Fish proteins as targets of ferrous-catalyzed oxidation: identification of protein carbonyls by fluorescent labeling on two-dimensional gels and MALDI-TOF/TOF mass spectrometry

Protein oxidation in fish meat is considered to affect negatively the muscle texture. An important source of free radicals taking part in this process is Fenton's reaction dependent on ferrous ions present in the tissue. The aim of this study was to investigate the susceptibility of cod muscle proteins in sarcoplasmic and myofibril fractions to in vitro metal-catalyzed oxidation and to point out protein candidates that might play a major role in the deterioration of fish quality. Extracted control proteins and proteins subjected to free radicals generated by Fe(II)/ascorbate mixture were labeled with fluorescein-5-thiosemicarbazide (FTSC) to tag carbonyl groups and separated by two-dimensional gel electrophoresis. Consecutive visualization of protein carbonyl levels by capturing the FTSC signal and total protein levels by capturing the SyproRuby staining signal allowed us to quantify the relative change in protein carbonyl levels corrected for changes in protein content. Proteins were identified using MALDI-TOF/TOF mass spectrometry and homology-based searches. The results show that freshly extracted cod muscle proteins exhibit a detectable carbonylation background and that the incubation with Fe(II)/ascorbate triggers a further oxidation of both sarcoplasmic and myofibril proteins. Different proteins exhibited various degrees of sensitivity to oxidation processes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), nucleoside diphosphate kinase B (NDK), triosephosphate isomerase, phosphoglycerate mutase, lactate dehydrogenase, creatine kinase, and enolase were the sarcoplasmic proteins most vulnerable to ferrous-catalyzed oxidation. Moreover, NDK, phosphoglycerate mutase, and GAPDH were identified in several spots differing by their pI, and those forms showed different susceptibilities to metal-catalyzed oxidation, indicating that post-translational modifications may change the resistance of proteins to oxidative damage. The Fe(II)/ascorbate treatment significantly increased carbonylation of important structural proteins in fish muscle, mainly actin and myosin, and degradation products of those proteins were observed, some of them exhibiting increased carbonylation levels.     

Functional Properties of White Shrimp (Litopenaeus vannamei) By-Products Protein Recovered by Isoelectric Solubilization/Precipitation

The processing of fish in general entails quantities of by-products, such as exoskeleton and cephalothorax of shrimp, which may reach 70%. Protein recovered from by-products has been investigated in recent years for its ability to offer more nutritious foods and improved functional properties. The objective of the present study was the use of the pH shifting process to recover proteins from solubilization and precipitation that are present in the by-products (heads and shells) of white shrimp (Litopenaeus vannamei). The proximal composition of the by-products presented 58.5% protein (d.b). The isoelectric point used to precipitate the protein was defined at pH 4. The protein concentrate presented 73.1% of protein. The yield of the protein concentrate mass resulted in 47.8% (w.b). The greatest solubility found for the protein concentrate was 51.3% at pH 8. The oil and water retention capacity resulted in values of 8.5 and 2.5 mL/g of protein, respectively, and the highest emulsifying capacity was found at pH 8. The by-products and concentrate amino acids profiles confirmed the presence of 16 amino acids. In general, the shrimp protein concentrate obtained showed the potential to be included as an ingredient in food formulations.     

DNA Fragmentation in Canine Immature Grade 1 Cumulus‐Oocyte Complexes

In this work, we studied the incidence of DNA fragmentation, interpreted as apoptotic changes and assessed by the TUNEL assay, in cumulus cells and oocytes of immature Grade 1 cumulus‐oocyte complexes (COCs) obtained from healthy bitches (n = 27) of three age groups: young (1–3 years; n = 13), adult (4–6 years; n = 8) and elderly (7–10 years; n = 6). Age affected (p < 0.05) Grade 1 COCs recovery rates, with young animals yielding more (p < 0.01) Grade 1 COCs than the other two age groups. Conversely, no differences were observed in the incidence of DNA fragmentation (TUNEL‐positive) in cumulus cells or oocytes between the three age groups. Overall, more than 80% of Grade 1 COCs presented <15% of TUNEL‐positive cumulus cells and enclosed TUNEL‐negative (intact DNA) oocytes. Despite a higher proportion of TUNEL‐negative oocytes being found in the germinal vesicle stage, most of the oocytes with nuclear material compatible with meiosis resumption (MR) or with non‐identifiable nuclear material (ND) did not present DNA fragmentation. No correlation was observed between DNA fragmentations in oocytes and in cumulus cells. We concluded that the morphological parameters used to classify canine Grade 1 COCs are reliable to select a homogeneous population of COCs with low incidence of DNA fragmentation. Furthermore, these results indicate that DNA fragmentation can only explain a minor proportion of the incidence of MR and degeneration in canine oocytes at collection.     

