A morphoanatomical approach to the adaptive features of the epidermis and proboscis of a marine Polychaeta: Eulalia viridis (Phyllodocida: Phyllodocidae).

Eulalia viridis is a marine Polychaeta of the rocky intertidal that, despite its simple anatomy, is an active predator of much larger invertebrates, from which it extracts pieces of soft tissue through suction. This uncanny feeding strategy triggered the pursuit for the morphological mechanisms that enable adaptation to its environment. The evaluation of the worm anatomy and microanatomy, combining electron and optical microscopy, revealed a series of particular adaptations in the epidermis and in the proboscis (the heavily muscled eversible pharynx). Besides its function in feeding, the proboscis is the main sensory organ, being equipped with numerous sensorial papillae holding chemoreceptors. Additionally, the proboscis possesses tentacles that become exposed when the organ is everted. These provide fast release of mucus and toxins, from mucocytes and special serous cells, respectively (the latter involving both merocrine and apocrine processes), whenever contact with a prey occurs. In its turn, the epidermis provides protection by cuticle and mucus secretion and has a sensorial function that may be associated to the worm's uncommon green pigment cells. Eulalia viridis presents a series of elegant adaptive tools to cope with its environment that are evolutionarily designed to counterbalance its relatively simple body plan.

Integration of sediment contamination with multi-biomarker responses in a novel potential bioindicator (Sepia officinalis) for risk assessment in impacted estuaries.

For the purpose of biomonitoring, species that combine ecological and commercial importance may provide a link between ecological and human health risk. The common cuttlefish, Sepia officinalis, holds both characteristics in south-western Europe, albeit remaining unsurveyed in ecotoxicological studies. Cuttlefish collected from an impacted estuary in SW Portugal and a reference location off the coast were analysed for a battery of biomarker responses in the digestive gland and gills. The contrast to reference animals revealed that biomarker responses, especially those related to oxidative stress, were consistent with sediment contamination by PAHs, even in a situation that combines complex toxicant mixtures, moderate levels of contamination and high ecotoxicological diversity. However, environmental parameters related to the differences between shore and estuarine habitats should not be overruled. Also, digestive gland metallothionein retained significant specificity to metals even though previous studies in the area with clams and fish failed to trigger a conclusive response. The highest net differences in biomarker responses were detected in the gills, likely indicating higher sensitivity to environmental stressors. Still, the digestive gland responses were overall the most consistent with sediment contamination and effectively differentiated between estuarine industrial- and rural-impacted sites. The results indicate that S. officinalis may be a candidate to meet the European Union's requirements for efficient biomonitoring programmes, with the additional importance of being cosmopolitan, abundant, commercially valuable and combining the molluscan biology that has been granting bivalves their high value for biomonitoring with foraging behaviour, thus better able to reflect anthropogenic stressors impacting a wider area than sedentary organisms. Nevertheless, further investigations in unpolluted sites are needed to better evaluate the background levels of biomarker responses in the species.

Ecotoxicological heterogeneity in transitional coastal habitats assessed through the integration of biomarkers and sediment-contamination profiles: a case study using a commercial clam.

Transitional waterbodies, such as estuaries, are highly diversified environments with respect to ecology, geophysics, and nature of anthropogenic impacts. This spatial heterogeneity may pose important constraints when developing monitoring programmes for aquatic pollution. The present study compared three distinct coastal ecosystems located in Southern Portugal (subjected to different anthropogenic stressors), namely, two estuaries and a coastal lagoon, through the characterisation of sediment contamination and a biomarker approach to an important commercial clam (Ruditapes decussatus) obtained from local fishing grounds. The results showed high heterogeneity of sediment contamination for both estuaries and a marked distinction between industrially and agriculturally influenced areas as well as between natural and artificialized sites. Hydrodynamics and oceanic influence (in essence dictating sediment type) play a major role in environmental quality. Environmental heterogeneity constituted an important confounding factor for biomarker analysis in the clams' digestive glands since the animals appeared to respond to their immediate surroundings' characteristics rather than the geographical area where they were collected from, despite the relative distance to pollution hot spots. Still, oxidative stress biomarkers (lipid peroxidation and catalase activity) could correlate with each other and to both organic and metallic contamination, whereas metallothionein-like protein induction failed to correlate to any class of sediment toxicants (albeit metals being the most representative pollutants) and appeared to be strongly affected (unlike the previous) by clam size and probably other unknown internal and external variables, among which contaminant interactions should play a major role.

