Recognition of single-stranded nucleic acids by small-molecule splicing modulators.

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.

Characterization of a highly neutralizing single monoclonal antibody to botulinum neurotoxin type A.

Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the HCN and HCC subdomains of the BoNT/A1 receptor-binding domain (HC ). The TA12-binding interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2- and the CR1/CR2-neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to HC confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1-binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.

Secreted Signaling Molecules at the Neuromuscular Junction in Physiology and Pathology.

: Signal transduction at the neuromuscular junction (NMJ) is affected in many human diseases, including congenital myasthenic syndromes (CMS), myasthenia gravis, Lambert-Eaton myasthenic syndrome, Isaacs' syndrome, Schwartz-Jampel syndrome, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. The NMJ is a prototypic cholinergic synapse between the motor neuron and the skeletal muscle. Synaptogenesis of the NMJ has been extensively studied, which has also been extrapolated to further understand synapse formation in the central nervous system. Studies of genetically engineered mice have disclosed crucial roles of secreted molecules in the development and maintenance of the NMJ. In this review, we focus on the secreted signaling molecules which regulate the clustering of acetylcholine receptors (AChRs) at the NMJ. We first discuss the signaling pathway comprised of neural agrin and its receptors, low-density lipoprotein receptor-related protein 4 (Lrp4) and muscle-specific receptor tyrosine kinase (MuSK). This pathway drives the clustering of acetylcholine receptors (AChRs) to ensure efficient signal transduction at the NMJ. We also discuss three secreted molecules (Rspo2, Fgf18, and connective tissue growth factor (Ctgf)) that we recently identified in the Wnt/ß-catenin and fibroblast growth factors (FGF) signaling pathways. The three secreted molecules facilitate the clustering of AChRs by enhancing the agrin-Lrp4-MuSK signaling pathway.

Chronic migraine and Botulinum Toxin Type A: Where do paths cross?

Migraine is a highly prevalent and disabling disorder accounted among the primary headaches. It is the expression of a complex, and not yet fully understood, pathophysiology involving the sensitization of peripheral and central nociceptive pathways. In this review we succinctly illustrate the molecular, anatomical, and functional abnormalities underlying the migraine attack that are relevant for understanding in more depth the neurobiology behind the therapeutic effect of Botulinum Toxin Type A (BoNT-A). BoNT-A has proved effective in several neurological conditions and, more recently, also in chronic migraine. Its antimigraine mechanism of action was initially thought to be limited to the periphery and interpreted as an inhibitory activity on the processes associated to the local release of neuropeptides, with subsequent induction of peripheral sensitization. Increasing experimental evidence has become available to suggest that additional mechanisms are possibly involved, including the direct/indirect inhibition of sensitization processes in central nociceptive pathways.

Effect of Prolonged Military Field Training on Neuromuscular and Hormonal Responses and Shooting Performance in Warfighters.