Characterization of Isoforms of the Lectin Isolated from the Red Algae Bryothamnion seaforthii and Its Pro-Healing Effect

Lectins are a structurally heterogeneous group of proteins that have specific binding sites for carbohydrates and glycoconjugates. Because of their biotechnological potential, lectins are widely used in biomedical research. The present study aimed to evaluate the healing potential of the lectin isolated from the marine red alga Bryothamnion seaforthii (BSL). The lectin was purified using ion exchange chromatography with DEAE cellulose and characterized using tandem mass spectrometry. For healing tests, skin wounds were induced in the dorsal thoracic region of mice. These animals were randomly divided into three groups and subjected to topical treatment for 12 days with BSL, bovine serum albumin and 150 mM NaCl. To evaluate the potential of each treatment, the animals were anesthetized and sacrificed on days 2, 7 and 12, respectively. The parameters evaluated included the wound area, the proportion of wound closure and the histological diagnosis. The wound closure was more effective with BSL (Postoperative Day 7 and 12) than controls. The luminal epithelium was completely restructured; the presence of collagen in the dermis and the strongly active presence of young skin annexes demonstrate the potential of treatment with BSL compared with controls. Our findings suggest that BSL has pro-healing properties and can be a potential medical process in the treatment of acute wounds.     

Exploiting genotypic variability from low-altitude Brazilian Savannah-adapted <Emphasis Type="Italic">Chenopodium quinoa</Emphasis>

Saponin-free quinoa genotypes were evaluated in summer and autumn–winter sowings, for phenotypic traits and yield stability. They were obtained by single plant-progeny selection from hybrids, as part of the breeding efforts in adapting the crop to the Brazilian Savannah environments. The soils (Ferralsols) were limed and fertilized prior to cultivation. The experiment was sown in two dates: 20th December 2006 (summer), and 30th April 2007 (autumn–winter), at 15°39′ and 16°14′ S latitude, 47°27′ and 47°44′ W longitude, with altitudes of 976 and 1,110 m, respectively. The mean temperature and rainfall for the summer sowing were 23.0°C and 723 mm. In autumn–winter, under controlled irrigation, the mean temperature was 2.9°C lower than in summer, when plants were exposed to water logging spells. The treatments consisted of fourteen selected genotypes and the varieties Kancolla, BRS Piabiru and Q4.5, used as controls. Comparisons were based on plant height, biomass and grain yield, harvest index (HI) and 1,000 seed weight. The experimental design was a complete randomized bloc, with three replications. Analysis of variance was performed for each sowing and jointly. Statistical differences for the environments were evident only for grain size and plant height. The performance of selected genotypes in each sowing date and in the joint analysis, allows saying that populations Q0779, Q0780 and Q0782 are potentially useful in the acquisition of stable genotypes for the Brazilian Savannahs.     

Influence of temperature on muscle fibre hyperplasia and hypertrophy in larvae of blackspot seabream,Pagellus bogaraveo

The effects of temperature on Pagellus bogaraveo muscle cellularity were investigated, by morphometry, throughout the endogenous feeding stage, testing two rearing temperatures: 14 and 18 °C. The following parameters were estimated in transversal body sections at post‐opercular and post‐anal body levels: the total cross‐sectional muscle area, the total number of fibres and the mean cross‐sectional fibre area. At hatching, no significant influence of the temperature was observed on the morphometric parameters measured in the white muscle. At mouth opening, an increase in the number of post‐opercular white fibres was promoted by the highest temperature. During embryonic development, the red muscle fibre number in the post‐anal part of the larvae increased with higher temperature, but it appears that the difference was no longer present at mouth opening. An increase in the fibre area and in the total cross‐sectional area of red muscle at the post‐anal level was promoted by a 4 °C increase in the temperature during the vitelline phase. In conclusion, the axial musculature of blackspot seabream embryos/larvae reacted differently to temperature influence according to the body location, strongly supporting the need to look at and account for different body locations when evaluating muscle cellularity in fish, namely in growth/aquaculture‐related studies.     

