Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells. Similarly, hydrogen peroxide induces VEGF synthesis and recent studies indicate the involvement of ROS in signaling downstream of VEGF stimulation. VEGF synthesis can not only be enhanced by gene transfer of VEGF but also by overexpression of NO synthase genes. Here, we examined the possibility of augmentation of VEGF production by gene transfer of copper/zinc superoxide dismutase (CuZnSOD, SOD1). Overexpression of human SOD1 in mouse NIH 3T3 fibroblasts increased SOD activity, enhanced intracellular generation of H2O2 and significantly stimulated VEGF production as determined by increase in VEGF promoter activity, VEGF mRNA expression and VEGF protein synthesis. The stimulatory effect on VEGF synthesis induced by SOD1 gene transfer was reverted by overexpression of human catalase. The effect of H2O2 produced by engineered cells is mediated by activation of hypoxia-inducible factor response element (HRE) as well as Sp1 recognition site of VEGF promoter. This data suggest the feasibility of stimulation of angiogenesis by overexpression of SOD1.

Introduction: The identification of mutations of the JAK2 gene is a useful marker in the diagnosis of polycythemia vera (PV) patients. We studied the frequency of JAK2 mutations in a group of PV patients because data are still very limited regarding this subject in Polish patients. Methods: The JAK2 V617F mutation was examined using the amplification refractory mutation system (ARMS)-PCR method. Direct sequencing and a cloning technique were performed to determine alternations in exon 12 of the JAK2 gene. Results: A group of 90 consecutive patients with a suspected diagnosis of polycythemia vera were investigated. In 91% of the cases, the JAK2 V617F mutation was identified. The remaining JAK2 V617F-negative patients were subjected to examination for JAK2 exon 12 by direct PCR product sequencing and the cloning technique. The following mutations were identified: H538-K539delinsL, E543-D544del and N542-E543del. These exon 12 mutants constituted 50% of PV JAK2 V617F-negative group and 4.4% (out of 90) of all PV patients (JAK2 V617F-positive and JAK2 V617F-negative). Conclusion: Our results demonstrate the prevalence of JAK2 mutations (V617F and in exon 12) in PV cases. Moreover, the data show that direct sequencing is not an adequate technique for exon 12 mutation identification; therefore, appropriate methodology should be considered for using this molecular marker in the process of diagnosis.

The predictive capability of the retention time prediction model based on quantitative structure-retention relationships (QSRR) was tested. QSRR model was derived with the use of set of peptides identified with the highest scores and originated from 8 known proteins annotated as model ones. The predictive ability of the QSRR model was verified with the use of a Bacillus subtilis proteome digest after separation and identification of the peptides by LC-ESI-MS/MS. That ability was tested with three sets of testing peptides assigned to the proteins identified with different levels of confidence. First, the set of peptides identified with the highest scores achieved in the search were considered. Hence, proteins identified on the basis of more than one peptide were taken into account. Furthermore, proteins identified on the basis of just one peptide were also considered and, depending on the possessed scores, both above and below the assumed threshold, were analyzed in two separated sets. The QSRR approach was applied as the additional constraint in proteomic research verifying results of MS/MS ion search and confirming the correctness of the peptides identifications along with the indication of the potential false positives.

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The exposure of HeLa cells to interleukin-1 alpha (IL-1α) in the presence of cycloheximide (CHX) leads to the release of active tumor necrosis factor alpha (TNF-α), eliciting cytocidal effect on these cells. A mass spectrometry (MS)-based analysis of the qualitative proteomic profiles of the HeLa cells treated only with IL-1α, CHX or simultaneously with IL-1α and CHX, in comparison to an untreated control, enabled to distinguish protein candidates possibly involved in this process. Among them protein disulphide isomerase (PDI) seemed to be particularly interesting for further research. Therefore, we focused on quantitative changes of PDI levels in HeLa cells subjected to IL-1α and CHX. Enzyme-linked immunosorbent assay (ELISA) was employed for determination of PDI concentrations in the investigated, differently treated HeLa cells. The obtained results confirmed up-regulation of PDI only in the cells stimulated with IL-1α alone. In contrary, the PDI levels in HeLa cells exposed to both IL-1α and CHX, where apoptotic process was intensive, did not increase significantly. Finally, we discuss how different expression levels of PDI together with other proteins, which were detected in this study, may influence the induction of cytotoxic effect and modulate sensitivity to cytotoxic action of IL1.Graphical Abstract Electronic supplementary materialThe online version of this article (doi:10.1007/s10337-017-3382-3) contains supplementary material, which is available to authorized users.