Introduction: Previous studies have shown that military field training (MFT) has effects on warfighters' hormonal responses, neuromuscular performance, and shooting accuracy. The aim of the present study was to investigate the changes in body composition, upper and lower body strength, serum hormone concentrations of testosterone (TES) and cortisol (COR), insulin-like growth factor-1 (IGF-1), and sex hormone binding globulin (SHBG) and shooting accuracy during prolonged MFT. Methods: Serum hormone concentrations, isometric strength of the upper and lower extremities, and shooting performance were measured four times during the study: before MFT (PRE), after 12 d (MID), at the end of MFT (POST) and after 4 d recovery (RECO). The study was approved by the Finnish Defence Forces and was granted ethical approval by the Ethical Committee of the University of Jyväskylä. Results: There was no change in prone shooting score between the measuring points. In the standing position, however, there was a significant (p ≤ 0.001) decrease from PRE 58.2 ± 12.3 points to MID 45.2 ± 10.4 points. Also POST 61.4 ± 10.8 points and RECO 56.8 ± 13.6 points were significantly (p ≤ 0.001) higher than MID 45.2 ± 10.4 points. Serum hormone concentrations of TES and IGF-1 decreased significantly during MFT. In COR and SHBG concentrations, significant increases were observed during MFT. Individual changes in lower body strength and changes in shooting standing score between the measurement points (PRE-MID/POST/RECO) correlated significantly (r = 0.332, p = 0.031; r = 0.335, p = 0.025; r = 0.489, p = 0.001, respectively). The similar finding was observed with changes in upper body strength and changes in standing shooting between the PRE and RECO measurement points (0.339, p = 0.010). The changes in COR and the changes in prone shooting showed a positive correlation in all measurement points (r = 0.531, p ≤ 0.001; r = 0.337, p = 0.024; r = 0.572, p ≤ 0.001). The changes in IGF-1 correlated negatively (r = -0.325, p = 0.038) with shooting prone between the PRE and MID measurement points. The changes in shooting standing and the changes in TES between PRE and POST correlated negatively (r = -0.378, p = 0.010). Conclusion: In this study, we observed a decrease in leg strength from the PRE to MID measurements. When the physical load requirements during the MFT decreased after the MID measurements, leg strength increased. In addition, the shooting score from the standing position decreased from the PRE to MID measurements and improved significantly from the MID to POST measurements. The prone shooting score did not show any significant changes during the study period. Significant positive correlations were found between the changes in standing shooting score and the changes in strength for the legs and upper body. There was a positive correlation between the changes in serum COR concentrations and changes in standing shooting score. Altogether, the present study showed that the prolonged MFT has adverse effect on the strength levels and the shooting ability in warfighters. This shows that ensuring warfighters get an appropriate amount of rest while performing their duties is important. Shooting from a prone position was not affected by the changing workloads and this result indicated that soldiers should shoot from a prone position, whenever possible, especially when fatigued.

Identification of enzymes responsible for dantrolene metabolism in the human liver: A clue to uncover the cause of liver injury.

Dantrolene is used for malignant hyperthermia during anesthesia, and it sometimes causes severe liver injury as a side effect. Dantrolene is metabolized to acetylaminodantrolene, which is formed via the reduction of dantrolene to aminodantrolene and subsequent acetylation. Formation of hydroxylamine during the metabolic process may be associated with liver injury. We identified the enzymes responsible for dantrolene metabolism in humans to elucidate the mechanism of liver injury. Dantrolene reductase activity was not detected in human liver microsomes, but it was detected in cytosol. Formation was increased in the presence of N1-methylnicotineamide, which is an electron donor to aldehyde oxidase 1 (AOX1). Potent inhibitors of AOX1 and a correlation study with a marker of AOX1 activity, namely phthalazine oxidase activity, in a panel of 28 human liver cytosol samples supported the role of AOX1 in dantrolene reduction. Acetylaminodantrolene formation from aminodantrolene was highly detected in recombinant N-acetyltransferase (NAT) 2 rather than NAT1. A glutathione trapping assay revealed the formation of hydroxylamine via an AOX1-dependent reduction of dantrolene but not via hydroxylation of aminodantrolene. In conclusion, we found that AOX1 and NAT2 were responsible for dantrolene metabolism in humans and that AOX1-dependent metabolism determines dantrolene-induced liver injury.

Electroestimulación neuromuscular intradiálisis, fuerza muscular, capacidad funcional y composición corporal / Intradialysis neuromuscular electro stimulation muscle strength, functional capacity and body composition

