Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening

<p>Abstract</p> <p>Background</p> <p>Deregulated activation of cyclin-dependent kinase-5 (Cdk5) is implicated in neurodegenerative disorders such as Alzheimer's disease. One of the restricting factors for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized cellular in vitro assay systems.</p> <p>Methods</p> <p>HEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we utilized an antibody that recognizes phospho serine in a consensus motif for Cdk substrates.</p> <p>Results</p> <p>Western blot analysis of transfected cells detected a 200 kDa band that was identified, by mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B). Phosphorylation of NMHC-B was evident only in cells that were double transfected with Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors. Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y cell line.</p> <p>Conclusion</p> <p>A novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.</p

Neurotrophin-3 regulates ribbon synapse density in the cochlea and induces synapse regeneration after acoustic trauma

Neurotrophin-3 (Ntf3) and brain derived neurotrophic factor (Bdnf) are critical for sensory neuron survival and establishment of neuronal projections to sensory epithelia in the embryonic inner ear, but their postnatal functions remain poorly understood. Using cell-specific inducible gene recombination in mice we found that, in the postnatal inner ear, Bbnf and Ntf3 are required for the formation and maintenance of hair cell ribbon synapses in the vestibular and cochlear epithelia, respectively. We also show that supporting cells in these epithelia are the key endogenous source of the neurotrophins. Using a new hair cell CreERT line with mosaic expression, we also found that Ntf3's effect on cochlear synaptogenesis is highly localized. Moreover, supporting cell-derived Ntf3, but not Bbnf, promoted recovery of cochlear function and ribbon synapse regeneration after acoustic trauma. These results indicate that glial-derived neurotrophins play critical roles in inner ear synapse density and synaptic regeneration after injury. DOI: http://dx.doi.org/10.7554/eLife.03564.00

Lingual deficits in neurotrophin double knockout mice

Brain-derived neurotrophic factor (BDNF) and Neurotrophin 3 (NT-3) are members of the neurotrophin family and are expressed in the developing and adult tongue papillae. BDNF null-mutated mice exhibit specific impairments related to innervation and development of the gustatory system while NT-3 null mice have deficits in their lingual somatosensory innervation. To further evaluate the functional specificity of these neurotrophins in the peripheral gustatory system, we generated double BDNF/NT-3 knockout mice and compared the phenotype to BDNF −/− and wild-type mice. Taste papillae morphology was severely distorted in BDNF −/− x NT-3 −/− mice compared to single BDNF −/− and wild-type mice. The deficits were found throughout the tongue and all gustatory papillae. There was a significant loss of fungiform papillae and the papillae were smaller in size compared to BDNF −/− and wild-type mice. Circumvallate papillae in the double knockouts were smaller and did not contain any intraepithelial nerve fibers. BDNF −/− x NT-3 −/− mice exhibited additive losses in both somatosensory and gustatory innervation indicating that BDNF and NT-3 exert specific roles in the innervation of the tongue. However, the additional loss of fungiform papillae and taste buds in BDNF −/− x NT-3 −/− mice compared to single BDNF knockout mice indicate a synergistic functional role for both BDNF-dependent gustatory and NT-3-dependent somatosensory innervations in taste bud and taste papillae innervation and development.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47465/1/11068_2005_Article_3330.pd

Building sensory receptors on the tongue

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro . Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds—the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47466/1/11068_2005_Article_3332.pd

Specificity of neurotrophins in the nervous system : A genetic approach to determine receptor engagement by neurotrophins

The classical neurotrophic factor hypothesis describes the neurotrophins as retrograde signalling factors supporting the survival of postmitotic neurons during the development. Knock-out mice for the neurotrophins and/or their receptors have been generated and their analyses gave new insights in the temporal expression and particular functions of these factors during development. Despite partially overlapping expression of neurotrophins and Trk receptor in both the central and peripheral nervous system, the different knock-outs display distinct phenotypes, demonstrating diverse and specific roles of the neurotrophins. Further analysis showed that several neurotrophins can function as survival factors for the same neurons, indicating a potential for the neurotrophins to display compensatory effects. Further comparison of the loss of neuronal numbers between neurotrophin and Trk receptor knock-outs have suggested a role for NT3 signalling through its non-preferred receptors, TrkA and TrkB. In this thesis the specificity and selective roles of BDNF and NT3 in the peripheral and central nervous system have been analysed. In order to analyse this, the coding part of the BDNF gene has been replaced by NT3, in genetically modified mice (BDNNT3/NT3). Analysis of the nervous system revealed striking differences in the ability of NT3 to replace BDNF. We conclude that there is no general mechanism by which neurotrophin specificity is attained and the specificity of neurotrophin signalling is set by several factors, which are dependent on the cellular system studied. These factors include a spatial and temporal expression of ligands and receptors and the activation of diverse intracellular pathways by different Trk receptors within the same neuronal population. Finally, we also investigated the ability of NT3 to signal through non-preferred receptors in vivo. We see that there is a high degree of receptor specificity between neurotrophins and their cognate receptors in vivo

