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Aims Acinetobacter baumannii is a nosocomial pathogen known to be multidrug-resistant (MDR), especially to drugs of the carbapenem class. Several factors contribute to resistance, including efflux pumps, β -lactamases, alteration of target sites, and permeability defects. In addition, outer membrane proteins (OMPs), like porins are involved in the passage of antibiotics, and their alteration could lead to resistance development. This study aimed to explore the possible involvement of porins and OMPs in developing carbapenem resistance due to differential expression. Methods and results The antibiotic-susceptible and MDR isolates of A. baumannii were first studied for differences in their transcriptional levels of OMP expression and OMP profiles. The antibiotic-susceptible isolates were further treated with imipenem, and it was found that the omp genes were differentially expressed. Six of the nine genes studied were upregulated at 1 h of exposure to imipenem. Their expression gradually decreased with time, further confirmed by their OMP profile and two-dimensional gel electrophoresis. Conclusions This study could identify OMPs that were differentially expressed on exposure to imipenem. Hence, this study provides insights into the role of specific OMPs in antibiotic resistance in A. baumannii. [ABSTRACT FROM AUTHOR]

Photosynthesis, an indispensable biological process of plants, produces organic substances for plant growth, during which photorespiration occurs to oxidize carbohydrates to achieve homeostasis. Although the molecular mechanism underlying photosynthesis and photorespiration has been widely explored, the crosstalk between the two processes remains largely unknown. In this study, we isolated and characterized a T-DNA insertion mutant of tomato (Solanum lycopersicum) named yellow leaf (yl) with yellowish leaves, retarded growth, and chloroplast collapse that hampered both photosynthesis and photorespiration. Genetic and expression analyses demonstrated that the phenotype of yl was caused by a loss-of-function mutation resulting from a single-copy T-DNA insertion in chaperonin 60α1 (SlCPN60α1). SlCPN60α1 showed high expression levels in leaves and was located in both chloroplasts and mitochondria. Silencing of SlCPN60α1 using virus-induced gene silencing and RNA interference mimicked the phenotype of yl. Results of two-dimensional electrophoresis and yeast two-hybrid assays suggest that SlCPN60α1 potentially interacts with proteins that are involved in chlorophyll synthesis, photosynthetic electron transport, and the Calvin cycle, and further affect photosynthesis. Moreover, SlCPN60α1 directly interacted with serine hydroxymethyltransferase (SlSHMT1) in mitochondria, thereby regulating photorespiration in tomato. This study outlines the importance of SlCPN60α1 for both photosynthesis and photorespiration, and provides molecular insights towards plant genetic improvement. [ABSTRACT FROM AUTHOR]

1-Deoxynojirimycin (DNJ) is the most abundant poly-hydroxylated alkaloid in the latex of mulberry leaves and it protects mulberry from insect predation. However, silkworms can survive the poisoning effect of DNJ and accumulate DNJ by consumption of the mulberry leaves. In order to determine the molecular mechanism of DNJ accumulation in silkworm, comparative proteomic analysis was employed to evaluate protein expression in two groups of silkworm bodies (the third instar silkworm bodies had the maximum content of DNJ throughout life, and the newly hatched silkworm bodies had no DNJ). Our results indicated some differentially expressed proteins in the third instar silkworm involved in material metabolism, energy metabolism, oxidation-reduction, detoxification, immune, and transport regulation may correspond to the accumulation of DNJ. Furthermore, the expression levels of five selected differentially expressed protein-encoding genes namely heat shock cognate protein (Hsp 70), glutathione S-transferase sigma 1 (GST), serine protease precursor (Ser), hemolymph protein (30K), and thiol peroxiredoxin (TPx) were investigated by quantitative real-time PCR and the accumulation of DNJ was measured by HPLC. Correlation analysis showed that the expression levels of Hsp70 and Ser were negatively correlated to DNJ accumulation with weak correlation, while 30K, GST, and TPx genes had positive correlation with DNJ accumulation. The findings suggested that these three proteins were probably important in the physiological process of DNJ accumulation in silkworm. [ABSTRACT FROM AUTHOR]

Meat color is the most important quality trait influencing consumer purchase decisions. The interinfluential interactions between myoglobin and biomolecules govern color stability in meat. The advances in proteomics, such as high throughput analytical tools in mass spectrometry, 2-dimensional electrophoresis, and bioinformatics, offer themselves as robust techniques to characterize the proteome basis of muscle- and species-specific meat color phenomena. Differential abundance of chaperones and antioxidant proteins contributes to muscle-specific color stability in beef; the greater abundance of chaperones and antioxidant proteins in color-stable Longissimus lumborum than in color-labile Psoas major protects myoglobin and contributes to superior color stability of beef Longissimus steaks. Lipid oxidation-induced myoglobin oxidation is more critical to beef color than pork color due to the inherent differences in myoglobin chemistry; the number of nucleophilic histidine residues adducted by reactive aldehydes is greater in beef myoglobin than in pork myoglobin. Preferential adduction of secondary products of lipid oxidation to beef myoglobin accelerates metmyoglobin formation at a greater degree than in its pork counterpart. Mass spectrometry investigations revealed that although cherry-red carboxymyoglobin is more stable than oxymyoglobin, both redox forms undergo lipid oxidation-induced oxidation in model systems. The accuracy of mass spectrometry to detect the molecular mass of proteins has been applied to differentiate myoglobins from closely related meat animals, such as goats and sheep or emu and ostrich. In addition, this approach indicated that turkey myoglobin is 350 Da greater in molecular mass than beef myoglobin, and the unique biochemistry of turkey myoglobin could be responsible for its greater thermostability in model systems as well as the pink color defect observed in fully cooked uncured turkey products. [ABSTRACT FROM AUTHOR]

Background: Two-dimensional electrophoresis is a crucial method in proteomics that allows the characterization of proteins’ function and expression. This usually implies the identification of proteins that are differentially expressed between two contrasting conditions, for example, healthy versus diseased in human proteomics biomarker discovery and stressful conditions versus control in animal experimentation. The statistical procedures that lead to such identifications are critical steps in the 2-DE analysis workflow. They include a normalization step and a test and probability correction for multiple testing. Statistical issues caused by the high dimensionality of the data and large-scale multiple testing have been a more active topic in transcriptomics than proteomics, especially in microarray analysis. We thus propose to adapt innovative statistical tools developed for microarray analysis and incorporate them in the 2-DE analysis pipeline.Results: In this article, we evaluate the performance of different normalization procedures, different statistical tests and false discovery rate calculation methods with both real and simulated datasets. We demonstrate that the use of statistical procedures adapted from microarrays lead to notable increase in power as well as a minimization of false-positive discovery rate. More specifically, we obtained the best results in terms of reliability and sensibility when using the ‘moderate t-test’ from Smyth in association with classic false discovery rate from Benjamini and Hochberg.Availability: The methods discussed are freely available in the ‘prot2D’ open source R-package from Bioconductor (http://www.bioconductor.org//) under the terms of the GNU General Public License (version 2 or later).Contact: sebastien.artigaud@univ-brest.fr or sebastien.artigaud@gmx.com [ABSTRACT FROM AUTHOR]

