RESUMO
The aim of the present work was the biophysical characterization of the Amynthas gracilis hemoglobin (HbAg). The oxy-HbAg optical absorption data, with Soret and Q bands centered at 415, 540 and 575 nm, were stable and unchanged at pH 7.0. An increase in pH promotes decrease in the intensity in the optical absorption bands, suggesting an oligomeric dissociation and partial oxidation. Identical stability at pH 7.0 was observed in DLS results that presented a hydrodynamic diameter of 28 nm, characteristic of the whole oligomer. DLS shows that HbAg undergoes oligomeric dissociation and an aggregation/denaturation process that corroborates spectroscopic data. Our results showed that the monomer d presents four isoforms with molecular mass (MM) ranging from 16,244 to 16,855 Da; the trimer subunit presents two isoforms, (abc)1 and (abc)2, with MM of 51,415 ± 20 Da and 51,610 ± 14 Da, respectively, and a less intense species, at 67,793 Da, assigned to the tetramer abcd. Monomeric chains a, obtained from reduction of the disulfide-bonded trimer abc, present four isoforms with MM 17,015 Da, 17,061 Da, 17,138 Da and 17,259 Da. DLS and LSI revealed an isoeletric point (pI) of oxy-HbAg of 6.0 ± 0.3 and 5.5, respectively. Data analysis by IEF-SDS-PAGE revealed that the pI of oxy-HbAg is 6.11, correlating with DLS and LSI data. These studies indicate that oxy-HbAg is very stable, at pH 7.0, and has differing properties from orthologous giant hemoglobins.
Assuntos
Espaço Extracelular/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Oligoquetos/citologia , Animais , Concentração de Íons de Hidrogênio , Peso MolecularRESUMO
The current use and development of applications with silver nanoparticles (Ag NPs) could lead to potential inputs of these NPs to soils. Consequently, it is crucial to understand the ecotoxicological risks posed by Ag NPs in the terrestrial compartment. In the present investigation, the effects produced by PVP-PEI coated Ag NPs were assessed in Eisenia fetida earthworms in comparison with the soluble form (AgNO3). Earthworms were exposed for 1, 3 and 14 days to a range of sublethal concentrations of Ag (0, 0.05 and 50mg/kg) and at each exposure time, apart from mortality and weight loss of individuals, metallothionein (MT) protein concentration and catalase (CAT) activity were quantified in earthworm tissues. In addition, cellular and molecular level endpoints (cell viability, absolute and relative trophic indices and transcription levels of catalase-cat- and metallothionein-mt-) were measured in coelomocytes extruded from exposed earthworms. Despite the lack of effects in traditional endpoints (mortality and weight loss), Ag NPs and AgNO3 posed changes at lower levels of biological complexity (biochemical, cellular and molecular levels). Both Ag forms induced similar changes in the metal detoxification mechanism (MT, mt) and in the antioxidant response system (CAT, cat) of E. fetida. In contrast, Ag form dependant cytotoxicity and subpopulation ratio alterations (eleocytes/amoebocytes) were recorded in extruded coelomocytes. Complementarily, the use of coelomocytes to assess molecular level endpoints represented a relevant alternative for development of non-invasive biomarkers.
Assuntos
Nanopartículas Metálicas/toxicidade , Oligoquetos/efeitos dos fármacos , Prata/toxicidade , Poluentes do Solo/toxicidade , Solo/química , Animais , Biomarcadores/metabolismo , Catalase/genética , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Nanopartículas Metálicas/química , Metalotioneína/genética , Metalotioneína/metabolismo , Oligoquetos/citologia , Oligoquetos/metabolismo , Prata/química , Nitrato de Prata/química , Nitrato de Prata/toxicidade , SolubilidadeRESUMO
Oxy-HbRa thermal stability was evaluated by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) at pH 5.0, 7.0, 8.0, and 9.0. DLS results show that oxy-HbRa, at pH 7.0 and 5.0, remains stable up to 56 °C, undergoing denaturation/aggregation in acidic media above 60 °C, followed by partial sedimentation of aggregates. At alkaline pH values 8.0 and 9.0, oxy-HbRa oligomeric dissociation is observed above 30 °C, before denaturation. SAXS data show that oxy-HbRa, at 20 °C, is in its native form, displaying radius of gyration (R g) and particle maximum dimension (D max) of 108 ± 1 and 300 ± 10 Å, respectively. Oxy-HbRa, at pH 7.0, undergoes denaturation/aggregation at 60 °C. At pH 5.0-6.0, HbRa thermal denaturation/aggregation start earlier, at 50 °C, accompanied by an increase of R g and D max values. However, an overlap of oligomeric dissociation and denaturation in the system is observed upon temperature increase, with an increase in R g and D max. Analysis of experimental p(r) curves as a linear combination of theoretical curves obtained for HbGp fragments from the crystal structure shows an increasing contribution of dodecamer (abcd)3 and tetramer (abcd) in solution, as a function of pH values (8.0 and 9.0) and temperature. Finally, our data show, for the first time, that oxy-HbRa, in neutral and acidic media, does not undergo oligomeric dissociation before denaturation, while in alkaline media the oligomeric dissociation process is an important step in the thermal denaturation.
