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One of the more prominent early childhood interventions focused on the development of executive function (EF) skills is Tools of the Mind (Tools; Bodrova and Leong, 2019). Intervention studies comparing Tools classrooms with control classrooms, however, reveal inconsistent findings for children’s EF outcomes. The current study utilizes Head Start CARES teachers assigned to the Tools of the Mind enhancement intervention (Tools; N = 75) and the children in their classrooms (N = 738). Relations between teachers’ characteristics (i.e., teaching experience, psychological well-being, and educational background), training attendance and implementation (i.e., coach rated fidelity and observed scaffolding), and the interaction among these factors were examined as predictors of classroom-level gains in EF. Results revealed several significant moderation effects indicating that Tools implementation is related to classroom EF gains for some but not all teachers.

Prof. Ugo Carraro reached 80 years of age on 23 February 2023, and we wish to celebrate him and his work by reviewing his lifetime of scientific achievements in Translational Myology. Currently, he is a Senior Scholar with the University of Padova, Italy, where, as a tenured faculty member, he founded the Interdepartmental Research Center of Myology. Prof. Carraro, a pioneer in skeletal muscle research, is a world-class expert in structural and molecular investigations of skeletal muscle biology, physiology, pathology, and care. An authority in bidimensional gel electrophoresis for myosin light chains, he was the first to separate mammalian muscle myosin heavy chain isoforms by SDS-gel electrophoresis. He has demonstrated that long-term denervated muscle can survive denervation by myofiber regeneration, and shown that an athletic lifestyle has beneficial impacts on muscle reinnervation. He has utilized his expertise in translational myology to develop and validate rehabilitative treatments for denervated and ageing skeletal muscle. He has authored more than 160 PubMed listed papers and numerous scholarly books, including his recent autobiography. Prof. Carraro founded and serves as Editor-in-Chief of the European Journal of Translational Myology and Mobility Medicine. He has organized more than 40 Padua Muscle Days Meetings and continues this, encouraging students and young scientists to participate. As he dreams endlessly, he is currently validating non-invasive analyses on saliva, a promising approach that will allow increased frequency sampling to analyze systemic factors during the transient effects of training and rehabilitation by his proposed Full-Body in- Bed Gym for bed-ridden elderly.

Methamphetamine (MA) abuse is related to risks to the cardiovascular system. The present study aimed to compare the effects of moderate-intensity aerobic training (MIAT) and vitamin E (Vit.E) supplementation on markers of cardiac apoptosis following MA exposure. Fifty-four rats were randomly divided into six groups. CON group did not receive MA, while the others received MA alone or in combination with MIAT, Vit. E, MIAT+Vit E, or paraffin (PAR). These groups received MA incrementally for 23 consecutive days. Vit.E and MIAT+Vit.E groups received vitamin E three times a week for six weeks. MIAT and MIAT+Vit.E groups exercised for 25-40 min. Immunohistochemical and gene expression analyses were performed on the heart tissues. Bax and TGF-β expression was significantly higher, while Bcl-2 and VEGF expression was significantly lower in the MA and PAR groups than in the other groups (p < 0.05). Bcl-2 and VEGF expression was higher, and Bax and TGF-β expression was significantly lower in the MIAT and MIAT+Vit.E groups than in the other groups (p < 0.05). In Vit.E treated groups, Bax and TGF-β expression were lower, and VEGF was higher than that in the MA and PAR groups, but higher than those in the CON, MIAT and MIAT+Vit.E groups. MA increased the expression of Bax and TGF-β, and decreased the expression of Bcl-2 and VEGF, suggesting increased cardiac apoptosis. In contrast, MIAT and Vit.E decreased the expression of Bax and TGF-β, suggesting a reduction in cardiac apoptosis induced by MA.

