RESUMO
The pathogenic fungus Exophiala jeanselmei (Ej4) was grown in submerged MM medium, glucose being consumed after six days with maximum biomass and EPS production. Cells were extracted with CHCl3-MeOH (2:1, v/v) yielding a product containing 10% lipid, with high levels of unsaturated C(18:1) (43.6%) and C(18:2) (21.0%), 2D-TLC showed the presence of PE (17.7%), PS (11.6%), PC (35.8%), PI (1.2%) and lyso-phospholipids, LPE (10.7%), LPC (2.0%), PA (10.4%), cardiolipin (10.5%) and glucosyl-ceramide. Analysis of EPS-1 (120 kDa) showed a galactomanan, containing a main chain of Manp-(1â2) (24.2%), substituted by side chains containing terminal Galf (16.8%) and Manp (3.5%) and acetyl groups attached at O-6 of terminal Galf. An immune response against antigens was obtained using Balb/C mice. Anti-EPS-1 antibodies recognized purified fraction containing cellular walls very titer and higher than 1:20,000 for EPS. The studied biomolecules showed biotechnological potential and point to important perspectives in diagnosis of fungi and immunomodulatory products.
Assuntos
Exophiala/imunologia , Exophiala/patogenicidade , Galactose/imunologia , Mananas/imunologia , Acetilação , Animais , Biomassa , Cromatografia em Gel , Ácidos Graxos/análise , Galactose/química , Glicolipídeos/análise , Espectroscopia de Ressonância Magnética , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Fosfolipídeos/análise , Polissacarídeos/imunologia , Polissacarídeos/isolamento & purificaçãoRESUMO
Here, we examined the impact of dichlorodiphenyltrichloroethane (DDT) and monomethyl mercury (MeHg) on the redox milieu and survival of hepatocytes from Hoplias malabaricus (traíra). After isolation and attachment of cells, we established one control and four treatments: DDT (50nM of DDT), MeHg I (0.25microM of MeHg), MeHg II (2.5microM of MeHg) and DDT * MeHg I (combination of 50nM of DDT and 0.25microM of MeHg). After four days the exposed hepatocytes presented significantly increased damage in lipids (all treatments), proteins (DDT * MeHg I and MeHg II) and reduced cell viability (all treatments). Also the antioxidant enzymes catalase, glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase and superoxide dismutase were affected. The current data showed that despite of some protective responses, the increased disturbs on membrane lipids and proteins, increased hydrogen peroxide levels, and decreased glutathione concentration and cell viability strongly indicate oxidative stress as the reason of hepatotoxicity due to DDT and MeHg exposure. In addition, DDT and MeHg together had greater effect than alone when G6PDH and glutathione-S-transferase activities and lipids damage were considered. These findings are indicative of hepatotoxicity occurring at realistic concentrations of DDT and MeHg found in Amazonian fish tissues.
Assuntos
DDT/toxicidade , Hepatócitos/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Brasil , Sobrevivência Celular/efeitos dos fármacos , Interações Medicamentosas , Peixes , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Rios , Testes de ToxicidadeRESUMO
Organisms are continuously exposed to a plethora of anthropogenic toxicants daily released to the environment. In the present study, the effects of a mixture of halogenated organic compounds (HOCs) extracted from hepatic lipids were evaluated on the primary hepatocyte culture from fish Hoplias malabaricus. Cells were isolated through non-enzymatic perfusion protocol and cultured during 3 days to allow attachment. Two concentrations of the mixture of HOCs (10 ng ml(-1) [Mix10] and 50 ng ml(-1) [Mix50]) were tested in cells for 2 days by medium replacement. The control groups, with and without solvent (DMSO) were run in the same conditions. Both tested concentrations of HOCs increased the catalase and GST activities, but only the Mix50 increase the DNA damage and decreased the GSH concentration and cell viability. Lipid peroxidation increased in the Mix10 group, but it seems to be more a consequence of DMSO presence than the HOCs themselves. The DMSO at 0.1% increased the lipid peroxidation, GSH concentration, apoptosis and DNA damage. The present data suggest that DMSO interferes with the hepatocytes of H. malabaricus in culture and that the mixture of HOCs tested alters the redox state of the hepatocytes.
Assuntos
Misturas Complexas/toxicidade , Peixes/fisiologia , Hepatócitos/efeitos dos fármacos , Hidrocarbonetos Halogenados/toxicidade , Animais , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fenômenos Químicos , Físico-Química , Cromatografia Gasosa , Ensaio Cometa , Misturas Complexas/química , Dano ao DNA , Glutationa Transferase/metabolismo , Hidrocarbonetos Halogenados/química , Peróxido de Hidrogênio/metabolismo , Marcação In Situ das Extremidades Cortadas , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos/química , OxirreduçãoRESUMO
The participation of thiol-oxidoreductases such as thioredoxin during implantation, embryogenesis and fetal development has been extensively studied. Here, we analyzed the expression of the thioredoxin superfamily enzyme quiescin Q6/sulfhydryl oxidase (QSOX) during development. Results show that QSOX is present in fetal bovine serum (4 months' gestation), but its levels decrease with time after birth (from P1 to P60). We also demonstrate that a sulfhydryl oxidase activity correlates with QSOX expression in such sera, suggesting a putative role in the redox modulation of developmental programs.
