| 1. |
- Beier, Frank, et al.
(författare)
-
Localization of silencer and enhancer elements in the human type X collagen gene.
- 1997
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 66:2, s. 210-218
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Tidskriftsartikel (refereegranskat)abstract
- Collagen type X is a short, network-forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5'-flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non-tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hypertrophic chondrocytes was located between -2.4 and -0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291-296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements.
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| 2. |
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| 3. |
- Lammi, Mikko, 1961-
(författare)
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Influences of in vivo and in vitro loading on the proteoglycan synthesis of articular cartilage chondrocytes
- 1993
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this study, the biosynthesis of proteoglycans (PGs) was examined in articular cartilage of canine hip joint after long-distance running experiment and in bovine chondrocyte cultures during in vitro loading with hydrostatic pressure. In addition, new assays were developed for more sensitive quantitation of glycosaminoglycans (GAGs) and PGs.Anterior (weight-bearing) and posterior (less weight-bearing) areas of the femoral head from young beagles were labeled after long-term, longdistance running exercise. Total sulpahte incorporation rates were determined and distribution of of the incorporated sulphate in the tissue was localized by quantitative autoradiography. Concentration and extractability of the PGs were determined, and PG structures were studied by gel filtration, agarose gel electrophoresis, and chemical determinations. In the less weight-bearing area, the amount of extractable PGs was decreased, simultaneously with an increased concentration of residual GAGs in the tissue after 4M GuCl extraction. In the weight-bearing area, no marked alterations were noticed. The congruency of the femoral head seems to protect the cartilage from untoward alterations that occur in the femoral head condyles subjected to the same running program.The effect of hydrostatic pressure on PG metabolism of chondrocyte cultures was examined during 20 hours' exposure of chondrocytes to 5 and 30 MPa pressures. The continuous 30 MPa pressure reduced total PG synthesis by 37 % as measured by [35S]sulphate incorporation, in contrast to the 5 MPa which had no effect. Continuous 30 MPa hydrostatic pressure also reduced the steady-state mRNA level of aggrecan. The cyclic pressures showed a frequency dependent stimulation (0.5 Hz, + 11 %) or inhibition (0.017 Hz, -17 %). Aggrecans secreted under continuous 30 MPa pressure showed a retarded migration in 0.75 % SDS-agarose gel electrophoresis and also eluted earlier on Sephacryl S-1000 gel filtration, indicative of larger molecular size. The results demonstrate that high hydostatic pressure can influence the synthesis of PGs in chondrocytes both at the transcriptionl and translational/posttranslational levels.
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| 4. |
- Nelimarkka, Lassi, et al.
(författare)
-
Expression of small extracellular chondroitin/dermatan sulfate proteoglycans is differentially regulated in human endothelial cells.
- 1997
-
Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 272:19, s. 12730-12737
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Tidskriftsartikel (refereegranskat)abstract
- We have examined the expression of the small extracellular chondroitin/dermatan sulfate proteoglycans (CS/DS PGs), biglycan, decorin, and PG-100, which is the proteoglycan form of colony stimulating factor-1, in the human endothelial cell line EA.hy 926. We have also examined whether modulation of the phenotype of EA.hy 926 cells by tumor necrosis factor-alpha (TNF-alpha) is associated with specific changes in the synthesis of these PGs. We demonstrate that EA.hy 926 cells, when they form monolayer cultures typical of macrovascular endothelial cells, express and synthesize detectable amounts of biglycan and PG-100, but not decorin. On SDS-polyacrylamide gel electrophoresis both PGs behave like proteins of the relative molecular weight of approximately 250,000. TNF-alpha that changed the morphology of the cells from a polygonal shape into a spindle shape and that also stimulated the detachment of the cells from culture dish, markedly decreased the net synthesis of biglycan, whereas the net synthesis of PG-100 was increased. These changes were parallel with those observed at the mRNA level of the corresponding PGs. The proportions of the different sulfated CS/DS disaccharide units of PGs were not affected by TNF-alpha. Several other growth factors/cytokines, such as interferon-gamma, fibroblast growth factors-2 (FGF-2) and -7 (FGF-7), interleukin-1beta, and transforming growth factor-beta, unlike TNF-alpha, modulated neither the morphology nor the biglycan expression of EA.hy 926 cells under the conditions used in the experiments. However, PG-100 expression was increased also in response to FGF-2 and -7 and transforming growth factor-beta. None of the above cytokines, including TNF-alpha, was able to induce decorin expression in the cells. Our results indicate that the regulatory elements controlling the expression of the small extracellular CS/DS PGs in human endothelial cells are different.
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| 5. |
- Rutberg, M, et al.
(författare)
-
Estramustine induces disorganization of microtubules, perinuclear retraction of vimentin and endoplasmatic reticulum, and inhibits cell migration.
- 1993
-
Ingår i: Acta histochemica. - 0065-1281. ; 95:2, s. 155-67
-
Tidskriftsartikel (refereegranskat)abstract
- The effects of the mitotic inhibitor estramustine on the cytoskeleton of DU 145 and AG 1518 cells were studied. Estramustine caused a partial disassembly of microtubules and withdrawal of microtubules from the cell periphery, disorganized microtubules and delayed regrowth of disassembled microtubules. It also induced a spheroid cellular morphology and affected cellular adhesion and survival. Sometimes microtubules seemed to be organized from several microtubule-organizing centers. The cytoskeleton-dependent cell migration was inhibited in the presence of estramustine and the microtubule-interacting vimentin and endoplasmatic reticulum retracted to the perinuclear area. Our results show that not only a complete disassembly of microtubules, but also disturbances of the microtubule network can have dramatic effects on microtubule-dependent processes and localization of cellular organelles. These effects could be of importance in the treatment of prostatic carcinoma with estramustine.
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| 6. |
- Pettersson, Jonas, et al.
(författare)
-
The V-antigen of Yersinia is surface exposed before target cell contact and involved in virulence protein translocation :
- 1999
-
Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 32:5, s. 961-976
-
Tidskriftsartikel (refereegranskat)abstract
- Type III-mediated translocation of Yop effectors is an essential virulence mechanism of pathogenic Yersinia. LcrV is the only protein secreted by the type III secretion system that induces protective immunity. LcrV also plays a significant role in the regulation of Yop expression and secretion. The role of LcrV in the virulence process has, however, remained elusive on account of its pleiotropic effects. Here, we show that anti-LcrV antibodies can block the delivery of Yop effectors into the target cell cytosol. This argues strongly for a critical role of LcrV in the Yop translocation process. Additional evidence supporting this role was obtained by genetic analysis. LcrV was found to be present on the bacterial surface before the establishment of bacteria target cell contact. These findings suggest that LcrV serves an important role in the initiation of the translocation process and provides one possible explanation for the mechanism of LcrV-induced protective immunity.
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| 7. |
- Los, Marek Jan, et al.
