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| 2. |
- Asker, Noomi, 1968, et al.
(författare)
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Human MUC5AC mucin dimerizes in the rough endoplasmic reticulum, similarly to the MUC2 mucin.
- 1998
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Ingår i: The Biochemical journal. - 0264-6021. ; 335:2, s. 381-7
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Tidskriftsartikel (refereegranskat)abstract
- Biosynthetic studies on the human MUC5AC mucin were performed by immunoprecipitations with antisera recognizing only the non-O-glycosylated apomucin in the colon adenocarcinoma cell line LS 174T. Pulse-chase studies and subcellular fractionations showed that MUC5AC formed dimers in the rough endoplasmic reticulum within 15 min of the initiation of biosynthesis. No non-O-glycosylated species larger than dimers were identified. The dimerization was N-glycosylation-dependent, because tunicamycin treatment significantly lowered the rate of dimerization. When the biosynthesis of MUC5AC apomucin was compared with that of MUC2 apomucin, also produced in the LS 174T cell line, both apomucins were assembled in similar ways with respect to their rates of dimerization with and without inhibition of N-glycosylation. No heterodimerization was observed between the human MUC5AC and the MUC2 apomucins despite the extensive sequence similarities in the positions of the cysteine residues in the C-termini proposed to be involved in mucin dimerization.
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| 3. |
- Asker, Noomi, 1968, et al.
(författare)
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The human MUC2 mucin apoprotein appears to dimerize before O-glycosylation and shares epitopes with the 'insoluble' mucin of rat small intestine.
- 1995
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Ingår i: The Biochemical journal. - 0264-6021. ; 308:3, s. 873-80
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Tidskriftsartikel (refereegranskat)abstract
- Rabbit antiserum against a synthetic peptide corresponding to a tandemly repeated amino acid sequence in the human intestinal mucin apoprotein MUC2 was used in immunoprecipitation to study the biosynthesis of MUC2 in the colon-carcinoma cell line LS 174T. Under non-reducing conditions, two bands were precipitated, the smaller with an apparent size of about 700 kDa on SDS/PAGE. When analysed by two-dimensional electrophoresis after reduction, the larger band migrated to the same position as the smaller band and was interpreted as a putative disulphide-bond-stabilized dimer. Pulse-chase experiments showed only the monomer after 5 min and the appearance of the putative dimer after 30 min. The MUC2 apoprotein was also precipitated by antisera against the HF-deglycosylated peptides of the two highly glycosylated domains of the 'insoluble' mucin complex of rat small intestine [Carlstedt, Herrmann, Karlsson, Sheehan, Fransson and Hansson (1993) J. Biol. Chem. 268, [18771-18781]. Endoprotease Lys-C cleavage of the immunopurified apoprotein gave a large fragment of about 250 kDa that was detected by both the antiserum against the MUC2 tandem repeat and one of the glycopeptide antisera. This supports the view that the 'insoluble' mucin of rat small intestine is encoded by the Muc2 gene, as recently indicated by a partial cDNA sequence [Hansson, Baeckström, Carlstedt and Klinga-Levan (1994) Biochem. Biophys. Res. Commun. 198, 181-190] and that parts of the apoprotein are conserved between the species. A lectin from the snail Helix pomatia that detects terminal alpha-GalNAc residues did not bind to the monomer or putative dimer, suggesting that O-glycosylation starts after dimerization. The results indicate that the biosynthetic pathway of the MUC2 mucin may be similar to that of the von Willebrand factor with which MUC2 shares sequence similarities at its C- and N-termini.
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| 4. |
- 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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| 5. |
- Chauhan, S., et al.
(författare)
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Cdk2 catalytic activity is essential for meiotic cell division in vivo
- 2016
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 473, s. 2783-2798
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Tidskriftsartikel (refereegranskat)abstract
- Cyclin-dependent kinases (Cdks) control the eukaryotic cell cycle by phosphorylating serine and threonine residues in key regulatory proteins, but some Cdk family members may exert kinase-independent functions that cannot easily be assessed using gene knockout approaches. While Cdk2-deficient mice display near-normal mitotic cell proliferation due to the compensatory activities of Cdk1 and Cdk4, they are unable to undergo meiotic generation of gametes and are consequently sterile. To investigate whether Cdk2 regulates meiosis via protein phosphorylation or by alternative kinase-independent mechanisms, we generated two different knockin mouse strains in which Cdk2 point mutations ablated enzyme activity without altering protein expression levels. Mice homozygous for the mutations Cdk2(D145N/D145N) or Cdk2(T160A/T160A) expressed only 'kinase-dead' variants of Cdk2 under the control of the endogenous promoter, and despite exhibiting normal expression of cell cycle regulatory proteins and complexes, both mutations rendered mice sterile. Mouse cells that expressed only 'kinase-dead' variants of Cdk2 displayed normal mitotic cell cycle progression and proliferation both in vitro and in vivo, indicating that loss of Cdk2 kinase activity exerted little effect on this mode of cell division. In contrast, the reproductive organs of Cdk2 mutant mice exhibited abnormal morphology and impaired function associated with defective meiotic cell division and inability to produce gametes. Cdk2 mutant animals were therefore comparable to gene knockout mice, which completely lack the Cdk2 protein. Together, our data indicate that the essential meiotic functions of Cdk2 depend on its kinase activity, without which the generation of haploid cells is disrupted, resulting in sterility of otherwise healthy animals.