We developed a method for detecting phosphatase activities in crude tissue extracts after separation of proteins by a novel non-denaturing two-dimensional electrophoresis. In the first dimension, protein samples were separated by a MicroRotofor, a liquid-phase isoelectric focusing, in the presence or absence of urea. In the second dimension, fractionated proteins by the MicroRotofor were resolved by a native polyacrylamide gel electrophoresis in the presence of 20 mM 2-mercaptoethanol. After electrophoresis, the polyacrylamide gel was directly immersed in a reaction mixture containing 4-methylumbelliferyl phosphate (MUP), a fluorogenic substrate, and phosphatase activities were detected as fluorescent bands. In this assay, a variety of phosphatase activities were clearly detected in gel when the tissue extracts were separated by the MicroRotofor in the presence of 1.5 M urea. Furthermore, after detecting phosphatase activities in polyacrylamide gel at neutral pH, its activities at acidic pH could be detected by immersing the gel in sodium citrate buffer (pH 3.0). Therefore, this method is a quite useful technique to analyze various phosphatases by sequential reactions with MUP under different conditions after sample separation by the two-dimensional electrophoresis. [ABSTRACT FROM AUTHOR]

Riemerella anatipestifer ( RA) is one of the most important bacterial pathogens of ducks and other avian species worldwide. Twenty-one serotypes of RA have been identified, with RA serotype two ( RA2) being reported as one of the most predominant serotypes underlying infections in China. Current approaches to the control of RA are hindered by the absence of effective vaccines, particularly those that exhibit cross-protection between different serotypes. In this study, a combination of two-dimensional electrophoresis, Western blot analysis and mass spectrometry were used to identify the antigenic proteins of RA2. A total of 16 immunoreactive proteins, representing 12 distinct proteins, were identified. These included OmpA, a known immunogenic protein of RA, as well as novel immunogens. PCR analysis also indicated that genes corresponding to each of the 12 distinct proteins were conserved among different RA serotypes. Eleven genes encoding these proteins were cloned and expressed in Escherichia coli BL21 ( DE3). Eight of the 11 expressed proteins were able to react with hyperimmune rabbit serum against RAf153. One of these, recombinant elongation factor G, responded to RA2 sera but not RA1, whereas recombinant OmpA responded to both RA1 and RA2 sera. These data form a basis for the development of vaccine for both homologous and heterogeneous RA serotypes in addition to the production of target antigens for the development of diagnostic antibodies with the potential to distinguish between RA serotypes. [ABSTRACT FROM AUTHOR]

Objective: Thyroid proteomics is a new direction in thyroid cancer research aiming at etiological understanding and biomarker identification for improved diagnosis. Methods: Two-dimensional electrophoresis was applied to cytosolic protein extracts from frozen thyroid samples (ten follicular adenomas, nine follicular carcinomas, ten papillary carcinomas, and ten reference thyroids). Spots with differential expression were revealed by image and multivariate statistical analyses, and identified by mass spectrometry. Results: Aset of 25 protein spots significant for discriminating between the sample groupswas identified. Proteins identified for nine of these spots were studied further including 14-3-3 protein beta/alpha, epsilon, and zeta/delta, peroxiredoxin 6, selenium-binding protein 1, protein disulfide-isomerase precursor, annexin A5 (ANXA5), tubulin alpha-1B chain, and a1-antitrypsin precursor. This subset of protein spots carried the same predictive power in differentiating between follicular carcinoma and adenoma or between follicular and papillary carcinoma, as compared with the larger set of 25 spots. Protein expression in the sample groups was demonstrated by western blot analyses. For ANXA5 and the 14-3-3 proteins, expression in tumor cell cytoplasm was demonstrated by immunohistochemistry both in the sample groups and an independent series of papillary thyroid carcinomas. Conclusion: The proteins identified confirm previous findings in thyroid proteomics, and suggest additional proteins as dysregulated in thyroid tumors. [ABSTRACT FROM AUTHOR]

The interindividual variation in the sensitivity to bitterness is attributed in part to genetic polymorphism at the taste receptor level, but other factors, such as saliva composition, might be involved. In order to investigate this, 2 groups of subjects (hyposensitive, hypersensitive) were selected from 29 healthy male volunteers based on their detection thresholds for caffeine, and their salivary proteome composition was compared. Abundance of 26 of the 255 spots detected on saliva electrophoretic patterns was significantly different between hypo- and hypersensitive subjects. Saliva of hypersensitive subjects contained higher levels of amylase fragments, immunoglobulins, and serum albumin and/or serum albumin fragments. It also contained lower levels of cystatin SN, an inhibitor of protease. The results suggest that proteolysis occurring within the oral cavity is an important perireceptor factor associated to the sensitivity to the bitter taste of caffeine. [ABSTRACT FROM AUTHOR]

The wide-scale analysis of protein expression provides a powerful strategy for the molecular exploration of complex pathophysiological mechanisms, such as the response to antidepressants. Using a 2D proteomic approach we investigated the Flinders Sensitive Line (FSL), a genetically selected rat model of depression, and the control Flinders Resistant Line (FRL). To evaluate gene–environment interactions, FSL and FRL pups were separated from their mothers for 3 h (maternal separation, MS), as early-life trauma is considered an important antecedent of depression. All groups were treated with either escitalopram (Esc) admixed to food (25 mg/kg.d) or vehicle for 1 month. At the week 3, forced swim tests were performed. Protein extracts from prefrontal/frontal cortex and hippocampus were separated by 2D electrophoresis. Proteins displaying statistically significant differences in expression levels were identified by mass spectrometry. Immobility time values in the forced swim test were higher in FSL rats and reduced by antidepressant treatment. Moreover, the Esc-induced reduction in immobility time was not detected in MS rats. The impact of genetic background in response to Esc was specifically investigated here. Bioinformatics analyses highlighted gene ontology terms showing tighter associations with the modulated proteins. Esc modulated protein belonging to cytoskeleton organization in FSL; carbohydrate metabolism and intracellular transport in FRL. Proteins differently modulated in the two strains after MS and Esc play a role in cytoskeleton organization, vesicle-mediated transport, apoptosis regulation and macromolecule catabolism. These findings suggest pathways involved in neuronal remodelling as molecular correlates of response to antidepressants in a model of vulnerability. [ABSTRACT FROM PUBLISHER]

