34 resultados para Catalase activity, unit per protein mass
Resumo:
The first cleavage divisions and preimplantation embryonic development are supported by mRNA and proteins synthesized and stored during oogenesis. Thus, mRNA molecules of maternal origin decrease and embryonic development becomes gradually dependent on expression of genetic information derived from the embryonic genome. However, it is still unclear what the role of the sperm cell is during this phase and whether the absence of the sperm cell during the artificial oocyte activation affects subsequent embryonic development. The objective of this study was to determine, in bovine embryos, changes in cell cycle-associated transcript levels (cyclin A, cyclin B, cyclin E, CDC2, CDK2, and CDK4) after oocyte activation in the presence or absence of the sperm cell. To evaluate that, in vitro-produced (IVP) and parthenogenetically activated (PA) embryos (2-4 cells (2-4C), 8-16 cells (8-16C) and blastocysts) were evaluated by real-time PCR. There was no difference in cleavage and blastocyst rates between IVP and PA groups. Transcript level was higher in oocytes than in IVP and PA embryos. Cleaved PA embryos showed higher expression of cyclin A, cyclin B, cyclin E, and CDK2 and lower expression of CDC2 when compared with that from the IVP group. At the time of activation, all transcripts were expressed less in PA than in IVP embryos, whereas at the blastocyst stage, almost all genes were expressed at a higher level in the PA group. These results suggest that in both groups there is an initial consumption of these transcripts in the early stages of embryonic development. Furthermore, 8-16C embryos seem to synthesize more cell cycle-related genes than 2-4C embryos. However, in PA embryos, activation of the cell cycle genes seems to occur after the 8- to 16-cell stage, suggesting a failure in the activation process.
Resumo:
Introduction: The cytolysis mediated by granules is one of the most important effector functions of cytotoxic T lymphocytes and natural killer cells. Recently, three single nucleotide polymorphisms (SNPs) were identified at exons 2, 3, and 5 of the granzyme B gene, resulting in a haplotype in which three amino acids of mature protein Q48P88Y245 are changed to R48A88H245, which leads to loss of cytotoxic activity of the protein. In this study, we evaluated the frequency of these polymorphisms in Brazilian populations. Methods: We evaluated the frequency of these polymorphisms in Brazilian ethnic groups (white, Afro-Brazilian, and Asian) by sequencing these regions. Results: The allelic and genotypic frequencies of SNP 2364A/G at exon 2 in Afro-Brazilian individuals (42.3% and 17.3%) were significantly higher when compared with those in whites and Asians (p < 0.0001 and p = 0.0007, respectively). The polymorphisms 2933C/G and 4243C/T also were more frequent in Afro-Brazilians but without any significant difference regarding the other groups. The Afro-Brazilian group presented greater diversity of haplotypes, and the RAH haplotype seemed to be more frequent in this group (25%), followed by the whites (20.7%) and by the Asians (11.9%), similar to the frequency presented in the literature. Conclusions: There is a higher frequency of polymorphisms in Afro-Brazilians, and the RAH haplotype was more frequent in these individuals. We believe that further studies should aim to investigate the correlation of this haplotype with diseases related to immunity mediated by cytotoxic lymphocytes, and if this correlation is confirmed, novel treatment strategies might be elaborated.
Resumo:
Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the h5PLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors. (C) 2012 Elsevier Inc. All rights reserved.
Resumo:
This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (gamma C-33) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K+, ATP and N-4(+)center dot K-0.5 for Na+ was unaffected. Exogenous pig FXYD2 increased the V-max for stimulation of gill Na,K-ATPase activity by Na+, K+ and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na, K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity. (C) 2012 Elsevier B.V. All rights reserved.
