| 1. |
- Baeckström, Dan, 1956, et al.
(author)
-
Expression of the leukocyte-associated sialoglycoprotein CD43 by a colon carcinoma cell line
- 1995
-
In: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258. ; 270, s. 13688-13692
-
Journal article (peer-reviewed)abstract
- The colon adenocarcinoma cell line COLO 205 secretes L-CanAg, a mucin- like glycoprotein carrying the carcinoma-associated sialyl-Lewis a carbohydrate epitope. In an attempt to identify its apoprotein, an NH2- terminal peptide sequence was obtained from purified L-CanAg. In all interpretable positions, this sequence showed 100% identity to the NH2- terminal of human CD43 (leukosialin, sialophorin), a plasma membrane-bound sialoglycoprotein hitherto only identified in leukocytes and other hematopoietic cells. An antiserum against deglycosylated L-CanAg and an anti- CD43 antiserum both immunoprecipitated a 61-kDa band, interpreted as the CD43 precursor, from COLO 205 cells as well as from the known CD43-expressing cell line HL-60. Results from immunoprecipitations following pulse-chase experiments and tunicamycin treatments were in agreement with earlier studies on the CD43 precursor. RNA blot analysis confirmed the expression of CD43 by the COLO 205 cell line, whereas three other colon carcinoma cell lines were negative. The glycosylation-dependent monoclonal antibody Leu-22, which recognizes leukocyte CD43, failed to bind L-CanAg, probably due to its much more extensive glycosylation. We conclude that L-CanAg is the secreted extracellular domain of a novel glycoform of CD43 and that CD43, if expressed in other carcinoma cells, may have escaped notice in studies relying on glycosylation-dependent monoclonal antibodies against leukocyte CD43.
|
|
| 2. |
- Boreström, Cecilia, 1974, et al.
(author)
-
E2F1, ARID3A/Bright and Oct-2 factors bind to the Epstein-Barr virus C promoter, EBNA1 and oriP, participating in long-distance promoter-enhancer interactions.
- 2012
-
In: The Journal of general virology. - : Microbiology Society. - 1465-2099 .- 0022-1317. ; 93, s. 1065-75
-
Journal article (peer-reviewed)abstract
- The Epstein-Barr virus (EBV) C promoter (Cp) regulates several genes required for B-cell proliferation in latent EBV infection. The family of repeats (FR) region of the latent origin of plasmid replication (oriP) functions as an Epstein-Barr nuclear antigen 1 (EBNA1)-dependent distant enhancer of Cp activity, and the enhancer-promoter interaction is mediated by a higher-order multi-protein complex containing several copies of EBNA1. Using DNA-affinity purification with a 170 bp region of the Cp in combination with mass spectrometry, we identified the cell cycle-regulatory protein E2F1, the E2F-binding protein ARID3A, and the B-cell-specific transcription factor Oct-2 as components of this multi-protein complex. Binding of the three factors to the FR region of oriP was determined by DNA-affinity and immunoblot analysis. Co-immunoprecipitation and proximity ligation analysis revealed that the three factors, E2F1, ARID3A and Oct-2, interact with each other as well as with EBNA1 in the nuclei of EBV-positive cells. Using the chromatin immunoprecipitation assay, we showed that E2F1 and Oct-2 interacted with the FR part of oriP and the Cp, but the ARID3A interaction was, however, only detected at the Cp. Our findings support the hypothesis that EBNA1 initiates transcription at the Cp via interactions between multiple EBNA1 homodimers and cellular transcription factors in a large molecular machinery that forms a dynamic interaction between Cp and FR.
|
|
| 3. |
- Brinkmalm, Gunnar, et al.
(author)
-
An online nano-LC-ESI-FTICR-MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid.
- 2012
-
In: Journal of mass spectrometry : JMS. - : Wiley. - 1096-9888 .- 1076-5174. ; 47:5, s. 591-603
-
Journal article (peer-reviewed)abstract
- Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), mainly long b-fragments are observed, limiting the possibility to determine variations such as amino acid variants or post-translational modifications (PTMs) within the N-terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann-Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O-glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top-down MS-based method.
|
|
| 4. |
- Brinkmalm-Westman, Ann, 1966, et al.
(author)
-
Proteomics/peptidomics tools to find CSF biomarkers for neurodegenerative diseases.
- 2009
-
In: Frontiers in bioscience : a journal and virtual library. - : IMR Press. - 1093-4715. ; 14, s. 1793-806
-
Research review (peer-reviewed)abstract
- Neurodegenerative diseases are characterized by premature neuronal loss in specific brain regions. During the past decades our knowledge on molecular mechanisms underlying neurodegeneration has increased immensely and resulted in promising drug candidates that might slow down or even stop the neuronal loss. These advances have put a strong focus on the development of diagnostic tools for early or pre-clinical detection of the disorders. In this review we discuss our experience in the field of neuroproteomics/peptidomics, with special focus on biomarker discovery studies that have been performed on CSF samples from well-defined patient and control populations.
|
|
| 5. |
- Constantinescu, Radu, 1966, et al.
(author)
-
Proteomic profiling of cerebrospinal fluid in parkinsonian disorders.
- 2010
-
In: Parkinsonism & related disorders. - : Elsevier BV. - 1873-5126 .- 1353-8020. ; :16, s. 545-49
-
Journal article (peer-reviewed)abstract
- Parkinson's disease (PD) and atypical parkinsonian disorders (APD), including multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD), are a group of neurodegenerative diseases sharing many similar signs and symptoms but distinguished by their particular clinical features, treatment response, prognosis and mortality. The differential diagnosis may be challenging, especially in early disease stages. Considering the importance of an accurate diagnosis both for clinical management and for research, new diagnostic tools are needed. In this study, we investigated 56 PD, 42 MSA, 39 PSP, 9 CBD patients, and 24 healthy controls. After screening the cerebrospinal fluid (CSF) proteome using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), we identified 4 proteins (ubiquitin [mass-to-charge ratio (m/z) 8590], beta2-microglobulin [m/z 11730], and 2 secretogranin 1 [chromogranin B] fragments [m/z 7260 and m/z 6250]) that differentiated healthy controls and PD patients from patients with APD. However, they could not differentiate PD patients from controls. As none of these changes were APD subgroup-specific, they most likely reflect the intensity and/or extent of the neurodegenerative process in general.
|
|
| 6. |
- Dufva, Martin, 1967, et al.
(author)
-
Epstein-Barr virus nuclear antigen 5 inhibits pre-mRNA cleavage and polyadenylation.
- 2002
-
In: Nucleic acids research. - 1362-4962. ; 30:10, s. 2131-43
-
Journal article (peer-reviewed)abstract
- The long-standing suspicion that Epstein-Barr virus nuclear antigen 5 (EBNA5) is involved in transcription regulation was recently confirmed by the observation by several groups that EBNA5 cooperates with EBNA2 in activation of the LMP1 promoter. In attempts to elucidate the molecular basis for the EBNA5-mediated enhancement of EBNA2 transactivation, we obtained evidence of an additional function of EBNA5: at high but still biologically relevant levels, EBNA5 acted as a repressor of gene expression by interfering with the processing of pre-mRNA. Transient transfections with reporter plasmids revealed that EBNA5 repressed reporter mRNA and protein expression in the cytoplasm, but did not lower the steady-state level of reporter RNA in the total cellular RNA fraction. We have excluded that repression occurred as a consequence of cell death induced by EBNA5. Using the RNase protection assay with a probe comprising the pre-mRNA cleavage and polyadenylation site, EBNA5 was found to inhibit 3'-end cleavage and polyadenylation of pre-mRNAs from the reporter plasmids investigated. The effect of inhibitory levels of EBNA5 on chromosomal genes was examined in transient transfections by expression profiling using a cDNA microarray panel containing 588 genes. The results showed that EBNA5 could also inhibit the expression of chromosomal genes and did it in a discriminatory manner. This is consistent with the notion that a regulatory mechanism exists in the cell that confers specificity to the selection by EBNA5 of target genes for repression.
|
|
| 7. |
- Ekman, Stina, et al.
