Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter

<p>Abstract</p> <p>Background</p> <p>Methionine sulfoxide reductases (Msrs) are enzymes that catalyze the reduction of oxidized methionine residues. Most organisms that were genetically modified to lack the <it>MsrA </it>gene have shown shortening of their life span. Methionine sulfoxide reductases B (MsrB) proteins codified by three separate genes, named <it>MsrB1</it>, <it>MsrB2</it>, and <it>MsrB3</it>, are included in the Msrs system. To date, the mechanisms responsible for the transcriptional regulation of <it>MsrB </it>genes have not been reported. The aim of this study was to investigate the regulation of MsrB1 selenoprotein levels through transcriptional regulation of the <it>MsrB1 </it>gene in MDA-MB231 and MCF-7 breast carcinoma cell lines.</p> <p>Results</p> <p>A <it>MsrB1 </it>gene promoter is located 169 base pairs upstream from the transcription start site. It contains three Sp1 binding sites which are sufficient for maximal promoter activity in transient transfection experiments.</p> <p>High levels of MsrB1 transcript, protein and promoter activity were detected in low metastatic MCF7 human breast cancer cells. On the contrary, very low levels of both MsrB1 transcript and promoter activity were detected in the highly metastatic counterpart MDA-MB231 cells.</p> <p>A pivotal role for Sp1 in the constitutive expression of the <it>MsrB1 </it>gene was demonstrated through transient expression of mutant <it>MsrB1 </it>promoter-reporter gene constructs and chromatin immunoprecipitation experiments.</p> <p>Since Sp1 is ubiquitously expressed, these sites, while necessary, are not sufficient to explain the patterns of gene expression of <it>MsrB1 </it>in various human breast cancer cells. MDA-MB231 cells can be induced to express MsrB1 by treatment with 5-Aza-2'-deoxycytidine, a demethylating agent. Therefore, the <it>MsrB1 </it>promoter is controlled by epigenetic modifications.</p> <p>Conclusion</p> <p>The results of this study provide the first insights into the transcriptional regulation of the human <it>MsrB1 </it>gene, including the discovery that the Sp1 transcription factor may play a central role in its expression. We also demonstrated that the <it>MsrB1 </it>promoter activity appears to be controlled by epigenetic modifications such as methylation.</p

Training Program at Medical School of Chieti, Italy

We describe the changes in medical training program offered at the G. D’Annunzio University Medical School in Chieti-Pescara, Italy, which took place over the last decade. The new curriculum differs from the previous one in several important aspects, including limited number of students admitted to school depending on the estimated needs for physicians, obligatory class attendance, student attendance in preclinical laboratories, formative credits as a measure of student activity, and elective subjects. Furthermore, all medical graduates are allowed to take the State exam to obtain the license to practice, which was not the case previously. As a result of these major changes, a higher number of students graduates in due time. The changes made in the medical education curriculum in Italy have enabled Italian medical graduates to work in European Community Hospitals, because their medical degree is recognized in other EU countries. The main motif that drives the Medical School in Chieti-Pescara is the achievement of high quality in medical education and biomedical research by creating as strong a relationship between education and research as possible

Training Program at Medical School of Chieti, Italy

We describe the changes in medical training program offered at the G. D’Annunzio University Medical School in Chieti-Pescara, Italy, which took place over the last decade. The new curriculum differs from the previous one in several important aspects, including limited number of students admitted to school depending on the estimated needs for physicians, obligatory class attendance, student attendance in preclinical laboratories, formative credits as a measure of student activity, and elective subjects. Furthermore, all medical graduates are allowed to take the State exam to obtain the license to practice, which was not the case previously. As a result of these major changes, a higher number of students graduates in due time. The changes made in the medical education curriculum in Italy have enabled Italian medical graduates to work in European Community Hospitals, because their medical degree is recognized in other EU countries. The main motif that drives the Medical School in Chieti-Pescara is the achievement of high quality in medical education and biomedical research by creating as strong a relationship between education and research as possible

Inhibition of P-glycoprotein-mediated multidrug resistance by unfractionated heparin: a new potential chemosensitizer for cancer therapy.

Anticoagulant treatment with heparins is frequently used to prevent venous thromboembolism in cancer patients. In the present study, we investigated the ability of unfractionated heparin (UFH) to inhibit P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) on human breast cancer cell line (MDA-MB231) and its doxo-resistant subline. Results were a compared to the classic reversing agent, Verapamil (Ver), used, as reference at 50 microM concentration. We analysed the Pgp function by calcein acetoxymethylester (calcein-AM) uptake, a fluorescent marker substrate, before and after in vitro exposure to UFH at clinically achievable dose of 20 U/ml. The mean percentage of calcein-AM retained into cancer cells after 3 and 12 h were 32 +/- 10.9 and 45 +/- 12.3, respectively, for UFH pretreated cells and 25.3 +/- 8.7 and 29.4 +/- 10.4, respectively, for Ver pretreated cells when compared to control cells, receiving only medium. Pgp activity was studied by measuring intracellular drug accumulation in doxo-resistant subline, treated (2 h) with either UFH or Ver, prior exposure (2 h) at different doxo concentrations (2, 4 and 8 microM). The mean percentage of remaining intracellular doxo were 55.4 +/- 4.5 , 51.4 +/- 3.9 and 50 +/- 1.8 percent, respectively for UFH treated cells, and 44.1 +/- 5.8, 39.3 +/- 4.4 and 19.4 +/- 8.6%, respectively, for Ver treated cells as compared with control cells, receiving only doxo. These results were consistent with the increase of sensitivity to doxo of the same doxo-resistant subline resulting in a 2.2, 2.6 and 2.2-fold increase, respectively, for UFH-doxo combination and 2.2, 2.5 and 2.0-fold respectively, for Ver-doxo combination respect to cells receiving doxo alone, as assessed by MTT test. In conclusion, these findings demonstrate the potentiating effect in vitro of UFH on doxo accumulation and cytotoxicity in the MDA-231 cell line and its doxo-resistant subline and suggest that UFH could to be used, as an potential chemosensitizer, in clinical chemotherapy for increasing in vivo, the efficacy of the anticancer treatment

