| 41. |
- Asker, Noomi, 1968, et al.
(author)
-
Human MUC5AC mucin dimerizes in the rough endoplasmic reticulum, similarly to the MUC2 mucin.
- 1998
-
In: The Biochemical journal. - 0264-6021. ; 335:2, s. 381-7
-
Journal article (peer-reviewed)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.
|
|
| 42. |
- Asker, Noomi, 1968, et al.
(author)
-
The human MUC2 mucin apoprotein appears to dimerize before O-glycosylation and shares epitopes with the 'insoluble' mucin of rat small intestine.
- 1995
-
In: The Biochemical journal. - 0264-6021. ; 308:3, s. 873-80
-
Journal article (peer-reviewed)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.
|
|
| 43. |
- Aspenström, Pontus, et al.
(author)
-
Rho GTPases have diverse effects on the organization of the actin filament system
- 2004
-
In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 377:Pt 2, s. 327-337
-
Journal article (peer-reviewed)abstract
- The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.
|
|
| 44. |
|
|
| 45. |
|
|
| 46. |
- Baird, Sarah K, et al.
(author)
-
Metallothionein protects against oxidative stress-induced lysosomal destabilization
- 2006
-
In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 394:1, s. 275-283
-
Journal article (peer-reviewed)abstract
- The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions. © 2006 Biochemical Society.
|
|
| 47. |
- Barderi, P, et al.
(author)
-
The NADP+ linked glutamate dehydrogenase from Trypanosoma cruzi : sequence, genomic organization and expression
- 1998
-
In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 330:2, s. 951-958
-
Journal article (peer-reviewed)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.
|
|
| 48. |
- BARKER, CJ, et al.
(author)
-
Inositol 1,2,3-trisphosphate and inositol 1,2- and/or 2,3-bisphosphate are normal constituents of mammalian cells
- 1995
-
In: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 306306 ( Pt 2), s. 557-564
-
Journal article (peer-reviewed)abstract
- 1. An inositol trisphosphate (InsP3) distinct from Ins(1,4,5)P3 and Ins(1,3,4)P3, which we previously observed in myeloid and lymphoid cells [French, Bunce, Stephens, Lord, McConnell, Brown, Creba and Michell (1991) Proc R. Soc. London B 245, 193-201; Bunce, French, Allen, Mountford, Moore, Greaves, Michell and Brown (1993) Biochem. J. 289, 667-673], is present in WRK1 rat mammary tumour cells and pancreatic endocrine beta-cells. 2. It has been identified as Ins(1,2,3)P3 by a combination of oxidation to ribitol, a structurally diagnostic polyol, and ammoniacal hydrolysis to identified inositol monophosphates. 3. Ins(1,2,3)P3 concentration in HL60 cells changed little during stimulation by ATP or fMetLeuPhe or during neutrophilic or monocytic differentiation, and Ins(1,2,3)P3 was unresponsive to vasopressin in WRK1 cells. 4. Ins(1,2,3)P3 was usually more abundant than Ins(1,4,5)P3, often being present at concentrations between approximately 1 microM and approximately 10 microM. 5. HL60, WRK-1 and lymphoid cells also contain Ins(1,2)P2 or Ins(2,3)P2, or a mixture of these two enantiomers, as a major InsP2 species. 6. Ins(1,2,3)P3 and Ins(1,2)P2/Ins(2,3)P2 are readily detected in cells labelled for long periods, but not in acutely labelled cells. This behaviour resembles that of InsP6, the most abundant cellular inositol polyphosphate that includes the 1,2,3-trisphosphate motif, which also achieves isotopic equilibrium with inositol only slowly. 7. Ins(1,2,3)P3 is the major InsP3 that accumulates during metabolism of InsP6 by WRK-1 cell homogenates. 8. Possible metabolic relationships between Ins(1,2,3)P3, Ins(1,2)P2/Ins(2,3)P2 and other inositol polyphosphates in cells, and a possible role for Ins(1,2,3)P3 in cellular iron handling, are considered.
|
|
| 49. |
- Barki-Harrington, Liza, et al.
(author)
-
Requirement for direct cross-talk between B1 and B2 kinin receptors for the proliferation of androgen-insensitive prostate cancer PC3 cells
- 2003
-
In: Biochemical Journal. - 0264-6021. ; 371, s. 581-587
-
Journal article (peer-reviewed)abstract
- Stimulation of endogenous kinin receptors promotes growth of androgen-independent prostate cancer PC3 cells via activation of the mitogenic extracellular-signal-regulated kinase (ERK) pathway. In the present study, we show that kinin-mediated mitogenic signalling and prostate-cell growth involves two subtypes of bradykinin (BK) receptors, B1R and B2R. Specific stimulation of either B1R or B2R by their respective agonists des-Arg(9)-BK and Lys-BK promoted ERK activation and cell growth, whereas selective blockade with specific antagonists des-Arg(9)-[Leu(8)]BK and Hoe 140 respectively obliterated this effect, indicating the presence of both receptor subtypes. However, blockade of B1R also inhibited B2R-mediated ERK activation and cell growth, and, similarly, antagonism of B2R inhibited the B1R-mediated response. Furthermore, both B1R and B2R agonists promoted internalization of B1R, whereas both receptor antagonists blocked this effect. The B1R ligands des-Arg(9)-BK and des-Arg(9)-[Leu(8)]BK had no effect on the binding of BK to B2R, as demonstrated by radioligand competitive binding studies. However, blockade of either B1R or B2R impaired the ability of the reciprocal receptor to produce inositol phosphates, suggesting that the interaction between B1R and B2R is proximal to activation of phospholipase C. These results provide evidence for the existence of B1R-B2R complexes in prostate cancer PC3 cells and demonstrate that antagonism of one receptor interferes with the signalling ability of the other, possibly at the level of receptor-Galpha(q) protein coupling. Selective inhibition of B1R, which is up-regulated in injured and cancerous tissue, may be beneficial for the treatment of advanced prostate cancer.
|
|
| 50. |
|
|
