| 1. |
- Elo, Mika, et al.
(författare)
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Differential regulation of stress proteins by high hydrostatic pressure, heat shock, and unbalanced calcium homeostasis in chondrocytic cells.
- 2000
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 79:4, s. 610-619
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Tidskriftsartikel (refereegranskat)abstract
- High hydrostatic pressure (HP) has recently been shown to increase cellular heat shock protein 70 (Hsp70) level in a specific way that does not involve transcriptional activation of the gene, but rather the stabilisation of the mRNA for Hsp70. In this study, we investigated whether there are other observable changes caused by HP stress, and compared them with those induced by certain other forms of stressors. A chondrocytic cell line T/C28a4 was exposed to 30 MPa continuous HP, heat shock at 43 degrees C, and increased cytosolic calcium concentration by the addition of sarco-endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin (25 nM) or calcium ionophore A23187 (1 microM) in the cultures. The protein synthesis was studied by in vitro metabolic labelling followed by one- and two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry was utilized to confirm the identity of the protein spots on two-dimensional gels. Continuous 30 MPa HP increased remarkably the relative labelling of Hsp70. Labelling of Hsp90 was also increased by 15-20%, although no clear change was evident at the protein level in Western blots. Elevated intracellular Ca(2+) concentration induced by thapsigargin and calcium ionophore A23187 increased mainly the synthesis of glucose-regulated protein 78 (Grp78/BiP), whereas Hsp70 and Hsp90 were decreased by the treatment. Heat shock was the strongest inducer of Hsp70 and Hsp90. This study further confirmed the induction of Hsp70 in chondrocytic cells exposed to high HP, but it also showed that calcium-mediated responses are unlikely to cause the stress response observed in the hydrostatically pressurized cells.
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| 2. |
- 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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| 3. |
- Sabaj, V, et al.
(författare)
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Histone genes expression during the cell cycle in Trypanosoma cruzi
- 2001
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Ingår i: Journal of Cellular Biochemistry. - 0730-2312 .- 1097-4644. ; 80:4, s. 617-624
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Tidskriftsartikel (refereegranskat)abstract
- Histones, the basic proteins which compact DNA into the nucleosomal and solenoidal fibers are synthesized in correlation with DNA replication during the S-phase of the cell cycle. This behavior is controlled both at transcriptional and postranscriptional levels in higher eukaryotes and yeasts. We have found that histone synthesis in synchronized trypanosomes is controlled by fluctuations on the levels of their mRNAs. Though we cannot preclude the existence of a transcriptional regulatory mechanism, our results point to the participation of changes in the stability of histone mRNAs as a regulatory mechanism of their levels during the cell cycle in Trypanosoma. We have also found a postranscriptional regulatory mechanism which could be acting at the translational level. These results show both similarities and differences between Trypanosoma and higher eukaryotes regarding the expression of their histone genes.
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| 4. |
- Schwartz, Yuri B, et al.
(författare)
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Interbands of Drosophila melanogaster polytene chromosomes contain matrix association regions.
- 1999
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Ingår i: Journal of Cellular Biochemistry. - 0730-2312 .- 1097-4644. ; 72:3, s. 368-372
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Tidskriftsartikel (refereegranskat)abstract
- The DNA of three previously cloned interband regions (85D9/D10, 86B4/B6, and 61C7/C8) of Drosophila melanogaster polytene chromosomes has been tested for the presence of matrix association regions (MAR), using the in vitro matrix-binding assay of Cockerill and Garrard. MARs were found in all three interband regions under study. These results are discussed in frames of a model postulating that interband regions of polytene chromosomes correspond to the chromosomal DNA loop borders, which can be identified in interphase nuclei using biochemical approaches.
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| 5. |
- Spillmann, Dorothe, et al.
(författare)
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Carbohydrate-carbohydrate Interactions in Adhesion
- 1996
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Ingår i: Journal of Cellular Biochemistry. - 0730-2312 .- 1097-4644. ; 61:4, s. 562-568
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Tidskriftsartikel (refereegranskat)abstract
- Cell-cell interactions play an important role in the development, maintenance, and pathogenesis of tissues. They are highly dynamic processes which include migration, recognition, signaling, adhesion, and finally attachment. Cells on their pathway to a final location have to pass and interact with their substratum formed of matrix and cell layers. Testing and recognition are important keys for the proper result of tissue formation. They can, however, also lead to diseases when they are misused in pathological situations, by microorganisms or malignant cells, for instance. Carbohydrates, which are the most prominent surface-exposed structures, must play an important role as recognition molecules in such processes. The rich variability of carbohydrate sequences which cell surfaces can present to lectins, adhesion molecules, and other ligands creates a refined pattern of potential attachment sites. The subtle control of the surface presentation density can provide variations in attachment strength. Not only the carbohydrate sequences but also the fact that carbohydrates can be branched while proteins cannot and that the oligosaccharide chains can be attached to the protein backbone in different densities and patterns will create yet more interaction possibilities. Maximal use of the combinatorial richness of carbohydrate molecules would be made when carbohydrate sequences could interact with other carbohydrate sequences. Such interactions have only very rarely been considered for biochemically and biologically relevant situations since they are difficult to measure. A few are known and will be summarized here with the hope that this wealth of possible chemical interactions may be considered more and more by surface cell biochemists when analyzing fine tuning in cellular interactions.
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| 6. |
- Parkkinen, Jyrki, et al.
(författare)
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Polyamine-dependent alterations in the structure of microfilaments, Golgi apparatus, endoplasmic reticulum, and proteoglycan synthesis in BHK cells.
- 1997
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 66:2, s. 165-174
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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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| 7. |
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| 8. |
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| 9. |
- Bironaite, Daiva, et al.
