Proteomic analysis of coronary sinus serum reveals leucine-rich α2-glycoprotein as a novel biomarker of ventricular dysfunction and heart failure

BACKGROUND: Heart failure (HF) prevention strategies require biomarkers that identify disease manifestation. Increases in B-type natriuretic peptide (BNP) correlate with increased risk of cardiovascular events and HF development. We hypothesize that coronary sinus serum from a high BNP hypertensive population reflects an active pathological process and can be used for biomarker exploration. Our aim was to discover differentially expressed disease-associated proteins that identify patients with ventricular dysfunction and HF.

METHODS AND RESULTS: Coronary sinus serum from 11 asymptomatic, hypertensive patients underwent quantitative differential protein expression analysis by 2-dimensional difference gel electrophoresis. Proteins were identified using mass spectrometry and then studied by enzyme-linked immunosorbent assay in sera from 40 asymptomatic, hypertensive patients and 105 patients across the spectrum of ventricular dysfunction (32 asymptomatic left ventricular diastolic dysfunction, 26 diastolic HF, and 47 systolic HF patients). Leucine-rich α2-glycoprotein (LRG) was consistently overexpressed in high BNP serum. LRG levels correlate significantly with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF, and systolic HF patient groups (P≤0.05). LRG levels were able to identify HF independent of BNP. LRG correlates with coronary sinus serum levels of tumor necrosis factor-α (P=0.009) and interleukin-6 (P=0.021). LRG is expressed in myocardial tissue and correlates with transforming growth factor-βR1 (P<0.001) and α-smooth muscle actin (P=0.025) expression.

CONCLUSIONS: LRG was identified as a serum biomarker that accurately identifies patients with HF. Multivariable modeling confirmed that LRG is a stronger identifier of HF than BNP and this is independent of age, sex, creatinine, ischemia, β-blocker therapy, and BNP.

Locomotor Muscle Fatigue Does Not Alter Oxygen Uptake Kinetics during High-Intensity Exercise

The V˙O2 slow component (V˙O2sc) that develops during high-intensity aerobic exercise is thought to be strongly associated with locomotor muscle fatigue. We sought to experimentally test this hypothesis by pre-fatiguing the locomotor muscles used during subsequent high-intensity cycling exercise. Over two separate visits, eight healthy male participants were asked to either perform a non-metabolically stressful 100 intermittent drop-jumps protocol (pre-fatigue condition) or rest for 33 min (control condition) according to a random and counterbalanced order. Locomotor muscle fatigue was quantified with 6-s maximal sprints at a fixed pedaling cadence of 90 rev·min−1. Oxygen kinetics and other responses (heart rate, capillary blood lactate concentration and rating of perceived exertion, RPE) were measured during two subsequent bouts of 6 min cycling exercise at 50% of the delta between the lactate threshold and V˙O2max determined during a preliminary incremental exercise test. All tests were performed on the same cycle ergometer. Despite significant locomotor muscle fatigue (P = 0.03), the V˙O2sc was not significantly different between the pre-fatigue (464 ± 301 mL·min−1) and the control (556 ± 223 mL·min−1) condition (P = 0.50). Blood lactate response was not significantly different between conditions (P = 0.48) but RPE was significantly higher following the pre-fatiguing exercise protocol compared with the control condition (P < 0.01) suggesting higher muscle recruitment. These results demonstrate experimentally that locomotor muscle fatigue does not significantly alter the V˙O2 kinetic response to high intensity aerobic exercise, and challenge the hypothesis that the V˙O2sc is strongly associated with locomotor muscle fatigue.