(författare)
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Hydrogen-Peroxide as a Potent Activator of T-Lymphocyte Functions
- 1995
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Ingår i: European Journal of Immunology. - : Wiley. - 0014-2980 .- 1521-4141. ; 25:1, s. 159-165
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Tidskriftsartikel (refereegranskat)abstract
- During inflammatory processes infiltrating cells produce large amounts of reactive oxygen intermediates (ROI). Increasing evidence suggests that ROI besides being cytotoxic may act as important mediators influencing various cellular and immunological processes. In this study, we have investigated the effects of hydrogen peroxide on several aspects of lymphocyte activation. In ESb-L T lymphoma cells, micromolar concentrations of hydrogen peroxide rapidly induced activation of the transcription factor NF-kappa B, whereas DNA-binding activity of the transcription factor AP-1 was virtually not affected. In addition, hydrogen peroxide induced early gene expression of interleukin-2 (IL-2) and the IL-2 receptor alpha chain. The stimulation of IL-2 expression was found to be conferred by a kappa B-like cis-regulatory region within the IL-2 gene promoter. In contrast to these activating effects, addition of hydrogen peroxide was largely inhibitory on cell proliferation which is consistent with a general requirement of thiol compounds for lymphocyte proliferation. However, hydrogen peroxide significantly increased T cell proliferation when applied for a short period under reducing conditions. These data indicate that ROI may act as an important competence signal in T lymphocytes inducing early gene expression as well as cell proliferation.
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| 8. |
- Björkegren Sjögren, Camilla
(författare)
-
Profilin in cell motility and signal transduction studies using site-specific mutagenesis
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- When a cell is stimulated by signalling molecules such as growth factors, one immediate response is an increased motile activity of the cell. Membrane lamella and spikes appear at the cell surface and perform undulating movements. This is caused by the activation and reorganisation of the highly dynamic microfilament system, present beneath the cell membrane. The major component of the microfilament system is actin, a protein that can polymerise into filaments -microfilaments-, and several actin-binding proteins which act in concert to organise actin into different supramolecular arrangements. To understand how a signal is transmitted form the exterior of the cell to the microfilament system, it is important to reveal how the activities of different actin-binding proteins are regulated.This thesis focus on the actin-binding protein profilin, which binds actin monomers and regulates actin polymerisation. Profilin also interacts with polyphosphoinositides, which are important signalling molecules, and this association appears to regulate both the function of profilin and the turnover of polyphosphoinositides. Furthermore, profilin binds poly(L-proline) and proline-rich proteins. The latter interaction seems to be involved in the positioning of profilin within a cell, and may also be important for signal transduction-related processes. In the current investigation site-directed mutagenesis was combined with biochemical methods to further investigate the function of profilin.The replacement of two amino acid residues located in a hydrophobic patch on the surface of the profilin molecule was shown to abolish the interaction with poly(L-proline), and enabled the localisation of the poly(L-proline) binding-site to this part of profilin. A new purification method was developed for these profilin mutants and further biochemical characterisation showed that the two mutations also decreased the affinity of profilin for actin. As the poly(L-proline)-binding site is separated from the actin-binding surface of profilin this showed that these amino acid replacements in the poly(L-proline)-binding site also introduced long-range alterations in the profilin molecule.Profilin was shown to be phosphorylated on a serine and a tyrosine residue by an epidermal growth factor receptor complex isolated from stimulated cells. The phosphorylated residues were located to the poly(L-proline) binding site of profilin, and the phosphorylation interfered with the binding of profilin to poly(L-proline). This suggests that this phosphorylation regulate interactions between profilin and proline-rich proteins.The simultaneous deletion of two amino acid residues adjacent to the actin-binding site of profilin was shown to reduce the affinity of profilin for actin, without affecting the interaction of profilin with other ligands. The effect of this mutant profilin on the organisation of the microfilament system in living cells was compared to that of wild type profilin in microinjection experiments. While wild type profilin caused disruption of actin bundles within the cell, the profilin mutant had no apparent effect, showing that the derangement of the actin bundles by profilin is dependent on the stability of the profilin:actin complex.
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| 9. |
- Lammi, Pirkko, et al.
(författare)
-
Localization of type X collagen in the intervertebral disc of mature beagle dogs.
- 1998
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Ingår i: Matrix Biology. - : Elsevier. - 0945-053X .- 1569-1802. ; 17:6, s. 449-453
-
Tidskriftsartikel (refereegranskat)abstract
- Type X collagen expression in intervertebral disc of young adult beagle dogs (n = 10) was studied. Type X collagen was immunostained mainly pericellularly in the central area of the vertebral endplate, but interterritorial staining there was also present. Annulus fibrosus and nucleus pulposus did not usually stain for type X collagen. However, immunostaining of nucleus pulposus for type X collagen with a simultaneous expression of collagen alpha1(X) mRNA was observed in one dog. A weak staining was observed in two other animals with a weak collagen alpha1(X) mRNA signal. In annulus fibrosus, lamellar staining was observed in two dogs. In three animals, type X collagen mRNAs were observed in the outer edge of the annulus fibrosus, but immunohistochemical staining did not always correlate with in situ hybridization signals. In conclusion, intervertebral disc type X collagen was mainly expressed in the cartilaginous endplate. In some apparently healthy animals there was type X collagen expression in the nucleus pulposus and also in the annulus fibrosus.
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| 10. |
- Frithz-Lindsten, Elisabet, et al.
(författare)
-
Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis.
- 1998
-
Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 29:5, s. 1155-1165
-
Tidskriftsartikel (refereegranskat)abstract
- Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa, on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia, YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa. Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants (yopB/yopD) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.
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| 11. |
- Leary, Sophie E. C., et al.
(författare)
-
Yersinia outer proteins (YOPS) E, K and N are antigenic but non-protective compared to V antigen, in a murine model of bubonic plague
- 1999
-
Ingår i: Microbial Pathogenesis. - : Elsevier. - 0882-4010 .- 1096-1208. ; 26:3, s. 159-169
-
Tidskriftsartikel (refereegranskat)abstract
- The pathogenic Yersiniae produce a range of virulence proteins, encoded by a 70 kb plasmid, which are essential for infection, and also form part of a contact-dependent virulence mechanism. One of these proteins, V antigen, has been shown to confer a high level of protection against parenteral infection with Y. pestis in murine models, and is considered to be a protective antigen. In this study, the protective efficacy of V antigen has been compared in the same model with that of other proteins (YopE, YopK and YopN), which are part of the contact-dependent virulence mechanism. Mice immunised with two intraperitoneal doses of V antigen or each of the Yops, administered with either Alhydrogel or interleukin-12, produced high antigen-specific serum IgG titres. As shown in previous studies, V+Alhydrogel was fully protective, and 5/5 mice survived a subcutaneous challenge with 90 or 9x10(3) LD50's of Y. pestis GB. In addition, these preliminary studies also showed that V+IL-12 was partially protective: 4/5 or 3/5 mice survived a challenge with 90 or 9x10(3) LD50's, respectively. In contrast, none of the mice immunised with the Yops survived the challenges, and there was no significant delay in the mean time to death compared to mice receiving a control protein. These results show that using two different vaccine regimens, Yops E, K and N, failed to elicit protective immune responses in a murine model of plague, whereas under the same conditions, V antigen was fully or partially protective.
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| 12. |
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| 13. |
- Valerio, M, et al.
