Isolation and characterisation of conomap-Vt, a D-amino acid containing excitatory peptide from the venom of a vermivorous cone snail

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a D-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture. (c) 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Dynamic training volume: A construct of both time under tension and volume load

The purpose of this study was to investigate the effects of three different weight training protocols, that varied in the way training volume was measured, on acute muscular fatigue. Ten resistance-trained males performed all three protocols which involved dynamic constant resistance exercise of the elbow flexors. Protocol A provided a standard for the time the muscle group was under tension (TUT) and volume load (VL), expressed as the product of the total number of repetitions and the load that was lifted. Protocol B involved 40% of the TUT but the same VL compared to protocol A; protocol C was equated with protocol A for TUT but only involved 50% of the VL. Fatigue was assessed by changes in maximum voluntary isometric force and integrated electromyography (iEMG) between the pre- and post-training protocols. The results of the study showed that, when equated for VL, greater TUT produced greater overall muscular fatigue ( p

Motor unit number estimation - A Bayesian approach

All muscle contractions are dependent on the functioning of motor units. In diseases such as amyotrophic lateral sclerosis (ALS), progressive loss of motor units leads to gradual paralysis. A major difficulty in the search for a treatment for these diseases has been the lack of a reliable measure of disease progression. One possible measure would be an estimate of the number of surviving motor units. Despite over 30 years of motor unit number estimation (MUNE), all proposed methods have been met with practical and theoretical objections. Our aim is to develop a method of MUNE that overcomes these objections. We record the compound muscle action potential (CMAP) from a selected muscle in response to a graded electrical stimulation applied to the nerve. As the stimulus increases, the threshold of each motor unit is exceeded, and the size of the CMAP increases until a maximum response is obtained. However, the threshold potential required to excite an axon is not a precise value but fluctuates over a small range leading to probabilistic activation of motor units in response to a given stimulus. When the threshold ranges of motor units overlap, there may be alternation where the number of motor units that fire in response to the stimulus is variable. This means that increments in the value of the CMAP correspond to the firing of different combinations of motor units. At a fixed stimulus, variability in the CMAP, measured as variance, can be used to conduct MUNE using the "statistical" or the "Poisson" method. However, this method relies on the assumptions that the numbers of motor units that are firing probabilistically have the Poisson distribution and that all single motor unit action potentials (MUAP) have a fixed and identical size. These assumptions are not necessarily correct. We propose to develop a Bayesian statistical methodology to analyze electrophysiological data to provide an estimate of motor unit numbers. Our method of MUNE incorporates the variability of the threshold, the variability between and within single MUAPs, and baseline variability. Our model not only gives the most probable number of motor units but also provides information about both the population of units and individual units. We use Markov chain Monte Carlo to obtain information about the characteristics of individual motor units and about the population of motor units and the Bayesian information criterion for MUNE. We test our method of MUNE on three subjects. Our method provides a reproducible estimate for a patient with stable but severe ALS. In a serial study, we demonstrate a decline in the number of motor unit numbers with a patient with rapidly advancing disease. Finally, with our last patient, we show that our method has the capacity to estimate a larger number of motor units.

Perindopril, an angiotensin converting enzyme inhibitor, in pulmonary hypertensive rats: comparative effects on pulmonary vascular structure and function