Background: The potassium channel encoded by the ether-a-gogo-related gene 1A ( erg1 a) has been detected in the atrophying skeletal muscle of mice experiencing either muscle disuse or cancer cachexia and further evidenced to contribute to muscle deterioration by enhancing ubiquitin proteolysis; however, to our knowledge, ERG1A has not been reported in human skeletal muscle., Methods and Results: Here, using immunohistochemistry, we detect ERG1A immunofluorescence in human Rectus abdominis skeletal muscle sarcolemma. Further, using single point brightness data, we report the detection of ERG1A immunofluorescence at low levels in the Rectus abdominis muscle sarcolemma of young adult humans and show that it trends toward greater levels (10.6%) in healthy aged adults. Interestingly, we detect ERG1A immunofluorescence at a statistically greater level (53.6%; p < 0.05) in the skeletal muscle of older cancer patients than in age-matched healthy adults. Importantly, using immunoblot, we reveal that lower mass ERG1A protein is 61.5% ( p < 0.05) more abundant in the skeletal muscle of cachectic older adults than in healthy age-matched controls. Additionally, we report that the ERG1A protein is detected in a cultured human rhabdomyosarcoma line that may be a good in vitro model for the study of ERG1A in muscle., Conclusions: The data demonstrate that ERG1A is detected more abundantly in the atrophied skeletal muscle of cancer patients, suggesting it may be related to muscle loss in humans as it has been shown to be in mice experiencing muscle atrophy as a result of malignant tumors.

Skeletal muscle atrophy may occur with disease, injury, decreased muscle use, starvation, and normal aging. No reliably effective treatments for atrophy are available, thus research into the mechanisms contributing to muscle loss is essential. The ERG1A K+ channel contributes to muscle loss by increasing ubiquitin proteasome proteolysis (UPP) in the skeletal muscle of both unweighted and cachectic mice. Because the mechanisms which produce atrophy vary based upon the initiating factor, here we investigate atrophy produced by denervation. Using immunohistochemistry and immunoblots, we demonstrate that ERG1A protein abundance increases significantly in the Gastrocnemius muscle of rodents 7 days after both sciatic nerve transection and hind limb unweighting. Further, we reveal that ectopic expression of a Merg1a encoded plasmid in normal mouse Gastrocnemius muscle has no effect on activity of the NFκB transcription factor family, a group of proteins which contribute to muscle atrophy by modulation of the UPP. Further, although NFκB activity increases significantly after denervation, we show that expression of a plasmid encoding a dominant negative Merg1a mutant in Gastrocnemius muscle prior to denervation, has no effect on NFκB activity. Thus, although the ERG1A K+ channel increases UPP, it does not do so through modulation of NFκB transcription factors., (© 2021 American Association of Neuropathologists, Inc. All rights reserved.)

Background: Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K + channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells., Methods: We transduced C 2 C 12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student's t test or two-way ANOVAs and SAS software as indicated., Results: Expression of human ERG1A in C 2 C 12 myotubes increased basal intracellular calcium concentration 51.7% (p < 0.0001; n = 177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p < 0.08; n = 24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p < 0.05; n = 6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p < 0.05; n = 6) in calpastatin which could contribute to enhanced calpain activity., Conclusions: Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

The ERG1A K + channel, which is partially responsible for repolarization of the cardiac action potential, has also been reported in skeletal muscle where it modulates ubiquitin proteolysis. Because ERG1A protein appears variably expressed in muscles composed of mixed fiber types, we hypothesized that its abundance in skeletal muscle might differ with fiber type. Indeed, skeletal muscle fibers vary in speed of contraction (fast or slow), which is mainly determined by myosin heavy chain (MyHC) isoform content, but a sarcolemmal K + channel might also modulate contraction speed. To test our hypothesis, we cryo-sectioned Soleus (SOL), Extensor Digitorum Longus (EDL), and Gastrocnemius muscles from five rats. These muscles were chosen because the SOL and EDL contain an abundance of slow- and fast-twitch fibers, respectively, while the Gastrocnemius has a more heterogeneous composition. The muscle sections were co-immunostained for the ERG1A protein and either the fast- or slow-twitch MyHC to identify fiber type. ERG1A fluorescence was then measured in the sarcolemma of each fiber type and compared. The data reveal that the ERG1A protein is more abundant in the fibers of the SOL than in the EDL muscles, suggesting ERG1A may be more abundant in the slow than the fast fibers, and this was confirmed with immunoblot. However, because of the homogeneity of fiber type within these muscles, it was not possible to get enough data from both fiber types within a single muscle to compare ERG1A composition within fiber type. However, immunohistochemistry of sections from the fiber type heterogeneous Gastrocnemius muscle reveals that slow fibers had, on average, a 17.2% greater ERG1A fluorescence intensity than fast fibers (p<0.03). Further, immunoblot reveals that ERG1A protein is 41.6% more abundant (p=0.051) in old than in young rat Gastrocnemius muscle. We postulate that this membrane bound voltage-gated channel may affect membrane characteristics, the duration of the action potential generated, and/or the speed of contraction. Indeed, ERG1A protein is more abundant in aged and atrophic skeletal muscle, both of which exhibit slower rates of contraction., Competing Interests: We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Aims: Previous studies have shown significant correlations between the acoustic characteristics of /w/ and speech intelligibility. To understand the articulatory pattern in /w/ production, the current study investigated the movement characteristics of the tongue and lower lip in whip productions in comparison to bib productions., Methods: Data from 19 typical adult male participants were collected using electromagnetic articulography. A number of kinematic measures of tongue body (approximately 25 mm posterior to the tongue apex) and lower lip movements were analyzed., Results: The findings suggested different movement patterns of the tongue body and lower lip in whip and bib productions. The amount of lip movement in the anterior-posterior plane exhibited by each speaker did not vary between whip and bib. However, speakers did exhibit more tongue retraction/advancement, greater overall tongue movement, and faster tongue movement speed in whip than in bib., Conclusion: Tongue movement in the anterior-posterior plane, such as tongue retraction and advancement, was found to be crucial for the production of /w/ in typical male speakers. Thus, the articulatory kinematics of the tongue in the anterior-posterior plane and its coordination with the lips need to be further examined in speakers with dysarthria to understand the underlying kinematic characteristics that influence speech intelligibility., (© 2016 S. Karger AG, Basel.)

