Jiří Stulík

Mapping of immunoreactive antigens of Francisella tularensis live vaccine strain

Francisella tularensis subsp. holarctica is the common causal agent of tularemia in Europe. Besides clinical signs, the diagnosis of the disease mostly depends on serological tests. To date, there is a lack of information about the F. tularensis antigens that induce antibody response. Therefore, we have started comprehensive mapping of immunoreactive antigens using the attenuated live vaccine strain of F. tularensis LVS originating from the European virulent strain. For this purpose, the immunoreactivity of sera collected from patients suffering from tularemia, together with the control sera of patients with Lyme disease and healthy blood donors, were examined by means of one‐dimensional and two‐dimensional immunoblotting. Furthermore, whole cell bacterial lysates, isolated integral membrane proteins and basic proteins were exploited as antigens. By this approach more than 80 different immunorelevant antigens were detected. Most of them came from whole cell bacterial lysate and integral membrane proteins. Conversely, only a negligible reaction was found in the case of basic proteins. Forty‐five spots were further selected for mass spectrometric analyses and 22 of them were annotated. Among the spots that provided characteristic reactions with sera from patients with tularemia, 60 kDa and 10 kDa chaperonins that occurred in several charge and mass variants, predominated.

Protein abundance alterations in matched sets of macroscopically normal colon mucosa and colorectal carcinoma

Our current results, aimed at the detection of protein abundance alterations that could be associated with the process of colon tumorigenesis, are summarized. The matched sets of macroscopically normal colon mucosa and colorectal carcinoma were examined by a one‐ or two‐dimensional electrophoretic approach and proteins were identified using immunoblotting or mass spectrometry. The following results were observed: The levels of liver fatty acid‐binding protein, actin‐binding protein/smooth muscle protein 22‐alpha and cyclooxygenase 2 were downregulated in colorectal carcinoma compared to normal colon mucosa. Conversely, the expression of a novel variant of heat shock protein70 and several members of the S100 protein family of calcium‐binding proteins (two isoforms of S100A9, S100A8, S100A11 and S100A6) were upregulated in transformed colon mucosa. Despite the variations of the levels of expression of given protein among analyzed samples, all quantitative changes were found to be sta tistically significant (Mann‐Whitney test assuming p ≤ 0.05). We conclude that the proteomic approach is useful for the study of complex biological events underlying the process of colorectal tumorigenesis.

Differential expression of the Ca2+ binding S100A6 protein in normal, preneoplastic and neoplastic colon mucosa

The expression of calcium-binding protein S100A6 was investigated in normal colon tissue, in colon adenomas and in colorectal carcinomas. Using an immunoblotting approach we detected four S100A6 variants with Mwt of 10 kDa and pI of 5.05 (isoform I), 5.15 (isoform II), 5.23 (isoform III) and 5.32 (isoform IV) that were differentially expressed in the analysed samples. The quantitative examination of S100A6 variant expression in 25 pairs of colorectal carcinoma and matched control mucosa proved a statistically significant increased abundance of S100A6 isoforms I (P = 0.004) and III (P = 0.025) in malignant tissue, and conversely, an increased level of S100A6 isoform IV in healthy tissue (P = 0.022). The expression of isoforms I and III and the loss of isoform IV were also observed in colon cancer cell lines. In addition, the immunohistochemical study of 16 primary colorectal carcinomas revealed both in the non-paired Student t-test and in the Mann Whitney test the statistically significant accumulation of S100A6 protein (P < 0.001) in the invasive margin of the tumour. The immunohistochemical analysis of S100A6 protein in polyps differing in clinical severity gave a strong staining that was maximal in dysplastic lesions. Thus, our results indicate a possible, statistically significant correlation (non-paired Student t-test P = 0.036) between S100A6 expression and colon carcinoma progression.

Proteomic insights into chronic anthracycline cardiotoxicity.

Chronic anthracycline cardiotoxicity is a feared complication of cancer chemotherapy. However, despite several decades of primarily hypothesis-driven research, the molecular basis of this phenomenon remains poorly understood. The aim of this study was to obtain integrative molecular insights into chronic anthracycline cardiotoxicity and the resulting heart failure. Cardiotoxicity was induced in rabbits (daunorubicin 3mg/kg, weekly, 10weeks) and changes in the left ventricular proteome were analyzed by 2D-DIGE. The protein spots with significant changes (p<0.01, >1.5-fold) were identified using MALDI-TOF/TOF. Key data were corroborated by immunohistochemistry, qRT-PCR and enzyme activity determination and compared with functional, morphological and biochemical data. The most important alterations were found in mitochondria - especially in proteins crucial for oxidative phosphorylation, energy channeling, antioxidant defense and mitochondrial stress. Furthermore, the intermediate filament desmin, which interacts with mitochondria, was determined to be distinctly up-regulated and disorganized in its expression pattern. Interestingly, the latter changes reflected the intensity of toxic damage in whole hearts as well as in individual cells. In addition, a marked drop in myosin light chain isoforms, activation of proteolytic machinery (including the proteasome system), increased abundance of chaperones and proteins involved in chaperone-mediated autophagy, membrane repair as well as apoptosis were found. In addition, dramatic changes in proteins of basement membrane and extracellular matrix were documented. In conclusion, for the first time, the complex proteomic signature of chronic anthracycline cardiotoxicity was revealed which enhances our understanding of the basis for this phenomenon and it may enhance efforts in targeting its reduction.

