| 1. |
- Ekberg, Jenny, et al.
(författare)
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Post-translational modification of cyclin A1 is associated with staurosporine and TNFalpha induced apoptosis in leukemic cells.
- 2009
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Ingår i: Molecular and Cellular Biochemistry. - : Springer Science and Business Media LLC. - 0300-8177 .- 1573-4919. ; 320:1-2, s. 115-24
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Tidskriftsartikel (refereegranskat)abstract
- Understanding of molecular mechanisms underlying the effects of cell cycle proteins in response to the chemotherapeutic agents is of great importance for improving the efficacy of targeted therapeutics and overcoming resistance to chemotherapeutic agents. Staurosporine and tumor necrosis factor alpha (TNFalpha) are the therapeutic agents that inhibit tumor cell growth by inducing cell death. Staurosporine induces apoptosis through the intrinsic pathway, while TNFalpha trigger the cell death via the extrinsic apoptotic pathway. We have previously demonstrated that the cell cycle regulatory protein, cyclin A1 played an important role in the development of acute myeloid leukemia (AML), and cyclin A1 expression correlated with disease characteristics and patient outcome in leukemia. However, it remains unknown how cyclin A1 expression is regulated in leukemic cells treated with the therapeutic agents. Here, we demonstrate that cyclin A1 protein is regulated by proteasome-mediated ubiquitination and degradation in untreated U-937 cells. Interestingly, ubiquitination- and proteasomal-mediated degradation of cyclin A1 is prevented in cells treated with staurosporine or TNFalpha. Induction of apoptosis in U-937 cells by staurosporine or TNFalpha resulted in an increase in cyclin A1 protein expression, which correlated well with cyclin A1 protein modification and the activation of caspase-3. Blocking caspases activity by Z-VAD-FMK had no effect on the increased cyclin A1 expression, suggesting that cyclin A1 might be regulated by caspase-3 independent pathways. We further propose that CDC25C may be associated with cyclin A1 protein modification in response to staurosporine or TNFalpha treatment. Our results suggest that cyclin A1 protein is stabilized via post-transcriptional modification in response to apoptosis induced by staurosporine or TNFalpha.
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| 2. |
- Heydarkhan-Hagvall, Sepideh, 1969, et al.
(författare)
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DNA microarray study on gene expression profiles in co-cultured endothelial and smooth muscle cells in response to 4- and 24-h shear stress
- 2006
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Ingår i: Molecular and cellular biochemistry. - : Springer Science and Business Media LLC. - 0300-8177 .- 1573-4919. ; 281:1-2, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Shear stress, a major hemodynamic force acting on the vessel wall, plays an important role in physiological processes such as cell growth, differentiation, remodelling, metabolism, morphology, and gene expression. We investigated the effect of shear stress on gene expression profiles in co-cultured vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Human aortic ECs were cultured as a confluent monolayer on top of confluent human aortic SMCs, and the EC side of the co-culture was exposed to a laminar shear stress of 12 dyn/cm(2) for 4 or 24 h. After shearing, the ECs and SMCs were separated and RNA was extracted from the cells. The RNA samples were labelled and hybridized with cDNA array slides that contained 8694 genes. Statistical analysis showed that shear stress caused the differential expression (p < or = 0.05) of a total of 1151 genes in ECs and SMCs. In the co-cultured ECs, shear stress caused the up-regulation of 403 genes and down-regulation of 470. In the co-cultured SMCs, shear stress caused the up-regulation of 152 genes and down-regulation of 126 genes. These results provide new information on the gene expression profile and its potential functional consequences in co-cultured ECs and SMCs exposed to a physiological level of laminar shear stress. Although the effects of shear stress on gene expression in monocultured and co-cultured EC are generally similar, the response of some genes to shear stress is opposite between these two types of culture (e.g., ICAM-1 is up-regulated in monoculture and down-regulated in co-culture), which strongly indicates that EC-SMC interactions affect EC responses to shear stress.
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| 3. |
- Kaczmarczyk, Aneta, et al.
(författare)
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Plasma bikunin : half-life and tissue uptake.
- 2005
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Ingår i: Molecular and Cellular Biochemistry. - : Springer Science and Business Media LLC. - 0300-8177 .- 1573-4919. ; 271:1-2, s. 61-67
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Tidskriftsartikel (refereegranskat)abstract
- Bikunin is a chondroitin sulfate-containing plasma protein synthesized in the liver. In vitro, it has been shown to inhibit proteases and to have additional activities, but its biological function is still unclear. Here we have studied the dynamics of plasma bikunin in rats and mice. A half-life of 7 +/- 2 min was obtained from the time course of the decrease of the plasma level of bikunin following hepatectomy. Clearance experiments with intravenously injected radiolabeled bikunin with or without the chondroitin sulfate chain showed that the polysaccharide had little influence on the elimination rate of the protein. The uptake of bikunin by different tissues was studied using bikunin labeled with the residualizing agent 125I-tyramine cellobiose; 60 min after intravenous injection, 49% of the radioactivity was recovered in the kidneys and 6-11% in the liver, bones, skin, intestine and skeletal muscle. The uptake in the liver was analyzed by intravenous injection of radiolabeled bikunin followed by collagenase perfusion and dispersion of the liver cells. These experiments indicated that bikunin is first trapped extracellularly within the liver before being internalized by the cells.