1 Hypoxic pulmonary hypertension in rats (10% O-2, 4 weeks) is characterized by changes in pulmonary vascular structure and function. The effects of the angiotensin converting enzyme inhibitor perindopril (oral gavage, once daily for the 4 weeks of hypoxia) on these changes were examined. 2 Perindopril (30 mg kg(-1) d(-1)) caused an 18% reduction in pulmonary artery pressure in hypoxic rats. 3 Structural changes (remodelling) in hypoxic rats included increases in (i) critical closing pressure in isolated perfused lungs (remodelling of arteries (50 mu m 0.d.) and (ii) medial wall thickness of intralobar pulmonary arteries, assessed histologically (vessels 30-100 and 101-500 mu m o.d.). Perindopril 10 and 30 mg kg(-1) d(-1) attenuated remodelling in vessels less than or equal to 100 mu m (lungs and histology), 30 mg kg(-1) d(-1) was effective in vessels 101-500 mu m but neither dose prevented hypertrophy of main pulmonary artery. 3 mg kg(-1) d(-1) was without effect. 4 Perindopril (30 mg kg(-1) d(-1)) prevented the exaggerated hypoxic pulmonary vasoconstrictor response seen in perfused lungs from hypoxic rats but did not prevent any of the functional changes (i.e. the increased contractions to 5-HT, U46619 (thromboxane-mimetic) and K+ and diminished contractions to angiotensins I and II) seen in isolated intralobar or main pulmonary arteries. Acetylcholine responses were unaltered in hypoxic rats. 5 We conclude that, in hypoxic rats, altered pulmonary vascular function is largely independent of remodelling. Hence any drug that affects only remodelling is unlikely to restore pulmonary vascular function to normal and, like perindopril, may have only a modest effect on pulmonary artery pressure.

Characterization of receptors for calcitonin gene-related peptide and adrenomedullin on the guinea-pig vas deferens

1. The receptors which mediate the effects of calcitonin gene-related peptide (CGRP), amylin and adrenomedullin on the guinea-pig vas deferens have been investigated. 2. All three peptides cause concentration dependant inhibitions of the electrically stimulated twitch response (pD 2s for CGRP, amylin and adrenomedullin of 7.90 ± 0.11, 7.70 ± 0.19 and 7.25 ± 0.10 respectively). 3. CGRP 8-37 (1 μM) and AC187 (10 μM) showed little antagonist activity against adrenomedullin. 4. Adrenomedullin 22-52 by itself inhibited the electrically stimulated contractions of the vas deferens and also antagonized the responses to CGRP, amylin and adrenomedullin. 5. [ 125I]-adrenomedullin labelled a single population of binding sites in vas deferens membranes with a pIC 50 of 8.91 and a capacity of 643 fmol mg -1. Its selectivity profile was adrenomedullin > AC187 > CGRP = amylin. It was clearly distinct from a site labelled by [ 125I]-CGRP (pIC 50 = 8.73, capacity = 114 fmol mg -1, selectivity CGRP > amylin = AC187 > adrenomedullin). [ 125I]-amylin bound to two sites with a total capacity of 882 fmol mg -1. 6. Although CGRP has been shown to act at a CGRP 2 receptor on the vas deferens with low sensitivity to CGRP 8-37, this antagonist displaced [ 125I]-CGRP with high affinity from vas deferens membranes. This affinity was unaltered by increasing the temperature from 4°C to 25°C, suggesting the anomalous behaviour of CGRP 8-37 is not due to temperature differences between binding and functional assays.

Multiple receptors for calcitonin gene-related peptide and amylin on guinea-pig ileum and vas deferens

1. The responses of the electrically stimulated guinea-pig ileum and vas deferens to human and rat calcitonin gene-related peptide (CGRP) and amylin were investigated. 2. The inhibition of contraction of the ileum produced by human alpha CGRP was antagonized by human alpha CGRP8-37 (apparent pA2 estimated at 7.15 +/- 0.23) > human alpha CGRP19-37 (apparent pA2 estimated as 6.67 +/- 0.33) > [Tyr0]-human alpha CGRP28-37. The amylin antagonist, AC187, was three fold less potent than CGRP8-37 in antagonizing human alpha CGRP. 3. Both human beta- and rat alpha CGRP inhibited contractions of the ileum, but this was less sensitive to inhibition by CGRP8-37 than the effect of human alpha CGRP. However, CGRP19-37 was twenty times more effective in inhibiting the response to rat alpha CGRP (apparent pA2 estimated as 8.0 +/- 0.1) compared to human alpha CGRP. 4. Rat amylin inhibited contractions in about 10% of ileal preparations; this effect was not antagonized by any CGRP fragment. Human amylin had no action on this preparation. 5. Both human and rat alpha CGRP inhibited electrically stimulated contractions of the vas deferens, which were not antagonized by 3 microM CGRP8-37 or 10 microM AC187. 6. Rat amylin inhibited the stimulated contractions of the vas deferens (EC50 = 77 +/- 9 nM); human amylin was less potent (EC50 = 213 +/- 22 nM). The response to rat amylin was antagonized by 10 microM CGRP8-37 (EC50 = 242 +/- 25 nM) and 10 microM AC187 (EC50 = 610 +/- 22 nM). 7. It is concluded that human alpha CGRP relaxes the guinea-pig ileum via CGRP1-like receptors, but that human beta CGRP and rat alpha CGRP may use additional receptors. These are distinct CGRP2-like and amylin receptors on guinea-pig vas deferens.

