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Alcoholic liver disease (ALD) progresses from steatosis to alcoholic hepatitis to fibrosis and cirrhosis. Liver biopsy is considered as the gold standard method for diagnosis of liver cirrhosis and provides useful information about damaging process which is an invasive procedure with complications. Existing biomarkers in clinical practice have narrow applicability due to lack of specificity and lack of sensitivity. The objective of this article is to identify proteomic biomarker candidates for alcoholic liver cirrhosis by differential expression analysis between alcoholic liver cirrhotic and healthy subjects. Blood samples were collected from 20 subjects (10 alcoholic liver cirrhosis and 10 healthy) from R. L. Jalapa Hospital and Research Centre, Kolar, Karnataka, India. Differential protein analysis was carried out by two-dimensional electrophoresis after albumin depletion, followed by liquid chromatography–mass spectrometry. The image analysis found 46 spots in cirrhotic gel and 69 spots in healthy gel, of which 14 spots were identified with significant altered expression levels. Based on the protein score and clinical significance, among 14 spots, a total of 28 protein biomarker candidates were identified: 13 with increased expression and 15 with decreased expression were categorized in alcoholic liver cirrhosis compared to healthy subjects. Protein biomarker candidates identified by “-omics” approach based on differential expression between alcoholic liver cirrhotic subjects and healthy subjects may give better insights for diagnosis of ALD. Prioritization of candidates identified is a prerequisite for validation regimen. Biomarker candidates require verification that demonstrates the differential expression will remain detectable by assay to be used for validation.

Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Currently prostate specific antigen (PSA) serum concentration is the most used prostate cancer marker, but it only shows limited specificity. Because PSA glycosylation is altered by prostate cancer, detecting glycosylation changes could increase PSA specificity as a prostate cancer marker. Changes in PSA glycosylation can modify its electrophoretic- behavior and techniques such as capillary zone electrophoresis (CZE) and two-dimensional electrophoresis (2-DE) could be applied to detect changes in PSA glycosylation. Most serum PSA is complexed with alpha-1 antichymotrypsin (ACT). To have access to most of the PSA, the complexed PSA has to be released as free PSA (fPSA); in addition, this total fPSA must be purified from the serum matrix so that it can be analyzed using CZE. In this work a methodology for isolating PSA from serum for its CZE analysis was established. By using PSA standard, the effect of this methodology, which combines conditions for dissociating complexed PSA and immunoaffinity chromatographic purification, was studied. It was seen that this highly repeatable sample treatment did not noticeably alter the circular dichroism (CD) spectrum or the CZE pattern of PSA standard. Therefore, as a proof-of-concept, the developed sample treatment was applied to serum from a cancer patient with a high PSA content. The following observations can be made from these experiments: first of all, the 2-DE pattern of serum PSA remained unchanged after sample treatment; second, as hypothesized, the established sample preparation methodology made it possible to obtain the CZE pattern of PSA from serum; and third, the CZE pattern of serum PSA and of PSA standard from seminal plasma of healthy individuals, both submitted to the sample treatment method, showed some differences regarding the proportion of CZE peaks of the glycoprotein. These differences could be related to possible changes in the

Serum levels of Prostate-Specific Antigen (PSA) are not fully specific for prostate cancer (PCa) diagnosis and several efforts are focused on searching to improve PCa markers through the study of PSA subforms that could be cancer associated. We have previously reported by 2DE a decrease in the sialic acid content of PSA from PCa compared to benign prostatic hyperplasia patients based on the different proportion of the PSA spots. However, faster and more quantitative techniques, easier to automate than 2DE, are desirable. In this study, we examined the potential of CE for resolving PSA subforms in different samples and compared the results with those obtained by 2DE. We first fractionated by OFFGEL the subforms of PSA from seminal plasma according to their pIs and analyzed each separated fraction by 2DE and CE. We also analyzed PSA and high pI PSA, both from seminal plasma, and PSA from urine of a PCa patient. These samples with different PSA spots proportions by 2DE, due to different posttranslational modifications, also presented different CE profiles. This study shows that CE is a useful and complementary technique to 2DE for analyzing samples with different PSA subforms, which is of high clinical interest.

Proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress were assessed on the basis of two-dimensional electrophoresis (2-DE) data. In this study, the bootstrap resampling statistical technique and a new measure of relative change of the volume of 2-DE protein spots are shown to be more efficient than commonly used statistics to reliably quantify changes in protein abundance in stress response. The data are supplied in this article and are related to “Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress” by Franco et al.

Tato práce se zabývá proteomickou analýzou membránových proteinů bakterie Mycoplasma hominis. Frakce obohacená o membránové proteiny byla separována dvourozměrnou elektroforézou a vybrané proteiny byly štěpeny proteolytickým enzymem trypsinem. Získané peptidy byly následně analyzovány hmotnostní spektrometrií. Z celkového počtu 31 analyzovaných proteinů se tímto postupem podařilo identifikovat 15 proteinových skvrn Mycoplasma hominis, které představují 6 proteinů. Jednalo se o P50 povrchový lipoproteinový adhezin, elongační faktor Tu, fosfoglycerátkinázu, DNA-dependentní RNA polymerázu podjenotka alfa, chaperon DnaK a jeden necharakterizovaný protein., This work deals with the proteomic analysis of membrane proteins of the bacterium Mycoplasma hominis. Fractions enriched membrane proteins were separated by two-dimensional electrophoresis and selected proteins were digested by the proteolytic enzyme trypsin. These peptides were analyzed by mass spectrometry. From a total of 31 proteins analyzed by this procedure were identified 15 protein spots Mycoplasma hominis which represent about 6 proteins. These proteins were P50 lipoprotein surface adhesin, elongation factor Tu, phosphoglyceratekinase, DNA-directed RNA polymerase subunit alpha, chaperone protein DnaK and uncharacterized protein., Katedra biologických a biochemických věd, Prezentace výsledků diplomové práce.Diskuze k posudku vedoucího a oponenta diplomové práce. Studentka zodpověděla vaechny dotazy a připomínky k diplomové práci.