(författare)
-
Tissue-specificity of the regulation of ATP hydrolysis by isolated plant-mitochondria
- 1993
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Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 318:2, s. 113-117
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Tidskriftsartikel (refereegranskat)abstract
- Pea leaf mitochondria had a high ATP hydrolase activity following the collapse of the membrane potential by addition of valinomycin in state 4. In mitochondria isolated from potato tubers such ATP hydrolase activity was not observed. Pea leaf mitochondria also had a DELTApH, in contrast to what was previously found for potato tuber mitochondria. This DELTApH could, however, not explain the different results on ATP hydrolysis since this activity was also observed in the presence of nigericin. The results suggest a tissue-specific regulation of ATP hydrolysis in resting organs (potato tubers) as compared to active organs (leaves).
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| 14. |
- Wang, Z. Q., et al.
(författare)
-
PARP is important for genomic stability but dispensable in apoptosis
- 1997
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Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory Press (CSHL). - 0890-9369 .- 1549-5477. ; 11:18, s. 2347-2358
-
Tidskriftsartikel (refereegranskat)abstract
- Mice lacking the gene encoding poly(ADP-ribosyl) transferase (PARP or ADPRT) display no phenotypic abnormalities, although aged mice are susceptible to epidermal hyperplasia and obesity in a mixed genetic background. Whereas embryonic fibroblasts lacking PARP exhibit normal DNA excision repair, they grow more slowly in vitro. Here we investigated the putative roles of PARP in cell proliferation, cell death, radiosensitivity, and DNA recombination, as well as chromosomal stability. We show that the proliferation deficiency in vitro and in vive is most likely caused by a hypersensitive response to environmental stress. Although PARP is specifically cleaved during apoptosis, cells Backing this molecule apoptosed normally in response to treatment with anti-Fas, tumor neurosis factor alpha, gamma-irradiation, and dexamethasone, indicating that PARP is dispensable in apoptosis and that PARP-/-thymocytes are not hypersensitive to ionizing radiation. Furthermore, the capacity of mutant cells to carry out immunoglobulin class switching and V(D)J recombination is normal. Finally, primary PARP mutant fibroblasts and splenocytes exhibited an elevated frequency of spontaneous sister chromatid exchanges and elevated micronuclei formation after treatment with genotoxic agents, establishing an important role for PARP in the maintenance of genomic integrity.
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| 15. |
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| 16. |
- Hakman, Inger
(författare)
-
Correction
- 1992
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Ingår i: Physiologia Plantarum. - 0031-9317 .- 1399-3054. ; 84:2, s. 318-318
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Tidskriftsartikel (refereegranskat)
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| 17. |
- Hakman, Inger, et al.
(författare)
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Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)
- 1990
-
Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 80:3, s. 441-445
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Tidskriftsartikel (refereegranskat)abstract
- Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 mM sucrose and 7.6 μM ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.
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| 18. |
- Högstrand, K., et al.
(författare)
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DNA damage caused by etoposide and γ-irradiation induces gene conversion of the MHC in a mouse non-germline testis cell line
- 1999
-
Ingår i: Mutation research. - 0027-5107 .- 1873-135X. ; 423:1-2, s. 155-169
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Tidskriftsartikel (refereegranskat)abstract
- We have explored the effects of γ-irradiation and etoposide on the gene conversion frequency between the endogenous major histocompatibility complex class II genes Abk and Ebd in a mouse testis cell line of non-germline origin with a polymerase chain reaction assay. Both γ-rays and etoposide were shown to increase the gene conversion frequency with up to 15-fold compared to untreated cells. Etoposide, which is an agent that stabilise a cleavable complex between DNA and DNA topoisomerase II, shows an increased induction of gene conversion events with increased dose of etoposide. Cells treated with γ-rays, which induce strand breaks, had an increased gene conversion frequency when they were subjected to low doses of irradiation, but increasing doses of irradiation did not lead to an increase of gene conversion events, which might reflect differences in the repair process depending on the extent and nature of the DNA damage. These results where DNA damage was shown to be able to induce gene conversion of endogenous genes in mouse testis cells suggests that the DNA repair system could be involved in the molecular genetic mechanism that results in gene conversion in higher eukaryotes like mammals.
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| 19. |
- Segura-Aguilar, Juan, et al.
(författare)
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The effect of 5OH-1,4-Naphthoquinone on Norway spruce seeds during germination
- 1992
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Ingår i: Plant Physiology. - 0032-0889 .- 1532-2548. ; 100:4, s. 1955-1961
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Tidskriftsartikel (refereegranskat)abstract
- The effect of 5-OH-1,4-naphthoquinone (50H-NQ), a knowninhibitor of germination and growth and an inducer of oxidativestress, on seeds from Norway spruce (Picea abies) during germinationwas studied. 50H-NQ was activated by homogenate fromseeds to reactive species that reduce oxygen to superoxide radicalsin vitro. Increasing concentrations of 50H-NQ increased lipidperoxidation during this activation. Small effects of 50H-NQ ongermination of seeds were observed at concentrations up to 200Mm. However, higher concentrations, e.g. 500 and 1000 AM, exertedmore pronounced effects on seeds. These results suggest that theeffect of 50H-NQ was a delay rather than an inhibition of germination.However, the effect of 50H-NQ on postgerminative growthwas more potent than that on germination, and higher concentrationsinhibited growth >97%. These results suggest that the seedshave a very effective defense system against quinone and reactiveoxygen species, since the small effects of 50H-NQ on germinationand postgermination at concentrations up to 200 AM can be explainedby the formation of a metabolite of 50H-NQ that is not asreactive with oxygen as the original quinone. The 50H-NQ metabolitecollected during germination experiments showed differencesin its absorption spectrum in comparison with 50H-NQ, whichsuggest changes in structure. This metabolite was reduced byquinone reductase, but reduction of oxygen to superoxide radicalswas not detected during its activation with homogenate from seeds.This metabolite may arise via a conjugation reaction, since theaddition of 500 Mm uridine 5'-diphosphoglucuronic acid or 3'-phosphoadenosine-5'-phosphosulfate to the incubation mixtureduring activation of this metabolite by homogenate from seeds invitro inhibited reduction of oxygen to superoxide radicals by 50and 64%, respectively. The constitutive levels of superoxide dismutaseand catalase were sufficient to prevent oxygen toxicityduring activation of 50H-NQ, since these enzymes were not inducedwhen the seeds were treated with 200 AM 50H-NQ.
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| 20. |
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| 21. |
- Beier, Frank, et al.
(författare)
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Variability in the upstream promoter and intron sequences of the human, mouse and chick type X collagen genes.
