Unbiased proteomic analysis of proteins interacting with the HIV-1 5'LTR sequence: role of the transcription factor Meis.

To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5'long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5'LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5'LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches.

Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory.

Worldwide, mass spectrometry is widely used to detect and quantify food allergens, especially in complex and processed food products. Yet, the absence of a regulatory framework for the developed methods has led to a lack of harmonization between laboratories. In this study, ten allergens were analyzed in eight food products by UHPLC-MS/MS, in order to establish criteria for the retention time, variation tolerance, the ion ratio deviation, and the signal-to-noise ratio for allergen detection. The set of criteria should help laboratories to compare results and avoid false positives and negatives. Furthermore, a strategy combining standard addition and labeled peptide correction was used to quantify milk, soy, peanut, and egg allergens in eight food products. This strategy is particularly interesting for routine laboratories, which receive hundreds of samples and cannot use an external calibration curve for each sample.

[Amiodarone-induced optic neuropathy: A rare side effect]. / Un effet indésirable rare de l'amiodarone : la neuropathie optique.

INTRODUCTION: The diagnosis of bilateral papilledema implies emergency medical care to look for intracranial hypertension and arteritic ischemic neuropathy. However, other causes must also be mentioned, including drugs. Too often underrated because of their usual benignity, drug side ophthalmological effects can be severe and are typically bilateral. CASE REPORT: An 80-year-old woman was hospitalized for bilateral papilledema, predominantly in the left eye, with lowered visual acuity. After ruling out intracranial hypertension, arteritic ischemic optic neuropathy, non-arteritic, and inflammatory bilateral papilledema, the diagnosis was toxic optic neuropathy. CONCLUSION: Bilateral edematous optic neuropathy is a known side effect of amiodarone, uncommon but to be known because of the large number of patients benefiting from this treatment.

Selection of egg peptide biomarkers in processed food products by high resolution mass spectrometry.

Food allergy is a growing health problem worldwide; thus, there is an urgent need for robust, specific, and sensitive analytical methods for detecting allergens. Mass spectrometry is an alternative to the existing methods, and it can overcome their limitations. One of the first steps in the development of any analytical method is the identification of the analytes to be further studied. In the case of allergen detection by mass spectrometry, the analytes are peptides. In this study, a strategy was developed for identifying potential peptide biomarkers in processed food products. This strategy was applied to processed egg matrices, and 16 potential peptide biomarkers were identified for the further detection and quantification of egg by means of mass spectrometry. With an empirical approach based on dedicated sample preparation, including tandem Lys-C/trypsin enzymatic digestion and high-resolution mass spectrometry analysis, hundreds of peptides from egg proteins were identified. This list of peptides was further refined with a series of criteria, obtained from empirical evidence, to identify the ideal biomarkers for the development of a quantitative method. These criteria include the resistance to food processing and the specificity of the peptides for eggs but also the effects of amino acid modifications and enzymatic digestion efficiency.

Cellular activation triggered by the autosomal dominant polycystic kidney disease gene product PKD2.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by germ line mutations in at least three ADPKD genes. Two recently isolated ADPKD genes, PKD1 and PKD2, encode integral membrane proteins of unknown function. We found that PKD2 upregulated AP-1-dependent transcription in human embryonic kidney 293T cells. The PKD2-mediated AP-1 activity was dependent upon activation of the mitogen-activated protein kinases p38 and JNK1 and protein kinase C (PKC) epsilon, a calcium-independent PKC isozyme. Staurosporine, but not the calcium chelator BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N', N'-tetraacetate], inhibited PKD2-mediated signaling, consistent with the involvement of a calcium-independent PKC isozyme. Coexpression of PKD2 with the interacting C terminus of PKD1 dramatically augmented PKD2-mediated AP-1 activation. The synergistic signaling between PKD1 and PKD2 involved the activation of two distinct PKC isozymes, PKC alpha and PKC epsilon, respectively. Our findings are consistent with others that support a functional connection between PKD1 and PKD2 involving multiple signaling pathways that converge to induce AP-1 activity, a transcription factor that regulates different cellular programs such as proliferation, differentiation, and apoptosis. Activation of these signaling cascades may promote the full maturation of developing tubular epithelial cells, while inactivation of these signaling cascades may impair terminal differentiation and facilitate the development of renal tubular cysts.

