In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.
The aim of this work was to identify a new protein that discriminated CD8 from CD4 and CD19 lymphocytes. Proteins were separated by high-resolution two-dimensional electrophoresis. After silver staining, the gel images were captured with a laser densitometer, and studied with a dedicated software. This study confirmed the presence of two spots that appeared to be preferentially associated with CD8 lymphocytes, and mass spectrometry analyzes (liquid chromatography-tandem mass spectrometry, LC-MS/MS) identified six peptides for one spot and four for the other. The peptide sequences corresponded to an unknown protein that we named swiprosin 1 (Swiss-Prot Q96C19). Molecular analysis (reverse transcriptase-polymerase chain reaction, RT-PCR) and Northern blots confirmed that the gene expression was increased in purified populations of CD8 lymphocytes, when compared to CD19 and CD4 lymphocytes. Database mining revealed that swiprosin 1 contains two potential EF-hand domains, and therefore may have a role in calcium signaling. Its predominant presence in CD8 lymphocytes suggests that it may be involved in functions that are important for cytotoxic lymphocytes.
In 1993, we reported the presence of an IgM-associated peptide (M(r) 44 kDa; pI 5.45) in all immunoglobulin M (IgM) fractions purified from plasma/serum by various methods. This peptide was absent in Ig fractions of non-IgM isotypes. The N-terminal sequence was determined as being APPSGVRLVGGLH. To gain insight into the nature of this peptide, we further analyzed, using modern proteomic tools, the IgM-associated peptide isolated from cryoglobulins. Mass spectrometry revealed three peptides of different masses: 2203.13 (ELGCGAASGTPSGILYEPPAEK), 1564.83 (KPIWLSQMSCSGR), and 1544.77 (EATLQDCPSGPWGK). Theses sequences together with the already known N-terminal sequence allowed us to identity the IgM-associated peptide as Sp alpha (O43866 in TrEMBL database; CD5 antigen-like). Sp alpha is a member of the scavenger receptor cysteine-rich superfamily of proteins. This family includes the T-and B-cell antigens CD5 and CD6, and several of its members influence immune cell fate. Our finding may have important implications in the understanding of the homeostasis of IgM antibodies.
In vertebrates, different isoforms of fibroblast growth factor 2 (FGF2) exist, which differ by their N-terminal extension. They show different localization and expression levels and exert distinct biological effects. Nevertheless, genetic inactivation of all FGF2 isoforms in the mouse results in only mild phenotypes. Here, we analyzed mouse FGF2, and show that, as in the human, mouse FGF2 contains CTG-initiated high molecular-weight (HMW) isoforms, which contain a nuclear localization signal, and which mediate localization of this isoform to the nucleus. Using green fluorescent protein-FGF2 fusions, we furthermore observed, that C-terminal deletions disable nuclear localization of the short low-molecular-weight (LMW) 18-kDa isoform. This loss of specific localization is accompanied by a loss in heparin binding. We therefore suggest that, first, localization of mouse FGF2 is comparable to that in other vertebrates and, second, FGF2 contains at least two sequences important for nuclear localization, a nuclear localization sequence at the N terminus which is only contained in the HMW isoform, and another sequence at the C terminus, which is only required for localization of the LMW 18-kDa isoform.
The solubilized proteins of purified CD19(+) (B), CD8(+) (T) as well as CD4(+) (T) lymphocytes were separated by high resolution two-dimensional polyacrylamide gel electrophoresis, and the gels were analyzed using Melanie 3.0. Nine gels were studied, three for each lymphocyte population. After image analysis, 1411 +/- 73 spots (mean + SD) were detected. The protein pattern of B lymphocytes segregated from the one of T lymphocytes by ascendant heuristic clustering analysis. In addition, computer analysis separated CD8(+) from CD4(+) lymphocytes. When a search was performed in order to detect subsets of specific spots (presence vs. absence), a group of three spots, detected in the area of the protein maps corresponding to isoelectric point (pI) of 5.2 to 5.4 and molecular weight (M(r)) of 50 to 51 kDa, were found in both CD8(+) and CD4(+) cells, but not in CD19(+) cells. Mass spectrometry analysis revealed that these spots were associated with several proteins such as vimentin, tubulin, desmin and cytokeratin. Two spots, located in the area of the gel corresponding to pI of about 5.0 and a M(r) of 30 kDa, appeared as CD8(+) cell associated. Mass spectrometry analysis showed that the two spots were related to the same non-identified protein. Moreover internal peptides sequences matched with two human expressed sequence tags: gi/9759776, gi/12798420. No spots were found as only B cell associated.
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