Role of clathrin- and actin-dependent endocytotic pathways in lung phospholipid uptake.
(57/347)
We evaluated the contribution of endocytotic pathways to pulmonary uptake of surfactant lipids from the alveolar space. Resting and stimulated 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) uptake of unilamellar liposomes labeled with either [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) or 1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl-phosphatidylcholine (NBD-PC) was studied in isolated perfused rat lungs and isolated type II cells. Amantadine and phenylarsine oxide, inhibitors of clathrin-mediated endocytosis, each decreased [(3)H]DPPC uptake under resting conditions by approximately 40%; their combination had no additional effect. Cytochalasin D, an inhibitor of actin-dependent processes, reduced liposome uptake by 55% and potentiated the effect of either clathrin inhibitor alone. Relative inhibition for all agents was higher in the presence of 8-Br-cAMP. The effect of inhibitors was similar for liposomes labeled with [(3)H]DPPC or NBD-PC. By fluorescence microscopy, NBD-PC taken up by lungs was localized primarily to alveolar type II cells and was localized to lamellar bodies in both lungs and isolated cells. These studies indicate that both clathrin-mediated and actin-mediated pathways are responsible for endocytosis of DPPC-labeled liposomes by alveolar type II cells in the intact lung. (+info)
Reversible labeling of tyrosine residue in peptide using 4-fluoro-7-nitro-2,1,3-benzoxadiazole and N-acetyl-L-cysteine.
(58/347)
The reversible labeling of tyrosine (Tyr)-containing peptide, which involves detection and recovery, is described in this paper. The phenolic-OH in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) under a mild condition at room temperature in alkaline medium. The resulting derivative absorbed at around 280 nm and 380 nm. However, the fluorescence was very weak. The NBD moiety in the derivative was efficiently removed by the treatment of N-acetyl-L-cysteine (N-AcCys), and the original peptide before the labeling was completely recovered. The proposed procedure was successfully applied to the reversible labeling of N-terminal amine-blocked peptides, i.e., N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Although the proposed method could not recover the N-terminal amine-free peptides without blocking, the selective detection and the recovery of Tyr-containing peptide fragments were possible by the combination with enzyme digestion. The reversible labeling of Tyr-containing peptide was demonstrated with [Tyr8]-bradykinin as a model for high-molecular-mass peptides and proteins. The peptide fragments containing NBD-O-Tyr moiety, obtained after the digestion, were easily discriminated from various peptides with the monitoring of UV and FL, because the target peptide did not fluoresce, but absorbed at both 280 nm and 380 nm. The peptide fragment containing Tyr was finally recovered from the de-labeling reaction with N-AcCys. The proposed method hence provides a novel technique for the reversible labeling of Tyr-containing peptides, which will enable the selective detection and the recovery of the original peptide. (+info)
Uptake and metabolism of low density lipoproteins with elevated ceramide content by human microvascular endothelial cells: implications for the regulation of apoptosis.
(59/347)
Ceramide is a bioactive molecule involved in cellular responses to stress and inflammation. The major pathway for ceramide accumulation is via agonist-induced activation of cellular sphingomyelinases. It has also been shown that the ceramide level in circulating low density lipoprotein (LDL) increases during systemic inflammation, hence it is of importance to understand whether LDL-derived ceramide also contributes to the cellular ceramide homeostasis and affects cell functions. This article provides evidence of uptake of ceramide-enriched LDL by human microvascular endothelial cells in a receptor-mediated fashion. This uptake can be competed by native LDL, indicating that the LDL-binding receptor may be involved. Following uptake, part of the LDL-derived ceramide is converted to sphingosine, but more importantly, part of it accumulates inside the cells (approximately 1.44 nmol/mg of cell protein). This accumulation of ceramide correlates with an increased incidence of apoptosis. The addition of tumor necrosis factor-alpha further enhances the accumulation of LDL-derived ceramide and the rate of apoptosis. In contrast, inhibitors of receptor-mediated endocytosis block both, the accumulation of LDL-derived ceramide and the concurrent increase in apoptosis. We also show that LDL-delivered ceramide is a more efficient inducer of apoptosis as compared with ethanol-delivered ceramide, the same apoptotic effect being achieved by substantially smaller increases in intracellular ceramide. Taken together, the presented data indicate that increases in lipoprotein ceramide concentration may result in changes in the bioactive properties of circulating lipoproteins such as the enhanced ability to induce apoptosis in the microvascular endothelium. (+info)
HDL-mediated cholesterol uptake and targeting to lipid droplets in adipocytes.
(60/347)
Adipocytes express high levels of the HDL scavenger receptor class B type I in a differentiation-dependent manner. We thus have analyzed the routes of HDL cholesterol trafficking at different phases of adipocyte differentiation in the 3T3-L1 cell line. One novel and salient feature of this paper is the observation of a widespread distribution in the cell cytoplasm of Golgi markers, caveolin-2, and a fluorescent cholesterol analog NBD-cholesterol (NBD-chol), observed in the early phases of adipocyte formation, clearly distinct from that observed in mature fat cells (i.e., with fully formed lipid vesicles). Thus, in cells without visible lipid droplets, Golgi markers (Golgi 58K, Golgin 97, trans-Golgi network 38, Rab 6, and BODIPY-ceramide), caveolin-2, and NBD-chol all colocalize in a widespread distribution in the cell. In contrast, when lipid droplets are fully formed at latter stages, these markers clearly are distributed to distinct cell compartments: a compact juxtanuclear structure for the Golgi markers and caveolin-2, while NDB-chol concentrates in lipid droplets. In addition, disorganization of the Golgi using three different agents (Brefeldin, monensin, and N-ethyl-maleimide) drastically reduces NBD-chol uptake at different phases of adipocyte formation, strongly suggesting that the Golgi apparatus plays a critical role in HDL-mediated NBD uptake and routing to lipid droplets. (+info)
Glutamate triggers rapid glucose transport stimulation in astrocytes as evidenced by real-time confocal microscopy.
