36 research outputs found
Targeting the mitochondrial inner membrane to improve bioenergetics in the diseased heart
Cardiovascular diseases continue to exact unparalleled economic and humanitarian costs across the globe. Manifestations of cardiovascular diseases include acute coronary syndromes and heart failure, both of which are exacerbated in diabetic patients. Although the underlying cellular culprits responsible for these cardiomyopathies are multi-factorial, aberrant cellular bioenergetics is emerging as a central component. Decrements in mitochondrial function impair cardiac function, and accordingly the development of novel therapies that improve cardiac function by targeting mitochondria has enormous therapeutic potential. In the work presented herein, we studied two diseases where impaired bioenergetics comprises a central component: diabetes, and ischemia/reperfusion injury. In diabetic heart studies, we determined the mechanisms responsible for the decline in mitochondrial bioenergetics of the diabetic heart. Comprehensive mitochondrial functional assays coupled with molecular techniques were employed. Our results showed that mitochondrial respiration and reactive oxygen species buffering capacity were significantly decreased in diabetic hearts. Diabetic mitochondria displayed aberrant mitochondrial calcium handling, post-translational oxidative modification of the adenine nucleotide translocase, increased sensitivity to permeability transition pore opening, and lowered overall expression of proteins involved in the electron transport system. These effects led to inefficient energy supply-demand matching and heightened reperfusion injury in intact heart studies. Treatment with several novel therapies that target the mitochondrial inner membrane reduced the extent of injury and restored mitochondrial function in the diabetic heart. In ischemia/reperfusion studies, we tested the hypothesis that aberrant respiration in the post-ischemic heart was due to impaired molecular organization along the inner mitochondrial membrane. Specifically, we used a novel respiratory substrate-inhibitor titration protocol to determine complex-specific changes, in the electron transport system, that lead to poor respiration. These respiratory studies were coupled with experiments using native gel electrophoresis, allowing us to link changes in respiration to altered expression of native respiratory "supercomplex" clusters. The decrease in mitochondrial respiration after ischemia/reperfusion was observed along several different sites of the electron transport system. These changes were associated with lower supercomplex expression, and altered levels of several native respiratory complexes. Post-ischemic treatment with a mitochondria-targeting peptide restored supercomplex assembly and was associated with improved respiration and a decreased extent of injury. Taken together, the results presented herein provide new insight into the molecular and functional alterations that occur along the mitochondrial inner membrane in diabetic and post-ischemic hearts. These data provide a basis for novel therapies targeting the inner mitochondrial membrane as viable pharmacological approaches to improving bioenergetics in diseased myocardium.Ă‚Â Ă‚Â Ph.D
The Unfolded Protein Response in Amelogenesis and Enamel Pathologies
During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other “professional” secretory cells, ameloblasts employ the unfolded protein response (UPR) to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum)/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI) and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed
Targeting the mitochondrial inner membrane to improve bioenergetics in the diseased heart
Cardiovascular diseases continue to exact unparalleled economic and humanitarian costs across the globe. Manifestations of cardiovascular diseases include acute coronary syndromes and heart failure, both of which are exacerbated in diabetic patients. Although the underlying cellular culprits responsible for these cardiomyopathies are multi-factorial, aberrant cellular bioenergetics is emerging as a central component. Decrements in mitochondrial function impair cardiac function, and accordingly the development of novel therapies that improve cardiac function by targeting mitochondria has enormous therapeutic potential. In the work presented herein, we studied two diseases where impaired bioenergetics comprises a central component: diabetes, and ischemia/reperfusion injury. In diabetic heart studies, we determined the mechanisms responsible for the decline in mitochondrial bioenergetics of the diabetic heart. Comprehensive mitochondrial functional assays coupled with molecular techniques were employed. Our results showed that mitochondrial respiration and reactive oxygen species buffering capacity were significantly decreased in diabetic hearts. Diabetic mitochondria displayed aberrant mitochondrial calcium handling, post-translational oxidative modification of the adenine nucleotide translocase, increased sensitivity to permeability transition pore opening, and lowered overall expression of proteins involved in the electron transport system. These effects led to inefficient energy supply-demand matching and heightened reperfusion injury in intact heart studies. Treatment with several novel therapies that target the mitochondrial inner membrane reduced the extent of injury and restored mitochondrial function in the diabetic heart. In ischemia/reperfusion studies, we tested the hypothesis that aberrant respiration in the post-ischemic heart was due to impaired molecular organization along the inner mitochondrial membrane. Specifically, we used a novel respiratory substrate-inhibitor titration protocol to determine complex-specific changes, in the electron transport system, that lead to poor respiration. These respiratory