Bidimensional Gel Research Articles

Periplasmic response upon disruption of transmembrane Cu transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen, has two transmembrane Cu(+) transport ATPases, CopA1 and CopA2. Both proteins export cytoplasmic Cu(+) into the periplasm and mutation of either gene leads to attenuation of virulence. CopA1 is required for maintaining cytoplasmic copper levels, while CopA2 provides copper for cytochrome c oxidase assembly. We hypothesized that transported Cu(+) ions would be directed to their destination via specific periplasmic partners and disruption of transport should affect the periplasmic copper homeostasis. Supporting this, mutation of either ATPase gene led to large increments in periplasmic cuproprotein levels. Toward identifying the proteins participating in this cellular response the periplasmic metalloproteome was resolved in non-denaturing bidimensional gel electrophoresis, followed by X-ray fluorescence visualization and identification by mass-spectrometry. A single spot containing the electron shuttle protein azurin was responsible for the observed increments in cuproprotein contents. In agreement, lack of either Cu(+)-ATPase induced an increase in azu transcription. This is associated with an increase in the expression of anr and rpoS oxidative stress response regulators, rather than cueR, a copper sensing regulator. We propose that azurin overexpression and accumulation in the periplasm is part of the cellular response to cytoplasmic oxidative stress in P. aeruginosa.

The response of Mucor plumbeus to pentachlorophenol: A toxicoproteomics study

Pentachlorophenol (PCP) represents a critical concern worldwide due to its toxicity and recalcitrance to degradation. The capacity of Mucor plumbeus to transform PCP into several detoxification metabolites, including tetrachlorohydroquinone and several phase II conjugates, was observed by LC-HRMS. The data obtained support the degradation pathway proposed previously. PCP effects in M. plumbeus, an unsequenced species, were investigated using a proteomics approach (bidimensional gel electrophoresis followed by MALDI TOF/TOF analyses). The mycelial proteins identified in the differentially accumulated spots allowed the identification of PCP responsive proteins. The presence of PCP increased the energy demand, altered the cell wall architecture and cytoskeleton and induced a significant stress response. The latter was emphasised by the up-accumulation of protein species associated with defence mechanisms (e.g. HSP70 and cytochrome c peroxidase). Overall the data produced corroborate the capability of PCP to uncouple oxidative-phosphorylation in mitochondria. Importantly, one of the identified mycelial protein species, a NAD- and Zn-dependent ADH, is likely to be involved in PCP degradation. Amongst the fungal secretome analysed, no putative PCP degradative enzymes were detected. This work constitutes the first toxicoproteomic study involving a Zygomycota fungus and the very first concerning the effect of PCP in a fungal proteome.

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

BackgroundThe unicellular parasite Trypanosoma cruzi is the causative agent of Chagaś disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored.Methodology/Principal FindingsViable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin.Conclusions/SignificanceHerein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

Occurrence of Major Whey Proteins in the pH 4.6 Insoluble Protein Fraction from UHT-Treated Milk

A clear picture of the protein rearrangement in milk following UHT-treatment was drawn by a comparative analysis of the pH 4.6 soluble protein fraction (SPF) and the pH 4.6 insoluble protein fraction (IPF) recovered from raw and UHT-treated milk samples. The two protein fractions were analyzed by mono- or bidimensional gel electrophoresis under reducing and nonreducing conditions, and protein bands were identified by specific immunostaining. Results showed that bovine serum albumin, β-lactoglobulin, and, to a lesser extent, α-lactalbumin coprecipitated with caseins in UHT-treated milk samples at pH 4.6. These proteins were almost exclusively involved in high molecular weight aggregates held together by disulfide bonds. Partition of α-lactalbumin and bovine serum albumin in the protein fractions obtained upon acidification of milk at pH 4.6 was evaluated by competitive immunoassays. The ELISA-based results suggested the possibility of using pH 4.6 insoluble α-lactalbumin and bovine serum albumin, in addition to pH 4.6 insoluble β-lactoglobulin, as indicators of the intensity of the heat treatment applied to milk.