Multi-organ histological observations on juvenile Senegalese soles exposed to low concentrations of waterborne cadmium.

A histopathological screening was performed on juvenile Senegalese soles exposed to environmentally realistic concentrations of waterborne Cd (0.5, 5 and 10 µg L(-1)) for 28 days. The severity and dissemination of histopathological changes were variable and limited to the kidney, liver, spleen, gills and skin goblet cells. Contradicting available literature that refers the liver as the most affected organ upon acute exposure and the kidney following chronic exposure, the liver was the most impacted organ (even at the lowest concentration), in a trend that could relate to the duration of exposure and Cd concentration. The most noticeable hepatic alterations related to inflammation, although hepatocellular alterations like lipidosis and eosinophilic foci also occurred. The trunk kidney of exposed fish endured moderate inflammation, apoptosis and necrosis, however, without a clear time-dependent effect. The spleen of fish subjected to the highest concentrations revealed diffuse necrotic foci accompanied by melanomacrophage intrusion. The gills, albeit the most important apical uptake organ of dissolved toxicants, sustained only moderate damage, from epithelial hyperplasia and pavement cell detachment to the potentially more severe chloride cell alterations. In the skin, an increase in goblet cell size occurred, most notoriously correlated to Cd concentration at earlier stages of exposure. The results show that a metal-naïve juvenile fish can endure deleterious effects when exposed to low, ecologically relevant, concentrations of a common toxic metal and that the pattern of Cd-induced histopathological alterations can be complex and linked to organ-specific responses and metal translocation within the organism.

Advances on assessing nanotoxicity in marine fish - the pros and cons of combining an ex vivo approach and histopathological analysis in gills.

The need to overcome logistic and ethical limitations of in vivo nanotoxicity evaluation in marine organisms is essential, mostly when dealing with fish. It is well established that medium/solvent conditions affect dispersion and agglomeration of nanoparticles (NPs), which represents a constraint towards a solid and realistic toxicity appraisal. In this way the pros and cons of an ex vivo approach, using a simplified exposure medium (seawater) and addressing gills histopathology, were explored. The nanotoxic potential of environmentally realistic concentrations of titanium dioxide NPs (TiO2 NPs) was also assessed, disclosing the morpho-functional effects on the gills and the possible uptake/elimination processes. Excised gills of the Senegalese sole (Solea senegalensis) were directly exposed in artificial seawater to 20 and 200 µg L-1 TiO2 NPs, for 2 h and 4 h. Semi-quantitative and quantitative histological analyses were applied. The normal morphology of the gill's epithelia was only slightly altered in the control, reflecting protective mechanisms against the artificiality of the experimental conditions, which, together with the absence of differences in the global histopathological index (Ih), corroborated that the gill's morpho-functional features were not compromised, thereby validating the proposed ex vivo approach. TiO2 NPs induced moderate severity and dissemination of histopathological lesions. After 2 h, a series of compensatory mechanisms occurred in NP treatments, implying an efficient response of the innate defense system (increasing number of goblet cells) and effective osmoregulatory ability (chloride cells proliferation). After 4 h, gills revealed signs of recovery (normalization of the number of chloride and goblet cells; similar Ih), highlighting the tissue viability and effective elimination and/or neutralization of NPs. The uptake of the TiO2 NPs seemed to be favored by the higher particle sizes. Overall, the proposed approach emerged as a high-throughput, reliable, accurate and ethically commendable methodology for nanotoxicity assessment in marine fish.

Modelling metallothionein induction in the liver of Sparus aurata exposed to metal-contaminated sediments.