(author)
-
Effect of circadian rhythm, age, training and acute lameness on serum concentrations of cartilage oligomeric matrix protein (COMP) neo-epitope in horses
- 2019
-
In: Equine Veterinary Journal. - : Wiley. - 0425-1644 .- 2042-3306. ; 51:5, s. 674-680
-
Journal article (peer-reviewed)abstract
- Background Molecular serum markers that can identify early reversible osteoarthritis (OA) in horses are lacking. Objectives We studied serum concentrations of a novel cartilage oligomeric matrix protein (COMP) neo-epitope in horses subjected to short-term exercise and with acute lameness. The effects of circadian rhythm and age were also evaluated. Study design Longitudinal studies in healthy horses and cross-sectional comparison of lame and non-lame horses. Methods Sera were collected from five horses before and after short-term interval exercise and during full-day box rest. Sera from 32 acutely lame horses were used to evaluate age-related effects. Independent samples from control horses (n = 41) and horses with acute lameness (n = 71) were included. COMP neo-epitope concentrations were analysed using custom-developed inhibition ELISAs validated for equine serum. The presence of COMP neo-epitope was delineated in healthy and osteoarthritic articular cartilage with immunohistochemistry. Results COMP neo-epitope concentrations decreased after speed training but returned to baseline levels post-exercise. No correlations between age and serum COMP neo-epitope concentrations were found (r = 0.0013). The mean (+/- s.d.) serum concentration of COMP neo-epitope in independent samples from non-lame horses was 0.84 +/- 0.38 mu g/mL, and for lame horses was 5.24 +/- 1.83 mu g/mL (P<0.001). Antibodies against COMP neo-epitope did not stain normal articular cartilage, but intracytoplasmic staining was found in superficial chondrocytes of mild OA cartilage and in the extracellular matrix of moderately osteoarthritic cartilage. Main limitations ELISA was based on polyclonal antisera rather than a monoclonal antibody. There is a sex and breed bias within the groups of horses, also it could have been of value to include horses with septic arthritis and tendonitis and investigated joint differences. Conclusions This COMP neo-epitope can be measured in sera, and results indicate that it could be a biomarker for pathologic fragmentation of cartilage in connection with acute joint lameness.
|
|
| 8. |
- Forsman, Alma, 1979, et al.
(author)
-
Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry.
- 2008
-
In: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:6, s. 2309-19
-
Journal article (peer-reviewed)abstract
- Epstein-Barr virus nuclear antigen 5 (EBNA5) is one of the first viral proteins detected after primary EBV infection and has been shown to be required for efficient transformation of B lymphocytes. EBNA5 is a protein that has many suggested functions but the underlying biology remains to be clarified. To gain further insight into the biological roles of the proposed multifunctional EBNA5, we isolated EBNA5 containing protein complexes using a modified tandem affinity purification (TAP) method and identified the protein components by LC-MS/MS analysis of tryptic digests on a LTQ-FT-ICR mass spectrometer. The modified TAP tag contained a Protein A domain and a StrepTagII sequence separated by two Tobacco Etch Virus protease cleavage sites and was fused to the C-terminus of EBNA5. Our results confirmed the wide applicability of this two-step affinity purification strategy for purification of protein complexes in mammalian cells. A total of 147 novel putative EBNA5 interaction partners were identified, 37 of which were validated with LC-MS/MS in split-tag experiments or in co-immuno precipitates from HEK293 cell extracts. This subgroup included the Bcl2-associated Athanogene 2 (BAG2) co-chaperone involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. These EBNA5 interactors were further verified by co-immunoprecipitations from cell extracts of three EBV-positive lymphoblastoid lines. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 interactors suggests that EBNA5 might have a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Our study also confirms previously identified interactors including HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, and alpha- and beta-tubulin.
|
|
| 9. |
- Granéli, Cecilia, 1981, et al.
(author)
-
Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach.
- 2014
-
In: Stem cell research. - : Elsevier BV. - 1876-7753 .- 1873-5061. ; 12:1, s. 153-165
-
Journal article (peer-reviewed)abstract
- Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue. Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed. The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages. It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.
|
|
| 10. |
- Halim, Adnan, et al.
(author)
-
Assignment of Saccharide Identities through Analysis of Oxonium Ion Fragmentation Profiles in LC-MS/MS of Glycopeptides.
- 2014
-
In: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 13:12, s. 6024-32
-
Journal article (peer-reviewed)abstract
- Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.
|
|
| 11. |
- Halim, Adnan, et al.
(author)
-
Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD.
- 2012
-
In: Molecular & cellular proteomics : MCP. - 1535-9484. ; 11:4
-
Journal article (peer-reviewed)abstract
- Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.
|
|
| 12. |
- Halim, Adnan, et al.
(author)
-
LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins.
- 2013
-
In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:2, s. 573-584
-
Journal article (peer-reviewed)abstract
- The GalNAc O-glycosylation on Ser/Thr residues of extracellular proteins has not been well characterized from a proteomics perspective. We previously reported a sialic acid capture-and-release protocol to enrich tryptic N- and O-glycopeptides from human cerebrospinal fluid glycoproteins using nanoLC-ESI-MS/MS with collision-induced dissociation (CID) for glycopeptide characterization. Here, we have introduced peptide N-glycosidase F (PNGase F) pre-treatment of CSF samples to remove the N-glycans facilitating the selective characterization of O-glycopeptides and enabling the use of an automated CID-MS2/MS3 search protocol for glycopeptide identification. We used electron capture and transfer dissociation (ECD/ETD) to pinpoint the glycosylation site(s) of the glycopeptides, identified as predominantly core 1 like HexHexNAc-O- glycans attached to 1-4 Ser/Thr residues. We characterized 108 O-glycosylations and found Pro residues preferentially in the n-1, n+1 and/or n+3 positions in relation to the Ser/Thr attachment site (n). The characterization of glycans and glycosylation sites in glycoproteins from human clinical samples provides a basis for future studies addressing the biological and diagnostic importance of specific protein glycosylations in relation to human disease.
|
|
| 13. |
- Halim, Adnan, et al.
(author)
-
Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid {beta}-peptides in human cerebrospinal fluid.