A regional analysis of contraction rate in written Standard American English

The goal of this study is to determine if various measures of contraction rate are regionally patterned in written Standard American English. In order to answer this question, this study employs a corpus-based approach to data collection and a statistical approach to data analysis. Based on a spatial autocorrelation analysis of the values of eleven measures of contraction across a 25 million word corpus of letters to the editor representing the language of 200 cities from across the contiguous United States, two primary regional patterns were identified: easterners tend to produce relatively few standard contractions (not contraction, verb contraction) compared to westerners, and northeasterners tend to produce relatively few non-standard contractions (to contraction, non-standard not contraction) compared to southeasterners. These findings demonstrate that regional linguistic variation exists in written Standard American English and that regional linguistic variation is more common than is generally assumed.

The effect of cis-5,8,11,14,17-eicosapentaenoic acid upon the contractile mechanisms linked to calcium influx and the mobilisation of intracellular calcium in aortic smooth muscle

Epidemiological studies previously identified cis-5,8,11,14,17-eicosapentaenoic acid (EPA) as the biologically active component of fish oil of benefit to the cardiovascular system. Although clinical investigations demonstrated its usefulness in surgical procedures, its mechanism of action still remained unclear. It was shown in this thesis, that EPA partially blocked the contraction of aortic smooth muscle cells to the vasoactive agents KCl and noradrenaline. The latter effect was likely caused by reducing calcium influx through receptor-operated channels, supporting a recent suggestion by Asano et al (1997). Consistently, EPA decreased noradrenaline-induced contractures in aortic tissue, in support of previous reports (Engler, 1992b). The observed effect of EPA on cell contractions to KCl was not simple due to blocking calcium influx through L-type channels, consistent with a previous suggestion by Hallaq et al (1992). Moreover, EPA caused a transient increase in [Ca2+]i in the absence of extracellular calcium. To resolve this it was shown that EPA increased inositol phosphate formation which, it is suggested, caused the release of calcium from an inositol phosphate-dependent internal binding site, possibly that of an intracellular membrane or superficial sarcoplasmic reticulum, producing the transient increase in [Ca2+]i. As it was shown that the cellular contractile filaments were not desensitised to calcium by EPA, it is suggested that the transient increase in [Ca2+]i subsequently blocks further cell contraction to KCl by activating membrane-associated potassium channels. Activation of potassium channels induces the cellular efflux of potassium ions, thereby hyperpolarising the plasma membrane and moving the membrane potential farther from the activation range for calcium channels. This would prevent calcium influx in the longer term and could explain the initial observed effect of EPA to block cell contraction to KCl.