In order to investigate the lesions and proteins with differential expression in cells infected with the 2009 A (H1N1) virus and to determine the specific proteins involved in cell damage, the present study has been performed. BEAS-2B cells were infected with the 2009 A (H1N1) influenza virus or the seasonal H1N1 influenza virus for 12, 24, 48, and 72 h, and cell cycle and apoptosis were analyzed with flow cytometry. Total cellular proteins were extracted and underwent two-dimensional gel electrophoresis. The differentially expressed proteins underwent mass spectrometry for identification. The results showed that after 12 h, cells infected with the virus strain sourced from severe cases had the highest apoptosis rate (P < 0.05). After 48 h, cells infected with the virus strain sourced from fatal cases and severe cases had the highest apoptosis rate (P < 0.05), and after 72 h, cells infected with virus strains from fatal cases and ordinary cases had the highest apoptosis rate (P < 0.05). All the four influenza virus strains induced cell cycle arrest mainly at the G0/G1 phase. Eighteen differentially expressed proteins were identified, including galectin-1, cofilin-1, protein DJ-1, proteasome subunit α type-5, macrophage migration inhibitory factor, translationally controlled tumor protein, profilin 1, and interferon α-2. Galectin-1 was specifically observed in BEAS-2B infected with 2009 A (H1N1) influenza viruses, and cofilin-1 was specifically observed in BEAS-2B cells in the late stage of 2009 A (H1N1) influenza virus infection. In conclusion, differential effects of the 2009 A (H1N1) influenza virus and seasonal H1N1 influenza virus were identified on the cell cycle and apoptosis, and galectin-1 may play a role in cell apoptosis induced by 2009 A (H1N1) influenza virus.

Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis

Tremendous progress in plant proteomics driven by mass spectrometry (MS) techniques has been made since 2000 when few proteomics reports were published and plant proteomics was in its infancy. These achievements include the refinement of existing techniques and the search for new techniques to address food security, safety, and health issues. It is projected that in 2050, the world's population will reach 9–12 billion people demanding a food production increase of 34–70% (FAO, 2009) from today's food production. Provision of food in a sustainable and environmentally committed manner for such a demand without threatening natural resources, requires that agricultural production increases significantly and that postharvest handling and food manufacturing systems become more efficient requiring lower energy expenditure, a decrease in postharvest losses, less waste generation and food with longer shelf life. There is also a need to look for alternative protein sources to animal based (i.e., plant based) to be able to fulfill the increase in protein demands by 2050. Thus, plant biology has a critical role to play as a science capable of addressing such challenges. In this review, we discuss proteomics especially MS, as a platform, being utilized in plant biology research for the past 10 years having the potential to expedite the process of understanding plant biology for human benefits. The increasing application of proteomics technologies in food security, analysis, and safety is emphasized in this review. But, we are aware that no unique approach/technology is capable to address the global food issues. Proteomics-generated information/resources must be integrated and correlated with other omics-based approaches, information, and conventional programs to ensure sufficient food and resources for human development now and in the future. © 2013 Wiley Periodicals, Inc. Mass Spec Rev

A two-dimensional gel electrophoresis (2-DE) based proteomic approach has been used to study the Medicago truncatula-Uromyces striatus interaction. The 2-DE leaf protein profile of three M. truncatula genotypes displaying different phenotypes (susceptible and showing prehaustorial and posthaustorial resistance) in both noninoculated and inoculated plants have been compared. Multivariate statistical analysis identified 63 differential protein spots under the experimental conditions (genotypes/treatments). Variable spots were subjected to tandem mass spectrometry (MS, matrix-assisted laser desorption ionisation time of flight, MALDI-TOF/TOF) analysis to identify their possible functions. A total of 27 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of these observed changes correspond to enzymes involved in photosynthesis, energy metabolic pathways and stress related, whose pattern expression was different in relation to susceptibility/resistance of the genotypes studied. Results obtained in this work suggest that differences observed could be related to efficiency in energy utilisation and the induction of proteins involved in defence mechanism operating during early stages of infection. © 2010 Association of Applied Biologists.

Cells are routinely exposed to hyperoxic conditions when cultured in the presence of 95% air and 5% carbon dioxide. Hyperoxic conditions can increase the generation of reactive oxygen species and cause oxidative stress. Oxidative stress has been proposed to cause cells in culture to behave differently from cells in vivo. One route by which oxidative stress could affect cellular function is through alterations in protein function caused by the oxidation of thiol groups (-SH) of redox-sensitive cysteine residues. To test whether changes in oxygen concentration were sufficient to cause changes in the thiol redox state of proteins, we developed a sensitive method involving the labeling of reduced and oxidized cysteine residues with fluorescent tags. Using this dual labeling method, we found 62 of 411 protein spots that were significantly more reduced following a 30 min decrease in oxygen concentration. We conclude that the elevated oxygen concentration characteristic of typical cell culture conditions has the potential to affect cellular behavior through changes in the thiol redox state of proteins.

