Relationship between cholesteryl ester transfer protein and LDL heterogeneity in familial hypercholesterolemia

Small, dense LDL particles have been associated with an increased risk of coronary artery disease, and cholesteryl ester transfer protein (CETP) has been suggested to play a role in LDL particle remodeling. We examined the relationship between LDL heterogeneity and plasma CETP mass in familial hypercholesterolemia (FH). LDL particles were characterized by polyacrylamide gradient gel electrophoresis in a total of 259 FH heterozygotes and 208 nonFH controls. CETP mass was measured by enzyme-linked immunosorbent assay in a subgroup of 240 participants, which included 120 FH patients matched with 120 controls. As compared with controls, FH subjects had an 11% higher CETP mass. Moreover, LDL-peak particle diameter (LDL-PPD) was significantly smaller in FH heterozygotes than in controls (258.1 +/- 4.8 vs. 259.2 +/- 4.1 A; P = 0.01) after adjustment for covariates. There was also an inverse relationship between LDL-PPD and CETP mass (R = -0.15; P = 0.02), and this relationship was abolished by adjustment for the FH/control status, indicating that LDL-PPD changes in FH are mediated, at least in part, by an increase in plasma CETP mass concentrations. These results suggest that increased plasma CETP mass concentrations could lead to significant LDL particle remodeling in FH heterozygotes and could contribute to the pathogenesis of atherosclerosis.     

Oxidation of CaMKII determines the cardiotoxic effects of aldosterone

Excessive activation of the β-adrenergic, angiotensin II (Ang II) and aldosterone signaling pathways promotes mortality after myocardial infarction, and antagonists targeting these pathways are core therapies for treating this condition. Catecholamines and Ang II activate the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), the inhibition of which prevents isoproterenol-mediated and Ang II-mediated cardiomyopathy. Here we show that aldosterone exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after myocardial infarction. Aldosterone induces CaMKII oxidation by recruiting NADPH oxidase, and this oxidized and activated CaMKII promotes matrix metalloproteinase 9 (MMP9) expression in cardiomyocytes. Myocardial CaMKII inhibition, overexpression of methionine sulfoxide reductase A (an enzyme that reduces oxidized CaMKII) or NADPH oxidase deficiency prevented aldosterone-enhanced cardiac rupture after myocardial infarction. These findings show that oxidized myocardial CaMKII mediates the cardiotoxic effects of aldosterone on the cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after myocardial infarction.     

Oleate-induced beta cell dysfunction and apoptosis: a proteomic approach to glucolipotoxicity by an unsaturated fatty acid

High levels of fatty acids contribute to loss of functional beta cell mass in type 2 diabetes, in particular in combination with high glucose levels. The aim of this study was to elucidate the role of the unsaturated free fatty acid oleate in glucolipotoxicity and to unravel the molecular pathways involved. INS-1E cells were exposed to 0.5 mM oleate, combined or not with 25 mM glucose, for 24 h. Protein profiling of INS-1E cells was done by 2D-DIGE, covering pH ranges 4-7 and 6-9 (n = 4). Identification of differentially expressed proteins (P < 0.05) was based on MALDI-TOF analysis using Peptide Mass Fingerprint (PMF) and fragmentation (MS/MS) of the most intense peaks of PMF and proteomic results were confirmed by functional assays. Oleate impaired glucose-stimulated insulin secretion and decreased insulin content. 2D-DIGE analysis revealed 53 and 54 differentially expressed proteins for oleate and the combination of oleate and high glucose, respectively. Exposure to oleate down-regulated chaperones, hampered insulin processing and ubiquitin-related proteasomal degradation, and induced perturbations in vesicle transport and budding. In combination with high glucose, shunting of excess amounts of glucose toward reactive oxygen species production worsened beta cell death. The present findings provide new insights in oleate-induced beta cell dysfunction and identify target proteins for preservation of functional beta cell mass in type 2 diabetes.     