Skeletal muscle (SKM) atrophy is a potentially debilitating condition induced by muscle disuse, denervation, many disease states, and aging. The ubiquitin proteasome pathway (UPP) contributes greatly to the protein loss suffered in muscle atrophy. The MERG1a K(+) channel is known to induce UPP activity and atrophy in SKM. It has been further demonstrated that the mouse ether-a-gogo-related gene (Merg)1a channel modulates expression of MURF1, an E3 ligase component of the UPP, while it does not affect expression of the UPP E3 ligase Mafbx/ATROGIN1. Because the UBR2 E3 ligase is known to participate in SKM atrophy, we have investigated the effect of Merg1a expression and hind limb suspension on Ubr2 expression. Here, we report that hind limb suspension results in a significant 25.6% decrease in mouse gastrocnemius muscle fiber cross sectional area (CSA) and that electro-transfer of Merg1a alone into gastrocnemius muscles yields a 15.3% decrease in CSA after 7 days. More interestingly, we discovered that hind limb suspension caused a significant 8-fold increase in Merg1a expression and a significant 4.7-fold increase in Ubr2 transcript after 4 days, while electro-transfer of Merg1a into gastrocnemius muscles resulted in a significant 6.2-fold increase in Merg1a transcript after 4 days but had no effect on Ubr2 expression. In summary, the MERG1a K(+) channel, known to induce atrophy and MURF1 E3 ligase expression, does not affect UBR2 E3 ligase transcript levels. Therefore, to date, the MERG1a channel's contribution to UPP activity appears mainly to be through up-regulation of Murf1 gene expression.

Introduction: We investigated the mechanism by which the MERG1a K+ channel increases ubiquitin proteasome proteolysis (UPP)., Methods: Hindlimb suspension and electro-transfer of Merg1a cDNA into mouse gastrocnemius muscles induced atrophy., Results: Atrophic gastrocnemius muscles of hindlimb-suspended mice express Merg1a, Murf1, and Mafbx genes. Electrotransfer of Merg1a significantly decreases muscle fiber size (12.6%) and increases UPP E3 ligase Murf1 mRNA (2.1-fold) and protein (23.7%), but does not affect Mafbx E3 ligase expression. Neither Merg1a-induced decreased fiber size nor Merg1a-induced increased Murf1 expression is curtailed significantly by coexpression of inactive HR-Foxo3a, a gene encoding a transcription factor known to induce Mafbx expression., Conclusions: The MERG1a K+ channel significantly increases expression of Murf1, but not Mafbx. We explored this expression pattern by expressing inactive Foxo3a and showing that it is not involved in MERG1a-mediated expression of Murf1. These findings suggest that MERG1a may not modulate Murf1 expression through the AKT/FOXO pathway., (Copyright © 2013 Wiley Periodicals, Inc.)

