Determinação eletroquímica da capacidade antioxidante para avaliação do exercício físico

Physical training can adapt or cause injury to skeletal muscles implicating metabolic alterations, which can be detected by biochemical analysis. Apparently the increase in the production of reactive oxygen species (ROS) is involved in both processes. Enzymatic and low molecular weight antioxidants (LMWA) minimize ROS's deleterious action through redox reactions. Cyclic voltammetry (CV) has been suggested as a tool to quantify the antioxidant capacity conferred by LMWA. The use of CV to evaluate the modulation of the antioxidant capacity conferred by LMWA in response to physical exercise is discussed here.

Experimental intoxication by the leaves of Melia azedarach (Meliaceae) in cattle

Green leaves of Melia azedarach were administered at single doses ranging from 5 to 30 g/kg bw to 11 calves. Clinical signs were depression, ruminal stasis, dry feces with blood, ataxia, muscle tremors, sternal recumbency, hypothermia and abdominal pain. Serum AST and CPK were increased. Signs appeared from 8 to 24 hours after dosing, and the clinical course lasted from 2 to 72 hours. Three calves dosed with 30g/kg bw died. The macroscopic findings included intestinal congestion, yellow discoloration of the liver, brain congestion and dry feces with blood in the rectum. The liver showed swollen and vacuolated hepatocytes. Necrotic hepatocytes were scattered throughout the parenchyma or concentrated in the periacinar zone. Degenerative and necrotic changes were observed in the epithelium of the forestomachs. There was also necrosis of the lymphoid tissue. Skeletal muscles showed hyaline degeneration and fiber necrosis. The necrotic fragments contained floccular or granular debris with infiltration by macrophages and satellite cells.

Novel Functions of ErbB4 and NRG-1 in Development and Cancer

Cells communicate, or signal, with each other constantly to ensure proper functioning of tissues and organs. Cell signaling is often performed by interplay of receptors and ligands that bind these receptors. ErbB receptors (epidermal growth factor receptors, EGFR, HER) bind extracellular growth factors and transduce these signals inside of cells. ErbB dysfunction promotes carcinogenesis, and also results in numerous defects during normal development. This study focused on the functions of one member of the ErbB receptor family, ErbB4, and growth factor, neuregulin-1 (NRG-1), that can bind and activate ErbB4. This study aimed to find novel functions of ErbB4 and NRG-1. Hypoxia, or deficiency of oxygen, is common in cancer and ischemic conditions. One of the key findings of the work was the identification and characterization of a cross-talk between ErbB4 and Hypoxia-inducible factor 1α (HIF-1α), the central mediator of hypoxia signaling. ErbB4 activation by NRG-1 was found to increase HIF-1α activity. Interestingly, this regulation occurred in reciprocal manner as HIF-1α was also able to increase protein levels of NRG-1 and ErbB4. Moreover, expression of NRG-1 and ErbB4 was associated with HIF activity in vivo in human clinical samples and in mice. Reduction of functional ErbB4 in developing zebrafish embryos resulted in defects in development of the skeletal muscles. To study ErbB4 functions in pathological situation in humans, clinical samples of serous ovarian carcinoma were analyzed using tissue microarrays and real-time RT-PCR. A specific isoform of ErbB4, CYT-1, was associated with poor survival in serous ovarian cancer and increased anchorage independent growth of ovarian cancer cells in vitro. These observations demonstrate that ErbB4 and NRG-1 are essential regulators of cellular response to hypoxia, of development, and of ovarian carcinogenesis.

Accidental and experimental salinomycin poisoning in rabbits

An outbreak of salinomycin poisoning in rabbits is described. At least 27 out of 2,000 rabbits reared on a farm died after the coccidiostatic drug sulfaquinoxaline was substituted by salinomycin in the feed. An average of 26.9ppm salinomycin was detected in the ration given to the rabbits. Clinical signs included anorexia, apathy and bradykinesia, which progressed to incoordination and recumbency. Gross lesions consisted of pale areas in the skeletal muscles. The histopathological findings showed severe necrotic degenerative myopathy in association with infiltration of neutrophils and macrophages. One rabbit exhibited similar alterations in the myocardium. Mineralization was observed in the affected skeletal muscles in some cases. In order to verify if the poisoning was due to salinomycin, 20 rabbits were divided into five groups and a ration containing the drug at doses of 10, 25, 50, 75 and 100ppm was given. The administration of doses higher than 50ppm resulted in manifestation of the clinical signs seen in the outbreak of poisoning. It was concluded, that probably an error related to the mixture of salinomycin in the feed was the cause of deaths in the spontaneous outbreak of poisoning on the rabbit farm.

