Correlation between turbidimetric and nephelometric methods of measuring C-reactive protein in patients with unstable angina or non-ST elevation acute myocardial infarction

OBJECTIVE: To evaluate the performance of the turbidimetric method of C-reactive protein (CRP) as a measure of low-grade inflammation in patients admitted with non-ST elevation acute coronary syndromes (ACS). METHODS: Serum samples obtained at hospital arrival from 68 patients (66±11 years, 40 men), admitted with unstable angina or non-ST elevation acute myocardial infarction were used to measure CRP by the methods of nephelometry and turbidimetry. RESULTS: The medians of C-reactive protein by the turbidimetric and nephelometric methods were 0.5 mg/dL and 0.47 mg/dL, respectively. A strong linear association existed between the 2 methods, according to the regression coefficient (b=0.75; 95% C.I.=0.70-0.80) and correlation coefficient (r=0.96; P<0.001). The mean difference between the nephelometric and turbidimetric CRP was 0.02 ± 0.91 mg/dL, and 100% agreement between the methods in the detection of high CRP was observed. CONCLUSION: In patients with non-ST elevation ACS, CRP values obtained by turbidimetry show a strong linear association with the method of nephelometry and perfect agreement in the detection of high CRP.

Einfluss von ZNS-Verletzung auf die m-RNA-Expression der Protease-Aktivierenden Rezeptoren (PARs) und Indentifizierung eines Interaktionspartners für PAR-2 in Retina

Protease-activated receptor, optic nerve crush, focal ischemia, protein-protein interaction, alpha crystallin A

Analysis of two newly identified protease-activated receptor-2-interacting proteins, Jab1 and p24A, and their role in receptor signalling

Protease-activated receptor, receptor signalling, receptor trafficking, receptor resensitization, protein interaction

Sex Differences in High Sensitivity C-Reactive Protein in Subjects with Risk Factors of Metabolic Syndrome

Abstract Background: Metabolic syndrome (MetS) is associated with a higher risk of all-cause mortality. High-sensitivity C-reactive protein (hsCRP) is a prototypic marker of inflammation usually increased in MetS. Women with MetS-related diseases present higher hsCRP levels than men with MetS-related diseases, suggesting sex differences in inflammatory markers. However, it is unclear whether serum hsCRP levels are already increased in men and/or women with MetS risk factors and without overt diseases or under pharmacological treatment. Objective: To determine the impact of the number of MetS risk factors on serum hsCRP levels in women and men. Methods One hundred and eighteen subjects (70 men and 48 women; 36 ± 1 years) were divided into four groups according to the number of MetS risk factors: healthy group (CT; no risk factors), MetS ≤ 2, MetS = 3, and MetS ≥ 4. Blood was drawn after 12 hours of fasting for measurement of biochemical variables and hsCRP levels, which were determined by immunoturbidimetric assay. Results: The groups with MetS risk factors presented higher serum hsCRP levels when compared with the CT group (p < 0.02). There were no differences in hsCRP levels among groups with MetS risk factors (p > 0.05). The best linear regression model to explain the association between MetS risk factors and hsCRP levels included waist circumference and HDL cholesterol (r = 0.40, p < 0.01). Women with MetS risk factors presented higher hsCRP levels when compared with men (psex < 0.01). Conclusions: Despite the absence of overt diseases and pharmacological treatment, subjects with MetS risk factors already presented increased hsCRP levels, which were significantly higher in women than men at similar conditions.

Development of metabolic labeling strategies to study changes in protein expression