- 2011
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490. ; 108:29, s. 11848-53
-
Journal article (peer-reviewed)abstract
- The proteolytic processing of human amyloid precursor protein (APP) into shorter aggregating amyloid β (Aβ)-peptides, e.g., Aβ1-42, is considered a critical step in the pathogenesis of Alzheimer's disease (AD). Although APP is a well-known membrane glycoprotein carrying both N- and O-glycans, nothing is known about the occurrence of released APP/Aβ glycopeptides in cerebrospinal fluid (CSF). We used the 6E10 antibody and immunopurified Aβ peptides and glycopeptides from CSF samples and then liquid chromatography-tandem mass spectrometry for structural analysis using collision-induced dissociation and electron capture dissociation. In addition to 33 unglycosylated APP/Aβ peptides, we identified 37 APP/Aβ glycopeptides with sialylated core 1 like O-glycans attached to Thr(-39, -21, -20, and -13), in a series of APP/AβX-15 glycopeptides, where X was -63, -57, -52, and -45, in relation to Asp1 of the Aβ sequence. Unexpectedly, we also identified a series of 27 glycopeptides, the Aβ1-X series, where X was 20 (DAEFRHDSGYEVHHQKLVFF), 19, 18, 17, 16, and 15, which were all uniquely glycosylated on Tyr10. The Tyr10 linked O-glycans were (Neu5Ac)(1-2)Hex(Neu5Ac)HexNAc-O- structures with the disialylated terminals occasionally O-acetylated or lactonized, indicating a terminal Neu5Acα2,8Neu5Ac linkage. We could not detect any glycosylation of the Aβ1-38/40/42 isoforms. We observed an increase of up to 2.5 times of Tyr10 glycosylated Aβ peptides in CSF in six AD patients compared to seven non-AD patients. APP/Aβ sialylated O-glycans, including that of a Tyr residue, the first in a mammalian protein, may modulate APP processing, inhibiting the amyloidogenic pathway associated with AD.
|
|
| 14. |
- Hansson, Sarah, 1976, et al.
(author)
-
Cystatin C in cerebrospinal fluid and multiple sclerosis.
- 2007
-
In: Annals of neurology. - : Wiley. - 0364-5134. ; 62:2, s. 193-196
-
Journal article (peer-reviewed)abstract
- OBJECTIVE: A recent study using surface-enhanced laser desorption/ionization time-of-flight analysis of cerebrospinal fluid identified a 12.5 kDa truncated isoform of cystatin C (CysC) as a specific biomarker for multiple sclerosis (MS). METHODS: Surface-enhanced laser desorption/ionization time-of-flight analysis of cerebrospinal fluid samples from 43 MS patients and 46 healthy control subjects. RESULTS: Full-length CysC (13.4 kDa) concentration was similar in MS and control samples. The 12.5 kDa CysC protein was produced from full-length CysC by N-terminal cleavage during storage at -20 degrees C. INTERPRETATION: The 12.5 kDa CysC isoform is a storage-related artifact and is not useful as a diagnostic marker for MS.
|
|
| 15. |
- Hölttä, Mikko, et al.
(author)
-
An Integrated Workflow for Multiplex CSF Proteomics and Peptidomics-Identification of Candidate Cerebrospinal Fluid Biomarkers of Alzheimer's Disease
- 2015
-
In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 14:2, s. 654-663
-
Journal article (peer-reviewed)abstract
- Many disease processes in the brain are reflected in the protein composition of the cerebrospinal fluid (CSF). In addition to proteins, CSF also contains a large number of endogenous peptides whose potential as disease biomarkers largely remains to be explored. We have developed a novel workflow in which multiplex isobaric labeling is used for simultaneous quantification of endogenous CSF peptides and proteins by liquid chromatography coupled with mass spectrometry. After the labeling of CSF samples, endogenous peptides are separated from proteins by ultrafiltration. The proteins retained on the filters are trypsinized, and the tryptic peptides are collected separately. We evaluated this technique in a comparative pilot study of CSF peptide and protein profiles in eight patients with Alzheimer's disease (AD) and eight nondemented controls. We identified several differences between the AD and control group among endogenous peptides derived from proteins known to be associated with AD, including neurosecretory protein VGF (ratios AD/controls 0.45-0.81), integral membrane protein 2B (ratios AD/controls 0.72-0.84), and metallothionein-3 (ratios AD/controls 0.51-0.61). Analysis of tryptic peptides identified several proteins that were altered in the AD group, some of which have previously been reported as changed in AD, for example, VGF (ratio AD/controls 0.70).
|
|
| 16. |
- Johansson, Pegah, 1978, et al.
(author)
-
Nuclear factor-kappaB binds to the Epstein-Barr Virus LMP1 promoter and upregulates its expression.
- 2009
-
In: Journal of virology. - 1098-5514. ; 83:3, s. 1393-401
-
Journal article (peer-reviewed)abstract
- The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-kappaB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-kappaB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-kappaB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-kappaB factors in both B cells and epithelial cells. Exogenous expression of NF-kappaB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-kappaB activation and LMP1 expression.
|
|
| 17. |
- Johansson, Pegah, 1978, et al.
(author)
-
The p38 signaling pathway upregulates expression of the Epstein-Barr virus LMP1 oncogene.
- 2010
-
In: Journal of virology. - 1098-5514. ; 84:6, s. 2787-97
-
Journal article (peer-reviewed)abstract
- The Epstein-Barr virus (EBV)-encoded LMP1 oncogene has a role in transformation, proliferation, and metastasis of several EBV-associated tumors. Furthermore, LMP1 is critically involved in transformation and growth of EBV-immortalized B cells in vitro. The oncogenic properties of LMP1 are attributed to its ability to upregulate anti-apoptotic proteins and growth signals. The transcriptional regulation of LMP1 is dependent on the context of cellular and viral proteins present in the cell. Here, we investigated the effect of several signaling pathways on the regulation of LMP1 expression. Inhibition of p38 signaling, using p38-specific inhibitors SB203580 and SB202190, downregulated LMP1 in estrogen-induced EREB2.5 cells. Similarly, p38 inhibition decreased trichostatin A-induced LMP1 expression in P3HR1 cells. Exogenous expression of p38 in lymphoblastoid cell lines (LCLs) led to an increase in LMP1 promoter activity in reporter assays, and this activation was mediated by the previously identified CRE site in the promoter. Inhibition of p38 by SB203580 and p38-specific small interfering RNA (siRNA) also led to a modest decrease in endogenous LMP1 expression in LCLs. Chromatin immunoprecipitation indicated decreased binding of CREB-ATF1 to the CRE site in the LMP1 promoter after inhibition of the p38 pathway in EREB2.5 cells. Taken together, our results suggest that an increase in p38 activation upregulates LMP1 expression. Since p38 is activated in response to stimuli such as stress or possibly primary infection, a transient upregulation of LMP1 in response to p38 may allow the cells to escape apoptosis. Since the p38 pathway itself is activated by LMP1, our results also suggest the presence of an autoregulatory loop in LMP1 upregulation.
|
|
| 18. |
- Lustig, Florentina, 1948, et al.
(author)
-
Alternative splicing determines the binding of platelet-derived growth factor (PDGF-AA) to glycosaminoglycans.
