Biomedical subjects

Henrik Vorum

Publications and source records attributed to Henrik Vorum.

At least 19 recordsLinked to original sources

PubMed2006

Locations, abundances, and possible functions of FXYD ion transport regulators in rat renal medulla.

The gamma-subunit of Na-K-ATPase (FXYD2) and corticosteroid hormone-induced factor (CHIF; FXYD4) are considered pump regulators in kidney tubules. The aim of this study was to expand the information about their locations in the kidney medulla and to evaluate their importance for electrolyte excretion in an animal model. The cellular and subcellular locations and abundances of gamma and CHIF in the medulla of control and sodium-depleted rats were analyzed by immunofluorescence and immunoelectron microscopy and semiquantitative Western blotting. The results showed that antibodies against the gamma-subunit COOH terminus and splice variant gamma(a), but not splice variant gamma(b), labeled intercalated cells, but not principal cells, in the initial part of the inner medullary collecting duct (IMCD1). In subsequent segments (IMCD2 and IMCD3), all principal cells exhibited distinct basolateral labeling for both the gamma-subunit COOH terminus, splice variant gamma(a), and CHIF. Splice variant gamma(b) was abundant in the inner stripe of the outer medulla but absent in the inner medulla (IM). Double labeling by high-resolution immunoelectron microscopy showed close structural association between CHIF and the Na-K-ATPase alpha(1)-subunit in basolateral membranes. The present observations provide new information about the cellular and subcellular locations of gamma and CHIF in the renal medulla and show a new gamma variant in the IM. Extensive NaCl depletion did not induce significant changes in the locations or abundances of the gamma-subunit COOH terminus and CHIF in different kidney zones. We conclude that the unchanged levels of these two FXYD proteins suggest that they are not primary determinants for urine electrolyte composition during NaCl depletion.

Animals↗

PubMed2006

Protein profiling the effects of in vitro hyperoxic exposure on fetal rabbit lung.

The aim of this study was to test the hypothesis that acute in vitro exposure of prematurely delivered fetal rabbit lungs to hyperoxic conditions will induce the expression of an adaptive cassette of proteins that mediates antioxidant and inflammatory processes. To test this hypothesis, ex situ fetal rabbit lung explants were prepared from New Zealand white rabbits delivered by cesarean section on day 29 of gestation and incubated under air (21% O2; 5% CO2) or hyperoxic (95% O2; 5% CO2) atmospheres. Total tissue protein was extracted following incubation and subjected to 2-DE. Using this technique, 1500-2000 protein spots were resolved per gel. Treatment-dependent, differentially expressed proteins were identified by image analysis (Melanie II) and MALDI-TOF MS and MALDI-MS/MS. The analysis identified 12 protein spots that were differentially expressed by 1.5-fold or more (p<0.05) by exposure to hyperoxic conditions. Six of these differentially expressed proteins were identified as vimentin, annexin I, inorganic pyrophosphatase, prohibitin, an N-terminal fragment of ATP synthase and heat shock protein 27. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation.

Animals↗

PubMed2006

Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton.

CREC proteins constitute a family of EF-hand calcium binding proteins localized to the secretory pathway. Calumenin is the only member known to be secreted. Recently, it was shown that thrombin-activated thrombocytes liberate calumenin, which also is found in atherosclerotic lesions but not in normal vasculature. To study the possible effects of calumenin extracellularly, we used proteomic profiling of fibroblasts cultured in absence and in presence of calumenin. Using 2-DE and MS/MS, we show that normal fibroblasts contain several 28-29-kDa N-terminal and a 16-kDa C-terminal fragment of beta- or gamma-actin. Extracellularly added calumenin decreases the levels of both the N-terminal and C-terminal actin fragments, and, in addition, decreases the expression level of septin 2, which interacts with the actin cytoskeleton and is involved in cytokinesis. Labeling of S-phase fibroblasts with bromo-2'deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin may have an autocrine or a paracrine effect on the cells in its vicinity, and, therefore, may be involved in the pathophysiology of thrombosis or in wound healing.

Actins↗

PubMed2005

Predictors of post-operative mortality following treatment for non-ruptured abdominal aortic aneurysm.