- 1996
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Ingår i: Matrix Biology. - : Elsevier. - 0945-053X .- 1569-1802. ; 15:6, s. 415-422
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Tidskriftsartikel (refereegranskat)abstract
- The type X collagen gene is specifically expressed in hypertrophic chondrocytes during endochondral ossification. Transcription of the type X collagen gene by these differentiated cells is turned on at the same time as transcription of several other cartilage specific genes is switched off and before mineralization of the matrix begins. Analysis of type X collagen promoters for regulatory regions in different cell culture systems and in transgenic mice has given contradictory results suggesting major differences among species. To approach this problem, we have determined the nucleotide sequences of the two introns and upstream promoter sequences of the human and mouse type X collagen genes and compared them with those of bovine and chick. Within the promoter regions, we found three boxes of homology which are nearly continuous in the human gene but have interruptions in the murine gene. One of these interruptions was identified as a complex 1.9 kb repetitive element with homology to LINE, B1, B2 and long terminal repeat sequences. Regulatory elements of the human type X collagen gene are located upstream of the region where the repetitive element is inserted in the mouse gene, making it likely that the repetitive element is inserted between the coding region and regulatory sequences of the murine gene without interfering with its expression pattern. We also compared the sequences of the introns of both genes and found strong conservation. Comparisons of the mammalian sequences with promoter and first intron sequences of the chicken type X collagen gene revealed that only the proximal 120 nucleotides of the promoter were conserved, whereas all other sequences displayed no obvious homology to the murine and human sequences.
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| 22. |
- Lammi, Mikko, 1961-, et al.
(författare)
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Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure.
- 1994
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 304, s. 723-730
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Tidskriftsartikel (refereegranskat)abstract
- The effect of hydrostatic pressure on proteoglycan (PG) metabolism of chondrocyte cultures was examined using a specially designed test chamber. Primary cultures of bovine articular chondrocytes at confluence were exposed for 20 h to 5 and 30 MPa continuous hydrostatic pressures and 5 MPa hydrostatic pulses (0.017, 0.25 and 0.5 Hz) in the presence of [35S]sulphate. Northern blot analyses showed that chondrocyte cultures used in this study expressed abundant mRNA transcripts of aggrecan, typical of chondrocytes, but not versican. The cultures also expressed biglycan and decorin. Enzymic digestions with keratanase and chondroitinases AC, ABC and B and subsequent SDS/agarose gel electrophoresis confirmed the synthesis of aggrecans and small dermatan sulphate PGs. The continuous 30 MPa pressure reduced total PG synthesis by 37% as measured by [35S]sulphate incorporation, in contrast to the 5 MPa continuous pressure which had no effect. The high static pressure also reduced total [3H]glucosamine incorporation by 63% and total [14C]leucine incorporation by 57%. The cyclic pressures showed a frequency-dependent stimulation (0.5 Hz, 11%) or inhibition (0.017 Hz, -17%) of [35S]sulphate incorporation. Aggrecans secreted under continuous 30 MPa pressure showed a retarded migration in 0.75% SDS/agarose gel electrophoresis and they also eluted earlier on Sephacryl S-1000 gel filtration, indicative of a larger molecular size. The increased size was consistent with an increase of average glycosaminoglycan chain length as determined by Sephacryl S-300 gel filtration. No change in aggrecan size was observed with the lower (5 MPa) static or cyclic pressures. Continuous 30 MPa hydrostatic pressure slightly reduced the steady-state mRNA level of aggrecan, in parallel with the decline in PG synthesis measured by [35S]sulphate incorporation. The results demonstrated that high hydrostatic pressure could influence the synthesis of PGs, especially of aggrecans, in chondrocytes both at the transcriptional and translational/post-translational levels.
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| 23. |
- Ferrari, D., et al.
(författare)
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Differential regulation and ATP requirement for caspase-8 and caspase-3 activation during CD95- and anticancer drug-induced apoptosis
- 1998
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 188:5, s. 979-984
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Tidskriftsartikel (refereegranskat)abstract
- Apoptosis is induced by different stimuli, among them triggering of the death receptor CD95, staurosporine, and chemotherapeutic drugs. In all cases, apoptosis is mediated by caspases, although it is unclear how these diverse apoptotic stimuli cause protease activation. Two regulatory pathways have been recently identified, but it remains unknown whether they are functionally independent or linked to each other. One is mediated by recruitment of the proximal regulator caspase-8 to the death receptor complex. The other pathway is controlled by the release of cytochrome c from mitochondria and the subsequent ATP-dependent activation of the death regulator apoptotic protease-activating factor 1 (Apaf-1). Here, we report that both pathways can be dissected by depletion of intracellular ATP. Prevention of ATP production completely inhibited caspase activation and apoptosis in response to chemotherapeutic drugs and staurosporine. Interestingly, caspase-8, whose function appeared to be restricted to death receptors, was also activated by these drugs under normal conditions, but not after ATP depletion. In contrast, inhibition of ATP production did not affect caspase activation after triggering of CD95. These results suggest that chemotherapeutic drug-induced caspase activation is entirely controlled by a receptor-independent mitochondrial pathway, whereas CD95-induced apoptosis can be regulated by a separate pathway not requiring Apaf-1 function.
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| 24. |
- Ekström, Per, et al.
(författare)
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A calmodulin inhibitor with high specificity, compound 48/80, inhibits axonal transport in frog nerves without disruption of axonal microtubules.
- 1991
-
Ingår i: Acta physiologica Scandinavica. - 0001-6772. ; 142:2, s. 181-9
-
Tidskriftsartikel (refereegranskat)abstract
- The calmodulin inhibitor compound 48/80 has previously been shown to arrest axonal transport in vitro in the regenerating frog sciatic nerve. The inhibition was limited to the outgrowth region of nerves, which had been allowed to regenerate in vivo for 6 days after a crush lesion, before they were incubated with or without drugs in vitro overnight. The effects of compound 48/80 on the regenerating nerve were further investigated. A concentration of compound 48/80 (50 micrograms ml-1), which effectively inhibits axonal transport, did not cause observable changes of the microtubules of regenerating axons in the outgrowth region as judged by electron microscopy. Furthermore, it was shown that also a lower concentration (25 micrograms ml-1) inhibited axonal transport. As a measure of possible metabolic effects, the level of ATP was assessed in the regenerating nerve after exposure to compound 48/80. Compound 48/80 at 25 micrograms ml-1 did not change the level of ATP in the nerve. The assembly of bovine brain microtubule proteins in a cell-free system was unaffected by 25 micrograms ml-1 of compound 48/80 and slightly inhibited by 50 micrograms ml-1. At higher concentrations (greater than 100 micrograms ml-1) assembly of microtubules appeared stimulated, and microtubule spirals as well as closely aligned microtubules could be seen. These effects appeared to be unrelated to the transport effects. The present results indicate that compound 48/80 arrests axonal transport via mechanisms other than destruction of axonal microtubules or interference with the energy metabolism. It is possible that these mechanisms involve inhibition of calmodulin-regulated events essential to the transport.
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| 25. |
- Los, Marek Jan, et al.
(författare)
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Requirement of an Ice/Ced-3 Protease for Fas/Apo-1-Mediated Apoptosis
- 1995
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 375:6526, s. 81-83
-
Tidskriftsartikel (refereegranskat)abstract
- THE Fas/APO-1 receptor is one of the major regulators of apoptosis(1-7). We report here that Fas/APO-1-mediated apoptosis requires the activation of a new class of cysteine proteases, including interleukin-1 beta-converting enzyme (ICE)(8-10) which are homologous to the product of the Caenorhabditis elegans cell-death gene ced-3 (refs 11, 12). Triggering of Fas/APO-1 rapidly stimulated the proteolytic activity of ICE. Overexpression of ICE, achieved by electroporation and microinjection, strongly potentiated Fas/APO-1-mediated cell death. In addition, inhibition of ICE activity by protease inhibitors, as well as by transient expression of the pox virus-derived serpin inhibitor CrmA or an antisense ICE construct, substantially suppressed Fas/APO-1-triggered cell death. We conclude that activation of ICE or an ICE-related protease is a critical event in Fas/APO-1-mediated cell death.
