Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor

LPL mediates the uptake of lipoproteins into different cell types independent of its catalytic activity. The mechanism of this process and its physiological relevance are not clear. Taking into account the importance of the endothelial barrier for lipoprotein uptake, in vitro studies with primary aortic endothelial cells from wild-type and low density lipoprotein receptor (LDLR)-deficient (LDLR(-/-)) mice were performed. Addition of LPL almost doubled the uptake of LDL into wild-type cells. However, there was virtually no LPL-mediated change of LDL uptake into LDLR(-/-) cells. Upregulation of LDLR by lipoprotein-deficient serum/lovastatin in wild-type cells resulted in a 7-fold increase of LPL-mediated LDL uptake. Uptake of chylomicron remnants was not affected by LDLR expression. In proteoglycan-deficient cells, LPL did not increase the uptake of lipoproteins. The physiological relevance of this pathway was studied in mice that were both LDLR(-/-) and transgenic for catalytically inactive LPL in muscle. In the presence of LDLR, inactive LPL reduced LDL cholesterol significantly (13-24%). In the absence of LDLR, LDL cholesterol was not affected by transgenic LPL. Metabolic studies showed that in the presence of LDLR, LPL increased the muscular uptake of LDL by 77%. In the absence of LDLR, transgenic LPL did not augment LDL uptake. Chylomicron uptake was not affected by the LDLR genotype. We conclude that LPL-mediated cellular uptake of LDL, but not of chylomicrons, is dependent on the presence of both LDLR and proteoglycans.     

Prominin-2 is a cholesterol-binding protein associated with apical and basolateral plasmalemmal protrusions in polarized epithelial cells and released into urine

Prominin-2 is a pentaspan membrane glycoprotein structurally related to the cholesterol-binding protein prominin-1, which is expressed in epithelial and non-epithelial cells. Although prominin-1 expression is widespread throughout the organism, the loss of its function solely causes retinal degeneration. The finding that prominin-2 appears to be restricted to epithelial cells, such as those found in kidney tubules, raises the possibility that prominin-2 functionally substitutes prominin-1 in tissues other than the retina and provokes a search for a definition of its morphological and biochemical characteristics. Here, we have investigated, by using MDCK cells as an epithelial cell model, whether prominin-2 shares the biochemical and morphological properties of prominin-1. Interestingly, we have found that, whereas prominin-2 is not restricted to the apical domain like prominin-1 but is distributed in a non-polarized fashion between the apical and basolateral plasma membranes, it retains the main feature of prominin-1, i.e. its selective concentration in plasmalemmal protrusions; prominin-2 is confined to microvilli, cilia and other acetylated tubulin-positive protruding structures. Similar to prominin-1, prominin-2 is partly associated with detergent-resistant membranes in a cholesterol-dependent manner, suggesting its incorporation into membrane microdomains, and binds directly to plasma membrane cholesterol. Finally, prominin-2 is also associated with small membrane particles that are released into the culture media and found in a physiological fluid, i.e. urine. Together, these data show that all the characteristics of prominin-1 are shared by prominin-2, which is in agreement with a possible redundancy in their role as potential organizers of plasma membrane protrusions.     

Hyperoxia modulates TGF-beta/BMP signaling in a mouse model of bronchopulmonary dysplasia

Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O(2) between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O(2), and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-beta type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-beta signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O(2) as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O(2). After exposure to 85% O(2), primary alveolar type II cells were more susceptible to TGF-beta-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O(2) significantly enhanced the TGF-beta-stimulated production of the alpha(1) subunit of type I collagen (Ialpha(1)), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-beta/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.     

Methodologies for examining problem solving success and failure

When designing research to examine the variables underlying creative thinking and problem solving success, one must not only consider (a) the demands of the task being performed, but (b) the characteristics of the individual performing the task and (c) the constraints of the skill execution environment. In the current paper we describe methodologies that allow one to effectively study creative thinking by capturing interactions among the individual, task, and problem solving situation. In doing so, we demonstrate that the relation between executive functioning and problem solving success is not always as straightforward as one might initially believe.     

The Antidepressant Fluoxetine Mobilizes Vesicles to the Recycling Pool of Rat Hippocampal Synapses During High Activity

Effects of the antidepressant fluoxetine in therapeutic concentration on stimulation-dependent synaptic vesicle recycling were examined in cultured rat hippocampal neurons using fluorescence microscopy. Short-term administration of fluoxetine neither inhibited exocytosis nor endocytosis of RRP vesicular membranes. On the contrary, acute application of the drug markedly increased the size of the recycling pool of hippocampal synapses. This increase in recycling pool size was corroborated using the styryl dye FM 1-43, antibody staining with αSyt1-CypHer?5E and overexpression of synapto-pHluorin, and was accompanied by an increase in the frequency of miniature postsynaptic currents. Analysis of axonal transport and fluorescence recovery after photobleaching excluded vesicles originating from the synapse-spanning superpool as a source, indicating that these new release-competent vesicles derived from the resting pool. Super resolution microscopy and ultrastructural analysis by electron microscopy revealed that short-term incubation with fluoxetine had no influence on the number of active synapses and synaptic morphology compared to controls. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recycling vesicle pool size and thus the recovery of neurotransmission from exhausting stimuli. The change in the recycling pool size is consistent with the plasticity hypothesis of the pathogenesis of major depressive disorder as stabilization of the vesicle recycling might be responsible for neural outgrowth and plasticity.     

Nitrate/oxygen co‐sensing by an NreA/NreB sensor complex of Staphylococcus carnosus

In Staphylococci maximal induction of nitrate reductase (narGHJI genes) requires anaerobic conditions, the presence of nitrate, and the NreABC regulatory system. Aerobic regulation is effected by the NreB/NreC two‐component system. The role of the nitrate receptor NreA in nitrate induction and its relation to aerobic regulation was analysed in Staphylococcus carnosus. Nitrate induction of a narG‐lip reporter gene required presence of NreB/NreC. When nreA was deleted, nitrate was no longer required for maximal induction, suggesting that NreA is a nitrate regulated inhibitor of NreB/NreC. In vitro, NreA and mutant NreA(Y95A) decreased NreB phosphorylation in part or completely, which was due to the inhibition of the autophosphorylating activity rather than an increase of phosphatase activity. Inhibition of phosphorylation was relieved completely when the nitrate‐bound NreA was used instead of the nitrate‐free form. In the bacterial two‐hybrid BACTH system and HPINE interaction assays, NreA interacted with NreB, but not with NreC, and the interaction was diminished by nitrate. In summary, NreA interacts with NreB and controls its phosphorylation level in a nitrate dependent manner. In this way nitrate and NreA modulate the function of the oxygen sensor NreB, resulting in nitrate/oxygen co‐sensing by an NreA/NreB sensor unit as part of the NreABC‐system.