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AbstractTime and resource constraints necessitate increasingly early decisions to advance or halt pre-clinical drug discovery programs. Early discovery or “tool” compounds may be potent inhibitors of new targets, but all too often they exhibit poor pharmaceutical and pharmacokinetic properties that make early assessment of in vivo efficacy difficult. 1,3-Dicyclohexylurea, a potent and selective inhibitor of soluble epoxide hydrolase (sEH), reduces blood pressure in hypertensive preclinical animal models when administered intraperitoneally using DMSO/corn oil as a delivery vehicle. However, the poor aqueous solubility of DCU poses a challenge for in vivo dosing in a multiple dose situation. Therefore, we developed a nanosuspension formulation of DCU to support oral, intravenous bolus and intravenous infusion dosing. Use of the nanosuspension formulation maintained DCU free plasma levels above the sEH IC50 and demonstrated that the application of formulation technology can accelerate in vivo evaluation of new targets by enabling pharmacodynamic studies of poorly soluble compounds.

Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: To evaluate the impact of pediatric sleep disturbances and night-time seizure monitoring of children with rare epilepsy syndromes on the sleep quality and mental health of caregivers., Study Design: A cross-sectional study was conducted using caregiver entered data from the Rare Epilepsy Network on pediatric sleep disturbances and Patient Reported Outcomes Measurement Information System measures for caregiver fatigue, sleep disturbance, sleep-related impairment, depression, anxiety, companionship, and cognition. Logistic regression was used to examine associations between risk factors and caregiver sleep quality., Results: Non-Hispanic white mothers comprised 83% of the 742 respondents in this study. After adjusting for covariates, difficulty falling asleep, excessive daytime sleepiness, frequent night-time awakenings, and very restless sleep in children were associated with fatigue (aOR 95% CI, 1.5-2.2), sleep-related disturbance (aOR 95% CI, 1.7-2.6) and sleep impairment (aOR 95% CI, 1.5-2.4) in caregivers. Caregiver anxiety (aOR 95% CI, 3.6-6.0) and depression (aOR 95% CI, 2.8-6.0) were also highly associated with their fatigue and sleep quality, whereas companionship (aOR 95% CI, 0.3-0.4) and higher caregiver cognition (aOR 95% CI, 0.1-0.2) were protective. In addition, sharing a room or bed or using methods that require listening for seizures were significantly related to sleep disturbance and fatigue in the caregivers., Conclusions: In rare epilepsies, pediatric sleep disturbances and night-time seizure monitoring are significantly associated with caregiver fatigue and poor sleep quality. In addition to the intense caregiving needs of children with rare epilepsies, fatigue and poor sleep quality in caregivers may contribute to or result from mental health problems., (© 2020 Published by Elsevier Inc.)

Asthma is a chronic inflammatory disease of the airways, the susceptibility to which is strongly influenced by genetics. Genomics, the study of the human genome, is redefining the process for rapidly identifying novel therapeutic targets for asthma and other diseases. One approach is to search for polymorphisms in genes that increase susceptibility to the disease in order to identify genes and cellular pathways relevant to the disease process. In asthma, for example, regardless of the genetic factors that contribute to susceptibility, good drug targets could be found that affect epithelial integrity, allergic response, and the recruitment or activity of inflammatory cells. Such targets may consist of proteins that are specifically expressed in certain cell types, proteins whose expression is regulated during the disease process or proteins involved in the destructive process. This review discusses some of the genomics tools that can be used to identify new molecular targets, which in turn are useful in screening for novel compounds likely to affect diseases such as asthma.

5'-mutations in the dystrophin gene can result in cardiomyopathy without clinically-apparent skeletal myopathy. The effect of dystrophin mutations on the assembly and stability of the dystrophin associated protein (DAP) complex in human heart are not fully understood. The molecular defect in the dystrophin complex was explored in a family with an X-linked pedigree and severe dilated cardiomyopathy. Dystrophin gene analysis demonstrated a 5' duplication involving exons 2-7, which encodes the N-terminal actin binding domain of dystrophin. Ribonuclease protection and PCR assays demonstrated a reduction in muscle promoter transcribed dystrophin mRNA in the heart compared to skeletal muscle. A deficiency of cardiac dystrophin protein was observed by Western blot and lack of membrane localization by immunocytochemistry. The cardiac expression of the dystrophin related protein utrophin was increased, and the 43 kDa (beta-dystroglycan), 50 kDa (alpha-sarcoglycan) and 59 kDa (syntrophin) dystrophin associated proteins (DAPs) were co-isolated and present in nearly normal amounts in the membrane. However, cardiac dystrophin deficiency and increased utrophin expression were associated with loss of extracellular 156 kDa dystrophin associated glycoprotein (alpha-dystroglycan) binding to the cardiomyocyte membrane. alpha-Dystroglycan is responsible for linkage of the dystrophin complex to the extracellular matrix protein laminin. Therefore, 5' dystrophin mutations can reduce cardiac dystrophin mRNA, protein expression, and dystrophin function in X-linked cardiomyopathy (XLCM). The presence of membrane-associated beta-dystroglycan, alpha-sarcoglycan, syntrophin, and utrophin are insufficient to maintain cardiac function. This XLCM family has a 5' dystrophin gene mutation resulting in cardiac dystrophin deficiency and a loss of alpha-dystroglycan membrane binding., (Copyright 1997 Academic Press Limited.)

