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Articles by Sebastien Planchon in JoVE
JoVE General
Toxinvapen Induktion och Protein Extraction från Fusarium SPP. Kulturer för proteomik studier
Protein extraktion för proteomik analyser i svamparter kräver höga nivåer av standardisering som skall utföras i enlighet med den minsta information om ett proteomik experiment (MIAPE) riktlinjer. Vi presenterar en video-protokoll som innehåller ett förfarande för att minimera experimentella partiskhet under toxin induktion och protein utvinnas ur
Other articles by Sebastien Planchon on PubMed
Proteomic Analysis of the Tetraspanin Web Using LC-ESI-MS/MS and MALDI-FTICR-MS
Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of a molecular network of interactions, the "tetraspanin web". Here, we have performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of two cell lines derived from primary tumor and metastasis from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification and the proteins were identified by MS using LC-ESI-MS/MS and MALDI-FTICR. The high resolution and mass accuracy of FTICR MS allowed reliable identification using mass finger printing with only two peptides. Thus, it could be used to resolve the composition of complex peptide mixtures from membrane proteins. Different types of membrane proteins were identified, including adhesion molecules (integrins, Lu/B-CAM, GA733 proteins), receptors and signaling molecules (BAI2, PKC, G proteins), proteases (ADAM10, TADG15), and membrane fusion proteins (syntaxins) as well as poorly characterized proteins (CDCP1, HEM-1, CTL1, and CTL2). Some components were differentially detected in the tetraspanin web of the two cell lines. These differences may be relevant for tumor progression and metastasis.
The Transferrin Receptor and the Tetraspanin Web Molecules CD9, CD81, and CD9P-1 Are Differentially Sorted into Exosomes After TPA Treatment of K562 Cells
Here we show that treatment of K562 cells with the phorbol ester TPA induces the down-modulation of various surface antigens. Among them, the transferrin receptor (TfR), the tetraspanin CD81, and a CD81-associated protein, CD9P-1, were unique in that their expression levels were lower after 24 h incubation than after 3 h. We demonstrated that like the TfR, CD81 was internalized at early times, and was less synthesized at latter times. Despite the association of a fraction of the TfR with CD81, these two molecules were subjected to different fates. TPA increased targeting of CD81 and CD9P-1 into exosomes but strongly reduced the localization of the TfR in these vesicles. Using this model we have shown that a fraction of CD81 and CD9P-1 in exosomes comes from a surface pool and that these molecules remain associated in exosomes. However, CD9P-1 could be targeted to exosomes in the absence of CD81 and of another tetraspanin, CD9. The targeting of CD9 into exosomes did not require palmitoylation of the protein. J. Cell. Biochem. 102: 650-664, 2007. (c) 2007 Wiley-Liss, Inc.
A DIGE Analysis of Developing Poplar Leaves Subjected to Ozone Reveals Major Changes in Carbon Metabolism
Tropospheric ozone pollution is described as having major negative effects on plants, compromising plant survival. Carbon metabolism is especially affected. In the present work, the effects of chronic ozone exposure were evaluated at the proteomic level in developing leaves of young poplar plants exposed to 120 ppb of ozone for 35 days. Soluble proteins (excluding intrinsic membrane proteins) were extracted from leaves after 3, 14 and 35 days of ozone exposure, as well as 10 days after a recovery period. Proteins (pI 4 to 7) were analyzed by 2-D DIGE experiments, followed by MALDI-TOF-TOF identification. Additional observations were obtained on growth, lesion formation, and leaf pigments analysis. Although treated plants showed large necrotic spots and chlorosis in mature leaves, growth decreased only slightly and plant height was not affected. The number of abscised leaves was higher in treated plants, but new leaf formation was not affected. A decrease in chlorophylls and lutein contents was recorded. A large number of proteins involved in carbon metabolism were identified. In particular, proteins associated with the Calvin cycle and electron transport in the chloroplast were down-regulated. In contrast, proteins associated with glucose catabolism increased in response to ozone exposure. Other identified enzymes are associated with protein folding, nitrogen metabolism and oxidoreductase activity.
