Inflammatory changes in the airways of mice caused by cigarette smoke exposure are only partially reversed after smoking cessation

Background

The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR), which is characterized by acute airway obstruction beginning within a few minutes. The EAR is the earliest indicator of the pathological progression of allergic asthma. Because the molecular mechanism underlying the EAR is not fully defined, this study will contribute to a better understanding of asthma.

Methods

In order to gain insight into the molecular basis of the EAR, we examined changes in protein expression patterns in the lung tissue of asthmatic rats during the EAR using 2-DE/MS-based proteomic techniques. Bioinformatic analysis of the proteomic data was then performed using PPI Spider and KEGG Spider to investigate the underlying molecular mechanism.

Results

In total, 44 differentially expressed protein spots were detected in the 2-DE gels. Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry. During subsequent bioinformatic analysis, the gene ontology classification, the protein-protein interaction networking and the biological pathway exploration demonstrated that the identified proteins were mainly involved in glycolysis, calcium binding and mitochondrial activity. Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses.

Conclusions

Our results reveal that the allergen-induced EAR in asthmatic rats is associated with glycolysis, calcium binding and mitochondrial activity, which could establish a functional network in which calcium binding may play a central role in promoting the progression of asthma.     

Highly sensitive proximity mediated immunoassay reveals HER2 status conversion in the circulating tumor cells of metastatic breast cancer patients

Background

Strawberries (Fragaria ananassa) reproduce asexually through stolons, which have strong tendencies to form adventitious roots at their second node. Understanding how the development of the proximal (I-1) and distal (I-2) internodes of stolons differ should facilitate nursery cultivation of strawberries.

Results

Herein, we compared the proteomic profiles of the strawberry stolon I-1 and I-2 internodes. Proteins extracted from the internodes were separated by two-dimensional gel electrophoresis, and 164 I-1 protein spots and 200 I-2 protein spots were examined further. Using mass spectrometry and database searches, 38 I-1 and 52 I-2 proteins were identified and categorized (8 and 10 groups, respectively) according to their cellular compartmentalization and functionality. Many of the identified proteins are enzymes necessary for carbohydrate metabolism and photosynthesis. Furthermore, identification of proteins that interact revealed that many of the I-2 proteins form a dynamic network during development. Finally, given our results, we present a mechanistic scheme for adventitious root formation of new clonal plants at the second node.

Conclusions

Comparative proteomic analysis of I-1 and I-2 proteins revealed that the ubiquitin-proteasome pathway and sugar-hormone pathways might be important during adventitious root formation at the second node of new clonal plants.     

Differential proteome analysis of mature and germinated seeds of Magnolia sieboldii K. Koch

Key message

This is the first reported proteomic analysis to study the dormancy breaking of Magnolia sieboldii seeds. Our results provide a fundamental reference for further studies on the regulation of protein expression during seed germination.

Abstract

Magnolia sieboldii K. Koch is an ornamental tree. The deep dormancy of its seeds hinders its cultivation for economic purposes. The biochemical basis of the regulation of seed germination remains poorly understood. The present study aimed to identify differentially expressed proteins in germinated seeds of M. sieboldii using polyethylene glycol fractionation. In total, 59 differentially expressed protein spots from two-dimensional gel maps were detected, 33 of which were identified by mass spectrometry. They were assigned to eight functional classes on the basis of their putative biological functions: photosynthesis (3 %), chaperonin/heat shock protein (9 %), protein and amino acid synthesis (9 %), stress/defense (18 %), cytoskeleton structure (3 %), metabolism (18 %), hormone and polyamine (9 %) and storage proteins (31 %). Among the other functions, the effects of plant hormones on seed germination may be one of the most important functions in plant growth. Gibberellins and ethylene positively regulate seed germination. The activities of several hormone-associated proteins possibly influencing seed germination were increased. The characterization of these proteins will be of great help in identifying the molecular mechanism underlying seed germination.     

