Monocyte cholesterol homeostasis correlates with the presence of detergent resistant membrane microdomains.

BACKGROUND: Lipid membrane microdomains are involved in the regulation of biological functions of monocyte membrane proteins. These microdomains show a relative resistance to non-ionic detergents providing an easy analytical tool to study them. METHODS: Here, we applied a rapid detergent-based flow cytometric assay to investigate microdomain association of proteins on monocytes from whole blood samples. The association of known surface antigens with detergent resistant fraction of membranes (DRMs) was compared using monocytes from healthy blood donors, patients with genetic disorders affecting cellular cholesterol traffic and patients with systemic inflammatory response. RESULTS: All investigated surface antigens of Niemann-Pick type C (NPC)-mutant monocytes with impaired cholesterol influx and defective late endosome cholesterol trafficking, presented a strongly increased DRM-association. Though, membrane antigens of ATP binding cassette transporter A1 (ABCA1)-mutant monocytes with impaired cholesterol efflux did not show alterations in DRM-association. Differential CD14-dependent receptor clustering within microdomains was also investigated in response to in vivo lipopolysaccharide (LPS) and/or atherogenic lipoprotein activation. Increased DRM-association of the GPI-anchored proteins CD14, CD55, the Fcgamma receptor CD64, the scavenger receptors CD36, CD91 and CD163, the integrin CD11a, and complement receptor 3 complex CD11b/CD18 were observed from patients with systemic inflammatory response syndrome (SIRS)/sepsis or coronary artery disease (CAD)/myocardial infarction. Interestingly, the tetraspanin CD81 presented increased DRM-association in SIRS/sepsis patients, but not in CAD patients. Moreover, the pentaspanin CD47 and the Fcgamma RIII CD16 showed an increased DRM partition in CAD patients but disassembled from DRMs in SIRS/sepsis patients. CONCLUSIONS: Our results demonstrate that flow cytometric analysis of short time in situ detergent extraction provides a powerful tool for rapid screening of blood monocyte DRMs to preselect patients with potential raft/microdomain abnormalities for more detailed analysis.     

Increased Levels of Sphingosylphosphorylcholine (SPC) in Plasma of Metabolic Syndrome Patients

Recent developments in lipid mass spectrometry enable extensive lipid class and species analysis in metabolic disorders such as diabesity and metabolic syndrome. The minor plasma lipid class sphingosylphosphorylcholine (SPC) was identified as a ligand for lipid sensitive G-protein coupled receptors playing a key role in cell growth, differentiation, motility, calcium signaling, tissue remodeling, vascular diseases and cancer. However, information about its role in diabesity patients is sparse. In this study, we analyzed plasma lipid species in patients at risk for diabesity and the metabolic syndrome and compared them with healthy controls. Our data show that SPC is significantly increased in plasma samples from metabolic syndrome patients but not in plasma from patients at risk for diabesity. Detailed SPC species analysis showed that the observed increase is due to a significant increase in all detected SPC subspecies. Moreover, a strong positive correlation is observed between total SPC and individual SPC species with both body mass index and the acute phase low grade inflammation marker soluble CD163 (sCD163). Collectively, our study provides new information on SPC plasma levels in metabolic syndrome and suggests new avenues for investigation.     

Using real-time impedance-based assays to monitor the effects of fibroblast-derived media on the adhesion,proliferation, migration and invasion of colon cancer cells

Increasing our knowledge of the mechanisms regulating cell proliferation, migration and invasion are central to understanding tumour progression and metastasis. The local tumour microenvironment contributes to the transformed phenotype in cancer by providing specific environmental cues that alter the cells behaviour and promotes metastasis. Fibroblasts have a strong association with cancer and in recent times there has been some emphasis in designing novel therapeutic strategies that alter fibroblast behaviour in the tumour microenvironment. Fibroblasts produce growth factors, chemokines and many of the proteins laid down in the ECM (extracellular matrix) that promote angiogenesis, inflammation and tumour progression. In this study, we use a label-free RTCA (real-time cell analysis) platform (xCELLigence) to investigate how media derived from human fibroblasts alters cancer cell behaviour. We used a series of complimentary and novel experimental approaches to show HCT116 cells adhere, proliferate and migrate significantly faster in the presence of media from human fibroblasts. As well as this, we used the xCELLigence CIM-plates system to show that HCT116 cells invade matrigel layers aggressively when migrating towards media derived from human fibroblasts. These data strongly suggest that fibroblasts have the ability to increase the migratory and invasive properties of HCT116 cells. This is the first study that provides real-time data on fibroblast-mediated migration and invasion kinetics of colon cancer cells.     

