• Development of Novel Inhibitors of HSP90

      Patwardhan, Chaitanya A.; Department of Biochemistry and Molecular Biology (2014-02)
      Pharmacological inhibition of the Hsp90 machinery is an exciting option for cancer therapy. Clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90, and induced overexpression of anti-apoptotic proteins, Hsp70 and Hsp27, is thought to contribute to this undesired outcome. We therefore face an urgent need to develop much better inhibitors of the Hsp90 machinery that can effectively kill cancer cells with minimal side effects. The goal of this dissertation is to identify novel inhibitors of Hsp90 chaperoning machinery to efficiently kill cancer cells with minimal side effects on normal cell survival. First, we report that the natural product, gedunin, may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to the co-chaperone p23 and inactivates it, without inducing over-expression of Hsp27 and only a relatively modest induction of Hsp70. Using molecular docking and mutational analyses; we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits p23 chaperoning activity, blocks its cellular interaction with Hsp90 and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the Cterminus. These results provide important insight into the molecular mechanism of action of this promising lead compound. Second, we report the development of a novel semi-high-throughput drugscreening assay to identify small molecule inhibitors of Hsp90 and its cochaperones. Our assay quantitatively measures the ability of Hsp90 and its cochaperones to refold the progesterone receptor (PR), a physiological client of Hsp90, in an in vitro assay performed in a 96-well plate format. We tested the NIH clinical collection drug library of 446 compounds and identified capsaicin as a “hit”. Our data show that capsaicin targets the Hsp70-Hsp90 chaperone complex in cells and alters Hsp70 multi-chaperone complexes. It induces cellular destabilization of Hsp90-Hsp70 client proteins and causes degradation of the Hsp70 (induced form) but not the Hsc70 (constitutive form) protein through lysosome-autophagy pathway. Cell survival assays showed that capsaicin selectively kills cancer cells by inducing mitophagy. Taken together, our data suggest that capsaicin could be used in combination with Hsp90 inhibitors for cancer treatment.
    • Development of the blood-thymus barrier and the effects of lipid RES blocking agents on the adult barrier

      Davenport, William Daniel; Department of Cell and Molecular Biology (1976-05)
    • The Development of the Human Field Image Metaphor Scale

      Johnston, Linda W.; Department of Physiological and Technological Nursing (1993-05)
      The purpose of this study was the theoretical development and operationalization of the concept, Human Field Image, within the framework of the Rogerian Science of Unitary Human Beings. Initial stages involved a review of the relevant literature, followed by a process of theoretical development and definition. Human Field Image was defined as an individual awareness of the infinite wholeness of the human field. Two areas of content domain, identified as field manifestations of Human Field Image, were specified; (1) one's individual perception of potential, (2) the perception of the integral nature of one's human and environmental fields. The process of definition was followed by a process of item development and refinement for the Human Field Image Metaphor Scale (HFIMS). The metaphor was chosen as the appropriate item form for this instrument. An initial pool of 105 metaphors was reviewed by prominent Rogerian scholars and reduced to 32 items for the pilot form of the instrument. A pilot study with a sample of 50 adults preceeded the major study and led to the deletion of two items. This thirty item form of the HFIMS was administered to a sample of 3 58 adults ranging in age from 17 to 85. Factor Analysis revealed the presence of five factors, with a total scale Cronbach's Alpha of .9211. Additional items were eliminated and the number of factors was decreased. Revision of the instrument resulted in a final form of 25 items with three factors and a Cronbach's Alpha of .9131. The three factors were labeled "expressions of clear images of human field", "expressions of blurred images of human field", and "Integrality." Content validity was established through consultation with Dr. Martha Rogers and other Rogerian scholars. Construct validity was established through the correlation of scores on the HFIMS with scores on the Pictorial Form of the Human Field Motion Tool (r .6647, p < .01). Results of this study indicate that the HFIMS is a valid and reliable instrument which will make a significant contribution to the Science of Unitary Human Beings.
    • Developmental and Behavioral Analyses of clarin-2: A Novel Somatosensory Neuron Subtype-Enriched Gene