Metallothionein (MT) in the liver of gilthead seabreams (Sparus aurata L., 1758) exposed to Sado estuary (Portugal) sediments was quantified to assess the MT induction potential as a biomarker of sediment-based contamination by copper (Cu), cadmium (Cd), lead (Pb) and arsenic (As). Sediments were collected from two control sites and four sites with different levels of contamination. Sediment Cu, Cd, Pb, As, total organic matter (TOM) and fine fraction (FF) levels were determined. Generalized linear models (GLM) allowed integration of sediment parameters with liver Cu, Cd, Pb, As and MT concentrations. Although sediment metal levels were lower than expected, we relate MT with liver Cd and also with interactions between liver and sediment Cu and between liver Cu and TOM. We suggest integrating biomarkers and environmental parameters using statistical models such as GLM as a more sensitive and reliable technique for sediment risk assessment than traditional isolated biomarker approaches.

Body fat and cholecalciferol supplementation in elderly homebound individuals.

Vitamin D deficiency, observed mainly in the geriatric population, is responsible for loss of bone mass and increased risk of bone fractures. Currently, recommended doses of cholecalciferol are advised, but since there are few studies evaluating the factors that influence the serum levels of 25-hydroxyvitamin D (25(OH)D) following supplementation, we analyzed the relationship between the increase in serum 25(OH)D after supplementation and body fat. We studied a group of 42 homebound elderly subjects over 65 years old (31 women) in order to assess whether there is a need for adjustment of the doses of cholecalciferol administered to this group according to their adipose mass. Baseline measurements of 25(OH)D, intact parathyroid hormone and bone remodeling markers (osteocalcin and carboxy-terminal fraction of type 1 collagen) were performed. Percent body fat was measured by dual-energy X-ray absorptiometry. The patients were divided into three groups according to their percent body fat index and were treated with cholecalciferol, 7,000 IU a week, for 12 weeks. The increases in serum levels of 25(OH)D were similar for all groups, averaging 7.46 ng/mL (P < 0.05). It is noteworthy that this increase only shifted these patients from the insufficiency category to hypovitaminosis. Peak levels of 25(OH)D were attained after only 6 weeks of treatment. This study demonstrated that adipose tissue mass does not influence the elevation of 25(OH)D levels following vitamin D supplementation, suggesting that there is no need to adjust vitamin D dose according to body fat in elderly homebound individuals.

Benthic biotope index for classifying habitats in the Sado Estuary: Portugal.

An integration of sediment physical, chemical, biological, and toxicity data is necessary for a meaningful interpretation of the complex sediment conditions in the marine environment. Assessment of benthic community is a vital component for that interpretation, yet their evaluation is complex and requires a large expenditure of time and funds. Thus, there is a need for new tools that are less expensive and more understandable for managers. This paper presents a benthic biotope index to predict from physical and chemical variables the occurrence of macrobenthic habitats. Parameters such as sediment type, organic matter, depth, and hydrodynamic parameters were selected, through a discriminant analysis, to compute the index. Other authors have used multivariate methods to determine the benthic biotopes for Sado Estuary. The index proved to be a valid tool to classify and assess the spatial patterns of benthic habitat and to synthesize stress biotope gradients.

Structure-activity relationships in the fusion of small unilamellar phosphatidylcholine vesicles induced by a model peptide.

Limited proteolysis of fatty acid-free bovine serum albumin by pepsin yields several well characterized peptides, one of which (P9, M(r) 9,000), induces fusion of small unilamellar vesicles (SUV) of phosphatidylcholine at pH 3.6. Circular dichroism (CD) of P9 solutions confirmed that the peptide undergoes a reversible transition between pH 7 and pH 3.6. The spectral changes observed with CD suggest that in the low pH conformation there is a decrease in the alpha-helical contents and an exposure of hydrophobic residues. CD and differential ultraviolet spectroscopy demonstrated that P9 binds to micelles of hexadecylphosphorylcholine and the binding produces changes in the tertiary structure of the peptide. Reduction and carboxymethylation of the two disulfide bridges of P9 produced loss of the ability to induce fusion of SUV, although the reduced peptide binds to vesicles, induces loss of entrapped marker and produces vesicle disruption. In the active form P9 exposes hydrophobic groups, one amphiphilic alpha-helix and requires the integrity of the disulfide bridge-stabilized tertiary structure.

Large unilamellar vesicles as trehalose-stabilised vehicles for vaccines: storage time and in vivo studies.

Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein from Mycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with large unilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehalose avoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrapped protein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on the liposomal membrane integrity.

Lactic acid triggers, in vitro, thiomersal to degrade protein in the presence of PLGA microspheres.

Microspheres of polymers like poly(lactic-co-glycolic acid) (PLGA) have been studied as a vehicle for controlled release vaccines. They require materials and processes that might change the protein antigenicity. Lactic acid is produced during microsphere degradation that occurs in tandem with protein liberation. In addition, most of the proteins that have been used in microencapsulation studies contain Thimerosal((R))(TM) and this can introduce another undesirable effect for their stability. We demonstrated in vitro that the thiosalycilic acid (TSA), produced after the reduction of TM by lactic acid, reduces the S-S bridge of the previously incubated diphtheric toxoid (Dtxd). This reduction is immediately followed by blocking the two -SH formed by the same TSA molecules. In the light of these conclusions it is necessary now, to reinterpret the in vitro protein degradation-stabilization data in the presence of PLGA microspheres, mainly for those proteins which contain S-S. We propose that all the PLGA microspheres microencapsulation studies and protein structural considerations should be done in the absence of TM as preservative.

Scaling behavior of diffusion and reaction processes in percolating porous media.

We investigate the diffusion-reaction behavior of two-dimensional pore networks at the critical percolation point. Our results indicate the existence of three distinct regimes of reactivity, determined by parameter xi[triple bond]D/(Kl2), where D is the molecular diffusivity of the reagent, K is its chemical reaction coefficient, and l is the length scale of the pore. First, when the diffusion transport is strongly limited by chemical reaction (i.e., D<>K), the flux of reagent reaches a saturation limit Phi(sat) that scales with the system size as Phi(sat) approximately L(alpha), with an exponent alpha approximately 1.89, corresponding to the fractal dimension of the sample-spanning cluster. We then show that the variation of flux Phi calculated for different network sizes at the second and third regimes can be adequately described in terms of the scaling relation, Phi approximately L(alpha)f(xi/L(z)), where the crossover exponent z approximately 2.69 is consistent with the predicted scaling law alpha=2betaz.

Characterization of different membrane forms of placental alkaline phosphatases.

We have extracted and purified four alkaline phosphatase forms from human term placenta. The enzymes are dependent on Mg2+ for their activity. They can be distinguished by different responses to Zn2+, vanadate and inositol derivatives.

Kinetic studies on the membrane form of intestinal alkaline phosphatase.

We have purified different membrane and soluble forms of alkaline phosphatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzyme studies. The membrane form of alkaline phosphatase extracted from bovine intestine was purified on Q-Sepharose and on L-histidyldiazobenzyl-phosphonic acid-agarose columns to remove phosphodiesterase activity. The purified enzyme had a molecular mass of 61 kDa, Km of 1208 microM, and Vmax 240 mumol pNP/min when assayed in 1 M diethanolamine, 0.5 mM MgCl2 buffer, pH 9.8, containing 10 to 2250 microM of pNPP at 37 degrees C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depend on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate (0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43% and 84%, respectively. Inositol (2 mM) and inositol-2-monophosphate (2 mM) reduced the activity by 23% and 17%. Inositol-1-monophosphate (0.5 mM) and cyclic-inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at least 30% compared to p-nitrophenylphosphate.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems.

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80 degrees C for 20 min). N-Acylation increased its ordered structure by 4% and decreased its beta-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

Effects of sediment geochemical properties on the toxicity of copper-spiked sediments to the marine amphipod Gammarus locusta.