Differential regulation of CFTRΔF508 degradation by ubiquitin ligases gp78 and Hrd1

The most common mutation associated with cystic fibrosis is the deletion of phenylalanine 508 of cystic fibrosis transmembrane conductance regulator (CFTRΔF508). This mutation renders otherwise functional protein susceptible to ER-associated degradation (ERAD) and prevents CFTR from exiting the ER and trafficking to the plasma membrane. In this study, we demonstrate that RNAi-mediated silencing of gp78, an established ubiquitin ligase (E3) involved in ERAD, leads to accumulation of CFTRΔF508 protein in cells. gp78 facilitates the degradation of CFTRΔF508 by enhancing both its ubiquitination and interaction with p97/VCP. SVIP, which is the inhibitor of gp78, causes accumulation of CFTRΔF508. We showed that endogenous gp78 co-immunoprecipitates with Hrd1. Furthermore, the results indicate that silencing the expression of another ERAD E3, Hrd1, leads to stabilization of gp78 and decline in gp78 ubiquitination; thereby enhancing CFTRΔF508 degradation. The results support that gp78 is an E3 targeting CFTRΔF508 for degradation and Hrd1 inhibits CFTRΔF508 degradation by acting as an E3 for gp78.     

ABC transporters in cellular lipid trafficking

ATP-binding cassette (ABC) transporters constitute a group of evolutionary highly conserved cellular transmembrane transport proteins. Recent work has implicated ABC transporters in cellular transmembrane lipid transport and hereditary diseases have been causatively linked to defective ABC transporters translocating lipid compounds. The emerging concept that a defined subset of ABC transporters is intimately involved in cellular lipid trafficking has recently been substantiated convincingly by the finding that ABCA1 plays a central role in the regulation of HDL metabolism and macrophage targeting to the RES or the vascular wall. Differentiation dependent expression of a large number of ABC transporters in monocytes/macrophages and their regulation by sterol flux render these transporter molecules potentially critical players in atherogenesis and other chronic inflammatory diseases.     

Identification of a novel human sterol-sensitive ATP-binding cassette transporter (ABCA7)

We report the identification of the full-length cDNA for a novel ATP-binding cassette (ABC) transporter from human macrophages. The mRNA is of 6.8 kb size and contains an open reading frame encoding a polypeptide of 2146 amino acids with a calculated molecular weight of 220 kDa. The predicted protein product is composed of two transmembrane domains and two nucleotide binding folds indicating that it pertains to the group of full-size ABC transporters. The novel transporter shows highest protein sequence homology with the recently cloned human cholesterol and phospholipid exporter ABCA1 (54%) and the human retinal transporter ABCR (49%), both members of the ABC transporter subfamily A. In accordance with the currently proposed classification, the novel transporter was designated ABCA7. ABCA7 mRNA was detected predominantly in myelo-lymphatic tissues with highest expression in peripheral leukocytes, thymus, spleen, and bone marrow. Expression of ABCA7 is induced during in vitro differentiation of human monocytes into macrophages. In macrophages, both the ABCA7 mRNA and protein expression are upregulated in the presence of modified low density lipoprotein and downregulated by HDL(3). Our results suggest a role for ABCA7 in macrophage transmembrane lipid transport.     

Monitoring of 7α-hydroxy-4-cholesten-3-one during therapy of cerebrotendinous xanthomatosis: a case report

Cerebrotendinous xanthomatosis (CTX) is a rare, inherited autosomal-recessive lipid-storage disorder caused by 27-hydroxylase deficiency. In this study, we report of a 30-year old man with this disorder who was treated using chenodeoxycholic acid, simvastatin, and low-density lipoprotein (LDL) apheresis. The LDL apheresis was performed weekly for nine months. The first subjective improvement was reported by the patient after his fourth LDL-apheresis. Spasticity, gait disturbances, and his entire psychomotoric test results had improved tremendously. His fine motoric skills have been regained. The efficacy of LDL-apheresis was monitored using quantitative determination of 7α-OH-4-cholesten-3-one in plasma based on a LC–MS/MS method. The clearance efficacy of 7α-hydroxy-4-cholesten-3-one from the patient's plasma per LDL-apheresis varied between 8% and 53% but returned to the initial high levels after seven days (mean value 241 ng/mL). A slight negative trend in the plasma concentration could be derived over the period of nine months.     

Determination of Physiological Indices and Some Antioxidant Enzymes of Chard Exposed to Nitric Oxide under Drought Stress

Russian Journal of Plant Physiology - This research was carried out to determine effectiveness of nitric oxide (NO) on some physiological and biochemical reactions, and water use efficiency (WUE)...