Introducción: La capacidad funcional disminuida y la importante atrofia muscular caracterizan a los pacientes en hemodiálisis (HD). El ejercicio físico intradiálisis y recientemente la electroestimulación neuromuscular (EMS), representan dos serias opciones terapéuticas para mejorar esta deteriorada condición física. Actualmente, no existen estudios publicados sobre el papel de la EMS y la composición corporal en los pacientes en HD. Objetivo: Analizar que efecto produce un programa de EMS sobre la fuerza muscular, capacidad funcional, parámetros nutricionales y composición corporal en nuestros pacientes en HD. Material y Métodos: Estudio unicéntrico, prospectivo de 12 semanas de duración. Los pacientes incluidos realizaron un programa adaptativo de EMS en ambos cuádriceps intradiálisis mediante el dispositivo Compex R Theta 500i. Analizamos: 1.- Parámetros nutricionales (Albumina, pre albúmina, triglicéridos, colesterol total y fracciones, ferritina y Proteína C reactiva). 2.- Datos musculares: Composición muscular cuadriceps, Fuerza extensión máxima cuádriceps (FEMQ) y handgrip (HG) brazo dominante. 3.- Test funcionales: “Sit to stand to sit” (STS10) y “six- minutes walking test” (6MWT). 4.- Composición corporal mediante biompedancia electrica (BIA). Resultados: 13 pacientes incluidos: (69.2% hombres). Edad media: 65.7 años y 33.9 meses en HD. I.Charlson medio 9.1. La principal etiología de la ERC fue la DM ( 38.5%). Al final del estudio se observó una mejoría en (*p<0.05): FEMQ* ( 11.7±7.1 vs 13.4±7.4 Kg), STS10 (39.3±15.5 vs 35.8±13.7 seg), 6MWT* (9.9%, 293.2 vs 325.2 m). En relación a la composición corporal, se observó únicamente un aumento significativo del área muscular (AMQ*: 128.6 ± 30.2 vs 144.6 ± 22.4 cm2) y una disminución del área grasa (AGQ*: 76.5 ± 26.9 vs 62.1 ± 20.1 cm2) a nivel quadricipital, sin cambios en el resto de datos analizados (% grasa abdominal, peso graso, peso magro, agua corporal total). No se objetivaron cambios relevantes en los parámetros nutricionales y de adecuación dialítica. Conclusiones: 1.- La electroestimulación neuromuscular intradialísis mejoró la fuerza muscular, la capacidad funcional y la composición muscular del cuadriceps de nuestros pacientes en HD. 2.- Nuestros resultados remarcan el carácter local de la electroes-timulación neuromuscular, dada la ausencia de cambios relevantes en el resto de los parámetros nutricionales y datos corporales analizados. 3.- No obstante, son necesarios futuros estudios mejor diseñados, de cara a discernir si la electroestimulación neuromuscular podría ser una nueva alternativa terapéutica para evitar la atrofia muscular y el deterioro progresivo de la condición física de éstos pacientes (AU)

Background: The reduced functional capacity and significant muscle atrophy characterized patients on hemodialysis. Intradialytic exercise and recently neuromuscular electrostimulation (EMS) represent two serious therapeutical options to improve the deteriorated physical condition. Until date, there are no published studies about the role of EMS and body composition in HD patients. Objectives: Analyze the effect a program of EMS on muscle strength, functional capacity, nutritional parameters and body composition in our HD patients. Methods: A 12 weeks single-center, prospective study. Patients included in the study performed an intradialysis EMS adaptive program in both quadriceps using the Compex R Theta 500i device. We analyzed: 1.- Nutritional parameters (albumin, pre-albumin, triglycerides, total cholesterol and fractions, ferritin and C-reactive protein). 2.- Muscular data: Muscular composition, Maximum length quadriceps strength (MLQS) and “hand-grip” (HG) dominant arm. 3.- Functional capacity test: “Sit to stand to sit” (STS10) and “six- minutes walking test” (6MWT). 4.- Body composition. Results: 13 HD patients included: 69.2 % men. Mean age 65.7 years and 33.9 months on HD. A significant (* p < 0,05) improvement was observed in MLQS* (11.7±7.1 vs 13.4±7.4 Kg), STS10* (39.3±15.5 vs 35.8±13.7 seg), 6MWT* (9.9%, 293.2 vs 325.2 m). There was a signi-ficant increase in the quadriceps muscular area (QMA*: 128.6 ± 30.2 vs 144.6 ± 22.4 cm2) and decrease of fat quadricipital area (FQA*: 76.5 ± 26.9 vs 62.1 ± 20.1 cm2). No significant changes were observed in nutritional parameters, body composition (body fat percentage, lean and fat mass, total body water) or dialysis adecuacy data. Conclusions: 1.- Intradialysis quadriceps EMS improved muscle strength, functional capacity and the quadriceps muscle composition in our HD patients. 2.- Our results underline the local aspects on EMS, given the absence of relevant changes on nutritional parameters and body composition. 3.- Future studies are manadatory in order to establish if EMS could be a new alternative to prevent muscle atrophy and the progressive deterioration of the physical condition of these patients (AU)

More than at the neuromuscular synapse: actions of botulinum neurotoxin A in the central nervous system.