Soil compaction caused by harvesting,skidding and wood processing in eucalyptus forests on coarse‐textured tropical soils

Eucalyptus forests play a major role in the world economy, providing raw materials for different purposes. In planted forests, harvest operations performed by heavy machinery may cause severe soil compaction. This study aimed to evaluate the impact of a full‐tree harvesting system in planted eucalyptus forests from Northeastern Brazil. Different soils were evaluated (two Hapludults and one Haplorthod) in two horizons (BA and Bt). We tested different equipment, namely feller buncher, skidder (with traffic intensities of 3, 6, 12 and 16 passes), flail (at different ground‐contact points), grapple saw and loader. The soil physical attributes reflected not only the impact of equipment traffic but also the intrinsic differences between the soils. Bulk density (ranging from 1.36 to 1.80 t m^?3 after trafficking) related well to soil class and horizon. Precompression stress (ranging from 203 to 430 kPa) and degree of compaction (76%–94%) following trafficking were well correlated, while increase in bulk density (reaching a maximum of 20%) related more strongly to soil moisture. A contingency table was constructed with the number of compacted samples and further examined by correspondence analysis. Compaction varied according to soil, horizon and equipment, indicating that machine–soil interactions are very specific and demand detailed research under different conditions. The Haplorthod experienced the greatest amount of compaction, whereas the Hapludult‐2 was more resistant (60% and 25% of compacted samples, respectively). The grapple saw and the skidder at higher traffic intensities (12 and 16 passes) exerted the highest mechanical impacts (81% and 67% of compacted samples, respectively).     

The bone degenerative processes in senile fishes from Holocene Brazilian shell mounds

Zooarchaeological collections from shell mounds in Rio de Janeiro (2,470–4,632 cal BP) contain a high prevalence of swollen fish bones belonging to the Atlantic spadefish (Chaetodipterus faber), crevalle jack (Caranx hippos) and fat snook (Centropomus parallelus). Given the lack of knowledge of the bone degenerative process in senile fishes, this study analysed hyperostotic bone in zooarchaeological and modern specimens to obtain high‐resolution morphology and microstructure reconstruction. We used microCT as well as X‐ray diffraction to characterize the crystallographic changes associated with fish senility. Our results showed that trabecular microstructures in hyperostotic bones were consistent with estimated values of the per cent bone volume‐to‐total volume ratio (BV/TV) and were greater than 60% in cortical bone. Hyperostotic bones indicated a high radiograph density, and X‐ray diffractograms showed a decrease in hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂] and calcite (CaCO₃) neocrystallization. These crystalline and density changes revealed an advanced stage of fish senile and indicate the vulnerability of ageing fish populations.     

Is the use of turmeric in the diet safe for fish?

With this study, we aimed to assess the safety of using Curcuma longa in fish feed. Juvenile Astyanax aff. bimaculatus (0.83 ± 0.04 g) were kept in 24 80‐L aquaria, at a density of 0.5 fish L^?1 for 60 days. Six diets supplemented with 0.0, 20.0, 40.0, 60.0, 80.0 and 100.0 g kg^?1 were evaluated. Histopathological analysis of the gills showed that there was an increasing linear effect of turmeric on the frequency of lamellar aneurysm, hypertrophy of mucous cells, oedema and necrosis. There was a quadratic effect of turmeric supplementation on the frequency of fusion and disruption of secondary lamellae, vacuolation and hypertrophy of the epithelial cells, and the estimated values to minimize these variables were 4.63, 25.93 and 24.46 g kg^?1 respectively. There was a quadratic effect of turmeric on the gills index (I_BRA), and the estimated value to minimize this index was 30.23 g kg^?1. No effect of turmeric on the frequency of histopathological changes and organ index in fish liver was observed. Thus, we conclude that turmeric is not hepatotoxic for A. aff. bimaculatus; however, its use is safe at concentrations up to 30.23 g kg^?1 diet as a function of its gill toxic effect at higher doses.     

Curcuma longa supplementation in the diet of Astyanax aff. bimaculatus in preparation for transport

The physiological state of fish before transportation is an important aspect to be observed when reduction in losses due to mortality is aimed. Various preparative procedures for transport are reported, but little is known about the use of additives in fish diet. In this study, we aimed to evaluate the effect of dietary turmeric (Curcuma longa) supplementation on the tolerance of juvenile Astyanax aff. bimaculatus to transportation stress. The experimental design consisted of six treatments: 0, 20, 40, 60, 80 and 100.0 g kg^?1 turmeric. Fish weighing 0.83 ± 0.04 g were distributed in 24 80‐L aquaria at a density of 0.5 fish L^?1 and were fed to satiation three times a day for 60 days. After this period, fish were transported for 24 h in plastic bags (15 fish per bag). Using a discontinuous linear response plateau (LRP) model, the lowest estimated concentration of turmeric that maximized the survival rate was 16.67 g kg^?1. Quadratic effects of turmeric on blood lactate levels were observed, and the estimated concentration of turmeric required to minimize blood lactate levels was 64.7 g kg^?1. We observed quadratic effects of turmeric on superoxide dismutase activity and the concentration of malondialdehyde in fish gills and the turmeric concentrations required to minimize the transport stress effects on these variables were 42.50 and 83.33 g kg^?1, respectively. Thus, dietary supplementation with turmeric enhanced the tolerance of juvenile A. aff. bimaculatus to transportation stress.