Large-scale investigations aimed at elucidating the molecular mechanism of action of antidepressant treatment are achievable through the application of proteomic technologies. We performed a proteomic study on the Flinders Sensitive Line (FSL), a genetically selected rat model of depression, and the control Flinders Resistant Line (FRL). To evaluate gene–environment interactions, FSL and FRL animals were separated from their mothers for 3 h from postnatal days 2 to 14 (maternal separation; MS), since early-life trauma is considered an important antecedent of depression. All groups received either escitalopram (Esc) admixed to food pellets (25 mg/kg.d) or vehicle for 1 month. Protein extracts from prefrontal/frontal cortex and hippocampus were separated by 2D electrophoresis. Proteins differentially modulated were identified by mass spectrometry. Bioinformatics analyses were performed to discover gene ontology terms associated with the modulated proteins. This paper was focused on the modifications induced by the environmental challenge of MS, both on the predisposed genetic background and on the resistant phenotype. The combination between Esc treatment and MS was investigated by comparing the MS, Esc-treated rats with rats subjected to each single procedure. In MS rats, antidepressant treatment influenced mainly proteins involved in carbohydrate metabolism in FSL rats and in vesicle-mediated transport in FRL rats. When studying the interaction between Esc and MS vs. non-separated rats, proteins playing a role in cytoskeleton organization, neuronal development, vesicle-mediated transport and synaptic plasticity were identified. The results provide further support to the available reports that antidepressant treatment affects intracellular pathways and also suggest new potential targets for future therapeutic intervention. [ABSTRACT FROM PUBLISHER]

Background. Depending on both membrane composition and solute transport rate across the membrane, protein composition of the dialysate of patients receiving peritoneal dialysis (PD) has recently become of great interest. Unfortunately, thus far few studies have focused on dialysate characterization, and further investigations are required to better understand the biological mechanisms influencing PD efficiency.Methods. Different classical proteomic approaches were combined with advanced mass spectrometric (MS) techniques to analyse peritoneal fluid (PF) protein composition of adult patients receiving PD. Characterization was performed by using 1D gel electrophoresis combined with nano-RP–HPLC–ESI–MS/MS and shotgun proteomics, while comparative analyses were performed coupling 2D gel electrophoresis with MALDI-TOF MS.Results. The study allowed the identification of 151 different proteins from PF, which are mainly of plasmatic origin. Comparison of PD effluents characterized by different glucose concentrations demonstrated four proteins (apolipoprotein A-IV, fibrinogen beta chain, transthyretin and alpha-1-antitrypsin) to be under-expressed in the highest osmolar solution having 4.25% compared to others having 1.5% and 2.5% glucose. All of them were found to be involved in the inflammatory processes.Conclusions. This study provides a possible platform for future diagnostic and therapeutic applications in the field of PD and allowed the identification of potential targets to be used in preventing inflammatory processes induced by the exposure to dialysis solutions. [ABSTRACT FROM AUTHOR]

Due to drawbacks of live attenuated vaccines, much attention has been focused on screening Brucella-protective antigens as subunit vaccine candidates. Here, an immunoproteomic assay was used to identify the immunogenic soluble proteins of Brucella melitensis 16M. In the present study, 27 unique immunogenic proteins were identified from the two-dimensional electrophoresis immunoblot profiles by liquid chromatography tandem MS (LC-MS/MS). From this set, the gene encoding one immunodominant protein of interest, S-adenosyl--homocysteine hydrolase (AdoHcyase), was expressed in Escherichia coli. The recombinant AdoHcyase induced a strong antibody response in BALB/c mice, and the polyclonal antibody could recognize a band of approximately 52 kDa in the immunoblots of soluble protein extracts from five Brucella strains. rAdoHcyase significantly stimulated the production of interferon-γ and interleukin-2, and induced a high level of protection against B. melitensis 16M challenge at 4 weeks postchallenge. Our results indicated that rAdoHcyase could be a useful candidate for the development of subunit vaccines against B. melitensis. [ABSTRACT FROM AUTHOR]

Motivation: Detection of protein spots in two-dimensional gel electrophoresis images (2-DE) is a very complex task and current approaches addressing this problem still suffer from significant shortcomings. When quantifying a spot, most of the current software applications include a lot of background due to poor segmentation. Other software applications use a fixed window for this task, resulting in omission of part of the protein spot, or including background in the quantification. The approach presented here for the segmentation and quantification of 2-DE aims to minimize these problems.Results: Five sections from different gels are used to test the performance of the presented method concerning the detection of protein spots, and three gel sections are used to test the quantification of sixty protein spots. Comparisons with a state-of-the-art commercial software and an academic state-of-the-art approach are presented. It is shown that the proposed approach for segmentation and quantification of 2-DE images can compete with the available commercial and academic software packages.Availability: A command-line prototype may be downloaded, for non-commercial use, from http://w3.ualg.pt/~aanjos/prototypes.html.Contact: antoniodosanjos@gmail.comSupplementary information: Supplementary data are available at Bioinformtaics online. [ABSTRACT FROM AUTHOR]

CONTEXT: Transsphenoidal adenomectomy is the primary treatment for acromegaly. However, assessment of the therapeutical outcome remains problematic since the existing biomarkers of disease activity frequently show discordant results. OBJECTIVE: To discover novel serum biomarkers of disease activity in acromegalic patients before and after surgery. DESIGN: Serum samples of eight newly diagnosed acromegaly patients before and after transsphenoidal surgery were analyzed for proteomic changes by two-dimensional gel electrophoresis. Protein spots displaying statistically significant changes, pre- versus post-surgery, were identified by mass spectrometry (MS), tandem MS (MS/MS), and western blot analysis. RESULTS: Six protein spots displaying decreased intensities after surgery were identified as transthyretin (two isoforms), haptoglobin α2, β-hemoglobin, and apolipoprotein A-1 (two isoforms). One protein spot, identified as complement C4B precursor, was increased after the surgery. CONCLUSIONS: Seven serum protein spots were differentially expressed following surgery in acromegalic patients. The identified proteins represent potential novel biomarkers to assess the effectiveness of surgical treatment in acromegalic individuals. Future studies will validate the use of the identified proteins as biomarkers of disease activity after medical treatment of acromegaly. [ABSTRACT FROM AUTHOR]

Transmission routes of noroviruses, leading aetiological agents of acute gastroenteritis, are rarely verified when outbreaks occur. Because the destination of norovirus particles being firmly captured by micro-organisms could be totally different from that of those particles moving freely, micro-organisms with natural affinity ligands such as virus-binding proteins would affect the fate of viruses in environment, if such microbial affinity ligands exist. The aim of this study is to identify norovirus-binding proteins (NoVBPs) that are presumably working as natural ligands for norovirus particles in water environments. NoVBPs were recovered from activated sludge micro-organisms by an affinity chromatography technique in which a capsid peptide of norovirus genogroup II (GII) was immobilized. The recovered NoVBPs bind to norovirus-like particles (NoVLPs) of norovirus GII, and this adsorption was stronger than that to NoVLPs of norovirus genogroup I. The profile of two-dimensional electrophoresis of NoVBPs showed that the recovered NoVBPs included at least seven spots of protein. The determination of N-terminal amino acid sequences of these NoVBPs revealed that hydrophobic interactions could contribute to the adsorption between NoVBPs and norovirus particles. NoVBPs conferring a high affinity to norovirus GII were successfully isolated from activated sludge micro-organisms. NoVBPs could be natural viral ligands and play an important role in the NoV transmission. [ABSTRACT FROM AUTHOR]

