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Tomas Bergman

Publications and source records attributed to Tomas Bergman.

At least 19 recordsLinked to original sources
PubMed2006

Proteomic comparison of prostate cancer cell lines LNCaP-FGC and LNCaP-r reveals heatshock protein 60 as a marker for prostate malignancy.

BACKGROUND: Androgen-sensitive prostate cancer cell-line LNCaP-FGC and androgen-resistant line LNCaP-r constitute a model for development of androgen resistance in prostate cancer. METHODS: Proteins differently expressed in the two cell-lines were identified by two-dimensional (2-D) electrophoresis and mass spectrometry. HSP60, more abundant in LNCaP-r, was studied by RT-PCR and immunohistochemistry in specimens of human prostate cancer. RESULTS: HSP60 was upregulated in LNCaP-r, nm23 in LNCaP-FGC, and titin (two isoforms) in either LNCaP-r or LNCaP-FGC. In non-malignant prostate, HSP60-staining was in the glandular compartment, particularly basal epithelial cells. In prostate cancer, most epithelial cells showed moderate-strong staining without apparent correlation between staining intensity and Gleason grade. CONCLUSIONS: The LNCaP-FGC/LNCaP-r model, characterized by 2-D electrophoresis, reveals distinct proteomic alterations. With HSP60, results from cell-lines correlated with clinical results, indicating that this model can be used for dissection of mechanisms involved in transformation to androgen resistance and assignment of protein markers in prostate cancer.

Biomarkers, Tumor↗
PubMed2006

Host-derived pentapeptide affecting adhesion, proliferation, and local pH in biofilm communities composed of Streptococcus and Actinomyces species.

Salivary proline-rich proteins (PRPs) attach commensal Actinomyces and Streptococcus species to teeth. Here, gel filtration, mass spectrometry and Edman degradation were applied to show the release of a pentapeptide, RGRPQ, from PRP-1 upon proteolysis by Streptococcus gordonii. Moreover, synthetic RGRPQ and derivatives were used to investigate associated innate properties and responsible motifs. The RGRPQ peptide increased 2.5-fold the growth rate of S. gordonii via a Q-dependent sequence motif and selectively stimulated oral colonization of this organism in a rat model in vivo. In contrast, the growth of Streptococcus mutans, implicated in caries, was not affected. While the entire RGRPQ sequence was required to block sucrose-induced pH-decrease by S. gordonii and S. mutans, the N-terminal Arg residue mediated the pH increase (i.e., ammonia production) by S. gordonii alone (which exhibits Arg catabolism to ammonia). Strains of commensal viridans streptococci exhibited PRP degradation and Arg catabolism, whereas cariogenic species did not. The RGRPQ peptide mediated via a differential Q-dependent sequence motif, adhesion inhibition, and desorption of PRP-1-binding strains of A. naeslundii genospecies 2 (5 of 10 strains) but not of S. gordonii (n=5). The inhibitable A. naeslundii strains alone displayed the same binding profile as S. gordonii to hybrid peptides terminating in RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1. The present findings indicate the presence of a host-bacterium interaction in which a host peptide released by bacterial proteolysis affects key properties in biofilm formation.

Actinomyces↗
PubMed2005

Microfluidic electrocapture for separation of peptides.

A separation method based on electroimmobilization and sequential release of captured molecules is reported. A microfluidic electrocapture device is utilized to immobilize peptides in a microflow stream. After capture, the electric field is decreased in a stepwise manner, causing sequential release of the captured peptides according to their electrophoretic mobility. Tryptic peptides were separated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The separation power was high enough to increase the ionization yield of several peptides not seen in the unprocessed sample. In addition to separation, simultaneous sample cleanup was demonstrated for peptides obtained by shotgun tryptic digestion of membrane protein extracts.

Amino Acid Sequence↗
PubMed2005

Determination of site-specificity of S-glutathionylated cellular proteins.