DATA DRIVEN APPROACHES TO IDENTIFY DETERMINANTS OF HEART DISEASES AND CANCER RESISTANCE

Cancer and cardio-vascular diseases are the leading causes of death world-wide. Caused by systemic genetic and molecular disruptions in cells, these disorders are the manifestation of profound disturbance of normal cellular homeostasis. People suffering or at high risk for these disorders need early diagnosis and personalized therapeutic intervention. Successful implementation of such clinical measures can significantly improve global health. However, development of effective therapies is hindered by the challenges in identifying genetic and molecular determinants of the onset of diseases; and in cases where therapies already exist, the main challenge is to identify molecular determinants that drive resistance to the therapies. Due to the progress in sequencing technologies, the access to a large genome-wide biological data is now extended far beyond few experimental labs to the global research community. The unprecedented availability of the data has revolutionized the capabilities of computational researchers, enabling them to collaboratively address the long standing problems from many different perspectives. Likewise, this thesis tackles the two main public health related challenges using data driven approaches. Numerous association studies have been proposed to identify genomic variants that determine disease. However, their clinical utility remains limited due to their inability to distinguish causal variants from associated variants. In the presented thesis, we first propose a simple scheme that improves association studies in supervised fashion and has shown its applicability in identifying genomic regulatory variants associated with hypertension. Next, we propose a coupled Bayesian regression approach -- eQTeL, which leverages epigenetic data to estimate regulatory and gene interaction potential, and identifies combinations of regulatory genomic variants that explain the gene expression variance. On human heart data, eQTeL not only explains a significantly greater proportion of expression variance in samples, but also predicts gene expression more accurately than other methods. We demonstrate that eQTeL accurately detects causal regulatory SNPs by simulation, particularly those with small effect sizes. Using various functional data, we show that SNPs detected by eQTeL are enriched for allele-specific protein binding and histone modifications, which potentially disrupt binding of core cardiac transcription factors and are spatially proximal to their target. eQTeL SNPs capture a substantial proportion of genetic determinants of expression variance and we estimate that 58% of these SNPs are putatively causal. The challenge of identifying molecular determinants of cancer resistance so far could only be dealt with labor intensive and costly experimental studies, and in case of experimental drugs such studies are infeasible. Here we take a fundamentally different data driven approach to understand the evolving landscape of emerging resistance. We introduce a novel class of genetic interactions termed synthetic rescues (SR) in cancer, which denotes a functional interaction between two genes where a change in the activity of one vulnerable gene (which may be a target of a cancer drug) is lethal, but subsequently altered activity of its partner rescuer gene restores cell viability. Next we describe a comprehensive computational framework --termed INCISOR-- for identifying SR underlying cancer resistance. Applying INCISOR to mine The Cancer Genome Atlas (TCGA), a large collection of cancer patient data, we identified the first pan-cancer SR networks, composed of interactions common to many cancer types. We experimentally test and validate a subset of these interactions involving the master regulator gene mTOR. We find that rescuer genes become increasingly activated as breast cancer progresses, testifying to pervasive ongoing rescue processes. We show that SRs can be utilized to successfully predict patients' survival and response to the majority of current cancer drugs, and importantly, for predicting the emergence of drug resistance from the initial tumor biopsy. Our analysis suggests a potential new strategy for enhancing the effectiveness of existing cancer therapies by targeting their rescuer genes to counteract resistance. The thesis provides statistical frameworks that can harness ever increasing high throughput genomic data to address challenges in determining the molecular underpinnings of hypertension, cardiovascular disease and cancer resistance. We discover novel molecular mechanistic insights that will advance the progress in early disease prevention and personalized therapeutics. Our analyses sheds light on the fundamental biological understanding of gene regulation and interaction, and opens up exciting avenues of translational applications in risk prediction and therapeutics.

The study of histopathological damage in heart and bulbus arteriosus in rainbow trout