Assuntos
Difusão Dinâmica da Luz , Espaço Extracelular , Hemoglobinas/química , Oligoquetos/citologia , Espalhamento a Baixo Ângulo , Temperatura , Difração de Raios X , Animais , Concentração de Íons de Hidrogênio , Oxiemoglobinas/química , Desnaturação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de ProteínaRESUMO
The earthworm (Eisenia foetida) is an attractive sentinel species for detecting genotoxicity in soil. In this study, an improved single-cell gel electrophoresis (SCGE) assay was developed for detecting DNA damage in the coelomocytes (lymphocytes) of earthworms. Coelomocytes were obtained from the coelomic fluid using a modified extrusion medium that did not include the mucolytic agent guaiacol. The extruded coelomocytes contained at least three types of cells: eleocytes (75% of the total), amoebocytes, and granulocytes. The DNA migration parameters were determined for untreated cells of each type in order that the assay could be performed with minimum inter- and intra-individual variation. In addition, lysis time was reduced to 10 min, and only one neutralization step was used. DNA damage was detected in isolated eleocytes treated with hydrogen peroxide and cadmium, and in eleocytes from earthworms exposed for up to 21 days to soil containing polycyclic aromatic hydrocarbons. The SCGE assay using earthworm eleocytes appears to be a sensitive biomarker for evaluating exposure to genotoxic compounds.
Assuntos
Ensaio Cometa/métodos , Dano ao DNA , Mutagênicos/toxicidade , Oligoquetos/efeitos dos fármacos , Oligoquetos/genética , Animais , Cádmio/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hidrocarbonetos Aromáticos/toxicidade , Peróxido de Hidrogênio/toxicidade , Testes de Mutagenicidade , Oligoquetos/citologia , Fatores de TempoRESUMO
Previous electrophysiological studies have suggested the presence of two types of muscular cells in the longitudinal muscle of A. hawayanus. However, a definite classification of such cells as muscles has been hindered by lack of morphological evidence. This study identified these cells electrophysiologically and morphologically by using the same intracellular microelectrode to record cell activity and inject Lucifer Yellow-CH. Electrophysiological measurements clearly differentiated the two groups of cells. One group did not respond to nerve stimulation but was spontaneously active, whereas the other responded to nerve stimulation with endplate potentials and did not discharge spontaneously. Morphological examination revealed two very similar types of longitudinal muscle cells. These results clearly show that, although morphologically similar, there are two distinct groups of muscle cells in the longitudinal muscle layer of the earthworm.
Assuntos
Músculos/citologia , Oligoquetos/citologia , Animais , Eletrofisiologia/métodos , Isoquinolinas , Microeletrodos , Microscopia de Fluorescência , Músculos/fisiologiaRESUMO
Histochemical study of mucopolysaccharides of both mucous secreting and subjacent granular cells of the epidermis of Glossoscolex uruguayensis L (Righi, 1978), has been done. Histochemical tests revealed two major types of mucous cells: large ortochromatic mucous cells and metachromatic mucous cells. The first ones possess alpha-amylase resistant, PAS-positive material, together with little mucic acid, and the latter present a very low rate of neutral mucopolysaccharides and to a variable degree, both carboxylate and sulphate mucopolysaccharides. A third cell type was also detected, presenting a frothy acid like material. Subjacent epidermal granular cells display alpha-amylase resistant, PAS-positive granules, with no acid mucus.