Proteome analysis of human Wharton's jelly cells during in vitro expansion

<p>Abstract</p> <p>Background</p> <p>The human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Wharton's jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine.</p> <p>Results</p> <p>To better understand their self-renewal and potential <it>in vitro </it>expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2^ndpassage of <it>in vitro </it>replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of <it>in vitro </it>growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time.</p> <p>Conclusions</p> <p>Our work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine.</p

An integrated metabolomics approach for the research of new cerebrospinal fluid biomarkers of multiple sclerosis

(1) Lipid profiling in MuS and OND patients. (2) Search of alterations associated with MuS. (3) Characterization of differences

LIMPIC: a computational method for the separation of protein MALDI-TOF-MS signals from noise

BACKGROUND: Mass spectrometry protein profiling is a promising tool for biomarker discovery in clinical proteomics. However, the development of a reliable approach for the separation of protein signals from noise is required. In this paper, LIMPIC, a computational method for the detection of protein peaks from linear-mode MALDI-TOF data is proposed. LIMPIC is based on novel techniques for background noise reduction and baseline removal. Peak detection is performed considering the presence of a non-homogeneous noise level in the mass spectrum. A comparison of the peaks collected from multiple spectra is used to classify them on the basis of a detection rate parameter, and hence to separate the protein signals from other disturbances. RESULTS: LIMPIC preprocessing proves to be superior than other classical preprocessing techniques, allowing for a reliable decomposition of the background noise and the baseline drift from the MALDI-TOF mass spectra. It provides lower coefficient of variation associated with the peak intensity, improving the reliability of the information that can be extracted from single spectra. Our results show that LIMPIC peak-picking is effective even in low protein concentration regimes. The analytical comparison with commercial and freeware peak-picking algorithms demonstrates its superior performances in terms of sensitivity and specificity, both on in-vitro purified protein samples and human plasma samples. CONCLUSION: The quantitative information on the peak intensity extracted with LIMPIC could be used for the recognition of significant protein profiles by means of advanced statistic tools: LIMPIC might be valuable in the perspective of biomarker discovery

Amphibian transition to the oxidant terrestrial environment affects the expression of glutathione S-transferases isoenzymatic pattern

AbstractIt has been postulated that glutathione S-transferases (GST; EC 2.5.1.18) may play a role in protecting against oxidative stress.In previous studies, we have purified and characterised from Bufo bufo embryos a GST isoenzyme (BbGSTP1-1), which falls at very low level in the adult liver, where a novel isoform (BbGSTP2-2), starts to be highly expressed. During transition to adult life, B. bufo leaves the aquatic environment to live predominantly in the terrestrial environment, characterised by higher oxygen concentration.It has been found that BbGSTP2-2 is more efficient in scavenging from organic hydroperoxides.Therefore, the appearance of BbGSTP2-2 may respond to the necessity of providing the adult toad with a more suitable protection against oxygen toxic by-products. In this work, we performed experiments aimed at verifying if oxidative stress (hyperoxic and H2O2 treatments) could act as a modulator of BbGSTP2-2 expression. Results show that: (a) BbGSTP2 mRNA starts to be expressed in the late embryonic period, while protein appears during metamorphosis; (b) oxidative stress induces anticipation of BbGSTP2 gene expression at both transcriptional and translational levels.These findings seem to indicate that the appearance of BbGSTP2-2 is aimed at endowing the adult toad with more efficient antioxidant defence in the terrestrial atmosphere

Cryopreservation influence in the WJCs Proteome

Cryopreservation is the only mode of long-term storage of viable cells and tissues for cellular therapy, stem cell transplantation and/or tissue engineering. However the freeze-thaw process strongly contributes to cell and tissue damage with several mechanism, including oxidative stress, intracellular ice formation (IIF) dependent cell injury and altered physical cellular properties, i.e. osmotic and ion homeostasis. Our previous proteomics investigation was carried on Wharton’s jelly cells (WJCs), fibroblastlike cells with similar properties to mesenchymal stem cells, therefore a rich source of primitive cells to be potentially used in regenerative medicine. The aim of the present work was to investigate molecular changes that occur in WJCs proteome at different culture conditions (freshly and post-frozen cell preparations) and to elucidate possible mechanism involved in maintaining active proliferation and maximal cellular plasticity in order to optimize in vitro culturing procedure. To analyze changes in protein expression of WJCs we performed a comparative proteomic analysis (2DE followed by MALDI–TOF MS) between fresh and post-frozen cell culturing. WJCs postfrozen showed a qualitative and quantitative changes compared to cells from fresh preparation, expressing proteins involved in replication, cellular defense mechanism and metabolism, that ensure freeze-thaw survival. However, further investigations are needed to clarify the biological mechanisms involved in maintaining active proliferation, plasticity, multipotency cell during in vitro expansio