Differential gene expression and developmental competence in in vitro produced bovine embryos

The embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (54) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.

The role of reactive oxygen species in the modulation of the contraction induced by angiotensin II in carotid artery from diabetic rat

The modulation played by reactive oxygen species on the angiotensin II-induced contraction in type I-diabetic rat carotid was investigated. Concentration-response curves for angiotensin II were obtained in endothelium-intact or endothelium-denuded carotid from control or streptozotocin-induced diabetic rats, pre-treated with tiron (superoxide scavenger), PEG-catalase (hydrogen peroxide scavenger), dimethylthiourea (hydroxyl scavenger), apocynin [NAD(P) H oxidase inhibitor], SC560 (cyclooxygenase-1 inhibitor), SC236 (cyclooxygenase-2 inhibitor) or Y-27632 (Rho-kinase inhibitor). Reactive oxygen species were measured by flow cytometry in dihydroethidium (DHE)-loaded endothelial cells. Cyclooxygenase and AT1-receptor expression was assessed by immunohistochemistry. Diabetes increased the angiotensin II-induced contraction but reduced the agonist potency in rat carotid. Endothelium removal, tiron or apocynin restored the angiotensin II-induced contraction in diabetic rat carotid to control levels. PEG-catalase, DMTU or SC560 reduced the angiotensin II-induced contraction in diabetic rat carotid at the same extent. SC236 restored the angiotensin II potency in diabetic rat carotid. Y-27632 reduced the angiotensin II-induced contraction in endothelium-intact or -denuded diabetic rat carotid. Diabetes increased the DHE-fluorescence of carotid endothelial cells. Apocynin reduced the DHE-fluorescence of endothelial cells from diabetic rat carotid to control levels. Diabetes increased the muscular cyclooxygenase-2 expression but reduced the muscular AT1-receptor expression in rat carotid. In summary, hydroxyl radical, hydrogen peroxide and superoxide anion-derived from endothelial NAD(P) H oxidase mediate the hyperreactivity to angiotensin II in type I-diabetic rat carotid, involving the participation of cyclooxygenase-1 and Rho-kinase. Moreover, increased muscular cyclooxygenase-2 expression in type I-diabetic rat carotid seems to be related to the local reduced AT1-receptor expression and the reduced angiotensin II potency. (C) 2011 Elsevier B. V. All rights reserved.

Alterations in vasoconstrictor responses to the endothelium-derived contracting factor uridine adenosine tetraphosphate are region specific in DOCA-salt hypertensive rats

Uridine adenosine tetraphosphate (Up(4)A) has been recently identified as a novel and potent endothelium-derived contracting factor and contains both purine and pyrimidine moieties, which activate purinergic P2X and P2Y receptors. The present study was designed to compare contractile responses to Up(4)A and other nucleotides such as ATP (P2X/P2Y agonist), UTP (P2Y(2)/P2Y(4) agonist), UDP (P2Y(6) agonist), and alpha,beta-methylene ATP (P2X(1) agonist) in different vascular regions [thoracic aorta, basilar, small mesenteric, and femoral arteries] from deoxycorticosterone acetate-salt (DOCA-salt) and control rats. In DOCA-salt rats [vs. control uninephrectomized (Uni) rats]: (1) in thoracic aorta, Up(4)A-, ATP-, and UP-induced contractions were unchanged; (2) in basilar artery, Up(4)A-, ATP-, UTP- and UDP-induced contractions were increased, and expression for P2X(1), but not P2Y(2) or P2Y(6) was decreased; (3) in small mesenteric artery, Up(4)A-induced contraction was decreased and UDP-induced contraction was increased; expression of P2Y(2) and P2X(1) was decreased whereas P2Y(6) expression was increased; (4) in femoral artery, Up(4)A-. UTP-, and UDP-induced contractions were increased, but expression of P2Y(2), P2Y(6) and P2X(1) was unchanged. The alpha,beta-methylene ATP-induced contraction was bell-shaped and the maximal contraction was reached at a lower concentration in basilar and mesenteric arteries from Uni rats, compared to arteries from DOCA-salt rats. These results suggest that Up(4)A-induced contraction is heterogenously affected among various vascular beds in arterial hypertension. P2Y receptor activation may contribute to enhancement of Up(4)A-induced contraction in basilar and femoral arteries. These changes in vascular reactivity to Up(4)A may be adaptive to the vascular alterations produced by hypertension. (C) 2011 Elsevier Ltd. All rights reserved.

