| 1. |
- Fredlund, J O, et al.
(författare)
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Ornithine decarboxylase and S-adenosylmethionine decarboxylase expression during the cell cycle of Chinese hamster ovary cells
- 1995
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 216:1, s. 86-92
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Tidskriftsartikel (refereegranskat)abstract
- Cells in mitosis were harvested from exponentially growing Chinese hamster ovary cells by the mitotic detachment technique. Immediately after harvesting, the mitotic cells were seeded in tissue culture flasks and incubated at 37 degrees C in a CO2 incubator. Care was taken not to perturb the progression of cells through the cell cycle. At every hour after seeding for 14 h, cells were collected for analysis of cell cycle distribution, cellular polyamine content, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities, and relative mRNA contents. The progression through the cell cycle was monitored by DNA flow cytometry. The putrescine, spermidine, and spermine levels were approximately doubled during the cell cycle: putrescine mainly during late S and G2, spermidine continuously during the entire cell cycle, and spermine mainly during G1 and S. The ODC activity was low in seeded mitotic cells and the enzyme was activated in late G1 and reached a plateau in S phase. A second burst in activity was observed during late S phase and maximal ODC activity was found at the S/G2 transition. The relative ODC mRNA level approximately doubled during the cell cycle and the increase in the relative level mainly took part during mid and late S phase. AdoMetDC activity increased in late G1 and a first maximum was observed during the G1/S transition. A second burst in activity was found in mid S phase. Maximal AdoMetDC activity was found in G2. The relative AdoMetDC mRNA approximately doubled during the cell cycle and the increase in the relative level mainly took place during late G1 and early S phase. Our results indicate that polyamine synthesis was regulated at transcriptional and translational/post-translational levels during the cell cycle of Chinese hamster ovary cells.
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| 2. |
- Hedman, H., et al.
(författare)
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Defective expression of beta 1-integrins in cells with constitutively active alpha L beta 2-integrins
- 1997
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Ingår i: Experimental Cell Research. - : Elsevier Science B.V., Amsterdam. - 0014-4827 .- 1090-2422. ; 232:2, s. 270-276
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated a potential relationship between expression of beta 1-integrins and adhesiveness of the beta 2-integrin LFA-1 (alpha L beta 2, CD11a/CD18). By an approach of random mutagenesis and selection we established clones from the human acute lymphatic leukemia cell line HPB-ALL with (i) constitutively active LFA-1 and (ii) with no apparent integrin-beta 1 cell surface expression. Thirty seven of 42 clones selected for activated LFA-1 were found to have lost apparent integrin-beta 1 expression. Conversely, 7 of 21 clones selected for lack of beta 1 expression were found to have activated LFA-1. Since this pointed toward a possible coupling between beta 1 expression and LFA-1 activity, we further analyzed at which level beta 1 expression was blocked. We focused on one clone, HAP4, with activated LFA-I and no detectable beta 1 cell surface expression and found, surprisingly, that it expressed wild-type levels of beta 1 mRNA and, in Western blots of whole cell lysates, apparently normal levels of beta 1 protein. However, in addition to beta 1 of the expected molecular weight, HAP4 expressed a unique 48-kDa band recognized by the polyclonal anti-beta 1 antiserum. Immunoprecipitation experiments revealed that the epitope recognized by the anti-beta 1 antibody 4B4 was hidden or lost. The alpha 4-chain was found in its precursor form but it did not associate with any beta-chain, and it was not processed to its mature form. Instead alpha 4-chains were eventually degraded. Taken together this showed that beta 1-chains were produced but not properly processed in HAP4. From this we propose that HAP4 is deficient in a gene product required both for proper beta 1 folding and for repression of LFA-1 adhesiveness.
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| 3. |
- Holmvall, K, et al.
