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Over the last two decades, a large number of non-communicable/chronic disorders reached an epidemic level on a global scale such as diabetes mellitus type 2, cardio-vascular disease, several types of malignancies, neurological and eye pathologies—all exerted system’s enormous socio-economic burden to primary, secondary, and tertiary healthcare. The paradigm change from reactive to predictive, preventive, and personalized medicine (3PM/PPPM) has been declared as an essential transformation of the overall healthcare approach to benefit the patient and society at large. To this end, specific biomarker panels are instrumental for a cost-effective predictive approach of individualized prevention and treatments tailored to the person. The source of biomarkers is crucial for specificity and reliability of diagnostic tests and treatment targets. Furthermore, any diagnostic approach preferentially should be noninvasive to increase availability of the biomaterial, and to decrease risks of potential complications as well as concomitant costs. These requirements are clearly fulfilled by tear fluid, which represents a precious source of biomarker panels. The well-justified principle of a “sick eye in a sick body” makes comprehensive tear fluid biomarker profiling highly relevant not only for diagnostics of eye pathologies but also for prediction, prognosis, and treatment monitoring of systemic diseases. One prominent example is the Sicca syndrome linked to a cascade of severe complications that include dry eye, neurologic, and oncologic diseases. In this review, protein profiles in tear fluid are highlighted and corresponding biomarkers are exemplified for several relevant pathologies, including dry eye disease, diabetic retinopathy, cancers, and neurological disorders. Corresponding analytical approaches such as sample pre-processing, differential proteomics, electrophoretic techniques, high-performance liquid chromatography (HPLC), enzyme-linked immuno-sorbent assay (ELISA), microarrays, and mass spectrometry (MS) methodology are detailed. Consequently, we proposed the overall strategies based on the tear fluid biomarkers application for 3P medicine practice. In the context of 3P medicine, tear fluid analytical pathways are considered to predict disease development, to target preventive measures, and to create treatment algorithms tailored to individual patient profiles.

Introduction Infective endocarditis (IE) has high mortality, partly due to delayed diagnosis. No biomarker can identify IE in patients with fever and clinical picture of infection. To find putative biomarkers we analyzed serum levels of two proteins found in cardiac valves, fibulin-1 (n = 696) and osteoprotegerin (n = 689) among patients on clinical suspicion of IE. Proteomic analyses were performed in 24 patients with bacteremia, 12 patients with definite IE and 12 patients with excluded IE. Methods Fibulin-1 and osteoprotegerin were studied by enzyme linked immunosorbent assay (ELISA). Proteomic analyses were conducted by 2-dimensional polyacrylamid gel electrophoresis (2D-PAGE) and label-free quantitative liquid chromatography - tandem mass spectrometry (LFQ LC-MS/MS). Controls for 2D 2D-PAGE and LFQ LC-MS/MS had bacteremia and excluded IE. Results Osteoprotegerin levels were significantly increased in IE patients compared with non-IE patients. Fibulin-1 showed no difference. 2D-PAGE showed significant differences of 6 proteoforms: haptoglobin, haptoglobin-related protein, α-2-macroglobulin, apolipoprotein A-I and ficolin-3. LFQ LC-MS/MS analysis revealed significant level changes of 7 proteins: apolipoprotein L1, complement C1q subcomponent B and C, leukocyte immunoglobulin-like receptor subfamily A member 3, neuropilin-2, multimerin-1 and adiponectin. Conclusions The concentration changes in a set of proteoforms/proteins suggest that stress and inflammation responses are perturbed in patients with IE compared to patients with bacteremia without IE.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide; it takes part in the regulation of various physiological processes, such as feeding, reproduction, catecholamine synthesis, thermoregulation, motor activity, brain development and neuronal survival. Since PACAP plays important regulatory roles, we hypothesized that the level of PACAP in blood is associated with expression of other proteins, which are involved in different metabolic pathways. The objective of the present study was to compare plasma protein profiles of cows with high and low plasma PACAP levels. Differential proteome analyses were performed by two-dimensional gel electrophoresis (2D-PAGE) followed by tryptic digestion and protein identification by liquid chromatography–mass spectrometry (LC-MS). A total of 210 protein spots were detected, and 16 protein spots showed statistically significant differences (p < 0.05) in the expression levels between groups. Ten spots showed a higher intensity in the high-PACAP-concentration group, while six spots were more abundant in the low-PACAP-concentration group. The functions of the differentially expressed proteins indicate that the PACAP level of plasma is related to the lipid metabolism and immune status of cattle.

Proteomics has been widely used to study muscle biology and meat quality traits from different species including beef. Beef proteomics studies allow a better understanding of the biological processes related to meat quality trait determination. This study aimed to decipher by means of two-dimensional electrophoresis (2D-PAGE), mass spectrometry and bioinformatics the changes in post-mortem muscle with a focus on proteins differentially expressed in the Longissimus thoracis (LT) muscle of immunocastrated young heifers and steers. Carcass traits, chemical composition, pH, instrumental color (L*, a*, b*), cooking loss and Warner-Bratzler shear force (WBSF) of meat from F1 Montana-Nellore cattle were also evaluated. Backfat thickness (BFT) and intramuscular fat content (IMF) were 46.8% and 63.6% higher in heifers (p < 0.05), respectively, while evaporation losses (EL) were 10.22% lower compared to steers. No differences (p > 0.05) were observed for tenderness evaluated by WBSF (3, 10, and 17 days post-mortem), pH, and color traits (L*, a* and b*) between the experimental groups. The study revealed several proteins to be differentially expressed proteins in heifers compared steers (p < 0.05). In heifers, proteins involved in nutrient transport (TF, ALB, and MB), energy metabolism (ALDOA, GAPDH, and PKM), and oxidative stress and response to stress (HSPA8 and CA3) were associated with a greater BFT and IMF deposition. The higher expression of these proteins indicated greater oxidative capacity and lower glycolytic activity in the LT muscle of heifers. In steers, there was greater abundance of protein expression related to muscle contraction and proteins of structure (ACTA1, TPM2 and TNNT3), energy metabolism (ENO1, ENO3, PYGM, PGM1 and TPI1) and ATP metabolism (ATP5F1B, PEBP1 and AK1), indicating greater glycogenolysis in LT muscle, suggesting a shift in the glycolytic/oxidative fibers of steers.

Made available in DSpace on 2019-10-06T16:27:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-06-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Protocols that improve growth performance in fish while assuring product quality are important for aquaculture. Fasting followed by refeeding may promote compensatory growth, thus optimizing growth performance. During fasting and refeeding, fast-twitch muscle, which comprises most of fish fillet, undergoes intense plasticity. In this work, we studied the proteome of pacu (Piaractus mesopotamicus)fast-twitch muscle after 30 days of fasting (D30), 30 days of refeeding (D60)and 60 days of refeeding (D90)with two-dimensional electrophoresis, mass spectrometry and bioinformatics. Body mass, growth rate and muscle histology were also assessed. At D30, fish presented muscle catabolism and decreased growth. Proteomic analysis showed that metabolism proteins were the most affected, up and downregulated. Cytoskeleton and amino acid biosynthesis proteins were downregulated, while nuclear and regulatory proteins were upregulated. At D60, fish showed accelerated growth, despite the body mass not completely recovering. Metabolism proteins were still the most affected. Amino acid biosynthesis proteins became upregulated, while cytoskeleton proteins remained downregulated. At D90, the fish presented total compensatory growth. Many metabolic proteins were up or downregulated. Few cytoskeleton proteins remained differentially expressed. Amino acid biosynthesis proteins were mostly upregulated, but less than at D60. Prolonged fasting followed by refeeding also led to the regulation of possible meat quality biomarkers, such as antioxidant enzymes. This fact suggests possible consequences of this protocol on fish meat quality. Our work also enriches our knowledge on proteomic changes during muscle plasticity that occur during fasting and refeeding diet protocols. Department of Morphology Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Chemistry and Biochemistry Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Biology University of Western São Paulo (UNOESTE) São Paulo Agency for Agribusiness Technology (APTA) Department of Morphology Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Chemistry and Biochemistry Institute of Biosciences of Botucatu São Paulo State University (UNESP) CNPq: 116795/2016 FAPESP: 2017/12237-6 CNPq: 302656/2015-4 CNPq: 447233/2014

Background The Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer’s disease. Methods The concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL. Results We report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker. Conclusions This demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.