Heterogeneous and Anisotropic Dynamics of a 2D Gel

We report x-ray photon correlation spectroscopy (XPCS) results on bidimensional (2D) gels formed by a Langmuir monolayer of gold nanoparticles. The system allows an experimental determination of the fourth order time correlation function, which is compared to the usual second order correlation function and to the mechanical response measured on macroscopic scale. The observed dynamics is anisotropic, heterogeneous and superdiffusive on the nanoscale. Different time scales, associated with fast heterogeneous dynamics inside 2D cages and slower motion of larger parts of the film, can be identified from the correlation functions. The XPCS results are discussed in view of other experimental results and models of three-dimensional gel dynamics.

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.

On the relation between hierarchical morphology and mechanical properties of a colloidal 2D gel system

We study a bidimensional gel system formed by a Langmuir film of gold nanoparticles. Its morphology is thoroughly characterized by AFM, SEM, and ellipsometric imaging techniques and shows a complex structure with features on a hierarchy of different sizes following a Levy distribution. The mechanical response arising after the gel point is investigated by Interfacial Shear Rheometry. The film is found to be mainly elastic, with the mechanical moduli scaling as a power law of the reduced concentration, in the same way as the fluctuation time which was measured in a recent X-ray Photon Correlation Spectroscopy experiment. The frequency dependence of the moduli is well described in the framework of the Soft Glass Rheology model (SGR) [Sollich et al. Phys Rev Lett 78, 2020 (1997)]. In this theory a power law distribution of relaxation times is postulated, whose exponent is experimentally determined for the present case. Such a distribution may reflect – in the dynamics – the hierarchical nature of the morphology of the film evidenced by microscopy. A mastercurve can be built, extending time–temperature to a time–concentration superposition principle as in [Cicuta et al. Phys Rev Lett 90, 116103(2003)]. This allows to describe the mechanical response over a frequency range slightly larger than that experimentally accessible. Besides the SGR component, a viscous term is always present, whose origin is investigated also taking into account samples with different preparation histories.

Expresión diferencial entre estadios de Trypanosoma cruzi I en el aislamiento de un paciente con cardiomiopatía chagásica crónica de zona endémica de Santander, Colombia

Introduction. Trypanosoma cruzi is the causative agent of Chagas disease. During infection in mammalian hosts two main forms of the parasite are observed: trypomastigotes and amastigotes. During differentiation each stage of the parasite expresses a specific proteins pattern of phase, which are responsible for their morphological, biochemical and biological properties that could play an important role in the infectivity, virulence and survival of the parasite.Objective. To realize an expression analysis differential between the stages tripomastigote and amastigote of isolated of T. cruzi I, using the electrophoresis in two dimensions and identification of proteins differential expressed by means of mass spectrometry.Materials and methods. A clone of the isolated MHOM/07/338 of T. cruzi I was used. The protein profiles of the stages of the parasite were obtained by electrophoresis in two dimensions, which were visualized in gels dyed with blue of Coomassie. The images were analyzed with software PDQuest and the proteins differential expressed were identified by MALDI TOF or LC MS/MS. Results. The bidimensional gels showed an average of 325 protein spots in each stage. The comparative analyses detected 21 spots over expressed in the stage tripomastigote and 30 in the stage amastigote. 16 proteins were selected for identification by mass spectrometry and classified in different functional categories.Conclusions. The findings show to differential expression between tripomastigotes and amastigotes of exclusive proteins of related T. cruzi, mainly with glucolític metabolism and cytoskeleton and therefore they would be target potentials for the drug design.