Prenatal and Neonatal Adaptations with a Focus on the Respiratory System

Contents Among the modifications that occur during the neonatal period, pulmonary development is the most critical. The neonate's lungs must be able to perform adequate gas exchange, which was previously accomplished by the placenta. Neonatal respiratory distress syndrome is defined as insufficient surfactant production or pulmonary structural immaturity and is specifically relevant to preterm newborns. Prenatal maternal betamethasone treatment of bitches at 55days of gestation leads to structural changes in the neonatal lung parenchyma and consequently an improvement in the preterm neonatal respiratory condition, but not to an increase in pulmonary surfactant production. Parturition represents an important challenge to neonatal adaptation, as the uterine and abdominal contractions during labour provoke intermittent hypoxia. Immediately after birth, puppies present venous mixed acidosis (low blood pH and high dioxide carbon saturation) and low but satisfactory Apgar scores. Thus, the combination of physiological hypoxia during birth and the initial effort of filling the pulmonary alveoli with oxygen results in anaerobiosis. As a neonatal adaptation follow-up, the Apgar analysis indicates a tachypnoea response after 1h of life, which leads to a shift in the blood acidbase status to metabolic acidosis. One hour is sufficient for canine neonates to achieve an ideal Apgar score; however, a haemogasometric imbalance persists. Dystocia promotes a long-lasting bradycardia effect, slows down Apgar score progression and aggravates metabolic acidosis and stress. The latest data reinforce the need to accurately intervene during canine parturition and offer adequate medical treatment to puppies that underwent a pathological labour.

The panicolytic-like effect of fluoxetine in the elevated T-maze is mediated by serotonin-induced activation of endogenous opioids in the dorsal periaqueductal grey

Serotonin (5-HT), opioids and the dorsal periaqueductal grey (DPAG) have been implicated in the pathophysiology of panic disorder. In order to study 5-HT-opioid interaction, the opioid antagonist naloxone was injected either systemically (1 mg/kg, i.p.) or intra-DPAG (0.2 mu g/0.5 mu L) to assess its interference with the effect of chronic fluoxetine (10 mg/kg, i.p., daily for 21 days) or of intra-DPAG 5-HT (8 mu g/0.5 mu L). Drug effects were measured in the one-escape task of the rat elevated T-maze, an animal model of panic. Pretreatment with systemic naloxone antagonized the lengthening of escape latency caused by chronic fluoxetine, considered a panicolytic-like effect that parallels the drug's therapeutic response in the clinics. Pretreatment with naloxone injected intra-DPAG antagonized both the panicolytic effect of chronic fluoxetine as well as that of 5-HT injected intra-DPAG. Neither the performance of the inhibitory avoidance task in the elevated T-maze, a model of generalized anxiety nor locomotion measured in a circular arena was affected by the above drug treatments. These results indicate that the panicolytic effect of fluoxetine is mediated by endogenous opioids that are activated by 5-HT in the DPAG. They also allow reconciliation between the serotonergic and opioidergic hypotheses of panic disorder pathophysiology.

The effect of hip abduction on the EMG activity of vastus medialis obliquus, vastus lateralis longus and vastus lateralis obliquus in healthy subjects