Glycosylation Status of the Membrane Protein CD9P-1
The membrane protein CD9P-1 is a major component of the tetraspanin web, a network of molecular interactions in the plasma membrane, in which it specifically associates with tetraspanins CD9 and CD81. The various functional effects of CD9 and CD81 may be related to their partners. Thus, we have addressed the characterization of the CD9P-1 glycosylation using stably transfected HEK-293 cells. After immunoprecipitation, CD9P-1 was subjected to enzymatic PNGase F cleavage of N-glycans, resulting in Asn to Asp conversion and increase in 1 mass unit. Thus, following protease digestion, deglycosylated peptides were selectively identified by high mass accuracy FTICR-MS, using this conversion as a signature. This has demonstrated that all nine potential N-glycosylation sites were actually engaged. On the other hand, the N-glycan structures were determined combining chemical derivatization and exoglycosidase digestions followed by MALDI-TOF MS, ESI-MS/MS, and GC-MS analysis. CD9P-1 was shown to exhibit more than 40 different N-glycans, essentially composed of complex and high mannose-type structures. Finally, 2-D PAGE and lectino-blot analyses have revealed the presence of at least 17 glycosylated isoforms of CD9P-1 at cell surface. All CD9P-1 isoforms associate with CD9 leading to additional level of complexity of this primary complex in the tetraspanin web.
Combining Proteomics and Metabolite Analyses to Unravel Cadmium Stress-response in Poplar Leaves
A proteomic analysis of poplar leaves exposed to cadmium, combined with biochemical analysis of pigments and carbohydrates revealed changes in primary carbon metabolism. Proteomic results suggested that photosynthesis was slightly affected. Together with a growth inhibition, photoassimilates were less needed for developmental processes and could be stored in the form of hexoses or complex sugars, acting also as osmoprotectants. Simultaneously, mitochondrial respiration was upregulated, providing energy needs of cadmium-exposed plants.
Proteomic Evaluation of Wound-healing Processes in Potato (Solanum Tuberosum L.) Tuber Tissue
Proteins from potato (Solanum tuberosum L.) tuber slices, related to the wound-healing process, were separated by 2-DE and identified by an MS analysis in MS and MS/MS mode. Slicing triggered differentiation processes that lead to changes in metabolism, activation of defence and cell-wall reinforcement. Proteins related to storage, cell growth and division, cell structure, signal transduction, energy production, disease/defence mechanisms and secondary metabolism were detected. Image analysis of the 2-DE gels revealed a time-dependent change in the complexity of the polypeptide patterns. By microscopic observation the polyalyphatic domain of suberin was clearly visible by D4, indicating that a closing layer (primary suberisation) was formed by then. A PCA of the six sampling dates revealed two time phases, D0-D2 and D4-D8, with a border position between D2 and D4. Moreover, a PCA of differentially expressed proteins indicated the existence of a succession of proteomic events leading to wound-periderm reconstruction. Some late-expressed proteins (D6-D8), including a suberisation-associated anionic peroxidase, have also been identified in the native periderm. Despite this, protein patterns of D8 slices and native periderm were still different, suggesting that the processes of wound-periderm formation are extended in time and not fully equivalent. The information presented in this study gives clues for further work on wound healing-periderm formation processes.
