| 1. |
- Banerjee, Debashish, et al.
(författare)
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Epithelial MUC1/Muc1 Promotes Cell migration, Reduces Apoptosis and Affects Levels of Mucosal Modulators During Acetylsalicylic Acid (Aspirin) Induced Gastropathy.
- 2015
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Ingår i: The Biochemical journal. - 1470-8728. ; 465:3, s. 423-431
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Tidskriftsartikel (refereegranskat)abstract
- MUC1 is a transmembrane mucin highly expressed in the stomach. While extensive research has uncovered many of its roles in cancer, knowledge about the functions of MUC1 in normal tissues is limited. Here we showed that acetylsalicylic acid (Aspirin, ASA) upregulated MUC1/Muc1 expression in the gastric mucosa of humans and wild type mice. ASA induced mucosal injury in all mice to a similar extent, however wild type animals and those chimeras with Muc1 on the epithelia recovered faster than Muc1 knock-out mice and chimeras carrying Muc1 on haematopoietic but not epithelial cells. MUC1 enhanced proliferation and migration of the human gastric cell line MKN-7, and increased resistance to apoptosis. The repeated treatment regime used caused a reduction in cyclooxygenase-1 expression, though wild type animals returned faster towards pre-treatment levels, and had increased cyclooxygenase-2 and vascular endothelial growth factor levels during recovery. Thus, we found that epithelial Muc1 is more important for the healing process than haematopoetic Muc1, and Muc1/MUC1 facilitates wound healing by enhancing cell migration and proliferation, protecting against apoptosis and mediating expression of mucosal modulators. Thus, MUC1 plays essential roles during wound healing, and development of treatment modalities targeting enhanced expression of MUC1 may be beneficial to treat mucosal wounds.
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| 2. |
- Carén, Helena, 1979, et al.
(författare)
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High incidence of DNA mutations and gene amplifications of the ALK gene in advanced sporadic neuroblastoma tumours.
- 2008
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 1470-8728 .- 0264-6021. ; 416:2, s. 153-9
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Tidskriftsartikel (refereegranskat)abstract
- ALK (anaplastic lymphoma kinase) is oncogenic in several tumours and has recently been identified as a predisposition gene for familial NB (neuroblastoma) harbouring mutations in the TKD (tyrosine kinase domain). We have analysed a large set of sporadic human NB primary tumours of all clinical stages for chromosomal re-arrangements using a CGH (comparative genomic hybridization) array (n=108) and mutations of the ALK gene (n=90), and expression of ALK and related genes (n=19). ALK amplification or in-gene re-arrangements were found in 5% of NB tumours and mutations were found in 11%, including two novel not previously published mutations in the TKD, c.3733T>A and c.3735C>A. DNA mutations in the TKD and gene amplifications were only found in advanced large primary tumours or metastatic tumours, and correlated with the expression levels of ALK and downstream genes as well as other unfavourable features, and poor outcome. The results of the present study support that the ALK protein contributes to NB oncogenesis providing a highly interesting putative therapeutic target in a subset of unfavourable NB tumours.
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| 3. |
- Fälker, Knut, 1971-, et al.
(författare)
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Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets
- 2011
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Ingår i: Biochemical Journal. - : Portland Press -- London. - 0264-6021 .- 1470-8728. ; 436:2, s. 469-480
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Tidskriftsartikel (refereegranskat)abstract
- PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(α12/13) and G(αq) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca²⁺ mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y₁₂ receptor-induced G(αi) signalling accounted for the loss of the aggregation response, as mimicking G(αi/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5'-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.
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| 4. |
- Liu, Ning, et al.
(författare)
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Transcriptional regulation mechanisms of hypoxia-induced neuroglobin gene expression.
- 2012
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Ingår i: The Biochemical journal. - 1470-8728 .- 0264-6021. ; 443:1, s. 153-164
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Tidskriftsartikel (refereegranskat)abstract
- Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554 bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor-1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions.
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| 5. |
- Schönherr, Christina, et al.
(författare)
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Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684
- 2011
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 440, s. 405-413
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in the kinase domain of ALK (anaplastic lymphoma kinase) have recently been shown to be important for the progression of the childhood tumour neuroblastoma. In the present study we investigate six of the putative reported constitutively active ALK mutations, in positions G1128A, I1171N, F1174L, R1192P, F1245C and R1275Q. Our analyses were performed in cell-culture-based systems with both mouse and human ALK mutant variants and subsequently in a Drosophila melanogaster model system. Our investigation addressed the transforming potential of the putative gain-of-function ALK mutations as well as their signalling potential and the ability of two ATP-competitive inhibitors, Crizotinib (PF-02341066) and NVP-TAE684, to abrogate the activity of ALK. The results of the present study indicate that all mutations tested are of an activating nature and thus are implicated in tumour initiation or progression of neuroblastoma. Importantly for neuroblastoma patients, all ALK mutations used in the present study can be blocked by the inhibitors, although some mutants exhibited higher levels of drug sensitivity than others.
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| 6. |
- Barderi, P, et al.
(författare)
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The NADP+ linked glutamate dehydrogenase from Trypanosoma cruzi : sequence, genomic organization and expression
- 1998
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 330:2, s. 951-958
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Tidskriftsartikel (refereegranskat)abstract
- NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.
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| 7. |
- Chevreuil, O, et al.