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| 26. |
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| 27. |
- Schulze-Osthoff, Klaus, et al.
(författare)
-
Apoptosis signaling by death receptors
- 1998
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 254:3, s. 439-459
-
Tidskriftsartikel (refereegranskat)abstract
- Death receptors have been recently identified as a subgroup of the TNF-receptor superfamily with a predominant function in induction of apoptosis. The receptors are characterized by an intracellular region, called the death domain, which is required for the transmission of the cytotoxic signal. Currently, five different such death receptors are known including tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2. The signaling pathways by which these receptors induce apoptosis are rather similar. Ligand binding induces receptor oligomerization, followed by the recruitment of an adaptor protein to the death domain through homophilic interaction. The adaptor protein then binds a proximal caspase, thereby connecting receptor signaling to the apoptotic effector machinery. In addition, further pathways have been linked to death receptor-mediated apoptosis, such as sphingomyelinases, JNK kinases and oxidative stress. These pro-apoptotic signals are counteracted by several mechanisms which inhibit apoptosis at different levels. This review summarizes the current and rapidly expanding knowledge about the biological functions of death receptors and the mechanisms to trigger or to counteract cell death.
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| 28. |
- Schulze-Osthoff, Klaus, et al.
(författare)
-
Role of ICE-related and other proteases in Fas-mediated apoptosis
- 1996
-
Ingår i: Cell Death and Differentiation. - 1350-9047 .- 1476-5403. ; 3:2, s. 177-184
-
Forskningsöversikt (refereegranskat)abstract
- Interleukin-1 beta-converting enzyme (ICE)-like proteases comprise a novel family of unusual cysteine proteases which have been implicated in programmed cell death in both invertebrates and mammals. Current available evidence indicates a role of ICE proteases as central executioners of apoptosis triggered by the cell surface receptor Fas (APO-1). The presence of multiple mammalian ICE proteases with partially overlapping but distinct activities suggests a complex proteolytic cascade which is induced upon Fas ligation. The precise role of single members of the ICE family in Fas-mediated apoptosis, however, is still unclear. Here, we summarize the present knowledge about the relevance of ICE proteases, their potential targets, and interaction with unrelated proteases in cell death mediated by Fas and other apoptotic stimuli.
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| 29. |
- Cerenius, Lage, et al.
(författare)
-
CRUSTACEAN IMMUNITY AND COMPLEMENT - A PREMATURE COMPARISON
- 1995
-
Ingår i: American Zoologist. - : Oxford University Press (OUP). - 0003-1569 .- 2162-4445. ; 35:1, s. 60-67
-
Tidskriftsartikel (refereegranskat)abstract
- The prophenoloxidase activating system constitutes a system for recognition of foreignness in several invertebrates. The system has been especially well studied in crustaceans and it will now be possible to begin structural comparisons between components
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| 30. |
- Erjefält, Jonas, et al.
(författare)
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In vivo restitution of airway epithelium
- 1995
-
Ingår i: Cell and Tissue Research. - 1432-0878. ; 281:2, s. 305-316
-
Tidskriftsartikel (refereegranskat)abstract
- Epithelial shedding occurs in health and, extensively, in inflammatory airway diseases. This study describes deepithelialisation, reepithelialisation and associated events in guinea-pig trachea after shedding-like epithelial denudation in vivo. Mechanical deepithelialisation of an 800-microns wide tracheal zone was carried out using an orotracheal steel probe without bleeding or damage to the basement membrane. Reepithelialisation was studied by scanning- and transmission electron microscopy and light microscopy. Nerve fibres were examined by immunostaining. Cell proliferation was analysed by [3H]-thymidine autoradiography. Immediately after epithelial removal secretory and ciliated (and presumably basal) epithelial cells at the wound margin dedifferentiated, flattened and migrated rapidly (2-3 microns/min) over the denuded basement membrane. Within 8-15 h a new, flattened epithelium covered the entire deepithelialised zone. At 30 h a tight epithelial barrier was established and after 5 days the epithelium was fully redifferentiated. After completed migration an increased mitotic activity occurred in the epithelium and in fibroblasts/smooth muscle beneath the restitution zone. Reinnervating intraepithelial calcitonin gene-related peptide-containing nerve fibres appeared within 30 h. We conclude that (1) reproducible shedding-like denudation, without bleeding or damage to the basement membrane, can be produced in vivo; (2) secretory and ciliated cells participate in reepithelialisation by dedifferentiation and migration; (3) the initial migration is very fast in vivo; (4) shedding-like denudation may cause strong secretory and exudative responses as well as proliferation of epithelium, and fibroblasts/smooth muscle. Rapid restitution of airway epithelium may depend on contributions from the microcirculation and innervation.
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| 31. |
- Hedengren, Marika, et al.
(författare)
-
Relish, a central factor in the control of humoral but not cellular immunity in Drosophila.
- 1999
-
Ingår i: Mol Cell. - : Elsevier. - 1097-2765. ; 4:5, s. 827-37
-
Tidskriftsartikel (refereegranskat)abstract
- The NF-kappa B-like Relish gene is complex, with four transcripts that are all located within an intron of the Nmdmc gene. Using deletion mutants, we show that Relish is specifically required for the induction of the humoral immune response, including both antibacterial and antifungal peptides. As a result, the Relish mutants are very sensitive to infection. A single cell of E. cloacae is sufficient to kill a mutant fly, and the mutants show increased susceptibility to fungal infection. In contrast, the blood cell population, the hematopoietic organs, and the phagocytic, encapsulation, and melanization responses are normal. Our results illustrate the importance of the humoral response in Drosophila immunity and demonstrate that Relish plays a key role in this response.
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| 32. |
- Larsson, K, et al.
(författare)
-
The Saccharomyces cerevisiae SOP1 and SOP2 genes, which act in cation homeostasis, can be functionally substituted by the Drosophila lethal(2)giant larvae tumor suppressor gene.
- 1998
-
Ingår i: The Journal of biological chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 273:50, s. 33610-8
-
Tidskriftsartikel (refereegranskat)abstract
- By complementation of a salt-sensitive mutant of Saccharomyces cerevisiae, we cloned the SOP1 gene, encoding a 114.5-kDa protein of 1033 amino acids. Cells deleted for SOP1 exhibited sensitivity to sodium stress, but showed no sensitivity to general osmotic stress. Following exposure of sop1Delta cells to NaCl stress, the intracellular Na+ level and the Na+/K+ ratio rose to values significantly higher than in wild type cells. Deletion of SOP2, encoding a protein sharing 54% amino acid identity with Sop1p, produced only slight Na+ sensitivity. Cells carrying a sop1Deltasop2Delta double deletion became, however, hypersensitive to Na+ and exhibited increased sensitivity also to Li+ and K+, suggesting involvement of both SOP1 and SOP2 in cation homeostasis. The predicted amino acid sequences of Sop1p and Sop2p show significant homologies with the cytoskeletal-associated protein encoded by the Drosophila lethal(2)giant larvae tumor suppressor gene. Immunolocalization of Sop1p revealed a cytoplasmic distribution and cell fractionation studies showed that a significant fraction of Sop1p was recovered in a sedimentable fraction of the cytosolic material. Expression of a Drosophila l(2)gl cDNA in the sop1Deltasop2Delta strain partially restored the Na+ tolerance of the cells, indicating a functional relationship between the Sop proteins and the tumor suppressor protein, and a novel function in cell homeostasis for this family of proteins extending from yeast to human.