Resumo:
Several biological and clinical studies have suggested that conjugated linoleic acid (CLA) prevents body fat accumulation and increases lean body mass. CLA is available as a concentrated dietary supplement and is purported to provide the aforementioned benefits for people who perform physical activity. This study was conducted to evaluate the effect of a CLA-supplemented diet combined with physical activity on the body composition of Wistar rats. Two groups of Wistar rats of both sexes, between 45 and 60 days old, were fed a diet containing 5.5% soybean oil (control group) or a CLA-supplemented diet (0.5% CLA and 5.0% soybean oil) (test group). Half the rats in both groups were assigned to exercise by running on a treadmill. The biochemical and anatomical body compositions were analyzed. In both groups, CLA had no effect on the dietary consumption or the weight of the liver, heart, and lungs. However, it did influence the overall weight gain of exercised male rats and the chemical and anatomical body composition in exercised and sedentary rats of both sexes. The results confirm that a CLA-supplemented diet with and without physical activity reduced body fat accumulation in rats of both sexes. However, there is no evidence of an increase in the lean body mass of the exercised rats.
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Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 mu M 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 mu M ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five mu M ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5X), perifosine (3X), and arsenic trioxide (8.5X). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019661, 1898-1912, 2012.
Resumo:
A mixed-valence complex, [Fe(III)Fe(II)L1(mu-OAc)(2)]BF4 center dot H2O, where the ligand H(2)L1 = 2-{[[3-[((bis-(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-methylbenzyl](pyridin-2-ylmethyl)amino]methyl]phenol}, has been studied with a range of techniques, and, where possible, its properties have been compared to those of the corresponding enzyme system purple acid phosphatase. The (FeFeII)-Fe-III and Fe-2(III) oxidized species were studied spectroelectrochemically. The temperature-dependent population of the S = 3/2 spin states of the heterovalent system, observed using magnetic circular dichroism, confirmed that the dinuclear center is weakly antiferromagnetically coupled (H = -2JS(1).S-2, where J = -5.6 cm(-1)) in a frozen solution. The ligand-to-metal charge-transfer transitions are correlated with density functional theory calculations. The (FeFeII)-Fe-III complex is electron paramagnetic resonance (EPR)-silent, except at very low temperatures (<2 K), because of the broadening caused by the exchange coupling and zero-field-splitting parameters being of comparable magnitude and rapid spin-lattice relaxation. However, a phosphate-bound Fe-2(III) complex showed an EPR spectrum due to population of the S-tot = 3 state (J= -3.5 cm(-1)). The phosphatase activity of the (FeFeII)-Fe-III complex in hydrolysis of bis(2,4-dinitrophenyl)phosphate (k(cat.) = 1.88 x 10(-3) s(-1); K-m = 4.63 x 10(-3) mol L-1) is similar to that of other bimetallic heterovalent complexes with the same ligand. Analysis of the kinetic data supports a mechanism where the initiating nucleophile in the phosphatase reaction is a hydroxide, terminally bound to Fe-III. It is interesting to note that aqueous solutions of [Fe(III)Fe(II)L1(mu-OAc)(2)](+) are also capable of protein cleavage, at mild temperature and pH conditions, thus further expanding the scope of this complex's catalytic promiscuity.
Resumo:
Heat shock proteins play a key regulatory role in cellular defense. To investigate the role of the inducible 70-kDa heat shock protein (HSP70) in skeletal muscle atrophy and subsequent recovery, soleus (SOL) and extensor digitorum longus (EDL) muscles from overexpressing HSP70 transgenic mice were immobilized for 7 days and subsequently released from immobilization and evaluated after 7 days. Histological analysis showed that there was a decrease in cross-sectional area of type II myofiber from EDL and types I and II myofiber from SOL muscles at 7-day immobilization in both wild-type and HSP70 mice. At 7-day recovery, EDL and SOL myofibers from HSP70 mice, but not from wild-type mice, recovered their size. Muscle tetanic contraction decreased only in SOL muscles from wild-type mice at both 7-day immobilization and 7-day recovery; however, it was unaltered in the respective groups from HSP70 mice. Although no effect in a fatigue protocol was observed among groups, we noticed a better contractile performance of EDL muscles from overexpressing HSP70 groups as compared to their matched wild-type groups. The number of NCAM positive-satellite cells reduced after immobilization and recovery in both EDL and SOL muscles from wild-type mice, but it was unchanged in the muscles from HSP70 mice. These results suggest that HSP70 improves structural and functional recovery of skeletal muscle after disuse atrophy, and this effect might be associated with preservation of satellite cell amount.