Redox modification by S-glutathionylation is an expanding field within cell signalling research. However, the methods available for analysis of S-glutathionylated proteins in complex mixtures are not sufficiently accurate to specifically and in a high-throughput manner on a structural level establish the effects of S-glutathionylation on the individual proteins. A method has been developed for rapid identification of the S-glutathionylation sites of proteins in diamide-treated ECV304 cells, through tagging of deglutathionylated proteins with a cysteine-reactive biotin-affinity tag, trypsinisation, avidin-affinity purification of tagged peptides, and subsequent analysis by liquid chromatography and quadrupole time-of-flight tandem mass spectrometry. The method has led to identification of the glutathionylation sites of gamma-actin (Cys(217)), heat shock protein 60 (Cys(447)), and elongation factor 1-alpha-1 (Cys(411)). Further developments of accuracy within the field of peptide-affinity capture and mass spectrometry are discussed.

Binding Sites↗
PubMed2005

Electroimmobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purification.

Proinsulin C-peptide was electroimmobilized to a quartz crystal microbalance sensor chip, localizing this low-pI peptide for covalent attachment to activated surface carboxyl groups. The resulting chip was used in a continuous flow biosensor to capture anti-C-peptide antibodies, which could subsequently be eluted in 5% formic acid between air bubbles for efficient recovery and mass spectrometric identification. The method is reproducible through repeated cycles, providing affinity purification of proteins under real-time monitoring of the binding and elution processes.

Amino Acid Sequence↗
PubMed2005

Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission.

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.

Animals↗
PubMed2005

Cloning of alkaline sphingomyelinase from rat intestinal mucosa and adjusting of the hypothetical protein XP_221184 in GenBank.

Intestinal alkaline sphingomyelinase (alk-SMase) digests sphingomyelin and the process may influence colonic tumorigenesis and cholesterol absorption. We recently identified the gene of human alk-SMase and cloned the cDNA. Cross-species screening of homology in GenBank found a hypothetical rat protein, XP_221184, with 491 amino acid residues, which shares 73% identity with human alk-SMase. Based on the cDNA sequence of this protein, we cloned a cDNA from rat intestinal mucosa by RT-PCR. The cloned cDNA encodes a protein with 439 amino acid residues and higher (85%) identity with human alk-SMase. The cloned cDNA differed from the XP_221184 cDNA in splice sites linking exons 2 and 3, and exons 3 and 4, respectively. In the sequence of the cloned protein, the predicted activity motif, sphingomyelin binding sites, and potential glycosylation sites in human alk-SMase are all conserved. To confirm the cloned protein is the real form of alk-SMase, native alk-SMase was purified from rat intestine and subjected to proteolytic digestion followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and electrospray ionization (ESI) tandem mass spectrometry. Seven tryptic peptides were found to match the cloned protein sequence. Transient expression of the cloned cDNA linked with a myc tag in COS-7 cells demonstrated high SMase activity, with an optimal pH at 9.0 and a specific dependence on taurocholate and taurochenodeoxycholate. The expressed protein reacted with both anti-myc and anti-human alk-SMase antibodies. Northern blotting of rat tissues revealed high levels of mRNA in jejunum but not in other tissues. In conclusion, we cloned rat alk-SMase cDNA from rat intestine, adjusted the putative rat alk-SMase protein in GenBank, and confirmed the specific expression of the gene in the small intestine.

Amino Acid Sequence↗
PubMed2005

Actin and hnRNP U cooperate for productive transcription by RNA polymerase II.

To determine the role of actin-ribonucleoprotein complexes in transcription, we set out to identify novel actin-binding proteins associated with RNA polymerase II (Pol II). Using affinity chromatography on fractionated HeLa cells, we found that hnRNP U binds actin through a short amino acid sequence in its C-terminal domain. Post-transcriptional gene silencing of hnRNP U and nuclear microinjections of a short peptide encompassing the hnRNP U actin-binding sequence inhibited BrUTP incorporation in vivo. In living cells, we found that both actin and hnRNP U are associated with the phosphorylated C-terminal domain of Pol II, and antibodies to actin and hnRNP U blocked Pol II-mediated transcription. Taken together, our results indicate that a general actin-based mechanism is implicated in the transcription of most Pol II genes. Actin in complex with hnRNP U may carry out its regulatory role during the initial phases of transcription activation.

Actins↗
PubMed2005

Differential protein expression in anatomical zones of the prostate.

