Inhibiting hexamer disassembly of human UDP-glucose dehydrogenase by photoactivated amino acid crosslinking

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD+-dependent oxidation steps. Human UGDH apoprotein purifies as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate crosslinking of monomeric subunits into predominantly obligate oligomeric species. Optimal crosslinking was achieved by encoding p-benzoyl-L-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing to long wavelength UV in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% tetramer and higher molecular mass species. Activity of the crosslinked enzyme was reduced by almost 60% relative to the uncrosslinked UGDH mutant, and UV exposure had no effect on activity of the wildtype enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer

Inhibiting hexamer disassembly of human UDP-glucose dehydrogenase by photoactivated amino acid crosslinking

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD+-dependent oxidation steps. Human UGDH apoprotein purifies as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate crosslinking of monomeric subunits into predominantly obligate oligomeric species. Optimal crosslinking was achieved by encoding p-benzoyl-L-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing to long wavelength UV in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% tetramer and higher molecular mass species. Activity of the crosslinked enzyme was reduced by almost 60% relative to the uncrosslinked UGDH mutant, and UV exposure had no effect on activity of the wildtype enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer

Absence of p55 TNF Receptor Reduces Atherosclerosis, but Has No Major Effect on Angiotensin II Induced Aneurysms in LDL Receptor Deficient Mice

The aim of the current study was to investigate the role of p55 TNF Receptor (p55 TNFR), the main signaling receptor for the pro-inflammatory cytokine tumor necrosis factor (TNF), in the development of two vascular disorders: atherosclerosis and angiotensin (Ang) II-induced abdominal aortic aneurysms (AAA). p55 TNFR deficient mice were crossed to an LDL receptor deficient background and were induced for the development of either atherosclerosis or AngII-induced AAA, and compared to littermate controls, wild-type for p55 TNFR expression. p55 TNFR deficient mice developed 43% smaller atherosclerotic lesions in the aortic sinuses compared to controls. Moreover, expression of CD68, a macrophage specific marker, exhibited a 50% reduction in the aortic arches. Decreased atherosclerosis correlated with a strong down-regulation in the expression of adhesion molecules, such as VCAM-1 and ICAM-1, by p55 TNFR deficient endothelium. In addition, expression levels of the pro-inflammatory cytokines and chemokines TNF, IL-6, MCP-1 and RANTES were significantly reduced in aortas of p55 TNFR deficient mice. In contrast, in the AngII-induced model of AAA, p55 TNFR deficiency correlated with a slight trend towards increased aneurismal lethality, but the incidence of aortic rupture due to a dissecting aneurysm, and the expansion of the suprarenal aorta were not significantly different compared to controls. We found that p55 TNFR expression promotes atherosclerosis, among other mechanisms, by enhancing expression of endothelial adhesion molecules, while it seems to have no major role in the development of AngII-induced AA

Eomes is sufficient to regulate IL-10 expression and cytotoxic effector molecules in murine CD4+ T cells

The T-box transcription factors T-bet and Eomesodermin regulate type 1 immune responses in innate and adaptive lymphocytes. T-bet is widely expressed in the immune system but was initially identified as the lineage-specifying transcription factor of Th1 CD4+ T cells, where it governs expression of the signature cytokine IFN- γ and represses alternative cell fates like Th2 and Th17. T-bet’s paralog Eomes is less abundantly expressed and Eomes+ CD4+ T cells are mostly found in the context of persistent antigen exposure, like bone marrow transplantation, chronic infection or inflammation as well as malignant disorders. However, it has remained unresolved whether Eomes executes similar transcriptional activities as T-bet in CD4+ T cells. Here we use a novel genetic approach to show that Eomes expression in CD4+ T cells drives a distinct transcriptional program that shows only partial overlap with T-bet. We found that Eomes is sufficient to induce the expression of the immunoregulatory cytokine IL-10 and, together with T-bet, promotes a cytotoxic effector profile, including Prf1, Gzmb, Gzmk, Nkg7 and Ccl5, while repressing alternative cell fates. Our results demonstrate that Eomes+ CD4+ T cells, which are often found in the context of chronic antigen stimulation, are likely to be a unique CD4+ T cell subset that limits inflammation and immunopathology as well as eliminates antigen-presenting and malignant cells

Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis

Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompanied by regional atrophy and pronounced neuron loss in the brain, is caused by mutations in the CLN6 gene. CLN6 is a non-glycosylated endoplasmic reticulum (ER)-resident membrane protein of unknown function. To investigate mechanisms contributing to neurodegeneration in CLN6 disease we examined the nclf mouse, a naturally occurring model of the human CLN6 disease. Prominent autofluorescent and electron-dense lysosomal storage material was found in cerebellar Purkinje cells, thalamus, hippocampus, olfactory bulb and in cortical layer II to V. Another prominent early feature of nclf pathogenesis was the localized astrocytosis that was evident in many brain regions and the more widespread microgliosis. Expression analysis of mutant Cln6 found in nclf mice demonstrated synthesis of a truncated protein with a reduced half-life. Whereas the rapid degradation of the mutant Cln6 protein can be inhibited by proteasomal inhibitors, there was no evidence for ER stress or activation of the unfolded protein response in various brain areas during postnatal development. Age-dependent increases in LC3-II, ubiquitinated proteins, and neuronal p62-positive aggregates were observed, indicating a disruption of the autophagy-lysosome degradation pathway of proteins in brains of nclf mice, most likely due to defective fusion between autophagosomes and lysosomes. These data suggest that proteasomal degradation of mutant Cln6 is sufficient to prevent the accumulation of misfolded Cln6 protein, whereas lysosomal dysfunction impairs constitutive autophagy promoting neurodegeneration