Effects of human and porcine bile on the proteome of Helicobacter hepaticus

Background

Helicobacter hepaticus colonizes the intestine and liver of mice causing hepatobiliary disorders such as hepatitis and hepatocellular carcinoma, and has also been associated with inflammatory bowel disease in children. In its habitat, H. hepaticus must encounter bile which has potent antibacterial properties. To elucidate virulence and host-specific adaptation mechanisms of H. hepaticus modulated by human or porcine bile, a proteomic study of its response to the two types of bile was performed employing two-dimensional gel electrophoresis (2-DE) and mass spectrometry.

Results

The 2-DE and mass spectrometry analyses of the proteome revealed that 46 proteins of H. hepaticus were differentially expressed in human bile, 18 up-regulated and 28 down-regulated. In the case of porcine bile, 32 proteins were differentially expressed of which 19 were up-regulated, and 13 were down-regulated. Functional classifications revealed that identified proteins participated in various biological functions including stress response, energy metabolism, membrane stability, motility, virulence and colonization. Selected genes were analyzed by RT-PCR to provide internal validation for the proteomic data as well as provide insight into specific expressions of motility, colonization and virulence genes of H. hepaticus in response to human or porcine bile.

Conclusions

Overall, the data suggested that bile is an important factor that determines virulence, host adaptation, localization and colonization of specific niches within host environment.     

Proteomic analysis of copper stress responses in the roots of two rice (Oryza sativa L.) varieties differing in Cu tolerance

Background and aims

Copper (Cu) is an essential micronutrient required for growth and development of plants. However, excess Cu is toxic to plants. To understand the mechanisms involved in copper stress response, a proteomic approach was used to investigate the differences in Cu stress-induced protein expression between a Cu-tolerant variety (B1139) and a Cu-sensitive one (B1195) of rice.

Methods

Rice seedlings were exposed to 8 μM Cu for 3 days, with plants grown in the normal nutrient solution containing 0.32 μM Cu serving as the control. Proteins were extracted from the roots and separated by two-dimensional PAGE. Thirty four proteins were identified using MALDI-TOF mass spectrometry.

Results

Thirty-four protein spots were found to be differently expressed in the Cu-stressed roots in at least one variety of rice, including those involved in antioxidative defense, redox regulation, stress response, sulfur and glutathione (GSH) metabolism, carbohydrate metabolism, signal transduction, and some other proteins with various functions. Nine proteins, including putative cysteine synthase, probable serine acetyltransferase 3, L-ascorbate peroxidase 1, putative glutathione S-transferase 2, and thioredoxin-like 3-3, exhibited a greater increase in response to Cu stress in the Cu-tolerant variety B1139 compared with the Cu–sensitive variety B1195.

Conclusion

The majority of the proteins showing differential expression in response to Cu exposure are involved in the redox regulation, and sulfur and GSH metabolism, suggesting that these proteins, together with antioxidant enzymes, play an important role in the detoxification of excess Cu and maintaining cellular homeostasis.     

Characterization of the Human Cervical Mucous Proteome

Introduction

Cervical cancer is among the most common cancers in women worldwide. Discovery of biomarkers for the early detection of cervical cancer would improve current screening practices and reduce the burden of disease.

Objective

In this study, we report characterization of the human cervical mucous proteome as the first step towards protein biomarker discovery.

Methods

The protein composition was characterized using one- and two-dimensional gel electrophoresis, and liquid chromatography coupled with mass spectrometry. We chose to use this combination of traditional biochemical techniques and proteomics to allow a more comprehensive analysis.

Results and Conclusion

A total of 107 unique proteins were identified, with plasma proteins being most abundant. These proteins represented the major functional categories of metabolism, immune response, and cellular transport. Removal of high molecular weight abundant proteins by immunoaffinity purification did not significantly increase the number of protein spots resolved. We also analyzed phosphorylated and glycosylated proteins by fluorescent post-staining procedures. The profiling of cervical mucous proteins and their post-translational modifications can be used to further our understanding of the cervical mucous proteome.     

18O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Carcinoma

Introduction

Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers.

Materials and Methods

In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent ¹⁸O/¹⁶O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to ¹⁸O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results.

Conclusion

This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.     

