Ischemic brain injury triggers an inflammatory response. tissue but can also exacerbate brain injury. Microglia are This response is necessary to clear damaged brain the innate immune cells of the brain that execute t...Ischemic brain injury triggers an inflammatory response. tissue but can also exacerbate brain injury. Microglia are This response is necessary to clear damaged brain the innate immune cells of the brain that execute this critical function. In healthy brain, microglia perform a housekeeping function, pruning unused syn- apses between neurons. However, microglia become activated to an inflammatory phenotype upon brain injury. Interferon regulatory factors modulate microglial activation and their production of inflammatory cytokines. This review briefly discusses recent findings pertaining to these regulatory mechanisms in the context of stroke recovery.展开更多
BACKGROUND Heart diseases are the primary cause of death all over the world.Following myocardial infarction,billions of cells die,resulting in a huge loss of cardiac function.Stem cell-based therapies have appeared as...BACKGROUND Heart diseases are the primary cause of death all over the world.Following myocardial infarction,billions of cells die,resulting in a huge loss of cardiac function.Stem cell-based therapies have appeared as a new area to support heart regeneration.The transcription factors GATA binding protein 4(GATA-4)and myocyte enhancer factor 2C(MEF2C)are considered prominent factors in the development of the cardiovascular system.AIM To explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells(hUC-MSCs).METHODS hUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry,and by their potential to differentiate into osteocytes and adipocytes.hUC-MSCs were transfected with GATA-4,MEF2C,and their combination to direct the differentiation.Cardiac differentiation was confirmed by semiquant itative real-time polymerase chain reaction and immunocytochemistry.RESULTS hUC-MSCs expressed specific cell surface markers CD105,CD90,CD44,and vimentin but lack the expression of CD45.The transcription factors GATA-4 and MEF2C,and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e.,GATA-4,MEF2C,NK2 homeobox 5(NKX2.5),MHC,and connexin-43,and cardiac proteins GATA-4,NKX2.5,cardiac troponin T,and connexin-43.CONCLUSION Transfection with GATA-4,MEF2C,and their combination effectively induces cardiac differentiation in hUC-MSCs.These genetically modified MSCs could be a promising treatment option for heart diseases in the future.展开更多
The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane pr...The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins aremembers of the basic helix-loop-helix-leucine zipper (bHLHZip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP). The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2 is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones, cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.展开更多
Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFNβ) gene regulation. Besides that IRF-2 is involved in immunomodulation,...Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFNβ) gene regulation. Besides that IRF-2 is involved in immunomodulation, hematopoietic differentiation, cell cycle regulation and oncogenesis. We have done molecular sub-cloning and expression of recombinant murine IRF-2 as GST (Glutathione-S-Transferase)- IRF-2 fusion protein in E. coli/XL-1blue cells. Recombinant IRF-2 with GST moiety at N-terminus expressed as GST-IRF-2 (~66 kd) in E. coli along with different low molecular mass degradation products revealed approximately 30, 42, 60 and 62 kd by SDS-PAGE and Western blot, respectively. We further confirm that degradation takes place at C-terminus of the fusion protein not at N-terminus as anti-GST antibody was detecting all bands in the immunoblot. The recombinant IRF-2 was biologically active along with their degradation products in terms of their DNA binding activity as assessed by Electrophoretically Mobility Shift Assay (EMSA). We observed three different molecular mass DNA/protein complexes (1 - 3) with Virus Response Element (VRE) derived from human Interferon IFNβ gene and five different molecular mass complexes (1 - 5) with IRF-E motif (GAAAGT)4 in EMSA gel. GST only expressed from empty vector did not bind to these DNA elements. To confirm that the binding is specific, all complexes were competed out completely when challenged with 100-X fold molar excess of IRF-E oligo under cold competition. It means degradation products along with full-length protein are able to interact with VREβ as well as IRF-E motif. This means degradation products may regulate the target gene (s) activation/repression via interacting with VRE/IRF-E.展开更多
基金supported by a grant from the Heart and Stroke Foundation of Canada(HHC,AFRS)a grant from the Natural Science&Engineering Research Council of Canada(HHC,AFRS)a Mid-Career Investigator Award from the Heart and Stroke Foundation of Ontario,Canada(HHC)
文摘Ischemic brain injury triggers an inflammatory response. tissue but can also exacerbate brain injury. Microglia are This response is necessary to clear damaged brain the innate immune cells of the brain that execute this critical function. In healthy brain, microglia perform a housekeeping function, pruning unused syn- apses between neurons. However, microglia become activated to an inflammatory phenotype upon brain injury. Interferon regulatory factors modulate microglial activation and their production of inflammatory cytokines. This review briefly discusses recent findings pertaining to these regulatory mechanisms in the context of stroke recovery.
