Erythropoietin enhances cell proliferation and survival of human fetal neuronal progenitors in normoxia.

Extensive data reporting the neurogenerative, neuroprotective and neuroregenerative potential of erythropoietin (EPO), mainly on RNA level, can be found in the literature. However, there is still a poor knowledge on the response of neuronal progenitor cells (NPC) upon stimulation with EPO in terms of the protein species involved. Herein, the effect of EPO on the proliferation of human mesencephalic NPC (hmNPC) under normoxia is monitored using cellular assays and proteomic analysis (two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry). The administration of EPO increased the proliferation of hmNPC within 4 days after application. It positively influenced the cell-cycle progression by affecting the G2 phase of the cell cycle. A proteomic analysis of the protein expression in hmNPC cultures 4 days after EPO treatment identified 8 proteins differentially expressed in EPO-treated cultures. It is likely that one or more of the identified proteins are involved in cellular pathways that promote cell proliferation and differentiation of hmNPC under normoxia. Their further characterization could provide cellular targets for the development of new therapeutic agents to treat CNS injury. Moreover, as EPO signaling is hypoxia-inducible, our findings may also indicate the beneficial effect of EPO to mimic hypoxia, while bypassing its negative effects, to culture human fetal midbrain-derived progenitor cells.

Identification of sulfoquinovosyldiacyglycerides from Phaeodactylum tricornutum by matrix-assisted laser desorption/ionization QTrap time-of-flight hybrid mass spectrometry.

The pharmaceutical industry is interested in identifying novel target compounds. Due to their versatile pharmacological activities (e.g. antiviral, anti-carcinogen and immunosuppressive) sulfoquinovosyldiacylglycerides (SQDGs) are potential drug candidates. The present publication deals with the purification and structural characterization of SQDGs from three different strains of Phaeodactylum tricornutum. Besides detection of SQDGs (sn-1: C16:1/sn-2: C16:0 and sn-1: C20:5/sn-2: C16:0), two novel 2'-O-acylsulfoquinovosyldiacylglyerides (Ac-SQDGs, sn-1: C16:0/ sn-2: C16:0/2' C20:5 and sn-1: C20:5/sn-2: C16:0/2' C20:5) were identified by using matrix-assisted laser desorption/ionization (MALDI) QTrap time-of-flight (ToF) hybrid mass spectrometry (MS) with multistage MS(n). The analytical method enables the sn-position verification of fatty acids (MS(2)) as well as the confirmation of the regioposition of eicospentanoic acid at the sulfoquinovose (MS(3)).

Proteomic analysis to display the effect of low doses of erythropoietin on rat liver regeneration.

AIMS: Several groups found different impact of erythropoietin (EPO) on liver regeneration. Both pro-proliferative as well as anti-proliferative and non-proliferative activities have been reported using high dosage of EPO. Systemic administration of high doses of this cytokine is a clinical concern due to risk of thrombosis. Herein, we applied EPO in low dosages and investigated whether it can stimulate liver regeneration after liver resection. MAIN METHODS: Parameters of liver regeneration were assessed 3 days after 70% hepatectomy by means of immunochemistry and proteomics. EPO was given twice in low dosages (200 and 600 IU/kg BW). KEY FINDINGS: We showed that EPO facilitated hepatic regeneration in rats. Enhanced hepatocyte proliferation (Ki67, BrdU-positive cells) was observed in all EPO-treated groups. By performing Differential Proteomic analysis, we identified two proteins which resulted sensitive to EPO treatment after hepatectomy: Peroxiredoxin-1 and glutathione S-transferase Mu 1. SIGNIFICANCE: Based on our results, low doses of rhEPO increase the hepatic regenerative capacity after partial hepatectomy in rats by enhancing hepatocyte proliferation and acting on antioxidant enzymes. Both proteins identified by proteomic analysis have not previously been associated with liver regeneration and will aid in the understanding of EPO's regenerative response having clinical implications to treat liver failure.

Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides.

BACKGROUND: Despite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach. RESULTS: The cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipµ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses. CONCLUSIONS: The proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis.

Mesenchymal stem cells restore lung function by recruiting resident and nonresident proteins.