Anti-endothelial cell antibodies (AECA) have been frequently detected in systemic vasculitis, which affects blood vessels of various sizes. To understand the pathogenic roles of AECA in systemic vasculitis, we attempted to identify target antigens for AECA comprehensively by a proteomic approach. Proteins extracted from human umbilical vein endothelial cells (HUVEC) were separated by two-dimensional electrophoresis, and Western blotting was subsequently conducted using sera from patients with systemic vasculitis. As a result, 53 autoantigenic protein spots for AECA were detected, nine of which were identified by mass spectrometry. One of the identified proteins was peroxiredoxin 2 (Prx2), an anti-oxidant enzyme. Frequency of anti-Prx2 autoantibodies, measured by enzyme-linked immunosorbent assay (ELISA), was significantly higher in systemic vasculitis (60%) compared to those in collagen diseases without clinical vasculitis (7%, P < 0·01) and healthy individuals (0%, P < 0·01). Further, the titres changed in parallel with the disease activity during time–courses. The presence of anti-Prx2 autoantibodies correlated significantly with elevation of serumd-dimers and thrombin–antithrombin complex ( P < 0·05). Immunocytochemical analysis revealed that live endothelial cells expressed Prx2 on their surface. Interestingly, stimulation of HUVEC with rabbit anti-Prx2 antibodies increased secretion of interleukin (IL)-6, IL-1β, IL-1ra, growth regulated oncogene (GRO)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM–CSF), IL-8 and monocyte chemoattractant protein (MCP)-1 more than twofold compared to that of with rabbit immunoglobulin (Ig)G. Taken together, our data suggest that anti-Prx2 autoantibodies would be a useful marker for systemic vasculitis and would be involved in the inflammatory processes of systemic vasculitis. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the influence of lifestyle on the aging alterations in skeletal muscle mitochondrial proteins. Thirty C57BL/6 strain mice (2 months) were randomly divided into three groups (young, Y; old sedentary, S; and old active, A). The S and A mice were individually placed into standard cages and in cages with running wheels for 25 months. Upon killing, mitochondria from the hind limb skeletal muscles were isolated for the evaluation of general proteome alterations, carbonylation, and electron transport chain (ETC) activity. We identified 77 different proteins mostly from the oxidative phosphorylation and mitochondrial metabolism. Sedentary mice presented a significant loss of ETC functionality in opposition to active mice. Although some proteins were found damaged in both A and S mice, damage to ETC proteins was more evident in S. Moreover, it is also possible to conclude that lifestyle is a key modulator for preventing the aging-induced protein expression and functionality in mitochondria. [ABSTRACT FROM PUBLISHER]

Motivation: The two-dimensional electrophoresis (2-DE) pattern of proteins is thought to be specifically related to the physiological or pathological condition at the moment of sample preparation. On this ground, most proteomic studies move to identify specific hallmarks for a number of different conditions. However, the information arising from these investigations is often incomplete due to inherent limitations of the technique, to extensive protein post-translational modifications and sometimes to the paucity of available samples. [ABSTRACT FROM PUBLISHER]

The purpose of this work was to produce and compare liver proteomic maps of cattle breeds that have been selected for different purposes: dairy (Holstein) and beef (Chianina). Liver was chosen because it displays functions of synthesis, homeostasis, excretion, and defense. Two dimensional electrophoresis analyses were performed on bovine livers collected from 4 individuals of each breed to evaluate differential protein expression. We found 9 spots differing in photodensity by ⩾2-fold between the 2 breeds. Spots were analyzed through matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDITOF MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). A search against protein sequences in the National Center for Biotechnology Information databases indicated that the differentially expressed proteins correspond to liver-specific enzymes, with a major role in the metabolism of fatty acids, carbohydrates, AA, and the urea cycle. [ABSTRACT FROM AUTHOR]

Black rot of cruciferous plants, caused by Xanthomonas campestris pv. campestris, causes severe losses in agriculture around the world. This disease affects several cultures, including cabbage and broccoli, among others. Proteome studies of this bacterium have been reported; however, most of them were performed using the bacterium grown under culture media conditions. Recently, we have analyzed the proteome of X. campestris pv. campestris during the interaction with the susceptible cultivar of Brassica oleracea and several proteins were identified. The objective of the present study was to analyze the expressed proteins of X. campestris pv. campestris during the interaction with the resistant cultivar of B. oleracea. The bacterium was infiltrated in the leaves of the resistant plant and recovered for protein extraction and two-dimensional electrophoresis. The protein profile was compared with that of the bacterium isolated from the susceptible host and the results obtained revealed a group of proteins exclusive to the resistant interaction. Among the proteins identified in this study were plant and bacterium proteins, some of which were exclusively expressed during the resistant interaction. [ABSTRACT FROM AUTHOR]

Apart from inhibiting RecA activity through protein–protein interactions, Deinococcus radiodurans RecX inhibits the expression of RecA and two other anti-oxidant proteins. To identify the repertoire of proteins regulated by RecX, comparative proteomic studies were undertaken on a wild-type strain (R1) and recX null mutant (RecX–). Two-dimensional electrophoresis followed by MALDI-TOF identification revealed 35 differentially expressed proteins, including 12 up-regulated and 23 down-regulated proteins in the mutant. The 12 up-regulated proteins are DNA repair proteins, stress response proteins, and metabolism-related proteins. Most of these have been previously characterized as ionizing radiation-induced proteins. The 23 down-regulated proteins are mainly involved in cellular metabolism, and some of these are key enzymes in the metabolic pathway. Thus, RecX is suggested to be involved in the switch between DNA damage response and normal metabolism in D. radiodurans. [ABSTRACT FROM PUBLISHER]

Summary: Dengue infection is a major public health problem affecting millions of people living in tropical countries. With no suitable vaccines and specific antiviral drugs, treatment for dengue is usually symptomatic and supportive. Early diagnosis and recognition of severe disease is therefore crucial for better management of the patient. Two-dimension electrophoresis was used to identify disease-associated proteins that can be used for diagnosis and as drug targets for treatment. Two markers, identified by mass spectrometry analysis as α1-antitrypsin and NS1 proteins were found to be upregulated in dengue fever (DF; n =10) and dengue haemorrhagic fever (DHF; n =10) patients compared with healthy individuals (n =8). Both α1-antitrypsin and NS1 proteins were overexpressed two-fold in DHF patients compared with DF patients. Our study suggests that α1-antitrypsin and NS1 protein could be used as biomarkers as early indicators of DHF risk among patients with suspected dengue infection. [Copyright &y& Elsevier]