To study the mechanism of heat-induced protein aggregates, skim milk was heated at 55, 65, 75, 85, and 95°C for 30 s. Then, the sulfhydryl content, surface hydrophobicity, and secondary structure of heat-treated skim milk were studied. Treating skim milk at different temperatures induced a decrease in sulfhydryl content (75.9% at 95°C) and an increase in surface hydrophobicity (44% at 95°C) with a disrupted secondary structure containing random coil, β-sheet, and β-turn of skim milk proteins. The change in these properties facilitated aggregate formation through disulfide bonds and hydrophobicity interaction. Microstructural observation also showed a higher degree of aggregation when skim milk was heated at 85 and 95°C. The result of two-dimensional polyacrylamide gel electrophoresis demonstrated that the aggregates consisted of a high proportion of κ-casein, β-lactoglobulin, and other whey proteins.

Recent weather fluctuations imposing heat stress at the time of wheat grain filling cause frequent losses in grain yield and quality. Field-based studies for understanding the effect of terminal heat stress on wheat are complicated by the effect of multiple confounding variables. In the present study, the effect of day and day–night combined heat stresses during the grain-filling stage was studied using gene expression and proteomics approaches. The gene expression analysis was performed by using real-time quantitative PCR (RT-qPCR). The expression of genes related to the starch biosynthetic pathway, starch transporters, transcription factors, and stress-responsive and storage proteins, at four different grain developmental stages, indicated the involvement of multiple pathways. Under the controlled conditions, their expression was observed until 28 days after anthesis (DAA). However, under the day stress and day–night stress, the expression of genes was initiated earlier and was observed until 14 DAA and 7 DAA, respectively. The protein profiles generated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS/MS) showed a differential expression of the proteins belonging to multiple pathways that included the upregulation of proteins related to the translation, gliadins, and low-molecular-weight (LMW) glutenins and the downregulation of proteins related to the glycolysis, photosynthesis, defense, and high-molecular-weight (HMW) glutenins. Overall, the defense response to the day heat stress caused early gene expression and day–night heat stress caused suppression of gene expression by activating multiple pathways, which ultimately led to the reduction in grain-filling duration, grain weight, yield, and processing quality.

In previous work, two independent Arabidopsis thaliana Ac/Ds transposon insertional mutant lines, atnp01 and atnp02, were identified that showed a higher level of tolerance than the wild type (wt) line to cadmium sulfide quantum dots (CdS QDs). The tolerance response was characterized at physiological, genetic and transcriptomic levels. In this work, a comparative analysis was performed on protein extracts from plantlets of the two mutants and of wt, each treated with 80 mg L-1 CdS QDs. A comparative protein analysis was performed by 2D-PAGE, and proteins were characterized by MALDI-TOF/TOF mass spectrometry. Of 250 proteins identified from all three lines, 98 showed significant changes in relative abundance between control and CdS QD-treated plantlets. The wt, atnp01, and atnp02 control-treated pairs respectively showed 61, 31, and 31 proteins with differential expression. The two mutants had a different response to treatment in terms of type and quantity of up- and downregulated proteins. This difference became more striking when compared to wt. A network analysis of the proteins differentially expressed in atnp01 and atnp02 included several of those encoded by putative genes accommodating the transposons, which were responsible for regulation of some proteins identified in this study. These included nifu-like protein 3 (Nfu3), involved in chloroplast assembly, elongator complex 3 (Elo3), involved in transcriptional elongation, magnesium-chelate subunit-2 (Chli2), involved in chlorophyll biosynthesis, and protein phosphatase 2C (PP2C) which mediates abiotic stress response.

Submitted by Mariane De Freitas Genari Severino (mariane.genari@unesp.br) on 2021-04-22T00:27:36Z No. of bitstreams: 1 Dissertação_Severino_Mariane.pdf: 616930 bytes, checksum: 150dc8f3a26c5672875832093238f033 (MD5) Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2021-04-22T13:57:53Z (GMT) No. of bitstreams: 1 severino_mfg_me_jabo_par.pdf: 509427 bytes, checksum: 5a7fc5aa080efe44584adf013f51cc9b (MD5) Made available in DSpace on 2021-04-22T13:57:54Z (GMT). No. of bitstreams: 1 severino_mfg_me_jabo_par.pdf: 509427 bytes, checksum: 5a7fc5aa080efe44584adf013f51cc9b (MD5) Previous issue date: 2021-02-26 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) RESUMO - Objetivou-se com esta pesquisa identificar proteínas diferencialmente expressas no músculo Longissimus thoracis et lumborum (LT) e sua associação com características físico-químicas encontradas da carne maturada de novilhas versus novilhos F1 Nelore-Montana, superprecoces e terminados em confinamento. Foram avaliadas características da carcaça (peso de carcaça quente, rendimento e espessura de gordura subcutânea), composição química (teor de umidade, gordura intramuscular, proteína, colágeno total) e características de qualidade da carne como força de cisalhamento (FC), perdas por cocção, coloração instrumental (L*; a*; b*) e pH, considerando-se três tempos de maturação (3, 10, 17 dias postmortem). Amostras individuais do músculo LT foram coletadas pré rigor mortis para estudo do proteoma utilizando eletroforese bidimensional (2D-PAGE) e espectrometria de massa por ionização por eletrospray (ESI-MS). Interações entre a classe sexual e o tempo de maturação foram analisadas sobre as características de qualidade da carne. Adicionalmente, estudos de imagem dos géis 2D-PAGE foram avaliados, sendo selecionados “spots” proteicos diferencialmente expressos (P0,05) sobre as demais características da carne (FC, L*, a*, b* e pH) entre as classes sexuais. No estudo do proteoma muscular, vinte e três proteínas foram identificadas entre os grupos biológicos (P0.05) regarding to other characteristics of the meat (SF, L *, a *, b * and pH) between sexual classes. In the study of muscle proteome, twenty-three proteins were identified between biological groups (P

Proteinopathy refers to a group of disorders defined by depositions of amyloids within living tissue. Neurodegenerative proteinopathies, including Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and others, constitute a large fraction of these disorders. Amyloids are highly insoluble, ordered, stable, beta-sheet rich proteins. The emerging theory about the pathophysiology of neurodegenerative proteinopathies suggests that the primary amyloid-forming proteins, also known as the prion-like proteins, may exist as multiple proteoforms that contribute differentially towards the disease prognosis. It is therefore necessary to resolve these disorders on the level of proteoforms rather than the proteome. The transient and hydrophobic nature of amyloid-forming proteins and the minor post-translational alterations that lead to the formation of proteoforms require the use of highly sensitive and specialized techniques. Several conventional techniques, like gel electrophoresis and conventional mass spectrometry, have been modified to accommodate the proteoform theory and prion-like proteins. Several new ones, like imaging mass spectrometry, have also emerged. This review aims to discuss the proteoform theory of neurodegenerative disorders along with the utility of these proteomic techniques for the study of highly insoluble proteins and their associated proteoforms.

International audience; Two-dimensional gel electrophoresis has been instrumental in the development of proteomics. Although it is no longer the exclusive scheme used for proteomics, its unique features make it a still highly valuable tool, especially when multiple quantitative comparisons of samples must be made, and even for large samples series. However, quantitative proteomics using two-dimensional gels is critically dependent on the performances of the protein detection methods used after the electrophoretic separations. This chapter therefore examines critically the various detection methods, (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of two-dimensional gels is performed.

Background Maize is one of the important cereal food crops in the world. High temperature stress causes adverse influence on plant growth. When plants are exposed to high temperatures, they produce heat shock proteins (HSPs), which may impart a generalized role in tolerance to heat stress. Proteome analysis was performed in plant to assess the changes in protein types and their expression levels under abiotic stress. The purpose of the study is to explore which proteins are involved in the response of the maize plant to heat shock treatment. Results We investigated the responses of abundant proteins of maize leaves, in an Egyptian inbred line of maize “K1”, upon heat stress through two-dimensional electrophoresis (2-DE) on samples of maize leaf proteome. 2-DE technique was used to recognize heat-responsive protein spots using Coomassie Brilliant Blue (CBB) and silver staining. In 2-D analysis of proteins from plants treated at 45 °C for 2 h, the results manifested 59 protein spots (4.3%) which were reproducibly detected as new spots where did not present in the control. In 2D for treated plants for 4 h, 104 protein spots (7.7%) were expressed only under heat stress. Quantification of spot intensities derived from heat treatment showed that twenty protein spots revealed clear differences between the control and the two heat treatments. Nine spots appeared with more intensity after heat treatments than the control, while four spots appeared only after heat treatments. Five spots were clearly induced after heat treatment either at 2 h or 4 h and were chosen for more analysis by LC-MSMS. They were identified as ATPase beta subunit, HSP26, HSP16.9, and unknown HSP/Chaperonin. Conclusion The results revealed that the expressive level of the four heat shock proteins that were detected in this study plays important roles to avoid heat stress in maize plants.

