Signalling pathways regulating muscle mass in ageing skeletal muscle: the role of the IGF1-Akt-mTOR-FoxO pathway.

During ageing skeletal muscles undergo a process of structural and functional remodelling that leads to sarcopenia, a syndrome characterized by loss of muscle mass and force and a major cause of physical frailty. To determine the causes of sarcopenia and identify potential targets for interventions aimed at mitigating ageing-dependent muscle wasting, we focussed on the main signalling pathway known to control protein turnover in skeletal muscle, consisting of the insulin-like growth factor 1 (IGF1), the kinase Akt and its downstream effectors, the mammalian target of rapamycin (mTOR) and the transcription factor FoxO. Expression analyses at the transcript and protein level, carried out on well-characterized cohorts of young, old sedentary and old active individuals and on mice aged 200, 500 and 800 days, revealed only modest age-related differences in this pathway. Our findings suggest that during ageing there is no downregulation of IGF1/Akt pathway and that sarcopenia is not due to FoxO activation and upregulation of the proteolytic systems. A potentially interesting result was the increased phosphorylation of the ribosomal protein S6, indicative of increased activation of mTOR complex1 (mTORC1), in aged mice. This result may provide the rationale why rapamycin treatment and caloric restriction promote longevity, since both interventions blunt activation of mTORC1; however, this change was not statistically significant in humans. Finally, genetic perturbation of these pathways in old mice aimed at promoting muscle hypertrophy via Akt overexpression or preventing muscle loss through inactivation of the ubiquitin ligase atrogin1 were found to paradoxically cause muscle pathology and reduce lifespan, suggesting that drastic activation of the IGF1-Akt pathway may be counterproductive, and that sarcopenia is accelerated, not delayed, when protein degradation pathways are impaired.

Meningoencefalite por Herpesvirus bovino 5 em Minas Gerais: relato de caso clínico / Meningoencephalitis by Bovine herpesvirus 5 in Minas Gerais state: clinical case report

Relata-se um caso de meningoencefalite causada por Herpesvirus bovino 5 (BoHV-5) heritabilityem uma vaca com cinco anos de idade. O animal manifestou quadro clínico inicial de síndrome medular baixa, caracterizada por incoordenação dos membros pélvicos, sinais estes ainda não descritos para a enfermidade. Dentro de pouco tempo a doença evoluiu para síndrome cerebral, e o óbito ocorreu seis dias após o inicio dos sintomas. Na histopatologia, evidenciou-se meningoencefalite difusa, não supurada, e a confirmação do diagnóstico foi feita por reação em cadeia de polimerase e sequenciamento do segmento parcial da glicoproteína G do vírus. O trabalho confirma a presença do BoHV-5 em Minas Gerais, descreve características clínicas novas para a enfermidade e ressalta sua importância no diagnóstico diferencial das neuropatias bovinas.

A clinical case of meningoencephalitis by Bovine herpesvirus 5 (BoHV-5) in a five-year-old cow was reported. The disease began with low spinal cord signs, characterized by incoordination, and these symptoms had never been related to this illness before. Signs of a brain syndrome were observed and the cow died in six days. At the histopathology, a spread non-supurative meningoencephalitis was diagnosed, and the virus identification was made by PCR and partial sequence of the glycoprotein G. This study confirm the BoHV-5 presence in the State of Minas Gerais, Brazil, describes new clinic characteristics, and show the importance of the disease in the differentiate diagnosis with others bovine central nervous system affections.

Avaliação clínica, ultra-sonográfica e teloscópica das papilas mamárias de búfalas / Clinical, ultrasonographic, and theloscopic evaluations of the water buffaloes mammary papillae

As papilas mamárias de 23 búfalas foram avaliadas por meio de exame clínico e ultra-sonográfico. Em oito animais do mesmo grupo, as papilas mamárias foram submetidas ao exame teloscópico. Os resultados mostraram que o exame ultra-sonográfico e a teloscopia podem ser utilizados para a avaliação das papilas mamárias na espécie bubalina. As restrições ao uso desses exames, com a metodologia e os equipamentos empregados, foram: a identificação ultra-sonográfica dos ductos papilares foi precária para as papilas mais curtas, e em matrizes jovens, a resistência do ducto papilar limita o uso de telescopia axial.