During post-harvest storage, potato tubers age as they undergo an evolution of their physiological state influencing their sprouting pattern. In the present study, physiological and biochemical approaches were combined to provide new insights on potato (Solanum tuberosum L. cv. Désirée) tuber ageing. An increase in the physiological age index (PAI) value from 0.14 to 0.83 occurred during storage at 4 °C over 270 d. Using this reference frame, a proteomic approach was followed based on two-dimensional electrophoresis. In the experimental conditions of this study, a marked proteolysis of patatin occurred after the PAI reached a value of 0.6. In parallel, several glycolytic enzymes were up-regulated and cellular components influencing protein conformation and the response to stress were altered. The equilibrium between the 20S and 26S forms of the proteasome was modified, the 20S form that recycles oxidized proteins being up-regulated. Two proteins belonging to the cytoskeleton were also differentially expressed during ageing. As most of these changes are also observed in an oxidative stress context, an approach focused on antioxidant compounds and enzymes as well as oxidative damage on polyunsaturated fatty acids and proteins was conducted. All the changes observed during ageing seemed to allow the potato tubers to maintain their radical scavenging activity until the end of the storage period as no accumulation of oxidative damage was observed. These data are interpreted considering the impact of reactive oxygen species on the development and the behaviour of other plant systems undergoing ageing or senescence processes. [ABSTRACT FROM PUBLISHER]

To better understand the metabolic processes of seed filling in soybean (Glycine mcix), two complementary proteomic approaches, two-dimensional gel electrophoresis (2-DGE) and semicontinuous multidimensional protein identification technology (Sec-MudPIT) coupled with liquid chromatography-mass spectrometry, were employed to analyze whole seed proteins at five developmental stages. 2-DGE and Sec-MudPIT analyses collectively identified 478 nonredundant proteins with only 70 proteins common to both datasets. 2-DGE data revealed that 38% of identified proteins were represented by multiple 2-DGE species. Identified proteins belonged to 13 (2-DGE) and 15 (Sec-MudPIT) functional classes. Proteins involved in metabolism, protein destination and storage, and energy were highly represented, collectively accounting for 61.1% (2-DGE) and 42.2% (Sec-MudPIT) of total identified proteins. Membrane proteins, based upon transmembrane predictions, were 3-fold more prominent in Sec-MudPIT than 2-DGE. Data were integrated into an existing soybean proteome database (www.oilseedproteomics.missouri.edu). The integrated quantitative soybean database was compared to a parallel study of rapeseed (Brassicci napus) to further understand the regulation of intermediary metabolism in protein-rich versus oil-rich seeds. Comparative analyses revealed (1) up to 3-fold higher expression of fatty acid biosynthetic proteins during seed filling in rapeseed compared to soybean; and (2) approximately a 48% higher number of protein species and a net 80% higher protein abundance for carbon assimilatory and glycolytic pathways leading to fatty acid synthesis in rapeseed versus soybean. Increased expression of glycolytic and fatty acid biosynthetic proteins in rapeseed compared to soybean suggests that a possible mechanistic basis for higher oil in rapeseed involves the concerted commitment of hexoses to glycolysis and eventual de novo fatty acid synthesis pathways. [ABSTRACT FROM AUTHOR]

High purity berry plasma membranes (PMs) of Vitis vinifera L. cv. Cabernet Sauvignon were isolated by two-phase partitioning of microsome fractions at different stages of berry ripening. PM proteins resolvable by the detergent cocktail of CHAPS and ASB-14 were separated by two-dimensional electrophoresis. A total of 119 protein spots from pre-véraison berry PMs on 2-D gels detected with silver staining were subjected to MALDI-TOF mass spectrometry analysis. Sixty-two spots were identified as putative PM proteins, with 1–6 predicted transmembrane helices, including true PM proteins such as ATP synthase, ABC transporters, and GTP-binding proteins reported in plants. They were then grouped into eight functional categories, mainly involved in transport, metabolism, signal transduction, and protein synthesis. Another 11 spots were identified as proteins of unknown function. The véraison and post-véraison samples stained 98 and 86 spots on the gels, respectively. During the berry ripening process, total PM protein content gradually decreased. Among all identified proteins, 12 showed significant differences in terms of their relative abundance. Increasing ubiquitin proteolysis and cytoskeleton proteins were observed from pre-véraison to post-véraison. Zeatin O-glucosyltransferase peaked at véraison, while ubiquitin-conjugating enzyme E2-21 was down-regulated at this stage. This proteome research provides the first information on PM protein characterization during the grape berry ripening process. [ABSTRACT FROM PUBLISHER]

Proteomic research has proved valuable for understanding the molecular mechanisms of biological processes, as well as in the search for biomarkers for a variety of diseases which lack a molecular diagnostic. While several new approaches are being developed, two-dimensional (2-DE) gel electrophoresis is still one of the most commonly used techniques, despite its many limitations. However, for biomarker research, 2-DE gel electrophoresis alone does not fulfill the necessary pre-requisites. If such a technique is utilized exclusively, a great part of a given proteome remains unseen. Therefore, very precise and sensitive techniques are needed. Here, we present a brief review of known methodologies that try to overcome the limitations of conventional proteome analysis as well as their respective advantages and limitations. [ABSTRACT FROM AUTHOR]

The genus Xanthomonas is composed of several species that cause severe crop losses around the world. In Latin America, one of the most relevant species is Xanthomonas campestris pv. c ampestris, which is responsible for black rot in cruciferous plants. This pathogen causes yield losses in several cultures, including cabbage, cauliflower and broccoli. Although the complete structural genome of X. campestris pv. c ampestris has been elucidated, little is known about the protein expression of this pathogen in close interaction with the host plant. Recently, a method for in vivo analysis of Xanthomonas axonopodis pv. citri was developed. In the present study, this technique was employed for the characterization of the protein expression of X. campestris pv. campestris in close interaction with the host plant Brassica oleracea. The bacterium was infiltrated into leaves of the susceptible cultivar and later recovered for proteome analysis. Recovered cells were used for protein extraction and separated by two-dimensional electrophoresis. Proteins were analysed by peptide mass fingerprinting or de novo sequencing and identified by searches in public databases. The approach used in this study may be extremely useful in further analyses in order to develop novel strategies to control this important plant pathogen. [ABSTRACT FROM AUTHOR]

The fission yeast lsd1/fas2 strain carries a temperature-sensitive mutation of the fatty-acid-synthase α-subunit, exhibiting an aberrant mitosis lsd phenotype, with accumulation of very-long-chain fatty-acid-containing phospholipid (VLCFA-PL). A novel 90-kDa protein, Lsd90 (SPBC16E9.16c), was found to be newly expressed in small particle-like structures in lsd1/fas2 cells under restrictive conditions. Two mismatches leading to a double frame shift were found between the sequences of the lsd90+ gene registered in the genomic database and the sequences determined experimentally at the amino acid, cDNA and genomic DNA levels. Unexpectedly, overexpression and disruption of the lsd90+ gene in either lsd1/fas2 or wild-type cells did not affect either cell growth or expression of the lsd phenotype. The amounts of VLCFA-PL that accumulated in lsd90-overexpressing lsd1/fas2 cells were significantly lower than those in lsd1/fas2 cells, suggesting the involvement of Lsd90 in the metabolism of VLCFA-PL. [ABSTRACT FROM PUBLISHER]