Liquid chromatography coupled to tandem mass spectrometry for detecting ten allergens in complex and incurred foodstuffs.

Food allergy is a considerable heath problem, as undesirable contaminations by allergens during food production are still widespread and may be dangerous for human health. To protect the population, laboratories need to develop reliable analytical methods in order to detect allergens in various food products. Currently, a large majority of allergen-related food recalls concern bakery products. It is therefore essential to detect allergens in unprocessed and processed foodstuffs. In this study, we developed a method for detecting ten allergens in complex (chocolate, ice cream) and processed (cookie, sauce) foodstuffs, based on ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Using a single protocol and considering a signal-to-noise ratio higher than 10 for the most abundant multiple reaction monitoring (MRM) transition, we were able to detect target allergens at 0.5mg/kg for milk proteins, 2.5mg/kg for peanut, hazelnut, pistachio, and cashew proteins, 3mg/kg for egg proteins, and 5mg/kg for soy, almond, walnut, and pecan proteins. The ability of the method to detect 10 allergens with a single protocol in complex and incurred food products makes it an attractive alternative to the ELISA method for routine laboratories.

Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs.

Sensitive detection of food allergens is affected by food processing and foodstuff complexity. It is therefore a challenge to detect cross-contamination in food production that could endanger an allergic customer's life. Here we used ultra-high performance liquid chromatography coupled to tandem mass spectrometry for simultaneous detection of traces of milk (casein, whey protein), egg (yolk, white), soybean, and peanut allergens in different complex and/or heat-processed foodstuffs. The method is based on a single protocol (extraction, trypsin digestion, and purification) applicable to the different tested foodstuffs: chocolate, ice cream, tomato sauce, and processed cookies. The determined limits of quantitation, expressed in total milk, egg, peanut, or soy proteins (and not soluble proteins) per kilogram of food, are: 0.5mg/kg for milk (detection of caseins), 5mg/kg for milk (detection of whey), 2.5mg/kg for peanut, 5mg/kg for soy, 3.4mg/kg for egg (detection of egg white), and 30.8mg/kg for egg (detection of egg yolk). The main advantage is the ability of the method to detect four major food allergens simultaneously in processed and complex matrices with very high sensitivity and specificity.

Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions.

The origin of several vascular pathologies involves sudden or recurrent oxygen deficiency. In this review, we examine what the biochemical and molecular responses of the endothelial cells to the lack of oxygen are and how these responses may account for the features observed in pathological situations, mainly by modifications of cell-cell interactions. Two major responses of the endothelial cells have been observed depending on the degree and duration of the oxygen deficiency. Firstly, acute hypoxia rapidly activates the endothelial cells to release inflammatory mediators and growth factors. These inflammatory mediators are able to recruit and promote the adherence of neutrophils to the endothelium where they become activated. The synthesis of platelet-activating factor plays a key role in this adherence process. Secondly, longer periods of hypoxia increase the expression of specific genes such as those encoding some cytokines as well as for the growth factors platelet-derived growth factor and vascular endothelial growth factor. The transcriptional induction of these genes is mediated through the activation of several transcription factors, the most important one being hypoxia inducible factor-1. The link between our knowledge of the signalling cascade of the cellular and molecular events initiated by hypoxia and their involvement in several vascular pathological situations, varicose veins, tumor angiogenesis and pulmonary hypertension is discussed briefly.

mtCLIC is up-regulated and maintains a mitochondrial membrane potential in mtDNA-depleted L929 cells.

To explain why mitochondrial DNA (mtDNA)-depleted or rho0 cells still keep a mitochondrial membrane potential (Delta(psi)m) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1-ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase-polymerase chain reaction on L929 mtDNA-depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up-regulated in mtDNA-depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor alpha. Interestingly, up- or down-regulation of mtCLIC protein expression changes Delta(psi)m whereas the chloride channel inhibitor NPPB reduces the Delta(psi)m in mtDNA-depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA-depleted cells incorporate, in a NPPB-sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into mitochondria.

