Overexpression or ablation of JNK in skeletal muscle has no effect on glycogen synthase activity

c-Jun NH₂-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle. electroporation; gene delivery; muscle contraction; exercise     

Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Organization of model helical peptides in lipid bilayers: insight into the behavior of single-span protein transmembrane domains

Selectively deuterated transmembrane peptides comprising alternating leucine-alanine subunits were examined in fluid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy in an effort to gain insight into the behavior of membrane proteins. Two groups of peptides were studied: 21-mers having a 17-amino-acid hydrophobic domain calculated to be close in length to the hydrophobic thickness of 1-palmitoyl-2-oleoyl phosphatidylcholine and 26-mers having a 22-amino-acid hydrophobic domain calculated to exceed the membrane hydrophobic thickness. (2)H NMR spectral features similar to ones observed for transmembrane peptides from single-span receptors of higher animal cells were identified which apparently correspond to effectively monomeric peptide. Spectral observations suggested significant distortion of the transmembrane alpha-helix, and/or potential for restriction of rotation about the tilted helix long axis for even simple peptides. Quadrupole splittings arising from the 26-mer were consistent with greater peptide "tilt" than were those of the analogous 21-mer. Quadrupole splittings associated with monomeric peptide were relatively insensitive to concentration and temperature over the range studied, indicating stable average conformations, and a well-ordered rotation axis. At high peptide concentration (6 mol% relative to phospholipid) it appeared that the peptide predicted to be longer than the membrane thickness had a particular tendency toward reversible peptide-peptide interactions occurring on a timescale comparable with or faster than approximately 10(-5) s. This interaction may be direct or lipid-mediated and was manifest as line broadening. Peptide rotational diffusion rates within the membrane, calculated from quadrupolar relaxation times, T(2e), were consistent with such interactions. In the case of the peptide predicted to be equal to the membrane thickness, at low peptide concentration spectral lineshape indicated the additional presence of a population of peptide having rotational motion that was restricted on a timescale of 10(-5) s.     

AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle

The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-beta-D-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPK alpha 1 and AMPK alpha 2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-beta-D-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3-O-methyl-D-glucose (3-MG) uptake. There were dose-dependent increases in AMPK alpha 2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPK alpha1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPK alpha 2 activity and 3-MG uptake but had little effect on AMPK alpha 1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPK alpha 1 and -alpha 2 activity and 3-MG uptake. Although the AMPK alpha 1 and -alpha 2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPK alpha 2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Ablation of LKB1 in the heart leads to energy deprivation and impaired cardiac function

Energy deprivation in the myocardium is associated with impaired heart function and increased morbidity. LKB1 is a kinase that is required for activation of AMP-activated protein kinase (AMPK) as well as 13 AMPK-related protein kinases. AMPK stimulates ATP production during ischemia and prevents post-ischemic dysfunction. We used the Cre–Lox system to generate mice where LKB1 was selectively knocked out in cardiomyocytes and muscle cells (LKB1-KO) to assess the role of LKB1 on cardiac function in these mice.Heart rates of LKB1-KO mice were reduced and ventricle diameter was increased. Ex vivo, cardiac function was impaired during aerobic perfusion of isolated working hearts, and recovery of function after ischemia was reduced. Although oxidative metabolism and mitochondrial function were normal, the AMP/ATP ratio was increased in LKB1-KO hearts. This was associated with a complete ablation of AMPKα2 activity, and a stimulation of signaling through the mammalian target of rapamycin. Our results establish a critical role for LKB1 for normal cardiac function under both aerobic conditions and during recovery after ischemia. Ablation of LKB1 leads to a decreased cardiac efficiency despite normal mitochondrial oxidative metabolism.     

A mutation in CCDC50, a gene encoding an effector of epidermal growth factor-mediated cell signaling, causes progressive hearing loss

We previously mapped a novel autosomal dominant deafness locus, DFNA44, by studying a family with postlingual, progressive, nonsyndromic hearing loss. We report here on the identification of a mutation in CCDC50 as the cause of hearing loss in the family. CCDC50 encodes Ymer, an effector of epidermal growth factor (EGF)-mediated cell signaling that is ubiquitously expressed in different organs and has been suggested to inhibit down-regulation of the EGF receptor. We have examined its expression pattern in mouse inner ear. Western blotting and cell transfection results indicate that Ymer is a soluble, cytoplasmic protein, and immunostaining shows that Ymer is expressed in a complex spatiotemporal pattern during inner ear development. In adult inner ear, the expression of Ymer is restricted to the pillar cells of the cochlea, the stria vascularis, and the vestibular sensory epithelia, where it shows spatial overlap with the microtubule-based cytoskeleton. In dividing cells, Ymer colocalizes with microtubules of the mitotic apparatus. We suggest that DFNA44 hearing loss may result from a time-dependent disorganization of the microtubule-based cytoskeleton in the pillar cells and stria vascularis of the adult auditory system.     

In vivo VL-targeted activation-induced apoptotic supraclonal deletion by a microbial B cell toxin

To interfere with host immune responses, some microbial pathogens produce proteins with the properties of superantigens, which can interact via conserved V region framework subdomains of the Ag receptors of lymphocytes rather than the complementarity-determining region involved in the binding of conventional Ags. In recent studies, we have elucidated how a model B cell superantigen affects the host immune system by targeting a conserved V(H) site on the Ag receptors of B lymphocytes. To determine whether these findings represent a general paradigm, we investigated the in vivo immunobiologic properties of protein L of Peptostreptococcus magnus (PpL), a microbial Ig-binding protein specific for a V region site on Ig L chains. Our studies confirmed that PpL binding is restricted to a subset of murine Vkappa-expressing B cells, and found that B cells with stronger PpL-binding activity are associated with certain B cell subsets: splenic marginal zone (CD21(high) CD23(low)), splenic CD1(+), peritoneal B-1a (IgD(low) CD5(+)), and CD21(high) CD24(high) B cells in peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. Infusion of PpL triggered a sequence of events in B cell receptor (BCR)-targeted B cells, with rapid down-regulation of BCR, the induction of an activation phenotype, and limited rounds of proliferation. Apoptosis followed through a process heralded by the dissipation of mitochondrial membrane potential, the induction of the caspase pathway, DNA fragmentation, and the deposition of B cell apoptotic bodies. These studies define a common pathway by which microbial toxins that target V region-associated BCR sites induce programmed cell death.