(författare)
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Protective Induction of Hsp70 in Heat-Stressed Primary Myoblasts: Involvement of MAPKs
- 2013
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Ingår i: Journal of Cellular Biochemistry. - : Wiley-Blackwell. - 0730-2312 .- 1097-4644. ; 114:9, s. 2024-2031
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Tidskriftsartikel (refereegranskat)abstract
- The involvement of extracellular signal-regulated kinases 1 and 2 (ERK1,2), stress kinase p38 and c-Jun NH2-terminal kinases 1 and 2 (JNK1,2) on Hsp70-upregulation following mild heat shock, and resulting cell protection, was studied on rabbit primary myoblasts. Cells subjected to heat stress (42 degrees C; 60min) showed a significantly enhanced amount of heat-shock-induced protein 70 (Hsp70), correlating with sustained phosphorylation of MAP kinases ERK1,2, inhibition of p38 and JNK1,2 activation. Induced Hsp70 did not autocrinally suppress activation of transcription factor c-Jun, suggesting involvement of the latter in the protection of myoblasts following heat shock. The inhibition of stress kinases p38, JNK1,2, and MEK1,2 by SP600125, SB203580, and UO126, respectively, established the involvement of JNK1,2 and p38 as upstream, and ERK1,2 as downstream targets of Hsp70 induction. Moreover, the effect of the MEK1,2 inhibitor UO126 revealed a new pathway of c-Jun activation by ERK1,2 in myogenic heat-stressed stem cells. The presented data show that transient activation of JNK1, JNK2, and p38 is necessary for Hsp70 induction and ensuing cell protection. In conclusion, affecting myogenic stem cell protective mechanisms might be a useful strategy in improving stem cell survival and their expanded application in therapy.
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| 10. |
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| 11. |
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| 12. |
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| 13. |
- Carthy, Jon M., et al.
(författare)
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Commercially Available Preparations of Recombinant Wnt3a Contain Non-Wnt Related Activities Which May Activate TGF- Signaling
- 2016
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 117:4, s. 938-945
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Tidskriftsartikel (refereegranskat)abstract
- The Wnt ligands are a family of secreted signaling proteins which play key roles in a number of cellular processes under physiological and pathological conditions. Wnts bind to their membrane receptors and initiate a signaling cascade which leads to the nuclear localization and transcriptional activity of -catenin. The development of purified recombinant Wnt ligands has greatly aided in our understanding of Wnt signaling and its functions in development and disease. In the current study, we identified non-Wnt related signaling activities which were present in commercially available preparations of recombinant Wnt3a. Specifically, we found that treatment of cultured fibroblasts with recombinant Wnt3a induced immediate activation of TGF- and BMP signaling and this activity appeared to be independent of the Wnt ligand itself. Therefore, while purified recombinant Wnt ligands continue to be a useful tool for studying this signaling pathway, one must exercise a degree of caution when analyzing the results of experiments that utilize purified recombinant Wnt ligands.
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| 14. |
- Cheng, Fang, et al.
(författare)
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Differences in the uptake and nuclear localization of anti-proliferative heparan sulfate between human lung fibroblasts and human lung carcinoma cells
- 2001
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 83:4, s. 597-606
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate inhibits the proliferation of normal human lung fibroblasts (HFL-1) but not of a human lung carcinoma cell-line (A549). in this study we investigated possible mechanisms and structural requirements by which anti proliferative heparan sulfates exerts its effects on binding, uptake and subcellular localisation. Both HFL-1 and A549 cells were incubated with I-125- or rhodamine-labeled L-iduronate-rich antiproliferative heparan sulfate species as well as L-iduronate-poor inactive ones. The anti proliferative heparan sulfate was bound to the cell surface on both HFL-1 and A549 cells, but to a lesser extent and with less affinity to A549 cells. Both cell types bound the anti proliferative heparan sulfate with one high- and with one low affinity site. The L-iduronate-poor heparan sulfate bound to a lesser extent and with less affinity to both cell types compared to the anti proliferative heparan sulfate. The antiproliferative heparan sulfate accumulated in the cytoplasm of HFL-1 cells after 24 h incubation, but after 72 h it was found evenly distributed in the nucleus. The time-scale for anti proliferative activity correlated with nuclear localization. In contrast, in A549 cells it was only found near the nuclear membrane. The inactive heparan sulfate was taken up in considerably smaller amounts compared to the antiproliferative heparan sulfate and could not be detected in the nucleus of either HFL-1 or A549 cells. Our data suggest that the anti proliferative activity of L-iduronate-rich heparan sulfate on normal fibroblasts may be due to direct effects on nuclear processes, such as gene transcription.
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| 15. |
- Dalby, Matthew J, et al.
(författare)
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Nanomechanotransduction and interphase nuclear organization influence on genomic control.
- 2007
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 102:5, s. 1234-44
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Tidskriftsartikel (refereegranskat)abstract
- The ability of cells to alter their genomic regulation in response to mechanical conditioning or through changes in morphology and the organization of the interphase nuclei are key questions in cell biology. Here, two nanotopographies have been used as a model surfaces to change cell morphology in order to investigate spatial genomic changes within the nuclei of fibroblasts. Initially, centromeres for chromosome pairs were labeled and the average distance on different substrates calculated. Further to this, Affymetrix whole genome GeneChips were used to rank genomic changes in response to topography and plot the whereabouts on the chromosomes these changes were occurring. It was seen that as cell spreading was changed, so were the positions along the chromosomes that gene regulations were being observed. We hypothesize that as changes in cell and thus nuclear morphology occur, that this may alter the probability of transcription through opening or closing areas of the chromosomes to transcription factors.
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| 16. |
- Elo, Mika, et al.
(författare)
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High hydrostatic pressure inhibits the biosynthesis of eukaryotic elongation factor-2.
- 2005
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 94:3, s. 497-507
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Tidskriftsartikel (refereegranskat)abstract
- High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure-regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor-2 (eEF-2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF-2 decreased by 40%-50% within 12 h of the pressure treatment. However, the steady-state mRNA level of eEF-2 was not affected by the pressure. Cycloheximide addition after 4 h-pressure treatment suggested that the half-life of eEF-2 protein was shorter in pressurized cells. eEF-2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF-2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56-phosphorylated form of eEF-2 showed that phosphorylation of eEF-2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF-2. However, this inhibition may result from the decrease of cellular eEF-2 protein.