The distribution and expression of dystrophin and three of the dystrophin-associated glycoproteins (DAG), alpha-dystroglycan (156 kDa DAG), beta-dystroglycan (43 kDa DAG) and adhalin (50 kDa DAG) in rat skeletal muscle were studied during a controlled cycle of degeneration and regeneration induced by the injection of a snake venom. Cryosections of muscle at various stages of degeneration and regeneration were labeled using monoclonal antibodies to the three glycoproteins and examined at fixed time points after venom injection. Adhalin and alpha-dystroglycan remained present at the sarcolemma throughout the entire cycle of degeneration and regeneration. beta-Dystroglycan, on the other hand, was lost from the sarcolemma by 12 hours and reappeared 2 days after venom injection when new muscle fibers were being formed. Dystrophin was not lost from the sarcolemma until 24 hours after venom injection and did not reappear at the membrane until 4 days. It is suggested that dystrophin and the glycoprotein complex are synthesized separately, both temporally and spatially, and only become associated at the plasma membrane during the later stages of regeneration. The expression of beta-dystroglycan in the regenerating muscle fibers was first seen at sites of newly forming plasma membrane that were closely associated with the old basal lamina tube. The basal lamina may therefore have a regulatory or modulatory role in the expression of the DAG.

The 43 kDa AChR-associated protein rapsyn is required for the clustering of nicotinic acetylcholine receptors (AChRs) at the developing neuromuscular junction, but the functions of other postsynaptic proteins colocalized with the AChR are less clear. Here we use a fibroblast expression system to investigate the role of the dystrophin-glycoprotein complex (DGC) in AChR clustering. The agrin-binding component of the DGC, dystroglycan, is found evenly distributed across the cell surface when expressed in fibroblasts. However, dystroglycan colocalizes with AChR-rapsyn clusters when these proteins are coexpressed. Furthermore, dystroglycan colocalizes with rapsyn clusters even in the absence of AChR, indicating that rapsyn can cluster dystroglycan and AChR independently. Immunofluorescence staining using a polyclonal antibody to utrophin reveals a lack of staining of clusters, suggesting that the immunoreactive species, like the AChR, does not mediate the observed rapsyndystroglycan interaction. Rapsyn may therefore be a molecular link connecting the AChR to the DGC. At the neuromuscular synapse, rapsyn-mediated linkage of the AChR to the cytoskeleton-anchored DGC may underlie AChR cluster stabilization.

Marked deficiency of muscle adhalin, a 50 kDa sarcolemmal dystrophin-associated glycoprotein, has been reported in severe childhood autosomal recessive muscular dystrophy (SCARMD). This is a Duchenne-like disease affecting both males and females first described in Tunisian families. Adhalin deficiency has been found in SCARMD patients from North Africa Europe, Brazil, Japan and North America (SLR & KPC, unpublished data). The disease was initially linked to an unidentified gene on chromosome 13 in families from North Africa, and to the adhalin gene itself on chromosome 17q in one French family in which missense mutations were identified. Thus there are two kinds of myopathies with adhalin deficiency: one with a primary defect of adhalin (primary adhalinopathies), and one in which absence of adhalin is secondary to a separate gene defect on chromosome 13. We have examined the importance of primary adhalinopathies among myopathies with adhalin deficiency, and describe several additional mutations (null and missense) in the adhalin gene in 10 new families from Europe and North Africa. Disease severity varies in age of onset and rate of progression, and patients with null mutations are the most severely affected.