The accurate quantification of changes in the abundance of proteins is one of the main applications of proteomics. The maintenance of accuracy can be affected by bias and error that can occur at many points in the experimental process, and normalization strategies are crucial to attempt to overcome this bias and return the sample to its regular biological condition, or normal state. Much work has been published on performing normalization on data post-acquisition with many algorithms and statistical processes available. However, there are many other sources of bias that can occur during experimental design and sample handling that are currently unaddressed. This article aims to cast light on the potential sources of bias and where normalization could be applied to return the sample to its normal state. Throughout we suggest solutions where possible but, in some cases, solutions are not available. Thus, we see this article as a starting point for discussion of the definition of and the issues surrounding the concept of normalization as it applies to the proteomic analysis of biological samples. Specifically, we discuss a wide range of different normalization techniques that can occur at each stage of the sample preparation and analysis process.

Made available in DSpace on 2019-10-06T15:49:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) High concentrations of mercury found in soils, sediments, fish, and humans of the Amazon region have gained prominence in scientific studies during the last decade. However, studies related to the elucidation of mercury toxicity mechanisms in ichthyofauna at the molecular and metallomic levels that seek to elucidate physiological and functional aspects, as well as the search for biomarkers of mercury exposure, are still sparse. In the search for these answers, the present study analyzed the hepatic tissue proteome of the Arapaima gigas (pirarucu) fish species collected in the Jirau hydroelectric power plant reservoir in the state of Rondônia state, Brazil, in order to identify mercury-related metal-binding proteins and to elucidate their physiological and functional aspects. The proteomic profile of the hepatic tissue of Arapaima gigas was obtained by two-dimensional electrophoresis (2D-PAGE) and the presence of mercury was mapped in the protein SPOTS by graphite furnace atomic absorption spectrometry(GFAAS). Mercury was detected in 18 protein SPOTS with concentrations ranging from 0.13 ± 0.003 to 131.00 ± 3 mg kg−1. The characterization of the protein SPOTS associated with mercury was performed by electrospray ionisation tandem mass spectrometry (ESI–MS/MS), and 10 proteins were identified. Bioinformatics analyses showed that most of the proteins found linked to mercury were involved in cellular component processes and biological processes. For the most part, protein sequences have cellular functions comprising catalytic, binding, sense of localization, and metabolic processes. São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Institute of Biosciences University of São Paulo (USP) Institute of Chemistry (Inqui) Federal University of Mato Grosso do Sul, Brazil (UFMS) University of Nebraska (UNL) University of Brasília (UNB) College of Planaltina São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Institute of Biosciences FAPESP: 2016/19404-2 FAPESP: 2017/09466-3 CNPq: 304768/2018-19 CNPq: 404485/2016-2

The impact of gender and diet on the proteome of Longissimus dorsi was addressed by 2D-PAGE analysis of male and female pigs, fed with a barley-based control diet and a diet enriched with extruded linseed and plant extracts. No statistically significant difference in protein number between female and male samples was found. Furthermore, PCA excluded gender-dependent protein clusters. For both the control and enriched diet, several spots exhibited at least a 1.5-fold intensity difference, but none showed a statistically relevant variation. Protein profiles PCA for both diets indicated that the first two principal components account up to 47% of total variance, with two diet-dependent separated clusters. Among 176 common spots, 29 exhibited >1.5 fold change, mostly more abundant in the control diet. PMF identified 14 distinct proteins, including myofibrillar proteins, glycolytic enzymes and myoglobin, thus suggesting a diet-dependent meat quality. A statistically significant increase in carbonylated proteins of enriched diet samples was detected using the 2,4-dinitrophenylhydrazine method but not using fluorescein-5-thiosemicarbazide-labeled bands. ROS induction and DNA oxidative damage, detected in a human cell line exposed to digested meat from both diets, further support the notion that the enriched diet does not protect against oxidative stress. SIGNIFICANCE: The comparison of the protein profile of female and male Longissimus dorsi from pigs fed by a control diet and a diet enriched with polyphenols, indicate no gender effect, whereas diet affects the abundance of several proteins, possibly linked to meat quality. Protein carbonylation was statistically higher in meat from the enriched diet, suggesting that polyphenols at the concentration present in the diet did not exert a protective effect against oxidation.

A proteomic method combining two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF) was used to compare the protein expression profiles of phosphine-resistant (PH3-R) and -susceptible (PH3-S) strains of Sitophilus oryzae. Thirty-nine differentially expressed protein spots were identified between the PH3-R and PH3-S strains; 20 protein spots were upregulated, and 19 protein spots were downregulated in the PH3-R strain compared with their expression in the PH3-S strain. In particular, cytochrome oxidase subunit I showed 15-fold higher expression in the PH3-R strain than in the PH3-S strain. Additionally, citrate synthase 2, delta-1-pyrolline-5-carboxylate dehydrogenase, and triose-phosphate isomerase were highly expressed in the PH3-R strain. In summary, our study has improved understanding of the molecular mechanisms of phosphine resistance in the rice weevil.

Increasing evidence indicates that tumor-derived endothelial cells (TECs) are more relevant for the study of tumor angiogenesis and for screening antiangiogenic drugs than normal ECs (NECs). In this data article, high-purity (>98%) primary CD105+ NECs and TECs purified from a mouse Lewis lung carcinoma model bearing 0.5 cm tumors were identified using 2D-PAGE and Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). All the identified proteins were categorized functionally by Gene Ontology (GO) analysis, and gene-pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, protein–protein interaction networks were also built. The proteomics and bioinformatics data presented here provide novel insights into the molecular characteristics and the early modulation of the TEC proteome in the tumor microenvironment. Keywords: Tumor-derived endothelial cells, Lung cancer, 2D-PAGE, MALDI-TOF, MS/MS

Scorpions belonging to the Tityus genus are of medical interest in Brazil. Among them, Tityus stigmurus is the main scorpion responsible for stings in the Northeast region. After a sting, the scorpion venom distributes rapidly to the organs, reaching the kidneys quickly. However, there are few studies concerning the renal pathophysiology of scorpion poisoning. In this study, we evaluated the effects of T. stigmurus venom (TsV) on renal parameters in isolated rat kidneys. Wistar rats (n = 6), weighing 250-300 g, were perfused with Krebs-Henseleit solution containing 6 g/100 mL bovine serum albumin. TsV at 0.3 and 1.0 μg/mL was tested, and the effects on perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and electrolyte excretion were analyzed. Effects were observed only at TsV concentration of 1.0 μg/mL, which increased PP (controlPP40' = 92.7 ± 1.95; TsVPP40' = 182.0 ± 4.70* mmHg, *p < 0.05), RVR (controlRVR40' = 3.28 ± 0.23 mmHg; TstRVR40' = 6.76 ± 0.45* mmHg, *p < 0.05), UF (controlUF50' = 0.16 ± 0.04; TstUF50' = 0.60 ± 0.10* mL/g/min,*p < 0.05), GFR and electrolyte excretion, with histological changes that indicate renal tubular injury. In conclusion, T. stigmurus venom induces a transient increase in PP with tubular injury, both of which lead to an augmented electrolyte excretion. RESUMO Os escorpiões de interesse médico no Brasil pertencem ao gênero Tityus. Dentre eles, Tityus stigmurus é o principal responsável por picadas de escorpião na região Nordeste. Após uma picada, a peçonha de escorpião se distribui rapidamente para os órgãos, atingindo os rins depressa. Entretanto, há poucos trabalhos sobre fisiopatologia renal do envenenamento por escorpiões. Neste trabalho, avaliamos os efeitos da peçonha de T. stigmurus (TsV) sobre parâmetros renais em rins de ratos isolados. Ratos Wistar (n = 6), pesando 250-300 g, foram perfundidos com solução de Krebs-Henseleit contendo 6 g/100 mL de albumina de soro bovino. TsV nas concentrações de 0,3 e 1,0 μg/mL foi testado e os efeitos sobre a pressão de perfusão (PP), resistência vascular renal (RVR), fluxo urinário (FU), taxa de filtração glomerular (TFG) e excreção de eletrólitos foram analisados. Apenas na concentração de 1,0 μg/mL de TsV foram observados efeitos que aumentaram PP (controlePP40' = 92,7 ± 1,95; TsVPP40' = 182,0 ± 4,70 mmHg*, *p < 0,05), RVR (controleRVR40' = 3,28 ± 0,23 mmHg; TstRVR40' = 6,76 ± 0,45 mmHg*, *p < 0,05), FU (controleFU50' = 0,16 ± 0,04; TstFU50' = 0,60 ± 0,10 mL/g/min*, *p < 0,05), a TFG e excreção de eletrólitos, com alterações histológicas que indicam de lesão renal tubular. Em conclusão, peçonha de T. stigmurus induz um aumento transiente da PP com lesão tubular, ambos os quais conduzem a uma excreção elevada de eletrólitos.