A reference growth curve for nutritional experiments in zebrafish (Danio rerio) and changes in whole body proteome during development

Zebrafish is one of the most used vertebrate model organisms in molecular and developmental biology, recently gaining popularity also in medical research. However, very little work has been done to assess zebrafish as a model species in nutritional studies in aquaculture in order to utilize the methodological toolbox that this species represents. As a starting point to acquire some baseline data for further nutritional studies, growth of a population of zebrafish was followed for 15weeks. Furthermore, whole body proteome was screened during development by means of bi-dimensional gel electrophoresis and mass spectrometry. Fish were reared under best practice laboratory conditions from hatching until 103days post-fertilization (dpf) and regularly fed ad libitum with Artemia nauplii from 12 dpf. A growth burst occurred within 9-51 dpf, reaching a plateau after 65 dpf. Fork length and body weight were significantly lower in males than in females from 58 dpf onwards. Proteomics analysis showed 28 spot proteins differently expressed through development and according to sex. Of these proteins, 20 were successfully identified revealing proteins involved in energy production, muscle development, eye lens differentiation, and sexual maturation. In summary, zebrafish exhibited a rapid growth until approximately 50 dpf, when most individuals started to allocate part of the dietary energy intake for sexual maturation. However, proteomic analysis revealed that some individuals reached sexual maturity earlier and already from 30 dpf onwards. Thus, in order to design nutritional studies with zebrafish fed Artemia nauplii, it is recommended to select a period between 20 and 40 dpf, when fish allocate most of the ingested energy for non-reproductive growth purposes.

Effect of fresh dietary garlic powder on some of the serum biochemical parameters in broiler chicks

The aim of the present study was to investigate the effect of garlic, in powder form, on protein fractions of broiler chicks' sera. Two hundred and eighty, 2-day-old Ross chicks were randomly divided into four groups: A and B (52 each) and C and D (88 each). The chicks in groups A and B were fed control diet, but those in groups C and D received diet supplemented with 1% and 3% garlic powder, respectively. To evaluate the effect of consumption period of garlic on immune response, half of the chicks in groups C and D were separated after the second bleeding as groups E and F and were fed control diet until the end of the experiment (6 weeks). All groups except A were inoculated against Newcastle disease, avian influenza, and infectious bursal disease. Fifteen birds were bled from each group on days 21, 32, and 42 and also five birds on day 2. The sera were assayed for prealbumin (pre-ALB), ALB, α-1, α-2, β, and γ globulin using bidimensional gel electrophoresis. The results showed that the aforementioned parameters, except γ globulin, were not affected by the diet (p > 0.05). The level of γ globulin had a decline with age in group A, whereas it increased in all vaccinated groups. Furthermore, when compared with group B, treated groups had a significantly higher amount of γ globulin from day 21 onwards, and the increase was dose dependent. Also, the removal of garlic from the diet of group E resulted in a significant drop in γ globulin, in relation to group C, on day 42. It is concluded that garlic powder has the potential to increase serum γ globulins in broiler chicks vaccinated against common poultry pathogens.

Protein modification and replicative senescence of WI‐38 human embryonic fibroblasts

Oxidized proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro. To better understand the mechanisms by which these damaged proteins build up and potentially affect cellular function during replicative senescence of WI-38 fibroblasts, proteins targeted by these modifications have been identified using a bidimensional gel electrophoresis-based proteomic approach coupled with immunodetection of HNE-, AGE-modified and carbonylated proteins. Thirty-seven proteins targeted for either one of these modifications were identified by mass spectrometry and are involved in different cellular functions such as protein quality control, energy metabolism and cytoskeleton. Almost half of the identified proteins were found to be mitochondrial, which reflects a preferential accumulation of damaged proteins within the mitochondria during cellular senescence. Accumulation of AGE-modified proteins could be explained by the senescence-associated decreased activity of glyoxalase-I, the major enzyme involved in the detoxification of the glycating agents methylglyoxal and glyoxal, in both cytosol and mitochondria. This finding suggests a role of detoxification systems in the age-related build-up of damaged proteins. Moreover, the oxidized protein repair system methionine sulfoxide reductase was more affected in the mitochondria than in the cytosol during cellular senescence. Finally, in contrast to the proteasome, the activity of which is decreased in senescent fibroblasts, the mitochondrial matrix ATP-stimulated Lon-like proteolytic activity is increased in senescent cells but does not seem to be sufficient to cope with the increased load of modified mitochondrial proteins.