(författare)
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Chondrocyte and chondrosarcoma cell integrins with affinity for collagen type II and their response to mechanical stress
- 1995
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 221:2, s. 496-503
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Tidskriftsartikel (refereegranskat)abstract
- Mechanical stress is an important regulator of chondrocyte functions but the mechanisms by which chondrocytes sense mechanical signals are unknown. Receptors for matrix molecules are likely involved in the mechanical signaling. In the first part of this study we identified integrins with affinity for the cartilage-specific collagen type II. We report that the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 isolated from bovine chondrocytes or human chondrosarcoma cells bound collagen type II as judged from affinity chromatography. The integrins alpha 3 beta 1 or alpha 9 beta 1 did not bind collagen type II-Sepharose. In the second part of the study we investigated the effect of mechanical stress on expression of matrix molecules and integrin subunits. Chondrocytes and chondrosarcoma cells, cultured on uncoated flexible silicone membranes in the presence of serum, were exposed to mechanical stress by the Flexercell system. Dynamic stimulation of chondrocytes for 3 h increased the mRNA expression of collagen type II and aggrecan as judged by Northern blotting, while the beta 1-integrin subunit was not changed. When chondrosarcoma cells were exposed to mechanical stimulation under the same conditions, mRNA expression of alpha 5 was found to increase while beta 1, alpha 2, and alpha v did not increase to significant levels. In another study the effect of mechanical stress on integrins was investigated when the cells were cultured on collagen type II-coated flex-dishes. Three hours of dynamic stress increased the mRNA expression of alpha 2-integrin subunit while the level of mRNA for integrin subunits beta 1, alpha 1, alpha 5, and alpha v showed no or small changes, indicating that matrix components may modulate the expression of integrins during mechanical stress.
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| 4. |
- Imreh, Gabriela, et al.
(författare)
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Noninvasive monitoring of apoptosis versus necrosis in a neuroblastoma cell line expressing a nuclear pore protein tagged with the green fluorescent protein
- 1998
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 238:2, s. 371-376
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Tidskriftsartikel (refereegranskat)abstract
- A fusion chimera between the integral nuclear pore membrane protein POM121 and GFP (green fluorescent protein) has been shown to correctly target to the nuclear pores when transiently expressed in a number of mammalian cell types. POM121-GFP is therefore an excellent marker for the noninvasive studies of the nuclear pores in living cells using fluorescence microscopy. We have established a line of neuroblastoma cells stably expressing the POM121-GFP fusion protein. We also monitored the nuclear envelope in living cells after induction of apoptosis or necrosis using 1 μM staurosporine or 100 μM p-benzoquinone, respectively. Interestingly, the POM121-GFP fluorescence was weaker or missing in the apoptotic cells. The disappearance of the nuclear pore marker accompanied apoptotic progression as judged by the degree of chromatin condensation and DNA fragmentation as analyzed by DNA staining and TUNEL assay, respectively. In contrast, the intensity of the nuclear rim fluorescence was unaffected in necrotic cells displaying an abnormal morphology with tilted nuclei. Thus, it was possible to distinguish between apoptotic and necrotic development in living cells using fluorescence microscopy. This cell line provides a fast and convenient model for screening suspected toxic xenobiotics.
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| 5. |
- Karlsson, Torbjörn, et al.
(författare)
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Modulation of Src homology 3 proteins by the proline-rich adaptor protein Shb
- 1997
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 231:2, s. 269-275
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Tidskriftsartikel (refereegranskat)abstract
- In the present study we have investigated a possible role for the proline-rich SH2 domain protein Shb as a regulator of expression or activity of certain SH3 domain proteins and MAP kinase. The expression of the Shb binding proteins Eps8, Src, and p85 PI3-kinase, PI3-kinase activity, and MAP kinase activation were assessed in wild-type NIH3T3 cells and in NIH3T3 cells overexpressing the Shb cDNA. In addition, the expression of the SH3 domain STAT1 proteins was assessed in wild-type and Shb overexpressing cells. The Eps8 protein content and Eps8 mRNA steady-state levels were downregulated, whereas the protein contents of Src and p85 PI3-kinase were unaffected by Shb overexpression. There was, however, an increased basal PI3-kinase activity in Shb transfected cells after a 3-h serum starvation. Increased steady-state levels of STAT1 mRNA were accompanied by an increased STAT1 protein content in Shb overexpressing cells. Shb overexpression was not associated with an altered activation of p44 or p42 MAP kinases in response to PDGF stimulation. The data presented in this study suggest novel functions for the adaptor protein Shb regulating the expression of certain signal-transducing SH3 domain proteins and modulating PI3-kinase activity.
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| 6. |
- Onyango, I, et al.
(författare)
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Effects of extracellular calcium on the subcellular translocation of bovine parathyroid PKC isozymes.