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| 17. |
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| 18. |
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| 19. |
- Granholm, Susanne, et al.
(författare)
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Comparisons between the effects of calcitonin receptor-stimulating peptide and intermedin and other peptides in the calcitonin family on bone resorption and osteoclastogenesis
- 2011
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 112:11, s. 3300-3312
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Tidskriftsartikel (refereegranskat)abstract
- Calcitonin receptor-stimulating peptide (CRSP) and intermedin (IMD) are two recently discovered peptides in the calcitonin (CT) family of peptides. CRSP and IMD, similar to CT, calcitonin gene-related peptide (CGRP) and amylin (AMY), but in contrast to adrenomedullin (ADM), inhibited bone resorption in mouse calvarial bones. CRSP and IMD, similar to CT, CGRP, AMY, but in contrast to ADM, decreased formation of osteoclasts and number of pits in bone marrow macrophage cultures stimulated by M-CSF and RANKL, with no effect on the expression of a number of genes associated with osteoclast progenitor cell differentiation. CRSP and IMD inhibited osteoclastogenesis at a late stage but had no effect on DC-STAMP mRNA. IMD, similar to CGRP, AMY and ADM stimulated cyclic AMP formation in M-CSF expanded osteoclast progenitor cells lacking CT receptors. RANKL induced CT receptors and a cyclic AMP response also to CT and CRSP, and increased the cyclic AMP response to CGRP, AMY and IMD but decreased the response to ADM. Our data demonstrate that CRSP and IMD share several functional properties of peptides in the CT family of peptides, including inhibition of bone resorption and osteoclast formation. The data also show that the reason why ADM does not inhibit osteoclast activity or formation is related to the fact that RANKL decreases ADM receptor signalling through the adenylate cyclase-cyclic AMP pathway. Finally, the findings indicate that activation by CGRP, AMY and IMD may include activation of both CT and CT receptor-like receptors. J. Cell. Biochem. © 2011 Wiley-Liss, Inc.
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| 20. |
- Granholm, Susanne, et al.
(författare)
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Expression of the calcitonin receptor, calcitonin receptor-like receptor, and receptor activity modifying proteins during osteoclast differentiation.
- 2008
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Ingår i: Journal of cellular biochemistry. - : Wiley. - 1097-4644 .- 0730-2312. ; 104:3, s. 920-33
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Tidskriftsartikel (refereegranskat)abstract
- The expressions of the calcitonin receptor (CTR), the calcitonin receptor-like receptor (CLR), the receptor activity-modifying proteins (RAMP) 1-3, and of the receptor component protein (RCP) have been studied in mouse bone marrow macrophages (BMM) during osteoclast differentiation, induced by treatment with M-CSF and RANKL. Analyses of mRNA showed that CLR and RAMP1-3, but not CTR, were expressed in M-CSF stimulated BMM. RANKL gradually increased CTR mRNA, transiently enhanced CLR and transiently decreased RAMP1 mRNA, but did not affect RAMP2, RAMP3, or RCP mRNA. However, RANKL did not affect protein levels of CLR or RAMP1-3 as assessed by Western blots or FACS analyses, whereas immunocytochemistry showed enhanced CTR protein. Analyses of cAMP production showed that BMM cells expressed functional receptors for calcitonin gene-related peptide (CGRP), amylin, adrenomedullin, and intermedin, but not for calcitonin and calcitonin receptor stimulating peptide (CRSP), but that RANKL induced the expression of receptors for calcitonin and CRSP as well. Calcitonin, CGRP, amylin, adrenomedullin, intermedin, and CRSP all down regulated the CTR mRNA, but none of the peptides caused any effects on the expression of CLR or any of the RAMPs. Our data show that BMM cells express receptors for CGRP, amylin, adrenomedullin, and intermedin and that RANKL induces the formation of receptors for calcitonin and CRSP in these cells. We also show, for the first time, that the CTR is not only down regulated by signaling through the CTR but also by the peptides signaling through CLR/RAMPs.
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| 21. |
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| 22. |
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| 23. |
- Koskela von Sydow, Anita, 1979-, et al.
(författare)
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IL-1α Counteract TGF-β Regulated Genes and Pathways in Human Fibroblasts
- 2016
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Ingår i: Journal of Cellular Biochemistry. - Hoboken, USA : Wiley-Blackwell. - 0730-2312 .- 1097-4644. ; 117:7, s. 1622-1632
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Tidskriftsartikel (refereegranskat)abstract
- Dysregulated wound healing is commonly associated with excessive fibrosis. Connective tissue growth factor (CTGF/CCN2) is characteristically overexpressed in fibrotic diseases and stimulated by transforming growth factor-β (TGF-β) in dermal fibroblasts. We previously showed that interleukin-1 (IL-1α) counteracts TGF-β-stimulated CTGF mRNA and protein expression in these cells. The aim of this study was to explore the effects of IL-1α on further genes and pathways in TGF-β regulated fibroblasts. Transcriptional microarray and multiple comparison analysis showed that the antagonizing effects of IL-1α was much more prominent than the synergistic effects, both with respect to number of genes and extent of changes in gene expression. Moreover, comparing canonical pathways by gene set enrichment analysis and the Ingenuity Pathway Analysis tool revealed that IL-1α counteracted TGF-β in the top six most confident pathways regulated by both cytokines. Interferon and IL-1 signaling, as well as two pathways involved in apoptosis signaling were suppressed by TGF-β and activated by IL-1α. Pathways involving actin remodeling and focal adhesion dynamics were activated by TGF-β and suppressed by IL-1α. Analyzing upstream regulators in part corroborate the comparison of canonical pathways and added cell cycle regulators as another functional group regulated by IL-1α. Finally, gene set enrichment analysis of fibrosis-related genes indicated that IL-1 moderately counteracts the collective effect of TGF-β on these genes. Microarray results were validated by qPCR. Taken together, the results indicate prominent antagonistic effects of IL-1α on TGF-β regulated interferon signaling, as well as on a wide variety of other genes and pathways in fibroblasts. This article is protected by copyright. All rights reserved.