This study investigated the pathological changes of heart and bulbus artrius of rainbow trout breeders in several group of ages and density. The aim of study was to consider the process and the intensity of the heart and bulbus arteriosus damages in accordance to gender, age and stocking density of trout in three fish culture center (Zarghezel, Niyak in Haraz Region,Mazandaran and Espiran in Tabriz city environs). In field research, the all records the feed and feeding type, rate of mortality, stocking density of spawners and per spawners fishes, water chemical and physical specification was screened. Stocking density was considered as the most important stressor. 10 fish specimens from 7 weight groups (less than 90g, 90 to 300g, 300 to 500 g, 500 to 1000g, 1 to 3 kg, 3 to 5 kg, over 5 kg), totally 210 specimens were sampled and heart and bulbus arteriosus were taken. Samples were fixed in 10 % formalin and transferred to pathology laboratory of veterinary faculty of Tabriz Azad University. Histopathological slides and H&E staining were prepared from these samples. In total, 47 male and 73 female samples showed cardiovascular injury (29 cases in extensive system, 41 cases in semi intensive system, 50 cases in intensive system). The most important was damages, edema and hyperemia in spongy layer of atrium and ventricle muscles, but degeneration the muscle fibers, moderate edema , minor vascular damage. Hemorrhage as the effect of severs vascular damage, thrombus, sever inflammation, sever degeneration in muscle fiber, necrosis and fibrose were further pathological changed. The results of this study showed that the severity of damage increased by increasing the age (weight) of fishes. This situation was seen in all three culturing system (extensive system, semi intensive system, recirculation system). Histopathological changes is obviously seen in samples over 500g, therefore the damages were found to be important (P<0.05). Pathological effects and its severity in recirculation system was significantly high (P<0.05). Comparison with two other culturing system, histopathological changed in heart and bulbus arterius between male and female was significantly different.

Non-invasive detection of acute cell-mediated graft rejection in paediatric heart transplant recipients: the role of cardiovascular magnetic resonance

Background and Aim: Acute cardiac rejection is currently diagnosed by endomyocardial biopsy (EMB), but multiparametric cardiac magnetic resonance (CMR) may be a non-invasive alternative by its capacity for myocardial structure and function characterization. Our primary aim was to determine the utility of multiparametric CMR in identifying acute graft rejection in paediatric heart transplant recipients. The second aim was to compare textural features of parametric maps in cases of rejection versus those without rejection. Methods: Fifteen patients were prospectively enrolled for contrast-enhanced CMR followed by EMB and right heart catheterization. Images were acquired on a 1,5 Tesla scanner including T1 mapping (modified Look-Locker inversion recovery sequence – MOLLI) and T2 mapping (modified GraSE sequence). The extracellular volume (ECV) was calculated using pre- and post-gadolinium T1 times of blood and myocardium and the patient’s hematocrit. Markers of graft dysfunction including hemodynamic measurements from echocardiography, catheterization and CMR were collated. Patients were divided into two groups based on degree of rejection at EMB: no rejection with no change in treatment (Group A) and acute rejection requiring new therapy (Group B). Statistical analysis included student’t t test and Pearson correlation. Results: Acute rejection was diagnosed in five patients. Mean T1 values were significantly associated with acute rejection. A monotonic, increasing trend was noted in both mean and peak T1 values, with increasing degree of rejection. ECV was significantly higher in Group B. There was no difference in T2 signal between two groups. Conclusion: Multiparametric CMR serves as a noninvasive screening tool during surveillance encounters and may be used to identify those patients that may be at higher risk of rejection and therefore require further evaluation. Future and multicenter studies are necessary to confirm these results and explore whether multiparametric CMR can decrease the number of surveillance EMBs in paediatric heart transplant recipients.

The Effect Of Pelvic Floor Muscle Training Alone Or In Combination With Electrostimulation In The Treatment Of Sexual Dysfunction In Women With Multiple Sclerosis.

Sexual dysfunction (SD) affects up to 80% of multiple sclerosis (MS) patients and pelvic floor muscles (PFMs) play an important role in the sexual function of these patients. The objective of this paper is to evaluate the impact of a rehabilitation program to treat lower urinary tract symptoms on SD of women with MS. Thirty MS women were randomly allocated to one of three groups: pelvic floor muscle training (PFMT) with electromyographic (EMG) biofeedback and sham neuromuscular electrostimulation (NMES) (Group I), PFMT with EMG biofeedback and intravaginal NMES (Group II), and PFMT with EMG biofeedback and transcutaneous tibial nerve stimulation (TTNS) (Group III). Assessments, before and after the treatment, included: PFM function, PFM tone, flexibility of the vaginal opening and ability to relax the PFMs, and the Female Sexual Function Index (FSFI) questionnaire. After treatment, all groups showed improvements in all domains of the PERFECT scheme. PFM tone and flexibility of the vaginal opening was lower after the intervention only for Group II. All groups improved in arousal, lubrication, satisfaction and total score domains of the FSFI questionnaire. This study indicates that PFMT alone or in combination with intravaginal NMES or TTNS contributes to the improvement of SD.