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that produces a sustained yet transitory blockade of transmitter release from peripheral nerve terminals. Local delivery of this neurotoxin is successfully employed in clinical practice to reduce muscle hyperactivity such as in spasticity and dystonia, and to relieve pain with long-lasting therapeutic effects. However, not all BoNT/A effects can be explained by an action at peripheral nerve terminals. Indeed, it appears that BoNT/A is endowed with trafficking properties similar to the parental tetanus neurotoxin and thus be able to directly affect the CNS. In this review, we present and discuss novel compelling evidence for a direct central effect of BoNT/A in both dorsal and ventral horns of the animal and human spinal cord after peripheral injection of the neurotoxin, with important consequences on pain and motor control. This new knowledge is expected to radically change the approach to the use of BoNT/A in the future. As BoNT/A central action appears to also contribute to functional improvement, for instance in human spastic gait, the challenge will be to develop new subtypes or BoNT derivatives with deliberate, cell-specific central effects in order to fully exploit the spectrum of BoNT/A therapeutic activity.

Postoperative respiratory outcomes in laparoscopic bariatric surgery: Comparison of a prospective group of patients whose neuromuscular blockade was reverted with sugammadex and a historical one reverted with neostigmine / Resultados respiratorios postoperatorios en cirugía bariátrica laparoscópica: comparación entre un grupo prospectivo de pacientes cuyo bloqueo neuromuscular fue revertido con sugammadex frente a uno histórico revertido con neostigmina

Purpose. Bariatric surgery patients are at high risk of perioperative respiratory adverse events. We hypothesized that the use of sugammadex to reverse neuromuscular blockade could improve postoperative respiratory outcomes. Methods. Prospective observational series of consecutive patients scheduled for laparoscopic bariatric surgery in whom neuromuscular blockade was reverted with sugammadex were compared with a historical matched cohort of patients reverted with neostigmines. The necessity of postoperative mechanical ventilation or pathological changes in postoperative chest X-ray were two of the comparisons done. Results. We enrolled 160 patients in each group (Sugammadex - SG and Historical - HG). Two patients (mean, CI 95%), (1.25, 0.34-4.4) in the SG and five patients in the HG (mean, CI 95%), (3.13, 1.34-7.11) required mechanical ventilation immediately after surgery (p = 0.38, chi-square test). Significantly less chest X-ray postoperative changes were observed in the SG: 11 patients (6.9%) versus 26 patients (16.3%) in the HG (Odds ratio OR, CI 95%) (0.36, 0.18-0.8). Conclusion. Requirement of mechanical ventilation is not associated to the reversal agent employed. Less pathological postoperative chest X-ray changes were found in the group of patients whose neuromuscular blockade was reverted with sugammadex (AU)

Objetivos. Los pacientes candidatos a cirugía bariátrica presentan mayor riesgo de eventos respiratorios adversos. Nuestra hipótesis fue que la utilización de sugammadex para revertir el bloqueo neuromuscular podría mejorar los resultados postoperatorios desde el punto de vista respiratorio. Métodos. Se comparó una serie prospectiva de pacientes sometidos a cirugía bariátrica laparoscópica revertidos con sugammadex con una cohorte histórica cotejada de pacientes sometidos a la misma cirugía revertidos con neostigmina. Se compararon, entre otros datos, la necesidad de ventilación mecánica postoperatoria y la aparición de cambios radiológicos patológicos tras la operación. Resultados. Se incluyeron 160 pacientes en cada grupo (Sugammadex [SG] e Histórico HG). Dos pacientes (media 1,25%, IC 95% 0,34-4,4) en el SG y 5 en el HG (media 3,13%, IC 95% 1,34-7,11) precisaron ventilación mecánica inmediatamente después de la cirugía (p = 0,38 test chi-cuadrado). Se observaron significativamente menos cambios en la radiografía posoperatoria de tórax en el grupo SG que en el HG: 11 pacientes (6,9%) frente a 26 (16,3%) (odds ratio 0,36, IC 95% 0,18-0,8). Conclusión. Las necesidades de ventilación mecánica no se asocian al agente reversor empleado. Se observaron menos cambios patológicos en la radiografía postoperatoria de tórax en el grupo de pacientes cuyo bloqueo neuromuscular fue revertido con sugammadex (AU)

Botulinum toxin type A in pregnancy.