- 1999
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 247:1, s. 9-16
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Tidskriftsartikel (refereegranskat)abstract
- The release of parathyroid hormone is regulated by the extracellular concentration of Ca2+ through a sensor(s) on the surface of the parathyroid cells, but few details are known on the further relay of the signal inside the cell. Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell soluble fraction to the particulate fraction of the cell. Therefore, identification of a subcellular localization of a PKC isozyme in parathyroid cells as a response to changes in extracellular Ca2+ should be an indication for its putative role in signal transduction coupled to the Ca2+ sensor. We have determined the subcellular localization of six PKC isozymes (alpha, betaI, betaII, epsilon, zeta, and iota) in nonstimulated parathyroid cells and in those treated with low (0.5 mM) and high (3.0 mM) extracellular Ca2+ by confocal microscopy. At the physiological concentration of serum Ca2+, all PKC isozymes studied were localized mainly to the cytosol, although to different extents. Low extracellular Ca2+ caused a redistribution of PKCalpha to the periphery of the cells. In contrast, PKCbetaI, -epsilon, -zeta, and -iota were translocated to the periphery of the cells at high extracellular Ca2+. These results indicate that PKCalpha, -betaI, -epsilon, -zeta, and -iota are involved in the response of parathyroid cells to changes in extracellular Ca2+.
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| 7. |
- Petrova, T V, et al.
(författare)
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Signaling via vascular endothelial growth factor receptors.
- 1999
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 253:1
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Tidskriftsartikel (refereegranskat)abstract
- Angiogenesis, or development of blood vessels from preexisting vasculature, has important functions under both normal and pathophysiological conditions. Vascular endothelial growth factor receptors 1-3, also known as flt-1, KDR, and flt-4, are endothelial cell-specific receptor tyrosine kinases which serve as key mediators of the angiogenic responses. The review focuses on the signaling pathways that are initiated from these receptors and the recently identified VEGF coreceptor neuroplilin-1.
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| 8. |
- Sahaf, Bita, et al.
(författare)
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Thioredoxin Expression and Localization in Human Cell Lines : Detection of Full-Length and Truncated Species
- 1997
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 236:1, s. 181-192
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Tidskriftsartikel (refereegranskat)abstract
- Thioredoxin (Trx) is an intracellular multifunctional 12-kDa protein with a reduction/oxidation (redox) active disulfide constitutively expressed by most cells of the human body. Trx can also be released by cells such as lymphocytes upon activation or oxidative stress exposure and exert a cocytokine and cytoprotective activity. In addition, a truncated 10-kDa form of Trx has been reported. In order to better understand the function of full-length and truncated Trx, we have produced, for the first time, specific monoclonal antibodies, which can discriminate between the two forms. Using these novel antibodies, designated αTrx1 to αTrx4, a panel of cell lines derived from human B and T lymphocytes, monocytes, granulocytes, and melanomas was analyzed by immunochemical techniques. The cellular distribution differed between the two forms. All lines contained full-length Trx, also located to a minor extent on the cell surface. One exception was the melanoma cell line FM28.4, which did not show any Trx expression. Truncated Trx was present in most cells in minimal amounts only, whereas the monocytic cell lines THP-1 and U-937 expressed high amounts on the cell surface, as shown by flow cytometric analysis of living cells and confocal laser-scanning microscopy. The biological importance and function of the short versus long forms of Trx as detected by the antibodies are discussed.
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| 9. |
- Stigson, Michael, et al.
(författare)
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Reduced epidermal expression of a PG-M/versican-like proteoglycan in embryos of the white mutant axolotl.
- 1997
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 236:1, s. 57-65
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Tidskriftsartikel (refereegranskat)abstract
- Axolotl embryos have previously been used to study neural crest cell migration. In embryos of the normal wild type, neural crest cells migrate subepidermally to form pigment cells. In the trunk of the white mutant embryo, these cells are unable to migrate, possibly due to an inherited delay in the maturation of the local extracellular matrix. The present investigation reveals a reduced incorporation of [35S]sulfate into PG-M/versican-like proteoglycans synthesized in epidermal explants from the dorsal trunk of white mutant embryos during stages pertinent to migration. This is the major form of proteoglycans in the subepidermal matrix, where they are assembled in large disulfide-stabilized supramolecular complexes. The reduction in [35S]sulfate incorporation is not due to qualitative differences between wild-type and white mutant proteoglycans but is paralleled by a reduced expression of mRNA for the core protein of the PG-M/versican-like proteoglycan. We conclude that a reduced amount of these proteoglycans is produced by the white mutant embryo during the period critical for migration.
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| 10. |
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