Two-dimensional electrophoresis (2-DE) is the most commonly-used technique for large-scale identification of proteins in clinical samples. Here, we review the use of this method to improve prostate cancer diagnostics, especially on the analysis of prostate-specific antigen (PSA). Two-dimensional electrophoresis is a powerful method, which makes possible the detection and analysis of thousands of proteins and their post-translational modifications. However, it is a sophisticated technique, which does not resolve all kind of proteins. Some groups were able to improve prostate cancer diagnostics using the 2-DE pattern of free PSA isoforms on prostate cancer and non-prostate cancer sera. To this end, it would be very interesting to further investigate and develop these new diagnostic approaches. [ABSTRACT FROM AUTHOR]

The article discusses the application of quantitative real-time polymerase chain reaction analysis of three genes, to investigate the correlation between messenger RNA and protein level in response to salinity. There was no concordance detected between the changes in levels of gene and protein expression. The results indicate that the proteomics approach is suitable for expression analysis of membrane associated proteins under salt stress.

The purpose of this study was to investigate the effect of oxidative stress on physiological and genetic characteristics of Fusobacterium nucleatum and its interference on this microbial identification methods. Methods and Results: Fus. nucleatum ssp. nucleatum ATCC 25586 (wt-strain) and an oxidative-stress-adapted strain derived from the wt-strain (aero-strain) were employed in the study. Cell-free crude protein extracts were obtained from both strains and differentially expressed proteins were identified by two-dimensional electrophoresis. Bacterium identification was performed by conventional biochemical tests, automated Rapid ID 32A system and specific PCR analysis. Genetic diversity between wt-and aero-strain was assessed by arbitrarily-primed (AP)-PCR. There were significant changes in the protein profile of aero-strain. The identification of the wt-strain was confirmed by all methods employed. Similar results were obtained for aero-strain when conventional biochemical tests and PCR were used. However, aero-strain was identified as Fusobacterium varium when submitted to Rapid ID 32A system. According to AP-PCR analysis, no significant genetic alteration was detected in aero-strain. Conclusions: The adaptive response of Fus. nucleatum to oxidative stress is associated with changes on its biology, which may lead to misidentification of the organism, according to the conventional identification methods. Significance and Impact of the Study: Oxidative stress may act as a cause of adaptive response in Fus. nucleatum with consequences to its biology, such as alterations on biochemical and physiological profile. [ABSTRACT FROM AUTHOR]

Background Skin develops through a process of epidermal proliferation, maturation, and remodelling of the epidermis and dermis. This period also involves the maturation of the skin immune system, such that antigen applied though the skin of a neonatal mouse always results in immunosuppression, whereas in adults, immunity will occur. Objectives Using proteomics, to identify proteins uniquely involved in the development of the skin and skin immune system. Methods Proteins were extracted from whole skin of mice aged 4 and 21 days, and separated using two-dimensional electrophoresis. Results Of the 25 proteins that were sequenced by peptide mass fingerprinting with matrix-assisted laser desorption/ionization–time of flight–mass spectrometry, three were known markers of keratinocyte differentiation and proliferation. These were cyclophilin A, epidermal fatty acid binding protein 5 and stefin A. Of interest were the two isoforms of stefin A, an intracellular protease inhibitor, found in neonatal skin. The strong expression of stefin A in neonates was confirmed by immunohistochemical analysis, suggesting an important role in the development of the epidermis. Additionally, Western blotting identified two larger isoforms in adult skin, revealing a change in the stefin A during development. Conclusions We propose that stefin A is involved in development of the skin, that development of the skin and of immune function is linked, and that stefin A has an important function in neonatal skin and potentially the neonatal immune response. [ABSTRACT FROM AUTHOR]

Staphylococcus epidermidis is one of the major causative agents for nosocomial infections. To reveal the pathogenesis factors, we performed the comparative proteomic analysis of invasive ATCC35984 and commensal ATCC12228 strains by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. The differentially expressed proteins were involved in carbohydrate metabolism, sugar binding, lipid degradation and amino acid binding. In addition, we demonstrated that the trap gene was transcribed by 3.657±0.156 ( P<0.01) -fold higher in ATCC35984 than in ATCC12228. Levels of accumulation-associated protein (AAP) were found to be low by the immuno-dot blotting assay in ATCC12228, which is unable to form biofilm. Our results suggest that the target of RNAIII activating protein and AAP may contribute to S. epidermidis virulence and biofilm formation. [ABSTRACT FROM AUTHOR]

Two-dimensional gel electrophoresis coupled to mass spectrometry analysis was used to examine for the first time the effect of a herbicide (flumioxazin) on a crop species (Vitis vinifera L.) at the proteome level. Examination of 2-D maps derived from chemically stressed tissues revealed the presence of 33 spots displaying a differential expression pattern. The presence of stress responsive proteins in the different plant organs analysed suggests that flumioxazin could act systemically. Among the responsive proteins, some photosynthesis-related proteins, including several fragments of the enzyme Rubisco, were identified. This effect suggests that photosynthesis could be impaired by the herbicide. The induction of several enzymatic antioxidant systems was also observed, probably as a result of an oxidative stress. Moreover, the photorespiration pathway was stimulated, as suggested by the induction of some key enzymes involved in this process. Changes in carbon metabolism-associated proteins presumably reflect altered patterns of carbon flux in response to impaired photosynthesis and an increased need for osmotic adjustment in affected tissues. Finally, plant defences were stimulated as revealed by the induction of a set of proteins belonging to the pathogenesis-related 10 class, suggesting that they could play an essential role in cell defence mechanisms against flumioxazin. [ABSTRACT FROM PUBLISHER]

Sample complexity frequently interferes with the analysis of low-abundance proteins by two-dimensional gel electrophoresis (2DGE). Ideally, high abundance proteins should be removed, allowing low-abundance proteins to be applied at much higher concentrations than is possible with the unfractionated sample. One approach is to partition the sample in a manner that segregates the bulk of extraneous proteins from the protein(s) of interest. Solution phase isoelectric focusing in the multi-compartment electrolyser generates fractions of discrete isoelectric point (pl) intervals allowing isolated narrow segments of a proteome to be analysed individually by 2DGE. It is particularly useful for the isolation of low-abundance proteins of extremely basic or acidic pl. [ABSTRACT FROM AUTHOR]