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| 24. |
- Kumar, Rajender
(författare)
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Structural dynamics and mechanistic action guided engineering of lipolytic enzymes
- 2023
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 124:6, s. 877-888
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Tidskriftsartikel (refereegranskat)abstract
- Lipases have been established as important biocatalysts in several industrial applications, owing to their diverse substrate specificity. The availability of data on three-dimensional crystal structures for various lipases offers an opportunity for modulating their structural and functional aspects to design and engineer better versions of lipases. With the aim of investigating the structural components governing the extremophilic behavior of lipases, structural analysis of microbial lipases was performed using advanced bioinformatics and molecular dynamics simulation approaches. In sequences and functionally distinct alkaliphilic and thermophilic lipases were investigated for their functional properties to understand the distinguishing features of their structures. The alkaliphilic lipase from Bacillus subtilis (LipA) showed conformational changes in the loop region Ala132–Met137, subsequently, the active site residue His156 shows two conformations, toward the active site nucleophilic residues Ser77 and away from the Ser77. Interestingly, the active site of LipA is more solvent-exposed and can be correlated with the adoption of an open conformation which might extend and expose the active site region to solvents during the catalysis process. Furthermore, the MD simulation of thermophilic lipase from marine Streptomyces (MAS1) revealed the role of N- and C-terminal regions with disulfide bridges and identified a metal ion binding site that facilitates the enzyme stability. The novel thermo-alkaliphilic lipase can be designed to integrate the stability features of MAS1 into the alkaliphilic LipA. These structural-level intrinsic characteristics can be used for lipase engineering to amend the lipase activity and stability as per the requirements of the industrial processes.
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| 25. |
- Larsson, Dennis, et al.
(författare)
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24,25-Dihydroxyvitamin D3 binds to catalase
- 2006
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 97:6, s. 1259-1266
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Tidskriftsartikel (refereegranskat)abstract
- There is increasing evidence that the vitamin D metabolite, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) has endocrine actions. In the current work, we report that an endogenous binding protein for 24,25(OH)2D3 is catalase, based on sequence analysis of the isolated protein. An antibody (Ab 365) generated against equivalent protein recognized bovine catalase and a 64 kDa band in subcellular fractions of chick intestine. A commercially available anti-catalase antibody reduced specific [3H]24,25(OH)2D3 binding in subcellular fractions of chick intestine by greater than 65%, relative to the same fractions treated with an unrelated antibody (Ab 099). The same commercially available anti-catalase was able to block the inhibitory actions of 24,25(OH)2D3 on 32P uptake in isolated intestinal epithelial cell suspensions. We subsequently characterized binding of steroid to commercially available catalase, and found that between 0 and 5 nM of enzyme added to subcellular fraction P2 (20,000g, 10-min post-nuclear pellet) resulted in a linear increase in the amount of [3H]24,25(OH)2D3 specifically bound. Additional studies indicated that 25(OH)D3 was an effective competitor for binding, whereas 1,25(OH)2D3 only poorly displaced [3H]24,25(OH)2D3. Saturation analyses with added catalase yielded a physiologically relevant affinity constant (KD = 5.6 ± 2.7 nM) and a Bmax = 209 ± 34 fmols/mg protein, comparable to previous studies using purified basal lateral membranes or vesicular fractions. Moreover, in a study on subcellular fractions isolated from chickens of varying ages, we found that in females, both specific [3H]24,25(OH)2D3 binding and catalase activity increased from 7- to 58-week-old birds, whereas in males, elevated levels of both parameters were expressed in preparations of 7- and 58-week-old birds. The data suggest that signal transduction may occur through modulation of hydrogen peroxide production.
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| 26. |
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| 27. |
- Liu, Kaifei, et al.
(författare)
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Regulatory role of Golgi brefeldin A resistance factor-1 in amyloid precursor protein trafficking, cleavage and Aβ formation
- 2019
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Ingår i: Journal of Cellular Biochemistry. - Hoboken : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 120:9, s. 15604-15615
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Tidskriftsartikel (refereegranskat)abstract
- β-amyloid peptide (Aβ) deposition derived from sequential cleavage of the amyloid precursor protein (APP) through the amyloidogenic pathway is an important characteristic feature of Alzheimer's disease (AD). During this process, cellular trafficking plays a crucial role. A large Sec7-domain containing ADP-ribosylation factor guanine nucleotide exchange factor (ARF-GEF), Golgi brefeldin A resistance factor 1 (GBF1) has been reported to initiate the ADP-ribosylation factor (Arf) activation cascade at trans-Golgi network, which plays a crucial function at the endoplasmic reticulum-Golgi interface. In this study, we investigated the role of GBF1 in APP transmembrane transport and Aβ formation. Using APP/PS1 (presenilin 1) overexpressing transgenic mice, we demonstrate that GBF1 has upregulated the expression of APP, indicating a role for GBF1 in APP physiological process. Knocking down of GBF1 using small interfering has significantly increased the intracellular but not the surface expression of APP. In contrast, overexpression of wild-type (WT) and guanine nucleotide exchange factor (GEF) in the activated form but not the GEF deficient mutation induced continuous activation of GBF1, which subsequently increased the surface level of APP. Interestingly, inhibition of GBF1 by c(BFA) also impaired APP trafficking and induced endoplasmic reticulum (ER) stress in SH-SY5Y cells. Our results thus for identified the role of GBF1 in APP trafficking and cleavage, and provide evidence for GBF1 as a possible therapeutic target in AD. © 2019 Wiley Periodicals, Inc.
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| 28. |
- Loskutoff, D J, et al.
(författare)
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Fibrinolytic system of cultured endothelial cells : regulation by plasminogen activator inhibitor.
