Modification of LDL with human secretory phospholipase A(2) or sphingomyelinase promotes its arachidonic acid-releasing propensity

Oxidation and lipolytic remodeling of LDL are believed to stimulate LDL entrapment in the arterial wall, expanding the inflammatory response and promoting atherosclerosis. However, the cellular responses and molecular mechanisms underlying the atherogenic effects of lipolytically modified LDL are incompletely understood. Human THP-1 monocytes were prelabeled with [(3)H]arachidonic acid (AA) before incubation with LDL or LDL lipolytically modified by secretory PLA(2) (sPLA(2)) or bacterial sphingomyelinase (SMase). LDL elicited rapid and dose-dependent extracellular release of AA in monocytes. Interestingly, LDL modified by sPLA(2) or SMase displayed a marked increase in AA mobilization relative to native LDL, and this increase correlated with enhanced activity of cytosolic PLA(2) (cPLA(2)) assayed in vitro as well as increased monocyte tumor necrosis factor-alpha secretion. The AA liberation was attenuated by inhibitors toward cPLA(2) and sPLA(2), indicating that both PLA(2) enzymes participate in LDL-induced AA release. In conclusion, these results demonstrate that LDL lipolytically modified by sPLA(2) or SMase potentiates cellular AA release and cPLA(2) activation in human monocytes. From our results, we suggest novel atherogenic properties for LDL modified by sPLA(2) and SMase in AA release and signaling, which could contribute to the inflammatory gene expression observed in atherosclerosis.     

Multiple stages in codon-anticodon recognition: double-trigger mechanisms and geometric constraints

Thirty years of kinetic studies on tRNA selection in the elongation cycle are reviewed, and confronted with results derived from various sources, including structural studies on the ribosome, genetic observations on ribosome and EF-Tu accuracy mutants, and codon-specific elongation rates. A coherent framework is proposed, which gives meaning to many puzzling effects. Ribosomal accuracy would be governed by a "double-trigger" principle, according to which the ribosome uses energy in the forward direction to create new configurations for tRNA selection, and energy in the backward direction to regain its initial configuration, in particular after a premature dissociation event. The conformation energy would come in part, in Hopfield's mode, from GTP cleavage on the ternary complex (TC). The reset energy would be provided in part, in the author's mode, from GTP cleavage on a binary EF-Tu.GTP complex (BC). There would be several paths for amino acid incorporation. The path of highest accuracy would involve TC binding followed by BC binding, followed either by GTP hydrolysis on the TC, or by TC dissociation and GTP hydrolysis on the BC. Codon-anticodon recognition would occur in at least three kinetically and geometrically distinct stages. In a first stage, there would be a very rapid sorting of the TCs with unstrained anticodons contacting a loosely held mRNA. This stage ends with the anchoring of the codon-anticodon complex by a cluster of three nucleotides of 16S RNA. The second stage would be the most discriminative one. It would operate on the 5 ms time scale and terminate with GTP cleavage on the TC. The third stage would provide a last, crude selection involving "naked" aa-tRNA, partially held back by steric hindrance. Streptomycin and most EF-Tu mutants as well as high accuracy ribosomal mutants would produce specific alterations at stage 2, which are mapped on the stage 2 kinetic mechanism. The ram ribosomal ambiguity mutants, and anticodon position 37 modifications could be markers of stages 1 and 3 selection. Dissociation events at stage 2 or stage 3, when they are not immediately followed by reset events create a leaky state favorable to shortcut incorporation events. These events are equivalent to an "error-prone codon-anticodon mismatch repair". From the recent evidence on ribosome structure, it is conjectured that the L7/L12 flexible stalk of the large ribosome subunit acts as a proofreading gate, and that the alternation of its GTPase activation center between "TCase" competence and "BCase" competence is a main factor in the control of accuracy.     