Résumé : Les progrès techniques de la spectrométrie de masse (MS) ont contribué au récent développement de la protéomique. Cette technique peut actuellement détecter, identifier et quantifier des milliers de protéines. Toutefois, elle n'est pas encore assez puissante pour fournir une analyse complète des modifications du protéome corrélées à des phénomènes biologiques. Notre objectif était le développement d'une nouvelle stratégie pour la détection spécifique et la quantification des variations du protéome, basée sur la mesure de la synthèse des protéines plutôt que sur celle de la quantité de protéines totale. Pour cela, nous volions associer le marquage pulsé des protéines par des isotopes stables avec une méthode d'acquisition MS basée sur le balayage des ions précurseurs (precursor ion scan, ou PIS), afin de détecter spécifiquement les protéines ayant intégré les isotopes et d'estimer leur abondance par rapport aux protéines non marquées. Une telle approche peut identifier les protéines avec les plus hauts taux de synthèse dans une période de temps donnée, y compris les protéines dont l'expression augmente spécifiquement suite à un événement précis. Nous avons tout d'abord testé différents acides aminés marqués en combinaison avec des méthodes PIS spécifiques. Ces essais ont permis la détection spécifique des protéines marquées. Cependant, en raison des limitations instrumentales du spectromètre de masse utilisé pour les méthodes PIS, la sensibilité de cette approche s'est révélée être inférieure à une analyse non ciblée réalisée sur un instrument plus récent (Chapitre 2.1). Toutefois, pour l'analyse différentielle de deux milieux de culture conditionnés par des cellules cancéreuses humaines, nous avons utilisé le marquage métabolique pour distinguer les protéines d'origine cellulaire des protéines non marquées du sérum présentes dans les milieux de culture (Chapitre 2.2). Parallèlement, nous avons développé une nouvelle méthode de quantification nommée IBIS, qui utilise des paires d'isotopes stables d'acides aminés capables de produire des ions spécifiques qui peuvent être utilisés pour la quantification relative. La méthode IBIS a été appliquée à l'analyse de deux lignées cellulaires cancéreuses complètement marquées, mais de manière différenciée, par des paires d'acides aminés (Chapitre 2.3). Ensuite, conformément à l'objectif initial de cette thèse, nous avons utilisé une variante pulsée de l'IBIS pour détecter des modifications du protéome dans des cellules HeLa infectée par le virus humain Herpes Simplex-1 (Chapitre 2.4). Ce virus réprime la synthèse des protéines des cellules hôtes afin d'exploiter leur mécanisme de traduction pour la production massive de virions. Comme prévu, de hauts taux de synthèse ont été mesurés pour les protéines virales détectées, attestant de leur haut niveau d'expression. Nous avons de plus identifié un certain nombre de protéines humaines dont le rapport de synthèse et de dégradation (S/D) a été modifié par l'infection virale, ce qui peut donner des indications sur les stratégies utilisées par les virus pour détourner la machinerie cellulaire. En conclusion, nous avons montré dans ce travail que le marquage métabolique peut être employé de façon non conventionnelle pour étudier des dimensions peu explorées en protéomique. Summary : In recent years major technical advancements greatly supported the development of mass spectrometry (MS)-based proteomics. Currently, this technique can efficiently detect, identify and quantify thousands of proteins. However, it is not yet sufficiently powerful to provide a comprehensive analysis of the proteome changes correlated with biological phenomena. The aim of our project was the development of ~a new strategy for the specific detection and quantification of proteomé variations based on measurements of protein synthesis rather than total protein amounts. The rationale for this approach was that changes in protein synthesis more closely reflect dynamic cellular responses than changes in total protein concentrations. Our starting idea was to couple "pulsed" stable-isotope labeling of proteins with a specific MS acquisition method based on precursor ion scan (PIS), to specifically detect proteins that incorporated the label and to simultaneously estimate their abundance, relative to the unlabeled protein isoform. Such approach could highlight proteins with the highest synthesis rate in a given time frame, including proteins specifically up-regulated by a given biological stimulus. As a first step, we tested different isotope-labeled amino acids in combination with dedicated PIS methods and showed that this leads to specific detection of labeled proteins. Sensitivity, however, turned out to be lower than an untargeted analysis run on a more recent instrument, due to MS hardware limitations (Chapter 2.1). We next used metabolic labeling to distinguish the proteins of cellular origin from a high background of unlabeled (serum) proteins, for the differential analysis of two serum-containing culture media conditioned by labeled human cancer cells (Chapter 2.2). As a parallel project we developed a new quantification method (named ISIS), which uses pairs of stable-isotope labeled amino acids able to produce specific reporter ions, which can be used for relative quantification. The ISIS method was applied to the analysis of two fully, yet differentially labeled cancer cell lines, as described in Chapter 2.3. Next, in line with the original purpose of this thesis, we used a "pulsed" variant of ISIS to detect proteome changes in HeLa cells after the infection with human Herpes Simplex Virus-1 (Chapter 2.4). This virus is known to repress the synthesis of host cell proteins to exploit the translation machinery for the massive production of virions. As expected, high synthesis rates were measured for the detected viral proteins, confirming their up-regulation. Moreover, we identified a number of human proteins whose synthesis/degradation ratio (S/D) was affected by the viral infection and which could provide clues on the strategies used by the virus to hijack the cellular machinery. Overall, in this work, we showed that metabolic labeling can be employed in alternative ways to investigate poorly explored dimensions in proteomics.

Immunocytochemical localisation of microtubule-associated proteins 1b and 2 in the developing rat spinal cord.