Involvement of the cerebral cortex in Parkinson disease linked with G2019S LRRK2 mutation without cognitive impairment

Previous studies have shown altered synuclein, increased oxidative stress damage and increased oxidative stress responses in patients with sporadic Parkinson's disease (PD) without cognitive impairment. Yet no information exists about possible molecular alterations in the cerebral cortex in familial PD. The present study shows abnormal alpha-synuclein solubility and aggregation, and aggregated nitrated alpha-synuclein, in the cerebral cortex (area 8) in cases with long-lasting PD linked with the G2019S LRRK2 mutation, one of them with a few Lewy bodies (LBs) and the other two without LBs in the cerebral cortex. Increased expression of the oxidative stress marker malondialdehyde-lysine (MDAL), together with increased oxidative stress responses, AGE receptors (RAGE) and superoxide dismutase 2, occurred in the frontal cortex in the three LRRK2 cases compared with three controls processed in parallel. Bi-dimensional gel electrophoresis, western blotting, in-gel digestion and mass spectrometry disclosed glial fibrillary acidic protein as a target of MDAL adducts. Tubulin beta4 and enolase 2 were also identified as targets of oxidative damage. These results demonstrate biochemical abnormalities of alpha-synuclein, and increased oxidative stress damage and oxidative stress responses in the frontal cortex in PD linked with G2019S LRRK2 mutation not related with the presence of cortical LBs and in the absence of apparent cognitive deficits. These findings show that the cerebral cortex in familial PD linked with G2019S LRRK2 is affected in a similar way than that seen in sporadic PD without cognitive impairment.

Protein targets of oxidative damage in human neurodegenerative diseases with abnormal protein aggregates.

Human neurodegenerative diseases with abnormal protein aggregates are associated with aberrant post-translational modifications, solubility, aggregation and fibril formation of selected proteins which cannot be degraded by cytosolic proteases, ubiquitin-protesome system and autophagy, and, therefore, accumulate in cells and extracellular compartments as residual debris. In addition to the accumulation of "primary" proteins, several other mechanisms are involved in the degenerative process and probably may explain crucial aspects such as the timing, selective cellular vulnerability and progression of the disease in particular individuals. One of these mechanisms is oxidative stress, which occurs in the vast majority of, if not all, degenerative diseases of the nervous system. The present review covers most of the protein targets that have been recognized as modified proteins mainly using bidimensional gel electrophoresis, Western blotting with oxidative and nitrosative markers, and identified by mass spectrometry in Alzheimer disease; certain tauopathies such as progressive supranuclear palsy, Pick disease, argyrophilic grain disease and frontotemporal lobar degeneration linked to mutations in tau protein, for example, FTLD-tau, Parkinson disease and related alpha-synucleinopathies; Huntington disease; and amyotrophic lateral sclerosis, together with related animal and cellular models. Vulnerable proteins can be mostly grouped in defined metabolic pathways covering glycolysis and energy metabolism, cytoskeletal, chaperoning, cellular stress responses, and members of the ubiquitin-proteasome system. Available information points to the fact that vital metabolic pathways are hampered by protein oxidative damage in several human degenerative diseases and that oxidative damage occurs at very early stages of the disease. Yet parallel functional studies are limited and further work is needed to document whether protein oxidation results in loss of activity and impaired performance. A better understanding of proteins susceptible to oxidation and nitration may serve to define damaged metabolic networks at early stages of disease and to advance therapeutic interventions to attenuate disease progression.