(författare)
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Heparin-decasaccharides impair the catabolism of chylomicrons.
- 1996
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 320 ( Pt 2), s. 437-44
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Tidskriftsartikel (refereegranskat)abstract
- On intravenous injection to rats, decasaccharides gave rise to a short-lived peak of lipoprotein lipase (LPL) activity, whereas octa- and hexasaccharides caused only marginal increases. In isolated hearts perfused by a single pass, decasaccharides released LPL more efficiently than conventional heparin on a mass basis. Octa- and hexasaccharides were much less efficient. Similar results were obtained for hepatic lipase, which was studied both in vivo and by liver perfusion. In the intact rat, the heparin fragments themselves disappeared rapidly from the circulating blood. The decay of hepatic lipase activity after the early peak roughly paralleled the decay of decasaccharide concentration, but for LPL the decay was faster, presumably because the liver extracted this lipase from plasma. To assess the lipase activities remaining in contact with blood a large dose of conventional heparin was injected at a series of times after the decasaccharides. LPL was decreased by 40% after 1 h. At that time, the LPL activity that could be released from isolated hearts by single-pass perfusion with heparin for 2 min ("functional LPL') was decreased by 75%. Chylomicrons labelled in vivo with [14C]oleic acid (primarily in triacylglycerols, providing a tracer for lipolysis) and [3H]retinol (primarily in ester form, providing a tracer for the particles) were injected intravenously to explore the effects of the LPL depletion on lipoprotein metabolism. Triacylglycerol lipolysis and particle clearance was markedly delayed from 30 min to 2 h after injection of decasaccharides. After 1 h the fractional catabolic rate was only one-third of the control value and the catabolism of chylomicron triacylglycerols by perfused hearts was delayed to a similar extent. Thus injection of decasaccharides leads to accelerated turnover of LPL with loss of functional LPL from extrahepatic tissues. This in turn leads to a period of delayed lipolysis and removal of chylomicron particles.
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| 8. |
- Hultin, M, et al.
(författare)
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Effect of protamine on lipoprotein lipase and hepatic lipase in rats.
- 1994
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 304 ( Pt 3), s. 959-66
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Tidskriftsartikel (refereegranskat)abstract
- The polycation protamine impedes the catabolism of triglyceride-rich lipoproteins and this has been suggested to be due to intravascular inactivation of lipoprotein lipase. We have made intravenous injections of protamine to rats and found that both lipoprotein lipase and hepatic lipase activities were released to plasma. The effect of protamine was more short-lived than that obtained by injection of heparin. The release of hepatic lipase by protamine was as effective as the release by heparin, while the amount of lipoprotein lipase released by protamine was only about one-tenth of that released by heparin. This was not due to inactivation of lipoprotein lipase, since injection of an excess of heparin 10 min after injection of protamine released as much lipoprotein lipase activity to plasma as in controls. The results in vivo differed from those obtained in model experiments in vitro. Protamine was able to almost quantitatively release both lipoprotein lipase and hepatic lipase from columns of heparin-agarose. The displacement was dependent on the total amount of protamine that had passed over the column, indicating that it was due to occupation by protamine of all available binding sites. Our results in vivo showed that the binding sites for lipoprotein lipase were not blocked as efficiently as those for hepatic lipase, indicating that the binding structures were not identical. It was concluded that the impaired turnover of lipoproteins by protamine probably was due to prevention of binding of the lipoproteins to endothelial cell surfaces rather than to impaired lipase function.
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| 9. |
- Liu, G, et al.
(författare)
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Interaction of size-fractionated heparins with lipoprotein lipase and hepatic lipase in the rat.
- 1992
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 285 ( Pt 3), s. 731-6
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Tidskriftsartikel (refereegranskat)abstract
- Heparin and heparin partially depolymerized by enzymic digestion were separated into six size fractions. Hep 1 (tetrasaccharides), with a mean M(r) of 1200, did not release significant amounts of either lipoprotein lipase (LPL) or hepatic lipase (HL) on intravenous injection into rats. Hep 2 (mainly octa- and deca-saccharides), with a mean M(r) of 2400-3000, released both lipases. To evoke the same plasma activity of LPL and HL required about 10 times more by weight, or about 40 times more molecules, of this heparin than of hep 5 (mean M(r) 12,000, similar to conventional heparin). Hep 5 impeded binding and degradation of 125I-labelled bovine LPL by perfused rat livers. In contrast, hep 2 had no detectable effect on these processes. This demonstrates a difference between the sites in the liver that mediate binding, uptake and degradation of LPL, and the extrahepatic sites that bind functional LPL, and the hepatic sites that bind functional HL. After injection of 3.25 mg of hep 5/kg body weight, plasma LPL activity rapidly rose and then remained high for at least 1 h. With hep 2, plasma LPL also rose rapidly, but then decreased to almost basal by 1 h. When a labelled triacylglycerol emulsion was injected 1 h after the heparins, the fractional catabolic rate was enhanced in the rats that had received conventional heparin, as expected from the high plasma LPL activity, but decreased compared with controls in rats that had received hep 2, indicating that available LPL had been depleted through enhanced transport to and uptake in the liver.
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| 10. |
- Nilsson, B, et al.
(författare)
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Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression.
- 1992
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 282 ( Pt 3), s. 715-721
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Tidskriftsartikel (refereegranskat)abstract
- In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.
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