Lipopolysaccharide (LPS) administration down-regulates lipoprotein lipase (LPL) activity at the posttranscriptional level. Hypertriglyceridemia is the main metabolic consequence of this fall in LPL activity and is presumably involved in the innate immune response to infection. Nitric oxide (NO) has been implicated in LPS-induced down-regulation of LPL activity, but whether its effects are direct or indirect remains unclear. Here we examined the potential nitration of LPL in vivo in response to LPS challenge in rats. We found hypertriglyceridemia, iNOS expression, NO overproduction, and a generalized decrease in LPL activity in tissues 6 h after LPS administration. LPL sensitivity to nitration was first explored by in vitro exposure of bovine LPL to peroxynitrite, a reactive nitrogen species (RNS). Nitration was confirmed by anti-nitrotyrosine Western blot and subsequent identification of specific nitrotyrosine-containing LPL sequences by tandem mass spectrometry. Further analysis by targeted mass spectrometry revealed three in vivo-nitrated tyrosine residues in heart LPL from LPS-challenged rats. This is the first study to identify nitrated tyrosine residues in LPL, both in vitro and in vivo, and it demonstrates that LPL is a target for RNS in endotoxemia. These results indicate that LPL nitration may be a new mechanism of LPL activity regulation in vivo.

Cílem diplomové práce byla komparativní proteomová analýza divokého kmene bakterie Francisella tularensis ssp. holarctica FSC 200 a mutantních kmenů s deletovanými geny ?pilC, ?pilQ, ?pilO1, ?pilO. Z uvedených bakteriálních kmenů byly připraveny jak celobuněčné lyzáty, tak i frakce obohacené o membránové proteiny. Všechny vzorky byly děleny 2D elektroforézou v rozmezí pH 3-10 a pH 6-11, digitalizovány pomocí softwaru KODAK 2D Image Analysis. Komparativní analýza mutantních kmenů a divokého kmene byla provedena pomocí programu ImageMaster 2D Platinum 6.0. Byly sledovány kvalitativní a kvantitativní změny proteinových skvrn a dále změny v posunu jejich isoelektrických bodů (?pI) a molekulových hmotností (?MW). Největší rozdíly v proteinové expresi byly nalezeny u mutantního kmene ?pilO2 a proto byla provedena jeho detailní analýza ukončená identifikací změněných proteinů. Identifikace proteinů byla provedena dvěma metodami: MALDI-TOF MS a komparativní analýzou s 2D referenčními proteinovými mapami. V rámci komparativní proteomové analýzy mutantního kmene ?pilO2 a mateřského kmene FSC200 byly pomocí MS identifikovány čtyři proteiny a pomocí srovnávací analýzy s referenční 2D mapou bylo identifikováno 21 proteinů. Vybrané proteiny budou zkoumány v dalších studiích., The aim of the thesis was a comparative proteome analysis of a wild strain bacteria Francisella tularensis ssp. holarctica FSC 200 and mutant strains with of deletion genes ?pilC, ?pilQ, ?pilO1, ?pilO2. Whole cell lysates and fractions enriched in membrane proteins were prepared from these strains. All samples were separated by 2D electrophoresis in range 3-10 and 6-11 pH gradients and digitized by KODAK 2D Image Analysis Softwar. Comparative analyses of the mutant strains and the wild strain were performed with the help of ImageMaster 2D Platinum 6.0 Software. Qualitative and quantitative changes of the protein spots and changes in shift of their isoelectric points (?pI) and molecular weights (?MW) were detected. The biggest differences in protein expression were found in mutant strain ?pilO2 and therefore its detailed analysis was performed and finished by identification of changed proteins. Identification of proteins was done by two methods: by MALDI-TOF MS and according to reference standard 2D maps. Four qualitative changed proteins from comparative proteome analysis of mutant strain ?pilO2 and wild strain FSC 200 were identified by MS and twendy-one quantitative changed proteins were identified according to reference standard 2D maps. Selected proteins will be examined in next studies., Katedra biologických a biochemických věd, Dokončená práce s úspěšnou obhajobou

Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography ( LC) coupled with two-dimensional electrophoresis (2DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel "blue mussel" ( Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern ( PES) composed of 13 proteins. Principal component analysis ( PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry ( ESI-MS/MS), and they are involved in,beta-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.

In the present study, rice seedlings were exposed to a range of Cd concentrations (0.1 mu M, 1 mu M, 10 mu M, 100 mu M and 1 mM) for 15 days and a combination of different molecular approaches were used to evidence Cd effects and to assess the plants' ability to counteract metal toxicity. At a macroscopical level, only the highest Cd concentration (I mM) caused a complete plant growth inhibition, whereas the lowest concentrations seemed to stimulate growth. At genome level, the amplified fragment length polymorphism (AFLP) technique was applied to detect DNA sequence changes in root cells, showing that all the Cd concentrations induced significant DNA polymorphisms in a dose-dependent manner. Data also evidenced the absence of preferential mutation sites. Plant responses were analysed by measuring the levels of gluthatione (GSH) and phytochelatins (PCs), the thiol-peptides involved in heavy metal tolerance mechanisms. Results showed a progressive increase of GSH up to 10 mu M of Cd treatment, whereas a significant induction only of PC3 was detected in roots of plants exposed to 100 mu M of Cd. As suggested by the proteome analysis of root tissues, this last concentration strongly induced the expression of regulatory proteins and some metabolic enzymes. Furthermore, the treatment with 10 mu M of Cd induced changes in metabolic enzymes, but it mainly activated defence mechanisms by the induction of transporters and proteins involved in the degradation of oxidatively modified proteins. (c) 2006 Elsevier B.V. All rights reserved.

