Crystal Structure of the Complex between Calyculin A and the Catalytic Subunit of Protein Phosphatase 1

AbstractThe crystal structure of the catalytic subunit of the protein phosphatase 1 (PP1), PP1γ, in complex with a marine toxin, calyculin A, was determined at 2.0 Å resolution. The metal binding site contains the phosphate group of calyculin A and forms a tight network via the hydrophilic interactions between PP1 and calyculin A. Calyculin A is located in two of the three grooves, namely, in the hydrophobic groove and the acidic groove on the molecular surface. This is the first observation to note that the inhibitor adopts not a pseudocyclic conformation but an extended conformation in order to form a complex with the protein. The amino acid terminus of calyculin A contributes, in a limited manner, to the binding to PP1γ, which is consistent with findings from the studies of dose-inhibition analysis

Kheper, a Novel ZFH/δEF1 Family Member, Regulates the Development of the Neuroectoderm of Zebrafish (Danio rerio)

AbstractKheper is a novel member of the ZFH (zinc-finger and homeodomain protein)/δEF1 family in zebrafish. kheper transcripts are first detected in the epiblast of the dorsal blastoderm margin at the early gastrula stage and kheper is expressed in nearly all the neuroectoderm at later stages. kheper expression was expanded in noggin RNA-injected embryos and also in swirl mutant embryos and was reduced in bmp4 RNA-injected embryos and chordino mutant embryos, suggesting that kheper acts downstream of the neural inducers Noggin and Chordino. Overexpression of Kheper elicited ectopic expansion of the neuroectoderm-specific genes fkd3, hoxa-1, and eng3, and the ectopic expression of hoxa-1 was not inhibited by BMP4 overexpression. Kheper interacted with the transcriptional corepressors CtBP1 and CtBP2. Overexpression of a Kheper mutant lacking the homeodomain or of a VP16–Kheper fusion protein disturbed the development of the neuroectoderm and head structures. These data underscore the role of Kheper in the development of the neuroectoderm and indicate that Kheper acts as a transcriptional repressor

Membrane-anchored growth factor, HB-EGF, on the cell surface targeted to the inner nuclear membrane

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway

Donepezil reverses buprenorphine-induced central respiratory depression in anesthetized rabbits

Buprenorphine is a mixed opioid receptor agonist-antagonist used in acute and chronic pain management. Although this agent's analgesic effect increases in a dose-dependent manner, buprenorphine-induced respiratory depression shows a marked ceiling effect at higher doses, which is considered to be an indicator of safety. Nevertheless, cases of overdose mortality or severe respiratory depression associated with buprenorphine use have been reported. Naloxone can reverse buprenorphine-induced respiratory depression, but is slow-acting and unstable, meaning that new drug candidates able to specifically antagonize buprenorphine-induced respiratory depression are needed in order to enable maximal analgesic effect without respiratory depression. Acetylcholine is an excitatory neurotransmitter in central respiratory control. We previously showed that a long-acting acetylcholinesterase inhibitor, donepezil, antagonizes morphine-induced respiratory depression. We have now investigated how donepezil affects buprenorphine-induced respiratory depression in anesthetized, paralyzed, and artificially ventilated rabbits. We measured phrenic nerve discharge as an Índex of respiratory rate and amplitude, and compared discharges following the injection of buprenorphine with discharges following the injection of donepezil. Buprenorphine-induced suppression of the respiratory rate and respiratory amplitude was antagonized by donepezil (78.4 ± 4.8 %, 92.3% ± 22.8 % of control, respectively). These findings indicate that systemically administered donepezil restores buprenorphine-induced respiratory depression in anesthetized rabbits

CORRELATION BETWEEN THROWING MOTION AND MAXIMUM ELBOW VARUS TORQUE IN FEMALE PROFESSIONAL BASEBALL PITCHERS

The purpose of this study was to identify the correlation between throwing motion and maximum elbow varus torque (MEV) in female professional baseball pitchers. Twelve pitchers without pre-existing pain were recruited. Ball velocity and pitching motion were measured. Ball velocity and, kinematic and kinetic data from each joint during the pitch were extracted to evaluate the correlation with MEV. There was no correlation between the fastest ball velocity and MEV. Sixteen kinematic and kinetic parameters were found to have significant correlations with MEV. Particularly, as trunk rotation angle to the non-throwing direction before lead foot contact (FC) increased, the MEV decreased. Rotating the trunk in the non-throwing direction before FC and immediately in the throwing direction after FC could be a key component of the throwing motion

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.

International audienceThe molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC beta-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The beta construct beta-AID dominant negative, which disrupts the RIM1-beta association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with beta in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs

Mutation associated with an autosomal dominant cone-rod dystrophy CORD7 modifies RIM1-mediated modulation of voltage-dependent Ca2+ channels.

International audienceGenetic analyses have revealed an association between the gene encoding the Rab3A-interacting molecule (RIM1) and the autosomal dominant cone-rod dystrophy CORD7. However, the pathogenesis of CORD7 remains unclear. We recently revealed that RIM1 regulates voltage-dependent Ca(2+) channel (VDCC) currents and anchors neurotransmitter-containing vesicles to VDCCs, thereby controlling neurotransmitter release. We demonstrate here that the mouse RIM1 arginine-to-histidine substitution (R655H), which corresponds to the human CORD7 mutation, modifies RIM1 function in regulating VDCC currents elicited by the P/Q-type Ca(v)2.1 and L-type Ca(v)1.4 channels. Thus, our data can raise an interesting possibility that CORD7 phenotypes including retinal deficits and enhanced cognition are at least partly due to altered regulation of presynaptic VDCC currents

Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1

A disintegrin and metalloprotease 17 (ADAM17) is the major sheddase that processes more than 80 substrates, including tumour necrosis factor-α (TNFα). The homozygous genetic deficiency of ADAM17 causing a complete loss of ADAM17 expression was reported to be linked to neonatal inflammatory skin and bowel disease 1 (NISBD1). Here we report for the first time, a family with NISBD1 caused by functionally confirmed compound heterozygous missense variants of ADAM17, namely c.1699T>C (p.Cys567Arg) and c.1799G>A (p.Cys600Tyr). Both variants were detected in two siblings with clinical features of NISBD1, such as erythroderma with exudate in whole body, recurrent skin infection and sepsis and prolonged diarrhoea. In a cell-based assay using Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17−/− mEFs) exogenously expressing each of these mutants, phorbol 12-myristate 13-acetate-stimulated shedding was strongly reduced compared with wild-type ADAM17. Thus, in vitro functional assays demonstrated that both missense variants cause the loss-of-function of ADAM17, resulting in the development of NISBD1. Our study further expands the spectrum of genetic pathology underlying ADAM17 in NISBD1 and establishes functional assay systems for its missense variants