studies were coupled with experiments using native gel electrophoresis, allowing us to link changes in respiration to altered expression of native respiratory "supercomplex" clusters. The decrease in mitochondrial respiration after ischemia/reperfusion was observed along several different sites of the electron transport system. These changes were associated with lower supercomplex expression, and altered levels of several native respiratory complexes. Post-ischemic treatment with a mitochondria-targeting peptide restored supercomplex assembly and was associated with improved respiration and a decreased extent of injury. Taken together, the results presented herein provide new insight into the molecular and functional alterations that occur along the mitochondrial inner membrane in diabetic and post-ischemic hearts. These data provide a basis for novel therapies targeting the inner mitochondrial membrane as viable pharmacological approaches to improving bioenergetics in diseased myocardium
Cross-Subtype T-Cell Immune Responses Induced by a Human Immunodeficiency Virus Type 1 Group M Consensus Env Immunogen†0--
The genetic diversity among globally circulating human immunodeficiency virus type 1 (HIV-1) strains is a serious challenge for HIV-1 vaccine design. We have generated a synthetic groupMconsensus env gene (CON6) for induction of cross-subtype immune responses and report here a comparative study of T-cell responses to this and natural strain env immunogens in a murine model. Three different strains of mice were immunized with CON6 as well as subtype A, B, or C env immunogens, using a DNA prime-recombinant vaccinia virus boost strategy. T-cell epitopes were mapped by gamma interferon enzyme-linked immunospot analysis using five overlapping Env peptide sets from heterologous subtype A, B, and C viruses. The CON6-derived vaccine was immunogenic and induced a greater number of T-cell epitope responses than any single wild-type subtype A, B, and C env immunogen and similar T-cell responses to a polyvalent vaccine. The responses were comparable to within-clade responses but significantly more than between-clade responses. The magnitude of the T-cell responses induced by CON6 (measured by individual epitope peptides) was also greater than the magnitude of responses induced by individual wild-type env immunogens. Though the limited major histocompatibility complex repertoire in inbred mice does not necessarily predict responses in nonhuman primates and humans, these results suggest that synthetic centralized env immunogens represent a promising approach for HIV-1 vaccine design that merits further characterization
Cross-Subtype T-Cell Immune Responses Induced by a Human Immunodeficiency Virus Type 1 Group M Consensus Env Immunogen
The genetic diversity among globally circulating human immunodeficiency virus type 1 (HIV-1) strains is a serious challenge for HIV-1 vaccine design. We have generated a synthetic group M consensus env gene (CON6) for induction of cross-subtype immune responses and report here a comparative study of T-cell responses to this and natural strain env immunogens in a murine model. Three different strains of mice were immunized with CON6 as well as subtype A, B, or C env immunogens, using a DNA prime-recombinant vaccinia virus boost strategy. T-cell epitopes were mapped by gamma interferon enzyme-linked immunospot analysis using five overlapping Env peptide sets from heterologous subtype A, B, and C viruses. The CON6-derived vaccine was immunogenic and induced a greater number of T-cell epitope responses than any single wild-type subtype A, B, and C env immunogen and similar T-cell responses to a polyvalent vaccine. The responses were comparable to within-clade responses but significantly more than between-clade responses. The magnitude of the T-cell responses induced by CON6 (measured by individual epitope peptides) was also greater than the magnitude of responses induced by individual wild-type env immunogens. Though the limited major histocompatibility complex repertoire in inbred mice does not necessarily predict responses in nonhuman primates and humans, these results suggest that synthetic centralized env immunogens represent a promising approach for HIV-1 vaccine design that merits further characterization
Dysregulation of glucose homeostasis in nicotinamide nucleotide transhydrogenase knockout mice is independent of uncoupling protein 2
Glucose intolerance in C57Bl/6 mice has been associated with mutations in the nicotinamide nucleotide transhydrogenase (Nnt) gene. It has been proposed that the absence of NNT from mitochondria leads to increased mitochondrial reactive oxygen species production and subsequent activation of uncoupling protein 2 (UCP2). Activation of UCP2 has been suggested to uncouple electron transport from ATP synthesis in pancreatic beta cell mitochondria thereby decreasing glucose tolerance due to decreased insulin secretion through lower ATP/ADP ratios. The hypothesis tested in this paper is that UCP2 function is required for the dysregulation of glucose homeostasis observed in NNT ablated mice. Single and double Nnt and Ucp2 knockout mouse lines were used to measure glucose tolerance, whole animal energy balance and biochemical characteristics of mitochondrial uncoupling. As expected, glucose tolerance was diminished in mice lacking NNT. This was independent of UCP2 as it was observed either in the presence or absence of UCP2. The range of metabolic parameters examined in the mice and the proton conductance of isolated mitochondria remained unaltered in this double NNT and UCP2 knockout model. Ablation of UCP2 did not itself affect glucose tolerance and therefore previous observations of increased glucose tolerance of mice lacking UCP2 were not confirmed. We conclude that the decreased glucose tolerance in Nnt knockout mice observed in our experiments does not require UCP2