Amphoteric azo dyes were used for internal control of pI values in Comparative two-dimensional Fluorescence Gel Electrophoresis (CoFGE) [1]. The 2D-gel images of separated Escherichia coli proteins as well as those of colored amphoteric dyes separated by isoelectric focussing are presented. The latter were used to correct for variation in the first electrophoretic dimension and further improve protein coordinate assignment in 2D-gel electrophoresis. Data tables are supplied to demonstrate pI-value calibration and the effect on the assignment of protein spot coordinates.

Submitted by Jéssica Moraes Malheiros (jessicamalheiros@yahoo.com.br) on 2018-05-03T18:53:41Z No. of bitstreams: 1 TESE MALHEIROS, J. M..pdf: 1363679 bytes, checksum: 33291bc89815d196826e037eeda5b237 (MD5) Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-05-04T18:11:25Z (GMT) No. of bitstreams: 1 malheiros_jm_dr_jabo.pdf: 1400936 bytes, checksum: bf9a223495b522a13300edf9c4026a2e (MD5) Made available in DSpace on 2018-05-04T18:11:25Z (GMT). No. of bitstreams: 1 malheiros_jm_dr_jabo.pdf: 1400936 bytes, checksum: bf9a223495b522a13300edf9c4026a2e (MD5) Previous issue date: 2018-03-02 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) O presente trabalho teve como objetivo avaliar a associação da expressão gênica e proteômica com a maciez da carne de bovinos da raça Nelore. A partir de uma população de 90 animais foram selecionados três grupos experimentais por meio da análise de força de cisalhamento (FC) e índice de fragmentação miofibrilar (MFI), sendo: carne moderadamente macia, carne moderadamente dura e carne muito dura. A expressão dos genes foi avaliada por meio da análise de PCR em tempo real e a análise proteômica foi realizada com base na separação de proteínas por meio da eletroforese bidimensional (2D-PAGE) e caracterizção por espectrometria de massas com ionização eletrospray (ESI-MS/MS). A expressão da isoforma da calpastatina (CAST2) mostrou-se up regulated (P0,05) no mesmo grupo experimental. Estes resultados fornecem uma visão importante de novos possíveis marcadores biológicos atuantes no processo de amaciamento da carne, o que pode colaborar para melhor entender e gerar novas estratégias de seleção nos programas de melhoramento genético de bovinos Nelore. The objective of this study was to evaluate the association of gene expression and proteomics with meat tenderness in Nellore cattle. From population of 90 animals three experimental groups were selected by shear force (SF) and/or myofibrillar fragmentation index (MFI): moderately tender meat, moderately tough meat and very tough meat. Gene expression was evaluated by real-time PCR and proteomics analysis was performed based on protein separation by two-dimensional gel electrophoresis (2D-PAGE) and characterisation by eletrospray ionisation mass spectrometry (ESI-MS/MS). Expression of the calpastatin isoform (CAST2) was up-regulated (P0.05) in the same experimental group. The present results suggest an important view of possible new biological markers in the meat tenderization process, wich permit to unsderstand and generate new strategies for selection in Nellore cattle breeding programs. FAPESP: 15/13021-1

The vascular wilt of tomato caused by Fusarium oxysporum f.sp. lycopersici is an important soil borne pathogen causes severe yield loss. The molecular characterization and their interaction with its host is necessary to develop a protection strategy. 20 isolates of F. oxysporum f.sp. lycopersici (FOL) were isolated from wilt infected tomato plants across Tamil Nadu. They were subjected to cultural, morphological, molecular and virulence studies. The results revealed that all the isolates produced both micro and macro conidia with different size, number of cells. The colors of the culture and growth pattern were also varied. In addition, chlamydospores were observed terminally and intercalary. The PCR analysis with F. oxysporum species-specific primer significantly amplified an amplicon of 600 bp fragment in all the isolates. Based on the above characters and pathogenicity, isolate FOL-8 was considered as virulent and FOL-20 was considered as least virulent. Proteomics strategy was adopted to determine the virulence factors between the isolates of FOL-8 and FOL-20. The 2D analyses have showed the differential expression of 17 different proteins. Among them, three proteins were down regulated and 14 proteins were significantly up regulated in FOL-8 than FOL-20 isolate. Among the 17 proteins, 10 distinct spots were analyzed by MALDI-TOF. The functions of the analyzed proteins, suggested that they were involved in pathogenicity, symptom expression and disease development, sporulation, growth, and higher penetration rate on tomato root tissue. Overall, these experiments proves the role of proteome in pathogenicity of F. oxysporum f.sp. lycopersici in tomato and unravels the mechanism behinds the virulence of the pathogen in causing wilt disease.

Made available in DSpace on 2018-12-11T16:47:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-02-01 Mercury is a potentially toxic element that is present in the environment of the Brazilian Amazon and is responsible for adverse health effects in humans. This study sought to assess possible protein biomarkers of mercury exposure in breast milk samples from lactating women in the Madeira and Negro Rivers in the Brazilian Amazon. The mercury content of hair samples of lactating women was determined, and the proteome of breast milk samples was obtained using two-dimensional electrophoresis after protein precipitation with acetone. Mercury measurements of protein spots obtained via protein fractionation were performed by graphite furnace atomic absorption spectrometry (GFAAS), and it was observed that mercury is linked to proteins with molecular masses in the range of 14–26 kDa. The total mercury concentration was also determined by GFAAS in unprocessed milk, lyophilized milk, and protein pellets, with the purpose of determining the mercury mass balance in relation to the concentration of this element in milk and pellets. Approximately 85 to 97% of mercury present in the lyophilized milk from samples of lactating women of the Madeira River is bound in the protein fraction. From lactating women of the Negro River, approximately 49% of the total mercury is bound in the protein fraction, and a difference of 51% is bound in the lipid fraction. Pontifical Catholic University of Goiás (PUC) Institute of Biosciences São Paulo State University (UNESP) São Paulo State University (UNESP) College of Planaltina University of Brasília (UnB) Institute of Biosciences São Paulo State University (UNESP) São Paulo State University (UNESP)

Venomous animals use venom, a complex biofluid composed of unique mixtures of proteins and peptides, to act on vital systems of the prey or predator. In bees, venom is solely used for defense against predators. However, the venom composition of bumble bees ( Bombus sp.) is largely unknown. The Thoracobombus subgenus of Bombus sp. is a diverse subgenus represented by 14 members across Turkey. In this study, we sought out to proteomically characterize the venom of five Thoracobombus species by using bottom-up proteomic techniques. We have obtained two-dimensional polyacrylamide gel (2D-PAGE) images of each species' venom sample. We have subsequently identified the protein spots by using matrix assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS). We have identified 47 proteins for Bombus humilis , 32 for B. pascuorum , 60 for B. ruderarius , 39 for B. sylvarum , and 35 for B. zonatus . Moreover, we illustrated that intensities of 2DE protein spots corresponding to putative venom toxins vary in a species-specific manner. Our analyses provide the primary proteomic characterization of five bumble bee species' venom composition.

Made available in DSpace on 2018-12-11T16:48:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-01 This study presents data on the extraction and characterization of proteins associated with mercury in the muscle and liver tissues of jaraqui (Semaprochilodus spp.) from the Madeira River in the Brazilian Amazon. Protein fractionation was carried out by two-dimensional electrophoresis (2D-PAGE). Mercury determination in tissues, pellets, and protein spots was performed by graphite furnace atomic absorption spectrometry (GFAAS). Proteins in the spots that showed mercury were characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The highest mercury concentrations were found in liver tissues and pellets (426 ± 6 and 277 ± 4 μg kg−1), followed by muscle tissues and pellets (132 ± 4 and 86 ± 1 μg kg−1, respectively). Mercury quantification in the protein spots allowed us to propose stoichiometric ratios in the range of 1–4 mercury atoms per molecule of protein in the protein spots. The proteins characterized in the analysis by ESI-MS/MS were keratin, type II cytoskeletal 8, parvalbumin beta, parvalbumin-2, ubiquitin-40S ribosomal S27a, 39S ribosomal protein L36 mitochondrial, hemoglobin subunit beta, and hemoglobin subunit beta-A/B. The results suggest that proteins such as ubiquitin-40S ribosomal protein S27a, which have specific domains, possibly zinc finger, can be used as biomarkers of mercury, whereas mercury and zinc present characteristics of soft acids. Institute of Bioscience of Botucatu São Paulo State University (UNESP), Rubião Júnior Pontifical Catholic University of Goiás (PUC) Bauru Dental School University of São Paulo-USP Institute of Bioscience of Botucatu São Paulo State University (UNESP), Rubião Júnior

Previously, we identified protein spots with differential intensity between normal and tumor tissues of renal cell carcinoma (RCC) using 2D-DIGE. Here, we further examined two proteins, raf-1 kinase inhibitory protein (RKIP) and manganese superoxide dismutase (MnSOD), identified in one protein spot. Western blotting demonstrated that RKIP and MnSOD exhibited opposite expression patterns in normal and tumor tissues. Immunohistochemisry showed that MnSOD level significantly correlated with shorter progression-free survival. Gene-silencing assay demonstrated that RKIP and MnSOD had suppressive and promotive effects on tumor cell proliferation and invasion, respectively. Our findings reveal biological and clinical significance of RKIP and MnSOD in RCC.