Impact Of Benign Prostatic Hyperplasia Pharmacological Treatment On Transrectal Prostate Biopsy Adverse Effects.

Background. Benign prostatic hyperplasia (BPH) pharmacological treatment may promote a decrease in prostate vascularization and bladder neck relaxation with theoretical improvement in prostate biopsy morbidity, though never explored in the literature. Methods. Among 242 consecutive unselected patients who underwent prostate biopsy, after excluding those with history of prostate biopsy/surgery or using medications not for BPH, we studied 190 patients. On the 15th day after procedure patients were questioned about symptoms lasting over a week and classified according to pharmacological BPH treatment. Results. Thirty-three patients (17%) were using alpha-blocker exclusively, five (3%) 5-alpha-reductase inhibitor exclusively, twelve (6%) patients used both medications, and 140 (74%) patients used none. There was no difference in regard to age among groups (P = 0.5). Postbiopsy adverse effects occurred as follows: hematuria 96 (50%), hematospermia 53 (28%), hematochezia 22 (12%), urethrorrhagia 19 (10%), fever 5 (3%), and pain 20 (10%). There was a significant negative correlation between postbiopsy hematuria and BPH pharmacological treatment with stronger correlation for combined use of 5-alpha-reductase inhibitor and alpha-blocker over 6 months (P = 0.0027). Conclusion. BPH pharmacological treatment, mainly combined for at least 6 months seems to protect against prostate biopsy adverse effects. Future studies are necessary to confirm our novel results.

Rat Atrial Responses To Bothrops Jararacussu (jararacuçu) Snake Venom.

Envenoming by the pitviper Bothrops jararacussu produces cardiovascular alterations, including coagulopathy, systemic hemorrhage, hypotension, circulatory shock and renal failure. In this work, we examined the activity of this venom in rat isolated right atria. Incubation with venom (0.025, 0.05, 0.1 and 0.2mg/ml) caused concentration-dependent muscle contracture that was not reversed by washing. Muscle damage was seen histologically and confirmed by quantification of creatine kinase-MB (CK-MB) release. Heating and preincubation of venom with p-bromophenacyl bromide (a phospholipase A2 inhibitor) abolished the venom-induced contracture and muscle damage. In contrast, indomethacin, a non-selective inhibitor of cyclooxygenase, and verapamil, a voltage-gated Ca(2+) channel blocker, did not affect the responses to venom. Preincubation of venom with Bothrops or Bothrops/Crotalus antivenom or the addition of antivenom soon after venom attenuated the venom-induced changes in atrial function and tissue damage. These results indicate that B. jararacussu venom adversely affected rat atrial contractile activity and muscle organization through the action of venom PLA2; these venom-induced alterations were attenuated by antivenom.

Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, And Increased Expression Of Extracellular Matrix Proteins In Laminin α2 Chain-deficient Muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Effect Of Exercise Test On Pulmonary Function Of Obese Adolescents.

to investigate the pulmonary response to exercise of non-morbidly obese adolescents, considering the gender. a prospective cross-sectional study was conducted with 92 adolescents (47 obese and 45 eutrophic), divided in four groups according to obesity and gender. Anthropometric parameters, pulmonary function (spirometry and oxygen saturation [SatO2]), heart rate (HR), blood pressure (BP), respiratory rate (RR), and respiratory muscle strength were measured. Pulmonary function parameters were measured before, during, and after the exercise test. BP and HR were higher in obese individuals during the exercise test (p = 0.0001). SatO2 values decreased during exercise in obese adolescents (p = 0.0001). Obese males had higher levels of maximum inspiratory and expiratory pressures (p = 0.0002) when compared to obese and eutrophic females. Obese males showed lower values of maximum voluntary ventilation, forced vital capacity, and forced expiratory volume in the first second when compared to eutrophic males, before and after exercise (p = 0.0005). Obese females had greater inspiratory capacity compared to eutrophic females (p = 0.0001). Expiratory reserve volume was lower in obese subjects when compared to controls (p ≤ 0,05). obese adolescents presented changes in pulmonary function at rest and these changes remained present during exercise. The spirometric and cardiorespiratory values were different in the four study groups. The present data demonstrated that, in spite of differences in lung growth, the model of fat distribution alters pulmonary function differently in obese female and male adolescents.

