| 1. |
- 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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| 2. |
- Nelimarkka, Lassi, et al.
(författare)
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Expression of small extracellular chondroitin/dermatan sulfate proteoglycans is differentially regulated in human endothelial cells.
- 1997
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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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| 3. |
- Greiff, Lennart, et al.
(författare)
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Effects of topical platelet activating factor on the guinea-pig tracheobronchial mucosa in vivo
- 1997
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Ingår i: Acta Physiologica Scandinavica. - 0001-6772. ; 160:4, s. 387-391
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Tidskriftsartikel (refereegranskat)abstract
- Platelet activating factor (PAF) has been reported to produce a variety of airway effects including epithelial damage and increased airway-lung absorption of hydrophilic tracers. The present study examines effects of PAF on the guinea-pig tracheobronchial mucosa in vivo. Vehicle with and without PAF (4.0 and 8.0 nmol) was superfused onto the tracheobronchial mucosa. The levels of 125I-albumin, previously given intravenously, were determined in tracheobronchial lavage fluids as an index of mucosal exudation of plasma. The mucosa was also examined by scanning electron microscopy. In separate animals, 99mTc-DTPA (a low molecular weight, 492 Da, hydrophilic tracer) was superfused onto the mucosal surface through an oro-tracheal catheter, together with vehicle or PAF (8.0 nmol). A gamma camera determined the disappearance rate of 99mTc-DTPA from the airways as an index of mucosal absorption. PAF produced dose-dependent mucosal exudation of plasma up to 20-fold greater than control (P < 0.001). However, PAF did not damage the epithelium and the absorption ability of the airway mucosa was unaffected. The results, in contrast to previous reports, suggest that PAF may not readily damage the airway mucosa even at large exudative doses of the agent. The present finding support the view that the plasticity of the epithelial junctions allows the creation of valve-like paracellular pathways for unidirectional clearance of extravasated plasma into the airway lumen. We suggest that endogenous PAF may participate in first line respiratory defence reactions by causing lumenal entry of bulk plasma without harming the epithelium.
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| 4. |
- Amandusson, Åsa, et al.
(författare)
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Colocalization of oestrogen receptor immunoreactivity and preproenkephalin mRNA expression fo neurons in the superficial laminae of the spinal and medullary dorsal horn of rats
- 1996
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Ingår i: Eur J Neurosci. - : Wiley InterScience. ; 8:11, s. 2440-2445
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Tidskriftsartikel (refereegranskat)abstract
- A double-labelling procedure combining immunohistochemical staining with in situ hybridization using a radiolabelled cRNA probe was employed to demonstrate oestrogen receptor-like immunoreactivity and preproenkephalin-A mRNA in the medullary and spinal dorsal horn of female rats. Both markers labelled large numbers of neurons in the substantia gelatinosa and its trigeminal homologue. Many of these neurons were double-labelled, displaying both oestrogen receptor-like-immunoreactivity and preproenkephalin-A mRNA; cell counts showed that 40-60% of the of the oestrogen receptor-like-immunoreactive cells in the superficial laminae also were labelled for preproenkephalin-A mRNA, and that 60-70% of the preproenkephalin-A mRNA-labelled neurons in the same laminae displayed oestrogen receptor-like immunoreactivity. Previous studies have shown that oestrogen receptors can bind to the promoter region of the preproenkephalin-A gene, and studies on the hypothalamus have demonstrated that oestrogen regulates enkephalin expression in select neuronal populations. The present results demonstrate that enkephalinergic neurons in the superficial dorsal horn contain oestrogen receptors and suggest that oestrogen may play an important role in the modulation of sensory and nociceptive processing in the lower medulla and spinal cord.
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| 5. |
- Amandusson, Åsa, 1974-, et al.
(författare)
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Estrogen-induced alterations of spinal cord enkephalin gene expression
- 1999
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Ingår i: Pain. - : Elsevier. - 0304-3959 .- 1872-6623. ; 83:2, s. 243-248
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Tidskriftsartikel (refereegranskat)abstract
- Enkephalin-synthesizing neurons in the super®cial laminae of the spinal and trigeminal dorsal horn are critical components of the endogenous pain-modulatory system. We have previously demonstrated that these neurons display intracellular estrogen receptors, suggesting that estrogen can potentially influence their enkephalin expression. By using Northern blot, we now show that a bolus injection of estrogen results in a rapid increase in spinal cord enkephalin mRNA levels in ovariectomized female rats. Thus, 4 h after estrogen administration the enkephalin mRNA-expression in the lumbar spinal cord was on average 68% higher (P , 0:05) than in control animals injected with vehicle only. A small increase in the amount of enkephalin mRNA was also seen after 8 h (P , 0:05), whereas no difference between estrogen-injected and control animals was found after 24 h or at time periods shorter than 4 h. Taken together with the previous anatomical data, the present findings imply that estrogen has an acute effect on spinal opioid levels in areas involved in the transmission of nociceptive information.
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| 6. |
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| 7. |
- 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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| 8. |
- 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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| 9. |
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| 10. |
- Beier, Frank, et al.
(författare)
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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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