Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related disorders, through abnormal interactions with other proteins containing short tracts such as the transcriptional coactivator CREB binding protein, CBP. We found that CBP was depleted from its normal nuclear location present aggregates HD cell culture models, transgenic mice, human postmortem brain. specifically interfere CBP-activated gene transcription, overexpression of...

The syntrophins are a multigene family of intracellular dystrophin-associated proteins comprising three isoforms, α1, β1, and β2. Based on their domain organization association with neuronal nitric oxide synthase, thought to function as modular adapters that recruit signaling the membrane via dystrophin complex. Using sequences derived from new mouse β1-syntrophin cDNA, previously isolated cDNAs for α1- β2-syntrophins, we prepared isoform-specific antibodies study expression, skeletal muscle...

Huntington's disease is a neurodegenerative disorder resulting from expansion of the polyglutamine region in huntingtin. Although huntingtin normally cytoplasmic, affected brain regions proteolytic fragments mutant containing repeat form intranuclear inclusions. Here, we examine contribution nuclear localization to toxicity by transiently transfecting neuro-2a cells with an N-terminal fragment similar size that believed be present patients. The fragment, HD-N63, was targeted either cytoplasm...

Cardiovascular toxicity, a prominent reason for late-stage failures in drug development, has resulted demand vitro assays that can predict this liability early discovery. Current cardiovascular safety testing primarily focuses on ion channel modulation and low throughput cardiomyocyte (CM) contractility measurements. We evaluated both human induced pluripotent stem cell-derived CMs (hiPSC-CMs) rat neonatal (rat CMs) the xCELLigence Cardio system which uses impedance technology to quantify CM...

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), identified enzyme in complex, uniquely absent from skeletal muscle plasma membrane many human patients and mouse models dystrophinopathy. An NH2-terminal domain nNOS directly interacts with alpha 1-syntrophin but not other proteins complex analyzed. However, does associate on sarcolemma transgenic mdx mice expressing truncated proteins. This...

Dystrophin-related and -associated proteins are important in the formation maintenance of mammalian neuromuscular junction. We have characterized mouse cDNA clones encoding isoforms dystrophin-homologous 87-kDa postsynaptic protein, dystrobrevin. In Torpedo, protein is multiply phosphorylated closely associated with cytoskeleton, including acetylcholine receptor. contrast to where only a single transcript seen, expresses several mRNAs different isoforms. A 6.0-kilobase brain encodes 78-kDa...

THE syntrophins are a multigene family of proteins which bind C-terminal domains dystrophin, utrophin and homologs thereof. We report here that antibodies specific for one isoform, β2-syntrophin, labeled only the neuro-muscular junction (NMJ) in rat skeletal muscle. Anti-α1-syntrophin gave strong labeling sarcolemma NMJ normal mouse muscle, similar but much weaker dystrophin-minus mdx β2-Syntrophin therefore appears to be as is utrophin, may involved acetylcholine receptor clustering....

G-protein-coupled receptors can couple to different signal transduction pathways in cell types (termed cell-specific signaling) and activate signaling depending on the receptor conformation(s) stabilized by activating ligand (functional selectivity). These concepts offer potential for developing pathway-specific drugs that increase efficacy reduce side effects. Despite significant interest, functional selectivity has been difficult exploit drug discovery, part due burden of multiple assays....

α-Dystrobrevin is both a dystrophin homologue and component of the protein complex. Alternative splicing yields five forms, which two predominate in skeletal muscle: full-length α-dystrobrevin-1 (84 kD), COOH-terminal truncated α-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that localized on sarcolemma at neuromuscular synapse, where, like dystrophin, it most concentrated depths postjunctional folds. α-Dystrobrevin-2 preferentially copurifies with from muscle extracts....

Dystrophin, the protein disrupted in Duchenne muscular dystrophy, is one of several related proteins that are key components submembrane cytoskeleton. Three dystrophin-related (utrophin, protein-2 (DRP2), and dystrobrevin) have been described. Here, we identify a human gene on chromosome 2p22-23 encodes novel protein, beta-dystrobrevin, with significant homology to other known dystrobrevin (now termed alpha-dystrobrevin). Sequence alignments including this second strongly support concept two...

The complete nucleotide sequence of the gene encoding surface (hexagonally packed intermediate [HPI])-layer polypeptide Deinococcus radiodurans Sark was determined and found to encode a 1,036 amino acids. Amino acid analysis about 30% residues revealed that mature consists at least 978 N terminus blocked Edman degradation. results proteolytic modification HPI layer in situ Mr estimations expressed Escherichia coli indicated there is leader sequence. N-terminal region contained very high...

The diversity and impact of label-free technologies continues to expand in drug discovery. Two classes instruments, using either an electrical impedance-based or optical-based biosensor, are now available for investigating the effects ligands on cellular targets. Studies GPCR function have been especially prominent with these instruments due importance this target class Although both biosensors share similar high sensitivity changes cell shape structure, it is unknown whether yield results...

Drug-induced gastrointestinal toxicities (GITs) rank among the most common clinical side effects. Preclinical efforts to reduce incidence are limited by inadequate predictivity of in vitro assays. Recent breakthroughs culture methods support intestinal stem cell maintenance and continual differentiation into epithelial types resident intestine. These diverse cells self-assemble microtissues with vivo-like architecture. Here, we evaluate human GI grown transwell plates that allow apical...

Huntington's disease is caused by an expanded CAG trinucleotide repeat coding for a polyglutamine stretch within the huntingtin protein. Currently, function of normal and mechanism which causes selective neurotoxicity remain unknown. Clues may come from identification huntingtin-associated proteins (HAPs). Here, we show that copurifies with single novel 40-kDa protein termed HAP40. HAP40 encoded open reading frame factor VIII-associated gene A (F8A) located intron 22 VIII gene. In...

Cellular dielectric spectroscopy (CDS) is an emerging technology capable of detecting a range whole-cell responses in label-free manner. A new CDS-based instrument, CellKey, has been developed that optimized for G-protein coupled receptor (GPCR) detection and automated liquid handling microplate format, thereby making CDS accessible to lead generation/optimization drug discovery. In addition having sufficient throughput, assay technologies must pass rigorous standards development, signal...

Drug-induced gastrointestinal toxicity (GIT) is a common treatment-emergent adverse event that can negatively impact dosing, thereby limiting efficacy and treatment options for patients. An in vitro assay of GIT needed to address patient variability, mimic the microphysiology gut, accurately predict drug-induced GIT. Primary human ileal organoids (termed 'enteroids') have proven useful stimulating intestinal stem cell proliferation differentiation multiple types present gut epithelium....