The Kv11.1 (also ERG1) K(+) channel underlies cardiac I(Kr), a current that contributes to repolarization in mammalian heart. In mice, I(Kr) current density decreases with development and studies suggest that changes in the structure and/or properties of the heteromultimeric I(Kr)/Kv11.1 channel are responsible. Here, using immunohistochemistry, we report that total Kv11.1 alpha subunit protein is more abundant in neonatal heart and is distributed throughout both adult and neonatal ventricles with greater abundance in epicardia. Immunoblots reveal that the alpha subunit alternative splice variant, Kv11.1a, is more abundant in adult heart while the Kv11.1b variant is more abundant in neonatal heart. Additionally, MinK channel subunit protein is shown to co-assemble with Kv11.1 protein and is more abundant in neonatal heart. In summary, Kv11.1/I(Kr) channel composition varies developmentally and the higher I(Kr) current density in neonatal heart is likely attributable to higher abundance of Kv11.1/I(Kr) channels, more specifically, the Kv11.1b splice variant.

Skeletal muscle atrophy results from an imbalance in protein degradation and protein synthesis and occurs in response to injury, various disease states, disuse, and normal aging. Current treatments for this debilitating condition are inadequate. More information about mechanisms involved in the onset and progression of muscle atrophy is necessary for development of more effective therapies. Here we show that expression of the mouse ether-a-go-go related gene (Merg1a) K+ channel is up-regulated in skeletal muscle of mice experiencing atrophy as a result of both malignant tumor expression and disuse. Further, ectopic expression of Merg1a in vivo induces atrophy in healthy wt-bearing mice, while expression of a dysfunctional Merg1a mutant suppresses atrophy in hindlimb-suspended mice. Treatment of hindlimb-suspended mice with astemizole, a known Merg1a channel blocker, inhibits atrophy in these animals. Importantly, in vivo expression of Merg1a in mouse skeletal muscle activates the ubiquitin proteasome pathway that is responsible for the majority of protein degradation that causes muscle atrophy, yet expression of a dysfunctional Merg1a mutant decreases levels of ubiquitin-proteasome proteolysis. Thus, expression of Merg1a likely initiates atrophy by activating ubiquitin-proteasome proteolysis. This gene and its product are potential targets for prevention and treatment of muscle atrophy.

Background: Electroporation (EP) is a widely used non-viral gene transfer method. We have attempted to develop an exact protocol to maximize DNA expression while minimizing tissue damage following EP of skeletal muscle in vivo. Specifically, we investigated the effects of varying injection techniques, electrode surface geometry, and plasmid mediums., Results: We found that as the amount of damage increased in skeletal muscle in response to EP, the level of beta-galactosidase (beta-gal) expression drastically decreased and that there was no evidence of beta-gal expression in damaged fibers. Two specific types of electrodes yielded the greatest amount of expression. We also discovered that DNA uptake in skeletal muscle following intra-arterial injection of DNA was significantly enhanced by EP. Finally, we found that DMSO and LipoFECTAMINE, common enhancers of DNA electroporation in vitro, had no positive effect on DNA electroporation in vivo., Conclusions: When injecting DNA intramuscularly, a flat plate electrode without any plasmid enhancers is the best method to achieve high levels of gene expression.

Background: Mutations in KCNE2 have been linked to long-QT syndrome (LQT6), yet KCNE2 protein expression in the ventricle and its functional role in native channels are not clear., Methods and Results: We detected KCNE2 protein in human, dog, and rat ventricles in Western blot experiments. Immunocytochemistry confirmed KCNE2 protein expression in ventricular myocytes. To explore the functional role of KCNE2, we studied how its expression was altered in 2 models of cardiac pathology and whether these alterations could help explain observed changes in the function of native channels, for which KCNE2 is a putative auxiliary (beta) subunit. In canine ventricle injured by coronary microembolizations, the rapid delayed rectifier current (I(Kr)) density was increased. Although the protein level of ERG (I(Kr) pore-forming, alpha, subunit) was not altered, the KCNE2 protein level was markedly reduced. These data are consistent with the effect of heterologously expressed KCNE2 on ERG and suggest that in canine ventricle, KCNE2 may associate with ERG and suppress its current amplitude. In aging rat ventricle, the pacemaker current (I(f)) density was increased. There was a significant increase in the KCNE2 protein level, whereas changes in the alpha-subunit (HCN2) were not significant. These data are consistent with the effect of heterologously expressed KCNE2 on HCN2 and suggest that in aging rat ventricle, KCNE2 may associate with HCN2 and enhance its current amplitude., Conclusions: KCNE2 protein is expressed in ventricles, and it can play diverse roles in ventricular electrical activity under (patho)physiological conditions.

