A double-strain TM (gp45) polypeptide antigen and its application in the serodiagnosis of equine infectious anemia.

Lentiviruses, including equine infectious anemia virus (EIAV), are considered viral quasispecies because of their intrinsic genetic, structural and phenotypic variability. Immunoenzymatic tests (ELISA) for EIAV reported in the literature were obtained mainly by using the capsid protein p26, which is derived almost exclusively from a single strain (Wyoming), and do not reflect the great potential epitopic variability of the EIAV quasispecies. In this investigation, the GenBank database was exploited in a systematic approach to design a set of representative protein antigens useful for EIAV serodiagnosis. The main bioinformatic tools used were clustering, molecular modelling, epitope predictions and aggregative/ solubility predictions. This approach led to the design of two antigenic proteins, i.e. a full sequence p26 capsid protein and a doublestrain polypeptide derived from the gp45 transmembrane protein fused to Maltose Binding Protein (MBP) that were expressed by recombinant DNA technology starting from synthetic genes, and analyzed by circular dichroism (CD) spectroscopy. Both proteins were used in an indirect ELISA test that can address some of the high variability of EIAV. The novel addition of the gp45 double-strain antigen contributed to enhance the diagnostic sensitivity and could be also useful for immunoblotting application.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Spermiotoxicity of nickel nanoparticles in the marine invertebrate Ciona intestinalis (ascidians).

Nickel nanoparticles (Ni NPs) are increasingly used in modern industries as catalysts, sensors, and in electronic applications. Due to this large use, their inputs into marine environment have significantly increased; however, the potential ecotoxicological effects in marine environment have so far received little attention. In particular, little is known on the impact of NPs on gamete quality of marine organisms and on the consequences on fertility potential. The present study examines, for the first time, the impact of Ni NPs exposure on sperm quality of the marine invertebrate Ciona intestinalis (ascidian). Several parameters related with sperm status such as plasma membrane lipid peroxidation, mitochondrial membrane potential (MMP), intracellular pH, DNA integrity, and fertilizing ability were assessed as toxicity end points after exposure to different Ni NPs concentrations. Ni NPs generate oxidative stress that in turn induces lipid peroxidation and DNA fragmentation, and alters MMP and sperm morphology. Furthermore, sperm exposure to Ni NPs affects their fertilizing ability and causes developmental anomalies in the offspring. All together, these results reveal a spermiotoxicity of Ni NPs in ascidians suggesting that the application of these NPs should be carefully assessed as to their potential toxic effects on the health of marine organisms that, in turn, may influence the ecological system. This study shows that ascidian sperm represent a suitable and sensitive tool for the investigation of the toxicity of NPs entered into marine environment, for defining the mechanisms of toxic action and for the environmental monitoring purpose.

17beta-Estradiol nitration by peroxidase/H2O2/NO2-: a chemical assessment.

Nitration of 17beta-estradiol by H(2)O(2) and nitrite in the presence of various peroxidases, viz. horseradish peroxidase, lactoperoxidase, and peroxidase-containing homogenates from bovine uteri, was systematically investigated to assess on a chemical basis its potential relevance to the mechanisms of impairment of estrogen functions under oxidative/nitrosative stress conditions. In the presence of excess nitrite 17beta-estradiol reacted smoothly to give 2-nitroestradiol (1), 4-nitroestradiol (2), and 2,4-dinitroestradiol (3). With 10-300 microM estradiol, formation yields of 1-3 were 12-55%, but dropped to 1% or less at lower estrogen concentration, for example, 1 microM, or in plasma as the reaction medium. Time course analysis showed that 2 is the prevalent nitration product under conditions of slow generation of nitrating species, suggesting some regioselectivity for estradiol nitration at C-4, whereas 1 prevails with bolus addition of reagents, due to faster degradation of 2. Competition experiments carried out with (15)NO(2)- showed that 2 is about twice more susceptible to nitration than 1 as determined by (15)N NMR analysis of the resulting 3. The biological effects of 1 and 2 were preliminarily tested on in vitro bovine embryo cultures. When 1 and 2 were substituted to the standard 17beta-estradiol in the oocyte maturation, a significant decrease in both cleavage and blastocyst efficiency was observed in the case of 1 but not 2. Overall, these results suggest that estradiol nitration is a potential pathway of hormonal dysfunction and toxicity but would require elevated estrogen levels of questionable physiological relevance.