The application of combined nitrogen, especially nitrate, to soybean plants is known to strongly inhibit nodule formation, growth and nitrogen fixation. In the present study, we measured the effects of supplying 5 mM nitrate on the growth of nodules, primary root, and lateral roots under light at 28 °C or dark at 18 °C conditions. Photographs of the nodulated roots were periodically taken by a digital camera at 1-h intervals, and the size of the nodules was measured with newly developed computer software. Nodule growth was depressed approximately 7 h after the addition of nitrate under light conditions. The nodule growth rate under dark conditions was almost half that under light conditions, and nodule growth was further suppressed by the addition of 5 mM nitrate. Similar results were observed for the extending growth rate of the primary root as those for nodule growth supplied with 5 mM nitrate under light/dark conditions. In contrast, the growth of lateral roots was promoted by the addition of 5 mM nitrate. The 2D-PAGE profiles of nodule protein showed similar patterns between the 0 and 5 mM nitrate treatments, which suggested that metabolic integrity may be maintained with the 5 mM nitrate treatment. Further studies are required to confirm whether light or temperature condition may give the primary effect on the growth of nodules and roots.

It is becoming increasingly evident that the RNA degradome is a crucial component of the total cellular RNA pool. Here, we present an analysis of the medium-sized RNAs (midi RNAs) that form in Arabidopsis thaliana. Our analyses revealed that the midi RNA fraction contained mostly 20–70-nt-long fragments derived from various RNA species, including tRNA, rRNA, mRNA and snRNA. The majority of these fragments could be classified as stable RNA degradation intermediates (RNA degradants). Using two dimensional polyacrylamide gel electrophoresis, we demonstrated that high copy number RNA (hcn RNA) degradants appear in plant cells not only during stress, as it was earlier suggested. They are continuously produced also under physiological conditions. The data collected indicated that the accumulation pattern of the hcn RNA degradants is organ-specific and can be affected by various endogenous and exogenous factors. In addition, we demonstrated that selected degradants efficiently inhibit translation in vitro. Thus, the results of our studies suggest that hcn RNA degradants are likely to be involved in the regulation of gene expression in plants. Electronic supplementary material The online version of this article (doi:10.1007/s11103-013-0079-3) contains supplementary material, which is available to authorized users.

Differently expressed proteins in probiotic Escherichia coli nissle 1917 under mercury stress identified by using a proteomic approach. We applied to separate proteins by using two-dimensional gel electrophoresis and proteins were identified using MALDI-TOF-MS using PMF, by mascot database search using the NCBI database. we identified six proteins after exposure to mercury stress with respect to different functional classes. It is useful to understand the molecular insights into mercury stress in probiotic E. coli. Next we describe a structure generated by homology modelling and functional domain identification; it is interesting to study the impact of stress on protein structures. MS characterization and computational methods together provide the opportunity to examine the impact of stress arising from mercury. The role of these proteins in metal tolerance and structure relation is discussed. To the best of our knowledge, proteomics of E. coli nissle 1917 overview of mercury stress has been reported for the first time.

Submitted by JOSÉ CAVALCANTE SOUZA VIEIRA null (jcavalcante-s@bol.com.br) on 2017-05-24T15:26:51Z No. of bitstreams: 1 Tese de Doutorado de José Cavalcante Souza Vieira 2017.pdf: 4955826 bytes, checksum: 47f49ca728d16c4dd16ffa14155cf4bd (MD5) Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-05-24T16:32:04Z (GMT) No. of bitstreams: 1 vieira_jcs_dr_bot_par.pdf: 1838076 bytes, checksum: 9c1f7a5bc01905022b7c61be22750c8c (MD5) Made available in DSpace on 2017-05-24T16:32:05Z (GMT). No. of bitstreams: 1 vieira_jcs_dr_bot_par.pdf: 1838076 bytes, checksum: 9c1f7a5bc01905022b7c61be22750c8c (MD5) Previous issue date: 2017-03-24 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Devido a sua grande concentração de nutrientes, tais como proteínas, vitaminas e minerais, o peixe é considerado um dos alimentos mais saudáveis que se pode encontrar na natureza. No entanto, a ingestão de peixes é considerada a forma predominante de via de exposição do ser humano ao mercúrio (Hg), principalmente para as populações que vivem às margens dos rios, onde o peixe constitui a principal fonte de proteína. Na tentativa de elucidar os mecanismos de toxicidade das espécies mercuriais, o teor desse metal tem sido estudado intensamente pela comunidade científica nas últimas décadas em amostras de solo, sedimentos, humanos e peixes na Amazônia brasileira. Sabe-se que as espécies mercuriais bioacumuladas nos tecidos dos seres vivos ligam-se a metaloproteínas, e quando há uma concentração alta de metal tóxico nos organismos, esses passam a expressar proteínas de defesa, denominadas metalotioneínas (MTs) responsáveis pelo transporte e eliminação de metais tóxicos. Apesar de estudos mostrarem o aumento das metalotioneínas em animais expostos a metais potencialmente tóxicos, essas proteínas não foram caracterizadas para confirmação de sua veridicidade, são analisadas por métodos indiretos, esse fato leva a necessidade de técnicas mais precisas na identificação de metalotioneínas. Levando em consideração o exposto esse estudo teve como objetivo otimizar métodos de quantificação de mercúrio e técnica de eletroforese para identificação de possíveis metalotioneínas biomarcadoras de mercúrio em tecido muscular e hepático de peixes de interesse econômico, Tucunaré (Cichla spp.), Filhote (Brachyplatystoma filamentosum), Jaraqui (Semaprochilodus sp.) e Barba chata (Pinirampus pirinampu). Através da Espectrometria de Absorção Atômica em Forno de Grafite (GFAAS) foi demonstrado que as quantificações dos níveis de mercúrio foram eficientes, mostrando boa recuperação do metal nos pools de tecidos, pellets e spots proteicos do músculo e fígado das espécies coletadas no reservatório da Usina Hidrelétrica de Jirau (UHE Jirau) - Bacia do rio Madeira. A separação das proteínas por eletroforese bidimensional (2D-PAGE) mostrou grande eficiência, com spots proteicos bem definidos, boa repetibilidade e reprodutibilidade nas corridas. Posteriormente a técnica de espectrometria de massas com ionização por eletrospray (ESI MS/MS) foi utilizada para identificação das proteínas obtidas por 2D-PAGE. Foi encontrado mercúrio em 27 spots proteicos e destes caracterizadas 23 proteínas ligadas ao mercúrio. No entanto, não foi identificada presença de metalotioneínas nas espécies estudadas, sugerindo que as concentrações de mercúrio não foram suficientes para provocar a produção destas proteínas de defesa contra intoxicação do organismo por este metal. Já nas análises comparando a atividade de indicadoras de estresse oxidativo glutationa peroxidase (GSH-Px), catalase (CAT), superóxido dismutase (SOD) e hidroperóxido de lipídios (HP) com a concentração de mercúrio encontrada em cada espécie de peixes, foi verificado aumento do estresse oxidativo em relação ao aumento da concentração de mercúrio nos tecidos. Due to its high concentration of nutrients, such as proteins, vitamins and minerals, fish is considered one of the healthiest foods that one can find in nature. However, fish intake is considered to be the predominant human exposure pathway to mercury (Hg), especially for populations living along riverbanks where fish are the main source of protein. In the attempt to elucidate the toxicity mechanisms of mercurial species, the content of this metal has been intensively studied by the scientific community in recent decades in soil, sediment, human and fish samples in the Brazilian Amazon. It is known that mercurial species bioaccumulated in the tissues of living beings bind to metalloproteins, and when there is a high concentration of toxic metal in organisms, they begin to express defense proteins, called metallothioneins (MTs) responsible for the transport and elimination of Toxic metals. Although studies have shown the increase of metallothioneins in animals exposed to potentially toxic metals, these proteins have not been characterized to confirm their veridicity, are analyzed by indirect methods, this fact leads to the need for more precise techniques in the identification of metallothioneins. Taking into account the above, this study aimed to optimize mercury quantification methods and electrophoresis technique for identification of possible mercury biomarkers metallothionein in muscular and hepatic tissue of fish of economic interest, Tucunaré (Cichla spp.), Filhote (Brachyplatystoma filamentosum), Jaraqui (Semaprochilodus sp.) and Barba-chata (Pinirampus pirinampu). Through Graphite Furnace Atomic Absorption Spectrometry (GFAAS) it was demonstrated that the quantification of mercury levels were efficient, showing good recovery of the metal in the pools of tissue, pellets and protein spots of the muscle and liver of the species collected in the Plant's reservoir Jirau Hydroelectric Plant (UHE Jirau) - Madeira River Basin. Separation of proteins by two-dimensional electrophoresis (2D-PAGE) showed high efficiency, with well-defined protein spots, good repeatability and reproducibility in the runs. Subsequently the mass spectroscopy technique with electrospray ionization (ESI MS/MS) was used to identify the proteins obtained by 2D-PAGE. Mercury was found in 27 protein spots and of these 23 mercury - bound proteins were characterized. However, the presence of metallothioneins in the species studied was not identified, suggesting that the concentrations of mercury were not enough to provoke the production of these defense proteins against intoxication of the organism by this metal. In the analyzes comparing the activity of oxidative stress indicators glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD) and lipid hydroperoxide (HP) with the concentration of mercury found in each species of fish, Increased oxidative stress in relation to the increase in the concentration of mercury in the tissues. FAPESP: 2014/02668-1

Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short chain fatty acids (propionic and acetic acids) as well as vitamin B12. In food applications, it is a promising organism for in situ fortification with B12 vitamin since it is generally recognized as safe (GRAS) and it is able to synthesize biologically active form of the vitamin. In the present study, vitamin B12 and pseudovitamin biosynthesis by P. freudenreichii was monitored by UHPLC as a function of growth in food-like conditions using a medium mimicking cheese environment, without cobalt or 5,6-dimethylbenzimidazole (DMBI) supplementation. Parallel growth experiments were performed in industrial-type medium known to support the biosynthesis of vitamin B12. The production of other key metabolites in the two media were determined by HPLC, while the global protein production was compared by gel-based proteomics to assess the effect of growth conditions on the physiological status of the strain and on the synthesis of different forms of vitamin. The results revealed distinct protein andmetabolite production, which reflected the growth conditions and the potential of P. freudenreichii for synthesizing nutritionally relevant amounts of active vitamin B12 regardless of the metabolic state of the cells.

Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short chain fatty acids (propionic and acetic acids) as well as vitamin B12. In food applications, it is a promising organism for in situ fortification with B12 vitamin since it is generally recognized as safe (GRAS) and it is able to synthesize biologically active form of the vitamin. In the present study, vitamin B12 and pseudovitamin biosynthesis by P. freudenreichii was monitored by UHPLC as a function of growth in food-like conditions using a medium mimicking cheese environment, without cobalt or 5,6-dimethylbenzimidazole (DMBI) supplementation. Parallel growth experiments were performed in industrial-type medium known to support the biosynthesis of vitamin B12. The production of other key metabolites in the two media were determined by HPLC, while the global protein production was compared by gel-based proteomics to assess the effect of growth conditions on the physiological status of the strain and on the synthesis of different forms of vitamin. The results revealed distinct protein and metabolite production, which reflected the growth conditions and the potential of P. freudenreichii for synthesizing nutritionally relevant amounts of active vitamin B12 regardless of the metabolic state of the cells.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a key technique for the separation of complex protein samples to survey the protein inventory of prokaryotic micro-organisms. Although the preparation of proteins is a critical step in 2D-gel experiments, its effect on the outcome of proteome data is often underestimated. In this work, we show that the choice of protein isolation method can have a profound impact on the quality of 2D-gels of protein extracts from prokaryotes. Based on Xanthomonas campestris, a commercially relevant producer of the thickening agent xanthan, we validated a phenol extraction protocol for the purification of bacterial proteins that provides excellent 2D-gel separation. As a proof of concept, this method was used to study the effect of methionine – a medium compound that reduces the xanthan output of industrial fermentations – on the cellular proteome of Xanthomonas. The detection of nine regulated proteins associated with sulfur metabolism (Cgl, CysI, CysJ, CysK, MetH1, MetY) and sugar nucleotide biosynthesis (Pgi, Ugd, XanA) proved the efficiency of phenol extraction for the screening of statistically significant abundance changes in 2D-gel spots and MALDI-TOF-MS based identification in bacteria. Since this method is very robust, it may be useful for the study of other prokaryotes that are relevant in industrial biotechnology. This article is protected by copyright. All rights reserved

© 2017 American Chemical Society. Ants have evolved venoms rich in peptides and proteins used for predation, defense, and communication. However, they remain extremely understudied due to the minimal amount of venom secreted by each ant. The present study investigated the differences in the proteome and peptidome of the venom from the bullet ant, Paraponera clavata. Venom samples were collected from a single colony either by manual venom gland dissection or by electrical stimulation and were compared using proteomic methods. Venom proteins were separated by 2D-PAGE and identified by nanoLC-ESI-QTOF MS/MS. Venom peptides were initially separated using C18 reversed-phase high-performance liquid chromatography, then analyzed by MALDI-TOF MS. The proteomic analysis revealed numerous proteins that could be assigned a biological function (total 94), mainly as toxins, or roles in cell regulation and transport. This investigation found that ca. 73% of the proteins were common to venoms collected by the two methods. The peptidomic analysis revealed a large number of peptides (total 309) but with

We report the genetic similarity changes between a mutant mushroom (Pleurotus florida, designated as PfCM4) having increased cellulolytic activity developed through radiation mutagenesis and its wild type by amplified fragment length polymorphism (AFLP). On average, 23 AFLP fragments were amplified per primer combination, and a total of 286 polymorphic fragments (78.57% polymorphism) with maximal fragment length of 1365 base pairs (bp) were obtained. The genetic similarity between wild type and PfCM4 was found to be 22.30%. In addition, mycelial and secreted protein profiling by 2D-PAGE showed at least three and five different protein spots in the range of 25 kD to 100 kD, respectively, in PfCM4. It seems that the variation in genetic similarity and different expression of both mycelial and secreted proteins in PfCM4 in comparison to the wild type could likely be correlated with its increased cellulolytic activity effected by the irradiation.

Allergic contact dermatitis is a widespread disease with high clinical relevance affecting approximately 20% of the general population. Typically, contact allergens are low molecular weight electrophilic compounds which can activate the Keap1/Nrf2 pathway. We performed a proteomics study to reveal possible biomarkers for dendritic cell (DC) activation by contact allergens and to further elucidate the role of Keap1/Nrf2 signaling in this process. We used bone marrow derived dendritic cells (BMDCs) of wild-type (nrf2+/+) and Nrf2 knockout (nrf2−/−) mice and studied their response against the model contact sensitizers 2,4-dinitrochlorobenzene (DNCB), cinnamaldehyde (CA) and nickel(II) sulfate by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS). Sodium dodecyl sulfate (SDS, 100 μM) served as irritant control. While treatment with nickel(II) sulfate and SDS had only little effects, CA and DNCB led to significant changes in protein expression. We found 18 and 30 protein spots up-regulated in wild-type cells treated with 50 and 100 μM CA, respectively. For 5 and 10 μM DNCB, 32 and 37 spots were up-regulated, respectively. Almost all of these proteins were not differentially expressed in nrf2−/− BMDCs, indicating an Nrf2-dependent regulation. Among them proteins were detected which are involved in oxidative stress and heat shock responses, as well as in signal transduction or basic cellular pathways. The applied approach allowed us to differentiate between Nrf2-dependent and Nrf2-independent cellular biomarkers differentially regulated upon allergen-induced DC activation. The data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization.