Although electrophysiological remodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for a large population of mouse genes encoding ion channel subunits ("IonChips") were developed and exploited to investigate remodeling of ion channel transcripts associated with altered thyroid status in adult mouse ventricle. Functional consequences of hypo- and hyperthyroidism were evaluated with patch-clamp and ECG recordings. Hypothyroidism decreased heart rate and prolonged QTc duration. Opposite changes were observed in hyperthyroidism. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNA5, KCNB1, KCND2, and KCNK2 transcripts, whereas KCNQ1 and KCNE1 expression is increased. In hyperthyroidism, in contrast, KCNA5 and KCNB1 expression is increased and KCNQ1 and KCNE1 expression is decreased. Real-time RT-PCR validated these results. Consistent with microarray analysis, Western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in I(to,f) and I(K,slow) densities, and increased I(Ks) density in hypothyroid myocytes. In addition to effects on K+ channel transcripts, transcripts for the pacemaker channel HCN2 were decreased and those encoding the alpha1C Ca2+ channel (CaCNA1C) were increased in hypothyroid animals. The expression of Na+, Cl-, and inwardly rectifying K+ channel subunits, in contrast, were unaffected by thyroid hormone status. Taken together, these data demonstrate that thyroid hormone levels selectively and differentially regulate transcript expression for at least nine ion channel alpha- and beta-subunits. Our results also document the potential of cDNA microarray analysis for the simultaneous examination of ion channel transcript expression levels in the diseased/remodeled myocardium.

The voltage-gated K(+) channel (Kv) pore forming alpha subunit, ERG1 (KCNH2), has been identified as the locus of mutations in one type of inherited long QT syndrome, LQT2. Heterologous expression of ERG1 reveals rapidly activating and inactivating K(+) currents, characterized by marked inward rectification at potentials positive to 0 mV, which are similar to the rapid component of cardiac delayed rectification I(Kr). There are, however, marked differences in the properties of expressed ERG1 and endogenous cardiac I(Kr), suggesting that functional I(Kr) channels reflect the coassembly of full-length ERG1 with splice variants and /or accessory subunits. Consistent with these hypotheses, N- and C-terminal variants of ERG1 have been identified, and it has been demonstrated that heterologously expressed ERG1 and minK (or MiRP1) coimmunoprecipitate. Recent biochemical studies, however, suggest that only full-length ERG1 is expressed in adult mouse, rat, or human heart. Clearly, further studies, focused on identifying the subunits that coassemble with ERG1 in vivo, as well as on post-translational processing of the full-length ERG1 protein will be necessary to define the molecular composition of functional cardiac I(Kr) channels.

One form of inherited long QT syndrome, LQT2, results from mutations in HERG1, the human ether-a-go-go-related gene, which encodes a voltage-gated K(+) channel alpha subunit. Heterologous expression of HERG1 gives rise to K(+) currents that are similar (but not identical) to the rapid component of delayed rectification, I(Kr), in cardiac myocytes. In addition, N-terminal splice variants of HERG1 and MERG1 (mouse ERG1) referred to as HERG1b and MERG1b have been cloned and suggested to play roles in the generation of functional I(Kr) channels. In the experiments here, antibodies generated against HERG1 were used to examine ERG1 protein expression in heart and in brain. In Western blots of extracts of QT-6 cells expressing HERG1, MERG1, or RERG1 (rat ERG1) probed with antibodies targeted against the C terminus of HERG1, a single 155-kDa protein is identified, whereas a 95-kDa band is evident in blots of extracts from cells expressing MERG1b or HERG1b. In immunoblots of fractionated rat (and mouse) brain and heart membrane proteins, however, two prominent high molecular mass proteins of 165 and 205 kDa were detected. Following treatment with glycopeptidase F, the 165- and 205-kDa proteins were replaced by two new bands at 175 and 130 kDa, suggesting that ERG1 is differentially glycosylated in rat/mouse brain and heart. In human heart, a single HERG1 protein with an apparent molecular mass of 145 kDa is evident. In rats, ERG1 protein (and I(Kr)) expression is higher in atria than ventricles, whereas in humans, HERG1 expression is higher in ventricular, than atrial, tissue. Taken together, these results suggest that the N-terminal alternatively spliced variants of ERG1 (i.e. ERG1b) are not expressed at the protein level in rat, mouse, or human heart and that these variants do not, therefore, play roles in the generation of functional cardiac I(Kr) channels.