- 1996
-
In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 35:37, s. 12077-85
-
Journal article (peer-reviewed)abstract
- We have shown previously that the platelet-derived growth factor (PDGF) and a synthetic oligopeptide, corresponding to the basic carboxyl-terminal amino acid extension of the long PDGF-A isoform, bind to heparin. Here, we have expressed the long (rA125) and the short (rA109) variants of PDGF A-chains in Escherichia coli and produced the functional homodimers. Surface plasmon resonance analyses showed that while the dimeric rA125 bound with high affinity to low molecular weight heparin, the rA109, lacking the basic extension, did not. This strongly indicated that high affinity binding is due to the carboxyl-terminal extension. Investigations of kinetics and thermodynamics suggested an allosteric binding mechanism. Thus, dimeric rA125 contains two equivalent binding sites. Following low affinity binding of heparin to one binding site, the dimer undergoes a conformational change, increasing the affinity for heparin about 40 times. This positive cooperativity requires the basic amino acid extension in both monomers of the dimeric PDGF molecule. Thermodynamics of the reaction, showing an entropy-driven endothermic process, suggest the involvement of hydrophobic interactions in this rearrangement. Three amino acids in the basic carboxyl-terminal extension were essential for the interaction: the basic residues Arg111 and Lys116, and the polar Thr125. We also found that other glycosaminoglycan species, corresponding to those produced by human arterial smooth muscle cells, bound to dimeric rA125 and that heparan sulfate showed the highest affinity.
|
|
| 19. |
- Lustig, Florentina, 1948, et al.
(author)
-
Processing of PDGF gene products determines interactions with glycosaminoglycans.
- 1999
-
In: Journal of molecular recognition : JMR. - 0952-3499. ; 12:2, s. 112-20
-
Journal article (peer-reviewed)abstract
- The platelet derived growth factor (PDGF), a mitogen for mesenchymal cells, may be bound to and inhibited by heparin and other glycosaminoglycans. PDGF is a homo- or heterodimer of A- and B-chains. They occur as short (A109 and B110) and long (A125 and B160) isoforms. The latter contain basic carboxyl-terminal extensions. Dimeric A125 binds to heparin through its basic extension in a two-step reaction. The mechanism involves a conformational change and is consistent with a Monod-Wyman-Changeux allosteric model. Previous indirect experiments suggested that three critical amino acids (basic R111, K116 and polar T125) might be involved. Here, direct binding experiments using dimeric full-length mutants in surface plasmon resonanse analysis showed that all three critical amino acids in an R(X)4K(X)8T-motif contributed in a concerted manner to the high affinity binding. Mutations of these amino acids to alanine resulted in large thermodynamic changes, loss of the allosteric mechanism and order(s) of magnitude lower binding affinity. The binding mechanism and affinity of long dimeric rB were similar to the mutants. Short dimeric rA109 and rB110 showed 100 times lower binding affinity than rA125. Consequently, interactions with glycosaminoglycans in tissues varies between PDGF isoforms and may influence their local accumulation and activity.
|
|
| 20. |
- Mattsson, Niklas, 1979, et al.
(author)
-
Cerebrospinal fluid concentrations of peptides derived from chromogranin B and secretogranin II are decreased in multiple sclerosis
- 2007
-
In: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 103:5, s. 1932-1939
-
Journal article (peer-reviewed)abstract
- Novel biomarkers for multiple sclerosis (MS) could improve diagnosis and provide clues to pathogenesis. In this study surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to analyze protein expression in CSF from 46 MS patients, 46 healthy siblings to the patients, and 50 unrelated healthy controls. Twenty-four proteins in the mass range 2–10 kDa were expressed at significantly different levels (p < 0.01) in a robust manner when comparing the three groups. Identities of three proteins were determined using biochemical purification followed by tandem mass spectrometric analysis. Immunoprecipitation experiments confirmed the identities for two peptides derived from chromogranin B (m/z 6252) and from secretogranin II (m/z 3679). These peptides were all decreased in MS when compared with siblings or controls. Radioimmunoassays specific for each peptide confirmed these differences. The lowered concentrations did not correlate to the axonal damage marker neurofilament light protein and may thus reflect functional changes rather than neurodegeneration. Further studies will investigate the involvement of these peptides in MS pathogenesis.
|
|
| 21. |
- Mattsson, Niklas, 1979, et al.
(author)
-
Novel cerebrospinal fluid biomarkers of axonal degeneration in frontotemporal dementia.
- 2008
-
In: Molecular medicine reports. - : Spandidos Publications. - 1791-2997. ; 1:5, s. 757-61
-
Journal article (peer-reviewed)abstract
- Frontotemporal dementia (FTD) is a heterogeneous disease with substantial interpersonal variance in aggressiveness. Novel biomarkers for rapidly progressive FTD could improve diagnosis and provide clues regarding its pathogenesis. In this study, surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) was used to analyze peptide profiles in cerebrospinal fluid (CSF) from 24 FTD patients. Thirteen patients had rapidly progressive FTD with distinct pathology in a brain MRI after less than 3 years of disease duration. Eleven patients had slowly progressive FTD with a normal brain MRI, but had abnormal findings in SPECT/PET after more than 5 years of disease duration. The axonal damage marker CSF neurofilament light-chain (NF-L) was measured in all subjects to evaluate the amount of axonal degeneration. A CSF NF-L level of 150 ng/l was used as a cut-off point for high NF-L expression. SELDI-TOF analysis of peptides in the range of 2000-20000 m/z revealed one peak with m/z of 6378 that was expressed at a significantly different level (p<0.01) when rapidly versus slowly progressive cases of FTD were compared. Eleven peaks were expressed at different levels when high versus low CSF NF-L were compared. Using chromatographic purification followed by tandem mass spectrometric analysis, five of these peaks were identified as follows: C-terminal fragment of neuroendocrine protein 7B2 (3512.84 Da), C-terminal fragment of osteopontin (7658.19 Da) as well as its mono- and diphosphorylated forms (7738.16 Da and 7818.13 Da, respectively) and pancreatic ribonuclease (14566.33 Da). The peak intensity of pancreatic ribonuclease was higher in patients with low NF-L expression, while the other peptides had a lower peak intensity in this group. Altered levels of these peptides have also been described in other neurodegenerative diseases. Taken together, these data suggest that differentially-expressed peptides are general markers of axonal degeneration. Further studies are needed to verify their prognostic value in FTD.
|
|
| 22. |
- Mehmedbasic, Arnela, et al.
(author)
-
SorLA CR-Domains Protect the Amyloid Precursor Protein against Processing.
- 2015
-
In: The Journal of biological chemistry. - 1083-351X. ; 290, s. 3359-3376
-
Journal article (peer-reviewed)abstract
- SorLA is a neuronal sorting receptor that is genetically associated with Alzheimer's disease. SorLA interacts directly with the amyloid precursor protein (APP) and affects the processing of the precursor, leading to a decreased generation of the amyloid-β (Aβ) peptide. The sorLA complement-type repeat (CR)-domains associate in vitro with APP, but the precise molecular determinants of sorLA-APP complex formation and the mechanisms responsible for the effect of binding on APP processing have not yet been elucidated. Here, we have generated protein expression constructs for sorLA devoid of the 11 CR-domains and for two sorLA mutants harboring substitutions of the fingerprint residues in the central CR-domains. We generated SH-SY5Y cell lines that stably express these sorLA variants to study the binding and processing of APP using co-immunoprecipitation and western blotting/ELISA assays, respectively. We found that the sorLA CR-cluster is essential for interaction with APP and that deletion of the CR-cluster abolishes the protection against APP processing. Mutation of identified fingerprint residues in the sorLA CR-domains leads to changes in the O-linked glycosylation of APP when expressed in SH-SY5Y cells. Our results provide novel information on the mechanisms behind the influence of sorLA activity on APP metabolism by controlling post-translational glycosylation in the Golgi, suggesting new strategies against amyloidogenesis in Alzheimer's disease.
|
|
| 23. |
- Muslimovic, Aida, et al.