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| 33. |
- Eerola, Iiro, et al.
(författare)
-
Type X collagen, a natural component of mouse articular cartilage : association with growth, aging, and osteoarthritis.
- 1998
-
Ingår i: Arthritis and Rheumatism. - : John Wiley & Sons. - 0004-3591 .- 1529-0131. ; 41:7, s. 1287-1295
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To perform a systematic study on the production and deposition of type X collagen in developing, aging, and osteoarthritic (OA) mouse articular cartilage.METHODS: Immunohistochemistry was employed to define the distribution of type X collagen and Northern analyses to determine the messenger RNA levels as an indicator of the synthetic activity of the protein.RESULTS: Type X collagen was observed in the epiphyseal and articular cartilage of mouse knee joints throughout development and growth. Type X collagen deposition in the transitional zone of articular cartilage became evident toward cessation of growth, at the age of 2-3 months. The most intense staining for type X collagen was limited to the tidemark, the border between uncalcified and calcified cartilage. Northern analysis confirmed that the type X collagen gene is also transcribed by articular cartilage chondrocytes. Intense immunostaining was observed in the areas of OA lesions, specifically, at sites of osteophyte formation and surface fibrillation. Type X collagen deposition was also seen in degenerating menisci.CONCLUSION: This study demonstrates that type X collagen is a natural component of mouse articular cartilage throughout development, growth, and aging. This finding and the deposition of type X collagen at sites of OA lesions suggest that type X collagen may have a role in providing structural support for articular cartilage.
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| 34. |
- Haapala, Jussi, et al.
(författare)
-
Coordinated regulation of hyaluronan and aggrecan content in the articular cartilage of immobilized and exercised dogs.
- 1996
-
Ingår i: Journal of Rheumatology. - : Journal of Rheumatology. - 0315-162X .- 1499-2752. ; 23:9, s. 1586-1593
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To study the influence of joint loading and immobilization on articular cartilage hyaluronan concentration and histological distribution in the knee joints of young dogs subjected to 11 weeks' immobilization by splinting, and 15 weeks' running exercise at a rate of 40 km/day.METHODS: The amount of hyaluronan in articular cartilage was determined by a competitive binding assay using a biotinylated hyaluronan binding complex (HABC) of aggrecan and link protein. Histologic sections were stained for the localization of hyaluronan with the HABC probe. Extracted proteoglycans were characterized by sodium dodecyl sulfate agarose gel electrophoresis.RESULTS: Immobilization significantly reduced the concentration of hyaluronan in all sites studied (tibial and femoral condyles, patellar surface of femur). The proportion of hyaluronan to total uronic acid (mainly from aggrecan) remained unchanged because of a concurrent decrease in aggrecan. The ratio of hyaluronan and aggrecan remained constant also in runners. The staining pattern of free hyaluronan in the tissue sections and the electrophoretic mobility of the extracted proteoglycans were not affected by the different loading regimes.CONCLUSION: Reduced joint loading due to splint immobilization significantly decreases both hyaluronan and aggrecan in the articular cartilage. The remarkably parallel changes in aggrecan and hyaluronan content suggest that joint loading exerts a coordinated influence on their metabolism.
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| 35. |
- Inkinen, Ritva, et al.
(författare)
-
Hyaluronan distribution in the human and canine intervertebral disc and cartilage endplate.
- 1999
-
Ingår i: The Histochemical Journal. - 0018-2214 .- 1573-6865. ; 31:9, s. 579-587
-
Tidskriftsartikel (refereegranskat)abstract
- A biotinylated complex of aggrecan G1-domain and link protein was used to characterize the distribution of hyaluronan in paraffin-embedded sections of adult human and canine intervertebral disc and cartilage endplate. Limited chondroitinase ABC and trypsin digestions of the sections before staining was utilized to expose hyaluronan potentially masked by aggrecan. Hyaluronan concentration and hyaluronan to uronic acid ratio in different parts of the discs were measured as a background for the histological analysis. Hyaluronan staining was strong in the nucleus pulposus and inner parts of annulus fibrosus of both species, corroborated by biochemical assays of the same compartments. Particularly in human samples, hyaluronan in the interterritorial matrix of nucleus pulposus and annulus fibrosus was readily accessible to the probe without enzyme treatments. In contrast, the cell-associated hyaluronan signal was enhanced after trypsin or limited chondroitinase ABC-treatment of the sections, suggesting that pericellular hyaluronan was more masked by aggrecan than in the distant matrix. A puzzling feature of canine cartilage endplate cells was their intensive cell-associated hyaluronan signal, part of which appeared intracellular. Hyaluronan was abundant between the collagenous lamellae in annulus fibrosus, perhaps important in the plasticity of this tissue.
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| 36. |
- Kaarniranta, Kai, et al.
(författare)
-
Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation.
- 1998
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 95:5, s. 2319-2324
-
Tidskriftsartikel (refereegranskat)abstract
- In response to various stress stimuli, heat shock genes are induced to express heat shock proteins (Hsps). Previous studies have revealed that expression of heat shock genes is regulated both at transcriptional and posttranscriptional level, and the rapid transcriptional induction of heat shock genes involves activation of the specific transcription factor, heat shock factor 1 (HSF1). Furthermore, the transcriptional induction can vary in intensity and kinetics in a signal- and cell-type-dependent manner. In this study, we demonstrate that mechanical loading in the form of hydrostatic pressure increases heat shock gene expression in human chondrocyte-like cells. The response to continuous high hydrostatic pressure was characterized by elevated mRNA and protein levels of Hsp70, without activation of HSF1 and transcriptional induction of hsp70 gene. The increased expression of Hsp70 was mediated through stabilization of hsp70 mRNA molecules. Interestingly, in contrast to static pressurization, cyclic hydrostatic loading did not result in the induction of heat shock genes. Our findings show that hsp70 gene expression is regulated posttranscriptionally without transcriptional induction in chondrocyte-like cells upon exposure to high continuous hydrostatic pressure. We suggest that the posttranscriptional regulation in the form of hsp70 mRNA stabilization provides an additional mode of heat shock gene regulation that is likely to be of significant importance in certain forms of stress.
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| 37. |
- Király, Kari, et al.
(författare)
-
Safranin O reduces loss of glycosaminoglycans from bovine articular cartilage during histological specimen preparation.