Die vorliegende Arbeit befaßt sich mit Nachweismöglichkeiten von Helicobacter pylori und deren Vergleich. Hierfür ist eine genaue Kenntnis der Helicobacter-Proteine notwendig. Zu diesem Zweck wurde die humorale Immunantwort gegenüber Helicobacter pylori unter Anwendung der Methodik des zweidimensionalen Immunoblots analysiert. Zunächst wurden Proteine des autologen Helicobacter pylori-Stammes über zweidimensionale Gelelektrophorese aufgetrennt, auf Nitrocellulose-Membranen geblottet und sodann mit Antikörpern aus autologem Plasma sowie Antikörpern aus dem Überstand von in vitro kultiviertem autologem Biopsiematerial detektiert. Zur Bestimmung einer Helicobacter-Infektion wurden andere invasive und nicht invasive Tests genutzt. In einer prospektiven Untersuchung wurden über 200 konsekutive Patienten mit gastrointestinalen Beschwerden und unbekanntem H. pylori-Status, die für eine Gastroskopie vorgesehen waren, routinemäßig untersucht. Bei jeder Gastroskopie wurden zwei Antrum- und zwei Korpusbiopsien zur Gastritis-Diagnostik und zur Bestimmung des H. pylori-Status entnommen. Es wurden Assoziationen zwischen einer Infektion mit Helicobacter pylori und Erkrankungen wie akute und chronische Gastritis, gastraler und duodenaler Ulkus, Magenkarzinom und Folgeerkrankungen der Gastritis überprüft. Dabei wurden auch die eindeutig Helicobacter pylori-negativen Seren mit den positiven Seren verglichen. Trotz ungleichmäßiger Verteilung der Patientenzahlen über die einzelnen Krankheitsgruppen (Gastritis, Ulkus, Karzinom) wurden bestimmte Proteine nur bei einer der Erkrankungen erkannt. Einige Proteinspots kamen deutlich intensiver bei einer einzigen Krankheitsgruppe vor. Anzustreben sind Studien mit größeren Patientenzahlen innerhalb der einzelnen Krankheitsgruppen, um mögliche weitere Assoziationen bestimmter Helicobacter-Antigene mit Folgeerkrankungen zu analysieren und zu verifizieren. Ferner wurde das Vorliegen einer Assoziation des zweidimensionalen Immunoblots mit anderen i, The present study is concerned with practicable methods of detecting Helicobacter pylori and comparing them. As a precondition, a precise knowledge of the proteins of Helicobacter is necessary. To this end the humoral immune response of Helicobacter pylori was analysed by using the method of two-dimensional immuno blots. First, the proteins of each autologous Helicobacter pylori strain were separated by two-dimensional electrophoresis, then blotted onto nitrocellulose membranes and eventually detected by using antibodies from autologous plasma and antibodies taken from the overflow of in vitro cultivated autologous biopsy material. For determining a Helicobacter infection various invasive and non-invasive tests were carried out. In a prospective study on more than 200 patients with gastrointestianal disorders but unknown H. pylori status were consecutively tested. At each gastroscopy two bioptic specimen each were taken from the antrum and from the corpus region in order to determine the H. pylori status. Associations were assessed between Helicobacter pylori infections and manifestations such as acute or chronic gastritis, gastric or duodenal ulcers, gastric carcinoma and gastritis induced disorders. In the process, clearly Helicobacter pylori negative sera were also compared with positive sera. Inspite of the unequal distribution of numbers of patients over different groups of disorders (gastritis, ulcers, carcinoma) certain proteins were only detected in connection with one group of disorder. Several of the protein spots only occurred in a single group of disorders. More studies will be necessary using greater numbers of patients within each group of diseases in order to analyse and verify associations between Helicobacter antigenes and other disorders. Further, evidences of an association between two-dimensional immunoblots and other invasive and non-invasive methods of assessing H. pylori were analysed with regard to respective antigene profiles, qualitatively

Die vorliegende Arbeit befaßt sich mit Nachweismöglichkeiten von Helicobacter pylori und deren Vergleich. Hierfür ist eine genaue Kenntnis der Helicobacter-Proteine notwendig. Zu diesem Zweck wurde die humorale Immunantwort gegenüber Helicobacter pylori unter Anwendung der Methodik des zweidimensionalen Immunoblots analysiert. Zunächst wurden Proteine des autologen Helicobacter pylori-Stammes über zweidimensionale Gelelektrophorese aufgetrennt, auf Nitrocellulose-Membranen geblottet und sodann mit Antikörpern aus autologem Plasma sowie Antikörpern aus dem Überstand von in vitro kultiviertem autologem Biopsiematerial detektiert. Zur Bestimmung einer Helicobacter-Infektion wurden andere invasive und nicht invasive Tests genutzt. In einer prospektiven Untersuchung wurden über 200 konsekutive Patienten mit gastrointestinalen Beschwerden und unbekanntem H. pylori-Status, die für eine Gastroskopie vorgesehen waren, routinemäßig untersucht. Bei jeder Gastroskopie wurden zwei Antrum- und zwei Korpusbiopsien zur Gastritis-Diagnostik und zur Bestimmung des H. pylori-Status entnommen. Es wurden Assoziationen zwischen einer Infektion mit Helicobacter pylori und Erkrankungen wie akute und chronische Gastritis, gastraler und duodenaler Ulkus, Magenkarzinom und Folgeerkrankungen der Gastritis überprüft. Dabei wurden auch die eindeutig Helicobacter pylori-negativen Seren mit den positiven Seren verglichen. Trotz ungleichmäßiger Verteilung der Patientenzahlen über die einzelnen Krankheitsgruppen (Gastritis, Ulkus, Karzinom) wurden bestimmte Proteine nur bei einer der Erkrankungen erkannt. Einige Proteinspots kamen deutlich intensiver bei einer einzigen Krankheitsgruppe vor. Anzustreben sind Studien mit größeren Patientenzahlen innerhalb der einzelnen Krankheitsgruppen, um mögliche weitere Assoziationen bestimmter Helicobacter-Antigene mit Folgeerkrankungen zu analysieren und zu verifizieren. Ferner wurde das Vorliegen einer Assoziation des zweidimensionalen Immunoblots mit anderen i, The present study is concerned with practicable methods of detecting Helicobacter pylori and comparing them. As a precondition, a precise knowledge of the proteins of Helicobacter is necessary. To this end the humoral immune response of Helicobacter pylori was analysed by using the method of two-dimensional immuno blots. First, the proteins of each autologous Helicobacter pylori strain were separated by two-dimensional electrophoresis, then blotted onto nitrocellulose membranes and eventually detected by using antibodies from autologous plasma and antibodies taken from the overflow of in vitro cultivated autologous biopsy material. For determining a Helicobacter infection various invasive and non-invasive tests were carried out. In a prospective study on more than 200 patients with gastrointestianal disorders but unknown H. pylori status were consecutively tested. At each gastroscopy two bioptic specimen each were taken from the antrum and from the corpus region in order to determine the H. pylori status. Associations were assessed between Helicobacter pylori infections and manifestations such as acute or chronic gastritis, gastric or duodenal ulcers, gastric carcinoma and gastritis induced disorders. In the process, clearly Helicobacter pylori negative sera were also compared with positive sera. Inspite of the unequal distribution of numbers of patients over different groups of disorders (gastritis, ulcers, carcinoma) certain proteins were only detected in connection with one group of disorder. Several of the protein spots only occurred in a single group of disorders. More studies will be necessary using greater numbers of patients within each group of diseases in order to analyse and verify associations between Helicobacter antigenes and other disorders. Further, evidences of an association between two-dimensional immunoblots and other invasive and non-invasive methods of assessing H. pylori were analysed with regard to respective antigene profiles, qualitatively