Because human lungs are unlikely to repair or regenerate beyond the cellular level, cell therapy has not previously been considered for chronic irreversible obstructive lung diseases. To explore whether cell therapy can restore lung function, we administered allogenic intratracheal mesenchymal stem cells (MSCs) in the trachea of rats with chronic thromboembolic pulmonary hypertension (CTEPH), a disease characterized by single or recurrent pulmonary thromboembolic obliteration and progressive pulmonary vascular remodeling. MSCs were retrieved only in high pressure-exposed lungs recruited via a homing stromal derived factor-1α/CXCR4 pathway. After MSC administration, a marked and long-lasting improvement of all clinical parameters and a significant change of the proteome level were detected. Beside a variation of liver proteome, such as caspase-3, NF-κB, collagen1A1, and α-SMA, we also identified more than 300 resident and nonresident lung proteins [e.g., myosin light chain 3 (P16409) or mitochondrial ATP synthase subunit alpha (P15999)]. These results suggest that cell therapy restores lung function and the therapeutic effects of MSCs may be related to protein-based tissue reconstituting effects.

IDENTIFICATION OF COENZYME Q10 FROM PORPHYRIDIUM PURPUREUM (RHODOPHYTA) BY MATRIX-ASSISTED LASER DESORPTION IONIZATION CURVED FIELD REFLECTRON MASS SPECTROMETRY1.

Antioxidant agents from natural sources are currently the focus of scientific interest and are part of several natural product screenings. Coenzymes Q (CoQ, ubiquinones) are integral parts of the electron transport chain of the inner mitochondrial membrane. As antioxidants they protect phospholipids against peroxidation and are also involved in various processes of tissue protection. Their natural occurrence was validated for Saccharomyces cerevisiae as CoQ6 , for Escherichia coli as CoQ8 , and for humans as CoQ10 . After carrying out a preparative reversed-phase (RP)-HPLC separation of extracts isolated from unicellular red alga Porphyridium purpureum (Bory) K. M. Drew et R. Ross, it was possible to identify a 2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone (CoQ10 ) within these extracts using a matrix-assisted laser desorption ionization (MALDI) curved field reflectron (CFR) mass spectrometer. Detected mass fragments showed a high significance and could be structurally interpreted for both commercialized standard and CoQ10 isolated from P. purpureum.

Identification of sulfoglycolipids from the alga Porphyridium purpureum by matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight mass spectrometry.

Sulfoglycolipids, isolated from different phototrophic organisms, particularly plants and algae, have already been identified as bioactive compounds. In addition to their antiviral activity their influence on the immune response in mammalian cells is the focus of many studies. For the first time it has been possible to investigate purified sulfoquinovosyldiacylglycerols (SQDGs) from the microalga Porphyridium purpureum by matrix-assisted laser desorption/ionisation (MALDI) in the negative ion reflectron mode. Thereby, different solid and ionic liquid matrices have been tested to improve signal intensity during the laser ionisation. By using the MALDI Trap time-of-flight (ToF) multiple-stage (MS(n)) hybrid mass spectrometer the fatty acid compositions of the SQDGs were analysed by MS, and confirmed by MS(2) and MS(3) experiments. Thereby, hexadecanoic acid (C16:0), octadecadienoic acid (C18:2), eicosatetraenoic acid (C20:4), and eicosapentaenoic acid (C20:5) were detected in the purified fraction of SQDGs. The localisation of hexadecanoic acid (C16:0) at the sn-2 position, and unsaturated fatty acids at the sn-1 position of the SQDGs, determined by specific enzymatic hydrolysis, marks a procaryotic biosynthesis of SQDGs in the eucaryotic alga cells.

Analysis of a chlorosulfolipid from Ochromonas danica by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) is an established tool for analyzing high mass molecules, such as proteins, whereas it attracts far less interest in the field of lipid analysis. In the study reported here a new chlorosulfolipid (CSL), 3,8,12,15-tetrachloroeicosane-1,17,18-triyl tris(hydrogen sulfate), was identified from the alga Ochromonas danica and de novo characterized by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-ToF) MS in negative ion mode. This method provides an effective alternative for the analysis of compounds directly derived from organic cell extracts. For MALDI analyses several frequently used solid MALDI matrices as well as some ionic liquid matrices (ILMs) were tested to enhance the analyte response to UV-laser and its ionization. The molecular weight of the observed compound could be determined as Li-, Na- and K-adducts [M+Me-2H]-. The characteristic isotopic patterns of the measured ions and the well-allocated molecular fragments by MS1, MS2 and MS3 indicate the fourfold chlorination and threefold sulfation of the investigated compound. The MS fragmentation alongside of the chlorine-bearing C-atoms is accompanied by the generation of a double bond at the opposite fragment in MS1. This obtained fragmentation pattern provides an insight into the allocation of the chlorine-bearing C-atoms along the carbon chain.