Potential diagnostic markers in bronchial fluid of small cell lung cancer (SCLC)

e22221 Background: Lung cancer is a major cause of mortality worldwide and overall survival rate has not improved significantly over the past 20 years. An early diagnosis could improve prognosis and survival among these patients. The aim of this study was to identify protein markers obtained from bronchial fluids of SCLC patients which may differ from non-pathological bronchoscopy samples. Methods: We have included 43 patients with SCLC diagnosed using bronchoscope and 49 consecutive patients with non pathological bronchoscopy. Bronquial fluid was obtained from each patient and potential protein markers were studied. After being centrifuged, supernatant proteins were analysed using bidimensional electrophoresis with poliacrilamid gel stained with silver nitrate. Gel was scanned and analysed with Progenesis PG6220 program, which measures intensity of each spot. Resultant intensities in each group of patients (SCLC/non pathological bronchoscopy) were compared using T-Student method. We selected as potential markers those spots with a p value of less than 0.05. We calculated “fold change” of each spot as the ratio between mean intensity in SCLC bronchoscopy samples and non pathological bronchoscopy samples. Results: Optimal bidimensional gels of each sample were obtained. Among 300 comparable spots, 10 of them were expressed with a different intensity in both groups of patients; 6 of these potential markers were over expressed in SCLC samples, whereas 4 of them were underexpressed. The “fold change” of these 10 spots ranges from 1.5 to 8.67. Conclusions: Different protein markers can be detected in bronchial fluid obtained from SCLC samples. Significant differences in expression of these biomarkers were detected between SCLC patients and non pathological bronchoscopy patients. The development of an early diagnostic test using these proteins must be validated in future studies. No significant financial relationships to disclose.

Proteomics: A panel of seven serum biomarkers as a promising diagnostic tool for breast cancer

e22019 Background: Conventionals serum biomarkets have a low sensibility in breast cancer diagnosis. Proteomic is a promising tool to identify new proteins profiles to be used in screening and early diagnosed. Methods: 1)Biomarker identification: Serum proteins from 114 women were separated and analysed by bidimensional gel electrophoresis; data from 72 patients (p) were compared with those from 42 control women to search for differentially expressed proteins. Several comparisons were performed between controls and p regarding clinical parameters such as histological type, tumour stage and lymph node affection (-/+). A total of 53 spots were found to be differentially expressed and identified by mass spectrometry (MS) as potential breast cancer biomarkers.2) Protein Chip development: A Protein Chip was developed with antibodies against six biomarkers and three control proteins (an antibody for the detection of a spiked control, for data normalization and two proteins as positive and negative controls). An antibody against CA15–3 antigen was also included as a reference breast tumour marker to be compared with our biomarker panel. 3) Clinical validation of the Protein Chip: A clinical validation study was carried out with 75 healthy women and 125 breast cancer p. After multivariate statistical analysis of the biomarker data, CA15–3 was discarded due to its low significance while a panel of 5 biomarkers was obtained as the best predictive model to discriminate p from healthy subjects. Results: 53 diferentially expressed proteins have been identified as potential breast cancer serum biomarkers after analysing 114 serum samples by 2DE Technology. A Protein Chip has been developed for the simultaneous detection of five serum biomarkers and three control proteins. After clinical validation with 200 p and healthy women, the sensitivity of the Protein Chip to detect breast cancer was 95% and its specificity was 27%. Conclusions: The high sensitivity of the Protein Chip suggests that it could be a valid tool to complement mammography (sensitivity < 80%) in breast cancer screening programs, specially when its sensitivity tends to decrease, as it happens in young women and dense breast cases. Larger clinical validation trials are being developed. This work is supported by INDAS BIOTECH. No significant financial relationships to disclose.

