939 research outputs found

    Technical Note: The Use of RNA-interference as a Tool to Find Proteins Involved in Melanosome Formation or Transport

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    Melanosomes are lysosome-related organelles that produce and transport the pigment melanin within melanocytes. Mutations in proteins required for melanosome transport and formation lead to a range of pigmentation defects, manifested at the cellular level as perinuclear clustering of melanosomes, or reduced sorting of melanosomal cargo such as tyrosinase-related protein 1 (TYRP1). A pilot screen was carried out to investigate whether a combination of cellular imaging and RNA interference could be used to identify new proteins involved in pigmentation pathways. In this study, eleven genes known to play a role in melanosome transport/formation or other pigmentation properties were knocked down in mouse melanocytes with shRNAmir constructs. The investigated genes were TYRP1, pallidin, cappuccino, dysbindin, HPS5, LYST, Myosin Va, melanophilin, RhoA, UBPY and mahogunin. In a blinded confocal imaging experiment, the only reproducible change observed in cells in which these targets were knocked down was a decrease in TYRP1 levels upon transfection with knockdown constructs against TYRP1 itself, or one of three constructs targeting HPS5 (Hermansky-Pudlak Syndrome 5). Upon analysis with high-content imaging software, only the knockdown construct against TYRP1 itself was detected. RT-PCR analysis showed that many of the shRNAmir constructs did not reduce mRNA and proteins levels enough to detect effects on melanosome properties. This was further examined for melanophilin, a protein necessary for melanosome transport. Altogether, the data show that this system is currently not sensitive enough for use in a screen for unknown regulators of melanosome transport and formation. The main obstacle appears to be incomplete reduction of target protein levels. Our observation that a ~50% reduction in mRNA level is not sufficient to elicit an effect is supported by the fact that heterozygous carriers of melanosomal transport disorders (Griscelli Syndrome, Hermansky-Pudlak Syndrome) do not display diseases phenotypes. A further reduction in protein levels, for example by viral infection of shRNA, may be required

    Changing practice in dealing with the Sec examination course work through action research

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    The changes that were implemented in the course work part of the SEC examination of Home Economics in 1997 meant that teachers had to find ways of guiding students in building up a portfolio with tasks which they had to work out using different investigative techniques. Teachers were also given the responsibility of assessing and marking the students’ work at the end. Dedicating time for course work in the classroom by integrating it with the rest of the programme of work, as well as teaching students how to make use of different methods of research through experiential learning was important. The need for assessment criteria which are less vague and more clear for teachers and moderators to interpret also emerged, mainly as a result of problems that were encountered during the marking and moderation process because of the different ways in which the criteria were interpreted by teachers and the moderator. Finally, this article also presents the reader with an account of the experience of teachers who tried to be researchers in their own practice. While proving to be an enriching experience for teachers, it was also realised that the school in which the action research project was carried out lacked the structural and cultural framework within which action research projects could be carried out.peer-reviewe

    Role of the UPS in Liddle syndrome

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    Hypertension is a serious medical problem affecting a large population worldwide. Liddle syndrome is a hereditary form of early onset hypertension caused by mutations in the epithelial Na+ channel (ENaC). The mutated region, called the PY (Pro-Pro-x-Tyr) motif, serves as a binding site for Nedd4-2, an E3 ubiquitin ligase from the HECT family. Nedd4-2 binds the ENaC PY motif via its WW domains, normally leading to ENaC ubiquitylation and endocytosis, reducing the number of active channels at the plasma membrane. In Liddle syndrome, this endocytosis is impaired due to the inability of the mutated PY motif in ENaC to properly bind Nedd4-2. This leads to accumulation of active channels at the cell surface and increased Na+ (and fluid) absorption in the distal nephron, resulting in elevated blood volume and blood pressure. Small molecules/compounds that destabilize cell surface ENaC, or enhance Nedd4-2 activity in the kidney, could potentially serve to alleviate hypertension

    Role of the ubiquitin system in regulating ion transport

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    Ion channels and transporters play a critical role in ion and fluid homeostasis and thus in normal animal physiology and pathology. Tight regulation of these transmembrane proteins is therefore essential. In recent years, many studies have focused their attention on the role of the ubiquitin system in regulating ion channels and transporters, initialed by the discoveries of the role of this system in processing of Cystic Fibrosis Transmembrane Regulator (CFTR), and in regulating endocytosis of the epithelial Na+ channel (ENaC) by the Nedd4 family of ubiquitin ligases (mainly Nedd4-2). In this review, we discuss the role of the ubiquitin system in ER Associated Degradation (ERAD) of ion channels, and in the regulation of endocytosis and lysosomal sorting of ion channels and transporters, focusing primarily in mammalian cells. We also briefly discuss the role of ubiquitin like molecules (such as SUMO) in such regulation, for which much less is known so fa

    Nedd4-2 and the Regulation of Epithelial Sodium Transport

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    Nedd4-2 is a ubiquitin ligase previously demonstrated to regulate endocytosis and lysosomal degradation of the epithelial Na+ channel (ENaC) and other ion channels and transporters. Recent studies using Nedd4-2 knockout mice specifically in kidney or lung epithelia has revealed a critical role for this E3 ubiquitin ligase in regulating salt and fluid transport in these tissues/organs and in maintaining homeostasis of body blood pressure. Interestingly, the primary targets for Nedd4-2 may differ in these two organs: in the lung Nedd4-2 targets ENaC, and loss of Nedd4-2 leads to excessive ENaC function and to cystic fibrosis – like lung disease, whereas in the kidney, Nedd4-2 targets the Na+/Cl− cotransporter (NCC) in addition to targeting ENaC. In accord, loss of Nedd4-2 in the distal nephron leads to increased NCC abundance and function. The aldosterone-responsive kinase, Sgk1, appears to be involved in the regulation of NCC by Nedd4-2 in the kidney, similar to its regulation of ENaC. Collectively, these new findings underscore the physiological importance of Nedd4-2 in regulating epithelial salt and fluid transport and balance