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| 6. |
- Dahlgren, Claes, 1949, et al.
(författare)
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The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils.
- 1995
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Ingår i: The Biochemical journal. - 0264-6021. ; 311 ( Pt 2), s. 667-74
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Tidskriftsartikel (refereegranskat)abstract
- The subcellular localization of two members of a highly glycosylated protein group present in lysosomal membranes in most cells, the lysosome-associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2), was examined in human neutrophil granulocytes. Antibodies that were raised against purified Lamp-1 adn Lamp-2 gave a distinct granular staining of the cytoplasm upon immunostaining of neutrophils. Subcellular fractionation was used to separate the azurophil and specific granules from a light-membrane fraction containing plasma membranes and secretory vesicles, and Western blotting was used to determine the presence of the Lamps in these fractions. The results show that Lamp-1 and Lamp-2 are present in the specific-granule-enriched fraction and in the light-membrane fraction, but not in the azurophil granules. Separation of secretory vesicles from plasma membranes disclosed that the light-membrane Lamps were present primarily in the secretory-vesicle-enriched fraction. During phagocytosis both Lamp-1 and Lamp-2 became markedly concentrated around the ingested particle and they both appear on the cell surface when the secretory organelles are mobilized.
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| 7. |
- El Hachmane, Michael, et al.
(författare)
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Extracellular ATP activates store-operated Ca2+ entry in white adipocytes: functional evidence for STIM1 and ORAI1
- 2018
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 475, s. 691-704
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Tidskriftsartikel (refereegranskat)abstract
- In the present study, we have applied ratiometric measurements of intracellular Ca2+ concentrations ([Ca2+](i)) to show that extracellularly applied ATP (adenosine triphosphate) (100 mM) stimulates store-operated Ca2+ entry (SOCE) in 3T3-L1 adipocytes. ATP produced a rapid increase in [Ca2+](i) consisting of an initial transient elevation followed by a sustained elevated phase that could be observed only in the presence of extracellular Ca2+. Gene expression data and [Ca2+](i) recordings with uridine-50-triphosphate or with the phospholipase C (PLC) inhibitor U73122 demonstrated the involvement of purinergic P2Y2 receptors and the PLC/inositol trisphosphate pathway. The [Ca2+](i) elevation produced by reintroduction of a Ca2+-containing intracellular solution to adipocytes exposed to ATP in the absence of Ca2+ was diminished by known SOCE antagonists. The chief molecular components of SOCE, the stromal interaction molecule 1 (STIM1) and the calcium release-activated calcium channel protein 1 (ORAI1), were detected at the mRNA and protein level. Moreover, SOCE was largely diminished in cells where STIM1 and/or ORAI1 had been silenced by small interfering (si) RNA. We conclude that extracellular ATP activates SOCE in white adipocytes, an effect predominantly mediated by STIM1 and ORAI1.
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| 8. |
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| 9. |
- Frick, Anna, 1982, et al.
(författare)
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Mercury increases water permeability of a plant aquaporin through a non-cysteine-related mechanism
- 2013
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 454:pt 3, s. 491-499
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Tidskriftsartikel (refereegranskat)abstract
- Water transport across cellular membranes is mediated by a family of membrane proteins known as AQPs (aquaporins). AQPs were first discovered on the basis of their ability to be inhibited by mercurial compounds, an experiment which has followed the AQP field ever since. Although mercury inhibition is most common, many AQPs are mercury insensitive. In plants, regulation of AQPs is important in order to cope with environmental changes. Plant plasma membrane AQPs are known to be gated by phosphorylation, pH and Ca2+. We have previously solved the structure of the spinach AQP SoPIP2;1 (Spinacia oleracea plasma membrane intrinsic protein 2; 1) in closed and open conformations and proposed a mechanism for how this gating can be achieved. To study the effect of mercury on SoPIP2; 1 we solved the structure of the SoPIP2;1-mercury complex and characterized the water transport ability using proteoliposomes. The structure revealed mercury binding to three out of four cysteine residues. In contrast to what is normally seen for AQPs, mercury increased the water transport rate of SoPIP2; 1, an effect which could not be attributed to any of the cysteine residues. This indicates that other factors might influence the effect of mercury on SoPIP2; 1, one of which could be the properties of the lipid bilayer.
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| 10. |
- Kannius-Janson, Marie, 1969, et al.
(författare)
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The tissue-specific regulation of the carboxyl ester lipase gene in exocrine pancreas differs significantly between mouse and human.
- 2000
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Ingår i: The Biochemical journal. - 0264-6021. ; 351 Pt 2, s. 367-76
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Tidskriftsartikel (refereegranskat)abstract
- The carboxyl ester lipase (CEL) gene is highly expressed in exocrine pancreas and expression of the human CEL gene is mediated by a strong tissue-specific enhancer, which is absolutely necessary for high-level expression. The mouse promoter, on the other hand, does not contain a corresponding enhancer element, but instead is totally dependent on another pancreas-specific element. This element is identified as a pancreatic transcription factor 1 (PTF 1)-binding site. The human CEL promoter also contains a putative PTF 1 element located at a position corresponding to the essential PTF 1 site in the mouse promoter. However, nucleotide changes in the human promoter 5' flanking this PTF 1 site have created an overlapping CCAAT/enhancer-binding protein (C/EBP)-like binding motif, interfering with the binding of PTF 1. Hence, our findings provide an example of genetic divergence between species not accompanied by difference in function.
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