Effects of the Endocrine Disruptors Atrazine and PCB 153 on the Protein Expression of MCF-7 Human Cells
Polychlorinated biphenyls (PCBs) and a number of pesticides can act as endocrine disrupting compounds (EDCs). These molecules exhibit hormonal activity in vivo, and can therefore interact and perturb normal physiological functions. Many of these compounds are persistent in the environment, and their bioaccumulation may constitute a significant threat for human health. Physiological abnormalities following exposure to these xenobiotic compounds go along with alterations at the protein level of individual cells. In this study, MCF-7 cells were exposed to environmentally relevant concentrations of atrazine, PCB153 (100 ppb, respectively), 17-beta estradiol (positive control, 10 nM) and a negative control (solvent) for t = 24 h (n = 3 replicates/exposure group). After trizol extraction and protein solubilization, protein expression levels were studied by 2D-DIGE. Proteins differentially expressed were excised, trypsin-digested, and identified by MALDI-ToF-ToF, followed by NCBInr database search. 2D-DIGE experiments demonstrated that 49 spots corresponding to 29 proteins were significantly differentially expressed in MCF-7 cells (>1.5-fold, P < 0.05, Student's paired t test). These proteins belonged to various cellular compartments (nucleus, cytosol, membrane), and varied in function; 88% of proteins were down-regulated during atrazine exposure, whereas 75% of proteins were up-regulated by PCB153. Affected proteins included those regulating oxidative stress such as superoxide dismutase and structural proteins such as actin or tropomyosin, which may explain morphological changes of cells already observed under the microscope. This study highlights the susceptibility of human cells to compounds with endocrine disrupting properties.
Acute Metal Stress in Populus Tremula X P. Alba (717-1B4 Genotype): Leaf and Cambial Proteome Changes Induced by Cadmium 2+
The comprehension of metal homeostasis in plants requires the identification of molecular markers linked to stress tolerance. Proteomic changes in leaves and cambial zone of Populus tremula x P. alba (717-1B4 genotype) were analyzed after 61 days of exposure to cadmium (Cd) 360 mg/kg soil dry weight in pot-soil cultures. The treatment led to an acute Cd stress with a reduction of growth and photosynthesis. Cd stress induced changes in the display of 120 spots for leaf tissue and 153 spots for the cambial zone. It involved a reduced photosynthesis, resulting in a profound reorganisation of carbon and carbohydrate metabolisms in both tissues. Cambial cells underwent stress from the Cd actually present inside the tissue but also a deprivation of photosynthates caused by leaf stress. An important tissue specificity of the response was observed, according to the differences in cell structures and functions.
Alteration of Plasma Membrane-bound Redox Systems of Iron Deficient Pea Roots by Chitosan
Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed.
Poplar Under Drought: Comparison of Leaf and Cambial Proteomic Responses
The forest ecosystem is of particular importance from an economic and ecological perspective. However, the stress physiology of trees, perennial and woody plants, is far from being fully understood. For that purpose, poplar plants were exposed to drought; the plants exhibited commonly reported drought stress traits in the different plant tissues. Leafy rooted cuttings of poplar were investigated through a proteomic approach in order to compare the water constraint response of two plant tissues, namely leaf and cambium. Sampling was realized during the drought period at 2 time points with increased drought intensity and 7 days after rewatering. Our data show that there is a difference in the moment of response to the water constraint between the two tissues, cambium being affected later than leaves. In leaves, drought induced a decrease in rubisco content, and an increase in the abundance of light harvesting complex proteins as well as changes in membrane-related proteins. In the cambial tissue, the salient proteome pattern change was the decrease of multiple proteins identified as bark storage proteins. After rewatering, almost all changes in cambial proteome disappeared whereas a significant number of leaf proteins appeared to be differentially regulated only during the recovery from drought.