All the mammart papillae from 23 water buffaloes were clinically and ultrasonographically examined, and eight animals from the same herd were submitted to theloscopic examination. The results showed that ultrasonography and theloscopy are satisfactory methods to evaluate the mammary papillae of the water buffalo. However, the ultrasonographic identification of the streak canal of the shorter papillae was poor and, it was not possible to proceed with axial theloscopy in some animals, specially the primiparous.

Prevalencia de síndrome disfórico premenstrual en población general / Prevalence of premenstrual dysphoric syndrome in general population

OBJETIVOS. Medir la prevalencia de síndrome disfórico premenstrual mediante encuesta heteroadministrada con los criterios diagnósticos de la Asociación Americana de Psiquiatría presentes en el DSM-IV. MÉTODOS. Se diseña un estudio observacional, descriptivo y transversal, con emplazamiento en la Zona Básica de Salud de Porzuna (Ciudad Real). Ámbito: Atención Primaria de salud; medio rural. Población diana: todas las mujeres de entre 18 y 50 años de edad de la Zona Básica de Salud (948); se seleccionaron 226 por muestreo aleatorio simple, con reemplazo automático y consecutivo de aquellas que rehusaron participar (8), no fueron localizables (21) o reunían criterios de exclusión: menopausia, gestación, déficit mental (11), participando finalmente en el estudio 186 mujeres. A dichas mujeres se les aplicó una encuesta heteroadministrada recabando información referente a los criterios de la APA para el diagnóstico del síndrome disfórico premenstrual durante el último año, así como datos sociodemográficos, de comorbilidad y sobre tratamiento farmacológico. RESULTADOS. El 84,33% de las encuestadas presentaba algún tipo de molestia premenstrual, siendo moderada (al menos un síntoma durante todos o la mayoría de los ciclos del último año) en el 69,73% e intensa (5 o más síntomas la mayoría de los ciclos pero sin afectar a la vida laboral, social o familiar) en el 8,11%. La prevalencia encontrada de síndrome disfórico premenstrual según criterios de la Asociación Americana de Psiquiatría es del 6,49% (n = 12; IC 95%: 2,95-10,03%). CONCLUSIONES. La prevalencia encontrada de síndrome disfórico premenstrual en nuestra población concuerda con los datos publicados en estudios internacionales (2,5-14%) y se aleja de las prevalencias publicadas en nuestro país (20,6-28,6%) con encuestas autoadministradas

OBJECTIVES. Measure the prevalence of premenstrual dysphoric syndrome (PMDS) by heteroadministered survey with diagnostic criteria of the American Association of Psychiatry (AAP) present in the DSM-IV. METHODS. An observational, descriptive and cross-sectional study was designed with location in the Basic Health Area (BHA) of Porzuna (Ciudad Real). Scope: primary health care; rural setting. Target population: all women between 18 and 50 years of age from the BHA (948). A total of 226 were selected by simple random sampling, with automatic and consecutive replacement of those who refused to participate (8). Of these, 21 could not be located or fulfilled exclusion criteria: menopause, gestation, mental retardation (11), 186 women finally participating in the study. These women were administered a heteroadministered survey, collecting information regarding the AAP criteria for the diagnosis of PMDS during the last year and sociodemographic, comorbidity and pharmacological treatment data. RESULTS. A total of 84.33% of those surveyed had some type of premenstrual discomfort, these being moderate (at least one symptom during all or most of the cycles of the last year) in 69.73% and intense (5 or more symptoms in most of the cycles but without affecting work, social or family life) in 8.11% Prevalence of PMDS found according to AAP criteria is 6.49% (n = 12; 95% CI: 2.95 - 10.03%). CONCLUSIONS. Prevalence of PMDS found in our population agrees with the data published in international studies (2.5 - 14%) and moves away from the prevalences published in our country (20.6 - 28.6%) with self-applied surveys

Desempenho de bezerros alimentados com dietas líquidas à base de leite integral ou soro de leite / Performance of calves fed on liquid diets based on whole milk and whey