(61/347)
Glutamate stimulates glycolysis in astrocytes, a phenomenon that couples astrocytic metabolism with neuronal activity. However, it is not known whether glutamate also affects glucose transporter-1 (GLUT1), the transporter responsible for glucose entry into astrocytes. To address this question, two different real-time single-cell hexose uptake assays were applied to cultured hippocampal astrocytes using confocal epifluorescence microscopy. Glutamate caused a twofold to threefold increase in the zero-trans uptake rates of the fluorescent hexoses 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) and 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG). Galactose uptake, determined by the calcein volumetric assay, was stimulated to a similar extent, confirming the fluorescent hexose data, and also demonstrating that glutamate stimulation is a Vmax effect. Remarkably, glucose transport stimulation developed fully inside 10 sec, which is 100 times faster than acute stimulations of glucose transport in other cell types. Glutamate did not significantly affect the rate of 6-NBDG uptake by GLUT1-expressing epithelial Clone 9 cells, suggesting that an astrocyte-specific factor is required for transport stimulation. We conclude that glucose transport stimulation occurs early during astrocytic activation by glutamate, which provides a novel regulatory node to current models of brain energy metabolism. This mechanism should also be considered for the interpretation of functional imaging data based on hexoses. (+info)
Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol for their survival.
(62/347)
Ehrlichia chaffeensis and Anaplasma phagocytophilum are agents of human monocytic and granulocytic ehrlichioses, respectively. They are extremely sensitive to mechanical stress and are pleomorphic gram-negative bacteria. Membrane incorporation of cholesterol from the eukaryotic host is known to be essential for other fragile and pleomorphic bacteria and mycoplasmas that lack a cell wall. Thus, we tested whether cholesterol is required for E. chaffeensis and A. phagocytophilum. Using a freeze fracture technique and biochemical analysis, these bacteria were found to contain significant levels of membrane cholesterol. These bacteria lack genes for cholesterol biosynthesis or modification. However, host cell-free bacteria had the ability to take up directly exogenous cholesterol or NBD-cholesterol, a fluorescent cholesterol derivative. Treatment of the bacteria with cholesterol extraction reagent methyl-beta-cyclodextrin caused their ultrastructural changes. Furthermore, pretreatment of the bacteria with methyl-beta-cyclodextrin or NBD-cholesterol deprived these bacteria of the ability to infect leukocytes, thus killing these obligate intracellular bacteria. Analysis of E. chaffeensis and A. phagocytophilum genome sequences revealed that these bacteria lack all genes for the biosynthesis of lipid A and most genes for the biosynthesis of peptidoglycan, which confer structural strength to gram-negative bacteria. Taken together, these results suggest that human ehrlichiosis agents became cholesterol dependent due to the loss of these genes. As the first report of gram-negative bacteria incorporating cholesterol for survival, these findings offer insight into the unique nature of their parasitism and imply that cholesterol is important in the control of human ehrlichioses. (+info)
Role of prothrombin fragment 1 in the pathway of regulatory exosite I formation during conversion of human prothrombin to thrombin.
(63/347)
Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin(54-65) labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir(54-65)(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir(54-65)(SO3-)). [NBD]Hir(54-65)(SO3-) distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir(54-65)(SO3-). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir(54-65)(SO3-) by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin.F1.2 complex bound [5F]Hir(54-65)(SO3-) with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va. (+info)
Enhanced exposure of phosphatidylserine in human gastric carcinoma cells overexpressing the half-size ABC transporter BCRP (ABCG2).
(64/347)
Members of the ABC (ATP-binding cassette) transporter super-family are emerging to be involved in lipid transport. In the present study, we studied the organization of phospholipids in the plasma membrane of EPG85-257 human gastric carcinoma cells overexpressing BCRP (breast cancer resistance protein, ABCG-2), a half-size transporter belonging to the ABCG subfamily. A significantly increased plasma membrane association of the PS (phosphatidylserine)-binding probe FITC-Annexin V in comparison with control cells was observed. Treatment of BCRP -overexpressing cells with the inhibitor Tryprostatin A decreased FITC-Annexin V binding almost to the control level. This suggests an enhanced exposure of PS in BCRP -overexpressing cells, which is dependent on functional BCRP. A role of BCRP in the transverse distribution of lipids in the plasma membrane is supported by the increased outward transport of the lipid analogue C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PS in BCRP -overexpressing EPG85-257 cells and MCF-7 human breast cancer cells. As shown for BCRP -overexpressing EPG85-257 cells, enhanced outward redistribution of the lipid analogue is inhibited by Tryprostatin A as well as by reduction of BCRP expression on transfection with an anti- BCRP -ribozyme. We also observed an enhanced outward transport of C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylcholine in BCRP -overexpressing EPG85-257 cells, suggesting that the influence of BCRP on transverse lipid organization is not highly specific. (+info)