Background: In skeletal muscle, dystrophin exists in a large oligomeric complex tightly associated with several novel sarcolemmal proteins, including the 50-kDa transmembrane glycoprotein called adhalin. The dystrophin-glycoprotein complex links the subsarcolemmal actin cytoskeleton to the basal lamina component laminin, thus providing stability to the sarcolemma. Disturbance of this linkage due to the absence of dystrophin plays a crucial role in the molecular pathogenesis of muscle fiber necrosis in Duchenne muscular dystrophy. Severe childhood autosomal recessive muscular dystrophy (SCARMD) is similar to Duchenne muscular dystrophy in phenotype but is characterized by the deficiency of adhalin. At present, the status of the link between the dystrophin-glycoprotein complex and laminin is unclear in SCARMD., Experimental Design: We investigated, by immunohistochemistry using confocal laser scanning microscopy, the status of the expression of laminin subunits, A, M, B1, B2, and S chains, in skeletal muscle biopsy specimens of eight SCARMD patients from various human populations. In addition, we correlated the severity of laminin abnormality with the severity of both clinical symptoms and histopathologic changes in these patients., Results: The reduction of laminin B1 chain and the overexpression of the S chain, a homologue of B1, in the extrajunctional basal lamina were observed in the five patients who had advanced clinical symptoms and histopathologic changes. Abnormalities in the expression of laminin were not observed in the three less affected patients., Conclusions: The expression of laminin is greatly disturbed in severely diseased SCARMD muscle deficient in adhalin. Disturbance of sarcolemma-basal lamina interaction may play an important role in the molecular pathogenesis of muscle fiber necrosis in SCARMD.

The expression of full-length dystrophin and various dystrophin deletion mutants was monitored in mdx mouse muscle after intramuscular injection of dystrophin-encoding plasmid DNAs. Recombinant dystrophin proteins, including those lacking either the amino terminus, carboxyl terminus, or most of the central rod domain, showed localization to the plasma membrane. This suggests that there are multiple attachment sites for dystrophin to the plasma membrane. Only those constructs containing the carboxyl terminus were able to stabilize dystrophin-associated proteins (DAP) at the membrane, consistent with other studies that suggest that this domain is critical to DAP binding. Colocalization with DAP was not necessary for membrane localization of the various dystrophin molecules. However, stabilization and co-localization of the DAP did seem to be a prerequisite for expression and/or stabilization of mutant dystrophins beyond 1 wk and these same criteria seemed important for mitigating the histopathological consequences of dystrophin deficiency.

Adhalin, the 50 kDa dystrophin-associated glycoprotein, is deficient in skeletal muscle of patients having severe childhood autosomal recessive muscular dystrophy (SCARMD). In several North African families, SCARMD has been linked to chromosome 13q, but SCARMD has been excluded from linkage to this locus in other families. We have now cloned human adhalin cDNA and mapped the adhalin gene to chromosome 17q12-q21.33, excluding it from involvement in 13q-linked SCARMD. However, one allelic variant of a polymorphic microsatellite located within intron 6 of the adhalin gene cosegregated perfectly with the disease phenotype in a large family. Furthermore, missense mutations were identified within the adhalin gene that might cause SCARMD in this family. Thus, the adhalin gene is involved in at least one form of autosomal recessive muscular dystrophy.

The dystrophin-glycoprotein complex was examined in dystrophin-deficient dogs with golden retriever muscular dystrophy (GRMD) using immunoblot and immunofluorescence analysis. The dystrophin-associated proteins were substantially reduced in muscle from dogs with GRMD. Interestingly, regression analysis revealed a strong correlation between the amount of alpha-dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.

Synapse formation is characterized by the accumulation of molecules at the site of contact between pre- and postsynaptic cells. Agrin, a protein implicated in the regulation of this process, causes the clustering of acetylcholine receptors (AChRs). Here we characterize an agrin-binding site on the surface of muscle cells, show that this site corresponds to alpha-dystroglycan, and present evidence that alpha-dystroglycan is functionally related to agrin activity. Furthermore, we demonstrate that alpha-dystroglycan and adhalin, components of the dystrophin-associated glycoprotein complex, as well as utrophin, colocalize with agrin-induced AChR clusters. Thus, agrin may function by initiating or stabilizing a synapse-specific membrane cytoskeleton that in turn serves as a scaffold upon which synaptic molecules are concentrated.