Relationship Between Lower-limb Muscle Strength And Functional Independence Among Elderly People According To Frailty Criteria: A Cross-sectional Study.

Muscle strength and functional independence are considered to be determinants of frailty levels among elderly people. The aim here was to compare lower-limb muscle strength (LLMS) with functional independence in relation to sex, age and number of frailty criteria, and to ascertain the influence of these variables on elderly outpatients' independence. Quantitative cross-sectional study, in a tertiary hospital. The study was conducted on 150 elderly outpatients of both sexes who were in a cognitive condition allowing oral communication, between October 2005 and October 2007. The following instruments were used: five-times sit-to-stand test (FTSST), Functional Independence Measurement (FIM) and Lawton's Instrumental Activities of Daily Living Scale (IADL). Descriptive, comparative, multivariate, univariate and Cronbach alpha analyses were performed. The mean time taken in the FTSST was 21.7 seconds; the mean score for FIM was 82.2 and for IADL was 21.2; 44.7% of the subjects presented 1-2 frailty criteria and 55.3% > 3 criteria. There was a significant association between LLMS and functional independence in relation to the number of frailty criteria, without homogeneity regarding sex and age. Functional independence showed significant influence from sex and LLMS. Elderly individuals with 1 or 2 frailty criteria presented greater independence in all FTSST scores. The subjects with higher LLMS presented better functional independence.

Granulocyte Colony-stimulating Factor (g-csf) Positive Effects On Muscle Fiber Degeneration And Gait Recovery After Nerve Lesion In Mdx Mice.

G-CSF has been shown to decrease inflammatory processes and to act positively on the process of peripheral nerve regeneration during the course of muscular dystrophy. The aims of this study were to investigate the effects of treatment of G-CSF during sciatic nerve regeneration and histological analysis in the soleus muscle in MDX mice. Six-week-old male MDX mice underwent left sciatic nerve crush and were G-CSF treated at 7 days prior to and 21 days after crush. Ten and twenty-one days after surgery, the mice were euthanized, and the sciatic nerves were processed for immunohistochemistry (anti-p75(NTR) and anti-neurofilament) and transmission electron microscopy. The soleus muscles were dissected out and processed for H&E staining and subsequent morphologic analysis. Motor function analyses were performed at 7 days prior to and 21 days after sciatic crush using the CatWalk system and the sciatic nerve index. Both groups treated with G-CSF showed increased p75(NTR) and neurofilament expression after sciatic crush. G-CSF treatment decreased the number of degenerated and regenerated muscle fibers, thereby increasing the number of normal muscle fibers. The reduction in p75(NTR) and neurofilament indicates a decreased regenerative capacity in MDX mice following a lesion to a peripheral nerve. The reduction in motor function in the crushed group compared with the control groups may reflect the cycles of muscle degeneration/regeneration that occur postnatally. Thus, G-CSF treatment increases motor function in MDX mice. Nevertheless, the decrease in baseline motor function in these mice is not reversed completely by G-CSF.

Morphological Aspects And Cox-2 Expression After Exposure To 780-nm Laser Therapy In Injured Skeletal Muscle: An In Vivo Study.

The effectiveness of low-level laser therapy in muscle regeneration is still not well known. To investigate the effects of laser irradiation during muscle healing. For this purpose, 63 rats were distributed to 3 groups: non-irradiated control group (CG); group irradiated at 10 J/cm(2) (G10); and group irradiated at 50 J/cm(2) (G50). Each group was divided into 3 different subgroups (n=7), and on days 7, 14 and 21 post-injury the rats were sacrificed. Seven days post-surgery, the CG showed destroyed zones and extensive myofibrillar degeneration. For both treated groups, the necrosis area was smaller compared to the CG. On day 14 post-injury, treated groups demonstrated better tissue organization, with newly formed muscle fibers compared to the CG. On the 21(st) day, the irradiated groups showed similar patterns of tissue repair, with improved muscle structure at the site of the injury, resembling uninjured muscle tissue organization. Regarding collagen deposition, the G10 showed an increase in collagen synthesis. In the last period evaluated, both treated groups showed statistically higher values in comparison with the CG. Furthermore, laser irradiation at 10 J/cm(2) produced a down-regulation of cyclooxygenase 2 (Cox-2) immunoexpression on day 7 post-injury. Moreover, Cox-2 immunoexpression was decreased in both treated groups on day 14. Laser therapy at both fluencies stimulated muscle repair through the formation of new muscle fiber, increase in collagen synthesis, and down-regulation of Cox-2 expression.

