,ZnSOD is widely distributed, and the MnSOD is localised in the mitochondria. The SOD activities have been measured indirectly by inhibiting oxidation reactions. Most techniques assessing the hepatic SOD activity are adaptations of classical methods and differ significantly from each other. This work assessed the hepatic Cu,ZnSOD activity in the supernatants of two different centrifugations, using two isotonic medium and the pyrogallol method. In most studies conducted in rat liver, only the Cu,Zn or total SOD activities were assessed and sometimes the Cu,ZnSOD activity was calculated based on the inhibition by cyanide. But, as demonstrated here, this inhibition is not complete. Besides, the novelty of this work is that we presented a method for the evaluating the Cu,ZnSOD and MnSOD activities separately and from the same liver.
Assuntos
Ensaios Enzimáticos/métodos , Fígado/enzimologia , Superóxido Dismutase/metabolismo , Animais , RatosRESUMO
The cox3 gene, encoding subunit III of cytochrome c oxidase (Cox3) is in mitochondrial genomes except in chlorophycean algae, where it is localized in the nucleus. Therefore, algae like Chlamydomonas reinhardtii, Polytomella sp. and Volvox carteri, synthesize the Cox3 polypeptide in the cytosol, import it into mitochondria, and integrate it into the cytochrome c oxidase complex. In this work, we followed the in vitro internalization of the Cox3 precursor by isolated, import-competent mitochondria of Polytomella sp. In this colorless alga, the precursor Cox3 protein is synthesized with a long, cleavable, N-terminal mitochondrial targeting sequence (MTS) of 98 residues. In an import time course, a transient Cox3 intermediate was identified, suggesting that the long MTS is processed more than once. The first processing step is sensitive to the metalo-protease inhibitor 1,10-ortophenantroline, suggesting that it is probably carried out by the matrix-located Mitochondrial Processing Protease. Cox3 is readily imported through an energy-dependent import pathway and integrated into the inner mitochondrial membrane, becoming resistant to carbonate extraction. Furthermore, the imported Cox3 protein was assembled into cytochrome c oxidase, as judged by the presence of a labeled band co-migrating with complex IV in Blue Native Electrophoresis. A model for the biogenesis of Cox3 in chlorophycean algae is proposed. This is the first time that the in vitro mitochondrial import of a cytosol-synthesized Cox3 subunit is described.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Multimerização Proteica , Volvocida/enzimologia , Transporte Biológico Ativo , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Transporte ProteicoRESUMO
The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the efficiency of the water-water cycle and photo-oxidative protection under stress conditions. In this study, we have generated double knockdown rice (Oryza sativa L.) plants in both OsAPX7 (sAPX) and OsAPX8 (tAPX) genes, which encode chloroplastic APXs (chlAPXs). By employing an integrated approach involving gene expression, proteomics, biochemical and physiological analyses of photosynthesis, we have assessed the role of chlAPXs in the regulation of the protection of the photosystem II (PSII) activity and CO2 assimilation in rice plants exposed to high light (HL) and methyl violagen (MV). The chlAPX knockdown plants were affected more severely than the non-transformed (NT) plants in the activity and structure of PSII and CO2 assimilation in the presence of MV. Although MV induced significant increases in pigment content in the knockdown plants, the increases were apparently not sufficient for protection. Treatment with HL also caused generalized damage in PSII in both types of plants. The knockdown and NT plants exhibited differences in photosynthetic parameters related to efficiency of utilization of light and CO2. The knockdown plants overexpressed other antioxidant enzymes in response to the stresses and increased the GPX activity in the chloroplast-enriched fraction. Our data suggest that a partial deficiency of chlAPX expression modulate the PSII activity and integrity, reflecting the overall photosynthesis when rice plants are subjected to acute oxidative stress. However, under normal growth conditions, the knockdown plants exhibit normal phenotype, biochemical and physiological performance.