Accommodation of Experiment Conditions for the Space Experiment „Cellular Responses to Radiation in Space“

In the current work, preparatory experiments for the Space Experiment “Cellular Responses to Radiation in Space” that will be accomplished in the Biolab of the laboratory module Columbus on the International Space Station (ISS) were performed. This experiment will analyse the effects of combined radiation and microgravity effects on the behaviour of human cells, especially in respect to DNA damage and repair and activation of the NF-κB pathway. It was necessary to test the biological activity of the designated radiation source Promethium-147 regarding cell survival and NF-κB activation, in both points an agreement with the postulated qualities was reached. The experimental hardware consisting of microtiter plate units and fluid reservoirs has been tested and several aspects that have to be improved have been identified. Furthermore the evaluation of a suitable cytotoxicity test system as well as the testing of a possible method for analyzing the cells after return to earth was conducted. It turned out that there are more experiments necessary to find a suitable test system. After fixation of the cells for termination of the experiment, several procedures are earmarked. The green fluorescence of the reporter cell line HEK-pNF-κBd2EGFP/ Neo L2 will be detected by fluorescence microscopy or flow cytometry. The fixed cell layers will be subjected to immunfluorescence staining. After optimization of the conditions, the method of γH2AX immunofluorescence can be used for analysis of DNA double-strand breaks and their repair

La Ermita de San Antonio : comunidad, viviendas sociales y río buscando una relación

Tesis (Arquitecto, Licenciado en Arquitectura)A través de una investigación de tipo exploratoria, correlacionar; y mediante la utilización de instrumentos de recabación como la encuesta, la observación en terreno, la entrevista y la revisión critica de la información disponible, se pretenden llegar a determinar las relaciones que se producen o podrían producirse, entre un conjunto de viviendas sociales, la comunidad que lo habita y el entorno en el que se emplaza. A través del estudio exhaustivo del conjunto de viviendas sociales "La Ermita de San Antonio", (ubicado en la ribera sur del río Mapocho, entre los puentes La Dehesa y San Enrique, comuna de Lo Barnechea, Santiago) se pretende llegar a comprobar, que las espacialidades exteriores, son aquellas potencialmente capaces de lograr la interrelación de los tres niveles en estudio, insertando los volúmenes llenos en la trama urbana y prometiendo la satisfacción residencial de los habitantes del conjunto. El estudio se centra en dicho conjunto, debido a los diversos conflictos que se han generado en los últimos siete meses, entorno a la problemática de trasladar una comunidad establecida y con una manera específica de habitar el territorio, hacia un conjunto de viviendas sociales, que dispone una nueva manera de habitar y de hacer uso de los espacios. Además surge la problemática ocasionada por la ubicación del conjunto en la ribera del Mapocho, generando una serie de espacialidades públicas que relacionan las viviendas con el río, sin responder a las exigencias de un cauce tan variante como el del Mapocho, ni a las necesidades publicas de una comunidad ya conocida. Esta investigación pretende analizar el comportamiento de los habitantes, en cuanto al uso de los espacios exteriores, los cuales se definen como todas aquellas espacialidades delimitadas esencialmente por las fachadas de los asentamientos. Teniendo como objetivo principal: Determinar las necesidades y el carácter de las espacialidades exteriores para los habitantes de un conjunto de viviendas sociales, estudiando su óptima incorporación entre viviendas y ciudad. Para lograr este objetivo se ha dividido la investigación, en dos grandes etapas: • La primera, denominada etapa práctica, se refiere a la revisión crítica de la información disponible respecto a los tres principales sujetos u objetos de investigación (la comunidad, las viviendas sociales y el lugar). • La segunda, denominada etapa teórica, se refiere al estudio acabado de la interacción entre estos sujetos u objetos de investigación, en un plano de conclusiones. Pretendiendo establecer los tipos de relaciones que debieran producirse entre ellos. Finalmente se rematará la investigación en una tercera etapa de aplicación teórica, formulando durante el segundo semestre académico, un proyecto arquitectónico, urbano, que se convierta en un real aporte tanto para la comuna como para los pobladores; en la proyección de los espacios exteriores de las etapas futuras en el proyecto "La Ermita de San Antonio"

UDP-glucose Dehydrogenase Activity and Optimal Downstream Cellular Function Require Dynamic Reorganization at the Dimer-Dimer Subunit Interfaces

Background: UDP-glucose dehydrogenase (UGDH) mutants were engineered to perturb hexamer:dimer quaternary structure equilibrium. Results: Dimeric species of UGDH have reduced activity in vitro and in supporting hyaluronan production by cultured cells. Conclusion: Only dynamic UGDH hexamers support robust cellular function. Significance: Manipulation of UGDH activity by hexamer stabilization may offer new therapeutic options in cancer and other pathologies. SUMMARY UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium, and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study, we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132), to investigate the role of quaternary structure in regulation of the enzyme. Characterization of the purified enzymes revealed a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. Kinetic analysis of wild-type UGDH and the inducible hexamer, T325A, showed that upon increasing enzyme concentration, which favors the hexameric species, activity was modestly decreased and exhibited cooperativity. In contrast, cooperative kinetic behavior was not observed in the obligate dimer, T325D. These observations suggest that the regulation of the quaternary assembly of the enzyme is essential for optimal activity and allosteric regulation. Comparison of kinetic and thermal stability parameters among the hexameric wild-type enzyme and the engineered mutants revealed structurallydependent properties consistent with a role for controlled assembly and disassembly of the hexamer in the regulation of UGDH. Finally, both T325A and T325D mutants were significantly less efficient in promoting downstream hyaluronan production by HEK293 cells. These data support a model that requires an operational dimer-hexamer equilibrium in order to function efficiently and preserve regulated activity in the cell