Proteomic Analysis of Plasma Samples from Patients with Acute Myocardial Infarction Identifies Haptoglobin As a Potential Prognostic Biomarker
Prognosis of clinical outcome following myocardial infarction is variable and difficult to predict. We have analyzed the plasma proteome of thirty patients with acute myocardial infarction to search for new prognostic biomarkers. Proteomic analyses of blood samples were performed by 2-D-DiGE after plasma depletion of albumin and immunoglobulins G. New York Heart Association (NYHA) class determined at 1-year follow-up was used to identify patients with heart failure. Principal component analysis and hierarchical clustering of proteomic data revealed that patients could be separated into 3 groups. The 22 differentially expressed proteins involved in this grouping were identified as haptoglobin (Hp) and respective isoforms. The 3 groups of patients had distinct Hp isoforms: patients from group 1 had the α1-α1, patients from group 2 the α2-α1, and patients from group 3 the α2-α2 genotype. This classification was also associated with different total plasma levels of Hp. The presence of the α2 genotype and low plasma levels of Hp was associated with a higher NYHA class and therefore with a detrimental functional outcome after myocardial infarction. A plasma level of Hp below 1.4g/L predicted the occurrence of heart failure (NYHA 2, 3, 4) at 1-year with 100% sensitivity.
Analysis of Proteome and Frost Tolerance in Chromosome 5A and 5B Reciprocal Substitution Lines Between Two Winter Wheats During Long-term Cold Acclimation
Dynamics of cold tolerance and crown proteome composition has been analysed in a set of two winter wheat cultivars Mironovskaya 808 and Bezostaya 1 and four reciprocal substitution lines with interchanged chromosomes 5A and 5B during a long-term cold-acclimation (CA) treatment. Proteome analysis has revealed 298 differently abundant spots during experiment. Most of them (260) were changed due to CA process and only 52 spots displayed differences between genotypes. Two hundred and seven protein spots were successfully identified by tandem mass spectrometry. Comparison of samples before and after vernalization fulfillment by a combination of ANOVA and Student' T-test displayed ten differentially abundant protein spots (e.g. chopper chaperones). However, differences in the accumulation of these spots did not reflect differences in vernalization requirement of genotypes. Therefore, our results indicate that vernalization process has not influenced total proteome of CA wheat crowns. A few protein spots (14 spots; e.g. malate dehydrogenase) revealed differential accumulation levels between the individual genotypes or their groups possessing chromosome 5A or 5B from Mironovskaya 808 versus Bezostaya 1. The study has shown the effect of chromosome 5A on physiological traits and also proteome in winter wheat. Putative candidate protein markers for cold tolerance in wheat are discussed.
Characterization of Maize Allergens - MON810 Vs. Its Non-transgenic Counterpart
One of the main concerns about genetically modified foods and their potential impacts on human health is that the introduction of a new/ altered gene may putatively alter the expression of others, namely endogenous allergens. We intended to evaluate, and to compare, using quantitative real time RT-PCR technique, the expression of 5 already known maize allergens (Zea m14, Zea m25, Zea m27kD, 50kD Zein and trypsin inhibitor) in MON 810 vs. its non-transgenic counterpart, throughout seed development (10, 16 and 23days after pollination). We have shown that none of the tested allergen genes presented differential expression, with statistic significance, along all tested seed development stages, in MON810 vs. its conventional counterpart. We have also used bidimensional gel electrophoresis followed by Western blotting with plasma from two maize allergic subjects to characterize their immunologic responses against MON 810 vs. its non-transgenic control. Immunoreactive spots were characterized by MS. We have identified fourteen new IgE-binding proteins present in both transgenic and non-transgenic maize.
Continuous Thrombin Infusion Leads to a Bleeding Phenotype in Sheep
BACKGROUND: In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo. METHODS: Thrombin (0.0004-0.42IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n=5 animals; 15 experiments). In the dose verification study animals received 0.42IU/kg/min of thrombin vs. saline solution in a cross-over design (n=3 animals; 7 experiments). RESULTS: Thrombin at an infusion rate of 0.42IU/kg/min decreased fibrinogen levels by 75% (p<0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p=0.006), prolonged thrombin time by 70% (p=0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p<0.05) and decreased the activity of the coagulation factors VIII and XIII (p<0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p=0.019) and induced a 65% decrease in maximal clot firmness (p<0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism. CONCLUSION: Protracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species.