基金Supported by the Higher Education Commission(HEC),Pakistan Scholarship for Ph.D.Studies to Razzaq SS,No.520-148390-2BS6-011.
文摘BACKGROUND Heart diseases are the primary cause of death all over the world.Following myocardial infarction,billions of cells die,resulting in a huge loss of cardiac function.Stem cell-based therapies have appeared as a new area to support heart regeneration.The transcription factors GATA binding protein 4(GATA-4)and myocyte enhancer factor 2C(MEF2C)are considered prominent factors in the development of the cardiovascular system.AIM To explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells(hUC-MSCs).METHODS hUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry,and by their potential to differentiate into osteocytes and adipocytes.hUC-MSCs were transfected with GATA-4,MEF2C,and their combination to direct the differentiation.Cardiac differentiation was confirmed by semiquant itative real-time polymerase chain reaction and immunocytochemistry.RESULTS hUC-MSCs expressed specific cell surface markers CD105,CD90,CD44,and vimentin but lack the expression of CD45.The transcription factors GATA-4 and MEF2C,and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e.,GATA-4,MEF2C,NK2 homeobox 5(NKX2.5),MHC,and connexin-43,and cardiac proteins GATA-4,NKX2.5,cardiac troponin T,and connexin-43.CONCLUSION Transfection with GATA-4,MEF2C,and their combination effectively induces cardiac differentiation in hUC-MSCs.These genetically modified MSCs could be a promising treatment option for heart diseases in the future.
文摘The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins aremembers of the basic helix-loop-helix-leucine zipper (bHLHZip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP). The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2 is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones, cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.
文摘Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFNβ) gene regulation. Besides that IRF-2 is involved in immunomodulation, hematopoietic differentiation, cell cycle regulation and oncogenesis. We have done molecular sub-cloning and expression of recombinant murine IRF-2 as GST (Glutathione-S-Transferase)- IRF-2 fusion protein in E. coli/XL-1blue cells. Recombinant IRF-2 with GST moiety at N-terminus expressed as GST-IRF-2 (~66 kd) in E. coli along with different low molecular mass degradation products revealed approximately 30, 42, 60 and 62 kd by SDS-PAGE and Western blot, respectively. We further confirm that degradation takes place at C-terminus of the fusion protein not at N-terminus as anti-GST antibody was detecting all bands in the immunoblot. The recombinant IRF-2 was biologically active along with their degradation products in terms of their DNA binding activity as assessed by Electrophoretically Mobility Shift Assay (EMSA). We observed three different molecular mass DNA/protein complexes (1 - 3) with Virus Response Element (VRE) derived from human Interferon IFNβ gene and five different molecular mass complexes (1 - 5) with IRF-E motif (GAAAGT)4 in EMSA gel. GST only expressed from empty vector did not bind to these DNA elements. To confirm that the binding is specific, all complexes were competed out completely when challenged with 100-X fold molar excess of IRF-E oligo under cold competition. It means degradation products along with full-length protein are able to interact with VREβ as well as IRF-E motif. This means degradation products may regulate the target gene (s) activation/repression via interacting with VRE/IRF-E.