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| 4. |
- Lind, Thomas, et al.
(författare)
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Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities.
- 2006
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Ingår i: Molecular and Cellular Biochemistry. - : Springer Science and Business Media LLC. - 0300-8177 .- 1573-4919. ; 281:1-2, s. 95-102
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Tidskriftsartikel (refereegranskat)abstract
- ADAMTS-1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats) is a member of a family of secreted proteolytic enzymes with a complex modular structure. These enzymes are characterised by an N-terminal metalloproteinase domain, a disintegrin-like domain and a carboxyl terminal region containing variable numbers of a repeat sequence with homology to thrombospondin-1. The expression of the gene for ADAMTS-1 has been associated with inflammation, ovulation, angiogenesis, cellular proliferation and bone formation. ADAMTS-1 can proteolytically process large proteoglycans indicating a potential role in extracellular matrix turnover. In this study, we have tested ADAMTS-1 activity in gelatin zymogram assays. Since previous data demonstrate that ADAMTS-1 is a matrix metalloproteinase (MMP) substrate and is highly unstable in conditioned medium from eukaryotic cell types, we created an insect cell line expressing human ADAMTS-1. We isolated an epitope tagged full-length recombinant ADAMTS-1 from serum free insect cell conditioned medium. The purified protein had aggrecanase activity and appears as two major bands on the silver stained SDS-PAGE corresponding well to a pro-domain on form of 115 kDa and a pro-domain off form of 90 kDa. Using denatured type I collagen in zymographic analysis we demonstrate that ADAMTS-1 has a previously unreported gelatinolytic activity. Also, we notice that processing of its C-terminal region by an apparently autocatalytic process reveals a 27 kDa species with gelatinolytic activity. Furthermore, we show that MMP2 but not MMP13 remove ADAMTS-1 specific gelatin zymopraphic zones.
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| 5. |
- Manna, Sugata, et al.
(författare)
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Differential alterations in metabolic pattern of the spliceosomal UsnRNAs during pre-malignant lung lesions induced by benzo(a)pyrene: modulation by tea polyphenols
- 2006
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Ingår i: Molecular and Cellular Biochemistry. - : Springer Science and Business Media LLC. - 0300-8177 .- 1573-4919. ; 289:1-2, s. 149-157
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Tidskriftsartikel (refereegranskat)abstract
- The differential alterations of the spliceosomal UsnRNAs (U1, U2, U4, U5, and U6) were reported to be associated with cellular proliferation and development. The attempt was made in this study to analyze the metabolic pattern of the spliceosomal UsnRNAs during the development of pre-malignant lung lesions induced in experimental mice model system by benzo(a)pyrene (BP) and also to see how tea polyphenols, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), modulate the metabolism of these UsnRNAs during the lung carcinogenesis. No significant changes in the level of the UsnRNAs were seen in the inflammatory lung lesions at 9th week due to treatment of BP. However, there was significant increase in the level of U1 ( approximately 2.5 fold) and U5 ( approximately 47%) in the hyperplastic lung lesions at 17th week. But in the mild dysplastic lung lesions at 26th week, the level of UsnRNAs did not change significantly. Whereas, in the dysplastic lung lesions at 36th week there was significant increase in the level of the U2 ( approximately 2 fold), U4 ( approximately 2.5 fold) and U5 ( approximately 2 fold). Due to the EGCG and ECG treatment the lung lesions at 9th week appeared normal and in the 17th, 26th, and 36th week it appeared as hyperplasia. The level of the UsnRNAs was significantly low in the lung lesions at 9th week (only U2 and U4 by EGCG), at 17th week (only U1 by EGCG/ECG), at 26th week (U1 by ECG; U2, U4 and U5 by EGCG/ECG) and at 36th week (U1 by ECG, U2 and U4 by EGCG/ECG). Whereas, there was significant increase in the level of U5 (by EGCG/ECG) and U6 (by EGCG only) in the lung lesions at 36th and 26th week respectively. This indicates that the metabolism of the spliceosomal UsnRNAs differentially altered during the development of pre-malignant lung lesions by BP as well as during the modulation of the lung lesions by the tea polyphenols.
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| 6. |
- Masłyk, Maciej, et al.
(författare)
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Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2
- 2008
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Ingår i: Molecular and Cellular Biochemistry. - : Springer Science and Business Media LLC. - 0300-8177 .- 1573-4919. ; 312:1-2, s. 61-69
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Tidskriftsartikel (refereegranskat)abstract
- Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2alpha (K (m) 0.35 muM) and CK2alpha' (K (m) 0.18 muM) as well as CK2 holoenzyme (K (m) 1.1 muM). Different K (m) values argue that CK2beta(beta') subunit has an inhibitory effect on the activity of both CK2alpha and CK2alpha' towards surviving factor Svf1. Reconstitution of alpha'(2)betabeta' isoform of CK2 holoenzyme shows that beta/beta' subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of alpha(2)betabeta' isoform, which may be due to interaction between Svf1 and regulatory CK2beta subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K(199)EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED(248) of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.