The mode of action of bradykinin

The action of bradykinin on transepithelial transfer of sodium and water in isolated rat jejunum and on smooth muscle contraction of rat terminal ileum has been investigated. (1) Bradykinin was shown to stimulate transfer at low control transfer, inhibit transfer at high control transfer and have no effect at intermediate transfer in rat jejunal sacs. Stimulation of transfer occurred only when bradykinin was in the serosal solutiun while inhibition of transfer occurred whether bradykinin was in the aerosal or mucosal solution. Bradykinin-induced stimulation of transfer was not affected by adrenalectomy, nephrectomy, combined adrenalectomy-nephrectomy,  nor maintenance on 1% saline drinking solution or low sodium diet pretreatment. Meclofenamic acid abolished the bradykinin-induced inhibition of water transfer while prostaglandins A1, E1 aud F2α all potentiated this action. Theophylline inhibited water transfer and potentiated the bradykinin-induced inhibition of water transfer. Cyclic AMP and dibutyryl cyclic AMP both inhibited water transfer and the bradykinin-induced inhibition of water transfer was potentiated by the latter. ( 2 ) Bradykinin-induced contractions of rat terminal ileum were little affected by hyoscine while those of acetylcholine were abolished. Anoxia reduced markedly responses tv bradykinin while those of acetylcholine were little affected . Theophylline reduced the responses of rat terminal ileum to bradykinin significantly more than those to acetylcholine. Aspirin and indomethacin reduced markedly the responses to bradykinin while not affecting those to acetylcholine and PGT2. Meslofenamic acid at a concentration of 3.4 µM blocked bradykinin-induced contractions but had no effect on those to acctylcholine, PGE2 or PGF2 and at a concentration of 17. 0 µM drastically reduced bradykinin responses but also reduced those to acetylcholine, PGE2 and PGF2α• Flufenamic acid drastically reduced responses to bradykinin while not affecting those to acetylcholine and PGE2 and slightly affecting those to PGF2α. Polyphloretin phosphate reduced responses to bradykinin, PGF2α and PGE2 but not acetylcholine . Diphloretin phosphate reduced responses to bradykinin, PGF2 and PGE2 in a dose dependent manner but not those to acetylcholine. SC 19220 , in a dose dependent manner, inhibited responses to bradykinin and PGE2 but not to acetylcholine and PGF2. 7 oxa - 13 -prostynoic acid non specifically reduced responses to acetylcholine, bradykinin and PGE2. Bradykinin, in the presence of SQ 20881 , increased the release of prostaglandin-like activity from rat terminal ileum and this was reduced or abolished in the presence of indomethacin, aspirin, meclofenamic acid or flufenamio acid. The extract of PG-like activity did not appear as PGE, PGA or PGFon TLC, but included a substance with similar mobility as 15-Keto-prosta-glandin E2.

A comparative study of the effects of hypoxia upon isolated arterial muscle from the rat

Contractile response of rat aorta, mesenteric artery and femoral artery to noradrenaline and potassium chloride were studied under standard and hypoxic conditions and the effect of hypoxia was dependent upon both the vessel and the stimulant. Hypoxia had less effect upon contractions to potassium chloride than those to noradrenaline. The effects of hypoxia on potassium chloride induced responses in different vessels were relatively similar although responses to noradrenaline were vessel dependent. Noradrenaline induced contractions of the femoral artery were most affected by hypoxia whilst those of the mesenteric artery were least affected. Hypoxia changed the well maintained response of the femoral artery to noradrenaline to a transient form; this effect of hypoxia was not evident in the aorta or the mesenteric artery. The aorta and mesenteric artery contracted in calcium free EGTA PSS suggesting that these vessels displayed a release component. Hypoxia reduced the magnitude of this component. The effects of verapamil on noradrenaline and potassium chloride induced responses were investigated and were found to be different to those of hypoxia. Verapamil exerted a greater effect on contractions to potassium chloride than on those to noradrenaline. The effects of hypoxia on 45calcium flux were also vessel dependent. In the mesenteric and femoral arteries hypoxia increased basal 45calcium accumulation. However, the magnitude of noradrenaline stimulated 45calcium accumulation was reduced in the femoral artery and aorta but was unchanged in the mesenteric artery. The effects of hypoxia on 45calcium accumulation were similar to verapamil only in the aorta. The results provide evidence that the effects of hypoxia may arise from alterations in calcium mobilisation processes and that differences between vessels in these processes accounts for the heterogeneity between vessels in their response to hypoxia.