Hypoxia induces PMN adherence to umbilical vein endothelium.

OBJECTIVE: In vitro incubation of cultured endothelial cells under hypoxia leads to the activation of these cells and results in an increase of their adhesiveness for neutrophils (PMN). Because of the possible relevance of these observations for pathological situations, we investigated whether adherence of PMN also occurs in an entire vein after its incubation in hypoxic conditions. METHODS: Human umbilical veins in complete cords were incubated for 2 h in normoxic or hypoxic conditions and the adherence of unstimulated human 51Cr-labelled-PMN was measured under flow conditions. Experiments with human umbilical vein endothelial cells (HUVEC) were performed in parallel for comparison. Morphological studies in scanning electron microscopy were carried out in both in vitro and ex vivo situations. RESULTS: Hypoxia induced an increase in the adherence of PMN either to HUVEC or to the umbilical vein endothelium up to 5- to 6-fold when compared to normoxic conditions (P < 0.001). In both cases, this hypoxia-induced adherence was inhibited by anti-ICAM-1 antibodies or when the PAF (platelet-activating factor) synthesis was blocked during hypoxia by oleic acid. Furthermore, the adherence of PMN was inhibited when PMN were pre-incubated with WEB 2086 (a selective PAF receptor antagonist). These results indicate a crucial role of PAF in this process. Morphological studies confirmed that the number of PMN adherent to hypoxic HUVEC or to the hypoxic umbilical vein endothelium was much greater than the number of PMN on normoxic endothelial cells. Both in vitro and ex vivo, PMN adherent to the hypoxic endothelial cells to the contrary of the ones adherent to normoxic endothelial cells demonstrated membrane foldings typical of an activated state. CONCLUSION: These results show that in a complete vein, hypoxia induced an increased adhesiveness of endothelial cells for PMN by a similar mechanism to the one observed for cultured endothelial cells. They suggest an active role of endothelial cells in the initiation of the inflammatory response often described in ischemic-reperfused organs.

ERK activation upon hypoxia: involvement in HIF-1 activation.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor activated by hypoxia. The HIF-1 activation transduction pathway is poorly understood. In this report, we investigated the activation of extracellular regulated kinases (ERK) in hypoxia and their involvement in HIF-1 activation. We demonstrated that in human microvascular endothelial cells-1 (HMEC-1), ERK kinases are activated during hypoxia. Using dominant negative mutants, we showed that ERK1 is needed for hypoxia-induced HIF-1 transactivation activity. Moreover, using a kinase assay and Western blot experiments, we showed that HIF-1alpha is phosphorylated in hypoxia by an ERK-dependent pathway. These results evidence the role of mitogen-activated protein kinase in the transcriptional response to hypoxia.

Effects of hydroxyethylrutosides on hypoxia-induced activation of human endothelial cells in vitro.

1. A clinically available mixture of hydroxyethylrutosides (HR) was examined as inhibitors of endothelial cell activation by hypoxia in vitro. Thus, the effects of HR on ATP depletion, phospholipase A2 activation and neutrophil adherence were investigated in hypoxia-activated human umbilical vein endothelial cells in primary cell culture. 2. Our results show that HR inhibited two important steps of the activation of endothelial cells by hypoxia: the decrease in ATP content, which is the starting point of the process, and the activation of phospholipase A2 one enzyme responsible for the release of inflammatory mediators. This inhibition was dose-dependent with 70 to 90% inhibition at 500 micrograms ml-1 of HR. 3. In addition, hypoxia-activated endothelial cells increased their adhesiveness for neutrophils. This process could also be prevented in a dose-dependent manner if endothelial cells were incubated in the presence of HR. This inhibition was confirmed by a morphological study. 4. In conclusion, the results of this study suggest that a possible explanation for the improvement in venous insufficiency by HR observed clinically could be their ability to inhibit the activation of endothelial cells during blood stasis.

Effect of aescine on hypoxia-induced activation of human endothelial cells.