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| 7. |
- Pivoriunas, A., et al.
(författare)
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PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/ Cip1 in human leukemia cells
- 2007
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Ingår i: Molecular and Cellular Biochemistry. - : Springer Science and Business Media LLC. - 0300-8177 .- 1573-4919. ; 302:1-2
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Tidskriftsartikel (refereegranskat)abstract
- Despite the understanding of the importance of phosphoinositide 3-kinase (PI 3-K) signaling pathway in the regulation of cellular proliferation, little is known about its role during phorbol 12-myristate 13-acetate (PMA)-induced differentiation in human leukemia cells. Here, we report a novel finding that PI 3-K inhibition by LY294002 significantly increases p21WAF1/Cip1 expression in PMA-stimulated human leukemia cells NB4 and THP1. LY294002 potentiated expression of p21WAF1/Cip1 via a p53-independent mechanism and did not affect mitogen activated protein kinase (MAPK) activation. Electrophoretic mobility shift (EMSA) experiments revealed that blocking of PI 3-K was associated with increased binding of transcription factor Sp1 to the PMA-responsive sites on the p21WAF1/Cip1 promoter. Pretreatment with rapamycin, an inhibitor of mTOR kinase, decreased the expression of p21WAF1/Cip1 protein in PMA-stimulated NB4 cells. The level of PMA-induced p21WAF1/ Cip1 protein expression was lower in NB4 cells overexpressing wild type protein kinase C ? (PKC ?) compared to those transfected with empty vector or with kinase inactive PKC ?. Sp1 binding to the p21WAF1/Cip1 promoter was completely lost in a wild type PKC ? overexpressing and PMA-stimulated NB4 cells. We demonstrate that PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/Cip1 in human leukemia cells, and that this effect is partly mediated by PKC ?. © Springer Science+Business Media, LLC 2007.
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| 8. |
- Sobti, R. C., et al.
(författare)
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Overexpression of STAT3 in HPV-mediated cervical cancer in a north Indian population
- 2009
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Ingår i: Molecular and Cellular Biochemistry. - : Springer Science+Business Media B.V.. - 0300-8177 .- 1573-4919. ; 330:1-2, s. 193-9
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Tidskriftsartikel (refereegranskat)abstract
- The constitutively activated STAT family members, particularly STAT3, have been shown to possess transforming properties, and are strongly correlated with tumor development and progression. STAT3 transmits signals from many cytokines and growth factors to target genes in the nucleus through the Jak/Stat signaling pathway. HPV is the main etiological factor in the development of cervical cancer. In the current study, the expression of STAT3 was analyzed in various stages of HPV-mediated cervical carcinogenesis. Tissue biopsies from 100 patients with cervical cancer of different stages and normal tissues from patients undergoing hysterectomy were selected for studying the HPV status and STAT3 expression. HPV status of each corresponding biopsy was analyzed by PCR and typing. The mRNA expression was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR). HPV infection was detected in majority of cases: 75% (9/12) in precancer, 85% (34/40) stage I & II, and 95% (36/38) in stage III & IV of cervical cancer cases by L1 PCR. Further sub typing revealed HPV16 in 100% (9/9) of L1 positives in precancerous & 90% (63/70) in different stages of cancer. Significant level of STAT3 mRNA expression was predominantly found in cervical cancer cases as compared to normal controls (P = 0.001). We also found a significant correlation of STAT3 expression in cases infected with HPV (P = 0.001). Our results indicate a potentially interactive effect between HPV 16/18 and transcriptional activation of STAT3 gene in cervical carcinogenesis. To our knowledge, this is the first such study to be reported from India. Further investigations are needed to determine the influence of STAT3 expression on cervical carcinogenesis and its possible interaction with HPV infection status.
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| 9. |
- Zielin„ski, Rafal, et al.
(författare)
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Fip1 - an essential component of the Saccharomyces cerevisiae polyadenylation machinery is phosophorylated by protein kinase CK2
- 2006
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Ingår i: Molecular and Cellular Biochemistry. - : Springer Science and Business Media LLC. - 0300-8177 .- 1573-4919. ; 286:1-2, s. 191-197
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Tidskriftsartikel (refereegranskat)abstract
- Since Fip1 is phosphoprotein we investigated whether it is a substrate for protein kinase CK2. According to the amino acid sequence Fip1 harbours twenty putative CK2 phosphorylation sites. Here we have report characterization of Fip1 as a substrate for both forms of CK2. Fip1 serves as a substrate for both the recombinant CK2α ′ (K m 1.28 μM) and holoenzyme (K m 1.4 μM) but not for CK1. By MALDI-MS we identified the two serine residues at positions 73 and 77 as the possible in vitro phosphorylation sites. These data may help to elucidate the role of Fip1 in the mRNA 3'-OH polyadenylation process and the involvement of CK2 mediated phosphorylation in regulation of interactions and activity members of cleavage/polyadenylation factor (CPF) complex.
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