Evaluation of phosphoramidon and three synthetic phosphonates for inhibition of botulinum neurotoxin B catalytic activity

Three putative metalloprotease inhibitors were synthesized and tested for their ability to inhibit the catalytic activity of botulinum neurotoxin B light chain (BoNT/B LC). The compounds were designed to emulate the naturally occurring metalloprotease inhibitor phosphoramidon, which has been reported to be a weak antagonist of BoNT/B action. All three analogs contained the dipeptide Phe-Glu in place of Leu-Trp of phosphoramidon and possessed a phenyl, ethyl or methyl group in place of the rhamnose sugar of the parent compound. The inhibitors were evaluated in a cell-free assay based on the detection of a fluorescent product following cleavage of a 50-mer synaptobrevin peptide ([Pya(88)] S 39-88) by BoNT/B LC. This peptide corresponds to the hydrophilic core of synaptobrevin-2 and contains a fluorescent analog L-pyrenylalanine (Pya) in place of Tyr(88). Cleavage of [Pya(88)] S 39-88 by BoNT/B LC gives rise to fragments of 38 and 12 amino acid residues. Quantification of BoNT/B-mediated substrate cleavage was achieved by separating the 12-mer fragment (FETSAAKLKRK-Pya) that contains the C-terminal fluorophore and measuring fluorescence at 377 nm. The results indicate that the phenyl-substituted synthetic compound ICD 2821 was slightly more active than phosphoramidon, but analogs with methyl or ethyl substitutions were relatively inactive. These findings suggest that phosphonate monoesters may be useful for providing insights into the structural requirement of BoNT/B protease inhibitors.     

Sputum processing for evaluation of inflammatory mediators

Neutrophil-dominated inflammation is prominent in the cystic fibrosis (CF) and chronic bronchitis (CB) airways. We assessed the degree of airway inflammation by measuring the sputum concentrations of interleukin (IL)-8, myeloperoxidase (MPO), and deoxyribonucleic acid (DNA). We determined the relationship among the concentrations of these mediators and investigated methodological problems that may be responsible for reported variability in measurements. Sputa obtained from 31 patients were solubilized with phosphate-buffered saline, dithiothreitol (DTT) (0.1% or 1%), or dornase alfa (0.2 mg/mL). The sputum concentration of IL-8 and MPO was measured by enzyme-linked immunosorbent assay (ELISA), and DNA was measured using microfluorimetry. There was a significant relationship among sputum IL-8, MPO, and DNA. For MPO (means +/- SD), CF was 1,392 +/- 771 vs. CB at 75 +/- 65 mcg/mL; P < 0.0001. For IL-8: CF was 239 +/- 154 vs. CB at 121 +/- 108 ng/mL; P = 0.0002. For DNA, CF was 1.707 +/- 1.25 vs. CB at 0.184 +/- 0.272 mg/mL; P < 0.0001. The MPO concentration in CF sputum was approximately double after in vitro treatment with dornase alfa (P < 0.0001). There is a greater concentration of IL-8, MPO, and DNA in CF than in CB sputa. There is a significant relationship among these inflammatory markers in sputum. DNA polymers bind myeloperoxidase in the sputum, and we speculate that treatment with dornase alfa may remove a source of MPO inhibition.     

Cross-Modal Selective Attention Effects on Retinal, Myogenic, Brainstem, and Cerebral Evoked Potentials

Short latency evoked potentials were recorded during a cross-modal selective attention task to evaluate recent proposals that sensory transmission in the peripheral auditory and visual pathways can be modified selectively by centrifugal mechanisms in humans. Twenty young adult subjects attended in turn to either left-ear tones or right-field flashes presented in a randomized sequence, in order to detect infrequent, lower-intensity targets. Attention-related enhancement of longer-latency components, including the visual P105 and the auditory N1/Nd waves and T-complex, showed that subjects were able to adopt a selective sensory set toward either modality. Neither the auditory evoked brainstem potentials nor the early visual components (electroretinogram, occipito-temporal N40, P50, N70 waves) were significantly affected by attention. Measures of retinal B-waves were significantly reduced in amplitude when attention was directed to the flashes, but concurrent recordings of eyelid electromyographic activity and the electro-oculogram indicated that this effect may have resulted from contamination of the retinal recordings by blink microreflex activity. A trend toward greater positivity in the 15-50 ms latency range for auditory evoked potentials to attended tones was observed. These results provide further evidence that the earliest levels of sensory transmission are unaffected by cross-modal selective attention, but that longer latency exogenous and endogenous potentials are enhanced to stimuli in the attended modality.     

Reciprocal modulation of eye-blink and pinna-flexion components of startle during reward anticipation

Because expectancies play a central role in current theories of dopaminergic neuron function, it is important to develop measures of reward anticipation processes. In the present study, reflexogenic bursts of white noise were presented to 39 healthy young adults as they awaited rewards and punishments in a gambling-like task. The rewards were small pieces of chocolate; the punishments, segments of bitter-tasting banana peel. Consistent with prior research on affective valence, postauricular reflexes were larger prior to rewards than punishments, whereas the reverse was true for acoustic blink reflexes. We theorized that potentiation of the postauricular reflex prior to consuming appetizing food is related to the priming of ear-retraction musculature during nursing in our remote ancestors.     