Quantitative proteomics involves the identification and quantitation of protein components in various biological systems. Stable isotope labelling technology, by both metabolic and chemical methods, has been the most commonly used approach for global proteome-wide profiling. Recently, its capability has been extended from labelled pairs to multiple labels, allowing for the simultaneous quantification of multiplex samples. The ion intensity-based quantitative approach has progressively gained more popularity as mass spectrometry performance has improved significantly. Although some success has been reported, it remains difficult comprehensively to characterise the global proteome, due to its enormous complexity and dynamic range. The use of sub-proteome fractionation techniques permits a simplification of the proteome and provides a practical step towards the ultimate dissection of the entire proteome. Further development of the technology for targeting sub-proteomes on a functional basis -- such as selecting proteins with differential expression profiles from mass spectrometric analyses, for further mass spectrometric sequencing in an intelligent manner -- is expected in the near future. [ABSTRACT FROM AUTHOR]

Abstract: Francisella tularensis is a facultative intracellular pathogen. Its capacity to induce disease depends on the ability to invade and multiply within a wide range of eukaryotic cells, such as professional phagocytes. The comparative disinterest in tularemia in the past relative to other human bacterial pathogens is reflected in the paucity of information concerning the mechanisms of pathogenesis. Only a few genes and gene products associated with Francisella virulence are known to date. The aim of this study was to find and identify proteins of F. tularensis live vaccine strain induced in the presence of hydrogen peroxide, and to investigate the role of the IglC protein in the regulation of genes expressed upon peroxide stress. The [35S]-radiolabelled protein patterns were examined for both the wild live vaccine strain and its ¿iglC1+2 mutant defective in synthesis of the IglC protein that was found to be strongly up-regulated during intracellular growth in murine macrophages in vitro and upon exposure to hydrogen peroxide. Globally, we found 21 protein spots whose levels were significantly altered in the presence of hydrogen peroxide in both the wild-type and mutant strains. [Copyright &y& Elsevier]

Abstract: The first protein map of an ale-fermenting yeast is presented in this paper: 205 spots corresponding to 133 different proteins were identified. Comparison of the proteome of this ale strain with a lager brewing yeast and the Saccharomyces cerevisiae strain S288c confirmed that this ale strain is much closer to S288c than the lager strain at the proteome level. The dynamics of the ale-brewing yeast proteome during production-scale fermentation was analysed at the beginning and end of the first and the third usage of the yeast (called generation in the brewing industry). During the first generation, most changes were related to the switch from aerobic propagation to anaerobic fermentation. Fewer changes were observed during the third generation but certain stress-response proteins such as Hsp26p, Ssa4p and Pnc1p exhibited constitutive expression in subsequent generations. The ale brewing yeast strain appears to be quite well adapted to fermentation conditions and stresses. [Copyright &y& Elsevier]

To identify fungal proteins involved in the arbuscular mycorrhizal symbiosis, root-inducing transferred-DNA transformed roots of carrot (Daucus carota L.) were in vitro inoculated with Glomus intraradices. Proteins extracted from the extra-radical fungus were analysed by two-dimensional gel electrophoresis. A fungal reference map displaying 438 spots was set up. Four proteins, among the 14 selected for tandem mass spectrometry analysis, were identified including a NmrA-like protein, an oxido-reductase, a heat-shock protein and an ATP synthase β mitochondrial precursor. The possible fungal origin of a MYK15-like protein found in mycorrhizal roots was further discussed. This is the first report of arbuscular mycorrhizal fungal protein identifications by using a proteomic approach. [Copyright &y& Elsevier]

The proteome of a Listeria monocytogenes strain isolated from a food plant was investigated to study the differential protein pattern expressed by biofilms and planktonic bacteria. The approach used in this study was a combination of two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight and database searches for the protein identification. Thirty-one proteins varied significantly between the two growth conditions. Twenty-two and nine proteins were up- and down-regulated respectively and nine proteins were successfully identified. The variations of the protein patterns indicated that the biofilm development is probably controlled by specific regulation of protein expression involved at various levels of cellular physiology. [Copyright &y& Elsevier]

Enabling technologies for proteomics (studies examining the protein complement of the genome) have been in development for over 20 years. However, more recently, the field has become formalised by combining the techniques for large-scale protein separation (two-dimensional electrophoresis) with very precise, high fidelity approaches to the analysis and characterisation of the separated proteins (mass spectrometry). These approaches bring to reality the powerful scope ofproteomics, enabling the interpretation of cell physiology through profiling the molecular biodescriptors of gene expression, the proteins. Applications of proteomics are wide ranging and can be found within many disciplines, although one of the more developed fields that is examined here covers the arena of toxicological and pharmacological profiling. In this paper, the current technologies that make proteomics possible will be looked at. Some areas where proteomics has delivered valuable biological insight will also be considered, and there will be a look towards future areas of development. [ABSTRACT FROM PUBLISHER]

Examines the effects of anisotropy on the two-dimensional (2-D) inversion procedure. Effect of anisotropy strikes on the behavior of the induction arrows; Calculation of the intrinsic anisotropy ratio; Dependence of the strike direction of a 2-D model on the angle within the range of variation.

Hydrophobic interaction chromatography and two-dimensional electrophoresis were used to isolate and characterize mouse SAA, and to study the in vivo effect of separate or combined administrations of cytokines, dexamethasone (DEX) and LPS on mouse SAA. Four SAA spots containing partial amino acid sequence in accordance with mouse apoSAA1 and apoSAA2/SAASJL/J pI 5.9 were demonstrated in serum. One of these proteins represents a previously undescribed, acidic acute-phase mouse SAA protein. Both DEX and interferon-gamma (IFN-γ) proved to be capable of increasing SAA serum levels. In contrast to what has been shown in previous in vivo studies, administration of IL-6 did increase the SAA levels to nearly the same magnitude as IL-1, and the effect of IL-6 and LPS on SAA production was not significantly altered by the addition of DEX. Irrespective of the inflammatory stimuli that was administered, a non-selective production of SAA1 and SAA2 was observed in most groups, including the group that received IL-6. The results illustrate that data obtained about mouse SAA are highly dependent on which models, isolation and identification methods are used. [ABSTRACT FROM AUTHOR]

In order to identify alterations in the myocardial protein pattern that characterize dilated cardiomyopathy (DCM), we compared, by two-dimensional gel electrophoresis, right atrial protein patterns from five patients with DCM and four with normal left ventricular function (two gels per patient). Using computer assisted analysis (PDQUEST, 4.1) we found reproducible protein patterns in the 18 gels (23 × 30 cm, pH 4-9, molecular weight 10–150 kDa). In the two gels from the same patient, 91 % of proteins were identical in their position in the pattern and the relative intensities of these protein species correlated with r=0.85. Three hundred and two ± 50 protein species were found in several gels, 186 in all 18 gels. Seven proteins in the DCM group were decreased in their relative intensity by > 100%, six were increased by >100%. Significant quantitative differences between DCM and control patients were found for 25 protein species. Based on seven external marker proteins, a pH and molecular weight value could be calculated for each protein. So far, 30 protein species have been identified by antibodies, amino acid analysis or sequencing procedures. From the 25 proteins that are significantly different between DCM and controls, three have been identified. Expression of the mitochondrial creatine kinase and alpha cristallin B chain was significantly increased in DCM; the malate dehydrogenase family was also significantly decreased in DCM. Two-dimensional electrophoresis appears to be a powerful method for the detection of disease-associated alterations in the myocardial protein pattern. [ABSTRACT FROM PUBLISHER]

