Molecular mechanism of glutathione-mediated protection from oxidized low-density lipoprotein-induced cell injury in human macrophages: role of glutathione reductase and glutaredoxin

Macrophage death is a hallmark of advanced atherosclerotic plaque, and oxidized low-density lipoprotein (OxLDL) found in these lesions is believed to contribute to macrophage injury. However, the underlying mechanisms of this phenomenon are only poorly understood. Here we show that in human monocyte-derived macrophages, OxLDL depleted intracellular glutathione (GSH) and inhibited glutathione reductase, resulting in a marked diminution of the glutathione/glutathione disulfide ratio. In the absence of OxLDL, an 80% depletion of intracellular GSH levels did not affect cell viability, but glutathione depletion dramatically increased OxLDL-induced cell death. Conversely, supplementation of intracellular GSH stores with glutathione diethyl ester substantially diminished OxLDL toxicity. OxLDL also promoted protein-S-glutathionylation, which was increased in macrophages pretreated with the glutathione reductase inhibitor BCNU. Knockdown experiments with siRNA directed against glutathione reductase and glutaredoxin showed that both enzymes are essential for the protection of macrophages against OxLDL. Finally, the peroxyl-radical scavenger Trolox did not prevent GSH depletion but completely blocked OxLDL-induced protein-S-glutathionylation and cell death. These data suggest that OxLDL promotes ROS formation and protein-S-glutathionylation by a mechanism independent from its effect on GSH depletion. Neither mechanism was sufficient to induce macrophage injury, but when stimulated concurrently, these pathways promoted the accumulation of protein-glutathione mixed disulfides and cell death.     

Synthesis and activity of novel bile-acid conjugated glucocorticoid receptor antagonists

A series of potent steroidal glucocorticoid receptor antagonists has been discovered. After conjugation to cholic acid, the compounds retained an affinity for GR in vitro and had modest in vivo efficacy.     

Rapid adjustments of sperm characteristics in relation to social status

Sperm competition models predict that males typically mating in disfavoured roles should be selected to compensate for their disadvantage by investing more into sperm. We studied the effect of rapid changes in social status on ejaculate investments during experimental trials with an externally fertilizing teleost--the Arctic charr (Salvelinus alpinus). We document that males becoming dominant produce less sperm with lower velocity, but have higher sex steroid concentrations than subordinate males. These differences in sperm characteristics seem mainly to result from a decreased investment in sperm among fish that become dominant compared to pre-trial levels. Moreover, these adjustments of sperm production and sperm velocity seem not to be traded against sperm longevity. Our results support theoretical models of sperm competition, as males forced to mate in disfavoured roles seem to invest more into ejaculate quality than males in favoured roles. Additionally, we are the first to report that males, in a species with status-dependent shifts in reproductive tactics, have evolved rapid tactic specific adjustments of sperm production and sperm velocity corresponding to what could be predicted from their reproductive roles.     

Magnesium-induced assembly of a complete DNA polymerase catalytic complex

The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3'-OH and catalytic Mg2+, resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here, we present a crystal structure of a precatalytic complex of a DNA polymerase with bound substrates that include the primer 3'-OH and catalytic Mg2+. This catalytic intermediate was trapped with a nonhydrolyzable deoxynucleotide analog. Comparison with two new structures of DNA polymerase beta lacking the 3'-OH or catalytic Mg2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3' on the alphaP of the incoming nucleotide.     

CASPAR: a hierarchical bayesian approach to predict survival times in cancer from gene expression data

MOTIVATION: DNA microarrays allow the simultaneous measurement of thousands of gene expression levels in any given patient sample. Gene expression data have been shown to correlate with survival in several cancers, however, analysis of the data is difficult, since typically at most a few hundred patients are available, resulting in severely underdetermined regression or classification models. Several approaches exist to classify patients in different risk classes, however, relatively little has been done with respect to the prediction of actual survival times. We introduce CASPAR, a novel method to predict true survival times for the individual patient based on microarray measurements. CASPAR is based on a multivariate Cox regression model that is embedded in a Bayesian framework. A hierarchical prior distribution on the regression parameters is specifically designed to deal with high dimensionality (large number of genes) and low sample size settings, that are typical for microarray measurements. This enables CASPAR to automatically select small, most informative subsets of genes for prediction. RESULTS: Validity of the method is demonstrated on two publicly available datasets on diffuse large B-cell lymphoma (DLBCL) and on adenocarcinoma of the lung. The method successfully identifies long and short survivors, with high sensitivity and specificity. We compare our method with two alternative methods from the literature, demonstrating superior results of our approach. In addition, we show that CASPAR can further refine predictions made using clinical scoring systems such as the International Prognostic Index (IPI) for DLBCL and clinical staging for lung cancer, thus providing an additional tool for the clinician. An analysis of the genes identified confirms previously published results, and furthermore, new candidate genes correlated with survival are identified.     