Lead poisoning in pregnancy: a case study with implications for midwives

Lead poisoning remains a significant problem in the United States affecting the health of women and children. Although the damage is greatest at higher levels of accumulation, no level of lead has been found to be safe. Over the last 25 years, the blood level of lead thought to be associated with toxicity has dropped dramatically, from to 60 mcg/dL in 1960 to 10 mcg/dL today. Studies confirm that exposure to lead causes kidney damage, encephalopathy, and impaired cognitive function in children and in adults. Recent evidence indicates children with levels less than 10 mcg/dL may suffer from compromised development and intellectual performance later in life. This article discusses the case of a woman found to have lead poisoning during pregnancy. Environmental sources of lead, implications for the immediate and future health of the fetus and mother, and techniques clinicians can use in their practice to minimize the adverse effect of lead on their clients are reviewed.     

Identification of correlates of physical activity among Latino adults

127 Latino adults responded to a survey concerning physical activity. Respondents over-represented well educated and middle class Latinos. Subjects reported a mean of 48 minutes/week of walking for exercise. This sample reported less than two episodes per week of vigorous physical activity, again below the recommended 3/wk needed to insure cardiovascular fitness. We expect more representative samples to engage in less physical activity. Stepwise multiple regression analyses were conducted using 24 variables based on Social Learning Theory. A multiple R of 0.66 accounted for over 27% of the variance in walking for exercise (p<0.001). Older adults, those with a history of childhood injury, and those who reported friend support were more likely to walk for exercise. Respondents who participated in physical activity during childhood and adolescence (including formal physical education in school) and, paradoxically, those who had models for exercise in childhood were less likely to walk for exercise. A multiple R of 0.75 accounted for 43% of the variance in vigorous physical activity and reached significance (p<0.001). Self-efficacy, friends' support, childhood physical activity, and eating a heart healthy diet were positively related to vigorous activity. These results suggest that different correlates influence walking versus vigorous activity, and that correlates of physical activity are different for Latinos compared to Anglos. The findings emphasize the need for larger scale investigations of the determinants of activity within the Latino population.M. Hovell, E. Barrington, M. Hackley, J. Elder, F. Castro, Kristin Kilbourne; Division of Health Promotion, Graduate School of Public Health, San Diego State University, San Diego, CA 92182, J. Sallis; Department of Psychology, San Diego State University, San Diego, CA 92182, R. Hofstetter; Department of Political Science, San Diego State University, San Diego, CA 92182.This research was supported by grants to Drs. Hovell and Sallis: ASPH/CDC Cooperative Agreement Contract; NHLBI (40575).     

Prepulse inhibition and facilitation of the postauricular reflex,a vestigial remnant of pinna startle

If the postauricular reflex (PAR) is to be used effectively in studies of emotion and attention, its sensitivity to basic modulatory effects such as prepulse inhibition and facilitation must be determined. Two experiments were carried out with healthy young adults to assess the effects of transient and sustained visual prestimuli on the pinna‐flexion response to trains of startle probes. In the first experiment, participants passively viewed a small white square. It was displayed from 1,000 ms prior to onset of a train of noise bursts until the end of that train. Relative to no‐prepulse control trials, PAR amplitude was inhibited, possibly due to the withdrawal of attentional resources from the auditory modality. In the second experiment, participants performed a visual oddball task in which irrelevant trains of startle probes followed most briefly displayed task stimuli (checkerboards). Prepulse inhibition was observed when a transient stimulus preceded the first probe at a lead time of 100 ms. Amplitude facilitation was observed at longer lead times. In addition to documenting the existence of prepulse inhibition and facilitation, the data suggest that the PAR is not elicited by visual stimuli, that temporal expectancy does not influence its amplitude or latency, and that this vestigial microreflex is resistant to habituation. Results are interpreted in light of a recent theory that the human PAR is a highly degraded pinna startle, in which the reflex arc no longer includes the startle center (nucleus reticularis pontis caudalis).     

Changes in the stimulus‐preceding negativity and lateralized readiness potential during reinforcement learning

According to reinforcement learning theory, dopamine‐dependent anticipatory processes play a critical role in learning from action outcomes such as feedback or reward. To better understand outcome anticipation, we examined variation in slow cortical potentials and assessed their changes over the course of motor‐skill acquisition. Healthy young adults learned a series of precisely timed, key press sequences. Feedback was delivered at a delay of either 2.5 or 8 s, to encourage use of either the striatally mediated, habit learning system or the hippocampus‐dependent, episodic memory system, respectively. During the 2.5‐s delay, the stimulus‐preceding negativity (SPN) was shown to decline in amplitude across trials, confirming previous results from a perceptual categorization task (Morís, Luque, & Rodríguez‐Fornells, 2013). This falsifies the hypothesis that SPN reflects specific outcome predictions, on the assumption that the ability to make such predictions should improve as a task is mastered. An SPN was also evident during the 8‐s delay, but it increased in amplitude across trials. At the conclusion of the 8‐s but not the 2.5‐s prefeedback interval, a reversed‐polarity lateralized readiness potential (LRP) was noted. It was suggested that this might indicate maintenance of an action representation for comparison with the feedback display. If so, this would constitute the first direct psychophysiological evidence for a popular hypothetical construct in quantitative models of reinforcement learning, the so‐called eligibility trace.