Type-Dependent Oxidative Damage in Frontotemporal Lobar Degeneration: Cortical Astrocytes Are Targets of Oxidative Damage

Oxidative injury and stress responses are common features of many neurodegenerative diseases. To assess oxidative stress responses in frontotemporal lobar degeneration (FTLD), we identified increased 4-hydroxynonenal (HNE) adducts using gel electrophoresis and Western blotting in frontal cortex samples in 6 of 6 cases of FTLD with the P301L mutation in the tau gene (FTLD-tau), in 3 of 10 cases with tau-negative ubiquitin-immunoreactive inclusions, and in 2 of 3 cases associated with motor neuron disease. Selectively increased lipoxidation-derived protein damage associated with altered membrane unsaturation and fatty acid profiles was verified by mass spectrometry in FTLD-tau and FTLD associated with motor neuron disease. All FTLD-tau and most cases with increased HNE-positive bands had marked astrocytosis as determined by glial fibrillary acidic protein (GFAP) immunohistochemistry and increased GFAP expression on Western blotting; 2 FTLD cases with tau-negative ubiquitin-immunoreactive inclusions and with increased GFAP expression did not have increased HNE adducts. Bidimensional gel electrophoresis, Western blotting, in-gel digestion, and mass spectrometry identified GFAP as a major target of lipoxidation in all positive cases; confocal microscopy revealed colocalization of HNE and GFAP in cortical astrocytes, superoxide dismutase 1 in astrocytes, and superoxide dismutase 2 in astrocytes and neurons in all FTLD types. Thus, in FTLD, there is variable disease-dependent oxidative damage that is prominent in FTLD-tau, astrocytes are targets of oxidative damage, and GFAP is a target of lipoxidation. Astrocytes are, therefore, crucial elements of oxidative stress responses in FTLD.

Proteomic Analysis of the Role of STAT5A in the Apoptosis of Human Pre-B Cells : Evidence for a Connection to Daxx and the Oxidative Stress

It is well established that STAT5A and 5B transcription factors (TF) play a major role in hematopoiseis and oncogenesis. In particular, there is a body of evidence suggesting that they might be involved in B lymphocyte survival, proliferation and development as well as in B cell neoplastic transformation. To investigate the role of STAT5A in human precursor B cell survival, we stably transfected a dominant negative form of STAT5A (DN-STAT5A, deleted in its transactivation domain) in the human leukemic Nalm6 pre-B cell line. All clones expressing DN-STAT5A exhibited a lower proliferation rate associated with a higher spontaneous apoptosis and that was massively enhanced upon IL7 stimulation. They also were more sensitive to FAS (CD95)- and at a lesser extent to etoposide-induced cell death than cells transfected with the empty vector (Nalm6neo), suggesting a hitherto unknown link between STAT5A and apoptosis/survival pathways. There was no evidence for changes in the levels of expression of Bcl2, BclxL, Bad or –c-Myc in DN-STAT5A expressing cells whereas Bax and Bim expression was sharply up-regulated in these cells. In order to identify the proteins involved in the connection between Fas and STAT5A, we used a proteomic approach with differential analysis of cells expressing (Nalm6Δ5A749) or not (Nalm6neo) the DN-STAT5A. Statistical analysis of bidimensional (2D) gels and use of MALDI-TOF technique enabled us to identify 7 proteins down-regulated in the Nalm6Δ5A749 cells, including members of heat shock proteins such as hsp27 and hsp70, as well as proteins implied in the control of oxidative stress like glutathione synthetase and transaldolase. By contrast, whereas 6 proteins were shown to be up-regulated, including prohibitin which is a negative regulator of cellular proliferation. Nalm6Δ5A749 cells exhibited enhanced levels of reactive oxygen species in keeping with the idea that oxidative stress might be involved in the increased sensitivity of these cells to apoptotic signals. Although expression of all above mentioned molecules did not change upon Fas stimulation, addition of gluthatione (10mM) resulted in a complete inhibition of Fas-mediated apoptosis in Nalm6Δ5A749 cells. Whether or not prohibitin might be involved in the decreased proliferation rate in DN-STAT5A expressing cells needs further investigation. As Hsp27 has been previously shown to have the potential to sequestrate Daxx, a molecule that is involved in Fas receptor signaling, we investigated the subcellular location of this molecule in Nalm6Δ5A749 cells. We showed that Daxx was essentially expressed in the cytoplasm of these cells whereas it was mainly located in the nucleus in Nalm6neo cells. Our results bring evidence for a role of STAT5A in pre-B cell growth and survival and in particular for a connection between this TF and Fas signaling machinery that might involve Hsp27 and Daxx. They also point out to a so far undescribed link between STAT5 and oxidative stress.