Severe autosomal recessive muscular dystrophy (SCARMD), McKusick n. 253700, has been originally described in North-African populations, in which significant linkage has been established with DNA markers mapping to the proximal region of the long arm of chromosome 13, without evidence for heterogeneity of the SCARMD locus in these populations. A striking feature of this disease is the isolated deficiency of adhalin, a sarcolemmal 50 kDa dystrophin-associated glycoprotein. We report a non-inbred French family with a milder progressive form of muscular dystrophy affecting subjects of both sexes. The parents are not affected suggesting an autosomal recessive transmission. In 4 siblings displaying mild to overt clinical signs of muscular dystrophy, serum creatine kinase was high, and muscle specimens showed variable degree of necrosis-regeneration with little fibrosis. In the 4 cases adhalin was completely absent in muscle sections, whereas dystrophin and the other members of the dystrophin-associated protein complex were normal, except for the 35 kDa dystrophin-associated glycoprotein which was decreased as usually observed in SCARMD. Linkage and homogeneity analysis using 4 microsatellite markers of chromosome 13q that are linked to the North-African SCARMD locus were performed in this family. Results show that the morbid locus involved in this family does not map to the same region as the SCARMD locus. This second locus may be involved in sporadic cases of muscular dystrophy with adhalin deficiency that have been reported in Europe.

The four Shaker-like subfamilies of Shaker-, Shab-, Shaw-, and Shal-related K+ channels in mammals have been defined on the basis of their sequence homologies to the corresponding Drosophila genes. Using interspecific backcrosses between Mus musculus and Mus spretus, we have chromosomally mapped in the mouse the Shaker-related K(+)-channel genes Kcna1, Kcna2, Kcna4, Kcna5, and Kcna6; the Shab-related gene Kcnb1; the Shaw-related gene Kcnc4; and the Shal-related gene Kcnd2. The following localizations were determined: Chr 2, cen-Acra-Kcna4-Pax-6-a-Pck-1-Kras-3-Kcn b1 (corresponding human Chrs 11p and 20q, respectively); Chr 3, cen-Hao-2-(Kcna2, Kcnc4)-Amy-1 (human Chr 1); and Chr 6, cen-Cola-2-Met-Kcnd2-Cpa-Tcrb-adr/Clc-1-Hox-1.1-Myk - 103-Raf-1-(Tpi-1, Kcna1, Kcna5, Kcna6) (human Chrs 7q and 12p, respectively). Thus, there is a cluster of at least three Shaker-related K(+)-channel genes on distal mouse Chr 6 and a cluster on Chr 2 that at least consists of one Shaker-related and one Shaw-related gene. The three other K(+)-channel genes are not linked to each other. The map positions of the different types of K(+)-channel genes in the mouse are discussed in relation to those of their homologs in man and to hereditary diseases of mouse and man that might involve K+ channels.

The 50-kDa dystrophin-associated glycoprotein (50-DAG) is a component of the dystrophin-glycoprotein complex, which links the muscle cytoskeleton to the extracellular matrix. 50-DAG is specifically deficient in skeletal muscle of patients with severe childhood autosomal recessive muscular dystrophy and in skeletal and cardiac muscles of BIO 14.6 cardiomyopathic hamsters. The lack of 50-DAG leads to a disruption and dysfunction of the dystrophin-glycoprotein complex in these diseases. The cDNA encoding 50-DAG has now been cloned from rabbit skeletal muscle. The 50-DAG deduced amino acid sequence predicts a novel protein having 387 amino acids, a 17-amino acid signal sequence, one transmembrane domain, and two potential sites of N-linked glycosylation. Affinity-purified antibodies against rabbit 50-DAG fusion proteins or synthetic peptides specifically recognized a 50-kDa protein in skeletal muscle sarcolemma and the 50-kDa component of the dystrophin-glycoprotein complex. In contrast to dystroglycan, which is expressed in a wide variety of muscle and non-muscle tissues, 50-DAG is expressed only in skeletal and cardiac muscles and in selected smooth muscles. Finally, 50-DAG mRNA is present in mdx and Duchenne muscular dystrophy (DMD) muscle, indicating that the down-regulation of this protein in DMD and the mdx mouse is likely a post-translational event.

Duchenne-like muscular dystrophy (DLMD) is an autosomal recessive (AR) muscular dystrophy which presents a clinical course indistinguishable from the Xp21 Duchenne muscular dystrophy or DMD. Recently, Othmane et al., based on a linkage study with 13q12 markers in 3 highly inbred DLMD families from Tunisia, suggested that the gene for this myopathy lies in the pericentromeric region of chromosome 13q. It is unknown if there is genetic heterogeneity causing the DLMD phenotype. Therefore, the aim of the present report is to describe the results of linkage analysis in 4 Brazilian DLMD families with 13q12 markers (D13S115 and D13S120), which were also tested for 50DAG. It was possible to exclude the 13q gene at theta = 0.10 as responsible for the DLMD phenotype in our families using both 13q12 markers, if the lod scores of each family were added up. Interestingly, 3 families were deficient for 50 DAG while one showed a positive pattern for this glycoprotein. Therefore, these results suggest: a) the DLMD phenotype is caused by more than one recessive gene; b) a gene, not located at 13q, causes deficiency of 50 DAG as a primary or secondary defect.