QUESTION: My patient received 62 units of botulinum toxin type A (BTX-A) for facial lines. Two weeks later, she found out that she was pregnant. Will this cause any harm to her fetus? ANSWER: Botulinum toxin is not expected to be present in systemic circulation following proper intramuscular or intradermal injection. Moreover, BTX-A, which has a high molecular weight, does not appear to cross the placenta. From the 38 pregnancies reported in the literature, including women who had botulism poisoning during pregnancy, exposure to BTX-A does not appear to increase the risk of adverse outcome in the fetus.

The analgesic effect on neuropathic pain of retrogradely transported botulinum neurotoxin A involves Schwann cells and astrocytes.

In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life.

Spread of onabotulinumtoxinA after bladder injection. Experimental study using the distribution of cleaved SNAP-25 as the marker of the toxin action.

BACKGROUND: OnabotulinumtoxinA (Onabot/A) has been used to treat detrusor overactivity disorders. The treatment is based on several injections of toxin throughout the bladder wall. However, injection protocols are not well established among clinicians, varying in dose and dilution. OBJECTIVE: Study the distribution and neurochemistry of cleaved synaptosome-associated protein of 25 kDa (cSNAP-25) after Onabot/A administration in the guinea pig bladder. In addition, we analyzed which factor, dose or volume, contributes more to the diffusion of the toxin. DESIGN, SETTING, AND PARTICIPANTS: Guinea pig bladders were treated with Onabot/A via intramural injection or an instillation. MEASUREMENTS: Bladder cryostat sections were processed for single or dual immunohistochemistry staining with antibodies against cSNAP-25, vesicular acetylcholine transporter, tyrosine hydroxylase, and calcitonin gene-related peptide. Different administration methods and doses were analyzed. Statistical analysis was performed using the chi-square test for colocalization studies after multiple injections and the t test for the evaluation of affected fibers after a single injection. RESULTS AND LIMITATIONS: cSNAP-25 immunoreactive fibers were abundant throughout the bladder tissue in the mucosa and muscular layer. Double labeling showed that parasympathetic fibers are more affected than sympathetic or sensory. A single Onabot/A injection is more effective if diluted in a higher volume. Onabot/A instillation in the bladder does not cleave SNAP-25 protein. CONCLUSIONS: A single Onabot/A injection spreads the neurotoxin activity to the opposite side of the guinea pig bladder. This action is more evident when high saline volumes are used to dissolve Onabot/A. The toxin cleaves the SNAP-25 protein mainly in cholinergic but also in adrenergic and sensory fibers. In contrast with intramural injection, instillation of Onabot/A does not cleave SNAP-25 in nerve fibers.

Structural and functional studies of a bothropic myotoxin complexed to rosmarinic acid: new insights into Lys49-PLA2 inhibition.

Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy.

Roles of affinity and lipophilicity in the slow kinetics of prostanoid receptor antagonists on isolated smooth muscle preparations.

BACKGROUND AND PURPOSE: The highly lipophilic acyl-sulphonamides L-798106 and L-826266 showed surprisingly slow antagonism of the prostanoid EP3 receptor system in guinea-pig aorta. Roles of affinity and lipophilicity in the onset kinetics of these and other prostanoid ligands were investigated. EXPERIMENTAL APPROACH: Antagonist selectivity was assessed using a panel of human recombinant prostanoid receptor-fluorimetric imaging plate reader assays. Potencies/affinities and onset half-times of agonists and antagonists were obtained on guinea-pig-isolated aorta and vas deferens. n-Octanol-water partition coefficients were predicted. KEY RESULTS: L-798106, L-826266 and the less lipophilic congener (DG)-3ap appear to behave as selective, competitive-reversible EP3 antagonists. For ligands of low to moderate lipophilicity, potency increments for EP3 and TP (thromboxane-like) agonism on guinea-pig aorta (above pEC50 of 8.0) were associated with progressively longer onset half-times; similar trends were found for TP and histamine H1 antagonism above a pA2 limit of 8.0. In contrast, L-798106 (EP3), L-826266 (EP3, TP) and the lipophilic H1 antagonists astemizole and terfenadine exhibited very slow onset rates despite their moderate affinities; (DG)-3ap (EP3) had a faster onset. Agonism and antagonism on the vas deferens EP3 system were overall much faster, although trends were similar. CONCLUSIONS AND IMPLICATIONS: High affinity and high liphophilicity may contribute to the slow onsets of prostanoid ligands in some isolated smooth muscle preparations. Both relationships are explicable by tissue disposition under the limited diffusion model. EP3 antagonists used as research tools should have moderate lipophilicity. The influence of lipophilicity on the potential clinical use of EP3 antagonists is discussed.