Spontaneous coffee senna poisoning in cattle: report on 16 outbreaks

Sixteen outbreaks of Senna occidentalis (coffee senna) that occurred in cattle in the state of Rio Grande do Sul, Brazil, were reviewed. The great majority (75%) of the outbreaks occurred in adult cattle at pasture during the autumn and winter months with 50% in May, evidencing a striking seasonality. Mortality rates varied from 4.2% to 55.2% and cattle died 2 days up to 2 weeks after showing clinical signs that included dry feces (occasionally diarrhea), muscle weakness, reluctance to move, tachypnea, instability of the hind limbs with dragging of the toes, tremors in muscles of the thighs, neck, and head, ear dropping, sternal recumbency, lateral recumbency and death. Myoglobinuria characterized by a dark red or black discolored urine was a consistent finding in cattle affected at pasture but not in those poisoned by ration contaminated with coffee senna beans. Creatine phosphokinase serum activity was marked ly elevated. Main gross changes observed in 23 necropsies involved skeletal muscles of the hind limbs. These changes consisted of varying degrees of paleness of muscle groups. Subepicardial and subendocardial hemorrhages were present in the hearts of all affected cattle. Histologically a segmental degenerative myopathy of striated muscles was present in every case and had a multifocal polyphasic or monophasic character. Myocardial (3/23), hepatic (3/13), renal (3/10), and splenic (1/6) microscopic lesions were observed occasionally. Myocardial lesions were mild and consisted of vacuolation of cardiomyocytes or focal fibrosis. Hepatic changes consisted of diffuse hepatocelular vacuolation, cytosegrosomes within hepatocytes, and individual hepatocellular necrosis. Kidneys had vacuolar degeneration of tubular epithelium associated with acidophilic casts (proteinosis) within tubular lumina. In the spleen there was marked necrosis of lymphocytes of the white pulp. No histological changes were found in the brains of 13 affected cattle. The data of this study suggest that coffee senna poisoning is an important cause of death in cattle in southern Brazil.

Two research paths for probing the roles of oxygen in metabolic regulation

Tissues such as skeletal and cardiac muscles must sustain very large-scale changes in ATP turnover rate during equally large changes in work. In many skeletal muscles these changes can exceed 100-fold. Examination of a number of cell and whole-organism level systems identifies ATP concentration as a key parameter of the interior milieu that is nearly universally 'homeostatic'; it is common to observe no change in ATP concentration even while change in its turnover rate can increase or decrease by two orders of magnitude or more. A large number of other intermediates of cellular metabolism are also regulated within narrow concentration ranges, but none seemingly as precisely as is [ATP]. In fact, the only other metabolite in aerobic energy metabolism that is seemingly as 'homeostatic' is oxygen - at least in working muscles where myoglobin serves to buffer oxygen concentrations at stable and constant values at work rates up to the aerobic maximum. In contrast to intracellular oxygen concentration, a 1:1 relationship between oxygen delivery and metabolic rate is observed over biologically realistic and large-magnitude changes in work. The central regulatory question is how the oxygen delivery signal is transmitted to the intracellular metabolic machinery. Traditional explanations assume diffusion as the dominant mechanism, while proponents of an ultrastructurally dominated view of the cell assume an intracellular perfusion system to account for the data which have been most perplexing to metabolic biochemistry so far: the striking lack of correlation between changes in pathway reaction rates and changes in concentrations of pathway substrates, including oxygen and pathway intermediates.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 ± 70 vs 1451 ± 235 × 10² neutrophils/mg; P<0.01) and in the kidneys (526 ± 89 vs 852 ± 73 × 10² neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.