Cardiomyopathies are a diverse group of primary cardiac diseases, most of which have a poorly understood etiology. One type of hereditary cardiomyopathy is caused by defects in the dystrophin gene in Duchenne and Becker muscular dystrophy patients. Our laboratory has identified a complex of dystrophin-associated proteins in skeletal and cardiac muscle which span the sarcolemma, linking the subsarcolemmal cytoskeleton to the extracellular matrix. The absence of dystrophin in Duchenne muscular dystrophy patients leads to the loss of dystrophin-associated proteins in both skeletal and cardiac muscle, suggesting that a primary loss of one or more dystrophin-associated proteins might lead to other forms of cardiomyopathy. Here we report the specific deficiency of the 50-kDa dystrophin-associated glycoprotein in cardiac and skeletal muscles of the BIO 14.6 strain of cardiomyopathic hamsters, which experience both autosomal recessive cardiomyopathy and myopathy. Other dystrophin-associated proteins are well preserved in myopathic hamster skeletal muscle, but the link between dystrophin and dystroglycan is disrupted. All dystrophin-associated proteins are decreased in abundance in the cardiomyopathic hamster heart, perhaps explaining why the cardiomyopathy is more severe than the myopathy. Thus, the disruption of the dystrophin-glycoprotein complex may play a role in skeletal and cardiac myocyte necrosis of the cardiomyopathic hamster.

K+ channels represent the most diverse class of voltage-gated ion channels in terms of function and structure. Voltage-gated K+ channels in the heart establish the resting membrane K+ permeability, modulate the frequency and duration of action potentials, and are targets of several antiarrhythmic drugs. Consequently, an understanding of K+ channel structure-function relationships and pharmacology is of great practical interest. However, the presence of multiple overlapping currents in native cardiac myocytes complicates the study of basic K+ channel function and drug-channel interactions in these cells. The application of molecular cloning technology to cardiovascular K+ channels has identified the primary structure of these proteins, and heterologous expression systems have allowed a detailed analysis of channel function and pharmacology without contaminating currents. To date six different K+ channels have been cloned from rat and human heart, and all have been functionally characterized in either Xenopus oocytes or mammalian tissue culture systems. This initial research is an important step toward understanding the molecular basis of the action potential in the heart. An important challenge for the future is to determine the cell-specific expression and relative contribution of these cloned channels to cardiac excitability.

A voltage-sensitive K+ channel previously cloned from rat heart designated RK5 (rat Kv4.2) (Roberds and Tamkun, 1991, Proc. Natl. Acad. Sci. USA 88, 1798-1802) was functionally characterized in the Xenopus oocyte expression system. RK5 is a homolog of the Drosophila Shal K+ channel, activates with a rise time of 2.8 ms, has a midpoint for activation of -1 mV and rapidly inactivates with time constants of 15 and 60 ms. RK5 is sensitive to 4-AP, IC50 = 5 mM, and is insensitive to TEA and dendrotoxins. The voltage dependence and kinetics of the RK5 induced currents suggest this channel contributes to the Ito current in heart.

Cardiac K+ channels are responsible for repolarization of the action potential and are the targets of several antiarrhythmic drugs. This study examines the differential expression of six K+ channel mRNAs during rat heart development. RK1 and RK2 K+ channel transcripts were undetectable prior to 10 days after birth. In contrast, RK4 mRNA was present at equivalent levels from day 14 in utero to 20 days after birth. RK3 and RK5 were detected as early as 14 days in utero. These data indicate that K+ channel expression in the heart is closely regulated and further argue for physiologically distinct roles for K+ channel isoforms.

At physiologically relevant concentrations an antiviral compound should not perturb the host's ability to mount an immune response against the infecting virus or some other opportunistic pathogen. The purpose of this study was to evaluate the immunomodulatory activity of the antiviral compound MDL 20,610 using murine models. When tested in vitro at the limit of aqueous solubility (6 microM), MDL 20,610 has no significant effect on neutrophil function as assessed by cell migration against FMLP and LTB4 gradients, myeloperoxidase secretion or 0.-2 production. In addition, 6 microM MDL 20,610 has no significant effect on macrophage function as determined by 0.-2 production, Ia and Mac-1 antigen expression and expression of Fc gamma receptors. Finally, MDL 20,610 does not significantly affect in vivo (1-100 mg/kg/day) NK cell activity or DTH to oxazolone; but treatment of mice with 50 or 100 mg MDL 20,610/kg/day significantly (P less than 0.01) enhances SRBC IgM antibody synthesis. These data indicate that MDL 20,610 is relatively devoid of immunomodulatory activity.