(author)
-
Calibrators for Clinical Measurements of Phosphorylated H2AX in Patient Cells by Flow Cytometry
- 2012
-
In: The Open Clinical Chemistry Journal. - : Bentham Science Publishers Ltd.. - 2588-7785 .- 1874-2416. ; 5:1, s. 33-39
-
Journal article (peer-reviewed)abstract
- Abstract: Agents that induce DNA double-strand breaks (DSBs), such as ionizing radiation, are frequently used in cancer therapy. H2AX is rapidly phosphorylated in response to DSBs and serves as a way to measure the extent of DSBs induced in patient cells. We have previously reported a flow cytometry-based method for measuring H2AX phosphorylation in pa-tient cells undergoing radiotherapy. To be able to implement measurement of H2AX phosphorylation in clinical practice, we have characterized calibrators for the flow cytometry analysis based on phosphopeptide-coated beads and fixed cells. The calibrator beads and fixed cells lost less than 11% of the signal after storage for 40 days under optimal conditions and were able to correct for day-to-day variation in method performance.
|
|
| 24. |
- Nilsson, Jonas, 1970, et al.
(author)
-
Enrichment of glycopeptides for glycan structure and attachment site identification.
- 2009
-
In: Nature methods. - : Springer Science and Business Media LLC. - 1548-7105 .- 1548-7091. ; 6:11, s. 809-11
-
Journal article (peer-reviewed)abstract
- We present a method to enrich for glycoproteins from proteomic samples. Sialylated glycoproteins were selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of sialic acid glycosidic bonds. Mass spectrometric fragment analysis allowed identification of glycan structures, and additional fragmentation of deglycosylated ions yielded peptide sequence information, which allowed glycan attachment site and protein identification. We identified 36 N-linked and 44 O-linked glycosylation sites on glycoproteins from human cerebrospinal fluid.
|
|
| 25. |
- Portelius, Erik, 1977, et al.
(author)
-
Mass spectrometric characterization of amyloid-β species in the 7PA2 cell model of Alzheimer's disease.
- 2013
-
In: Journal of Alzheimer's disease : JAD. - 1875-8908. ; 33:1, s. 85-93
-
Journal article (peer-reviewed)abstract
- The Chinese hamster ovary cell line 7PA2, stably transfected with the 751 amino acid isoform of amyloid-β protein precursor (AβPP) containing the Val → Phe mutation at residue 717, is one of the most used models to study the biochemistry and toxicity of secreted amyloid-β (Aβ) peptides, particularly Aβ oligomers, which are considered to be of relevance to the pathogenesis of Alzheimer's disease. Here, we present a detailed immunochemical and mass spectrometric characterization of primary structures of Aβ peptides secreted by 7PA2 cells. Immunoprecipitation and western blot of 7PA2 cell culture media revealed abundant anti-Aβ immunoreactive bands in the molecular weight range of 4-20 kDa. Mass spectrometric analysis showed that these bands contain several AβPP/Aβ peptides, starting at the N-terminal of the Aβ sequence and extending across the BACE1 cleavage site. Treatment of cells with a BACE1 inhibitor decreased the abundance of the Aβ monomer band by western blot and resulted in lower levels of Aβ1-40, Aβ1-42, and sAβPPβ as measured by ELISA. However, western blot bands thought to represent oligomers of Aβ increased in response to BACE1 inhibition. This increase was paralleled by the emergence of N-terminally truncated Aβ species (Aβ5-40 in particular) and Aβ species that spanned the β-secretase site in AβPP according to mass spectrometric analyses. The formation of these AβPP/Aβ peptides may have implications for the use of the 7PA2 cell line as a model for Aβ pathology. The enzyme(s) responsible for this particular BACE1-independent AβPP-processing remains to be identified.
|
|
| 26. |
- Rüetschi, Ulla, 1962, et al.
(author)
-
Characterization of 4-hydroxyphenylpyruvate dioxygenase. Primary structure of the Pseudomonas enzyme.
- 1992
-
In: European journal of biochemistry / FEBS. - 0014-2956. ; 205:2, s. 459-66
-
Journal article (peer-reviewed)abstract
- The primary structure of Pseudomonas 4-hydroxyphenylpyruvate dioxygenase was determined. Sequence degradation of the intact protein and of peptides from three different digests of the carboxymethylated protein established a 357-residue polypeptide chain with a free alpha-amino group. Hydroxylamine cleavage at a single Asn-Gly sequence was useful. Comparisons with known structures in data banks revealed no close relationship with other characterized proteins. The human enzyme has a related composition, suggesting that also the eukaryotic form belongs to this protein type, but with a blocked N-terminus like in many other eukaryotic intracellular proteins. Secondary structure predictions suggest an alpha/beta mixed structure, fairly typical of globular proteins, without long segments of hydrophobicity or charge, although a region in the middle of the C-terminal third of the subunit appears to have the most extreme properties. A ferric centre, correlating with enzyme activity and absorbance at 595 nm, has previously been assigned to tyrosinate coordination. The Tyr and His distributions, and the position of a single Cys residue, all suggest a few likely sites, outside the C-terminal segment, for this centre.
|
|
| 27. |
- Rüetschi, Ulla, 1962, et al.
(author)
-
gamma-Butyrobetaine hydroxylase. Structural characterization of the Pseudomonas enzyme.
- 1993
-
In: European journal of biochemistry / FEBS. - 0014-2956. ; 213:3, s. 1075-80
-
Journal article (peer-reviewed)abstract
- gamma-Butyrobetaine hydroxylase is a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of gamma-butyrobetaine to carnitine, the last step in the biosynthesis of carnitine from lysine. The primary structure of the enzyme from Pseudomonas sp. AK1 has been determined. Sequence analysis of the intact protein and of peptides from essentially three different digests established the presence of a peptide chain containing 383 residues, and an N-terminal truncated form of 382 residues. The two chains have molecular masses of 43,321 Da and 43,207 Da, respectively, and are identical except for the presence or absence of an N-terminal asparagine residue; the shorter form starts with an alanine residue. In preparations of the dimeric protein, the two chains occur in an approximate ratio of 1:1. There are nine cysteine residues and 13 histidine residues, i.e. amino acids which have been postulated as ligands for iron binding. In spite of functional similarities, there appears to be no clear sequence similarities with any of the other mammalian 2-oxoglutarate-dependent dioxygenases so far characterized.
|
|
| 28. |
- Rüetschi, Ulla, 1962, et al.
(author)
-
Human 4-hydroxyphenylpyruvate dioxygenase gene (HPD).
- 1997
-
In: Genomics. - : Elsevier BV. - 0888-7543. ; 44:3, s. 292-9
-
Journal article (peer-reviewed)abstract
- Overlapping DNA fragments spanning approximately 21 kb of genomic DNA and encompassing the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) have been cloned by screening a human leukocyte genomic library and by PCR amplification of human fibroblastic DNA. A continuous gene sequence of 20,890 nucleotides was established, including 1957 bp of the 5'-flanking region. The 4-hydroxyphenylpyruvate dioxygenase gene is composed of 14 exons interrupted by 13 introns, all exhibiting conventional vertebrate splicing. Computer analysis of the DNA sequence revealed 12 complete repetitive Alu elements, 1 in the 5'-flanking region and 11 in the intervening segments of the gene. The transcriptional initiation site was mapped to a position 35 nt upstream of the translational start point. The computer analysis also identified several potential transcription regulatory elements, including one CRE site, two AP-2 sites, and two Sp1 sites, in the sequence upstream of the transcription initiation site. Functional analysis of promoter activity by transient transfection of chloramphenicolacetyl transferase reporter plasmids revealed a possible involvement of cyclic adenosine monophosphate in the regulation of transcription. The highest level of expression of 4-hydroxyphenylpyruvate dioxygenase was found in human liver tissue as demonstrated by Northern blot analysis.
|
|
| 29. |
- Rüetschi, Ulla, 1962, et al.