Increased oxidation of certain glycolysis and energy metabolism enzymes in the frontal cortex in Lewy body diseases

Lipoxidative damage of aldolase A, enolase 1, and glyceraldehyde dehydrogenase (GAPDH) was found in the frontal cortex in a percentage of aged controls by bidimensional gel electrophoresis, Western blot test, in-gel digestion, and mass spectrometry. Aldolase A and enolase 1 were altered in 12 of 19 cases, whereas oxidation of GAPDH was found in 6 of 19 controls. The three enzymes were oxidized in the frontal cortex in the majority of cases of incidental Parkinson's disease (iPD), PD, and dementia with Lewy bodies (DLB). Differences were statistically significant (chi(2) test) for GAPDH in PD and DLB. Densitometric studies have shown that the ratio of oxidized protein per spot is higher in iPD, PD, and DLB compared with controls. These findings show oxidation of three enzymes linked with glycolysis and energy metabolism in the adult human brain as well as increased oxidation of aldolase A, enolase 1, and GAPDH in the frontal cortex in Lewy body diseases. Modifications of these enzymes may result in decreased activity and may partly account for impaired metabolism and function of the frontal lobe in PD.

Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood

Proteomic analysis was performed to determine and differentiate the composition of the secretomes of Phanerochaete chrysosporium CIRM-BRFM41, a peroxidase hypersecretory strain grown under ligninolytic conditions and on softwood chips under biopulping conditions. Extracellular proteins from both cultures were analyzed by bidimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 37 spots were identified. The secretome in liquid synthetic medium comprised mainly peroxidases, while several wood-degrading enzymes and enzymes involved in fungal metabolism were detected in biopulping cultures on softwood. This prompted an analysis of the impact of secretome modulation in the presence of softwood chips. Biotreated wood was submitted to kraft cooking and chemical bleaching using chlorine dioxide. The fungal pre-treatment led to a significant increase in pulp yield and a better bleachability of the pulp. This bleachability improvement could be explained by the production of specific lignocellulose-degrading enzymes.

Proteomic analysis of an imatinib‐resistant K562 cell line highlights opposing roles of heat shock cognate 70 and heat shock 70 proteins in resistance

Understanding the molecular basis of resistance to imatinib, a tyrosine kinase inhibitor used as front-line therapy in chronic myeloid leukemia, remains a challenge for successful treatment. In an attempt to identify new mechanisms of resistance, we performed a comparative proteomic analysis of an imatinib-resistant cell line generated from the erythroblastic cell line K562 (K562-r) for which no known mechanism of resistance has been detected. Bidimensional gel electrophoresis was carried out to compare the protein expression pattern of imatinib-sensitive and of imatinib-resistant K562 cells. Among the 400 matched spots on five pairs of gels, only 14 spots had a significantly increased or decreased expression leading to the identification of 24 proteins identified as scaffold proteins, metabolic enzymes, DNA translation and maturation, and chaperon proteins. Among the chaperon family, only Hsp70 and Hsc70 are overexpressed in K562-r, results confirmed by Western blotting. We recently reported the participation of Hsp70 overexpression in imatinib resistance whereas a role for Hsc70 has yet to be determined. Hsc70 is not involved in imatinib resistance as the inhibition of its expression by siRNA does not restore sensitivity to imatinib. In contrast, the induced decreased expression of Hsc70 was accompanied by a greater overexpression of Hsp70. This proteomic study therefore suggests opposing roles of Hsp70 and Hsc70 in imatinib resistance.