Quinze bezerros da raça Holandesa, desaleitados aos 30 dias de idade, foram utilizados para avaliar os efeitos da inclusão de fontes protéicas alternativas, em substituição às proteínas do leite integral, na formulação de sucedâneos do leite. Os animais foram divididos em três grupos: o grupo controle (C) - recebeu leite em pó integral reconstituído; o grupo sucedâneo SL (soro/leite) recebeu 41,6 por cento da proteína bruta (PB) vinda do concentrado protéico de soro (CPS), 23,1 por cento do soro de leite e 35,3 por cento do leite em pó integral; e o grupo sucedâneo S (soro) recebeu 68 por cento da PB vinda do CPS e 32 por cento do soro de leite. O consumo de concentrado, feno, sal mineral e água foi mensurado diariamente, enquanto o ganho de peso foi avaliado semanalmente. Não houve diferença (P>0,05) para o consumo de concentrado, sal mineral e água entre os grupos, nos períodos avaliados. O consumo de feno e o ganho de peso foram inferiores nos animais do grupo S (P<0,05). A substituição de 100 por cento do leite integral por CPS e soro de leite influenciou negativamente o desempenho.

Sleep disorder in alien hand syndrome.

A 63-year-old right-handed woman developed an alien hand syndrome (AHS) after an acute infarction in the territory of the left anterior cerebral artery. The uncontrolled hand movements were present during the daytime and eventually disturbed sleep. Polysomnography revealed that these motor actions only appeared when the patient was awake. These awakenings emerged mostly from NREM sleep stage 2 during the first half of the night. There was no evidence of any epileptiform activity, dyssomnia or parasomnia. These movements were controlled making her wear an oven mitt during sleep. The temporal distribution of this motor activity seems to follow the progressive hyperpolarization of anterior horn neurons that occurs when sleep deepens. The accommodation of the grasp reflex in AHS probably helps control this unwanted motor activity.

Effects of transcutaneous short-term electrical stimulation on M. vastus lateralis characteristics of healthy young men.

Fifteen healthy, untrained male subjects (mean age +/- SD, 22 +/- 5 years) were used to examine the plasticity of myosin heavy chain phenotype, size, oxidative capacity and capillarization of skeletal muscle fibre types with short-term electrical stimulation (ES). Ten subjects were electro-stimulated on both quadriceps muscles with a frequency of 45-60 Hz, with 12 s of stimulation followed by 8 s of recovery for a total of 30 min per day, 3 days per week for 6 weeks. The remaining five subjects served as controls. Two vastus lateralis muscle biopsy samples were removed from each subject before (week 0) and after (week 6) ES training. A standardized exercise test on a cycle ergometer was performed by each subject before and after the experimental period and several indicators of whole-body aerobic capacity were estimated. The so-called electromyographic threshold was also determined during the tests. Muscle biopsy samples were analysed by electrophoresis, immunohistochemistry and quantitative histochemistry. Myosin heavy chain (MHC) composition, muscle fibre type distribution, fibre areas, oxidative capacity and capillaries of each fibre type were estimated. Muscular changes with ES revealed an increase of fibres expressing MHC-IIA, and a decrease of fibres expressing MHC-IIX and MHC-I, as well as an increase of the oxidative capacity and mean number of capillaries of fast-twitch (type II) fibres with minimal muscle fibre hypertrophy. These adaptations seem related to a bi-directional transformation from both MHC isoforms I and IIX towards the MHC-IIA isoform. The aerobic performance and electromyographic variables at the whole-body level were not altered by ES. These results indicate that the particular short-term ES training protocol tested in the present study induces significant adaptations in histochemical and metabolic machineries of human skeletal muscle. The results also offer new perspectives for realistic applications of ES in various clinical situations and sport training.

Coordinated changes of kinematics and muscle fibre properties with prolonged endurance training.

The objective of this study was to investigate the relationship between kinematic variables and muscle characteristics by determining (1) if heavy endurance training alters these variables and (2) if such modifications occur in a coordinated manner. Fifteen Andalusian stallions age 41-45 months were used. Five horses were used as controls and 10 horses underwent a training programme based on aerobic exercise for 8 months. Intensity of exercise was adjusted individually for each horse according to a standardised exercise test. Stride kinematic characteristics at the trot were analysed by videography and gluteus medius muscle biopsies were removed before and after the experiment. Muscle samples were analysed immunohistochemically for fibre types and fibre sizes. After training, stride frequency increased (6%, P < 0.05), whereas stride duration and stance time of the stride decreased (6 and 10% respectively, P < 0.05 in both). The percentage of type IIA fibres increased (25%, P < 0.01), whereas the percentage of type IIX fibres decreased (80%, P < 0.001). Training had no significant effect on the percentage of types IIAX and I fibres. After training, mean lesser fibre diameters of types I and IIA fibres increased (13 and 10%, respectively; P < 0.05). These results suggest that prolonged endurance training alters kinematic and muscle fibre properties in a coordinated manner, reflecting a greater stability and more efficient propulsion associated with an increase in muscle fatigue resistance and strength. This adaptive response has an impact on athletic performance.

Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth.

Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. Here, we report that the up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506.

Immunolabelling, histochemistry and in situ hybridisation in human skeletal muscle fibres to detect myosin heavy chain expression at the protein and mRNA level.

The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in vastus lateralis muscle biopsies of 15 young men (with an average age of 22 y) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry and in situ hybridisation with probes specific for MHC beta-slow, MHC-IIA and MHC-IIX. The characterisation of a large number of individual fibres was compared and correlated on a fibre-to-fibre basis. The panel of monoclonal antibodies used in the study allowed classification of human skeletal muscle fibres into 5 categories according to the MHC isoform they express at the protein level, types I, I+IIA, IIA, IIAX and IIX. Hybrid fibres coexpressing two isoforms represented a considerable proportion of the fibre composition (about 14%) and were clearly underestimated by mATPase histochemistry. For a very high percentage of fibres there was a precise correspondence between the MHC protein isoforms and mRNA transcripts. The integrated methods used demonstrate a high degree of precision of the immunohistochemical procedure used for the identification and quantification of human skeletal muscle fibre types. The monoclonal antibody S5-8H2 is particularly useful for identifying hybrid IIAX fibres. This protocol offers new prospects for muscle fibre classification in human experimental studies.

Regulatory elements governing transcription in specialized myofiber subtypes.

Skeletal myofibers of vertebrates acquire specialized metabolic and physiological properties as a consequence of developmental cues in the embryo and different patterns of contractile activity in the adult. The myoglobin gene is regulated stringently in muscle fibers, such that high myoglobin expression is observed in mitochondria-rich, oxidative myofibers (Types I and IIa) compared with glycolytic fibers (Type IIb). Using germ-line transgenesis and somatic cell gene transfer methods, we defined discrete regions of the murine and human genes encoding myoglobin that are sufficient to confer muscle- and fiber type-specific expression to reporter genes. Mutational analysis confirms the importance of A/T-rich, MEF2-binding motifs in myoglobin gene regulation, as suggested by previous studies using different experimental approaches. In addition, we demonstrated a previously unsuspected role for an intragenic E-box motif as a negative regulatory element contributing to the tightly regulated variation in myoglobin gene expression among particular myofiber subtypes.

Myosin heavy chain profile of equine gluteus medius muscle following prolonged draught-exercise training and detraining.

Fourteen 4-year old Andalusian mares were used to examine the plasticity of myosin heavy chain (MHC) composition in horse skeletal muscle with heavy draught-exercise training and detraining. Seven horses underwent a training programme based on carriage exercises for 8 months. Afterwards, they were kept in paddocks for 3 months. The remaining seven animals were used as control horses. Three gluteus medius muscle biopsies were removed at depths of 20, 40 and 60 mm from each horse before (month 0), during the training (months 3 and 8) and after detraining (month 11). Myosin heavy chain composition was analysed by electrophoresis and immunohistochemically with anti-MHC monoclonal antibodies. Fibre areas, oxidative capacity and capillaries were studied histochemically. After 8 months of training, MHC-IIX and IIX fibres decreased whereas MHC-I and type I and I + IIA fibres increased. Neither MHC-IIA nor the percentage of IIA fibres changed when the data were considered as a whole, but the proportion of MHC-IIA increased in the superficial region of the muscle after 8 months of training. Mean areas of type II fibres were not affected by training and detraining, but the cross-sectional of type I fibres increased after 3 month of training and not further increases were recorded afterward. The percentage of high-oxidative capacity fibres and the number of capillaries per mm2 increased with training. Most of these muscular adaptations reverted after detraining. These results indicate that long term draught-exercise training induces a reversible transition of MHC composition in equine muscle in the order IIX --> IIA --> I. The physiological implication of these changes is an impact on the velocity of shortening and fatigue resistance of muscle fibres.