Resumo:
The purpose of this study was to examine the relationship between cardiac autonomic control derived from heart rate variability (HRV), high-sensitivity C-reactive protein (hs-CRP) and physical activity (PA) levels measured using accelerometers. A total of 80 healthy university students volunteered to participate in this study (20.56 +/- 0.82 years, 1.36 +/- 1.5 mg/L of hs-CRP). The participants were divided into groups based on tertiles of hs-CRP. Analysis of covariance adjusted to PA was used to assess group differences in HRV. Associations between hs-CRP, HRV indices and PA were analyzed using Pearson's correlation. The participants at the highest tertile of hs-CRP (tertile 3) had lower cardiac vagal modulation (SDNN, tertile 1=78.05 +/- 5.9,tertile 2=82.43 +/- 5.9,tertile 3=56.03 +/- 6.1; SD1, tertile 1=61.27 +/- 5.3, tertile 2=62.93 +/- 5.4, tertile 3=40.03 +/- 5.5). In addition, vagal indices were inversely correlated with hs-CRP but positively correlated with PA (SDNN r=-0.320, SD1 r=-0.377; SDNN r=0.304, SD1 r=0.299; P<0.05). Furthermore, the most physically active subjects had lower levels of hs-CRP and the highest levels of vagal modulation.
Resumo:
The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Para) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164I165M166 motif that is associated with a "Met-turn" structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMP alpha-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 mu g, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both a and beta-chains of the fibrinogen molecule, and it can be inhibited by EDTA. EGTA and beta-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity. Published by Elsevier Ltd.
Resumo:
Objective: Aging is characterized by alterations in body composition such as an increase in body fat and decreases in muscle mass (sarcopenia) and bone density (osteopenia). Leucine supplementation has been shown to acutely stimulate protein synthesis and to decrease body fat. However, the long-term effect of consistent leucine supplementation is not well defined. This study investigated the effect of leucine supplementation during aging. Methods: Six-month-old rats were divided into three groups: an adult group (n = 10) euthanized at 6 mo of age, a leucine group (n = 16) that received a diet supplemented with 4% leucine for 40 wk, and a control group (n = 19) that received the control diet for 40 wk. The following parameters were evaluated: body weight, food intake, chemical carcass composition, indicators of acquired chronic diseases, and indicators of protein nutritional status. Results: Body weight and fat were lower in the leucine group after 40 wk of supplementation compared with the control group but still higher than in the adult group. The lipid and glycemic profiles were equally altered in the control and leucine groups because of aging. In addition, leucine supplementation did not affect the changes in protein status parameters associated with aging, such as decreases in body and muscle protein and total serum protein. Conclusion: The results indicate that leucine supplementation attenuates body fat gain during aging but does not affect risk indicators of acquired chronic diseases. Furthermore, supplemented animals did not show signs of a prevention of the decrease in lean mass associated with aging. (C) 2012 Elsevier Inc. All rights reserved.
Resumo:
Background: The prevalence of overweight individuals has increased in recent years. Moreover, the importance of a healthy diet is associated with the practice of physical activity and attempt to verify the achievement of physical exercise influences on food choice. However, it relationship between food intake and physical activity have not been studied. Aim: To evaluate if the period in which the trainings are conducted, morning and nocturne, interfere qualitatively and quantitatively in food consumption as well as verify possible associations between anthropometric profiles and dietary habits. Methods: We collected data from 33 adult volunteers, between men and women, practitioners of bodybuilding. Results: A total of 33 volunteers were interviewed (18 (54.5%) were men and 15 (45.5%) were women). Regarding anthropometric data, it turns out that that the volunteers of the two periods had similar characteristics, differentiating only weight. The consumption of nutritional supplements was observed in 30.77% of the practitioners in the morning period vs. 35% for the nocturne. Considering macronutrient intake, there was a significant difference in the consumption of protein between the periods. The consumption during nocturne period was greater (126 +/- 5% of the daily requirement) than the morning period (115.7 +/- 2%). As for micro-nutrients, calcium intake was greater among men when compared to women. There was a positive correlation between the BMI, and arm circumference for practitioners of the morning period. Conclusion: This study show that the practitioners who train in the morning have quietly better eating habits than those in the nocturne period, however both are inappropriate.