The aim of this prospective study of patients undergoing repair of non-ruptured abdominal aortic aneurysm between 1999 and 2003 was to evaluate and compare risk factors for mortality after surgery, to determine a complex of informative factors for lethal outcome, and to define patient risk groups. Logistic regression analysis revealed a complex of informative factors, including female gender, previous myocardial infarction, age greater than 75 years, and clinical course of abdominal aortic aneurysm as important indicators for lethal outcome. A risk score model identified low-, moderate- and high-risk groups with mortality rates of 2.9%, 8.0% and 44.4%, respectively.

Journal Article↗

PubMed2005

Proteome profiling of corneal epithelium and identification of marker proteins for keratoconus, a pilot study.

The purpose of this study is to identify corneal proteins differentially expressed between keratoconus and normal epithelial samples. Proteins from the corneal epithelium were isolated from 6 keratoconus and 6 myopia patients (controls) and separated by 2D-gel electrophoresis. Six % and 12% SDS-PAGE gels were used to separate low and high molecular weight proteins. Gels were silver stained and protein spots were defined by Melanie II software. The proteins that were most altered in expression comparing keratoconus and controls were extracted, trypsin-digested, and identified by mass spectroscopy. Approximately 200-500 protein spots were detected on each gel. Nineteen spots were identified as differentially expressed between keratoconus and reference epithelium including cytokeratin 3 (< 7.8 fold), gelsolin (1.6 fold), S100A4 (1.9 fold), and enolase 1 (0.72 fold). Another identified protein found at very high levels was cytokeratin 12. Gelsolin, cytokeratin 3, and cytokeratin 12 have previously been described to be involved in other corneal diseases. Three proteins, gelsolin, alpha enolase, and S100A4 were identified to be differentially expressed in keratoconus compared to reference epithelium and thus may be involved in the pathogenesis.

Biomarkers↗

PubMed2005

Interaction of FXYD10 (PLMS) with Na,K-ATPase from shark rectal glands. Close proximity of Cys74 of FXYD10 to Cys254 in the a domain of the alpha-subunit revealed by intermolecular thiol cross-linking.

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the alpha-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the alpha-subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the alpha-subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the alpha-subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the alpha-subunit comprising 33 amino acids, including a single Cys residue, Cys254. Thus, regulation of Na,K-ATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark alpha-subunit.

Adenosine Triphosphate↗

PubMed2005

Amnionless function is required for cubilin brush-border expression and intrinsic factor-cobalamin (vitamin B12) absorption in vivo.

Amnionless (AMN) and cubilin gene products appear to be essential functional subunits of an endocytic receptor called cubam. Mutation of either gene causes autosomal recessive Imerslund-Gräsbeck syndrome (I-GS, OMIM no. 261100) in humans, a disorder characterized by selective intestinal malabsorption of cobalamin (vitamin B12) and urinary loss of several specific low-molecular-weight proteins. Vital insight into the molecular pathology of I-GS has been obtained from studies of dogs with a similar syndrome. In this work, we show that I-GS segregates in a large canine kindred due to an in-frame deletion of 33 nucleotides in exon 10 of AMN. In a second, unrelated I-GS kindred, affected dogs exhibit a homozygous substitution in the AMN translation initiation codon. Studies in vivo demonstrated that both mutations abrogate AMN expression and block cubilin processing and targeting to the apical membrane. The essential features of AMN dysfunction observed in vivo are recapitulated in a heterologous cell-transfection system, thus validating the system for analysis of AMN-cubilin interactions. Characterization of canine AMN mutations that cause I-GS establishes the canine model as an ortholog of the human disorder well suited to studies of AMN function and coevolution with cubilin.

Animals↗

PubMed2005

Influence of oxygen concentration and pH on expression of hypoxia induced genes.