(author)
-
Human 4-hydroxyphenylpyruvate dioxygenase. Primary structure and chromosomal localization of the gene.
- 1993
-
In: European journal of biochemistry / FEBS. - 0014-2956. ; 213:3, s. 1081-9
-
Journal article (peer-reviewed)abstract
- We report the primary structure of 4-hydroxyphenylpyruvate dioxygenase [4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating)]. The work is based on the isolation of cDNA clones from human liver lambda gt11 libraries. Several overlapping clones covering the coding sequence were characterized. In parallel, peptides from four different digests of the purified protein were analysed for their amino-acid sequence. These peptide sequences covered 86% of the cDNA-derived amino-acid sequence. This gives the sequence for a polypeptide of 392 amino acids with a calculated molecular mass of 44.8 kDa. There is more than 80% identity between the human and the pig enzymes and also between these enzymes and the F antigen from rat and the two allelic forms of this antigen from mouse. The enzyme has 53% conserved amino acids and 27% identical amino acids in common with 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 and 52% conserved and 28% identical residues, with a protein from Shewanella colwelliana. At the C-terminus there is 61% identity between the seven proteins. These results indicate that these proteins are all 4-hydroxyphenylpyruvate dioxygenases. The identity of the C-terminus makes this part of the molecule a candidate for a functional role in the catalytic process. At conserved positions in all seven enzymes, there are two tyrosine residues and three histidine residues, i.e. amino acids which have been implicated as ligands for iron in 2-oxoacid-dependent dioxygenases. The gene encoding the enzyme was localized to chromosome 12q14-->qter by Southern-blot analysis of human-rodent somatic-cell hybrids.
|
|
| 30. |
- Rüetschi, Ulla, 1962, et al.
(author)
-
Identification of CSF biomarkers for frontotemporal dementia using SELDI-TOF.
- 2005
-
In: Experimental neurology. - : Elsevier BV. - 0014-4886. ; 196:2, s. 273-81
-
Journal article (peer-reviewed)abstract
- This investigation describes the discovery of novel possible cerebrospinal fluid (CSF) biomarkers for frontotemporal dementia (FTD) using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS). Sixteen clinically diagnosed FTD patients and 12 non-demented controls were included in the study. CSF was collected and analyzed for protein expression by SELDI-TOF MS. The samples were analyzed on four different array surfaces using two different energy-absorbing molecules as matrices. In total each sample was subjected to eight different surface/matrix conditions. About 2000 protein peaks (mass/charge ratios) were detected. Forty-two peaks were differentially expressed in FTD (P < 0.01). After exclusion of peaks with low signal-to-noise ratio and/or poor resolution and peaks representing differentially charged proteins, 10 peaks remained, five of which were increased and five decreased in FTD cases compared to controls. Using partial least square discriminant analysis (PLS-DA), the combination of these biomarkers discriminated FTD from non-demented controls with a sensitivity of 94%, a specificity of 83% and an accuracy of 89%. Five of the peaks were purified further and identified by tandem MS as a fragment of neurosecretory protein VGF, transthyretin, S-cysteinylated transthyretin, truncated cystatin C and a fragment of chromogranin B. With use of these potential biomarkers, FTD can be distinguished from control subjects with high accuracy in this pilot study.
|
|
| 31. |
- Rüetschi, Ulla, 1962, et al.
(author)
-
Mutations in the 4-hydroxyphenylpyruvate dioxygenase gene (HPD) in patients with tyrosinemia type III.
- 2000
-
In: Human genetics. - 0340-6717. ; 106:6, s. 654-62
-
Journal article (peer-reviewed)abstract
- Tyrosinemia type III (OMIM 276710) is an autosomal recessive disorder caused by the deficiency of 4-hydroxyphenylpyruvate dioxygenase (HPD), the second enzyme in the tyrosine catabolic pathway. The enzyme deficiency results in an accumulation and increased excretion of tyrosine and phenolic metabolites. Only a few cases with the disorder have been described, and the clinical spectrum of the disorder is unknown. Reported patients have presented with mental retardation or neurological symptoms or have been picked up by neonatal screening. We have identified four presumed pathogenic mutations (two missense and two nonsense mutations) in the HPD gene in three unrelated families encompassing four homozygous individuals and one compound heterozygous individual with tyrosinemia type III. Furthermore, a number of polymorphic mutations have been identified in the HPD gene. No correlation of the severity of the mutation and enzyme deficiency and mental function has been found; neither do the recorded tyrosine levels correlate with the clinical phenotype.
|
|
| 32. |
- Rüetschi, Ulla, 1962, et al.
(author)
-
Proteomic analysis using protein chips to detect biomarkers in cervical and amniotic fluid in women with intra-amniotic inflammation
- 2005
-
In: J Proteome Res. ; 4:6, s. 2236-42
-
Journal article (peer-reviewed)abstract
- Intra-amniotic inflammation (IAI) may cause preterm birth with poor neonatal out-come. To identify novel biomarkers for IAI, we analyzed amniotic and cervical fluid samples from 27 patients with signs of threatening preterm birth with or without IAI by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Seventeen proteins were significantly overexpressed in amniotic fluid from IAI cases and more often in women with preterm labor than those with rupture of membranes. Five of these were identified as human neutrophil protein 1-3, calgranulin A and B.
|
|
| 33. |
- Rüetschi, Ulla, 1962, et al.
(author)
-
SILAC-Based Quantitative Proteomic Analysis of Diffuse Large B-Cell Lymphoma Patients.
- 2015
-
In: International journal of proteomics. - : Hindawi Limited. - 2090-2166 .- 2090-2174. ; 2015
-
Journal article (peer-reviewed)abstract
- Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma, is a heterogeneous disease where the outcome for patients with early relapse or refractory disease is very poor, even in the era of immunochemotherapy. In order to describe possible differences in global protein expression and network patterns, we performed a SILAC-based shotgun (LC-MS/MS) quantitative proteomic analysis in fresh-frozen tumor tissue from two groups of DLBCL patients with totally different clinical outcome: (i) early relapsed or refractory and (ii) long-term progression-free patients. We could identify over 3,500 proteins; more than 1,300 were quantified in all patients and 87 were significantly differentially expressed. By functional annotation analysis on the 66 proteins overexpressed in the progression-free patient group, we found an enrichment of proteins involved in the regulation and organization of the actin cytoskeleton. Also, five proteins from actin cytoskeleton regulation, applied in a supervised regression analysis, could discriminate the two patient groups. In conclusion, SILAC-based shotgun quantitative proteomic analysis appears to be a powerful tool to explore the proteome in DLBCL tumor tissue. Also, as progression-free patients had a higher expression of proteins involved in the actin cytoskeleton protein network, such a pattern indicates a functional role in the sustained response to immunochemotherapy.
|
|
| 34. |
- Sandstedt, Joakim, et al.