Phlebotonic drugs are very often old drugs which improve symptoms in chronic venous insufficiency but their precise mechanism remains unclear. One reason for this lack of information is our poor understanding of the aetiology of the varicose vein. One hypothesis which is being more and more substantiated is that the origin of the disease lies in the activation of the endothelium during blood stasis, leading to a cascade of reactions which, in the long term, alter the structure of the vein wall. In this work, we tested aescine (Reparil i.v. form), a phlebotonic drug, in an in vitro model which mimics this situation, i.e. human endothelial cells exposed to hypoxic conditions. Aescine was shown to inhibit 2 important steps of the activation of endothelial cells incubated 120 min under hypoxia the decrease in ATP content, which is the starting point of the activation cascade, and the increase in the activity of phospholipase A2, an enzyme responsible for the release of precursors of inflammatory mediators. Hypoxia-activated endothelial cells also increase their adhesiveness for neutrophils. This process could also be prevented in a dose-dependent manner if endothelial cells were incubated in the presence of aescine. This inhibition was confirmed by morphological observations in scanning electron microscopy. All 3 effects were already evidenced at 100 ng/ml and were maximal at 750 ng/ml. These effects obtained at very low concentrations probably represent one of the main molecular and cellular mechanisms that underlie, among others, protection of the vessel wall. Objective criteria for our understanding of the preventive action of this phlebotonic drug are, thus, provided.

Effect of aescine on hypoxia-induced neutrophil adherence to umbilical vein endothelium.

Although venous stasis due to blood stagnation in lower limbs has been recognised as an important etiological factor for the development of varicose veins, the mechanism linking this ischemic situation to the modifications of the venous wall in varicose veins is still unclear. There is evidence that the activation of the endothelium during blood stasis and its subsequent cascade of interactions with other cell types could alter the structure of the vein wall and could possibly be at the origin of the disease. While phlebotonic drugs are often used to improve symptoms in chronic venous insufficiency, their precise mechanism of action is not well understood. We now tested aescine (Reparil i.v. form) in an ex vivo model which mimics this situation, i.e., perfused human umbilical vein exposed to hypoxic conditions. To study the effect of aescine on neutrophil activation and adhesion to the endothelium, human umbilical veins were incubated under hypoxic conditions with or without aescine and the interactions between the endothelium and neutrophil-like cells, HL60, were investigated. We observed that a large number of HL60 became adherent to the endothelium of veins after 2 h hypoxia and that these adherent HL60 were activated: they released high amounts of superoxide anion and of leukotriene B4. Aescine (250 ng/ml or 0.22 microM) was shown to markedly inhibit HL60 adherence to hypoxic endothelium. By decreasing the number of adherent HL60, aescine also decreased the subsequent production of superoxide anion and of leukotriene B4. Scanning electron microscopy confirmed the increased HL60 adherence to the endothelium, as well as the inhibitory effect of aescine. These results support results of in vitro studies on isolated endothelial cells in which aescine was shown to inhibit the hypoxia-induced activation of endothelial cells and the subsequent increased adherence of neutrophils. In vivo, the activated and infiltrated leukocytes release free radicals, chemotactic molecules such as leukotriene B4 and proteases which then can degrade the extracellular matrix. These processes could contribute to alterations of the venous wall similar to those observed in varicose veins. By maintaining an intact endothelium during in vivo blood stasis in the lower limbs and preventing neutrophil recruitment, adherence and activation, aescine could prevent the resulting alterations of the venous wall. These results could explain at least in part the potential benefit of the drug in the prevention of venous insufficiency.

Interactions between endothelial cells and smooth muscle cells after their activation by hypoxia. A possible etiology for venous disease.

Because of their localization at the interface between blood and tissue, endothelial cells are responsible for the maintenance of vascular homeostasis. They fulfil a series of various functions and constantly interact with circulating leukocytes and with the smooth muscle cells (SMC) present in the media. Any disturbance of their metabolism can thus lead to alterations of the blood vessel functions. We have shown that hypoxia, for example resulting from venous stasis, induces the activation of endothelial cells which then release inflammatory mediators able to activate neutrophils and to induce their infiltration as well as growth factors for SMC. We propose that these processes are the beginning of a cascade of events eventually leading to structural and functional modifications of the venous wall similar to the ones observed in varicose vein wall. The endothelium alterations resulting from venous stasis would thus be the origin of the development of the venous disease. Pharmacological and clinical evidence reinforce this hypothesis.

