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Articles by Gozde Colak in JoVE
JoVE Clinical and Translational Medicine
माउस में स्थायी मध्य सेरेब्रल धमनी Ligation के आवेदन
1Department of Pharmacology and Physiology, University of Rochester, 2Department of Neurology, University of Alabama at Birmingham, 3Departments of Anesthesiology, Pharmacology and Physiology, University of Rochester
मध्य मस्तिष्क धमनी ligation (एमसीए) के लिए पशु मॉडल में फोकल मस्तिष्क ischemia अध्ययन के लिए एक तकनीक है. इस विधि में, मध्य मस्तिष्क धमनी craniotomy से अवगत कराया है और दाग़ना द्वारा ligated. इस विधि में अत्यधिक प्रतिलिपि प्रस्तुत करने योग्य रोधगलितांश मात्रा और वृद्धि की पोस्ट ऑपरेटिव अस्तित्व अन्य तरीके उपलब्ध की तुलना में दरों देता है.
Other articles by Gozde Colak on PubMed
Cytosolic Guanine Nucledotide Binding Deficient Form of Transglutaminase 2 (R580a) Potentiates Cell Death in Oxygen Glucose Deprivation
Transglutaminase 2 (TG2) is a hypoxia-responsive protein that is a calcium-activated transamidating enzyme, a GTPase and a scaffolding/linker protein. Upon activation TG2 undergoes a large conformational change, which likely affects not only its enzymatic activities but its non-catalytic functions as well. The focus of this study was on the role of transamidating activity, conformation and localization of TG2 in ischemic cell death. Cells expressing a GTP binding deficient form of TG2 (TG2-R580A) with high basal transamidation activity and a more extended conformation showed significantly increased cell death in response to oxygen-glucose deprivation; however, targeting TG2-R580A to the nucleus abrogated its detrimental role in oxygen-glucose deprivation. Treatment of cells expressing wild type TG2, TG2-C277S (a transamidating inactive mutant) and TG2-R580A with Cp4d, a reversible TG2 inhibitor, did not affect cell death in response to oxygen-glucose deprivation. These findings indicate that the pro-cell death effects of TG2 are dependent on its localization to the cytosol and independent of its transamidation activity. Further, the conformational state of TG2 is likely an important determinant in cell survival and the prominent function of TG2 in ischemic cell death is as a scaffold to modulate cellular processes.
Transglutaminase 2: A Molecular Swiss Army Knife
Transglutaminase 2 (TG2) is the most widely distributed member of the transglutaminase family with almost all cell types in the body expressing TG2 to varying extents. In addition to being widely expressed, TG2 is an extremely versatile protein exhibiting transamidating, protein disulphide isomerase and guanine and adenine nucleotide binding and hydrolyzing activities. TG2 can also act as a protein scaffold or linker. This unique protein also undergoes extreme conformational changes and exhibits localization diversity. Being mainly a cytosolic protein; it is also found in the nucleus, associated with the cell membrane (inner and outer side) and with the mitochondria, and also in the extracellular matrix. These different activities, conformations and localization need to be carefully considered while assessing the role of TG2 in physiological and pathological processes. For example, it is becoming evident that the role of TG2 in cell death processes is dependent upon the cell type, stimuli, subcellular localization and conformational state of the protein. In this review we discuss in depth the conformational and functional diversity of TG2 in the context of its role in numerous cellular processes. In particular, we have highlighted how differential localization, conformation and activities of TG2 may distinctly mediate cell death processes.
Complete Transglutaminase 2 Ablation Results in Reduced Stroke Volumes and Astrocytes That Exhibit Increased Survival in Response to Ischemia
Transglutaminase 2 (TG2) is a very multifunctional protein that is ubiquitously expressed in the body. It is a Ca(2+)-dependent transamidating enzyme, a GTPase, as well as a scaffolding protein. TG2 is the predominant form of transglutaminase expressed in the mammalian nervous system. Previously, it was shown that TG2 can affect both cell death and cell survival mechanisms depending on the cell type and the stressor. In the case of ischemic stress, TG2 was previously shown to play a protective role in the models used. For example in hTG2 transgenic mice, where TG2 is overexpressed only in neurons, middle cerebral artery ligation (MCAL) resulted in smaller infarct volumes compared to wild type mice. In this study TG2 knock out mice were used to determine how endogenous TG2 affected stroke volumes. Intriguingly, infarct volumes in TG2 knock out mice were significantly smaller compared to wild type mice. As expected, primary neurons isolated from TG2 knock out mice showed decreased viability in response to oxygen-glucose deprivation. However, primary astrocytes that were isolated from TG2 knock out mice were resistant to oxygen-glucose deprivation in situ. Both wild type and knock out neurons were protected against oxygen glucose deprivation when they were co-cultured with astrocytes from TG2 knockout mice. Therefore, the decreased stroke volumes observed in TG2 knock out mice after MCAL, can be correlated with the protective effects of TG2 knock out in astrocytes in response to oxygen glucose deprivation in situ. These findings suggest that neuron-astrocyte crosstalk plays a significant role in mediating ischemic cell death and that TG2 differentially impacts cell survival depending on cell context.
Vena Cava and Aortic Smooth Muscle Cells Express Transglutaminases 1 and 4 in Addition to Transglutaminase 2
Transglutaminase (TG) function facilitates several vascular processes and diseases. Although many of these TG-dependent vascular processes have been ascribed to the function of TG2, TG2 knockout mice have a mild vascular phenotype. We hypothesized that TGs besides TG2 exist and function in the vasculature. Biotin-pentylamide incorporation, a measure of general TG activity, was similar in wild-type and TG2 knockout mouse aortae, and the general TG inhibitor cystamine reduced biotin-pentylamine incorporation to a greater extent than the TG2-specific inhibitor Z-DON, indicating the presence of other functional TGs. Additionally, 5-hydroxytryptamine-induced aortic contraction, a TG-activity dependent process, was decreased to a greater extent by general TG inhibitors versus Z-DON (maximum contraction: cystamine = abolished, monodansylcadaverine = 28.6 ± 14.9%, Z-DON = 60.2 ± 15.2% vehicle), providing evidence for the importance of TG2-independent activity in the vasculature. TG1, TG2, TG4 and Factor XIII (FXIII) mRNA in rat aortae and vena cavae was detected by RT-PCR. Western analysis detected TG1 and TG4, but not FXIII, in rat aortae and vena cavae, and in TG2 knockout and wild-type mouse aortae. Immunostaining confirmed the presence of TG1, TG2 and TG4 in rat aortae and vena cavae, notably in smooth muscle cells; FXIII was absent. K5 and T26, FITC-labeled peptide substrates specific for active TG1 and TG2, respectively, were incorporated into rat aortae and vena cavae, and wild-type, but not TG2 knockout, mouse aortae. These studies demonstrate that TG2-independent TG activity exists in the vasculature, and that TG1 and TG4 are expressed in vascular tissues.