(author)
-
COMP (Cartilage Oligomeric Matrix Protein) Neoepitope: A Novel Biomarker to Identify Symptomatic Carotid Stenosis
- 2021
-
In: Arteriosclerosis, thrombosis, and vascular biology. - 1524-4636 .- 1079-5642. ; 41:3, s. 1218-1228
-
Journal article (peer-reviewed)abstract
- OBJECTIVE: COMP (cartilage oligomeric matrix protein) is abundantly expressed in the cardiovascular system, cartilage, and atherosclerotic plaques. We investigated if the total COMP (COMPtotal) and COMP neoepitope (COMPneo) with other cardiovascular markers and clinical parameters could identify symptomatic carotid stenosis. Approach and Results: Blood samples were collected from patients with symptomatic carotid stenosis (stenosis, n=50), patients with stroke without carotid stenosis but small plaques (plaque, n=50), and control subjects (n=50). COMPtotal and COMPneo were measured using an ELISA. Ninety-two cardiovascular disease markers were measured by the Olink CVD kit. The presence of native COMP and COMPneo was determined by immunohistochemistry. The concentration of COMPneo was higher and COMPtotal was lower in the stenosis group. When the concentration was compared between the stenosis and control groups, IL-1ra (interleukin-1 receptor antagonist protein), IL6 (interleukin-6), REN (Renin), MMP1 (matrix metalloproteinase-1), TRAIL-R2 (tumor necrosis factor-related apoptosis-inducing ligand receptor 2), ITGB1BP2 (integrin beta 1 binding protein 2), and COMPneo were predictive of stenosis. Conversely, KLK6 (kallikrein-6), COMPtotal, NEMO (nuclear factor-kappa-B essential modulator), SRC (Proto-oncogene tyrosine-protein kinase Src), SIRT2 (SIR2-like protein), CD40 (cluster of differentiation 40), TF (tissue factor), MP (myoglobin), and RAGE (receptor for advanced glycation end-products) were predictive of the control group. Model reproducibility was good with the receiver operating characteristic plot area under the curve being 0.86. When comparing the plaque group and stenosis group, COMPneo, GAL (galanin), and PTX3 (pentraxin-related protein PTX3) were predictive of stenosis. Model reproducibility was excellent (receiver operating characteristic plot area under the curve 0.92). COMPneo was detected in smooth muscle-, endothelial-, and foam-cells in carotid stenosis. CONCLUSIONS: Degradation of COMP may be associated with atherosclerosis progression and generation of a specific COMP fragment-COMPneo. This may represent a novel biomarker that together with COMPtotal and other risk-markers could be used to identify symptomatic carotid stenosis. Graphic Abstract: A graphic abstract is available for this article.
|
|
| 35. |
- Simonsen, A H, et al.
(author)
-
Amyloid beta1-40 quantification in CSF: comparison between chromatographic and immunochemical methods.
- 2007
-
In: Dementia and geriatric cognitive disorders. - : S. Karger AG. - 1420-8008 .- 1421-9824. ; 23:4, s. 246-50
-
Journal article (peer-reviewed)abstract
- BACKGROUND/AIMS: Amyloid beta (Abeta) is the principal component of senile plaques, one of the hallmarks of Alzheimer's disease (AD). Evidence is accumulating that soluble aggregates (oligomers) of Abeta are important in the pathogenesis of AD. METHODS: We compared three different methods for quantification of the 40 amino acid form of Abeta (Abeta40) in CSF, two based on antibodies [ELISA and surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) with antibody-coated arrays] and one based on direct binding of proteins to a protein array [SELDI-TOF and immobilized metal affinity [copper] (IMAC30)]. RESULTS: CSF Abeta40 concentration was only found to be significantly elevated in AD (127% of control levels; p=0.0095) using SELDI-TOF with IMAC30 arrays. CONCLUSIONS: These data suggest that the measured Abeta level in CSF may differ depending on whether antibody-based methods are used or not, possibly caused by epitope masking due to Abeta oligomerization or to binding of Abeta to carrier proteins.
|
|
| 36. |
- Skiöldebrand, Eva, et al.
(author)
-
Cartilage oligomeric matrix protein neoepitope in the synovial fluid of horses with acute lameness: A new biomarker for the early stages of osteoarthritis
- 2017
-
In: Equine Veterinary Journal. - : Wiley. - 0425-1644 .- 2042-3306. ; 49:5, s. 662-667
-
Journal article (peer-reviewed)abstract
- BackgroundClinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking. ObjectivesWe sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA. Study designIn vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well-characterised horses. MethodsArticular cartilage explants were incubated with or without interleukin-1 for 25 days. Media were analysed via quantitative proteomics. Synovial fluid was obtained from either normal joints (n = 15) or joints causing lameness (n = 17) or with structural OA lesions (n = 7) and analysed for concentrations of the COMP neoepitope using a custom-developed inhibition enzyme-linked immunosorbent assay (ELISA). Explants were immunostained with polyclonal antibodies against COMP and the COMP neoepitopes. ResultsSemitryptic COMP peptides were identified and quantified in cell culture media from cartilage explants. A rabbit polyclonal antibody was raised against the neoepitope of the N-terminal portion of one COMP fragment (sequence SGPTHEGVC). An inhibition ELISA was developed to quantify the COMP neoepitope in synovial fluid. The mean concentration of the COMP neoepitope significantly increased in the synovial fluid from the joints responsible for acute lameness compared with normal joints and the joints of chronically lame horses and in joints with chronic structural OA. Immunolabelling for the COMP neoepitope revealed a pericellular staining in the interleukin-1-stimulated explants. Main limitationsThe ELISA is based on polyclonal antisera rather than a monoclonal antibody. ConclusionsThe increase in the COMP neoepitope in the synovial fluid from horses with acute lameness suggests that this neoepitope has the potential to be a unique candidate biomarker for the early molecular changes in articular cartilage associated with OA.
|
|
| 37. |
- Stenberg, Johan, et al.
(author)
-
Quantitative proteomics reveals regulatory differences in the chondrocyte secretome from human medial and lateral femoral condyles in osteoarthritic patients
- 2013
-
In: Proteome Science. - : Springer Science and Business Media LLC. - 1477-5956. ; 11:1
-
Journal article (peer-reviewed)abstract
- Background: Osteoarthritis (OA) is a destructive joint disease and there are no known biomarkers available for an early diagnosis. To identify potential disease biomarkers and gain further insight into the disease mechanisms of OA we applied quantitative proteomics with SILAC technology on the secretomes from chondrocytes of OA knees, designated as high Mankin (HM) scored secretome. A quantitative comparison was made between the secretomes of the medial and lateral femur condyle chondrocytes in the same knee since the medial femur condyle is usually more affected in OA than the lateral condyle, which was confirmed by Mankin scoring. The medial/lateral comparison was also made on the secretomes from chondrocytes taken from one individual with no clinically apparent joint-disease, designated as low Mankin (LM) scored secretome. Results: We identified 825 proteins in the HM secretome and 69 of these showed differential expression when comparing the medial and lateral femoral compartment. The LM scored femoral condyle showed early signs of OA in the medial compartment as assessed by Mankin score. We here report the identification and relative quantification of several proteins of interest for the OA disease mechanism e.g. CYTL1, DMD and STAB1 together with putative early disease markers e.g. TIMP1, PPP2CA and B2M. Conclusions: The present study reveals differences in protein abundance between medial/lateral femur condyles in OA patients. These regulatory differences expand the knowledge regarding OA disease markers and mechanisms.
|
|
| 38. |
- Stenman, Göran, 1953, et al.