- 1986
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 32:4, s. 273-80
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Tidskriftsartikel (refereegranskat)abstract
- Cultured bovine aortic endothelial cells have a relatively complex fibrinolytic system that is responsive to both the physiological state of the cell itself and to a variety of agents added to the culture medium. The fibrinolytic activity of these cells results from the production of both urokinase-type and tissue-type plasminogen activators and is regulated by an inhibitor capable of neutralizing their activities. The properties of these fibrinolytic components will be reviewed, and their respective roles in initiating and regulating the fibrinolytic activity of the cells will be summarized. A cDNA coding for the inhibitor has been isolated, and its sequence will be compared to that of other serine proteinase inhibitors.
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| 29. |
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| 30. |
- Madhu, Priyanka, et al.
(författare)
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Distinct types of amyloid-beta oligomers displaying diverse neurotoxicity mechanisms in Alzheimer's disease
- 2021
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 122:11, s. 1594-1608
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Forskningsöversikt (refereegranskat)abstract
- Soluble oligomers of amyloid-beta (A beta) are recognized as key pernicious species in Alzheimer's disease (AD) that cause synaptic dysfunction and memory impairments. Numerous studies have identified various types of A beta oligomers having heterogeneous peptide length, size distribution, structure, appearance, and toxicity. Here, we review the characteristics of soluble A beta oligomers based on their morphology, size, and structural reactivity toward the conformation-specific antibodies and then describe their formation, localization, and cellular effects in AD brains, in vivo and in vitro. We also summarize the mechanistic pathways by which these soluble A beta oligomers cause proteasomal impairment, calcium dyshomeostasis, inhibition of long-term potentiation, apoptosis, mitochondrial damage, and cognitive decline. These cellular events include three distinct molecular mechanisms: (i) high-affinity binding with the receptors for A beta oligomers such as N-methyl- d-aspartate receptors, cellular prion protein, nerve growth factor, insulin receptors, and frizzled receptors; (ii) the interaction of A beta oligomers with the lipid membranes; (iii) intraneuronal accumulation of A beta by alpha 7-nicotinic acetylcholine receptors, apolipoprotein E, and receptor for advanced glycation end products. These studies indicate that there is a pressing need to carefully examine the role of size, appearance, and the conformation of oligomers in identifying the specific mechanism of neurotoxicity that may uncover potential targets for designing AD therapeutics.
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| 31. |
- Nazir, S. U., et al.
(författare)
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Differential expression of Ets-1 in breast cancer among North Indian population
- 2019
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 120:9, s. 14552-14561
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Tidskriftsartikel (refereegranskat)abstract
- Breast cancer is a highly aggressive disease contributing to high mortality rate among females across the globe owing to wide geographical variations, change in lifestyle along with rapid tumor growth, drug resistance, and high metastasis rate. To understand the molecular and genetic basis of breast cancer progression; we studied the role of E26 transformation-specific-1 (Ets-1) transcription factor which is implicated to have a role in carcinogenesis like invasion, metastasis, angiogenesis, etc. Our findings revealed an overexpression of Ets-1 gene in 75 breast cancer tumors as compared with their normal adjacent tissues. The findings significantly established a co-relation between Ets-1 expression in breast cancer tissue with hormonal receptor profiles and ductal-lobular histological subtypes in Indian population. In addition, a differential expression pattern of Ets-1 was observed between high, moderate, and low grades of breast cancer patients. The present study demonstrates a crucial role of Ets-1 transcription factor which may serve as a potential biomarker for breast carcinogenesis. © 2019 Wiley Periodicals, Inc.
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| 32. |
- Negoita, Florentina, et al.
(författare)
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PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes
- 2019
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 120:1, s. 343-356
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism of how patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden-standard of LC3-II turnover in combination with the well characterized GFP-RFP-LC3 vector. To discriminate between, perturbed autophagic initiation and lysosome functionality, lysosomes were characterized by Lysotracker staining and LAMP1 protein levels as well as activity and activation of cathepsin B. For validation, human liver biopsies genotyped for I148 and 148M were analyzed for the presence of LC3-II and PNPLA3 on lipid droplets. We show that the M148-PNPLA3 variant is associated with lipid droplets that are resistant to starvation-mediated degradation. M148 expressing hepatocytes reveal decreased autophagic flux and reduced lipophagy. Both I148-PNPLA3 and M148-PNPLA3 colocalize and interact with LC3-II, but the M148-PNPLA3 variant has lower ability to bind LC3-II. Together, our data indicate that PNPLA3 might play an essential role in lipophagy in hepatocytes and furthermore that the M148-PNPLA3 variant appears to display a loss in this activity, leading to decreased lipophagy.
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| 33. |
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| 34. |
- Nilsson, Anna, et al.
(författare)
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Fluid pressure induces osteoclast differentiation comparably to titanium particles but through a molecular pathway only partly involving TNFa
- 2012
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Ingår i: Journal of Cellular Biochemistry. - : Wiley-Blackwell. - 0730-2312 .- 1097-4644. ; 113:4, s. 1224-1234
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Tidskriftsartikel (refereegranskat)abstract
- In contrast to the well-understood inflammatory pathway driven by TNFa, by which implant-derived particles induce bone resorption, little is known about the process in which loosening is generated as a result of force-induced mechanical stimulus at the boneimplant interface. Specifically, there is no knowledge as to what cells or signaling pathways couple mechanical stimuli to bone resorption in context of loosening. We hypothesized that different stimuli, i.e., fluid flow versus wear particles, act through different cytokine networks for activation and localization of osteoclasts. By using an animal model in which osteoclasts and bone resorption were induced by fluid pressure or particles, we were able to detect distinct differences in osteoclast localization and inflammatory gene expression between fluid pressure and titanium particles. Fluid pressure recruits and activates osteoclasts with bone marrow contact away from the fluid pressure exposure zone, whereas titanium particles recruit and activate osteoclasts in areas in direct contact to particles. Fluid pressure induced weaker expression of the selected inflammatory related genes, although the eventual degree of osteoclast induction was similar in both models. Using TNFaRa (4?mg/kg) (Enbrel) and dexamethasone (2?mg/kg) as specific and more general suppressors of inflammation we showed that the TNFaRa failed to generate statistically impaired osteoclast generation while dexamethasone was much more potent. These results demonstrate that fluid pressure induces osteoclasts at a different localization than titanium particles by a molecular pathway less associated with TNFa and the innate system, which open up for other pathways controlling pressure induced osteoclastogenesis.