Perfused human saphenous veins for the study of the origin of varicose veins: role of the endothelium and of hypoxia.

If venous stasis due to blood stagnation has been recognized to be involved in the development of varicose veins, the mechanism linking this situation to the modifications of the venous wall observed in varicoses is still unclear. In order to study this mechanism, human saphenous veins were incubated in normoxic or hypoxic conditions and the interactions between the endothelium and neutrophils were investigated. We observed that many neutrophils adhered to the endothelium of veins incubated in hypoxic conditions rather than in normoxia and that these adherent neutrophils were activated: they released high amounts of superoxide anion and of leukotriene B4. Studies in scanning electron microscopy confirmed the increased neutrophil adherence to the endothelium as well as their activation. These results were then related to the histological observation of varicose veins. These veins show a thickening of the media with extracellular matrix deposit as well as an alteration of the elastic lamina with the presence of smooth muscle cells in the intima. These results are in agreement with in vitro studies on isolated endothelial cells. They all show that hypoxia is able to activate endothelial cells: they release inflammatory mediators and become adhesive for neutrophils which are then activated. These activated leukocytes release free radicals and proteases which are able to degrade the extracellular matrix. In addition, hypoxia-activated endothelial cells secrete growth factors which will trigger smooth muscle cell proliferation and the synthesis of extracellular matrix components. Altogether and because they are frequently repeated, these processes could eventually lead to alterations of the venous wall similar to those observed in varicose veins.

Hypoxia-induced activation of endothelial cells as a possible cause of venous diseases: hypothesis.

Blood stasis in leg veins is a situation commonly linked to the development of venous diseases such as varicoses. Such a stasis will provoke an ischemia, thus decreasing oxygen availability to tissues. Owing to its localization between blood and tissue, endothelium is the first target of this insult. The authors develop here a hypothesis in which the effect of oxygen deprivation on the functional state of the endothelium is the starting point of a cascade of events leading to the disorganization of the vessel wall typical of these pathologies. When venous human endothelial cells obtained from umbilical cords (HUVEC) are exposed to hypoxic conditions they become activated without change in their viability. The synthesis of a proinflammatory molecule (PAF, platelet-activating factor) and the adhesion of human polymorphonuclear neutrophils (PMN) on HUVEC are markedly increased during hypoxia incubation. These two processes are related to a calcium-dependent activation of endothelial cells due to a decrease of adenosine triphosphate (ATP) availability during hypoxia. Adherence of neutrophils to endothelial cells is the first step of diapedesis, which leads to the infiltration of these cells in the media of the veins, where they affect the smooth muscle cells and the connective tissue, leading to tissue alterations typical of the venous pathologies. The authors propose that this sequential process which originates from a reduction in oxygen availability and which involves different cell type as one main cause of the venous disorders, in addition to genetic, hormonal, and mechanical factors.

Mass spectrometry-based identification of proteins interacting with nucleic acids.

The identification of the regulatory proteins that control DNA transcription as well as RNA stability and translation represents a key step in the comprehension of gene expression regulation. Those proteins can be purified by DNA- or RNA-affinity chromatography, followed by identification by mass spectrometry. Although very simple in the concept, this represents a real technological challenge due to the low abundance of regulatory proteins compared to the highly abundant proteins binding to nucleic acids in a nonsequence-specific manner. Here we review the different strategies that have been set up to reach this purpose, discussing the key parameters that should be considered to increase the chances of success. Typically, two categories of biological questions can be distinguished: the identification of proteins that specifically interact with a precisely defined binding site, mostly addressed by quantitative mass spectrometry, and the identification in a non-comparative manner of the protein complexes recruited by a poorly characterized long regulatory region of nucleic acids. Finally, beside the numerous studies devoted to in vitro-assembled nucleic acid-protein complexes, the scarce data reported on proteomic analyses of in vivo-assembled complexes are described, with a special emphasis on the associated challenges.