Clinical, hematological, total plasma protein and fibrinogen parameters of magellanic penguins ( Spheniscus magellanicus ) pre- and post-rehabilitation

Abstract: Magellanic penguins (Spheniscus magellanicus) usually arrive in poor body conditions at Brazilian beaches during the winter. Hematology provides valuable information about clinical and immunity status of the animals. The aims of this study were to determine the hematologic, total plasma protein (TPP) and fibrinogen profiles of young and adult magellanic penguins in PROAMAR and CETAS-SC, relating these results with the state of health and survival possibility of the animals. In Paraná 14 animals were evaluated in pre and eight in post-rehabilitation and 29 animals were evaluated in Santa Catarina after rehabilitation. Before rehabilitation, all animals showed weakness. In hematological exams of these animals, we found that anemia was present in 83% of the penguins that died and 50% of those which survived. The heterophils/lymphocytes (H/L) ratio was 3.87±0.57 in animals that died, significantly higher than the average of 2.20±0.30 for animals that survived. These two parameters are useful to assess the survival possibility of animals to rehabilitation. The body condition score was positively correlated with hematocrit and TPP, and negatively correlated with H/L ratio. After rehabilitation, the values were similar to other animals of the family Spheniscidae, with averages ranging from 1.64 to 1.90x106 erythrocytes/μL; 43.38 to 48.80% of hematocrit; 12.45 to 13.52g/dL of hemoglobin; 8,684 to 14,011 leukocytes/μL; 4,767 to 8,041 heterophils/μL; 3,215 to 4,951 lymphocytes/μL; 95 to 655 eosinophils/μl; 179.8 to 277.9 monocytes/μL; 141 to 184.9 basophils/μL; and 1.26 to 1.74 of H/L ratio. These parameters can therefore be used as reference values and release parameters for young and adult Magellanic penguins in captivity on the rehabilitation centers.

Retrospective canine skin peripheral nerve sheath tumors data with emphasis on histologic, immunohistochemical and prognostic factors

Abstract: In this retrospective study was determined the frequency of canine skin peripheral nerve sheath tumors (PNST) in cases diagnosed by the Setor de Patologia Veterinária of the Universidade Federal do Rio Grande do Sul (SPV-UFRGS), Brazil, between the years 2000 and 2012. The canine profiles, as well as histological, immunohistochemical and prognostic aspects of the tumors were based on 70 samples, comprising 40 females, 29 males and one unspecified sample. Between 2000 and 2012, 2,984 skin tumors of dogs were diagnosed in the SPV-UFRGS, totaling 2.34% of skin neoplasms in dogs. Animals that comprised the largest amount of samples (43%) were those with no breed (SRD), followed by German Shepherds (10%). Females were more affected than males (40/70 - 57% and 29/70 - 41% respectively). Skin PNST of this research showed predominant localization on the limbs (40% in the forelimbs and 29% in the hindlimbs); affecting adult dogs, mostly aged between 8 and 11 years (54%). The samples were routinely processed for hematoxylin and eosin, and were also evaluated by toluidine blue and Masson's trichrome staining, and immunohistochemistry (IHC) anti-vimentin, -S-100, -GFAP, -actin, von Willebrand factor and neurofilament. Anisocytosis and anisokaryosis, mitotic index, intratumoral necrosis, invasion of adjacent tissues, tumor location, local recurrence and metastasis were related to the diagnosis of benign (49/70) or malignant tumor (21/70). The Antoni A histological pattern was observed more frequently in benign tumors. The immunohistochemistry helped to diagnose PNST, and anti-vimentin and anti-protein S-100 showed the highest rates of immunostaining. Throughout statistical analysis of animals with tumor recurrence, it was found that the chance of an animal with a malignant peripheral nerve sheath tumor to develop recurrence is 4.61 times higher than in an animal that had a benign tumor.

Effect of nitrate and ammonium on the growth and protein concentration of Microcystis viridis Lemmermann (Cyanobacteria)

Cyanobacteria are a very important group in aquatic systems, particularly in eutrophic waters. Therefore studies about their success in the environment are essential. Many hypotheses have tried to explain the dominance of Cyanobacteria, and several emphasized the importance of various nitrogen sources for the success of the group. In this study, we measured the effect of ammonium and nitrate on the growth and protein concentration of Microcystis viridis (Cyanobacteria). This species is well-known because bloom formation in eutrophic waters. The study was carried out, in experimental batch cultures, using the WC medium with different nitrogen sources: ammonium, nitrate, ammonium + nitrate (50% ammonium + 50% nitrate) and ammonium at different concentrations (to test for possible NH4+ toxicity). Protein, ammonium and nitrate concentrations were measured at end of each experiment, whereas samples for cell counts were taken daily. Results showed that Microcystis viridis grew faster with ammonium (µ = 0.393 day-1) than with nitrate (µ = 0.263 day-1) and ammonium + nitrate (µ = 0.325 day-1). This pattern is explained by the metabolism of ammonium that presents higher uptake and assimilation rates than nitrate. Maximum cell concentration, however, was higher in the ammonium + nitrate treatment, followed by nitrate treatment. Higher protein concentration were observed in the treatment with nitrate. In the ammonium toxicity test, no difference between the control and NH4+ at 50% was found. Thus, the ammonium concentrations used in these experiments were not toxic. Our results suggest that Cyanobacteria is able to grow on both nitrogen sources even if ammonium may allow faster growth and bloom development.