Construction of a Francisella tularensis two-dimensional electrophoresis protein database.

We have started the construction of a two‐dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two‐dimensional gel electrophoresis with immobilized pH 3–10 gradient in the first dimension and 9–16% gradient or tricine SDS‐PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Towards proteome database of Francisella tularensis.

The accessibility of the partial genome sequence of Francisella tularensis strain Schu 4 was the starting point for a comprehensive proteome analysis of the intracellular pathogen F. tularensis. The main goal of this study is identification of protein candidates of value for the development of diagnostics, therapeutics and vaccines. In this review, the current status of 2-DE F. tularensis database building, approaches used for identification of biologically important subsets of F. tularensis proteins, and functional and topological assignments of identified proteins using various prediction programs and database homology searches are presented.

Francisella tularensis Strain LVS Resides in MHC II-Positive Autophagic Vacuoles in Macrophages

TheFrancisella tularensis strain LVS phagosome disintegrates during the first few hours after bacterial entry and microbes are released to the cytosol. Within 12 h both rapid multiplication of microbes and a steep increase of apoptosis of infected macrophages occur. We searched for signals involved in the death of macrophages and detected molecules associated with the autophagy machinery cathepsin D, PTEN, p53 and LC3, whose levels or modification were influenced by ongoingin vitro tularemic infection. The sequestration of cytoplasmicF. tularensis LVS into autophagosomes was confirmed by co-localization of the LVS strain containing vacuoles with LC3 (an autophagosomal marker). We also demonstrated the presence of MHC II antigens in these autophagosomes, indicating that they might act as a source of endogenous tularemic antigens for presentation to CD4+ T lymphocytes.

Overexpression of calcium-binding protein calgranulin B in colonic mucosal diseases

Subtractive two-dimensional gel electrophoresis (2-DE) has been used for the study of the protein patterns of the normal colonic mucosa and the specimens collected from patients diagnosed for inflammatory bowel disease (IBD), colonic polyps and colorectal cancer. We found a 13 kDa protein that was detected in five of seven adenomas and in 13 of 15 colorectal carcinomas while it was absent or only slightly expressed in normal colonic mucosa. Furthermore, this protein occurred in all specimens collected from patients suffering from IBD and its quantity reflected the increased severity of inflammation. The combination of microsequencing and mass spectrometry led to the identification of the 13 kDa spot as calgranulin B. Our results indicate that the production of calgranulin B is unregulated in inflammatory, preneoplastic and neoplastic lesions of colonic mucosa.

Protein Changes in HL60 Leukemia Cells Associated with 5-Aminolevulinic Acid-based Photodynamic Therapy. Early Effects on Endoplasmic Reticulum Chaperones ¶

Abstract Using two-dimensional electrophoresis we investigated the effect of 5-aminolevulinic acid (ALA)–based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm2) on the protein expression in HL60 leukemia cells. ALA-PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA-induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin presursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix-assisted laser desorption and ionization-mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca2+-binding chaperones and/or Ca2+ signaling in ALA-PDT mediated cytotoxicity.

Comparison of the abundance of 10 radiation‐induced proteins with their differential gene expression in L929 cells

Purpose: To determine whether radiation‐induced changes in protein abundance can be correlated with their differential gene expression in a murine fibroblast L929 cell line. Materials and methods: L929 cells were irradiated with 6 Gy. Cell lysates were collected at different points in time (20 min, 12, 24, 36, 48 and 72 h). The extracted proteins were separated by two‐dimensional gel electrophoresis and quantified using computerized image analysis. Proteins exhibiting a differential expression equal to or more than twofold were identified by mass spectrometry following trypsin digestion. From these, 10 proteins characterized by large changes of radiation‐induced abundance were selected in order to measure their corresponding gene expression using RTQ‐PCR (real‐time quantitative polymerase chain reaction). Results: Up to 15‐fold changes in the abundance of these 10 proteins were associated with no detectable changes more than twofold on the gene expression level. However, one gene (VEGF‐D) showed a significant (p=0.005) up‐regulation (1.8‐fold). Conclusions: Deducing protein abundance from mRNA expression levels and vice versa appears to be of limited use. Furthermore, examination of transcriptional and translational changes provides different but complementary information.