Quantitative evidence for neurofilament heavy subunit aggregation in motor neurons of spinal cords of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of unknown etiology, affects motor neurons leading to atrophy of skeletal muscles, paralysis and death. There is evidence for the accumulation of neurofilaments (NF) in motor neurons of the spinal cord in ALS cases. NF are major structural elements of the neuronal cytoskeleton. They play an important role in cell architecture and differentiation and in the determination and maintenance of fiber caliber. They are composed of three different polypeptides: light (NF-L), medium (NF-M) and heavy (NF-H) subunits. In the present study, we performed a morphological and quantitative immunohistochemical analysis to evaluate the accumulation of NF and the presence of each subunit in control and ALS cases. Spinal cords from patients without neurological disease and from ALS patients were obtained at autopsy. In all ALS cases there was a marked loss of motor neurons, besides atrophic neurons and preserved neurons with cytoplasmic inclusions, and extensive gliosis. In control cases, the immunoreaction in the cytoplasm of neurons was weak for phosphorylated NF-H, strong for NF-M and weak for NF-L. In ALS cases, anterior horn neurons showed intense immunoreactivity in focal regions of neuronal perikarya for all subunits, although the difference in the integrated optical density was statistically significant only for NF-H. Furthermore, we also observed dilated axons (spheroids), which were immunopositive for NF-H but negative for NF-M and NF-L. In conclusion, we present qualitative and quantitative evidence of NF-H subunit accumulation in neuronal perikarya and spheroids, which suggests a possible role of this subunit in the pathogenesis of ALS.

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

Erythropoietin reduces the expression of myostatin in mdx dystrophic mice

Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16) and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle.

Identification des bases génétiques des myopathies à multi-minicores avec ou sans cardiomyopathie

Thèse réalisée en cotutelle avec l'Université Pierre et Marie Curie, Paris 6(UPMC, Paris, France).

Identificación y caracterización de las mutaciones en el gen GAA en pacientes colombianos afectados por la enfermedad de Pompe

La Enfermedad de Pompe (EP) es un desorden metabólico caracterizado por la deficiencia de alfa-glucosidasa acida (GAA), una enzima que cataliza la hidrolisis de las uniones glucosidicas α-1.4 y α-1,6 de glucógeno. Esta deficiencia resulta en acumulación de glucógeno en todos los tejidos, especialmente en musculo esquelético. Los pacientes con EP muestran un gran espectro de fenotipos con respecto a la edad de inicio, progresión de la enfermedad, severidad y tasa de progresión a muerte. El curso clínico de la enfermedad es principalmente determinado por la naturaleza de las variaciones genéticas de GAA quellevan a diferentes grados de deficiencia enzimática. Hasta la fecha alrededor de 400 distintas variaciones en la secuencia de GAA han sido descritas, y en algunos casos la correlación genotipo-fenotipo no es claramente evidente. En este estudio se describe el primer análisis genético y clínico de pacientes colombianos con EP en 13 individuos afectados. La secuenciación directa del gen GAA, revelo ocho distintas mutaciones relacionadas con la etiología de EP incluyendo dos nuevas mutaciones missense c.1106T>C (p.Leu369Pro) y c.2236T >C (p. Trp746Arg). Estudios funcionales in vitro mostraron que los cambios estructurales conferidos por ambas mutaciones no inhiben la síntesis del precursor de GAA de 110 KD pero afectan el procesamiento y el transporte intra-celular de la proteína.

Relação das actividades desportivas não lectivas e a aptidão física nos alunos do Ensino Secundário (estudo efectuado em 4 escolas da rede de Estágios da ULHT 2010/211)