Value Of Sentinel Lymph Node Biopsy In Papillary Thyroid Cancer: Initial Results Of A Prospective Trial.

The objectives of the study were to evaluate the performance of sentinel lymph node biopsy (SLNB) in detecting occult metastases in papillary thyroid carcinoma (PTC) and to correlate their presence to tumor and patient characteristics. Twenty-three clinically node-negative PTC patients (21 females, mean age 48.4 years) were prospectively enrolled. Patients were submitted to sentinel lymph node (SLN) lymphoscintigraphy prior to total thyroidectomy. Ultrasound-guided peritumoral injections of (99m)Tc-phytate (7.4 MBq) were performed. Cervical single-photon emission computed tomography and computed tomography (SPECT/CT) images were acquired 15 min after radiotracer injection and 2 h prior to surgery. Intra-operatively, SLNs were located with a gamma probe and removed along with non-SLNs located in the same neck compartment. Papillary thyroid carcinoma, SLNs and non-SLNs were submitted to histopathology analysis. Sentinel lymph nodes were located in levels: II in 34.7 % of patients; III in 26 %; IV in 30.4 %; V in 4.3 %; VI in 82.6 % and VII in 4.3 %. Metastases in the SLN were noted in seven patients (30.4 %), in non-SLN in three patients (13.1 %), and in the lateral compartments in 20 % of patients. There were significant associations between lymph node (LN) metastases and the presence of angio-lymphatic invasion (p = 0.04), extra-thyroid extension (p = 0.03) and tumor size (p = 0.003). No correlations were noted among LN metastases and patient age, gender, stimulated thyroglobulin levels, positive surgical margins, aggressive histology and multifocal lesions. Sentinel lymph node biopsy can detect occult metastases in PTC. The risk of a metastatic SLN was associated with extra-thyroid extension, larger tumors and angio-lymphatic invasion. This may help guide future neck dissection, patient surveillance and radioiodine therapy doses.

The Neuromuscular Activity Of Bothriopsis Bilineata Smaragdina (forest Viper) Venom And Its Toxin Bbil-tx (asp49 Phospholipase A2) On Isolated Mouse Nerve-muscle Preparations.

The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA2 from this venom, was examined in detail in mouse phrenic nerve-muscle (PND) preparations in vitro and in a neuroblastoma cell line (SK-N-SH) in order to gain a better insight into the mechanism of action of the venom and associated Asp49 PLA2. In low Ca(2+) solution, venom (3μg/ml) caused a quadriphasic response in PND twitch height whilst at 10μg/ml the venom additionally induced an abrupt and marked initial contracture followed by neuromuscular facilitation, rhythmic oscillations of nerve-evoked twitches, alterations in baseline and progressive blockade. The venom slowed the relaxation phase of muscle twitches. In low Ca(2+), Bbil-TX [210nM (3μg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10μg/ml) and Bbil-TX (210nM) caused minor changes in the compound action potential (CAP) amplitude recorded from sciatic nerve preparations, with no significant effect on rise time and latency; tetrodotoxin (3.1nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle (TSn-m) preparations, venom (10μg/ml) and Bbil-TX (210nM) significantly reduced the perineural waveform associated with the outward K(+) current while the amplitude of the inward Na(+) current was not significantly affected. Bbil-TX (210nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca(2+) solution. Venom (3μg/ml) and toxin (210nM) increased the calcium fluorescence in SK-N-SH neuroblastoma cells loaded with Fluo3 AM and maintained in low or normal Ca(2+) solution. In normal Ca(2+), the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10μg/ml) caused an immediate increase in intracellular Ca(2+) followed by oscillations in fluorescence and muscle contracture; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K(+) channel activity and presynaptic protein expression.