Chronic atrial fibrillation (AF) is characterized by decreased atrial contractility, shortened action potential duration, and decreased accommodation of action potential duration to changes in activation rate. Studies on experimental animal models of AF implicate a reduction in L-type Ca2+ current (I(Ca)) density in these changes. To evaluate the effect of AF on human I(Ca), we compared I(Ca) in atrial myocytes isolated from 42 patients in normal sinus rhythm at the time of cardiac surgery with that of 11 chronic AF patients. I(Ca) was significantly reduced in the myocytes of patients with chronic AF (mean -3.35+/-0.5 pA/pF versus -9.13+/-1. 0 pA/pF in the controls), with no difference between groups in the voltage dependence of activation or steady-state inactivation. Although I(Ca) was lower in myocytes from the chronic AF patients, their response to maximal beta-adrenergic stimulation was not impaired. Postoperative AF frequently follows cardiac surgery. Half of the patients in the control group (19/38) of this study experienced postoperative AF. Whereas chronic AF is characterized by reduced atrial I(Ca), the patients with the greatest I(Ca) had an increased incidence of postoperative AF, independent of patient age or diagnosis. This observation is consistent with the concept that calcium overload may be an important factor in the initiation of AF. The reduction in functional I(Ca) density in myocytes from the atria of chronic AF patients may thus be an adaptive response to the arrhythmia-induced calcium overload.

A-esterases are calcium-dependent hydrolases that can detoxify the active metabolites (oxons) of organophosphorus insecticides such as chlorpyrifos and parathion. A-esterases from rat liver have previously been shown to hydrolyze chlorpyrifos-oxon but not paraoxon at low substrate concentrations. Two A-esterases were extracted by ammonium sulfate fractionation from solubilized rat liver microsomes followed by gel filtration chromatography and preparative scale isoelectric focusing. The proteins displayed similar characteristics and were difficult to separate; both had similar high molecular mass and isoelectric point range and exhibited A-esterase activity toward high and low concentrations of chlorpyrifos-oxon and high concentrations of paraoxon. Sufficient amounts of the higher molecular mass protein were obtained for kinetic studies, which yielded a Km of 0.93 mM toward high concentrations of chlorpyrifos-oxon and a Vmax of 369 nmoles product formed/mg protein-min. The protein hydrolyzed phenyl acetate, chlorpyrifos-oxon and paraoxon, suggesting that arylesterase and A-esterase activities are attributable to the same liver protein(s). Assays of purified protein and kinetic studies of microsomes suggested that the activity toward high (320 microM) and low (

Chronic atrial fibrillation is associated with a shortening of the atrial action potential duration and atrial refractory period. To test the hypothesis that these changes are mediated by changes in the density of specific atrial K+ currents, we compared the density of K+ currents in left and right atrial myocytes and the density of delayed rectifier K+ channel alpha-subunit proteins (Kv1.5 and Kv2.1) in left and right atrial appendages from patients (n = 28) in normal sinus rhythm with those from patients (n = 15) in chronic atrial fibrillation (AF). Contrary to our expectations, nystatin-perforated patch recordings of whole-cell K+ currents revealed significant reductions in both the inactivating (ITO) and sustained (IKsus) outward K+ current densities in left and right atrial myocytes isolated from patients in chronic AF, relative to the ITO and IKsus densities in myocytes isolated from patients in normal sinus rhythm. Quantitative Western blot analysis revealed that although there was no change in the expression of the Kv2.1 protein, the expression of Kv1.5 protein was reduced by > 50% in both the left and the right atrial appendages of AF patients. The finding that Kv1.5 expression is reduced in parallel with the reduction in delayed rectifier K+ current density is consistent with recent suggestions that Kv1.5 underlies the major component of the delayed rectifier K+ current in human atrial myocytes, the ultrarapid delayed rectifier K+ current, IKur. The unexpected finding of reduced voltage-gated outward K+ current densities in atrial myocytes from AF patients demonstrates the need to further examine the details of the electrophysiological remodeling that occurs during AF to enable more effective and safer therapeutic strategies to be developed.