Abstract Study design Controlled laboratory study. Objectives The purposes of this paper were to investigate (d) whether vastus medialis obliquus (VMO), vastus lateralis longus (VLL) and vastus lateralis obliquus (VLO) EMG activity can be influenced by hip abduction performed by healthy subjects. Background Some clinicians contraindicate hip abduction for patellofemoral patients (with) based on the premise that hip abduction could facilitate the VLL muscle activation leading to a VLL and VMO imbalance Methods and measures Twenty-one clinically healthy subjects were involved in the study, 10 women and 11 men (aged X = 23.3 ± 2.9). The EMG signals were collected using a computerized EMG VIKING II, with 8 channels and three pairs of surface electrodes. EMG activity was obtained from MVIC knee extension at 90° of flexion in a seated position and MVIC hip abduction at 0° and 30° with patients in side-lying position with the knee in full extension. The data were normalized in the MVIC knee extension at 50° of flexion in a seated position, and were submitted to ANOVA test with subsequent application of the Bonferroni multiple comparisons analysis test. The level of significance was defined as p ≤ 0.05. Results The VLO muscle demonstrated a similar pattern to the VMO muscle showing higher EMG activity in MVIC knee extension at 90° of flexion compared with MVIC hip abduction at 0° and 30° of abduction for male (p < 0.0007) and MVIC hip abduction at 0° of abduction for female subjects (p < 0.02196). There were no statistically significant differences in the VLL EMG activity among the three sets of exercises tested. Conclusion The results showed that no selective EMG activation was observed when comparison was made between the VMO, VLL and VLO muscles while performing MVIC hip abduction at 0° and 30° of abduction and MVIC knee extension at 90° of flexion in both male and female subjects. Our findings demonstrate that hip abduction do not facilitated VLL and VLO activity in relation to the VMO, however, this study included only healthy subjects performing maximum voluntary isometric contraction contractions, therefore much remains to be discovered by future research

Plantar flexion force induced by amplitude-modulated tendon vibration and associated soleus V/F-waves as an evidence of a centrally-mediated mechanism contributing to extra torque generation in humans

Background: High-frequency trains of electrical stimulation applied over the human muscles can generate forces higher than would be expected by direct activation of motor axons, as evidenced by an unexpected relation between the stimuli and the evoked contractions, originating what has been called “extra forces”. This phenomenon has been thought to reflect nonlinear input/output neural properties such as plateau potential activation in motoneurons. However, more recent evidence has indicated that extra forces generated during electrical stimulation are mediated primarily, if not exclusively, by an intrinsic muscle property, and not from a central mechanism as previously thought. Given the inherent differences between electrical and vibratory stimuli, this study aimed to investigate: (a) whether the generation of vibration-induced muscle forces results in an unexpected relation between the stimuli and the evoked contractions (i.e. extra forces generation) and (b) whether these extra forces are accompanied by signs of a centrally-mediated mechanism or whether intrinsic muscle properties are the redominant mechanisms. Methods: Six subjects had their Achilles tendon stimulated by 100 Hz vibratory stimuli that linearly increased in amplitude (with a peak-to-peak displacement varying from 0 to 5 mm) for 10 seconds and then linearly decreased to zero for the next 10 seconds. As a measure of motoneuron excitability taken at different times during the vibratory stimulation, short-latency compound muscle action potentials (V/F-waves) were recorded in the soleus muscle in response to supramaximal nerve stimulation. Results: Plantar flexion torque and soleus V/F-wave amplitudes were increased in the second half of the stimulation in comparison with the first half. Conclusion: The present findings provide evidence that vibratory stimuli may trigger a centrally-mediated mechanism that contributes to the generation of extra torques. The vibration-induced increased motoneuron excitability (leading to increased torque generation) presumably activates spinal motoneurons following the size principle, which is a desirable feature for stimulation paradigms involved in rehabilitation programs and exercise training.

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.

Verbal and visual stimulation effects on rectus femoris and biceps femoris muscles during isometric and concentric

Abstract Background: Coactivation may be both desirable (injury prevention) or undesirable (strength measurement). In this context, different styles of muscle strength stimulus have being investigated. In this study we evaluated the effects of verbal and visual stimulation on rectus femoris and biceps femoris muscles contraction during isometric and concentric. Methods: We investigated 13 men (age =23.1 ± 3.8 years old; body mass =75.6 ± 9.1 kg; height =1.8 ± 0.07 m). We used the isokinetic dynamometer BIODEX device and an electromyographic (EMG) system. We evaluated the maximum isometric and isokinetic knee extension and flexion at 60°/s. The following conditions were evaluated: without visual nor verbal command (control); verbal command; visual command and; verbal and visual command. In relation to the concentric contraction, the volunteers performed five reciprocal and continuous contractions at 60°/s. With respect to isometric contractions it was made three contractions of five seconds for flexion and extension in a period of one minute. Results: We found that the peak torque during isometric flexion was higher in the subjects in the VVC condition (p > 0.05). In relation to muscle coactivation, the subjects presented higher values at the control condition (p > 0.05). Conclusion We suggest that this type of stimulus is effective for the lower limbs.