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| 35. |
- Nowinski, Daniel, et al.
(författare)
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Inhibition of Connective Tissue Growth Factor/CCN2 Expression in Human Dermal Fibroblasts by Interleukin-1 alpha and beta
- 2010
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 110:5, s. 1226-1233
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Tidskriftsartikel (refereegranskat)abstract
- Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)-beta and intimately involved with tissue repair and overexpressed in various fibrotic conditions We previously showed that keratmocytes in vitro downregulate TGF-beta-induced expression of CTGF in fibroblasts by an interleukin (IL)-1 alpha-dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL-1 alpha and beta Human dermal fibroblasts and NIH 3T3 cells were treated with IL-1 alpha or beta in presence or absence of TGF-beta 1. IL-1 suppressed basal and TGF-beta-induced CTGF mRNA and protein expression. IL-1 alpha and beta inhibited TGF-beta-stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3-binding CAGA elements Furthermore. IL-1 alpha and beta inhibited TGF-beta-stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition. RNA interference suggested that TGF-beta activated kinase1 (TAK1) is necessary for IL-1 inhibition of TGF-beta-stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell Biochem. 110: 1226-1233, 2010. (C) 2010 Wiley-Liss. Inc
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| 36. |
- Ostrakhovitch, Elena A, et al.
(författare)
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P53 mediated regulation of metallothionein transcription in breast cancer cells
- 2007
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Ingår i: Journal of Cellular Biochemistry. - Hoboken : Wiley-Liss. - 0730-2312 .- 1097-4644. ; 102:6, s. 1571-1583
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Tidskriftsartikel (refereegranskat)abstract
- Recent studies have shown that only breast cancer epithelial cells with intact p53 can induce metallothionein (MT) synthesis after exposure to metals. In this study, the potential role of p53 on regulation of MT was investigated. Results demonstrate that zinc and copper increased metal response elements (MREs) activity and MTF-1 expression in p53 positive MN1 and parental MCF7 cells. However, inactivation of p53 by treatment with pifithrin- or the presence of inactive p53 inhibited MRE-dependent reporter gene expression in response to metals. MTF-1 levels remained unchanged after treatment with zinc in cells with nonfunctional p53. The introduction of wild-type p53 in MDD2 cells, containing nonfunctional p53, enhanced the ability of zinc to increase MRE-dependent reporter gene expression. The cellular level of p21Cip1/WAF1 was increased in MDD2 cells after p53 transfection, confirming the presence of active p53. The treatment of MN1 and parental MCF7 with trichostatin A led to a sixfold increase in the MRE activity in response to zinc. On the contrary, MRE activity remained unaltered in MDD2 cells with inactive p53. The above results demonstrate that activation of p53 is an important factor in metal regulation of MT. J. Cell. Biochem. 102: 1571-1583, 2007.
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| 37. |
- Paredes, Rodolfo, et al.
(författare)
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Bovine (Bos taurus) humoral immune response against Echinococcus granulosus and hydatid cyst infertility
- 2011
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Ingår i: Journal of Cellular Biochemistry. - : Wiley-Liss, Inc.. - 0730-2312 .- 1097-4644. ; 112:1, s. 189-199
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Tidskriftsartikel (refereegranskat)abstract
- Echinococcus granulosus, the agent of hydatid disease, presents an indirect life cycle, with canines (mainly dogs) as definitive hosts, and herbivores and human as intermediary ones. In intermediary hosts fertile and infertile cysts develop, but only the first ones develop protoscoleces, the parasite form infective to definitive hosts. We report the presence of bovine IgGs in the germinal layer from infertile cysts (GLIC), in an order of magnitude greater than in the germinal layer from fertile cysts (GLFC). When extracted with salt solutions, bovine IgGs from GLIC are associated with low or with high affinity (most likely corresponding to non specific and antigen specific antibodies, respectively). Specific IgGs penetrate both the cells of the germinal layer and HeLa cultured cells and recognize parasitic proteins. These results, taken together with previous ones from our laboratory, showing induction of apoptosis in the germinal layer of infertile hydatid cysts, provide the first coherent explanation of the infertility process. They also offer the possibility of identifying the parasite antigens recognized, as possible targets for immune modulation.
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| 38. |
- Paul, Alexandra, 1988, et al.
(författare)
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Comparing lipid remodeling of brown adipose tissue, white adipose tissue, and liver after one-week high fat diet intervention with quantitative Raman microscopy
- 2023
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 124:3, s. 382-395
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Tidskriftsartikel (refereegranskat)abstract
- Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, nonchronic dietary intervention (a 1-week interval) on WAT and interscapular BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that 1 week of high fat diet (HFD) intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after 1 week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance.
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| 39. |
- Persson, Emma, et al.
(författare)
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The neuropeptide VIP regulates the expression of osteoclastogenic factors in osteoblasts
- 2011
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 112:12, s. 3732-3741
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Tidskriftsartikel (refereegranskat)abstract
- Osteoclast formation is controlled by stromal cells/osteoblasts expressing macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), crucial for osteoclast progenitor cell proliferation, survival and differentiation, and osteoprotegerin (OPG) that inhibits the interaction between RANKL and its receptor RANK. Recent data have strongly indicated that the nervous system play an important role in bone biology. In the present study, the effects of the neuropeptide vasoactive intestinal peptide (VIP), present in peptidergic skeletal nerve fibers, on the expression of RANKL, OPG, and M-CSF in osteoblasts and stromal cells have been investigated. VIP and pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38), but not secretin, stimulated rankl mRNA expression in mouse calvarial osteoblasts. In contrast, VIP inhibited the mRNA expressions of opg and m-csf, effects shared by PACAP-38, but not by secretin. VIP did not affect rankl, opg or m-csf mRNA expression in mouse bone marrow stromal cells. The effects by VIP on the mRNA expression of rankl, opg and m-csf were all potentiated by the cyclic AMP phosphodiesterase inhibitor rolipram. In addition, VIP robustly enhanced the phosphorylation of ERK and the stimulatory effect by VIP on rankl mRNA was inhibited by the MEK1/2 inhibitor PD98059. These observations demonstrate that activation of VPAC(2) receptors in osteoblasts enhances the RANKL/OPG ratio by mechanisms mediated by cyclic AMP and ERK pathways suggesting an important role for VIP in bone remodelling. J. Cell. Biochem. © 2011 Wiley-Liss, Inc.