BACKGROUND AND PURPOSE: This study was designed to determine the oxygen dependency for expression of the endogenous hypoxic markers carbonic anhydrase IX (protein: CAIX/gene: CA9), glucose transporter 1 (GLUT1/GLUT1), osteopontin (OPN/OPN) and lactate dehydrogenase A (LDH-A/LDHA), and how this expression was influenced by extracellular pH (pHe). MATERIALS AND METHODS: Human cervix squamous cell carcinoma (SiHa) cells were used in all experiments. These cells were gassed in an enclosed environment under either anoxia (95% N2+5% CO2) for various times (0-30 h) or under different oxygen concentrations (0-21% O2) for 24 h at normal pHe (7.4) or low pHe (6.3). Response to radiation (7 Gy) was estimated using a clonogenic assay. Gene expression was determined by real-time PCR (normalized to the housekeeping gene, TFRC) and protein expression by Western blots. RESULTS: Under normal pHe conditions, CA9, GLUT1 and LDHA gene expression was upregulated within 1-3h of anoxia, reaching near maximal values by 6h. OPN showed a slow increase over 24 h. At 24 h the relative increase was 135, 12, 90 and 5 times for CA9, GLUT1, OPN and LDHA, respectively. No induction was seen with the EGF receptor (EGFR). Gassing cells with differing oxygen concentrations for 24h resulted in a maximum level of expression for CA9 at 1% oxygen, whereas with GLUT1 and LDHA maximal expression occurred at 0.01% oxygen, but at 0% oxygen with OPN. The oxygen dependency for radiation response was identical to that seen for GLUT1 and LDHA. Expression of CA9, GLUT1, OPN and LDHA was inhibited under hypoxic conditions when pHe was reduced to 6.3. Expression of CAIX protein mimicked the CA9 gene expression patterns. CONCLUSION: The expression of all the endogenous markers were upregulated by hypoxia, but the timing and oxygen dependencies were different and their expression was influenced by low pHe. This raises concerns about the generalised use of these agents as markers for hypoxia.

Antigens, Neoplasm↗

PubMed2005

Suppressed phospholamban levels differentiate irreversibly dysfunctional from hibernating myocardium in humans.

OBJECTIVES: We studied whether dysfunction of human hibernating (HIB) and irreversibly dysfunctional myocardium (IRDM) are associated with altered levels of the sarcoplasmatic reticulum calcium handling proteins Ca2+-ATPase (SERCA2a) and its inhibitor phospholamban (PLB). DESIGN: In 12 patients myocardial biopsies were taken during bypass surgery and analysed for contents of these proteins. We classified regions as control, HIB, or IRDM based on echocardiographic studies before and 6 months after surgery. RESULTS: SERCA2a content (mean+/-SEM) was similar to control in HIB and IRDM (2.6 +/- 1.7, 3.8 +/- 2.0, and 3.4 +/- 1.9 units/g non-collagen protein (NCP), p = 0.40). PLB content was similar to control in HIB (2.6 +/- 0.4 and 3.5 +/- 0.5 units/microg NCP) but reduced in IRDM (0.9 +/- 0.2 units/microg NCP, p < 0.05). SERCA2a:PLB ratio, an indicator of SERCA2a activity, did not differ between control and HIB (1.2 +/- 0.3 and 1.4 +/- 0.4 units/microg NCP) but was increased in IRDM (5.1 +/- 1.7 units/microg NCP, p < 0.05). CONCLUSIONS: Inappropriate SERCA2a activity due to suppressed PLB levels may represent a maladaptive mechanism in chronic ischemic myocardium being causally linked to irreversibility of left ventricular dysfunction.

Analysis of Variance↗

PubMed2005

Altered expression of CLC, DSG3, EMP3, S100A2, and SLPI in corneal epithelium from keratoconus patients.