Are digital HRM-tools like a bad Facebook? : A qualitative study about HR-practitioners’ conceptions of the usage of digital HRM-tools

Studien syftar till att bidra med kunskap om hur HR-medarbetare uppfattar användandet av digitala HRM-verktyg i sitt dagliga arbete. Syftet preciseras genom de tre frågeställningarna; Vad kännetecknar användandet av HRM-verktyg i en HR-medarbetares dagliga arbete? Vilka konsekvenser får användandet av digitala HRM-verktyg för HR-funktionens arbete? Vilka konsekvenser får användandet av digitala HRM-verktyg för anställda och chefer i organisationen? Studien är av kvalitativ karaktär och baseras på en fenomenografisk metodansats för att undersöka informanternas uppfattningar om digitala HRM-verktyg. Studiens upplägg är en intervjustudiedesign och tio halvstrukturerade intervjuer genomfördes med HR-medarbetare från fem olika företag. Resultatet visade att HR-medarbetarna har varierande uppfattningar om hur de digitala HRM-verktygen används, men att de används i både strategiska och operativa syften. Även implementeringen uppfattades som en viktig faktor för hur verktygen skulle komma att användas samt att HR-medarbetarna själva vara ansvariga för lärandet. HR-medarbetarna uppfattade även att de digitala HRM-verktygen leder till automatisering, professionalisering och ökad rollspecialisering för HR-funktionen

Are digital HRM-tools like a bad Facebook? : A qualitative study about HR-practitioners’ conceptions of the usage of digital HRM-tools

Studien syftar till att bidra med kunskap om hur HR-medarbetare uppfattar användandet av digitala HRM-verktyg i sitt dagliga arbete. Syftet preciseras genom de tre frågeställningarna; Vad kännetecknar användandet av HRM-verktyg i en HR-medarbetares dagliga arbete? Vilka konsekvenser får användandet av digitala HRM-verktyg för HR-funktionens arbete? Vilka konsekvenser får användandet av digitala HRM-verktyg för anställda och chefer i organisationen? Studien är av kvalitativ karaktär och baseras på en fenomenografisk metodansats för att undersöka informanternas uppfattningar om digitala HRM-verktyg. Studiens upplägg är en intervjustudiedesign och tio halvstrukturerade intervjuer genomfördes med HR-medarbetare från fem olika företag. Resultatet visade att HR-medarbetarna har varierande uppfattningar om hur de digitala HRM-verktygen används, men att de används i både strategiska och operativa syften. Även implementeringen uppfattades som en viktig faktor för hur verktygen skulle komma att användas samt att HR-medarbetarna själva vara ansvariga för lärandet. HR-medarbetarna uppfattade även att de digitala HRM-verktygen leder till automatisering, professionalisering och ökad rollspecialisering för HR-funktionen

Are digital HRM-tools like a bad Facebook? : A qualitative study about HR-practitioners’ conceptions of the usage of digital HRM-tools

Studien syftar till att bidra med kunskap om hur HR-medarbetare uppfattar användandet av digitala HRM-verktyg i sitt dagliga arbete. Syftet preciseras genom de tre frågeställningarna; Vad kännetecknar användandet av HRM-verktyg i en HR-medarbetares dagliga arbete? Vilka konsekvenser får användandet av digitala HRM-verktyg för HR-funktionens arbete? Vilka konsekvenser får användandet av digitala HRM-verktyg för anställda och chefer i organisationen? Studien är av kvalitativ karaktär och baseras på en fenomenografisk metodansats för att undersöka informanternas uppfattningar om digitala HRM-verktyg. Studiens upplägg är en intervjustudiedesign och tio halvstrukturerade intervjuer genomfördes med HR-medarbetare från fem olika företag. Resultatet visade att HR-medarbetarna har varierande uppfattningar om hur de digitala HRM-verktygen används, men att de används i både strategiska och operativa syften. Även implementeringen uppfattades som en viktig faktor för hur verktygen skulle komma att användas samt att HR-medarbetarna själva vara ansvariga för lärandet. HR-medarbetarna uppfattade även att de digitala HRM-verktygen leder till automatisering, professionalisering och ökad rollspecialisering för HR-funktionen

Customer-Based Design with Constraint Reasoning

This paper identifies some principles for computer aided customer based design. In this type of design. technical prerequisites and the range of customer requirements are typically the same over time. From time to time there are larger changes in manufacturing methods and customer needs though. Thus the degree of aiutomation in a design support system can be fairly large, but it still needs to be interactive and flexible for changes. The testcase presented is based on an industrial application, implemented using a commercial CAD system and a CLP (Constraint Logic Programming) system. Special emphasis is put on interactivity - the collaboration between a designer and automated design system.QC 20140317</p