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| 40. |
- Piltti, Juha, 1982-, et al.
(författare)
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Effects of long-term hypoxia in human chondrosarcoma cells
- 2018
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 119:2, s. 2320-2332
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Tidskriftsartikel (refereegranskat)abstract
- The cell-based therapies could be potential methods to treat damaged cartilage tissues. Instead of native hyaline cartilage, the current therapies generate mainly weaker fibrocartilage-type of repair tissue. A correct microenvironment influences the cellular phenotype, and together with external factors it can be used, e.g., to aid the differentiation of mesenchymal stem cells to defined types of differentiated adult cells. In this study, we investigated the effect of long-term exposure to 5% low oxygen atmosphere on human chondrosarcoma HCS-2/8 cells. This atmosphere is close to normal oxygen tension of cartilage tissue. The proteome was analyzed with label-free mass spectrometric method and further bioinformatic analysis. The qRT-PCR method was used to gene expression analysis, and ELISA and dimethylmethylene blue assays for type II collagen and sulfated glycosaminoglycan measurements. The hypoxic atmosphere did not influence cell proliferation, but enhanced slightly ACAN and COL2A1 gene expression. Proteomic screening revealed a number of hypoxia-induced protein level responses. Increased ones included NDUFA4L2, P4HA1, NDRG1, MIF, LDHA, PYGL, while TXNRD1, BAG2, TXN2, AQSTM1, TNFRSF1B and EPHX1 decreased during the long-term hypoxia. Also a number of proteins previously not related to hypoxia changed during the treatment. Of those S100P, RPSS26, NDUFB11, CDV3 and TUBB8 had elevated levels, while ALCAM, HLA-B, EIF1, and ACOT9 were lower in the hypoxia samples. In conclusion, low oxygen condition causes changes in the cellular amounts of several proteins.
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| 41. |
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| 42. |
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| 43. |
- Ryme, Jessica, et al.
(författare)
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Variations in the Composition of Mammalian SWI/SNF Chromatin Remodelling Complexes
- 2009
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 108:3, s. 565-576
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Tidskriftsartikel (refereegranskat)abstract
- The ATP-dependent chromatin remodelling complexes SWI/SNF alter the chromatin structure in transcriptional regulation. Several classes of mammalian SWI/SNF complex have been isolated biochemically, distinguished by a few specific subunits, such as the BAF-specific BAF250A, BAF250B and BRM, and the PBAF-specific BAF 180. We have determined the complex compositions using low stringency immunoprecipitation (IP) and shown that the pattern of subunit interactions was more diverse than previously defined classes had predicted. The subunit association at five gene promoters that depend on the SWI/SNF activity varied and the sequential chromatin immunoprecipitations revealed that different class-specific subunits occupied the promoters at the same time. The low-stringency IP showed that the BAF-specific BAF250A and BAF250B and the PBAF-specific BAF180 co-exist in a subset of SWI/SNF complexes, and fractionation of nuclear extract on size-exclusion chromatography demonstrated that sub-complexes with unorthodox subunit compositions were present in the cell. We propose a model in which the constellations of SWI/SNF complexes are ""tailored"" for each specific chromatin target and depend on the local chromatin environment to which complexes and sub-complexes are recruited.
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| 44. |
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| 45. |
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| 46. |
- Sogaard, Peter, et al.
(författare)
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Spatial Insulin Signalling in Isolated Skeletal Muscle Preparations
- 2010
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Ingår i: Journal of Cellular Biochemistry. - : Wiley-Liss, Inc.. - 0730-2312 .- 1097-4644. ; 109:5, s. 943-949
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Tidskriftsartikel (refereegranskat)abstract
- During in vitro incubation in the absence or presence of insulin, glycogen depletion occurs in the inner core of the muscle specimen, concomitant with increased staining of hypoxia-induced-factor-1-alpha and caspase-3, markers of hypoxia and apoptosis, respectively. The aim of this study was to determine whether insulin is able to diffuse across the entire muscle specimen in sufficient amounts to activate signalling cascades to promote glucose uptake and glycogenesis within isolated mouse skeletal muscle. Phosphoprotein multiplex assay on lysates from muscle preparation was performed to detect phosphorylation of insulin-receptor on Tyr1146, Akt on Ser473 and glycogen-synthases-kinase-3 on Ser21/Ser9. To address the spatial resolution of insulin signalling, immunohistochemistry studies on cryosections were performed. Our results provide evidence to suggest that during the in vitro incubation, insulin sufficiently diffuses into the centre of tubular mouse muscles to promote phosphorylation of these signalling events. Interestingly, increased insulin signalling was observed in the core of the incubated muscle specimens, correlating with the location of oxidative fibres. In conclusion, insulin action was not restricted due to insufficient diffusion of the hormone during in vitro incubation in either extensor digitorum longus or soleus muscles from mouse under the specific experimental settings employed in this study. Hence, we suggest that the glycogen depleted core as earlier observed is not due to insufficient insulin action.
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| 47. |
- Svartz, Jesper, 1972-, et al.