The prostate has three anatomical zones: the peripheral (PZ), the transition (TZ), and the central (CZ) zone. It is proposed that the CZ may be of mesodermal origin, whereas the other two are of endodermal origin. Proteome patterns in the zones were characterized to test for differences. Cells were scraped from macroscopically normal areas of PZ, TZ, and CZ in radical prostatectomy specimens. After exclusion of samples with cancer or prostatic intraepithelial neoplasia, 18 cases remained for analysis. Cells were collected in a medium with protease inhibitors, and the protein material was prepared for two-dimensional gel electrophoresis. The proteins in spots that differed quantitatively between regions were identified via mass spectrometric fingerprinting of tryptic fragments and selected tandem mass spectrometry sequence analysis. Ten proteins with significant zonal differential expression were identified, eight with underexpression in the CZ versus the PZ and the TZ (arginase II, ATP synthase, cytokeratin 8, lamin A/C, peroxiredoxin 4, protein disulfide isomerase A3, tropomyosin, and vimentin), and two with overexpression in the CZ (peroxiredoxin 2 and creatine kinase B). The PZ and TZ, although differing in terms of incidence of cancer and hyperplasia, have epithelium with highly similar major protein expression profiles. However, the protein profile of the CZ differs from that of the other regions, suggesting functional differences.

Aged↗
PubMed2005

Large-surface biosensor technology for enhanced recovery in protein characterization.

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.

Adsorption↗
PubMed2004

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology.

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.

Alkaline Phosphatase↗
PubMed2004

Increased levels of insulin and insulin-like growth factor-1 hybrid receptors and decreased glycosylation of the insulin receptor alpha- and beta-subunits in scrapie-infected neuroblastoma N2a cells.

We have previously shown that ScN2a cells (scrapie-infected neuroblastoma N2a cells) express 2-fold- and 4-fold-increased levels of IR (insulin receptor) and IGF-1R (insulin-like growth factor-1 receptor) respectively. In addition, the IR alpha- and beta-subunits are aberrantly processed, with apparent molecular masses of 128 and 85 kDa respectively, as compared with 136 and 95 kDa in uninfected N2a cells. Despite the 2-fold increase in IR protein, the number of (125)I-insulin-binding sites was slightly decreased in ScN2a cells [Ostlund, Lindegren, Pettersson and Bedecs (2001) Brain Res. 97, 161-170]. In order to determine the cellular localization of IR in ScN2a cells, surface biotinylation was performed, showing a correct IR trafficking and localization to the cell surface. The present study shows for the first time that neuroblastoma N2a cells express significant levels of IR-IGF-1R hybrid receptors, and in ScN2a cells the number of hybrid receptors was 2-fold higher than that found in N2a cells, potentially explaining the apparent loss of insulin-binding sites due to a lower affinity for insulin compared with the homotypic IR. Furthermore, the decreased molecular mass of IR subunits in ScN2a cells is not caused by altered phosphorylation or proteolytic processing, but rather by altered glycosylation. Enzymic deglycosylation of immunoprecipitated IR from N2a and ScN2a cells with endoglycosidase H, peptide N-glycosidase F and neuraminidase all resulted in subunits with increased electrophoretic mobility; however, the 8-10 kDa shift remained. Combined enzymic or chemical deglycosylation using anhydrous trifluoromethane sulphonic acid treatment ultimately showed that the IR alpha- and beta-subunits from ScN2a cells are aberrantly glycosylated. The increased formation of IR-IGF-1R hybrids in ScN2a cells may be part of a neuroprotective response to prion infection. The degree and functional significance of aberrantly glycosylated proteins in ScN2a cells remain to be determined.

Animals↗
PubMed2004

Multistep microreactions with proteins using electrocapture technology.

A method to perform multistep reactions by means of electroimmobilization of a target molecule in a microflow stream is presented. A target protein is captured by the opposing effects between the hydrodynamic and electric forces, after which another medium is injected into the system. The second medium carries enzymes or other reagents, which are brought into contact with the target protein and react. The immobilization is reversed by disconnecting the electric field, upon which products are collected at the outlet of the device for analysis. On-line reduction, alkylation, and trypsin digestion of proteins is demonstrated and was monitored by MALDI mass spectrometry.

Animals↗
PubMed2004

A cellular UDP-glucose deficiency causes overexpression of glucose/oxygen-regulated proteins independent of the endoplasmic reticulum stress elements.