RESUMO: Actividade Física, é qualquer movimento corporal produzido pelos músculos esqueléticos que resultam em energia despendida, acima do nível de repouso (Caspersen et al1985; Baranowski et al 1992). Um indivíduo sedentário que comece uma Actividade Física regular desencadeia um conjunto de adaptações ao esforço, em que algumas destas adaptações serão benéficas em termos de saúde, ajudando na prevenção inicial de várias doenças (Haskell et al 1965; Barata et al 1997). A Aptidão Física é um conjunto de atributos que as pessoas têm ou alcançam, que estão relacionados com os movimentos que as pessoas desempenham (Caspersen et al 1985; Baranowski et al 1992). O objectivo central do presente estudo consistiu em verificar se existem diferenças significativas entre os Praticantes e Não Praticantes de ADEC e a Aptidão Física dos alunos. A amostra foi constituída por 310 sujeitos, 165 rapazes e 145 raparigas pertencentes ao Ensino Secundário de 4 escolas da rede de estágios da ULHT 2010/2011. As Capacidades Físicas foram avaliadas através dos testes do Fitnessgram, a Prática de ADEC e o Índice de Actividade Física foram obtidos através do Questionário de Actividade Desportiva. Conclusões: Os alunos que praticam ADEC são mais aptos do que os alunos que não praticam ADEC. Estes Praticantes apresentam valores mais elevados na Aptidão Aeróbia e no VO2máx., quando comparados com os Não Praticantes. Nas restantes Capacidades Físicas analisadas (Força de Braços, Força Abdominal e Flexibilidade) não foram encontradas diferenças significativas. Não foram encontradas diferenças significativas entre a Prática de ADEC e o IMC. Os Praticantes de ADEC demonstraram ter um IAF superior aos Não Praticantes de ADEC. O Género Feminino apresenta melhores resultados do que o Género Masculino para a Força de Braços. Nas restantes Capacidades Físicas não foram encontradas diferenças significativas entre Géneros. Quando analisado o IMC, o Género Feminino apresenta percentagens superiores de Peso Normal relativamente ao Género Masculino. Não foram encontradas diferenças significativas entre o Género e a Aptidão Física. Verificou-se que existe diferenças significativas na relação entre o Género e o IAF, o Género Masculino tem um IAF superior ao Género Feminino. Os alunos mais aptos fisicamente são os alunos com maior IAF. Não foram encontradas diferenças significativas entre as Modalidades praticadas pelos alunos e o desenvolvimento qualquer das Capacidades Físicas estudadas. ABSTRACT: Physical Activity is any body movement produced by skeletal muscles resulting in energy expenditure above the resting level (Caspersen et al1985; Baranowski et al 1992). A sedentary individual, who starts a regular Physical Activity, triggers a set of adaptations to stress, in which some of these changes will be helpful in terms of health, aiding in primary prevention of several diseases (Haskell et al 1965; Barata et al 1997). Physical Fitness is a set of attributes that people have or achieve, and are related with the movements that people perform (Caspersen et al 1985; Baranowski et al 1992). The main purpose of this study was to determine whether there are significant differences between Practicing and Non-practitioners of ADEC and Physical Fitness of students. The sample consisted of 310 subjects, 165 boys and 145 girls belonging to high school students of 4 schools integrated in the ULHT 2010/2011. The Physical Abilities were assessed through Fitnessgram tests, ADEC practice and the Index of Physical Activity were obtained through the Physical Activity Questionnaire. Conclusions: Students that practice ADEC are more able than students who do not practice ADEC. These students that practice ADEC have higher values in Aerobic Fitness and VO2max when compared to non-practicing students. In the remaining Physical Abilities analyzed (Arm Strength, Abdominal Strength and Flexibility) were not considered significant differences. No significant differences were found between ADEC practice and the BMI. The students that practice ADEC showed higher Physical Activity levels than students who do not practice. Female gender presents better results than the male gender in Arms strength. In the remaining components of Physical Fitness were not found significant differences between genders. When analyzed BMI, female gender has higher percentages of normal weight compared to male gender. No significant differences were found between Gender and Physical Fitness. It was verified that there are significant differences in the relation between gender and Physical Activity levels, the male gender have a higher level of Physical Activity than female gender. The most able students are the ones with higher levels of Physical Activity. There were no significant differences between the sports practiced by students and the development of the studied components of physical fitness.

Potranskrypcyjna regulacja ekspresji genu CELF1 - wpływ na patomechanizm dystrofii miotonicznej

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii

Discovery of a mammalian splice variant of myostatin that stimulates myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P,0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.