To characterize the sequence features surrounding the translation initiation sites on the genome of Synechocystis sp. strain 6803, the total proteins extracted from the cell were resolved by two-dimensional electrophoresis, and the amino-terminal sequences of the relatively abundant protein spots were determined. By comparison of the determined amino-terminal sequences with the nucleotide sequence of the entire genome, the translation initiation sites of a total of 72 proteins were successfully assigned on the genome. The sequence features emerged from the nucleotide sequences at and surrounding the translation initiation sites were as follows: (1) In addition to the three initiation codons, ATG, GTG, and TTG, evidence was obtained that ATT was also used as a rare initiation codon; (2) the core sequences (GAGG, GGAG and AGGA) of the Shine-Dalgarno sequence were identified in the appropriate position preceding the 35 initiation sites (48.6%); and (3) the preferential sequence surrounding the initiation codons was formulated as 5′-YY[· · ·]R-3′ where Y and R denote pyrimidine and purine nucleotides, respectively, and three dots represent the initiation codons. The result obtained would provide valuable information for improvement of the gene-finding software, and the approach used in this study should be applicable for comprehensive analysis of the expression profiles of cellular proteins. [ABSTRACT FROM PUBLISHER]

We describe a novel method, two-dimensional electrophoresis/phage panning (2D-PP), for the generation of antibodies against proteins in crude biochemical samples, such as cellular membrane fractions. These sources have traditionally presented problems as to the development of antibodies by conventional techniques. 2D-PP involves two-dimensional resolution of proteins, blotting of the proteins onto a nitrocellulose membrane, and screening of a phage antibody library and isolation of corresponding antibodies. By 2D-PP with detergent-insoluble “lipid rafts” as a target protein complex, we obtained specific phage pools against eight antigen spots (from a total of 39 spots). These antibodies were functional in Western blotting, enzyme-linked immunosorbent assaying (ELISA), and immunoscreening of a cDNA expression library. Propagation of anti-nitrocellulose phages was the major problem in 2D-PP, but was overcome by the use of the soluble anti-nitrocellulose antibody fragment. 2D-PP constitutes a key tool for functional analysis of proteins in complex fractions. [ABSTRACT FROM AUTHOR]

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and in wildlife, and it has the potential for developmental toxicity. However, the molecular mechanisms that lead to these toxic effects are not well known. In the present study, proteomic analysis has been performed to investigate the proteins that are differentially expressed in zebrafish embryos exposed to 0.5 mg/l PFOS until 192 h postfertilization. Two-dimensional electrophoresis coupled with mass spectrometry was employed to detect and identify the protein profiles. The analysis revealed that 69 proteins showed altered expression in the treatment group compared to the control group with either increase or decrease in expression levels (more than twofold difference). Of the 69 spots corresponding to the proteins with altered expression, 38 were selected and subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (TOF/TOF) analysis; 18 proteins were identified in this analysis. These proteins can be categorized into diverse functional classes such as detoxification, energy metabolism, lipid transport/steroid metabolic process, cell structure, signal transduction, and apoptosis. Overall, proteomic analysis using zebrafish embryos serves as an in vivo model in environmental risk assessment and provides insight into the molecular events in PFOS-induced developmental toxicity. [ABSTRACT FROM AUTHOR]

Endometrial cancer, the most common gynecological malignancy in western countries, is characterized by a favorable prognosis. Nonetheless, deep myometrial invasion correlates with more undifferentiated tumors, lymph-vascular invasion, node involvement and decreased global survival. We have described previously the Ets family member ERM/ETV5 specifically upregulated in endometrial endometrioid carcinoma (EEC) associated with myometrial infiltration. To understand the role of this transcription factor during myometrial infiltration, we analyzed by two-dimension differential gel electrophoresis (2D-DIGE) technology those proteins whose expression was altered in endometrial cell lines stably overexpressing ERM/ETV5. Pathway analysis pointed to actin regulation and transforming growth factor beta and progesterone signaling as processes regulated by ERM/ETV5. In addition, we characterized the specific upregulation of the nuclear dehydrogenase/reductase Hep27 as well as its ERM/ETV5-dependent mitochondrial localization. Further functional studies demonstrated a protective role of Hep 27 against apoptosis induced by oxidative stress. Overall, the ETV5-related proteomic approach performed in the Hec-1A cell line reinforces a role of this transcription factor in the regulation of the migratory and invasive tumor behavior and points to a modulated response to oxidative stress associated with the promotion of invasion in endometrial cancer. Unraveling the molecular events in EEC associated with the initiation of tumor invasion would represent an obvious improvement in the pursuit of rational targets for the onset of metastasis. This knowledge would also be a valuable tool for the molecular stratification of patients since myometrial affectation determines an increase in the rate of recurrence after a first surgical treatment and a decrease in 5 year survival. [ABSTRACT FROM AUTHOR]

To explore the proteins regulated by cyclooxygenase-2 (COX-2) in gastric cancer, the expression plasmid of COX-2siRNA was constructed and transfected into gastric cancer cell line SGC7901. Then, two-dimensional electrophoresis and the PDQuest software analysis were applied to discover the differentially expressed proteins. The differential protein spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Fourteen differentially expressed proteins between the two cell lines were identified. 15-Hydroxyprostaglandin dehydrogenase [NAD+] (15-PGDH), a key enzyme in prostaglandin degradation, was identified as an upregulated protein in SGC7901 cells transfected with the COX-2siRNA plasmid. To further explore whether the 15-PGDH is regulated by COX-2, western blotting and immunocytochemical assay were performed to detect the expression of 15-PGDH in different cell lines with different expression level of COX-2. The results showed that the expression of 15-PGDH was upregulated (128.57%) as COX-2 was suppressed by small interfering RNA and downregulated (51.72%) as COX-2 was enhanced by COX-2 cDNA transfection in gastric cancer cells. In tissue specimens with gastric cancer, there was a decreased expression of 15-PGDH and an increased expression of COX-2 simultaneously. A significantly negative correlation of 15-PGDH expression was found to COX-2 level, tumor differentiation, tumor, lymph node, metastasis (TNM) staging and lymph node metastasis of gastric cancer. All the results suggest that 15-PGDH is downregulated by COX-2 in human gastric cancer and may contribute to the carcinogenesis and development of human gastric cancer in combination with COX-2. [ABSTRACT FROM AUTHOR]