The proteome is described as the entirety of all proteins expressed within a cell at a given moment. In contrast to the stability of the genome, the proteome is highly dynamic and reflects the cell's current status. Since proteins carry out almost all biological functions, the proteome stands in direct relation to cellular functions. Proteomic analysis (i.e., two-dimensional electrophoresis, mass spectrometry and bioinformatics) aims at identifying changes in the composition of the proteome associated to pathophysiologic events that affect basic cellular functions. Functional proteomics expands to understanding the connection between proteomic changes and the state of a cell, taken into account that the observed modifications can be either cause or consequence of the pathological state. Proteomics will not only improve our basic understanding of the factors and molecular mechanisms underlying cardiovascular disease, but also will help identifying novel diagnostic markers and fuel the rational design and discovery of new drugs for medical intervention. This review will discuss basic proteomic approaches relevant to cardiovascular disease, as well as their applications for the identification of biomarkers and drug design.

Palliative Therapie inklusive Chemotherapie und andere Behandlungsmethoden wie Hyperthermie ist oftmals die einzig verbleibende Option bei der Behandlung von bestimmten soliden Tumoren wie Magen- und Pankreaskarzinom. Leider ist der Erfolg dieser Therapien limitiert durch geringes Ansprechen der Tumoren auf die Behandlung und die Entwicklung einer Therapieresistenz. In dieser Arbeit wurde die globale Proteinexpression von chemo- und thermoresistenten Varianten der Magenkarzinomzellinie EPG85-257 und der Pankreaskarzinomzellinie EPP85-181 mit Hilfe von Proteomics (Kombination aus zweidimensionaler Elektrophorese, computergestützter Gel-Analyse und Massenspektrometrie) in vitro untersucht, um Kandidatenproteine zu finden, die potentiell mit Thermoresistenz bzw. Chemoresistenz assoziiert sind. In diesem Zusammenhang wurde eine mit Maldi-TOF kompatible Spezialsilberfärbung neu entwickelt. Es zeigte sich eine differentielle Expression einer Vielzahl von Proteinen in den thermoresistenten Zellen, darunter eine Hochregulation von Proteinen mit Chaperonaktivität aus nahezu allen subzellulären Kompartimenten sowie eine Überexpression von Enzymen des Arzneimittelmetabolismus in Zellen mit sowohl chemo- als auch thermoresistentem Phänotyp. Darüber hinaus wurden weitere Proteine identifiziert, welche hinsichtlich ihrer Beteiligung an Resistenzphänomenen im Vorfeld noch gar nicht charakterisiert worden sind (u.a. Aldehyd-Dehydrogenase 1, Transgelin, Phosphoglyceromutase)., Palliative treatment including chemotherapy and other modes of treatment, e.g. hyperthermia, is often the only remaining option in the management of certain solid tumours including gastric and pancreatic carcinoma. Unfortunately, its efficacy is poor due to low tumour sensitivity and the development of therapy resistance. The aim was to study in vitro global protein expression of chemo- and thermoresistant variants of the stomach cancer cell line EPG85-257 and the pancreatic cancer cell line EPP85-181 using proteomics (two-dimensional electrophoresis in combination with computer-assisted image analysis and mass spectrometry) to identify candidate proteins potentially associated with thermoresistance alone or in combination with chemoresistance. In this context, a special silver stain compatible with Maldi-TOF was developed. A large number of proteins was found to be differentially expressed in thermoresistant cells including up-regulation of molecular chaperones at practically every sub-cellular level as well as over-expression of enzymes involved in drug metabolism in both chemo- and thermoresistant cells. Furthermore, other proteins were identified that have not yet been linked to resistance phenomena (e.g. aldehyde dehydrogenase 1, transgelin, phosphoglycerate mutase).