E. coli a-hemolysin: a membrane-active protein toxin

Alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation. A second activation step takes place in the extracellular medium through binding of Ca2+ ions. Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation. However, Ca2+-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes. The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can a-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues. Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension. Protein or glycoprotein receptors for a-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.

Effects of pregnancy and protein-energy malnutrition on monooxygenase O-dealkylation activity in rat liver microsomes

Xenobiotic metabolism is influenced by a variety of physiological and environmental factors including pregnancy and nutritional status of the individual. Pregnancy has generally been reported to cause a depression of hepatic monooxygenase activities. Low-protein diets and protein-energy malnutrition have also been associated with a reduced activity of monooxygenases in nonpregnant animals. We investigated the combined effects of pregnancy and protein-energy malnutrition on liver monooxygenase O-dealkylation activity. On pregnancy day 0 rats were assigned at random to a group fed ad libitum (well-nourished, WN) or to a malnourished group (MN) which received half of the WN food intake (12 g/day). WN and MN rats were killed on days 0 (nonpregnant), 11 or 20 of pregnancy and ethoxy- (EROD), methoxy- (MROD) and penthoxy- (PROD) resorufin O-dealkylation activities were measured in liver microsomes. Only minor changes in enzyme activities were observed on pregnancy day 11, but a clear-cut reduction of monooxygenase activities (pmol resorufin min-1 mg protein-1) was noted near term (day 0 vs 20, means ± SD, Student t-test, P<0.05) in WN (EROD: 78.9 ± 15.1 vs 54.6 ± 10.2; MROD: 67.8 ± 10.0 vs 40.9 ± 7.2; PROD: 6.6 ± 0.9 vs 4.3 ± 0.8) and in MN (EROD: 89.2 ± 23.9 vs 46.9 ± 15.0; MROD: 66.8 ± 13.8 vs 27.9 ± 4.4; PROD: 6.3 ± 1.0 vs 4.1 ± 0.6) dams. On pregnancy day 20 MROD was lower in MN than in WN dams. Malnutrition did not increase the pregnancy-induced reduction of EROD and PROD activities. Thus, the present results suggest that the activities of liver monooxygenases are reduced in near-term pregnancy and that protein-energy malnutrition does not alter EROD or PROD in pregnant rats.

Activity and functional interaction of alternative oxidase and uncoupling protein in mitochondria from tomato fruit

Cyanide-resistant alternative oxidase (AOX) is not limited to plant mitochondria and is widespread among several types of protists. The uncoupling protein (UCP) is much more widespread than previously believed, not only in tissues of higher animals but also in plants and in an amoeboid protozoan. The redox energy-dissipating pathway (AOX) and the proton electrochemical gradient energy-dissipating pathway (UCP) lead to the same final effect, i.e., a decrease in ATP synthesis and an increase in heat production. Studies with green tomato fruit mitochondria show that both proteins are present simultaneously in the membrane. This raises the question of a specific physiological role for each energy-dissipating system and of a possible functional connection between them (shared regulation). Linoleic acid, an abundant free fatty acid in plants which activates UCP, strongly inhibits cyanide-resistant respiration mediated by AOX. Moreover, studies of the evolution of AOX and UCP protein expression and of their activities during post-harvest ripening of tomato fruit show that AOX and plant UCP work sequentially: AOX activity decreases in early post-growing stages and UCP activity is decreased in late ripening stages. Electron partitioning between the alternative oxidase and the cytochrome pathway as well as H+ gradient partitioning between ATP synthase and UCP can be evaluated by the ADP/O method. This method facilitates description of the kinetics of energy-dissipating pathways and of ATP synthase when state 3 respiration is decreased by limitation of oxidizable substrate.

Can organic and transgenic soy be used as a substitute for animal protein by rats?

We evaluated the protein quality of organic and transgenic soy fed to rats throughout life. Thirty female Wistar rats were divided into three groups (N = 10): organic soy group (OSG) receiving organic soy-based diet, genetically modified soy group (GMSG) receiving transgenic soy-based diet, and a control group (CG) receiving casein-based diet. All animals received water and isocaloric diet (10% protein), ad libitum for 291 days. After this, the weight of GMSG animals (290.9 ± 9.1 g) was significantly lower (P <= 0.04) than CG (323.2 ± 7.9 g). The weight of OSG (302.2 ± 8.7 g) was between that of the GMSG and the CG. Protein intake was similar for OSG (308.4 ± 6.8 g) and GMSG (301.5 ± 2.5 g), and significantly lower (P <= 0.0005) than the CG (358.4 ± 8.1 g). Growth rate was similar for all groups: OSG (0.80 ± 0.02 g), GMSG (0.81 ± 0.03 g) and CG (0.75 ± 0.02 g). In addition to providing a good protein intake and inducing less weight gain, both types of soy were utilized in a manner similar to that of casein, suggesting that the protein quality of soy is similar to that of the standard protein casein. The groups fed soy-based diet gained less weight, which may be considered to be beneficial for health. We conclude that organic and transgenic soy can be fed throughout life to rats in place of animal protein, because contain high quality protein and do not cause a marked increase in body weight.

