| 1. |
- Jankowski, V, et al.
(författare)
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Detection of angiotensin II in supernatants of stimulated mononuclear leukocytes by MALDI-TOF-TOF-mass spectrometric analysis
- 2005
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Ingår i: Hypertension. - 1524-4563. ; 46:3, s. 591-597
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Tidskriftsartikel (refereegranskat)abstract
- Angiotensin II ( Ang II) is the major vasoactive component of the renin- angiotensin system. Several components of the renin- angiotensin system have been demonstrated in different tissues. Whereas the roles of tissue and renal renin- angiotensin system have been studied in detail, much less is known on whether the corpuscular elements of circulating blood contribute to Ang II production. Here we examined whether, in addition to vasculature, blood cells also contribute to the circulating Ang II levels. Mononuclear leukocytes were obtained from healthy subjects and were incubated. The resulting supernatant was chromatographed using different chromatographic methods. The vasoconstrictive effects of aliquots of the resulting fractions were tested. Each fraction with a vasoconstrictive effect was analyzed by mass spectrometry. In one fraction with a strong vasoconstrictive effect, Ang II was identified. Mononuclear lymphocytes produced Ang II in amounts sufficient to stimulate Ang II type 1 receptors. Moreover, in mononuclear leukocytes, renin as well as angiotensin- converting enzyme mRNA expression was detectable by RT- PCR. These findings demonstrate that mononuclear leukocytes are a source of Ang II. Ang II secretion by these cells may play a significant role in humoral vascular regulation. In conclusion, the isolation of Ang II in supernatants of mononuclear leukocytes adds a further physiological source of Ang II to the current view of angiotensin metabolism. The quantitative role of lymphocyte- derived Ang II secretion compared with the other sources of Ang II should be defined further, but the release found under the present conditions is at least sufficient to elicit vasoconstrictive effects
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| 2. |
- Gustavsson, Niklas, et al.
(författare)
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A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry
- 2005
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Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 5:14, s. 3563-3570
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Tidskriftsartikel (refereegranskat)abstract
- While several techniques exist for assessing quantitative differences among proteomes representing different cell states, methods for assessing how these differences are mediated are largely missing. We present a method that allows one to differentiate between cellular processes, such as protein synthesis, degradation and PTMs which affect protein concentrations. An induced systemic perturbation of a cell culture was coupled to a replacement of the growth medium to one highly enriched in the stable isotope N-15. The relative abundance of the N-15- and N-14-enriched forms of proteins, isolated from cell cultures harvested at time points following the onset of the perturbation, were determined by MS. Alterations in protein synthesis and degradation were quantified by comparing proteins isolated from perturbed and unperturbed cultures, respectively. The method was evaluated by subjecting HeLa cells to heat stress. As expected, a number of known heat shock proteins (Hsp) increased in concentration during heat stress. For Hsp27, increased de novo synthesis accounted for the concentration increase, while for Hsp70, decreased degradation accounted for the increase. A protein that was detected only after prolonged heat stress, vimentin, was not primarily synthesized de novo, but appeared rather as a result of PTM.
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| 3. |
- Otterhag, L, et al.
(författare)
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Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase
- 2006
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Ingår i: Biochimie. - : Elsevier BV. - 1638-6183 .- 0300-9084. ; 88:1, s. 11-21
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Tidskriftsartikel (refereegranskat)abstract
- The A rabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1 expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp- 167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro tar et of AtPDK1. Our data clearly show that Asp- 167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident. (C) 2005 Elsevier SAS. All rights reserved.
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| 4. |
- Weichart, D, et al.
(författare)
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Analysis of NOD2-mediated proteome response to muramyl dipeptide in HEK293 cells
- 2006
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 281:4, s. 2380-2389
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Tidskriftsartikel (refereegranskat)abstract
- NOD2, a cytosolic receptor for the bacterial proteoglycan fragment muramyl dipeptide (MDP), plays an important role in the recognition of intracellular pathogens. Variants in the bacterial sensor domain of NOD2 are genetically associated with an increased risk for the development of Crohn disease, a human chronic inflammatory bowel disease. In the present study, global protein expression changes after MDP stimulation were analyzed by two-dimensional PAGE of total protein extracts of human cultured cells stably transfected with expression constructs encoding for wild type NOD2 (NOD2(WT)) or the disease-associated NOD2 L1007fsinsC (NOD2(SNP13)) variant. Differentially regulated proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) peptide mass fingerprinting and MALDI MS/MS. The limited overlap in the responses of the NOD2-overexpressing cell lines to MDP included a down-regulation of heat shock 70-kDa protein 4. A complex pro-inflammatory program regulated by NOD2(WT) that encompasses a regulation of key genes involved in protein folding, DNA repair, cellular redox homeostasis, and metabolism was observed both under normal growth conditions and after stimulation with MDP. By using the comparison of NOD2(WT) and disease-associated NOD2(SNP13) variant, we have identified a proteomic signature pattern that may further our understanding of the influence of genetic variations in the NOD2 gene in the pathophysiology of chronic inflammatory bowel disease.
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