Dynamic expression of synemin isoforms in mouse embryonic stem cells and neural derivatives

Background

Nucleolus is the most prominent mammalian organelle within the nucleus which is also the site for ribosomal biogenesis. There have been many reports indicating the involvement of nucleolus in the process of aging. Several proteins related to aging have been shown to localize in the nucleolus, which suggests the role of this organelle in senescence.

Results

In this study, we used quantitative mass spectrometry to map the flux of proteins into and out of the nucleolus during the induction of senescence in cultured mammalian cells. Changes in the abundance of 344 nucleolar proteins in sodium butyrate-induced senescence in NIH3T3 cells were studied by SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry. Biochemically, we have validated the proteomic results and confirmed that B23 (nucleophosmin) protein was down-regulated, while poly (ADP-ribose) polymerase (PARP) and nuclear DNA helicase II (NDH II/DHX9/RHA) were up-regulated in the nucleolus upon treatment with sodium butyrate. Accumulation of chromatin in the nucleolus was also observed, by both proteomics and microscopy, in sodium butyrate-treated cells. Similar observations were found in other models of senescence, namely, in mitoxantrone- (MTX) treated cells and primary fibroblasts from the Lamin A knockout mice.

Conclusion

Our data indicate an extensive nuclear organization during senescence and suggest that the redistribution of B23 protein and chromatin can be used as an important marker for senescence.     

Effect of glutathione reductase knockdown in response to UVB-induced oxidative stress in human lung adenocarcinoma

Background

Glutathione reductase (GR) plays a critical role in the maintenance of physiological redox status in cells. However, the comprehensive investigations of GR-modulated oxidative stress have not been reported.

Methods

In the present study, we cultured a human lung adenocarcinoma line CL1-0 and its GR-knockdown derivative CL1-0ΔGR to evaluate their differential responses to UVB-irradiation.

Results

We identified 18 proteins that showed significant changes under UVB-irradiation in CL1-0ΔGR cells rather than in CL1-0 cells. Several proteins involving protein folding, metabolism, protein biosynthesis and redox regulation showed significant changes in expression.

Conclusions

In summary, the current study used a comprehensive lung adenocarcinoma-based proteomic approach for the identification of GR-modulated protein expression in response to UVB-irradiation. To our knowledge, this is the first global proteomic analysis to investigate the role of GR under UVB-irradiation in mammalian cell model.     

Cloning mature holm oak trees by somatic embryogenesis

Key message

Integuments from holm oak developing ovules were suitable initial explants to obtain embryogenic lines from which plants could be regenerated.

Abstract

The implementation of multivarietal forestry as part of breeding strategies is expected to provide more productive forest plantations. To achieve this, a reliable and effective method for mass production of clonal plants is needed. Somatic embryogenesis is considered the enabling technology for implementing multivarietal forestry. The holm oak (Quercus ilex L.) is a Mediterranean evergreen tree of economic interests because of the acorn production for animal feed and edible fungi mycorrhization. The aim of this work was to obtain clonal plants by inducing somatic embryogenesis in tissues of female flowers from mature trees. The influence of the developmental stage of the explant, the genotype and medium composition, and the effect of arabinogalactan proteins on the induction frequency, were assessed. Somatic embryogenesis induction (frequency ranging from 1.2 to 3.2 %) was restricted to ovules excised at an advanced stage of development, when they were at least 3–4 mm wide and the rest of the ovules within the ovary had aborted. Somatic embryos arose from the integuments of those fertilized ovules. Embryogenic response was obtained on media with and without plant growth regulators. All the genotypes that were cultured on medium containing “as reported by Schenk and Hildebrandt (Can J Bot 50:199–204, 1972)” SH macronutrients could be captured. Treatments including Larix arabinogalactan proteins did not improve induction, while those from Acacia inhibited the embryogenic response. Several embryogenic lines were multiplied by repetitive embryogenesis on medium lacking plant growth regulators. Mature somatic embryos of three genotypes were germinated at frequencies ranging from 41 to 58 %, and converted into plants at frequencies from 11 to 30 %, depending on the genotype.     