(author)
-
Regional assignment of the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) to 12q24-->qter by fluorescence in situ hybridization.
- 1995
-
In: Cytogenetics and cell genetics. - 0301-0171. ; 71:4, s. 374-6
-
Journal article (peer-reviewed)abstract
- Using a panel of human-rodent somatic cell hybrids, we have previously mapped the gene (HPD, previously called PPD) encoding 4-hydroxyphenylpyruvate dioxygenase to the distal half of the long arm of human chromosome 12, region q14-->qter. To obtain a genomic probe useful for fluorescence in situ hybridization (FISH) analysis we screened a human leukocyte genomic library and isolated a 13.4-kb phage clone, which by restriction fragment and sequence analyses was shown to contain exons 1-10 of HPD and approximately 2-kb upstream sequences. We now report the subregional localization of HPD to 12q24-->qter based on two color FISH analysis employing this clone.
|
|
| 39. |
- Svala, Emilia, et al.
(author)
-
An inflammatory equine model demonstrates dynamic changes of immune response and cartilage matrix molecule degradation in vitro
- 2015
-
In: Connective Tissue Research. - : Informa UK Limited. - 0300-8207 .- 1607-8438. ; 56:4, s. 315-325
-
Journal article (peer-reviewed)abstract
- The molecular aspects of inflammation were investigated in equine articular cartilage explants using quantitative proteomics. Articular cartilage explants were stimulated with interleukin (IL)-1 beta in vitro for 25 days, and proteins released into cell culture media were chemically labeled with isobaric mass tags and analyzed by liquid chromatography-tandem mass spectrometry. A total of 127 proteins were identified and quantified in media from explants. IL-1 beta-stimulation resulted in an abundance of proteins related to inflammation, including matrix metalloproteinases, acute phase proteins, complement components and IL-6. Extracellular matrix (ECM) molecules were released at different time points, and fragmentation of aggrecan and cartilage oligomeric matrix protein was observed at days 3 and 6, similar to early-stage OA in vivo. Degradation products of the collagenous network were observed at days 18 and 22, similar to late-stage OA. This model displays a longitudinal quantification of released molecules from the ECM of articular cartilage. Identification of dynamic changes of extracellular matrix molecules in the secretome of equine explants stimulated with IL-1 beta over time may be useful for identifying components released at different time points during the spontaneous OA process.
|
|
| 40. |
- Svala, Emilia, et al.
(author)
-
Characterisation of lubricin in synovial fluid from horses with osteoarthritis.
- 2017
-
In: Equine veterinary journal. - : Wiley. - 2042-3306 .- 0425-1644. ; 49:1, s. 116-123
-
Journal article (peer-reviewed)abstract
- The glycoprotein lubricin contributes to the boundary lubrication of the articular cartilage surface. The early events of osteoarthritis involve the superficial layer where lubricin is synthesised.To characterise the glycosylation profile of lubricin in synovial fluid from horses with osteoarthritis and study secretion and degradation of lubricin in an in vitro inflammation cartilage model.In vitro study.Synovial fluid samples collected from horses with joints with normal articular cartilage and structural osteoarthritic lesions; with and without osteochondral fragments, were analysed for the lubricin glycosylation profiles. Articular cartilage explants were stimulated with or without interleukin-1β for 25 days. Media samples collected at 3-day intervals were analysed by quantitative proteomics, western blot and enzyme-linked immunosorbent assay.O-glycosylation profiles in synovial fluid revealed both Core 1 and 2 O-glycans, with Core 1 O-glycans predominating. Synovial fluid from normal joints (49.5 ± 1.9%) contained significantly lower amounts of monosialylated Core 1 O-glycans compared with joints with osteoarthritis (53.8 ± 7.8%, P = 0.03) or joints with osteochondral fragments (57.3 ± 8.8%, P = 0.001). Additionally, synovial fluid from normal joints (26.7 ± 6.7%) showed higher amounts of disialylated Core 1 O-glycan than from joints with osteochondral fragments (21.2 ± 4.9%, P = 0.03). A C-terminal proteolytic cleavage site in lubricin was found in synovial fluid from normal and osteochondral fragment joints and in media from interleukin-1β stimulated and unstimulated articular cartilage explants.This is the first demonstration of a change in the glycosylation profile of lubricin in synovial fluid from diseased equine joints compared with that from normal joints. We demonstrate an identical proteolytic cleavage site of lubricin both in vitro and in vivo. The reduced sialation of lubricin in synovial fluid from diseased joints may affect the boundary lubricating ability of the superficial layer of articular cartilage and could be one of the early events in the progression of osteoarthritis.
|
|
| 41. |
- Toledo, Alejandro Gomez, et al.
(author)
-
O-Mannose and O-N-acetyl galactosamine glycosylation of mammalian alpha-dystroglycan is conserved in a region-specific manner
- 2012
-
In: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 22:11, s. 1413-1423
-
Journal article (peer-reviewed)abstract
- Defects in the O-linked glycosylation of the peripheral membrane protein alpha-dystroglycan (alpha-DG) are the main cause of several forms of congenital muscular dystrophies and thus the characterization of the glycosylation of alpha-DG is of great medical importance. A detailed investigation of the glycosylation pattern of the native alpha-DG protein is essential for the understanding of the biological processes related to human disease in which the protein is involved. To date, several studies have reported novel O-glycans and attachment sites on the mucin-like domain of mammalian alpha-DG with both similar and contradicting glycosylation patterns, indicating the species-specific O-glycosylation of mammalian alpha-DG. By applying a standardized purification scheme and subsequent glycoproteomic analysis of native alpha-DG from rabbit and human skeletal muscle biopsies and from cultured mouse C2C12 myotubes, we show that the O-glycosylation patterns of the mucin-like domain of native alpha-DG are conserved among mammalians in a region-specific manner.
|
|
| 42. |
- Zetterberg, Henrik, 1973, et al.
(author)
-
Clinical proteomics in neurodegenerative disorders.
- 2008
-
In: Acta neurologica Scandinavica. - : Hindawi Limited. - 1600-0404 .- 0001-6314. ; 118:1, s. 1-11
-
Research review (peer-reviewed)abstract
- Neurodegenerative disorders are characterized by neuronal impairment that eventually leads to neuronal death. In spite of the brain's known capacity for regeneration, lost neurons are difficult to replace. Therefore, drugs aimed at inhibiting neurodegenerative processes are likely to be most effective if the treatment is initiated as early as possible. However, clinical manifestations in early disease stages are often numerous, subtle and difficult to diagnose. This is where biomarkers that specifically reflect onset of pathology, directly or indirectly, may have a profound impact on diagnosis making in the future. A triplet of biomarkers for Alzheimer's disease (AD), total and hyperphosphorylated tau and the 42 amino acid isoform of beta-amyloid, has already been established for early detection of AD before the onset of dementia. However, more biomarkers are needed both for AD and for other neurodegenerative disorders, such as Parkinson's disease, frontotemporal dementia and amyotrophic lateral sclerosis. This review provides an update on recent advances in clinical neuroproteomics, a biomarker discovery field that has expanded immensely during the last decade, and gives an overview of the most commonly used techniques and the major clinically relevant findings these techniques have lead to.
|
|