(författare)
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Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization
- 2006
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 98:6, s. 1517-1527
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Tidskriftsartikel (refereegranskat)abstract
- Leukotrienes (LTs) are fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope (NE) where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 is further converted to LTC4 by conjugation with glutathione, a reaction catalyzed by the integral membrane protein LTC4 synthase (LTC4S), which is localized on the NE and endoplasmic reticulum (ER). We now report the mapping of regions of LTC4S that are important for its subcellular localization. Multiple constructs encoding fusion proteins of green fluorescent protein (GFP) as the N-terminal part and various truncated variants of human LTC4S as C-terminal part were prepared and transfected into HEK 293/T or COS-7 cells. Constructs encoding hydrophobic region 1 of LTC4S (amino acids 6–27) did not give distinct membrane localized fluorescence. In contrast hydrophobic region 2 (amino acids 60–89) gave a localization pattern similar to that of full length LTC4S. Hydrophobic region 3 (amino acids 114–135) directed GFP to a localization indistinguishable from that of full length LTC4S. A minimal directing sequence, amino acids 117–132, was identified by further truncation. The involvement of the hydrophobic regions in the homo-oligomerization of LTC4S was investigated using bioluminescence resonance energy transfer (BRET) analysis in living cells. BRET data showed that hydrophobic regions 1 and 3 each allowed oligomerization to occur. These regions most likely form transmembrane helices, suggesting that homo-oligomerization of LTC4S is due to helix–helix interactions in the membrane.
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| 48. |
- Sögaard, Peter, et al.
(författare)
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Effects of fibre type and diffusion distance on mouse skeletal muscle glycogen content in vitro
- 2009
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 107:6, s. 1189-1197
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Tidskriftsartikel (refereegranskat)abstract
- In vitro incubation of isolated rodent skeletal muscle is a widely used procedure in metabolic research. One concern with this method is the development of an anoxic state during the incubation period that can cause muscle glycogen depletion. Our aim was to investigate whether in vitro incubation conditions influence glycogen concentration in glycolytic extensor digitorum longus (EDL) and oxidative soleus mouse muscle. Quantitative immunohistochemistry was applied to assess glycogen content in incubated skeletal muscle. Glycogen concentration was depleted, independent of insulin-stimulation in the incubated skeletal muscle. The extent of glycogen depletion was correlated with the oxidative fibre distribution and with the induction of hypoxia-induced-factor-1-alpha. Insulin exposure partially prevented glycogen depletion in soleus, but not in EDL muscle, providing evidence that glucose diffusion is not a limiting step to maintain glycogen content. Our results provide evidence to suggest that the anoxic milieu and the intrinsic characteristics of the skeletal muscle fibre type play a major role in inducing glycogen depletion in during in vitro incubations.
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| 49. |
- Tarhriz, Vahideh, et al.
(författare)
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CDK9 Regulates Apoptosis of Myoblast Cells by Modulation of microRNA-1 Expression
- 2018
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 119:1, s. 547-554
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Tidskriftsartikel (refereegranskat)abstract
- Cdk9 is the catalytic core of the positive transcription elongation factor b (P-TEFb) and regulates transcriptional elongation factors by phosphorylation of RNA pol II. Apart from its role on myogenic gene expression, Cdk9 regulation of muscle-specific microRNAs in the early stage of cardiomyogenesis is poorly understood. Here we demonstrate that Cdk9 not only regulates myogenic transcription factors, but also controls muscle-specific microRNAs. During cardiac differentiation of mouse embryonic stem cells, high Cdk9 expression preceded up-regulation of miR-1. To investigate potential regulatory roles of Cdk9 on cardiac microRNAs and myogenesis genes, we overexpressed Cdk9 in myoblast C2C12 cells, which resulted in significant induction of miR-1 and miR-206, while miR-133 was downregulated. Moreover, expression levels of MyoD and Srf, key regulators of myogenesis, also increased in cells with overexpression of Cdk9. We further observed Cdk9-mediated apoptosis in C2C12 cells corresponding to induction of miR-1 expression levels. Thus, Cdk9 plays a complex role in myocyte progenitor differentiation and apoptosis by regulating myogenic protein and muscle-specific microRNA expression. J. Cell. Biochem. 119: 547–554, 2018.
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| 50. |
- Thörn, Kristofer, 1981-, et al.
(författare)
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Fatty acid-induced oxidation and triglyceride formation is higher in insulin-producing MIN6 cells exposed to oleate compared to palmitate
- 2010
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 111:2, s. 497-507
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Tidskriftsartikel (refereegranskat)abstract
- Palmitate negatively affects insulin secretion and apoptosis in the pancreatic β-cell. The detrimental effects are abolished by elongating and desaturating the fatty acid into oleate. To investigate mechanisms of how the two fatty acids differently affect β-cell function and apoptosis, lipid handling was determined in MIN6 cells cultured in the presence of the fatty acids palmitate (16:0) and oleate (18:1) and also corresponding monounsaturated fatty acid palmitoleate (16:1) and saturated fatty acid stearate (18:0). Insulin secretion was impaired and apoptosis accentuated in palmitate-, and to some extent, stearate-treated cells. Small or no changes in secretion or apoptosis were observed in cells exposed to palmitoleate or oleate. Expressions of genes associated with fatty acid esterification (SCD1, DGAT1, DGAT2 and FAS) were augmented in cells exposed to palmitate or stearate but only partially (DGAT2) in palmitoleate- or oleate-treated cells. Nevertheless, levels of triglycerides were highest in cells exposed to oleate. Similarly, fatty acid oxidation was most pronounced in oleate-treated cells despite comparable up-regulation of CPT1 after treatment of cells with the four different fatty acids. The difference in apoptosis between palmitate and stearate was paralleled by similar differences in levels of markers of endoplasmic reticulum (ER) stress in cells exposed to the two fatty acids. Palmitate-induced ER stress was not accounted for by ceramide de novo synthesis. In conclusion, although palmitate initiated stronger expression changes consistent with lipid accumulation and combustion in MIN6 cells, rise in triglyceride levels and fatty acid oxidation was favored specifically in cells exposed to oleate.
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