Characterisation of calcitonin generelated peptide (CGRP) and amylin receptors

The aims of this study were to examine the binding characteristics of the rat CGRP receptor and to further the classification of CGRP and amylin receptors in guinea-pig tissue preparations. Binding characteristics of CGRP were investigated on rat splenic, cerebellar and liver membrane preparations. Human-α-CGRP, rat-α-CGRP and the CGRP receptor analogues Tyrº -CGRPC28-37) and [Cys (ACM)2,7 ]-human CGRP and the CGRP receptor antagonist CGRPC8-37) were utilised in competitive radioligand binding experiments to identify possible CGRP receptor subtypes in these tissues. There appeared to be no significant differences between the rat CGRP receptors examined. A panel of monoclonal antibodies (Mabs) raised against CGRP were employed to investigate the structure-activity relationships of CGRP and its receptor. No differences between the tissue receptors were observed using this panel of Mabs. The effects of human-α, human-β, rat-α-CGRP, human and rat amylin and adrenomedullin(13-52) were examined on the spontaneously beating right atria and on electrically evoked twitch contractions of isolated guinea-pig ileum, vas deferens and left atria. All of the peptides caused concentration-dependent inhibition of twitch amplitude in the ileum and vas deferens. CGRP produced positive inotropic effects in the right and left atria and positive chronotropic effects in the right atria. A variety of CGRP receptor antagonists and putative amylin receptor antagonists were used to antagonise these effects. CGRP(8-37) is currently used as a basis for CGRP receptor classification (Dennis, et al., 1989). Based upon results obtained using CGRP(8-37) it has been shown that the guinea-pig ileum contains mainly CGRP 1 receptors and the vas deferens contain CGRP2 receptors. Amylin was shown to act at receptors distinct from those for CGRP and it is postulated that amylin has its own receptors in these preparations. Experiments using CGRP (19-37) and Tyrº -CGRP(28-37) indicate that human and rat CGRP act at distinct receptors in guinea-pig ileum and vas deferens. The amylin receptor antagonist amylin(8-37) and the putative antagonist AC187 provide evidence to suggest human and rat amylin also act at receptors able to distinguish between the two types of amylin.

PSD modifications of FHRV due to CTG storage rate

Cardiotocographic data provide physicians information about foetal development and, through assessment of specific parameters (like accelerations, uterine contractions, ...), permit to assess conditions such as foetal distress. An incorrect evaluation of foetal status can be of course very dangerous. In the last decades, to improve interpretation of cardiotocographic recordings, great interest has been dedicated to FHRV spectral analysis. It is worth reminding that FHR is intrinsically an uneven series and that to obtain evenly sampled series, many commercial cardiotocographs use a zero-order interpolation (storage rate of CTG data equal to 4 Hz). This is not suitable for frequency analyses because interpolation introduces alterations in the FHR power spectrum. In particular, this interpolation process can produce artifacts and an attenuation of the high-frequency components of the PSD that, for example, affects the estimation of the sympatho-vagal balance (SVB - computed as low-frequency/high-frequency ratio), which represents an important clinical parameter. In order to estimate the frequency spectrum alterations due to zero-order interpolation and other CTG storage rates, in this work, we simulated uneven FHR series with set characteristics, their evenly spaced versions (with different storage rates) and computed SVB values by PSD. For PSD estimation, we chose the Lomb method, as suggested by other authors in application to uneven HR series. ©2009 IEEE.

Efeitos do Kinesio Taping® sobre os marcadores clínicos indiretos de dano muscular induzido pelo exercício excêntrico: ensaio clínico randomizado