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

Submitted by admin tede (tede@pucgoias.edu.br) on 2016-11-30T18:10:51Z No. of bitstreams: 1 MARIA ROSA CERBINO.pdf: 2627014 bytes, checksum: 98680184bb60e636de01507e3f1eeec4 (MD5) Made available in DSpace on 2016-11-30T18:10:51Z (GMT). No. of bitstreams: 1 MARIA ROSA CERBINO.pdf: 2627014 bytes, checksum: 98680184bb60e636de01507e3f1eeec4 (MD5) Previous issue date: 2016-04-12 Mercury is a potentially toxic element with a wide distribution on the Amazonian environment. This metal is dangerous and responsible for environmental contaminations and human intoxications as it is capable to biomagnifications and bioaccumulate throughout the food chains becoming the main way of exposing the riverine Amazonian communities to methylmercury to whom the main diet is fish. Therefore, studies related to mercury toxicity are of fundamental importance to health and life quality of the Amazonian communities. This study aimed to detect and evaluate possible proteic biomarkers of mercury toxicity in samples of human milk collected in riverine populations of Madeira and Negro rivers in the Brazilian Amazon. Initially total mercury was determined in the hair of breast feeding women to identify who were contaminated with mercury followed by the obtaining of the proteome of milk samples by two-dimensional electrophoresis (2D PAGE) after precipitation of proteins in half acetone. On the proteic spots obtained in the process of protein fractionation of milk samples, detection of mercury was carried out by atomic absorption spectrometry in graphite furnace(GFAAS), where the results showed that mercury was bonded in proteins of molecular weight around 14-26 kDa. The concentration determination of total mercury by GFAAS was also carried out with milk in natura, lyophilized milk and the proteic pellets aiming a mass balance of mercury related to the concentration of this element on milk and pellets. The measurements of mass balance permitted to observe that, in relation to the milk samples from Madeira River, about 85 to 95%of the mercury present in the lyophilized milk is on the proteic fraction. In relation to the breastfeeding women of the Negro River, about 50% of the total mercury is bound in the proteic fraction and the difference of 51% might be bound to the lipidic fraction. However, more studies in this line of research need to be pursued to achieve more robust conclusions. O mercúrio é um elemento potencialmente tóxico com ampla distribuição no ambiente amazônico. Este metal é perigoso e responsável por contaminações ambientais e intoxicações humanas, já que é capaz de biomagnificar e bioacumular através das cadeias alimentares, tornando-se assim a principal via de exposição às comunidades amazônicas ribeirinhas do metilmercúrio, cuja dieta é baseada em peixes. Sendo assim estudos relacionados à toxicidade do mercúrio são de fundamental importância para a saúde e a qualidade de vida das comunidades amazônicas. Este estudo buscou detectar e avaliar possíveis biomarcadores proteicos da toxicidade do mercúrio em amostras de leite materno coletadas de populações ribeirinhas do rio Madeira e do rio Negro, na Amazônia brasileira. Inicialmente, determinou-se mercúrio total no cabelo das lactantes para identificar quais estavam contaminadas com mercúrio, em seguida obteve-se o proteoma das amostras de leite por eletroforese bidimensional (2D-PAGE) após precipitação das proteínas em meio acetônico. Nos spotsproteicos obtidos no processo de fracionamento das proteínas, nas amostras leite, foram feitas determinações de mercúrio por espectrometria de absorção atômica em forno de grafite (GFAAS), onde os resultados mostraram que o mercúrio se encontra ligado em proteínas de massa molecular na faixa de 14-26 kDa. A determinação da concentração de mercúrio total por GFAAS foi feita também no leite in natura, leite liofilizado e nos pelletsproteicos, com o objetivo de se fazer um balanço de massa de mercúrio em relação à concentração deste elemento no leite e pellets. As medidas de balanço de massa permitiram observar que, em relação às amostras de leite do rio Madeira, cerca de 85 a 95% do mercúrio presente no leite liofilizado encontrase na fração proteica. Em relação às lactantes do rio Negro, cerca de 50% do mercúrio total está ligado na fração proteica e a diferença de 51% pode estar ligado na fração lipídica. Contudo, mais estudos nesta linha de pesquisa devem ser desenvolvidos, para que se possam ter conclusões mais robustas.

Breast cancer is the most common cancer among women worldwide. Breast cancer metastasis primarily happens through lymphatic system, where the extent of lymph node metastasis is the major factor influencing staging, prognosis and therapeutic decision of the disease. We aimed to study the protein expression changes in different N (regional lymph nodes) stages of breast cancer. Protein expression profiles of breast cancerous and adjacent normal tissues were mapped by proteomics approach that comprises of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and tandem mass spectrometry (LC-MS/MS) analysis. Calreticulin and tropomyosin alpha 3 chains were the common up-regulated proteins in N0, N1 and N2 stages of breast cancer. Potential biomarker for each N stage was HSP 70 for N0, 80 k protein H precursor and PDI for N1 stage while 78 kDa glucose-regulated protein was found useful for N2 stage. In addition, significant up-regulation of PDI A3 was detected only in the metastasized breast cancer. The up-regulation expression of these proteins in cancerous tissues can potentially use as indicators for diagnosis, treatment and prognosis of different N stages of breast cancer.

Oxidation of serum proteins can lead to carbonyl formation that alters their function and is often associated with stress-related diseases. As it is recommended that all pigs reared in modern production facilities be given supplemental iron at birth to prevent anemia, and metals can catalyze the carbonylation of proteins, the primary objective of this study was to determine whether standard iron dextran treatment was associated with enhanced serum protein oxidation in newborn piglets. Piglets were treated with 100 mg of iron dextran intramuscularly either on the day of birth, or on the third day after birth. Blood samples were collected from piglets 48 or 96 h after treatment and serum was harvested. For quantification, serum protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and analyzed spectrophotometrically. To identify and determine relative distribution of carbonylated proteins, serum protein carbonyls were derivatized with biotin hydrazide, separated by two-dimensional polyacrylamide gel electrophoresis, stained with avidin-fluorescein and identified by mass spectrometry. The standard iron dextran treatment was associated with no increase in total oxidized proteins if given either on the first or third day of life. In addition, with a few noted exceptions, the overall distribution and identification of oxidized proteins were similar between control and iron dextran-treated pigs. These results indicate that while iron dextran treatment is associated with a marked increase in circulating iron, it does not appear to specifically induce the oxidation of serum proteins.

An initial profiling of the intracellular proteome of Aspergillus niger ATCC 10864 biofilm cultures developed on polyester cloth was carried out by using 2D-PAGE and MS-TOF analysis and it was compared to the proteome of conventionally grown free-living submerged cultures. A number of 2D-PAGE protein spots from both types of cultures were subjected to MS-TOF analysis and data interrogation of the NCBI nr database available for this species. Proteomic maps showed different expression patterns in both culture systems with differentially expressed proteins in each case. In biofilm cultures, 19% and 32% of the selected protein spots were over- expressed and differentially expressed, respectively. On the contrary, in free-living cultures, 44% and 7% of the selected protein spots were over-expressed and differentially expressed, respectively. Although preliminary, results presented in this paper show that there are significant differences between the proteomes of A. niger biofilm and free-living mycelia. It seems that cell adhesion is the most important stimulus responsible for biofilm development which is the basis of Surface Adhesion Fermentation., Se realizó un perfil inicial del proteoma de biopelículas de Aspergillus niger ATCC 10864 desarrolladas sobre tela de poliéster mediante 2D-PAGE y análisis MS-TOF y comparado con el proteoma de cultivos sumergidos convencionales de micelio libre. De ambos tipos de cultivo se analizó un número de muestras proteicas de geles 2D-PAGE mediante MS-TOF y los resultados se compararon con la base de datos NCBI nr disponible para esta especie. Los mapas proteómicos mostraron patrones diferentes de expresión en cada caso. En cultivo de biopelículas, el 19% y el 32% de las muestras seleccionadas fueron sobre-expresadas y diferencialmente expresadas, respectivamente. Por el contrario, en cultivos sumergidos en micelio libre el 44% y el 7% de las muestras seleccionadas fueron sobre-expresadas y diferencialmente expresadas, respectivamente. Aunque preliminares, los resultados presentados en este trabajo muestran que existen diferencias significativas entre los proteomas de biopelículas y micelio libre de A. niger. Parece ser que la adhesión celular es el estímulo más importante para el desarrollo de biopelículas, las cuales son la base de la Fermentación por Adhesión a Superficies.

The continuously growing interest in small regulatory RNA exploration is one of the important factors that have inspired the recent development of new high throughput techniques such as DNA microarrays or next generation sequencing. Each of these methods offers some significant advantages but at the same time each of them is expensive, laborious and challenging especially in terms of data analysis. Therefore, there is still a need to develop new analytical methods enabling the fast, simple and cost-effective examination of the complex RNA mixtures. Recently, increasing attention has been focused on the RNA degradome as a potential source of riboregulators. Accordingly, we attempted to employ a two-dimensional gel electrophoresis as a quick and uncomplicated method of profiling RNA degradome in plant or human cells. This technique has been successfully used in proteome analysis. However, its application in nucleic acids studies has been very limited. Here we demonstrate that two dimensional electrophoresis is a technique which allows one to quickly and cost-effectively identify and compare the profiles of 10–90 nucleotide long RNA accumulation in various cells and organs. Electronic supplementary material The online version of this article (doi:10.1007/s11033-011-0718-1) contains supplementary material, which is available to authorized users.