- 1996
-
Ingår i: The Histochemical Journal. - : Chapman & Hill. - 0018-2214 .- 1573-6865. ; 28:2, s. 99-107
-
Tidskriftsartikel (refereegranskat)abstract
- The ability of Safranin O, added to fixation and decalcification solutions, to prevent the escape of glycosaminoglycans (GAGs) from small cartilage tissue blocks during histological processing of cartilage has been studied. GAGs in the fixatives and decalcifying solutions used and those remaining in the 1 mm3 cubes of cartilage were assayed biochemically. The quantity of GAGs remaining in the cartilage cubes were determined from Safranin O-stained sections using videomicroscopy or microspectrophotometry. A quantity (10.6%) of GAGs were lost during a conventional 4% buffered formaldehyde fixation (48 h) and a subsequent decalcification in 10% EDTA (12 days) at 4 degrees C. Roughly one-quarter of the total GAG loss occurred during the 48 h fixation, and three-quarters during the 12 days of decalcification. Inclusion of 4% formaldehyde in the decalcification fluid decreased the loss of GAGs to 6.2%. The presence of 0.5% Safranin O in the fixative reduced this loss to 3.4%. When 0.5% Safranin O was included in the fixative and 4% formaldehyde in the decalcification solution, Safranin O staining of the histological sections increased on average by 13.5%. After fixation in the presence of 0.5% Safranin O, there was no difference in the staining intensities when decalcification was carried out in the presence of either Safranin O or formaldehyde, or both. It took 24 h for Safranin O to penetrate into the deep zone of articular cartilage, warranting a fixation period of at least this long. In conclusion, the addition of Safranin O to the fixative and either Safranin O or formaldehyde in the following decalcification fluid, markedly reduces the loss of GAGs from small articular cartilage explants during histological processing. However, for immunohistochemical studies, Safranin O cannot be included in the processing solutions, because it may interfere.
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| 38. |
- Moses, J, et al.
(författare)
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Biosynthesis of the proteoglycan decorin -- identification of intermediates in galactosaminoglycan assembly
- 1997
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 248:3, s. 767-774
-
Tidskriftsartikel (refereegranskat)abstract
- Biosynthesis of decorin was investigated by incubating a rat fibroblast cell line with various radiolabelled protein and galactosaminoglycan precursors. The following cell-associated and distinct intermediates were isolated and identified: a pool of non-glycosylated core protein, two pools of decorin with incomplete chains, one with three sulphated disaccharide repeats and another with five or more sulphated disaccharide repeats, as well as decorin with mature chains. Results of pulse/chase experiments indicated that these pools represented discrete stages in chain growth. Treatment with brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi, resulted in accumulation of decorin with an incomplete chain containing six or seven largely unsulphated disaccharide repeats. During recovery from drug treatment, 4-sulfation reappeared earlier than 6-sulfation. The results suggest that the galactosaminoglycan assembly-line consists of separate multienzyme complexes that build only a limited section of the chain. Furthermore, brefeldin A causes segregation of compartments involved in separate stages of the assembly line. In an earlier report [Moses, J., Oldberg. A., Cheng, F. & Fransson, L.-A. (1997) Eur. J. Biochem. 248, 521-526] we took advantage of such segregation to identify and characterize a transient 2-phosphorylation of xylose in the linkage region.
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| 39. |
- Parkkinen, Jyrki, et al.
(författare)
-
Altered Golgi apparatus in hydrostatically loaded articular cartilage chondrocytes.
- 1993
-
Ingår i: Annals of the Rheumatic Diseases. - : British Medical Journal. - 0003-4967 .- 1468-2060. ; 52:3, s. 192-198
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Articular cartilage proteoglycan content is controlled by joint loading. This study aimed to elucidate the role of hydrostatic pressure in this regulation.METHODS: Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure, applied continuously or cyclically at 0.125 or 0.05 Hz. The Golgi apparatus was visualised either by a fluorochrome coupled wheat germ agglutinin or by transmission electron microscopy. Proteoglycan synthesis was studied by the incorporation of sulphur-35 labelled sulphate.RESULTS: After 30 MPa continuous hydrostatic pressure, the Golgi apparatus was observed in a compact form with a concomitant decrease in proteoglycan synthesis. The normal stacked appearance of the Golgi apparatus was no more visible in the electron microscopy preparation of the pressurised chondrocytes. This effect was reversible and was also noticed after 15 MPa continuous load, though to a minor extent. Cyclic pressures (5-30 MPa) caused no apparent change in the Golgi apparatus. The shape of some cells changed to a more retracted form after 30 MPa continuous pressure. Nocodazole, which causes disassembly of the microtubules, blocked the compacting influence of pressurisation on the Golgi apparatus, and reduced proteoglycan synthesis to about half of the control level.CONCLUSIONS: The packing of the Golgi apparatus is dependent on microtubules and may contribute to the inhibition of proteoglycan synthesis observed in articular cartilage subjected to high hydrostatic pressure.
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| 40. |
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| 41. |
- Parkkinen, Jyrki, et al.
(författare)
-
Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan.
- 1996
-
Ingår i: Histochemistry and Cell Biology. - : Springer. - 0948-6143 .- 1432-119X. ; 105:3, s. 187-194
-
Tidskriftsartikel (refereegranskat)abstract
- The proportion of total tissue hyaluronan involved in interactions with aggrecan and link protein was estimated from extracts of canine knee articular cartilages using a biotinylated hyaluronan binding region-link protein complex (bHABC) of proteoglycan aggregate as a probe in an ELISA-like assay. Microscopic sections were stained with bHABC to reveal free hyaluronan in various sites and zones of the cartilages. Articular cartilage, cut into 20 microns-thick sections, was extracted with 4 M guanidinium chloride (GuCl). Aliquots of the extract (after removing GuCl) were assayed for hyaluronan, before and after papain digestion. The GuCl extraction residues were analyzed after solubilization by papain. It was found that 47-51% of total hyaluronan remained in the GuCl extraction residue, in contrast to the 8-15% of total proteoglycans. Analysis of the extract revealed that 24-50% of its hyaluronan was directly detectable with the probe, while 50-76% became available only after protease digestion. The extracellular matrix in cartilage sections was stained with the bHABC probe only in the superficial zone and the periphery of the articular surfaces, both sites known to have a relatively low proteoglycan concentration. Trypsin pretreatment of the sections enhanced the staining of the intermediate and deep zones, presumably by removing the steric obstruction caused by the chondroitin sulfate binding region of aggrecans. Enhanced matrix staining in these zones was also obtained by a limited digestion with chondroitinase ABC. The results indicate that a part of cartilage hyaluronan is free from endogenous binding proteins, such as aggrecan and link protein, but that the chondroitin sulfate-rich region of aggrecan inhibits its probing in intact tissue sections. Therefore, hyaluronan staining was more intense in cartilage areas with lower aggrecan content. A large proportion of hyaluronan resists GuCl extraction, even from 20-micrograms-thick tissue sections.
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| 42. |
- Parkkinen, Jyrki, et al.
(författare)
-
Effects of cyclic hydrostatic pressure on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants.