PURPOSE: This investigation was designed to determine whether the five genes, CLC, DSG3, EMP3, S100A2 and SLPI, are differentially expressed in keratoconus, as indicated from another study. METHODS: Gene expression was monitored using quantitative real-time PCR on 14 keratoconus samples and 16 controls, and normalized to GAPDH and B2M. The DSG3, S100A2, and SLPI proteins were quantified by Western blotting, and the cellular localization was determined by immunohistochemistry. One of the genes, CLC, was reduced in gene expression and its four exons were sequenced. RESULTS: The five genes were all differentially expressed in keratoconus (P < 0.04) and so were at least three of the encoded proteins (P = 0.009). DSG3 was expressed in association with the cell membrane of the basal and suprabasal epithelial cells, and S100A2 was expressed in the nucleus and cytoplasm, often as intracellular granules. Two SNPs (rs374185 and rs384138) were observed in the CLC gene, each with an allele frequency of 68%. No other mutations were detected. CONCLUSIONS: The five genes, and three of the encoded proteins, were shown differentially expressed between a group of keratoconus patients and a reference group using different techniques. These alterations, in combination with earlier findings, strongly demonstrate the genes to be involved in the corneal disease. We suggest the unambiguously expressed DSG3 protein to be used as a marker for keratoconus.

Biomarkers↗

PubMed2005

Proteomic analysis of aqueous humour from patients with acute corneal rejection.

PURPOSE: To compare the basic proteomic composition of aqueous humour (AH) from patients with corneal rejection (patients) with AH from patients with cataract (controls). METHODS: Aqueous humour was analysed for total protein concentration using Bradford's method and for protein composition using two-dimensional (2D) gel electrophoresis. Image analysis was used to detect protein spots in 2D gels that were increased by more than factor 2 in patients as compared with controls. Increased spots were identified by immunoblotting and mass spectrometry. RESULTS: Aqueous humour from patients contained significantly higher total protein concentration than did AH from controls. A total of 31 spots were significantly increased in 2D gels from patients. The spots were derived from albumin, alpha1-antitrypsin, apolipoprotein J, cytokeratin type II, serin proteinase inhibitor and transthyretin. After correction of spot volumes by total protein concentrations, 10 spots derived from albumin, cytokeratin type II and alpha1-antitrypsin remained significantly increased. CONCLUSION: The proteomic composition of AH differed significantly between patients and controls. The identified proteins suggest that the changes in AH are due to at least three different mechanisms: breakdown of the aqueous-blood barrier, enzymatic degradation, and liberation of locally synthesized proteins.

Aged↗

PubMed2005

Soluble CD163 and interleukin-6 are increased in aqueous humour from patients with endothelial rejection of corneal grafts.

PURPOSE: To evaluate soluble CD163 (sCD163) as a new marker of macrophage activity in aqueous humour from patients with corneal rejection and to investigate correlations between sCD163, the CD163 inducing interleukin-6 (IL-6), and albumin; to investigate whether increases in sCD163 and IL-6 levels in aqueous humour were results of intra- or extraocular production, and to investigate the impact of sCD163 and IL-6 levels on graft outcome. METHODS: Aqueous humour was obtained from 19 patients with endothelial rejection of corneal grafts, seven cataract patients and five cataract patients with uncomplicated corneal grafts. The presence of sCD163 was investigated by immunoprecipitation and immunoblotting. The concentrations of sCD163, IL-6 and albumin were measured by ELISA. RESULTS: Soluble CD163 was detected in aqueous humour from patients with corneal rejection. Soluble CD163, IL-6 and albumin were significantly increased in aqueous humour from patients with corneal rejection when compared with control patients. When normalizing sCD163 and IL-6 with albumin, the differences between rejection patients and control groups became insignificant for sCD163 but remained significant for IL-6. Neither IL-6 nor sCD163 were related to the outcome of the corneal rejection. CONCLUSIONS: Both sCD163 and IL-6 are present in high levels in aqueous humour from patients with rejection of corneal grafts. Our data suggest that IL-6 is produced locally, whereas sCD163 is at least partly derived from influx from plasma.

Adult↗

PubMed2005

Alpha-1--antitrypsin in aqueous humour from patients with corneal allograft rejection.