Neurobehavioral effects of acute exposure to aromatic hydrocarbons.

This article reports the results of neurobehavioral tests on representative aromatic constituents, specifically C(9) to C(11) species. The testing evaluated effects in several domains including clinical effects, motor activity, functional observations, and visual discrimination performance. Exposures ranging from 600 to 5000 mg/m(3), depending on the molecular weights of the specific aromatic constituents, produced minor, reversible effects on the central nervous system (CNS), particularly in the domains of gait and visual discrimination. There was little evidence of effects at lower exposure levels. There was some evidence of respiratory effects at 5000 mg/m(3) in 1 study, and there were also minor changes in body weight and temperature. The CNS effects became less pronounced with repeated exposures, corresponding to lower concentrations in the brain of 1 representative substance, 1,2,4-trimethyl benzene (TMB). At high exposure levels, the alkyl benzenes apparently induced their own metabolism, increasing elimination rates.

Papel del género en la potencia y curso de acción del bromuro de rocuronio / Influence of gender on the potency and course of action of rocuronium bromide

OBJETIVOS: La correlación entre el género y la potenciao efecto de ciertos bloqueantes neuromuscularesincluyendo rocuronio ha sido estudiada. Sin embargo, lametodología empleada pudiera adolecer de inconsistencias.Nuestro objetivo es analizar con amplitud esa relacióny aportar conclusiones definitivas.PACIENTES Y MÉTODOS: En la primera y segunda etapas,se construyeron las curvas dosis respuesta al rocuronioen seis grupos de pacientes: dos mixtos, dos exclusivamentefemeninos y dos masculinos que recibierondosis únicas o acumulativas. Estas fueron transformadasen logaritmos y el efecto, monitorizado por electromiografía,en probits, calculándose las DE50, 90 y 95 para cadapaciente. En la tercera etapa se administró un bolo de400 μg.Kg-1 a otros tres grupos determinándose: el periodode comienzo inicial, máximo efecto, tiempo decomienzo, velocidad de acción, recuperación entre 10 y25%, duración clínica y velocidad de recuperación. Enla cuarta etapa sólo se obtuvieron los parámetros quemiden la instalación del bloqueo en el grupo anterior utilizandoel método restrictivo.RESULTADOS: No se observaron diferencias significativasen las DE50, 90 y 95 entre los grupos, después de dosisúnicas: 176±68, 252±97 y 285±110 (μg.Kg-1) para el grupofemenino, 187±69, 271±100 y 307±114 para el masculinoy 172±73, 233±98 y 258±109 para el mixto respectivamentey en el mismo orden 249±63, 310±79, 334±85,(μg.Kg-1) para el grupo femenino 261±60, 327±75,354±81, para el masculino y 242±70 305±88, 330±95 parael mixto, para la técnica acumulativa. Después del bolono se detectó ninguna diferencia estadística.CONCLUSIONES: Estos resultados sugieren que no existeinfluencia significativa del género sobre la potencia yacción del bromuro de rocuronio. Los hallazgos de otrasinvestigaciones que apuntan en dirección contrariapudieran estar afectados en su metodología y resultados(AU)