The renin–angiotensin system plays a major role in voiding dysfunction of ovariectomized rats

Aims: The renin–angiotensin system (RAS) plays a major role in cardiovascular diseases in postmenopausal women, but little is known about its importance to lower urinary tract symptoms. In this study we have used the model of ovariectomized (OVX) estrogen-deficient rats to investigate the role of RAS in functional and molecular alterations in the urethra and bladder. Main methods: Responses to contractile and relaxant agents in isolated urethra and bladder, as well as cystometry were evaluated in 4-month OVX Sprague–Dawley rats. Angiotensin-converting enzyme activity and Western blotting for AT1/AT2 receptors were examined. Key findings: Cystometric evaluations in OVX rats showed increases in basal pressure, capacity and micturition frequency, as well as decreased voiding pressure. Angiotensin II and phenylephrine produced greater urethral contractions in OVX compared with Sham group. Carbachol-induced bladder contractions were significantly reduced in OVX group. Relaxations of urethra and bladder to sodium nitroprusside and BAY 41-2272 were unaffected by OVX. Angiotensin-converting enzyme activity was 2.6-fold greater (p < 0.05) in urethral tissue of OVX group,whereas enzyme activity in plasma and bladder remained unchanged. Expressions of AT1 and AT2 receptors in the urethra were markedly higher in OVX group. In bladder, AT1 receptors were not detected, whereas AT2 receptor expression was unchanged between groups. 17β-Estradiol replacement (0.1 mg/kg, weekly) or losartan (30 mg/kg/day) largely attenuated most of the alterations seen in OVX group. Significance: Prolonged estrogen deprivation leads to voiding dysfunction and urethral hypercontractility that are associated with increased ACE activity and up-regulation of angiotensin AT1/AT2 receptor in the urethral tissue.

Convective oxygen transport and fatigue

[EN] During exercise, fatigue is defined as a reversible reduction in force- or power-generating capacity and can be elicited by "central" and/or "peripheral" mechanisms. During skeletal muscle contractions, both aspects of fatigue may develop independent of alterations in convective O(2) delivery; however, reductions in O(2) supply exacerbate and increases attenuate the rate of accumulation. In this regard, peripheral fatigue development is mediated via the O(2)-dependent rate of accumulation of metabolic by-products (e.g., inorganic phosphate) and their interference with excitation-contraction coupling within the myocyte. In contrast, the development of O(2)-dependent central fatigue is elicited 1) by interference with the development of central command and/or 2) via inhibitory feedback on central motor drive secondary to the peripheral effects of low convective O(2) transport. Changes in convective O(2) delivery in the healthy human can result from modifications in arterial O(2) content, blood flow, or a combination of both, and they can be induced via heavy exercise even at sea level; these changes are exacerbated during acute and chronic exposure to altitude. This review focuses on the effects of changes in convective O(2) delivery on the development of central and peripheral fatigue.

Contractive-like mapping principles in ordered metric spaces and application to ordinary differential equations

[EN] The purpose of this paper is to present a fixed point theorem for generalized contractions in partially ordered complete metric spaces. We also present an application to first-order ordinary differential equations.

On some Banach space constants arising in nonlinear fixed point and eigenvalue theory

[EN] As is well known, in any infinite-dimensional Banach space one may find fixed point free self-maps of the unit ball, retractions of the unit ball onto its boundary, contractions of the unit sphere, and nonzero maps without positive eigenvalues and normalized eigenvectors. In this paper, we give upper and lower estimates, or even explicit formulas, for the minimal Lipschitz constant and measure of noncompactness of such maps.