Objective: analyze the effect of Kinesio Taping (KT) on the indirect clinical markers of muscle damage induced by eccentric exercises in the elbow flexors in healthy individuals. Materials and methods: It is a randomized controlled trial involving sixty volunteers at age group between 18 and 28 years randomly selected. The sample into three groups with twenty participants: control group (CG) – eccentric protocol without KT, KT group – eccentric with tensioned KT, placebo group – eccentric protocol KT with no tension. The evaluations took place at four moments; the first one was the basis line (AV1), after the second protocol (AV2) and the following two groups 24 (AV3) and 48 hours (AV4) after the intervention protocol. The muscle damage was induced by sixteen maximum eccentric contractions of the elbow flexors from the non-dominant limb, divided in two sets of eight repetitions, at 60º/s, with two minutes interval. The variables analyzed were: the joint amplitude in rest, the level of pain, the joint position sense (JPS) followed of isokinetic checking with electromyographic sign capitation. These data were analyzed in software SPSS 20.0. The normality was identified by Kolmogorov-Smimov examination and then, being used the ANOVA mixed model with significance of 5%. Outcomes: a decrease was observed at joint amplitude moreover, an immediate increase of pain wich increased after 24 and remained until 48 hours at all groups searched. There was not difference at the JPS. The variables peak torque, average peak torque, total work and mean power mean reduced until 48 hours after muscle lesion in all groups. Among the groups, there was no difference in EMG values and for any of the variables. Conclusion: The KT did not influence at the indirect clinical markers of muscle lesion induced by eccentric exercises in the elbow flexors in healthy people.

Efeitos do Kinesio Taping® sobre os marcadores clínicos indiretos de dano muscular induzido pelo exercício excêntrico: ensaio clínico randomizado

Objective: analyze the effect of Kinesio Taping (KT) on the indirect clinical markers of muscle damage induced by eccentric exercises in the elbow flexors in healthy individuals. Materials and methods: It is a randomized controlled trial involving sixty volunteers at age group between 18 and 28 years randomly selected. The sample into three groups with twenty participants: control group (CG) – eccentric protocol without KT, KT group – eccentric with tensioned KT, placebo group – eccentric protocol KT with no tension. The evaluations took place at four moments; the first one was the basis line (AV1), after the second protocol (AV2) and the following two groups 24 (AV3) and 48 hours (AV4) after the intervention protocol. The muscle damage was induced by sixteen maximum eccentric contractions of the elbow flexors from the non-dominant limb, divided in two sets of eight repetitions, at 60º/s, with two minutes interval. The variables analyzed were: the joint amplitude in rest, the level of pain, the joint position sense (JPS) followed of isokinetic checking with electromyographic sign capitation. These data were analyzed in software SPSS 20.0. The normality was identified by Kolmogorov-Smimov examination and then, being used the ANOVA mixed model with significance of 5%. Outcomes: a decrease was observed at joint amplitude moreover, an immediate increase of pain wich increased after 24 and remained until 48 hours at all groups searched. There was not difference at the JPS. The variables peak torque, average peak torque, total work and mean power mean reduced until 48 hours after muscle lesion in all groups. Among the groups, there was no difference in EMG values and for any of the variables. Conclusion: The KT did not influence at the indirect clinical markers of muscle lesion induced by eccentric exercises in the elbow flexors in healthy people.

Effects of pelvic floor muscle rehabilitation on PFM function and morphology in older women

Aims The purpose of this study was to examine the effect of a pelvic floor muscle (PFM) rehabilitation program on incontinence symptoms, PFM function, and morphology in older women with SUI. Methods Women 60 years old and older with at least weekly episodes of SUI were recruited. Participants were evaluated before and after a 12-week group PFM rehabilitation intervention. The evaluations included 3-day bladder diaries, symptom, and quality of life questionnaires, PFM function testing with dynamometry (force) and electromyography (activation) during seven tasks: rest, PFM maximum voluntary contraction (MVC), straining, rapid-repeated PFM contractions, a 60 sec sustained PFM contraction, a single cough and three repeated coughs, and sagittal MRI recorded at rest, during PFM MVCs and during straining to assess PFM morphology. Results Seventeen women (68.9 ± 5.5 years) participated. Following the intervention the frequency of urine leakage decreased and disease-specific quality of life improved significantly. PFM function improved significantly: the participants were able to perform more rapid-repeated PFM contractions; they activated their PFMs sooner when coughing and they were better able to maintain a PFM contraction between repeated coughs. Pelvic organ support improved significantly: the anorectal angle was decreased and the urethrovescial junction was higher at rest, during contraction and while straining. Conclusions This study indicated that improvements in urine leakage were produced along with improvements in PFM co-ordination (demonstrated by the increased number of rapid PFM contractions and the earlier PFM activation when coughing), motor-control, pelvic organ support.