On the characterization and selection of diverse conformational ensembles with applications to flexible docking

To address challenging flexible docking problems, a number of docking algorithms pregenerate large collections of candidate conformers. To remove the redundancy from such ensembles, a central problem in this context is to report a selection of conformers maximizing some geometric diversity criterion. We make three contributions to this problem. First, we resort to geometric optimization so as to report selections maximizing the molecular volume or molecular surface area (MSA) of the selection. Greedy strategies are developed, together with approximation bounds. Second, to assess the efficacy of our algorithms, we investigate two conformer ensembles corresponding to a flexible loop of four protein complexes. By focusing on the MSA of the selection, we show that our strategy matches the MSA of standard selection methods, but resorting to a number of conformers between one and two orders of magnitude smaller. This observation is qualitatively explained using the Betti numbers of the union of balls of the selection. Finally, we replace the conformer selection problem in the context of multiple-copy flexible docking. On the aforementioned systems, we show that using the loops selected by our strategy can improve the result of the docking process.     

IRGM is a common target of RNA viruses that subvert the autophagy network

Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity.     

Two-dimensional gel proteome reference map of INS-1E cells

The insulin-producing INS-1E rat cell line is widely used as a model for studying β-cells. It is a well-characterized cell line, mainly used in diabetes research. We established a 2-DE reference map for INS-1E cells. Using MALDI-TOF/TOF-MS/MS, we identified 546 spots. These included various proteins with an important role in β-cell physiology and with known roles as crucial proteins for diabetes development. We believe that the availability of this reference map will enhance our knowledge of β-cell physiology.     

Differential reproductive timing in Echinocardium spp.: the first Mediterranean survey allows interoceanic and interspecific comparisons

Echinocardium cordatum had long been considered as cosmopolitan, but molecular data revealed it is a complex of cryptic species, with two non-hybridizing species (B1 & B2) in the Mediterranean Sea living in syntopy with Echinocardium mediterraneum. Histological analyses of the gonads from a 17-month sampling period revealed a statistically significant time lag between the Maturity Indices of E.?cordatum and E.?mediterraneum. The main environmental stimulus may be different for the two nominal species, possibly seawater temperature for E.?cordatum and chlorophyll a concentration for E.?mediterraneum. Within the E.?cordatum complex, spawning timing and synchrony are different according to major geographic areas (Atlantic/Pacific/Mediterranean) and/or the corresponding genetic subdivision [A/P/(B1 & B2)]. In contrast, the effects of temperature on the reproductive cycle seem rather to mirror the genetic lineages than environmental similarities of the different localities. Between the sister species (B1 & B2) no differences could be detected, maybe due to small sample sizes.     

Sphingosine kinase 1 and sphingosine 1-phosphate receptor 3 are functionally upregulated on astrocytes under pro-inflammatory conditions

Background

Reactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS). The sphingosine kinase 1 (SphK1)/sphingosine1-phosphate (S1P) receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S1P receptor subtype 3 (S1P₃) signaling and SphK1 in activated rat astrocytes has not been defined.

Methodology/Principal Findings

Using immunohistochemistry we observed the upregulation of S1P₃ and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P₃ and SphK1, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS). Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino)-4-(p-chlorphenyl) thiazole. Treatment of astrocytes with a selective S1P₃ agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2), which was further elevated with a LPS pre-challenge, suggesting that S1P₃ upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S1P and LPS and this LPS-induced migration was sensitive to inhibition of SphK1, and independent of cell proliferation. In addition, S1P induced secretion of the potentially neuroprotective chemokine CXCL1, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S1P₃ signaling compared to S1P₁ signaling was demonstrated by the use of selective S1P₃ or S1P₁ agonists.

Conclusion/Significance

In summary, our data demonstrate that the SphK1/S1P₃ signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment and maintenance of astrocyte activation. Upregulation of the pathway in MS may be detrimental, e.g. through enhancing astrogliosis, or beneficial through increased remyelination via CXCL1.