PURPOSE: To investigate the presence and concentration of alpha1-antitrypsin in aqueous humour at the time of corneal rejection and to compare results obtained from patients with reversible and irreversible rejection. METHODS: Samples of aqueous humour were obtained from 17 patients with acute corneal endothelial allograft rejection. The presence of alpha1-antitrypsin in aqueous humour was confirmed by immunoblotting and measured employing a sandwich ELISA. Total protein concentrations in aqueous humour were measured using Bradford's method. The outcome of corneal rejection episodes was determined 1 month after diagnosing corneal rejection and described as reversible or irreversible rejection. RESULTS: alpha1-antitrypsin was detected in aqueous humour. Patients with reversible rejection had significantly higher alpha1-antitrypsin concentration than patients with irreversible rejection (p = 0.044). There was no significant difference in total protein concentrations (p = 0.745), and no correlation was found between alpha1-antitrypsin and total protein concentrations (p = 0.368). CONCLUSIONS: alpha1-antitrypsin in aqueous humour seems to signal a favourable outcome of corneal rejection. The possible mechanism is discussed.

Acute Disease↗

PubMed2005

Altered calmodulin response to light in the suprachiasmatic nucleus of PAC1 receptor knockout mice revealed by proteomic analysis.

In mammals circadian rhythms are generated by a light-entrainable oscillator located in the hypothalamic suprachiasmatic nucleus (SCN). Light signals reach the SCN via a monosynaptic neuronal pathway, the retinohypothalamic tract, originating in a subset of retinal ganglion cells. The nerve terminals of these cells contain the classical neurotransmitter glutamate and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), and there is evidence that these two transmitters interact to mediate photoentrainment of the oscillator in the SCN. To elucidate light-provoked PACAP receptor signaling we used proteomic analysis. Wild-type mice and mice lacking the PAC1 receptor (PAC1-/-) were light stimulated at early night, and the SCN was examined for proteins that were differentially expressed using two-dimensional gel electrophoresis and identification by tandem mass spectrometry. The most striking finding, which was subsequently confirmed by Western blotting, was a significant reduction of calmodulin (CaM) in wild-type mice as compared with PAC1-/- mice. Analysis at the mRNA level by quantitative in situ hybridization histochemistry was inconclusive, indicating that a translational mechanism might be involved. The findings indicate that PAC1 receptor signaling in the SCN in response to light stimulation induces a down-regulation of CaM expression and that CaM is involved in the photic-entrainment mechanism.

Animals↗

PubMed2004

The gamma-subunit of Na-K-ATPase is incorporated into plasma membranes of mouse IMCD3 cells in response to hypertonicity.

Hypertonicity mediated by chloride upregulates the expression of the gamma-subunit of Na-K-ATPase in cultured cells derived from the murine inner medullary collecting duct (IMCD3; Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Proc Natl Acad Sci USA 100: 6428-6433, 2003). The purpose of this study was to examine the cellular locations and the time course of gamma-subunit expression after long-term adaptation and acute hypertonic challenges induced with different salts. Cells were analyzed by confocal immunofluorescence and immunoelectron microscopy with antibodies against the COOH terminus of the Na-K-ATPase gamma-subunit or the gamma(b) splice variant. Cells grown in 300 mosmol/kgH(2)O showed no immunoreactivity for the gamma-subunit, whereas cells adapted to 600 or 900 mosmol/kgH(2)O demonstrated distinct reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mosmol/kgH(2)O with sodium chloride or choline chloride showed incorporation of gamma into plasma membrane 12 h after osmotic challenge and distinct membrane staining in approximately 40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mosmol/kgH(2)O by addition of sodium acetate did not result in expression of the gamma-subunit in the membranes of surviving cells after 48 h. The present results demonstrate that the Na-K-ATPase gamma-subunit becomes incorporated into the basolateral membrane of IMCD3 cells after both acute hyperosmotic challenge and hyperosmotic adaptation. We conclude that the gamma-subunit has an important role in the function of Na-K-ATPase to sustain the cellular cation balance over the plasma membrane in a hypertonic environment.

Adaptation, Physiological↗

PubMed2004

Non-muscle myosin II and myosin light chain kinase are downstream targets for vasopressin signaling in the renal collecting duct.