Ras is involved in nerve-activity-dependent regulation of muscle genes.

Gene expression in skeletal muscle is regulated by the firing pattern of motor neurons, but the signalling systems involved in excitation-transcription coupling are unknown. Here, using in vivo transfection in regenerating muscle, we show that constitutively active Ras and a Ras mutant that selectively activates the MAPK(ERK) pathway are able to mimic the effects of slow motor neurons on expression of myosin genes. Conversely, the effect of slow motor neurons is inhibited by a dominant-negative Ras mutant. MAPK(ERK) activity is increased by innervation and by low-frequency electrical stimulation. These results indicate that Ras-MAPK signalling is involved in promoting nerve-activity-dependent differentiation of slow muscle fibres in vivo.

Early and long-term changes of equine skeletal muscle in response to endurance training and detraining.

Twenty-four 4-year-old Andalusian (Spanish breed) stallions were used to examine the plasticity of myosin heavy chain (MHC) phenotype and the metabolic profile in horse skeletal muscle with long-term endurance-exercise training and detraining. Sixteen horses underwent a training programme based on aerobic exercises for 8 months. Afterwards, they were kept in paddocks for 3 months. The remaining eight horses were used as controls. Three gluteus medius muscle biopsy samples were removed at depths of 20, 40 and 60 mm from each horse before (month 0), during (month 3) and after (month 8) training, and again after 3 months of detraining (month 11). MHC composition was analysed by electrophoresis and immunohistochemistry with anti-MHC monoclonal antibodies. Fibre areas, oxidative capacity and capillaries were studied histochemically. The activities of key muscle enzymes of aerobic (citrate synthase and 3-hydroxy-acyl-CoA-dehydrogenase) and anaerobic (phosphofructokinase and lactic dehydrogenase) metabolism and the intramuscular glycogen and triglyceride contents were also biochemically analysed. Early changes with training (3 months) included hypertrophy of type IIA fibres, a reduction of MHC-IIX with a concomitant increase of MHC-IIA, a rise in the number of high-oxidative fibres and in the activities of aerobic muscle enzymes and glycogen content. Long-term changes with training (8 months) were a further decline in the expression of MHC-IIX, an increase of slow MHC-I, additional increases of high-oxidative fibres, capillary density, activities of aerobic enzymes and endogenous glycogen; intramuscular lipid deposits also increased after 8 months of training whereas the activities of anaerobic enzymes declined. Most of exercise-induced alterations reverted after 3 months of detraining. These results indicate that endurance-exercise training induces a reversible transition of MHC composition in equine muscle in the order IIX-->IIA-->I, which is coordinated with changes in the metabolic properties of the muscle. Furthermore, a dose-response relationship was evident between the duration (in total) of training and the magnitude of muscle adaptations.

Analysis of myosin heavy chains at the protein level in horse skeletal muscle.

Combined methodologies of enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting, traditional myofibrillar ATPase (mATPase) histochemistry and immunocytochemistry of whole biopsied samples were used to study myosin heavy chain (MHC) isoforms in the equine gluteus medius muscle. The ELISA technique allowed the quantification of the three MHC isoforms known to be present in different horse muscles: slow (MHC-I) and two fast (termed MHC-IIA and MCH-IIX). The SDS-PAGE method resolved MHCs in three bands: MHC-I, MHC-IIX and MHC-IIA from the fastest to the slowest migrating band and a quantification by densitometry for each MHC isoform was also possible. The identity of these three MHCs was confirmed by immunoblots with specific monoclonal antibodies. Five fibre types were defined immunohistochemically according to their MHC content: I, I + IIA, IIA, the hybrid IIAX and IIX. When quantitative data obtained with the four different methodologies were combined and compared, they were consistent and, when considered together, showed significant correlation. Nevertheless, the percentage of MHC-IIA histochemically derived was underestimated, while that of MHC-IIX was overestimated in comparison with the immunocytochemical determination of these MHC isoforms. The percentage of MHC-I obtained by ELISA technique was underestimated. In short, these integrated methods for the analysis of MHCs at the protein level demonstrate that equine skeletal muscle does not express the MHC-IIB, so type II fibres have been misclassified in numerous previous studies based upon the vary traditional mATPase histochemistry. They also offer new prospects for muscle fibre typing in equine experimental studies and veterinary medicine.