Coupling astogenic aging in the colonial tunicate Botryllus schlosseri with the stress protein mortalin

Botryllus schlosseri, a colonial marine invertebrate, exhibits three generations of short-lived astogenic modules that continuously grow and die throughout the colony's entire lifespan, within week-long repeating budding cycles (blastogenesis), each consisting of four stages (A-D). At stage D, aging is followed by the complete absorption of adult modules (zooids) via a massive apoptotic process. Here we studied in Botryllus the protein mortalin (HSP70s member), a molecule largely known for its association with aging and proliferation. In-situ hybridization and qPCR assays reveal that mortalin follows the cyclic pattern of blastogenesis. Colonies at blastogenic stage D display the highest mortalin levels, and young modules exhibit elevated mortalin levels compared to old modules. Manipulations of mortalin with the specific allosteric inhibitor MKT-077 has led to a decrease in the modules’ growth rate and the development of abnormal somatic/germinal morphologies (primarily in vasculature and in organs such as the endostyle, the stomach and gonads). We therefore propose that mortalin plays a significant role in the astogeny and aging of colonial modules in B. schlosseri, by direct involvement in the regulation of blastogenesis.     

Plasma PAF-acetylhydrolase in patients with coronary artery disease: results of a cross-sectional analysis

Inflammation underlies both onset and perpetuation of atherosclerosis. Plasma lipoproteins transport the platelet-activating factor-acetylhydrolase (PAF-AH) with potentially anti-inflammatory activities. Our aim was to determine whether PAF-AH activity was associated with inflammatory markers and with coronary artery disease (CAD). PAF-AH activity and a panel of inflammatory mediators were measured in plasma of 496 patients with CAD and in 477 controls; 276 patients presented with stable angina pectoris and 220 with acute coronary syndrome (ACS). Individuals within the highest quartile of PAF-AH activity had an 1.8-fold increase in CAD risk [95% confidence interval (CI), 1.01 to 3.2; P = 0.048] compared with those in the first quartile (adjusted for clinical and metabolic factors). When excluding individuals receiving statin and angiotensin-converting enzyme-inhibitor medication, individuals within the highest quartile of PAF-AH activity revealed a 3.9-fold increase in CAD risk (95% CI, 2.0 to 7.7; P < 0.0001). In these subjects, the plasma PAF-AH activity increased gradually in stable angina and in ACS both in men (P < 0.0001) and in women (P < 0.001), as compared with controls.No correlation was found between PAF-AH levels and those of common markers of inflammation. This study and the previous ones raise the important issue of whether PAF-AH is simply a marker of risk or directly promotes atherosclerosis.     

The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation

The intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella, the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two-hybrid screen. It was determined that the IcmS-IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS-IcmW complex being involved in the recognition and Dot/Icm-dependent translocation of substrate proteins during Legionella infection of host cells.     

Graphical coding of nucleic acid sequences

When, in a nucleic acid sequence, the four letters C, G, A, T (or U) are replaced by suitable graphical symbols, some patterns become immediately apparent. Two sets of symbols, constructed for the analysis of either purine/pyrimidine alternations, or of regions of complementarity within a sequence are shown. In addition, another mode of coding is presented, in which the four letters are represented by vectors. The sequence is thus transformed into a planar trajectory. We show, in the case of the gene for human beta hemoglobin, that such a coding enables an easy discrimination between introns and exons.     

Plasma PAF-acetylhydrolase: an unfulfilled promise?

Plasma Platelet-activating-Factor (PAF)-acetylhydrolase (PAF-AH also named lipoprotein-PLA(2) or PLA(2)G7 gene) is secreted by macrophages, it degrades PAF and oxidation products of phosphatidylcholine produced upon LDL oxidation and/or oxidative stress, and thus is considered as a potentially anti-inflammatory enzyme. Cloning of PAF-AH has sustained tremendous promises towards the use of PAF-AH recombinant protein in clinical situations. The reason for that stems from the numerous animal models of inflammation, atherosclerosis or sepsis, where raising the levels of circulating PAF-AH either through recombinant protein infusion or through the adenoviral gene transfer showed to be beneficial. Unfortunately, neither in human asthma nor in sepsis the recombinant PAF-AH showed sufficient efficacy. One of the most challenging questions nowadays is as to whether PAF-AH is pro- or anti-atherogenic in humans, as PAF-AH may possess a dual pro- and anti-inflammatory role, depending on the concentration and the availability of potential substrates. It is equally possible that the plasma level of PAF-AH is a diagnostic marker of ongoing atherosclerosis.