The effects of exogenous naloxone and adrenocorticotropin (ACTH) on circulating concentrations of corticosterone and glucose in broilers were determined. Birds were injected i.m. at 0 and 2 h with either saline or naloxone, then i.v. at 2.5 h with either saline or ACTH. Control birds received saline at each injection. Blood samples were taken before the experiment started (0 min) and 30, 60, and 90 min after the last injection. Intramuscular injections of naloxone significantly reduced subsequent ACTH-stimulated increases in serum corticosterone; however, when followed by saline, naloxone elevated corticosterone by 90 min after the final injection of saline. Glucose levels were significantly elevated at 60 min in birds receiving ACTH i.v., but remained elevated through 90 min in birds pretreated with naloxone. Naloxone pretreatment attenuated serum corticosterone but augmented serum glucose concentrations in ACTH-stimulated broilers.

The purpose of the present study was to investigate the effects of elevated corticosterone (CORT) on circulating lipoprotein cholesterol during a 1-wk period. For this study, 15 commercial broilers were randomly assigned to one of three treatment groups. Group 1 served as the control (CON) and received no treatment, whereas Groups 2 and 3 received subcutaneous mini-osmotic pumps containing either physiological saline (PS) or adrenocorticotropin (ACTH), respectively. The ACTH was delivered at a rate of 8 IU/kg of BW/d. Blood samples were taken at Time 0 (before implants) and on Days 2, 4, and 7 postimplantation. Continuous infusion of ACTH increased plasma glucose, cholesterol, triglycerides, very low density lipoprotein cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, and CORT during the postimplantation period. The group treated with ACTH also exhibited a decrease in BW during the last 2 sampling d. There were no differences in any of the serum constituents measured between CON and PS birds, which suggest that CON birds can serve as useful controls. These data suggest that birds given a continuous infusion of ACTH at 8 IU/kg of BW/d can experience changes in plasma lipoprotein cholesterol concentrations along with changes in other blood parameters and may serve as a useful model in accelerated lipoprotein production.

The active metabolites (oxons) of phosphorothionate insecticides can be detoxified via A-esterase hydrolysis. Two enzymes with A-esterase activity have been isolated from rat serum. Whole serum was applied to anion exchange gel (DEAE Sepharose Fast Flow) and incubated (1 hr). Tris-HCl buffer (0.05 M; pH 7.7, at 5 degrees) containing 0.25 M NaCl was added to the slurry and incubated. The decant, containing low A-esterase activity but a high protein concentration, was discarded. Further displacement of A-esterase from DEAE gel was achieved with 1.0 M NaCl in 0.05 M Tris-HCl buffer (Ph 7.7 at 5 degrees). Following desalting and concentration, further separation was achieved by gel filtration (Sephacryl S-100 HR) and two sequential preparative scale isoelectric focusings. Final fractions contained two proteins of high molecular mass (one about 200 kDa and one between 137 and 200 kDa). The apparent range of isoelectric points for the two enzymes was 4.5 to 5.6. Following native-PAGE analysis, activity stains with beta-naphthyl acetate and Fast Garnet GBC in the presence of paraoxon (10-5 M) verified that A-esterase activity was associated with both proteins. Spectropho-tometric assay detected A-esterase activity toward paraoxon, chlorpyrifos-oxon, and phenyl acetate in the final preparation.

Paraoxon and chlorpyrifos-oxon, active metabolites of the organophosphorus insecticides parathion and chlorpyrifos, can be detoxified via A-esterases and aliesterases. These enzyme activities were measured in various tissues of Sprague-Dawley rats. High A-esterase activities were detected in liver, serum and liver mitochondrial/microsomal fractions. Low or no A-esterase activities were detected in other tissues and tissue fractions. A-Esterase substrate:substrate activity ratios suggest that the substrates are probably not degraded by the same enzyme. Highest aliesterase activities were observed in the small intestine and liver with moderate activity in kidney, serum and lungs. Low activities were noted in brain, spleen and skeletal muscle.