A low level of UDP-Glc occurs in cells exposed to hypoxia or glucose starvation. This work reveals that a 65% reduction in the cellular UDP-Glc level causes up-regulation of the mitochondrial chaperone GRP75 and the endoplasmic reticulum (ER) resident chaperones GRP58, ERp72, GRP78, GRP94, GRP170, and calreticulin. Conditions that cause misfolding of proteins within the ER activate the transcription factors ATF6alpha/beta and induce translation of the transcription factors XBP-1/TREB5 and ATF4/CREB2. These transcription factors induce the overexpression of ER chaperones and CHOP/GADD153. However, the 65% decrease in the cellular UDP-Glc level does not cause activation of ATF6alpha, splicing of XBP-1/TREB5, induction of ATF4/CREB2, or expression of CHOP/GADD153. The activity of the promoters of the ER chaperones is increased in UDP-Glc-deficient cells, but the activity of the CHOP/GADD153 promoter is not affected, in comparison with their respective activities in cells having compensated for the UDP-Glc deficiency. The results demonstrate that the unfolded protein response remains functionally intact in cells with a 65% decrease in the cellular UDP-Glc level and provide evidence that this decrease is a stress signal in mammalian cells, which triggers the coordinate overexpression of mitochondrial and ER chaperones, independently of the ER stress elements.

Activating Transcription Factor 6↗
PubMed2004

Integrated sample preparation and MALDI mass spectrometry on a microfluidic compact disk.

High-throughput microfluidic processing of protein digests integrated with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on a compact disk (CD) is described. Centrifugal force moves liquid through multiple microstructures, each containing a 10-nL reversed-phase chromatography column. The CD enables parallel preparation of 96 samples with volumes ranging from one to several microliters. The peptides in the digests are concentrated, desalted, and subsequently eluted from the columns directly into MALDI target areas (200 x 400 microm) on the CD using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into the MALDI instrument for peptide mass fingerprinting and database identification at a routine sensitivity down to the 200-amol level. Detection of proteolytic peptides down to the 50-amol level is demonstrated. The success rate of the CD technology in protein identification is about twice that of the C(18) ZipTips and standard MALDI steel targets. The CDs are operated using robotics to transfer samples and reagents from microcontainers to the processing inlets on the disposable CD and spinning to control the movement of liquid through the microstructures.

Journal Article↗
PubMed2004

Identification of endothelial proteins by MALDI-MS using a compact disc microfluidic system.

Vascular endothelial proteins have been analyzed using two-dimensional (2D) gel electrophoresis and subsequent mass spectrometry, with separate methods for the intervening sample preparations. Compact disc (CD) technology was found to be rapid, giving high overall yield both with ordinary Coomassie staining and with Sypro Ruby staining. Combined with automatic in-gel digestion, the CD technology has great capacity for large numbers of protein analysis, although for limited sample numbers, manual methods can give similar sequence coverage. In a test set of 48 samples, 45 proteins were identified using the CD preparation technique, 32 identified with higher sequence coverage using the CD technique, 7 with higher using ZipTips in a robotic workstation, and 5 with higher coverage using dried droplets of unpurified samples. In the process of these methodological comparisons, basic patterns for 116 endothelial proteins were defined, representing 297 separate protein spots on the 2D gels.

Compact Disks↗
PubMed2003

A microfluidic electrocapture device in sample preparation for protein analysis by MALDI mass spectrometry.

The design and operation of a microfluidic device for sample preparation in MALDI mass spectrometry of peptides and proteins is described. It is particularly useful for proteomics applications and for mass determination of proteins in salt- and detergent-containing solutions. The system consists of a flow channel with two conductive areas or electrical junctions where proteins and peptides are retained by means of an electric field. The microfluidic device is made of PEEK tubing, and the junctions are covered with a conductive polymeric membrane. A syringe pump connected to the device produces a flow stream, and injection of sample is carried out manually via hydrodynamic pressure. Proteolytic peptides and intact proteins in salt- and detergent-containing acidic media were captured at the cathode junction followed by exchange of the original solution to a solvent suitable for subsequent mass spectrometry. Using this principle, a significant desalting effect was obtained for tryptic peptides in mass-mapping experiments. Protein sequence coverages were high (up to 40%) at subpicomole levels with results better than those obtained using reversed-phase solid-phase extraction. In contrast to the latter technique, the microfluidic device has the capacity to efficiently remove detergents such as CHAPS before peptide mapping and protein analysis.

Animals↗