Resumo:
Purpose: To discharge a patient from the intensive care unit (ICU) is a complex decision-making process because in-hospital mortality after critical illness may be as high as up to 27%. Static C-reactive protein (CRP) values have been previously evaluated as a predictor of post-ICU mortality with conflicting results. Therefore, we evaluated the CRP ratio in the last 24 hours before ICU discharge as a predictor of in-hospital outcomes. Methods: A retrospective cohort study was performed in 409 patients from a 6-bed ICU of a university hospital. Data were prospectively collected during a 4-year period. Only patients discharged alive from the ICU with at least 72 hours of ICU length of stay were evaluated. Results: In-hospital mortality was 18.3% (75/409). Patients with reduction less than 25% in CRP concentrations at 24 hours as compared with 48 hours before ICU discharge had a worse prognosis, with increased mortality (23% vs 11%, P = .002) and post-ICU length of stay (26 [7-43] vs 11 [5-27] days, P = .036). Moreover, among hospital survivors (n = 334), patients with CRP reduction less than 25% were discharged later (hazard ratio, 0.750; 95% confidence interval, 0.602-0.935; P = .011). Conclusions: In this large cohort of critically ill patients, failure to reduce CRP values more than 25% in the last 24 hours of ICU stay is a strong predictor of worse in-hospital outcomes. (C) 2012 Elsevier Inc. All rights reserved.
New Protein Sources in Adults Diet for Mass-rearing of Anastrepha fraterculus (Diptera: Tephritidae)
Resumo:
The aim of this study was to find alternatives to reduce the cost of mass production of the South American fruit fly (A. fraterculus) by looking for locally available products as protein source in the diet of adults to replace the imported product without changing the quality parameters. Two yeast from a Brazilian company were evaluated. The quality parameters showed that the imported hydrolyzed yeast used in the adult diet could be perfectly replaced by the local products tested, with a reduction of over 80% of the cost of the diet. The quality of the produced insects remained the same and there were improvements in some quality parameters such as the volume of eggs produced, number of adults flying and longevity under the stress.
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Lasiodiplodan, an exopolysaccharide of the (1 -> 6)-beta-d-glucan type, is produced by Lasiodiplodia theobromae MMPI when grown under submerged culture on glucose. The objective of this study was to evaluate lasiodiplodan production by examining the effects of carbon (glucose, fructose, maltose, sucrose) and nitrogen sources (KNO3, (NH4)(2)SO4, urea, yeast extract, peptone), its production in shake flasks compared to a stirred-tank bioreactor, and to study the rheology of lasiodiplodan, and lasiodiplodan's anti-proliferative effect on breast cancer MCF-7 cells. Although glucose (2.05 +/- A 0.05 g L-1), maltose (2.08 +/- A 0.04 g L-1) and yeast extract (2.46 +/- A 0.06 g L-1) produced the highest amounts of lasiodiplodan, urea as N source resulted in more lasiodiplodan per unit biomass than yeast extract (0.74 +/- A 0.006 vs. 0.22 +/- A 0.008 g g(-1)). A comparison of the fermentative parameters of L. theobromae MMPI in shake flasks and a stirred-tank bioreactor at 120 h on glucose as carbon source showed maximum lasiodiplodan production in agitated flasks (7.01 +/- A 0.07 g L-1) with a specific yield of 0.25 +/- A 0.57 g g(-1) and a volumetric productivity of 0.06 +/- A 0.001 g L-1 h(-1). A factorial 2(2) statistical design developed to evaluate the effect of glucose concentration (20-60 g L-1) and impeller speed (100-200 rpm) on lasiodiplodan production in the bioreactor showed the highest production (6.32 g L-1) at 72 h. Lasiodiplodan presented pseudoplastic behaviour, and the apparent viscosity increased at 60A degrees C in the presence of CaCl2. Anti-proliferative activity of lasiodiplodan was demonstrated in MCF-7 cells, which was time- and dose-dependent with an IC50 of 100 mu g lasiodiplodan mL(-1).