The contribution of some sediment features, namely acid-volatile sulfide (AVS) and total organic carbon (TOC) in order to explain the toxicity of copper-spiked sediments was investigated. A 10-day assay with the marine amphipod Gammarus locusta was performed with three selected sediment types that consisted of different proportions of fine fractions (FF) and TOC (0.5% FF/1% TOC; 25% FF/2% TOC; and 75% FF/4% TOC). Copper toxic effects were observed for 0.5% FF/25% FF sediments when simultaneously extracted copper (SEM(Cu)) minus acid volatile sulfide (SEM(Cu) - AVS) were 0.3 and 3.4 micromol/g, respectively. However, no significant mortality was observed for 75% FF sediment even when the sulfide binding capacity was exceeded (1.1 micromol/g < or = SEM(Cu) - AVS < or = 8.9 micromol/g). These results indicate that mortality could not be described solely as a function of the SEM(Cu) - AVS, suggesting the presence of other binding phases in addition to AVS. Results of the normalized LC50 values to total organic carbon indicated that this component probably also interferes in the amphipod survival, although not completely. The estimated LC50 values were close (2 mg Cu/g C and 8 mg Cu/g C, respectively for 0% FF and 25% FF sediment) although there was still a fourfold difference between them. Considering that none of these sediment features by itself could fully explain copper toxicity, an attempt was made to describe G. locusta mortality as a function of simultaneous SEM(Cu) - AVS and copper concentrations normalized to TOC. Nevertheless, the limited set of data available from this study prevented an attempt to model toxicity as a function of both of these factors. These findings reinforce the need to develop integrated models that include more than one binding phase, sulfide and organic carbon, and also other compartments of the system (e.g. interstitial water) to improve current methodologies for predicting copper toxicity based on sediment geochemical features.

Conformational stability and antibody response to the 18kDa heat-shock protein formulated into different vehicles.

Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines. Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid. The biologically active conformation of the protein was preserved after chemical modification. The immune responses to the recombinant 18kDa-hsp from Mycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes. Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer. Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes. The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization. This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.

[Natural history of myocardial hypertrophy and its association with hyperinsulinism in infants of diabetic mothers]. / Estudo da história natural da hipertrofia miocárdica e sua associação com hiperinsulinismo em filhos de mães diabéticas.

PURPOSE: To test the hypothesis that myocardial hypertrophy is associated with increased serum insulin levels in children of diabetic mothers and to determine the frequency and timing of this spontaneous regression. METHODS: Seventy-two patients were studied (54 children of diabetic mothers and 18 controls). Diagnosis of myocardial hypertrophy was made by fetal echocardiography. Amniotic fluid insulin levels were obtained from a previous prenatal study on hypertrophic cardiomyopathy, since the participation of the mothers was common to both projects. RESULTS: There were 10 cases of myocardial hypertrophy among children of diabetic mothers (18.52%). Septal thickness was significantly different between the 2 groups (children of diabetic mothers and controls) in the evaluation performed at the age of 1 month (p = 0.04). Insulin levels were still increased in children of diabetic mothers until the age of 3 months. During this period insulin levels were significantly higher than those of controls (p = 0.003 and p = 0.001, at 1 and 3 months, respectively). The association between regression of ventricular septum thickness and the decrease of insulin levels occurred up to the age of 1 month. CONCLUSION: There was spontaneous regression of ventricular septum thickness in children of diabetic mothers during the first 6 months of life. The association between hyperinsulinism and hypertrophic cardiomyopathy was present up to the first month of life.

Composition and dietary effects of the fish oil from "mandi" (Pinelodus clarias).

A study was carried out to determine the chemical composition and dietary effects of the oil from "mandi" (Pimelodus clarias). Findings revealed that it had a low proportion of essential C18:2 fatty acids and a high percentage of oleic acid (20.93%), as well as polyunsaturated fatty acids with more than 18 carbon atoms. Of the long-chain fatty acids, C22:6 was present in the highest percentage (1.94%). When rats were fed a diet the lipid source of which was the oil of mandi, they showed a reduced growth rate as compared to animals receiving the control diet (corn oil). The fatty acid composition of the liver and heart of rats from the experimental group was modified. The greatest variation occurred in the percentage of C22:6 in the heart muscle, wherein a five-fold increase was observed. Reduction of growth and alteration in the polyunsaturated fatty acids levels may be due to a deficiency in C18:2-w6, or to a possible imbalance between C18:2-w6 and C18:3-w3.

[Hypereosinophilic syndrome. Clinical case]. / Síndrome hipereosinofilico. Caso clínico.

We report the case of a 16 years old girl with hypereosinophilic syndrome, presenting with fever, abdominal pain, skin lesions and peripheral neuropathy, which responded successfully to treatment with prednisone.