ITRAQ-based quantitative proteomic analysis of processed <Emphasis Type="Italic">Euphorbia lathyris</Emphasis> L. for reducing the intestinal toxicity

Background

Euphorbia lathyris L., a Traditional Chinese medicine (TCM), is commonly used for the treatment of hydropsy, ascites, constipation, amenorrhea, and scabies. Semen Euphorbiae Pulveratum, which is another type of Euphorbia lathyris that is commonly used in TCM practice and is obtained by removing the oil from the seed that is called paozhi, has been known to ease diarrhea. Whereas, the mechanisms of reducing intestinal toxicity have not been clearly investigated yet.

Methods

In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic method was applied to investigate the effects of Euphorbia lathyris L. on the protein expression involved in intestinal metabolism, in order to illustrate the potential attenuated mechanism of Euphorbia lathyris L. processing. Differentially expressed proteins (DEPs) in the intestine after treated with Semen Euphorbiae (SE), Semen Euphorbiae Pulveratum (SEP) and Euphorbiae Factor 1 (EFL₁) were identified. The bioinformatics analysis including GO analysis, pathway analysis, and network analysis were done to analyze the key metabolic pathways underlying the attenuation mechanism through protein network in diarrhea. Western blot were performed to validate selected protein and the related pathways.

Results

A number of differentially expressed proteins that may be associated with intestinal inflammation were identified. They mainly constituted by part of the cell. The expression sites of them located within cells and organelles. G protein and Eph/Ephrin signal pathway were controlled jointly by SEP and SE. After processing, the extraction of SEP were mainly reflected in the process of cytoskeleton, glycolysis and gluconeogenesis.

Conclusions

These findings suggest that SE induced an inflammatory response, and activated the Interleukin signaling pathway, such as the Ang/Tie 2 and JAK2/ STAT signaling pathways, which may eventually contribute to injury result from intestinal inflammatory, while SEP could alleviate this injury by down-regulating STAT1 and activating Ang-4 that might reduce the inflammatory response. Our results demonstrated the importance of Ang-4 and STAT1 expression, which are the target proteins in the attenuated of SE after processing based on proteomic investigation. Thus iTRAQ might be a novel candidate method to study scientific connotation of hypothesis that the attenuated of SE after processing expressed lower toxicity from cellular levels.

     

Identification of differentially regulated proteins in a patient with Leber's Congenital Amaurosis – a proteomic study

Background

Proteomics continues to play a critical role in post-genomic science as continued advances in mass spectrometry and analytical chemistry support the separation and identification of increasing numbers of peptides and proteins from their characteristic mass spectra. In order to facilitate the sharing of this data, various standard formats have been, and continue to be, developed. Still not fully mature however, these are not yet able to cope with the increasing number of quantitative proteomic technologies that are being developed.

Results

We propose an extension to the PRIDE and mzData XML schema to accommodate the concept of multiple samples per experiment, and in addition, capture the intensities of the iTRAQ ^TM reporter ions in the entry. A simple Java-client has been developed to capture and convert the raw data from common spectral file formats, which also uses a third-party open source tool for the generation of iTRAQ ^TM reported intensities from Mascot output, into a valid PRIDE XML entry.

Conclusion

We describe an extension to the PRIDE and mzData schemas to enable the capture of quantitative data. Currently this is limited to iTRAQ ^TM data but is readily extensible for other quantitative proteomic technologies. Furthermore, a software tool has been developed which enables conversion from various mass spectrum file formats and corresponding Mascot peptide identifications to PRIDE formatted XML. The tool represents a simple approach to preparing quantitative and qualitative data for submission to repositories such as PRIDE, which is necessary to facilitate data deposition and sharing in public domain database. The software is freely available from http://www.mcisb.org/software/PrideWizard.     

Biomarkers for ragwort poisoning in horses: identification of protein targets

Background

Ingestion of the poisonous weed ragwort (Senecio jacobea) by horses leads to irreversible liver damage. The principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. In this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods.

Results

One dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. Using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin.

Conclusion

These findings demonstrate that pyrrolic metabolites can modify equine plasma proteins. The high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole. This model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock.