Sphingolipid Transport to the Apical Plasma Membrane Domain in Human Hepatoma Cells Is Controlled by PKC and PKA Activity: A Correlation with Cell Polarity in HepG2 Cells

The regulation of sphingolipid transport to the bile canalicular apical membrane in the well differentiated HepG2 hepatoma cells was studied. By employing fluorescent lipid analogs, trafficking in a transcytosis-dependent pathway and a transcytosis-independent (‘direct') route between the trans-Golgi network and the apical membrane were examined. The two lipid transport routes were shown to operate independently, and both were regulated by kinase activity. The kinase inhibitor staurosporine inhibited the direct lipid transport route but slightly stimulated the transcytosis-dependent route. The protein kinase C (PKC) activator phorbol-12 myristate-13 acetate (PMA) inhibited apical lipid transport via both transport routes, while a specific inhibitor of this kinase stimulated apical lipid transport. Activation of protein kinase A (PKA) had opposing effects, in that a stimulation of apical lipid transport via both transport routes was seen. Interestingly, the regulatory effects of either kinase activity in sphingolipid transport correlated with changes in cell polarity. Stimulation of PKC activity resulted in a disappearance of the bile canalicular structures, as evidenced by the redistribution of several apical markers upon PMA treatment, which was accompanied by an inhibition of apical sphingolipid transport. By contrast, activation of PKA resulted in an increase in the number and size of bile canaliculi and a concomitant enhancement of apical sphingolipid transport. Taken together, our data indicate that apical membrane-directed sphingolipid transport in HepG2 cells is regulated by kinases, which could play a role in the biogenesis of the apical plasma membrane domain

Patient safety in Dutch primary care: a study protocol

<p>Abstract</p> <p>Background</p> <p>Insight into the frequency and seriousness of potentially unsafe situations may be the first step towards improving patient safety. Most patient safety attention has been paid to patient safety in hospitals. However, in many countries, patients receive most of their healthcare in primary care settings. There is little concrete information about patient safety in primary care in the Netherlands. The overall aim of this study was to provide insight into the current patient safety issues in Dutch general practices, out-of-hours primary care centres, general dental practices, midwifery practices, and allied healthcare practices. The objectives of this study are: to determine the frequency, type, impact, and causes of incidents found in the records of primary care patients; to determine the type, impact, and causes of incidents reported by Dutch healthcare professionals; and to provide insight into patient safety management in primary care practices.</p> <p>Design and methods</p> <p>The study consists of three parts: a retrospective patient record study of 1,000 records per practice type was conducted to determine the frequency, type, impact, and causes of incidents found in the records of primary care patients (objective one); a prospective component concerns an incident-reporting study in each of the participating practices, during two successive weeks, to determine the type, impact, and causes of incidents reported by Dutch healthcare professionals (objective two); to provide insight into patient safety management in Dutch primary care practices (objective three), we surveyed organizational and cultural items relating to patient safety. We analysed the incidents found in the retrospective patient record study and the prospective incident-reporting study by type of incident, causes (Eindhoven Classification Model), actual harm (severity-of-outcome domain of the International Taxonomy of Medical Errors in Primary Care), and probability of severe harm or death.</p> <p>Discussion</p> <p>To estimate the frequency of incidents was difficult. Much depended on the accuracy of the patient records and the professionals' consensus about which types of adverse events have to be recognized as incidents.</p

Regulation of sphingolipid transport in mammalian cells

Het onderzoek dat in dit proefschrift is beschreven heeft zich gericht op de regulering van intracellulair sfingolipiden transport in gekweekte zoogdiercellen. ... Zie: Samenvatting

Scrib regulates HGF-mediated epithelial morphogenesis and is stabilized by Sgt1-HSP90.

Scribble was originally identified as a Drosophila protein that regulates epithelial polarity and formation of the basolateral surface. The mammalian orthologue, Scrib, is evolutionarily conserved, but does not appear to be necessary for apical-basolateral epithelial polarity. Instead, it is implicated in the regulation of cell survival, protein trafficking, adhesion and migration. A key issue is to understand the molecular pathway by which Scrib participates in these processes. We have investigated Scrib using a three-dimensional epithelial cell culture system. We show a novel association between the leucine-rich repeat domain of Scrib and the co-chaperone Sgt1 and demonstrate that these proteins are necessary for epithelial morphogenesis and tubulogenesis following hepatocyte growth factor (HGF) stimulation. The molecular chaperone HSP90 is also required for Sgt1 association with Scrib, and both Sgt1 and HSP90 are needed to ensure proper Scrib protein levels. Furthermore, reduced Scrib stability, following inhibition of Sgt1-HSP90, lowers the cellular abundance of the Scrib-βPix-PAK complex. Inhibition of any member of this complex, Scrib, βPix or PAK, is sufficient to block HGF-mediated epithelial morphogenesis. The identification of Scrib as an Sgt1-HSP90 client protein required for three-dimensional cell migration suggests that chaperone-mediated regulation of polarity protein stability and homeostasis is an unappreciated mechanism underlying dynamic rearrangements during morphogenesis