Mixing properties of ester-containing solutions: a study on the ternary (methyl alkanoate (pentanoate and methanoate)+methanol and the corresponding binaries. New contributions to the (ester+ester) interactions

[EN]This work studies the volumetric (VE m) and energetic (HE m) properties resulting from the mixing processes of binary systems and the corresponding ternary of two methyl esters (methanoate and pentanoate) with methanol. The three binaries produce net endothermic mixing effects, with important energetic interactions, with maximum values of HE m ffi 400 J mol 1 , for the (ester + ester) system. This produces expansive effects VE m > 0, but the binaries of the (methyl esters + methanol) give rise to contractions VE m < 0, due to the formation of molecular aggregates.

Mechanosensitivity in the myenteric plexus of the guinea pig ileum

The enteric nervous system regulates autonomously from the central nervous system all the reflex pathways that control blood flow, motility, water and electrolyte transport and acid secretion. The ability of the gut to function in isolation is one of the most intriguing phenomenons in neurogastroenterology. This requires coding of sensory stimuli by cells in the gut wall. Enteric neurons are prominent candidates to relay mechanosensitivity. Surprisingly, the identity of mechanosensitive neurons in the enteric nervous system as well as the appropriate stimulus modality is unknown despite the evidence that enteric neurons respond to sustained distension. Objectives: The aim of our study was to record from mechanosensitive neurons using physiological stimulus modalities. Identification of sensory neurons is of central importance to understand sensory transmission under normal conditions and in gut diseases associated with sensorimotor dysfunctions, such as Irritable Bowel Syndrome. Only then it will be possible to identify novel targets that help to normalise sensory functions. Methods: We used guinea-pig ileum myenteric plexus preparations and recorded responses of all neurons in a given ganglion with a fast neuroimaging technique based on voltage sensitive dyes. To evoke a mechanical response we used two different kinds of stimuli: firstly we applied a local mechanical distortion of the ganglion surface with von Frey hair. Secondarily we mimic the ganglia deformation during physiological movements of myenteric ganglia in a freely contracting ileal preparation. We were able to reliably and reproducibly mimic this distortion by intraganglionic injections of small volumes of oxygenated and buffered Krebs solution using stimulus parameters that correspond to single contractions. We also performed in every ganglion tested, electrical stimulations to evoke fast excitatory postsynaptic potentials. Immunohistochemistry reactions were done with antibodies against Calbindin and NeuN, considered markers for sensory neurons. Results: Recordings were performed in 46 ganglia from 31 guinea pigs. In every ganglion tested we found from 1 to 21 (from 3% to 62%) responding cells with a median value of 7 (24% of the total number of neurons). The response consisted of an almost instantaneous spike discharge that showed adaptation. The median value of the action potential frequency in the responding neurons was 2.0 Hz, with a recording time of 1255 ms. The spike discharge lasted for 302 ± 231 ms and occurred only during the initial deformation phase. During sustained deformation no spike discharge was observed. The response was reproducible and was a direct activation of the enteric neurons since it remained after synaptic blockade with hexamethonium or ω-conotoxin and after long time perfusion with capsaicin. Muscle tone appears not to be required for activation of mechanosensory neurons. Mechanosensory neurons showed a response to mechanical stimulation related to the stimulus strength. All mechanosensory neurons received fast synaptic inputs. There was no correlation between mechanosensitivity and Calbindin-IR and NeuN-IR (44% of mechanosensitive neurones Calb-IR-/NeuN-IR-). Conclusions: We identified mechanosensitive neurons in the myenteric plexus of the guinea pig ileum which responded to brief deformation. These cells appear to be rapidly accommodating neurons which respond to dynamic change. All mechanosensitive neurons received fast synaptic input suggesting that their activity can be highly modulated by other neurons and hence there is a low stimulus fidelity which allows adjusting the gain in a sensory network. Mechanosensitivity appears to be a common feature of many enteric neurons belonging to different functional classes. This supports the existence of multifunctional enteric neurons which may fulfil sensory, integrative and motor functions.