- 1993
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 300:1, s. 458-465
-
Tidskriftsartikel (refereegranskat)abstract
- Primary chondrocyte cell cultures and explants of bovine articular cartilage were subjected to cyclic hydrostatic pressure in a novel computer-controlled pressure chamber designed for this purpose. The cultures were labeled with 5 microCi/ml 35SO4 and simultaneously pressurized with 5 MPa load for 1.5 or 20 h with pressure cycles of 0.0167, 0.05, 0.25, and 0.5 Hz. The chondrocyte cell cultures were also subjected to 0.0082 and 0.0034 Hz cycles. Sulfate incorporation was significantly inhibited in cell cultures subjected to the 0.5, 0.25, or 0.05 Hz cyclic loads for 1.5 h, but stimulated in explant cultures with a 0.5 Hz cyclic 1.5-h load. Chondrocyte cultures subjected to longer (20 h) loading showed a stimulation of sulfate incorporation with 0.5 and 0.25 Hz cycles, but an inhibition with 0.0167 Hz. The results indicate that cyclic hydrostatic pressures of presumably physiological magnitude have significant influences on proteoglycan synthesis in articular cartilage chondrocytes. Comparison of the cell and explant cultures under identical pressure conditions suggested that chondrocyte interactions with extracellular matrix are involved in this regulation by cyclic hydrostatic pressure. The responses of the chondrocytes to pressurization also varied according to the total length of the treatment, a finding compatible with the idea of multiple metabolic steps in chondrocytes, both pre- and post-translational, controlled by the ambient hydrostatic pressure.
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| 43. |
- Parkkinen, Jyrki, et al.
(författare)
-
Polyamine-dependent alterations in the structure of microfilaments, Golgi apparatus, endoplasmic reticulum, and proteoglycan synthesis in BHK cells.
- 1997
-
Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 66:2, s. 165-174
-
Tidskriftsartikel (refereegranskat)abstract
- The activity of ornithine decarboxylase, the key enzyme in the synthesis of polyamines, is essential for proliferation and differentiation of all living cells. Two inhibitors of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA), caused swelling of endoplasmic reticulum (ER) and medial and trans Golgi cisternae, and the disappearance of stress fibers, as visualized by staining with fluorescent concanavalin A (ConA), C6-NBD-ceramide or wheat germ agglutinin (WGA), and phalloidin, respectively. In contrast, the pattern of microtubules, stained with a beta-tubulin antibody, was not affected. Rough ER seemed to be especially affected in polyamine deprivation forming whorls and involutions, which were observed by transmission electron microscopy. Since ER and Golgi apparatus are vital parts of the glycosylation and secretory machinery of the cell, we tested the ability of these structurally altered cell organelles to synthesize proteoglycans using [3H]glucosamine and [35S]sulfate as precursors. The total incorporation rate into proteoglycans and hyaluronan was not reduced in polyamine-deprived cells, suggesting that the total glycosylation capacity of cells was not affected. However, the synthesis of a high molecular weight proteoglycan containing chondroitin and keratan sulfate was completely inhibited. The remodeling of cytoskeleton and rough endoplasmic reticulum in polyamine deprivation may perturb the synthesis and secretion of the components of membrane skeleton and of the extracellular matrix, e.g., proteoglycans. Rough ER and cytoskeleton may be the targets where polyamines affect cell proliferation and differentiation.
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| 44. |
- Visser, Nanette, et al.
(författare)
-
Increase of decorin content in articular cartilage following running.
- 1998
-
Ingår i: Connective Tissue Research. - : Informa Healthcare. - 0300-8207 .- 1607-8438. ; 37:3-4, s. 295-302
-
Tidskriftsartikel (refereegranskat)abstract
- The effect of long distance running exercise (40 km/day for 15 weeks, five days a week) on the decorin content of articular cartilage from the knee joint was studied in female beagle dogs. Samples from load bearing sites on the lateral plateau of the tibia (TL), and pooled material from two minimum load bearing sites on the posterior section of lateral (FLP) and medial (FMP) femoral condyles were analyzed. The running exercise protocol did not lead to significant changes in the overall glycosaminoglycan content of the cartilage. However, the amount of decorin significantly increased in the TL samples, and also in the FMP pool. These results support earlier in vitro observations that decorin synthesis is stimulated by loading, independent of the synthesis of aggrecan.
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| 45. |
- Edström, Anders, et al.
(författare)
-
Adenosine inhibition of the regeneration in vitro of adult frog sciatic sensory axons
- 1992
-
Ingår i: Brain Research. - 0006-8993. ; 570:1-2, s. 35-41
-
Tidskriftsartikel (refereegranskat)abstract
- The sensory axons of the adult frog sciatic nerve have earlier been shown to regenerate in vitro. If a local test crush is made at the initiation of culturing, regeneration starts after 3.4 days and proceeds at a rate of about 0.8-0.9 mm/day for several days. In the present experiments regeneration was inhibited by adenosine in a reversible and dose-dependent fashion. Similarly, both an adenosine analogue, 2-chloroadenosine (2-CA), and a non-hydrolyzable ATP analogue, AMP-PNP, reduced the outgrowth of sensory axons. The effect of adenosine was partially antagonized by theophylline at a critical concentration. Using a compartmental system, it could clearly be shown that adenosine exerted its effects at the outgrowth region. Adenosine, 2-CA, and AMP-PNP were also found to inhibit the proliferation of Schwann cells in the regenerating nerve. Various experiments showed that the latter can not explain the outgrowth inhibitory effects, which could be mediated by adenosine receptors associated with the elongating axons.
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| 46. |
|
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| 47. |
|
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| 48. |
- Mincheva-Nilsson, Lucia, et al.
(författare)
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Immunomorphologic studies of human decidua-associated lymphoid cells in normal early pregnancy.
- 1994
-
Ingår i: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 152:4, s. 2020-32
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Tidskriftsartikel (refereegranskat)abstract
- Human decidual lymphocytes from early, normal pregnancy were characterized in situ with respect to ultrastructure and distribution of subsets. The ultrastructure of isolated decidual gamma delta T cells was also studied. CD45+ cells comprised 11 +/- 2% of all decidual cells. The majority were localized in large lymphoid cell clusters (LCC), near endometrial glands, or as intraepithelial lymphocytes (IEL) in glandular epithelium. The major cell populations in LCC were CD56+TCR-gamma delta+ cells, CD56+ cells, TCR-alpha beta+CD4+ cells, and TCR-alpha beta+CD8+ cells. All expressed activation markers (CD45RO, Kp43, and/or HML-1) and MHC class II Ag (HLA-DR, HLA-DP, and/or HLA-DQ). No B cells were found. Almost all IEL were activated TCR-gamma delta+ cells (CD56+ and CD56-). The glandular epithelial cells expressed heat shock protein 60 at the basolateral side facing the TCR-gamma delta+ IEL. Decidual lymphocytes displayed cytoplasmic processes, microvilli, characteristic cytoplasmic granules, and had intimate contact with neighboring cells. Lymphocytes in the outer rim of LCC and the stroma showed signs of cellular movement. Two main morphotypes of gamma delta T cells could be distinguished. One had single microvilli, membrane-bound granules, and nuclear inclusions. The other had many microvilli, nonmembrane-bound granules and cytoplasmic multivesicular bodies. Our data suggest that LCC are centers of immune reactivity where T and NK cells become activated. The activated cells may guard against infections and undue trophoblast invasion and/or be involved in modulating the local maternal immune system toward unresponsiveness against the semiallogeneic fetus.
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