2D-immunomics may be useful in the identification of autoantigens in neurological autoimmune diseases, but its application may be limited by denaturation of target proteins. Here we compared the capacity of a single or multiple antigens to elicit autoantibodies targeting multiple neural autoantigens by ELISA and 2D-immunomics. We induced experimental autoimmune encephalomyelitis (EAE) with MBP peptide 89–104 , total MBP or spinal cord homogenate. Both techniques showed anti-MBP IgG only after immunization with total MBP. In addition, 2D-immunomics revealed the presence in EAE mice of autoantibodies targeting other neural proteins, some displaying partial sequence homology with MBP. The present finding by 2D-immunomics of multiple neural proteins targeted by autoantibodies generated by a single antigen may help to explain the complex autoimmune response observed in multiple sclerosis.

Soybean cyst nematode (Heterodera glycines, SCN) is the most destructive pathogen of soybean around the world. Crop rotation and resistant cultivars are used to mitigate the damage of SCN, but these approaches are not completely successful because of the varied SCN populations. Thus, the limitations of these practices with soybean dictate investigation of other avenues of protection of soybean against SCN, perhaps through genetically engineering of broad resistance to SCN. For better understanding of the consequences of genetic manipulation, elucidation of SCN protein composition at the subunit level is necessary. We have conducted studies to determine the composition of SCN proteins using a proteomics approach in our laboratory using twodimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate SCN proteins and to characterize the proteins further using mass spectrometry. Our analysis resulted in the identification of several hundred proteins. In this investigation, we developed a web based database (SCNProDB) containing protein information obtained from our previous published studies. This database will be useful to scientists who wish to develop SCN resistant soybean varieties through genetic manipulation and breeding efforts. The database is freely accessible from: http://bioinformatics.towson.edu/Soybean_SCN_proteins_2D_Gel_DB/Gel1.aspx.

International audience; Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2-DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo-natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.

Epidermal growth factor (EGF) is an important mitogen for hepatocytes. Its targeted overexpression induced hepatocellular carcinomas (HCC), as recently reported by us (Borlak et al. 2005). Early detection of disease is essential for successful therapy and overall survival. Here we describe our efforts in identifying serum biomarkers of liver cancer in a transgenic disease model that mimics effectively the consequence of exaggerated EGF signaling. We report a reference 2-DE map of mouse serum proteins. About 180 proteins were detected per gel and 130 proteins were identified by 2-DE-MALDI-MS analysis. We compared serum proteins of healthy nontransgenic and HCC tumor-bearing mice and identified 25 regulated proteins, of which n = 7 reached statistical significance ( p < 0.05). Furthermore, we identified several fragments of fibrinogens and of the alpha-2-macroglobulin to be disease-associated. We also found immunoglobulins to be repressed or absent in serum samples of tumor-bearing mice, and this included, among others, the Ig K and L class. In contrast, amyloid component P and apolipoprotein M were highly significantly increased by 10- and 8-fold, respectively, in serum samples of HCC mice. Overall, our findings provide a rationale for further clinical evaluation of the herein identified biomarker candidates.

Na cana-de-açúcar, a conversão de meristema vegetativo para reprodutivo é uma etapa importante para o melhoramento genético, no entanto, é indesejável na produção comercial por consumir sacarose para o desenvolvimento da inflorescência. Este estudo teve como objetivo identificar proteínas diferencialmente acumuladas no meristema apical da variedade de cana RB867515 sob aplicação foliar de sulfato de cálcio, através de eletroforese 2D e espectrometria de massas. Foi testado o efeito do cálcio em parâmetros morfofisiológicos, concentração de macronutrientes e anatomia do ápice meristemático por microscopia ótica. Adicionalmente, foi avaliado o efeito do cálcio no proteoma do ápice meristemático durante as fases de pré e pós-indução do florescimento. A aplicação foliar de cálcio aumentou a altura de colmos, e a abertura estomática também foi alterada. A composição dos macronutrientes mostrou maiores níveis de cálcio e menores teores de potássio e magnésio. A aplicação foliar de cálcio reduziu em cerca de 20% o florescimento da cana planta e 35% na cana soca. Na análise proteômica, um total de 60 DEPs foram identificados por PMF a partir de perfis 2D de meristemas em pré ou pós indução floral, dos quais 14 foram observadas exclusivamente em meristemas tratados com cálcio, 11 foram identificadas exclusivamente após a indução floral, e 29 foram comuns em ambas as situações, porém mais abundantes em meristemas sem aplicação de cálcio antes da indução floral. A aplicação foliar de cálcio alterou significativamente o proteoma meristemático da cana-de-açúcar. O cálcio parece também melhorar a sinalização celular e a atividade antioxidante em meristemas. O efeito da aplicação foliar de cálcio no proteoma parece ser atenuado pelo tempo. As proteínas identificadas são fortes candidatas a estudos futuros envolvendo o controle do florescimento da cana, visando sua aplicação como marcador molecular funcional associadas ao cálcio, podendo auxiliar os programas de melhoramento genético desta cultura. In sugarcane (Saccharum spp.), the conversion of apical meristem to breeding is an important step for the genetic improvement, however, it is undesirable in commercial production due to consuming sucrose to develop inflorescence. This study aimed to identify differentially accumulated proteins in the apical meristem of the RB867515 variety under foliar application of calcium sulfate through 2D electrophoresis and mass spectrometry. It has been tested the calcium effect on morphophysiological parameters, macronutrient contents and anatomy of the shoot apical meristem by optical microscopy. Additionally, we evaluated the effect of calcium on the proteome of sugarcane apical meristem during the phases of pre and post-induction of flowering. Foliar applications of calcium increased the stalk height, and stomatal opening was also measured. The macronutrient composition showed higher calcium levels and lower levels of potassium and magnesium. Leaf applications of calcium reduces flowering by 20% in sugarcane plant, and 35% reduction in the ratoon cane. In the proteome analysis, a total of 60 DEPs have been identified by PMF from 2D meristems profiles in pre and post-floral induction, of which 14 were found exclusively in meristems treated with calcium, 11 were identified only after floral induction, and 29 were common in both cases, but more abundant in meristems without application of calcium before floral induction. Foliar applications of calcium significantly altered the meristematic proteome of sugarcane. Calcium also appears to enhance cell signaling and antioxidant activity in meristems. It was observed that the effect of foliar calcium application in proteome appears to be attenuated by time. The proteins identified are strong candidates for future studies aiming its use as a functional molecular marker involving control of flowering of sugarcane associated with calcium, can help breeding programs of this culture.

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two- dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan- Brucella, B. abortus- and B. melitensis-specific antibodies.

To survive under highly variable environmental conditions, higher plants have acquired a large variety of acclimation responses. Different strategies are used to cope with changes in light intensity with the common goal of modulating the functional antenna size of Photosystem II. Here we use a combination of biochemical and biophysical methods to study these changes in response to acclimation to high light. After two hours of exposure, a decrease in the amount of the large PSII supercomplexes is observed indicating that plants are already acclimating to high light at this stage. It is also shown that in high light the relative amount of antenna proteins decreases but this decrease is far less than the observed decrease of the functional antenna size, suggesting that part of the antenna present in the membranes in high light does not transfer energy efficiently to the reaction center. Finally, we observed LHCII monomers in all conditions. As the solubilization conditions used do not lead to monomerization of purified LHCII trimers, we should conclude that a population of LHCII monomers exists in the membrane. The relative amount of LHCII monomers strongly increases in plants acclimated to high light, while no changes in the trimer to monomer ratio are observed upon short exposure to stress.

We present a web-based integrated proteome database, termed 2DBase of Escherichia coli which was designed to store, compare, analyse, and retrieve various information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry. The main objectives of this database are (1) to provide the features for query and data-mining applications to access the stored proteomics data (2) to efficiently compare the specific protein spots present in the comparable proteome maps and (3) to analyse the data with the integrated classification for cellular functions of gene products of E. coli. This database currently contains 12 gels consisting of 1185 protein spots information in which 723 proteins were identified and annotated. Individual protein spots in the existing gels can be displayed, queried, analyzed, and compared in a tabular format based on various functional categories enabling quick and subsequent analyses. Our database satisfies the requirement to be a federated 2-DE database by accomplishing various tasks through a web interface providing access to a relational database system. The 2DBase of E. coli database can be accessed at http://2dbase.techfak.uni-bielefeld.de/.