Palliative Therapie inklusive Chemotherapie und andere Behandlungsmethoden wie Hyperthermie ist oftmals die einzig verbleibende Option bei der Behandlung von bestimmten soliden Tumoren wie Magen- und Pankreaskarzinom. Leider ist der Erfolg dieser Therapien limitiert durch geringes Ansprechen der Tumoren auf die Behandlung und die Entwicklung einer Therapieresistenz. In dieser Arbeit wurde die globale Proteinexpression von chemo- und thermoresistenten Varianten der Magenkarzinomzellinie EPG85-257 und der Pankreaskarzinomzellinie EPP85-181 mit Hilfe von Proteomics (Kombination aus zweidimensionaler Elektrophorese, computergestützter Gel-Analyse und Massenspektrometrie) in vitro untersucht, um Kandidatenproteine zu finden, die potentiell mit Thermoresistenz bzw. Chemoresistenz assoziiert sind. In diesem Zusammenhang wurde eine mit Maldi-TOF kompatible Spezialsilberfärbung neu entwickelt. Es zeigte sich eine differentielle Expression einer Vielzahl von Proteinen in den thermoresistenten Zellen, darunter eine Hochregulation von Proteinen mit Chaperonaktivität aus nahezu allen subzellulären Kompartimenten sowie eine Überexpression von Enzymen des Arzneimittelmetabolismus in Zellen mit sowohl chemo- als auch thermoresistentem Phänotyp. Darüber hinaus wurden weitere Proteine identifiziert, welche hinsichtlich ihrer Beteiligung an Resistenzphänomenen im Vorfeld noch gar nicht charakterisiert worden sind (u.a. Aldehyd-Dehydrogenase 1, Transgelin, Phosphoglyceromutase)., Palliative treatment including chemotherapy and other modes of treatment, e.g. hyperthermia, is often the only remaining option in the management of certain solid tumours including gastric and pancreatic carcinoma. Unfortunately, its efficacy is poor due to low tumour sensitivity and the development of therapy resistance. The aim was to study in vitro global protein expression of chemo- and thermoresistant variants of the stomach cancer cell line EPG85-257 and the pancreatic cancer cell line EPP85-181 using proteomics (two-dimensional electrophoresis in combination with computer-assisted image analysis and mass spectrometry) to identify candidate proteins potentially associated with thermoresistance alone or in combination with chemoresistance. In this context, a special silver stain compatible with Maldi-TOF was developed. A large number of proteins was found to be differentially expressed in thermoresistant cells including up-regulation of molecular chaperones at practically every sub-cellular level as well as over-expression of enzymes involved in drug metabolism in both chemo- and thermoresistant cells. Furthermore, other proteins were identified that have not yet been linked to resistance phenomena (e.g. aldehyde dehydrogenase 1, transgelin, phosphoglycerate mutase).

The reconstruction of Giardia lamblia life cycle in vitro is an excellent tool for the study of the parasite's molecular biology. The present work describes techniques developed that better define parasite differentiation. An encystation protocol is presented along with a method for isolation and purification of the produced cysts. The cyst morphology at the light microscopy level is identical to that of in vivo cysts. A two-dimension protein map obtained by high-resolution electrophoresis indicated that most of the parasite's proteins are acid. Based on this result, the two dimension gel electrophoresis used a pH 4-7 gradient in the first, isoelectric focusing dimension. Differences in protein expression during the stages of encystation were clearly discerned, as well as images of the parasite obtained by light and by transmission electron microscopy that describe the morphological and the ultrastructural changes that occur as the cysts are produced in vitro., La reconstrucción in vitro del ciclo de vida de Giardia lamblia es un excelente instrumento para el estudio de la biología molecular del parásito. El presente trabajo pretende contribuir al desarrollo de algunas técnicas por las cuales se puede definir mejor un modelo de diferenciación celular del parásito. El estudio presenta un protocolo de enquistación in vitro y establece un método para el aislamiento y la purificación de los quistes producidos, cuyas características morfológicas, por microscopía de luz, coinciden con las de los quistes obtenidos in vivo. Se estudió el mapa de las proteínas de G. lamblia por medio de electroforesis bidimensional de alta resolución y de electroforesis en una dimensión. Estos estudios mostraron que la mayoría de las proteínas del parásito son de carácter ácido. Se amplió, con base en ese resultado, la resolución de la región ácida por medio de un isoelectroenfoque de pH 4-7 en la primera dimensión. Se encontraron diferencias en la expresión de las proteínas durante el proceso de enquistación. Se obtuvieron, además, imágenes por microscopía de luz y electrónica de transmisión que permitieron observar morfológica y ultraestructuralmente las células producidas durante el proceso de la enquistación.

The Merluccidae family comprises marine species, some of them of high commercial value and others less appreciated, whose commercialization in Europe under the generic name of “hake” is highly remarkable. The potential of proteomics was employed in this study with the aim of achieving the differential characterization of five different hake species: Merluccius merluccius (European hake), M. australis (Southern hake), M. hubbsi (Argentinian hake), M. gayi (Chilean hake), and M. capensis (Cape hake), some of them very closely related. Species-specific polypeptides were observed for the five hake species studied in isoelectric focusing (IEF) and/or two-dimensional electrophoresis (2-DE) high-resolution gels. The peptide mass maps of two polypeptide groups, previously selected by 2-DE analysis as potentially species-specific, were obtained by “in-gel” tryptic digestion, followed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Analysis of group A polypeptides (with pI in the range of 5.0–5.5 and molecular mass of 17 kDa), allowed the differential classification of the hake species into two groups: the East Atlantic coast group and the West Atlantic coast group. Moreover, the peptide mass-maps from the heat-resistant parvalbumin fraction (pI below 4.5; molecular mass <12 kDa) allowed the detection of a peptide characteristic of M. australis not present in the other four hake species tested. A specific 17 kDa protein from M. merluccius was also partially sequenced by nanospray-ion trap-tandem MS, revealing a high homology with rat nucleoside diphosphate kinase A (NDKA). This work opens the way to the application of proteomics to the differential characterization of commercial hake species at the molecular leve

The water-soluble proteins of nine flat fish species of high commercial value, belonging to the Pleuronectidae, Scophtalmidae, and Soleidae families, were analyzed by two-dimensional (2D) electrophoresis, this being carried out by nondenaturing isoelectric focusing (IEF) in the 3.5–9.5 pH range and gradient SDS-PAGE in the 12–14% range. Most of the major proteins fell in the 3.5–6.9 pH range. From these, the most specific proteins were in the acidic fraction (pI~5.2; MW~16 kDa). Species-specific 2D protein patterns were observed in all the nine species, and more than 25 proteins could be individualized. The combination of nondenaturing IEF and gradient SDSPAGE, both in the absence of urea, allowed detailed characterization of both the isoelectric points and molecular weights of the major water-soluble proteins and proved to be a valuable tool for the differential characterization of the flat fish species studied.