We have previously demonstrated that vasopressin increases the water permeability of the inner medullary collecting duct (IMCD) by inducing trafficking of aquaporin-2 to the apical plasma membrane and that this response is dependent on intracellular calcium mobilization and calmodulin activation. Here, we address the hypothesis that this water permeability response is mediated in part through activation of the calcium/calmodulin-dependent myosin light chain kinase (MLCK) and regulation of non-muscle myosin II. Immunoblotting and immunocytochemistry demonstrated the presence of MLCK, the myosin regulatory light chain (MLC), and the IIA and IIB isoforms of the non-muscle myosin heavy chain in rat IMCD cells. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified two isoforms of MLC, both of which also exist in phosphorylated and non-phosphorylated forms. 32P incubation of the inner medulla followed by autoradiography of two-dimensional gels demonstrated increased 32P labeling of both isoforms in response to the V2 receptor agonist [deamino-Cys1,D-Arg8]vasopressin (DDAVP). Time course studies of MLC phosphorylation in IMCD suspensions (using immunoblotting with anti-phospho-MLC antibodies) showed that the increase in phosphorylation could be detected as early as 30 s after exposure to vasopressin. The MLCK inhibitor ML-7 blocked the DDAVP-induced MLC phosphorylation and substantially reduced [Arg8]vasopressin (AVP)-stimulated water permeability. AVP-induced MLC phosphorylation was associated with a rearrangement of actin filaments (Alexa Fluor 568-phalloidin) in primary cultures of IMCD cells. These results demonstrate that MLC phosphorylation by MLCK represents a downstream effect of AVP-activated calcium/calmodulin signaling in IMCD cells and point to a role for non-muscle myosin II in regulation of water permeability by vasopressin.

Actins↗

PubMed2004

Serum antibodies against Chlamydia pneumoniae outer membrane protein cross-react with the heavy chain of immunoglobulin in the wall of abdominal aortic aneurysms.

BACKGROUND: Chlamydia pneumoniae (Cp) has been demonstrated in arteries and abdominal aortic aneurysms (AAAs). However, the validity of the methods used is questioned, and antibiotic treatment trials have thus far shown disappointing results. Nevertheless, antibodies against the Cp outer membrane proteins (OMPs) have been associated with progression of atherosclerosis and AAAs. The aim of this study was to detect Cp OMPs in the wall of AAA patients by use of purified serum antibodies directed against Cp OMP and to assess potential cross-reacting proteins in AAA walls. METHODS AND RESULTS: Seventeen patients undergoing infrarenal AAA repair were studied. Full AAA thickness tissue was collected from the anterior wall of the aneurysm. Anti-OMP was extracted from seropositive AAA patients by use of an ELISA kit (Labsystems). Analysis was performed by use of 2D polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometric protein identification. OMP antigens were not detected in 16 of 17 AAA walls. However, 3 major AAA proteins cross-reacted with anti-OMP. The proteins were all identified as heavy chains of human immunoglobulin. CONCLUSIONS: We could not find evidence of Cp OMP in 16 of 17 AAA walls, but instead, all samples showed a strong cross-reaction between Cp OMP antibodies and human immunoglobulin. This might indicate that AAA is an autoimmune disease, perhaps triggered by an initial Cp infection.

Aged↗

PubMed2004

Changes in protein expression in p53 deleted spontaneous thymic lymphomas.

By the use of high-resolution two-dimensional gel electrophoresis and computerized image analysis we investigated and compared the expression of cellular proteins from p53 positive (+/+) mouse thymocytes, p53-/- thymocytes before neoplastic transformation, and from cell lines derived from two spontaneous p53-/- thymic lymphomas, SM5 and SM7. A total of around 1500 proteins were detected on individual gels. Only changes in protein expression by a factor of 2 or more were considered. In the thymic lymphoma cells 3-5% of the proteins were found to be differentially regulated when compared with the protein expression in p53+/+ and p53-/- thymocytes. Only a minority (13 proteins) of the quantitatively changed proteins were common for the two thymic lymphoma cell lines, suggesting that the p53 deficiency mainly results in genetic dysfunctions which are individual for a given tumor. Two of the detected proteins increased their expression levels by more than 10 times from the p53+/+ to the p53-/- thymocytes and these high expression levels were also found in thymic lymphomas. The two proteins were identified by mass spectrometry as acidic ribosomal phosphoprotein P0 and a 33-kDa protein with a primary structure containing motifs of the glyoxalase-bleomycin resistance protein family (MDR) as deduced from the cDNA.

Animals↗