The straightforward anatomical organisation of the developing and mature rat spinal cord was used to determine and interpret the time of appearance and expression patterns of microtubule-associated proteins (MAP) 1b and 2. Immunoblots revealed the presence of MAP1b and 2 in the early embryonic rat spinal cord and confirmed the specificity of the used anti-MAP mouse monoclonal antibodies. The immunocytochemical data demonstrated a rostral-to-caudal and ventral-to-dorsal gradient in the expression of MAP1b/2 within the developing spinal cord. In the matrix layer, MAP1b was found in a distinct radial pattern distributed between the membrana limitans interna and externa between embryonal day (E)12 and E15. Immunostaining for vimentin revealed that this MAP1b pattern was morphologically and topographically different from the radial glial pattern which was present in the matrix layer between E13 and E19. The ventral-to-dorsal developmental gradient of the MAP1b staining in the spinal cord matrix layer indicates a close involvement of MAP1b either in the organisation of the microtubules in the cytoplasmatic extensions of the proliferating neuroblasts or neuroblast mitosis. MAP2 could not be detected in the developing matrix layer. In the mantle and marginal layer, MAP1b was abundantly present between E12 and postnatal day (P)0. After birth, the staining intensity for MAP1b gradually decreased in both layers towards a faint appearance at maturity. The distribution patterns suggest an involvement of MAP1b in the maturation of the motor neurons, the contralaterally and ipsilaterally projecting axons and the ascending and descending long axons of the rat spinal cord. MAP2 was present in the spinal cord grey matter between E12 and maturity, which reflects a role for MAP2 in the development as well as in the maintenance of microtubules. The present description of the expression patterns of MAP1b and 2 in the developing spinal cord suggests important roles of the two proteins in various morphogenetic events. The findings may serve as the basis for future studies on the function of MAP1b and 2 in the development of the central nervous system.

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1.

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

The protein phosphatase 7 regulates phytochrome signaling in Arabidopsis.

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.

Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2.

FGF-2 has been implicated in the cardiac response to hypertrophic stimuli. Angiotensin II (Ang II) contributes to maintain elevated blood pressure in hypertensive individuals and exerts direct trophic effects on cardiac cells. However, the role of FGF-2 in Ang II-induced cardiac hypertrophy has not been established. Therefore, mice deficient in FGF-2 expression were studied using a model of Ang II-dependent hypertension and cardiac hypertrophy. Echocardiographic measurements show the presence of dilated cardiomyopathy in normotensive mice lacking FGF-2. Moreover, hypertensive mice without FGF-2 developed no compensatory cardiac hypertrophy. In wild-type mice, hypertrophy was associated with a stimulation of the c-Jun N-terminal kinase, the extracellular signal regulated kinase, and the p38 kinase pathways. In contrast, mitogen-activated protein kinase (MAPK) activation was markedly attenuated in FGF-2-deficient mice. In vitro, FGF-2 of fibroblast origin was demonstrated to be essential in the paracrine stimulation of MAPK activation in cardiomyocytes. Indeed, fibroblasts lacking FGF-2 expression have a defective capacity for releasing growth factors to induce hypertrophic responses in cardiomyocytes. Therefore, these results identify the cardiac fibroblast population as a primary integrator of hypertrophic stimuli in the heart, and suggest that FGF-2 is a crucial mediator of cardiac hypertrophy via autocrine/paracrine actions on cardiac cells.

Profiling of T-cell receptor signaling complex assembly in human CD4 T-lymphocytes using RP protein arrays.

The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC-coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5 x 10(7)) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T-cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T-cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T-cell activation in as few as 0.5 million of cells. Second- to fourth-order TCR interacting proteins were accurately quantified, making this strategy specially well-suited to the analysis of membrane-associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.

Structure-function relationships for the interleukin 2 receptor system

Receptors for interleukin 2 (IL-2) esit in at least three forms which differ in their subunit compositio, their affinity for ligand and their ability to mediate a cellular reponse. Type I receptors occur following cellular acitivation and consist of the 55,000 m. w. glycoprotein Tac. These receptors bind IL-2 with a low affinity, do not internalize ligand and have not been definitively associated with any response. Type II receptors, on the other hand, conssit of one or more glycoproteins of 70,000 m. w. which have been termed "beta ([beta]) chains." They bind IL-2 with an intermediate affinity and rapidly internalize the ligand. [Beta] proteins mediate many cellular IL-2-dependent reponses, including the short-term activation of natural killer cells and the induction of Tac protein expression. Type III receptors consist of a ternary complex of the Tac protein, the [beta] chain(s) and IL-2. They are characterized by a paricularly high affinity for ligand association. Type III receptors also internalize ligand and mediate IL-2-dependent responses at low factor concentrations. The identification of two independent IL-2-binding molecules, Tac and [beta], thus provides the elusive molecular explanation for the differences in IL-2 receptor affinity and suggests the potential for selective therapeutic manipulation of IL-2 reponses.

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.