The water-soluble proteins of nine flat fish species of high commercial value, belonging to the Pleuronectidae, Scophtalmidae, and Soleidae families, were analyzed by two-dimensional (2D) electrophoresis, this being carried out by nondenaturing isoelectric focusing (IEF) in the 3.5–9.5 pH range and gradient SDS-PAGE in the 12–14% range. Most of the major proteins fell in the 3.5–6.9 pH range. From these, the most specific proteins were in the acidic fraction (pI~5.2; MW~16 kDa). Species-specific 2D protein patterns were observed in all the nine species, and more than 25 proteins could be individualized. The combination of nondenaturing IEF and gradient SDSPAGE, both in the absence of urea, allowed detailed characterization of both the isoelectric points and molecular weights of the major water-soluble proteins and proved to be a valuable tool for the differential characterization of the flat fish species studied.

Interferons comprise a class of structurally related proteins which exert several regulatory effects in responsive cells. These effects include the establishment of an antiviral state, the inhibition of cellular proliferation and the alteration of different immune reactions. In particular, the IFN:s rapidly augment the lytic activity of the natural killer (NK) cells. In the present thesis, some of the functional and molecular mechanisms by which IFN:s act on NK cells and other leucocytes are studied. A good correlation is found between the ability of different tumor cell lines to induce IFN production among peripheral blood lymphocytes and their sensitivity to NK cell cytotoxicity, indicating that IFN might regulate the activity of NK cells through a positive feed-back mechanism. When studying the interaction between the NK cells and two target cell lines it is demonstrated that the two cell lines are not recognized by the same receptors. The augmentation of NK cell cytotoxicity by IFN is shown to involve both alteration of receptor structures on the NK cell and enhancement of steps in their lytic machinery. The effects of IFN on the synthesis of individual proteins is then studied by two-dimensional electrophoresis. It is demonstrated that IFN-a and IFN-ß within 1.5 hours induce the synthesis of nine proteins (Mf80, 75, 62, 58, 53, 38, 36, 33 and 30 kD) in human lymphocytes. Tne induction is dependent on a rapid de novo RNA synthesis, which is initiated less than 30 minutes after the addition of IFN. The expression of the nine proteins is well correlated to the development of augmented NK cell cytotoxicity. Four of the proteins (Mr 80, 62, 38 and 33 kD) are found to be expressed in a panel of ten hematopoetic and two anchorage-dependent cell lines, whereas the remaining proteins seem to be expressed in leucocytes only. IFN induce the synthesis of the same proteins in both purified large granular lymphocytes (responsible for the main NK cell activity in man), T cell, Diss. (sammanfattning) Umeå : Umeå universitet, 1985, härtill 5 uppsatser., digitalisering@umu

Interferons comprise a class of structurally related proteins which exert several regulatory effects in responsive cells. These effects include the establishment of an antiviral state, the inhibition of cellular proliferation and the alteration of different immune reactions. In particular, the IFN:s rapidly augment the lytic activity of the natural killer (NK) cells. In the present thesis, some of the functional and molecular mechanisms by which IFN:s act on NK cells and other leucocytes are studied. A good correlation is found between the ability of different tumor cell lines to induce IFN production among peripheral blood lymphocytes and their sensitivity to NK cell cytotoxicity, indicating that IFN might regulate the activity of NK cells through a positive feed-back mechanism. When studying the interaction between the NK cells and two target cell lines it is demonstrated that the two cell lines are not recognized by the same receptors. The augmentation of NK cell cytotoxicity by IFN is shown to involve both alteration of receptor structures on the NK cell and enhancement of steps in their lytic machinery. The effects of IFN on the synthesis of individual proteins is then studied by two-dimensional electrophoresis. It is demonstrated that IFN-a and IFN-ß within 1.5 hours induce the synthesis of nine proteins (Mf80, 75, 62, 58, 53, 38, 36, 33 and 30 kD) in human lymphocytes. Tne induction is dependent on a rapid de novo RNA synthesis, which is initiated less than 30 minutes after the addition of IFN. The expression of the nine proteins is well correlated to the development of augmented NK cell cytotoxicity. Four of the proteins (Mr 80, 62, 38 and 33 kD) are found to be expressed in a panel of ten hematopoetic and two anchorage-dependent cell lines, whereas the remaining proteins seem to be expressed in leucocytes only. IFN induce the synthesis of the same proteins in both purified large granular lymphocytes (responsible for the main NK cell activity in man), T cell, Diss. (sammanfattning) Umeå : Umeå universitet, 1985, härtill 5 uppsatser., digitalisering@umu

Ribosomal basic proteins of Artemia salina and rat liver were analyzed by using two different systems of tow-dimensional acrylamide gel electrophoresis. In system I, the first dimension was run at pH 8.6 and the 2nd at pH 4.9, and in system II, the first dimension at pH 8.6 and the 2nd in the presence of SDS. Artemia salina 40S proteins were separated into 27 proteins and their tow-dimensional electrophoretogram was similar to that of rat liver except that in the basic region near the origin. Therefore, individual Artemia salina 40S proteins were designated by comparing the location of proteins of Artemia salina with those of rat liver on the two kinds of two-dimensional gels. Artemia salina 60S proteins were separated into 38 proteins and their pattern of tow-dimensional electrophoretogram was somewhat different from that of rat liver. Correspondence of individual proteins between two species was more difficult as compared with the case of 40S proteins, I tentatively designated Artemia salina 60S proteins by considering the mutual locations on the two kinds of gels between Artemia and rat liver. The number average of molecular weights of 40S proteins was 18,600 for Artemia salina (27 proteins) and 18,500 for rat liver (29 proteins). That of 60S proteins for Artemia salina was 21,600 (38 proteins) and 21,800 (38 proteins) for rat liver. The averages of relative basicities of 40S proteins were similar in both species, whereras the average of those of 60S proteins of Artemia salina was lower than that of rat liver. The protein moiety of 40S ribosomes is more conservative than that of 60S ribosomes during the evolution of animal species.