Assuntos
Sangue Fetal/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Tiorredoxinas/biossíntese , Animais , Bovinos , Oxirredução , Oxirredutases/biossíntese , Fatores de TempoRESUMO
The cellular prion protein (PrP(c)) is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein whose conformational altered forms (PrP(sc)) are known to cause neurodegenerative diseases in mammals. In order to investigate the intracellular traffic of mammalian PrP(c) in living cells, we have generated a green fluorescent protein (GFP) tagged version of PrP(c). The recombinant protein was properly anchored at the cell surface and its distribution pattern was similar to that of the endogenous PrP(c), with labeling at the plasma membrane and in an intracellular perinuclear compartment. Comparison of the steady-state distribution of GFP-PrP(c) and two N-terminal deletion mutants (Delta32-121 and Delta32-134), that cause neurological symptoms when expressed in PrP knockout mice, was carried out. The mutant proteins accumulated in the plasma membrane at the expense of decreased labeling in the perinuclear region when compared with GFP-PrP(c). In addition, GFP-PrP(c), but not the two mutants, internalized from the plasma membrane in response to Cu2+ treatment and accumulated at a perinuclear region in SN56 cells. Our data suggest that GFP-PrP(c) can be used to follow constitutive and induced PrP(c) traffic in living cells.
Assuntos
Proteínas Luminescentes/genética , Proteínas PrPC/genética , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Clonagem Molecular , Cobre/farmacologia , Imunofluorescência , Deleção de Genes , Proteínas de Fluorescência Verde , Immunoblotting , Camundongos , Camundongos Knockout , Microscopia Confocal , Mutagênese , Proteínas PrPC/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC-12 cell model to explore the biological role of LN-PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN-coated culture plaques. Furthermore, chromophore-assisted laser inactivation of cell surface PrPc perturbs LN-induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.
Assuntos
Diferenciação Celular/fisiologia , Laminina/fisiologia , Príons/fisiologia , Animais , Anticorpos/imunologia , Adesão Celular/fisiologia , Adesão Celular/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Lasers , Células PC12 , Príons/imunologia , Príons/efeitos da radiação , RatosRESUMO
Laminin (LN) plays a major role in neuronal differentiation, migration and survival. Here, we show that the cellular prion protein (PrPc) is a saturable, specific, high-affinity receptor for LN. The PrPc-LN interaction is involved in the neuritogenesis induced by NGF plus LN in the PC-12 cell line and the binding site resides in a carboxy-terminal decapeptide from the gamma-1 LN chain. Neuritogenesis induced by LN or its gamma-1-derived peptide in primary cultures from rat or either wild type or PrP null mice hippocampal neurons, indicated that PrPc is the main cellular receptor for that particular LN domain. These results point out to the importance of the PrPc-LN interaction for the neuronal plasticity mechanism.
Assuntos
Laminina/metabolismo , Neuritos/fisiologia , Proteínas PrPC/metabolismo , Animais , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Camundongos , Neurônios/metabolismo , Neurônios/ultraestrutura , Fragmentos de Peptídeos/metabolismo , Proteínas PrPSc/genética , Ligação Proteica , Ratos , Células Tumorais CultivadasRESUMO
Prions are the causative agents of transmissible spongiform encephalopathies. The transmissible agent (PrP(Sc)) is an abnormal form of PrP(C), a normal neuronal protein. The physiological role of PrP(C) remains unclear. In the present report, we evaluated behavioral parameters in Prnp(0/0) mice devoid of PrP(C). Prnp(0/0) mice showed normal short- and long-term retention of a step-down inhibitory avoidance task and normal behavior in an elevated plus maze test of anxiety. During a 5-min exploration of an open field, Prnp(0/0) mice showed normal number of rearings, defecation, and latency to initiate locomotion, but a significant increase in the number of crossings. The results suggest that Prnp(0/0) mice show normal fear-motivated memory, anxiety and exploratory behavior, and a slight increase in locomotor activity during exploration of a novel environment.
Assuntos
Ansiedade , Aprendizagem da Esquiva/fisiologia , Locomoção/fisiologia , Proteínas PrPC/fisiologia , Animais , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos KnockoutRESUMO
Prions, the etiological agents for infectious degenerative encephalopathies, act by entering the cell and inducing conformational changes in PrPC (a normal cell membrane sialoglycoprotein), which result in cell death. A specific cell-surface receptor to mediate PrPC and prion endocytosis has been predicted. Complementary hydropathy let us generate a hypothetical peptide mimicking the receptor binding site. Antibodies raised against this peptide stain the surface of mouse neurons and recognize a 66-kDa membrane protein that binds PrPC both in vitro and in vivo. Furthermore, both the complementary prion peptide and antiserum against it inhibit the toxicity of a prion-derived peptide toward neuronal cells in culture. Such reagents might therefore have therapeutic applications.