Stabilization of partially folded states in protein folding/misfolding transitions by hydrostatic pressure

In the last few years, hydrostatic pressure has been extensively used in the study of both protein folding and misfolding/aggregation. Compared to other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, which allow the stabilization of partially folded intermediate states that are usually not significantly populated under more drastic conditions (e.g., in the presence of chemical denaturants or at high temperatures). Much of the recent research in the field of protein folding has focused on the characterization of folding intermediates since these species appear to be involved in a variety of disease-causing protein misfolding and aggregation events. The exact mechanisms of these biologicalphenomena, however, are still poorly understood. Here, we review recent examples of the use of hydrostatic pressure as a tool to obtain insight into the forces and energetics governing the productive folding or the misfolding and aggregation of proteins.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-α-induced myocardial hypertrophy

We investigated whether Ca2+/calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor α (TNF-α). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-α (100 μg/L), and Ca2+ signal transduction was blocked by several antagonists, including BAPTA (4 µM), KN-93 (0.2 µM) and cyclosporin A (CsA, 0.2 µM). Protein content, protein synthesis, cardiomyocyte volumes, [Ca2+]i transients, CaMKIIδB and CaN were evaluated by the Lowry method, [³H]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-α induced a significant increase in protein content in a dose-dependent manner from 10 µg/L (53.56 µg protein/well) to 100 μg/L (72.18 µg protein/well), and in a time-dependent manner from 12 h (37.42 µg protein/well) to 72 h (42.81 µg protein/well). TNF-α (100 μg/L) significantly increased the amplitude of spontaneous [Ca2+]i transients, the total protein content, cell size, and [³H]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 µM BAPTA, an intracellular Ca2+ chelator. The increases in protein content, cell size and [³H]-leucine incorporation were abolished by 0.2 µM KN-93 or 0.2 µM CsA. TNF-α increased the expression of CaMKIIδB by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 µM BAPTA, which itself had no effect. These results suggest that TNF-α induces increases in [Ca2+]i, CaMKIIδB and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca2+/CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-α.

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

Correlation of cadherin-17 protein expression with clinicopathological features and prognosis of patients with sporadic gastric cancer

This study aimed to explore the correlations between cadherin-17 (CDH17) protein expression and the clinicopathological features and prognosis of patients with sporadic gastric cancer (GC). Nine relevant studies of 1,960 patients were identified using electronic database searches supplemented with a manual search in strict accordance with inclusion and exclusion criteria. Statistical analyses were conducted using STATA 12.0 statistical software. Relative risks and 95% confidence intervals were determined, and Z test was used to measure the significance of the overall effect size. A total of nine eligible cohort studies were included in this meta-analysis. The expression of CDH17 in patients with diffuse GC was significantly higher than in those with intestinal-type GC. Moreover, the tumor depth of invasion differed significantly between patients with positive CDH17 (CDH17+) and negative CDH17 (CDH17-) GC. However, there were no significant differences between CDH17+ and CDH17- GC patients with respect to tumor node metastasis clinical stages, histological grades, or lymph node metastasis. Despite the differences in invasive depth, there was no significant difference in 5-year survival rates between CDH17+ and CDH17- GC patients. Our meta-analysis provides evidence that CDH17 protein expression may be associated with the development of GC, suggesting that CDH17 is an important biomarker that could be useful for the early diagnosis of GC. However, CDH17 levels do not appear to impact overall survival.

Effect of wheat flour protein variations on sensory attributes, texture and staling of Taftoon bread

The quality of flat breads depends in part on the textural properties of breads during storage. These properties are largely affected by flour protein quality and quantity. The present study aimed to examine differences between sensory properties, textural and staling of Tandoori breads made from flours of different quality and different quantities of protein. This was implemented by using three flours with 9.4, 11.5 and 13.5% protein contents and different protein qualities shown by Zeleney sedimentation volume 16.25, 22.75 and 23.25 mL respectively. Bread strips were submitted to uniaxial compression between two parallel plates on an Instron Universal Testing machine, and firmness of the breads was determined. Results indicated the differences in the sensory attributes of breads produced by flours of different protein content and quality, demonstrating that high protein high quality flours are not able to sheet and expand under the high temperature - short time conditions employed in Taftoon bread production and are therefore not suitable for this kind of bread. Results showed that flour with 11.5% protein content, produced bread with better sensory characteristics and acceptable storage time.