OBJECTIVE: The correlation between gender and thepotency of certain neuromuscular blockers, includingrocuronium, has been investigated. However, themethods used have been inconsistent. This study aimedto look further at the role of gender in order to arrive atdefinitive conclusions.PATIENTS AND METHODS: In a first and second phases of thestudy, rocuronium dose-response curves were constructedfor patient groups as follows: 2 mixed-gender groups, 2female groups, and 2 male groups. One group in each setreceived a single dose. In the other group the effect of theaccumulated dose was measured after each of 3 fractions.The doses were transformed logarithmically. The effect wasmonitored by electromyography, and probit analysis wasused to calculate the effective dose to induce 50%, 90% and95% of the maximum effect (ED50, ED90 and ED95,respectively) in each patient. In the third phase, a bolus doseof 400 μg·Kg-1 was administered to 3 additional groups.Onset time, maximum effect, speed of onset, duration ofeffect as the 10%-25% recovery index, and speed of recoverywere recorded. In the fourth phase analysis was restricted tomeasurements during the period of onset.RESULTS: No significant between-group differences inmean (SD) ED50, ED90, or ED95 values were observedafter single doses (female group: 176 [68], 252 [97], and285 [110] μg·Kg-1, respectively; male group: 187 [69], 271[100], and 307 [114] μg·Kg-1; mixed group: 172 [73], 233[98], and 258 [109] μg·Kg-1). Assessment of effect after cumulative fracional doses gave the following mean values for ED50, ED90, and ED95: 249 (63), 310 (79), and 334 (85) μg·Kg-1 for the female group; 261 (60), 327 (75), and 354 (81) μg·Kg-1 for the male group; and 242 (70), 305 (88), and 330 (95) μg·Kg-1 for the mixed group. No significant differences in measures of effect were found after bolus administration in the third phase...(AU)

[Mechanisms of action of botulinum toxins and neurotoxins]. / Mécanismes d'action des toxines et neurotoxines botuliques.

Several bacteria of the Clostridium genus (C. botulinum) produce 150 kDa di-chainal protein toxins referred as botulinum neurotoxins or BoNTs. They associate with non-toxic companion proteins and form a complex termed botulinum toxin. BoNTs specifically inhibit vesicular neurotransmitter release. The cellular action of BoNTs can be depicted according to a multi-step model : The toxin's heavy chain mediates binding to specific receptors comprised of a ganglioside moiety and a vesicular protein (SV2 for BoNT type A, synaptotagmin for BoNT type B), followed by endocytotic internalisation of the BoNT/receptor complex. Vesicle recycling induces BoNT internalisation. Upon acidification of vesicles, the light chain of the neurotoxin is translocated into the cytosol. Here, this zinc-endopeptidase cleaves one or two among three synaptic proteins (VAMP-synapto-brevin, SNAP25, and syntaxin). As the three protein targets of BoNT play major role in fusion of synaptic vesicles at the release sites, their cleavage is followed by blockade of neurotransmitter exocytosis. Importantly, as the BoNT receptors and intracellular targets are present in all nerve terminals, the BoNTs are not specific for cholinergic transmission. Duration of their inhibitory action is mainly determined by the the life-time of the toxin's light chain in the cytosol. Sprouting of new nerve-endings, which are retracted when the poisoned nerve terminals have recovered full functionality, may lead to anticipated recovery of the poisoned nerve terminals.

Assay of diffusion of different botulinum neurotoxin type a formulations injected in the mouse leg.

Botulinum neurotoxin type-A (BoNT/A) is very effective in the therapy of a wide range of human syndromes characterized by hyperactivity of peripheral cholinergic nerve terminals. Little diffusion of this toxin from the site of injection is commonly observed, but even minor changes in this property would greatly affect the validity of the treatment. Different pharmacological formulations of BoNT/A are available, and they may have different diffusion characteristics due to protein complex size, product format, and pharmacological properties. Here we assessed the extent of diffusion of three commercial preparations of BoNT/A: Botox (Allergan), Dysport (Ipsen), and Xeomin (Merz Pharmaceuticals) using a novel and highly sensitive test based on neural cell adhesion molecule (N-CAM) expression in muscle. N-CAM is a membrane glycoprotein that accumulates on muscle fibers after denervation and is not expressed in untreated adult muscle. This allows fine monitoring of the functional diffusion of this toxin, and the sensitivity of this assay is emphasized by the use of the mouse model because of the small muscle dimensions. The results presented here indicate that there is no significant difference between Botox, Dysport, and Xeomin with respect to diffusion into adjacent muscles in the mouse leg.