Interferons comprise a class of structurally related proteins which exert several regulatory effects in responsive cells. These effects include the establishment of an antiviral state, the inhibition of cellular proliferation and the alteration of different immune reactions. In particular, the IFN:s rapidly augment the lytic activity of the natural killer (NK) cells. In the present thesis, some of the functional and molecular mechanisms by which IFN:s act on NK cells and other leucocytes are studied. A good correlation is found between the ability of different tumor cell lines to induce IFN production among peripheral blood lymphocytes and their sensitivity to NK cell cytotoxicity, indicating that IFN might regulate the activity of NK cells through a positive feed-back mechanism. When studying the interaction between the NK cells and two target cell lines it is demonstrated that the two cell lines are not recognized by the same receptors. The augmentation of NK cell cytotoxicity by IFN is shown to involve both alteration of receptor structures on the NK cell and enhancement of steps in their lytic machinery. The effects of IFN on the synthesis of individual proteins is then studied by two-dimensional electrophoresis. It is demonstrated that IFN-a and IFN-ß within 1.5 hours induce the synthesis of nine proteins (Mf80, 75, 62, 58, 53, 38, 36, 33 and 30 kD) in human lymphocytes. Tne induction is dependent on a rapid de novo RNA synthesis, which is initiated less than 30 minutes after the addition of IFN. The expression of the nine proteins is well correlated to the development of augmented NK cell cytotoxicity. Four of the proteins (Mr 80, 62, 38 and 33 kD) are found to be expressed in a panel of ten hematopoetic and two anchorage-dependent cell lines, whereas the remaining proteins seem to be expressed in leucocytes only. IFN induce the synthesis of the same proteins in both purified large granular lymphocytes (responsible for the main NK cell activity in man), T cell, Diss. (sammanfattning) Umeå : Umeå universitet, 1985, härtill 5 uppsatser., digitalisering@umu

Interferons comprise a class of structurally related proteins which exert several regulatory effects in responsive cells. These effects include the establishment of an antiviral state, the inhibition of cellular proliferation and the alteration of different immune reactions. In particular, the IFN:s rapidly augment the lytic activity of the natural killer (NK) cells. In the present thesis, some of the functional and molecular mechanisms by which IFN:s act on NK cells and other leucocytes are studied. A good correlation is found between the ability of different tumor cell lines to induce IFN production among peripheral blood lymphocytes and their sensitivity to NK cell cytotoxicity, indicating that IFN might regulate the activity of NK cells through a positive feed-back mechanism. When studying the interaction between the NK cells and two target cell lines it is demonstrated that the two cell lines are not recognized by the same receptors. The augmentation of NK cell cytotoxicity by IFN is shown to involve both alteration of receptor structures on the NK cell and enhancement of steps in their lytic machinery. The effects of IFN on the synthesis of individual proteins is then studied by two-dimensional electrophoresis. It is demonstrated that IFN-a and IFN-ß within 1.5 hours induce the synthesis of nine proteins (Mf80, 75, 62, 58, 53, 38, 36, 33 and 30 kD) in human lymphocytes. Tne induction is dependent on a rapid de novo RNA synthesis, which is initiated less than 30 minutes after the addition of IFN. The expression of the nine proteins is well correlated to the development of augmented NK cell cytotoxicity. Four of the proteins (Mr 80, 62, 38 and 33 kD) are found to be expressed in a panel of ten hematopoetic and two anchorage-dependent cell lines, whereas the remaining proteins seem to be expressed in leucocytes only. IFN induce the synthesis of the same proteins in both purified large granular lymphocytes (responsible for the main NK cell activity in man), T cell, Diss. (sammanfattning) Umeå : Umeå universitet, 1985, härtill 5 uppsatser., digitalisering@umu

This project is part of a pilot study designed to establish a two dimensional gel electrophoresis-based protocol, which can be utilized in screening mutagencity/carcinogencity of uncharacterized substances. Currently, the Ames test is the most commonly used method, which is solely based on mutation at the genome level. In contrast to the genome, which is static, protein expression in a cell under a given set of conditions (the proteome) is dynamic. A screening method based on the proteome rather than the genome may prove more reliable and quantitative in predicting mutagenicity and carcinogenicity. In this project, Pseudomonas putida KT2440 was employed as the biological model because its genome has been completely sequenced and the strain is already known to be able to grow on several aromatic carbon sources. Two carbon sources were selected, succinic acid and phenylethylamine. The strain grew in the non-aromatic and aromatic carbon source respectively. Proteins were extracted at a fixed growth stage (mid-log phase) so that protein expression is consistent. Protein expression was then analyzed by two dimensional gel electrophoresis, with the hope of identifying proteins that are expressed in response to phenylethylamine ? the proteomic signature for phenylethylamine. In total, six signature protein spots were found. In addition to identifying these signature protein spots, a major goal of this project (to establish standard protocols for protein expression and analysis) was accomplished. Based on the results obtained in this project, several directions for further improvement in later stages of the procedure are also discussed.

A new version of a two-dimensional electrophoretic catalog of proteins of the human kidney and of various morphological and functional structures of the human kidney is presented; it contains information about 179 polypeptides. The following proteins were identified: crystallin, albumin, mitochondrial Superoxide dismutase, actin, fatty acid-binding protein, alpha-ATP-synthase, and transferrin. Some protein groups are specific for certain morphological structures.

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.

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.