Definition, expression, and characterization of a protein domain in the N-terminus of pregnancy-associated plasma protein-A distantly related to the family of laminin G-like modules

Although pregnancy-associated plasma protein-A (PAPP-A), a modulator of insulin-like growth factor (IGF) activity through its cleavage of IGF-binding protein (IGFBP)-4 and -5, has been known for more than two decades, knowledge about its domain architecture is still incomplete. Using position-specific iterative BLAST, we have identified distant relatives of the PAPP-A N-terminal sequence stretch of 250 residues. We present evidence that a protein domain with weak similarity to known laminin G-like (LG) modules is contained within this region, and we propose that PAPP-A and PAPP-A2 are new and unique members in the group of LG proteins as the pappalysins represent the first examples where LG modules are associated with proteinases. Fourteen beta-strands characteristic for the LG structure were tentatively located within the PAPP-A LG (PA-LG) module using secondary structure prediction and sequence alignment. Upon mammalian expression of PAPP-A truncation mutants, we defined domain boundaries showing that PA-LG is an autonomously folding unit, which spans the first 243 residues. We were unable to express PAPP-A variants which lack the PA-LG module, suggesting a possible role in stabilization of the proteolytic domain. To obtain larger amounts of protein for functional and structural analysis, the defined PA-LG domain was expressed in bacteria and folded in vitro. In addition, the availability of recombinant PA-LG module may potentially improve diagnostic assays based on the measurement of PAPP-A antigen, and also facilitate the study of PAPP-A in animal model systems.     

Analyzing proteome topology and function by automated multidimensional fluorescence microscopy

Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipf's law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.     

A wiring of the human nucleolus

Recent proteomic efforts have created an extensive inventory of the human nucleolar proteome. However, approximately 30% of the identified proteins lack functional annotation. We present an approach of assigning function to uncharacterized nucleolar proteins by data integration coupled to a machine-learning method. By assembling protein complexes, we present a first draft of the human ribosome biogenesis pathway encompassing 74 proteins and hereby assign function to 49 previously uncharacterized proteins. Moreover, the functional diversity of the nucleolus is underlined by the identification of a number of protein complexes with functions beyond ribosome biogenesis. Finally, we were able to obtain experimental evidence of nucleolar localization of 11 proteins, which were predicted by our platform to be associates of nucleolar complexes. We believe other biological organelles or systems could be "wired" in a similar fashion, integrating different types of data with high-throughput proteomics, followed by a detailed biological analysis and experimental validation.     

Energy expenditure, insulin, and VLDL-triglyceride production in humans

Hypertriglyceridemia is considered a cardiovascular risk factor in diabetic and nondiabetic subjects. In this study, we aimed to determine potential regulators of very low density lipoprotein-triglyceride (TG) production. VLDL-TG kinetics were measured in 13 men and 12 women [body mass index [mean (range)]: 24.8 (20.2-35.6) kg/m(2)]. VLDL-TG production was assessed from the plasma decay of a bolus injection of ex vivo labeled VLDL particles ([1-(14)C]triolein-VLDL-TG). Similar VLDL-TG production (micromol/min) was found in men and women. VLDL-TG production was not significantly correlated with palmitate flux ([9,10-(3)H]palmitate) (r = 0.09, P = 0.67) or palmitate concentration (r = -0.29, P = 0.2) but was correlated significantly with fasting insulin concentration (r = 0.46, P < 0.05) and resting energy expenditure (REE) (r = 0.45, P < 0.05). The latter correlation improved when adjusted for sex. The best multivariate model with VLDL-TG production as the dependent variable and REE, body composition, hormones, and substrate levels as independent variables included fasting insulin (P = 0.02) and REE (P = 0.02) (r(2) = 0.32, P < 0.001). We conclude that VLDL kinetics are similar in men and women and that REE and plasma insulin are significant independent predictors of VLDL-TG production. FFA availability and body fat distribution are unrelated to VLDL production. We suggest that REE plays a greater role in VLDL-TG production than previously anticipated. REE and insulin should be taken into account when VLDL-TG production comparisons between groups are made.