      Roberts, Rachel; Department of Neuroscience and Regenerative Medicine (2017-06)
      The trigeminal ganglion (TG) is a somatosensory organ that relays stimuli in the head to the hindbrain and spinal cord, and it comprises multiple subtypes of sensory neurons that respond to different somatosensory stimuli and establish distinct neuronal circuits. The Trpa1b subtype of TG sensory neurons (TGSNs) are responsible for sensing noxious chemicals, but the molecular cues that specify the development of this neuronal subtype remain poorly understood. Zebrafish were previously established as a robust model for studying the development of TGSNs due to its small size, translucency, and robust somatosensory behaviors. A previous microarray study in zebrafish found a novel four transmembrane-domain protein, clarin-2, to be enriched in Trpa1b-expressing cells. Nothing is known about the function of clarin-2, but a close homolog, clarin-1, is one of the causative genes for Usher Syndrome Type 3, a disorder characterized by progressive hearing and vision loss. We hypothesize that clarin-2 may play a role in the development and sensory function of TGSNs. To test this hypothesis, we examined the expression of clarin-2 within the TG during development and used clarin-2 knockout (KO) fish to study the genesis and neurite outgrowth of Trpa1b TGSNs. We found that clarin-2 is indeed enriched in a subset of TGSNs but is not required for the morphogenesis of the TG or the specification of nociceptive sensory neurons. Furthermore, axon projections from Trpa1b neurons were normal in clarin-2 KO fish, compared to control siblings. To test whether clarin-2 is required for the function of TGSNs, we tested somatosensory behaviors in larval zebrafish, including chemo-, thermo-, and mechanosensation. Behavioral analyses showed that clarin-2 is not required for the ability of Trpa1b neurons to detect the chemical irritant mustard oil. Additionally, the detection of heat or vibration was not affected in clarin-2 KO fish. Together, these results suggest that although clarin-2 is enriched in a subset of TGSNs, it is not required for the general morphogenesis of TGSNs or for somatosensation.
    • Diabetes-Induced Cholinergic Supersensitivity of Rat Heart

      Edwards, Ashley; Department of Pharacology & Toxicology (1984-11)
    • Diabetic Membrane Repair Deficiency and Repair Promotion By Vitamin E

      Howard, Amber Cyran; Department of Cellular Biology and Anatomy (5/30/2014)
      Myopathy, characterized by muscle necrosis and atrophy, is a diabetic complication. The myopathy of at least one muscular dystrophy is linked to defective membrane repair. We hypothesized that defective membrane repair is also associated with diabetic myopathy. To test this hypothesis, we monitored repair in intact muscle from diabetic type 1 (INS2Akita+/-) and type 2 (db/db) mouse models. Myocytes were laser injured in the presence of a membrane impermeant dye, and cellular dye uptake through the disruption site was monitored. Dye influx of diabetic myocytes was significantly increased, compared to controls, indicating repair deficiency. This defect was mimicked in cultured cell models by high (30 mM) glucose exposure. Inhibiting the high glucose formation of advanced glycation endproducts (AGE) prevented this repair defect, but was induced in the absence of high glucose exposure by enhanced AGE receptor (RAGE) binding. We conclude that high glucose exposure leads to defective membrane repair in skeletal muscle, and that AGE/RAGE interactions underlie this defect. AGE/RAGE binding also induces generation of reactive oxygen species (ROS), which is increased in diabetes. ROS are also produced in skeletal muscle during eccentric contracts, an act that creates muscle membrane disruptions. Using a potent antioxidant, vitamin E (α-tocopherol), we were able to reverse the high glucose exposure repair defect. Interestingly, diets deficient in vitamin E results in a lethal muscular dystrophy. α-Tocopherol partitions into membrane bilayers where it is thought to act as a membrane stabilizer and/or as an antioxidant. We hypothesize that one important biological role of vitamin E is to promote muscle membrane repair. To test this hypothesis, cultured muscle cells were loaded with α-tocopherol and repair assessed with the laser assay. α-Tocopherol loading significantly decreased cellular dye influx, indicating that repair had been promoted. Strikingly, the HeLa cell, a non-muscle cell that normally displays unrestricted dye influx after laser disruption, e.g. not capable of repair via this form of injury, became repair competent after loading with α-tocopherol. Vitamin C, another antioxidant that can be loaded into cells, also significantly decreased dye influx after laser injury. However, horseradish peroxidase, an antioxidant that lacks transport across the plasma membrane was found to be ineffective in promoting repair. Cells injured in the presence of H2O2, displayed significantly more dye influx than controls injured in physiological saline lacking this oxidant. If however cells were loaded with vitamin E the H2O2 did not affect repair. We further tested H2O2 exposure in intact mouse skeletal muscle, and found repair to be significantly impaired. However, comparable to vitamin E loading in the cell model, Trolox (a water soluble analog of vitamin E) pretreatment prevented the H2O2 muscle membrane repair defect. We conclude that vitamin E promotes plasma membrane repair, and that its capacity as an anti-oxidant is crucial in this role.
    • Diabetic membrane repair deficiency and repair promotion by vitamin E

      Howard, Amber Cyran; School of Graduate Studies (2011-01)
      Myopathy, characterized by muscle necrosis and atrophy, is a diabetic complication. The myopathy of at least one muscular dystrophy is linked to defective membrane repair. We hypothesized that defective membrane repair is also associated with diabetic myopathy. To test this hypothesis, we monitored repair in intact muscle from diabetic type 1 (INS2Akita+t) and type 2 (db/db) mouse models. Myocytes were laser injured in the presence of a membrane impermeant dye, and cellular dye uptake through the disruption site was monitored. Dye influx of diabetic myocytes was significantly increased, compared to controls, indicating repair deficiency. This defect was mimicked in cultured cell models by high (30 mM) glucose exposure. Inhibiting the high glucose formation of advanced glycation endproducts (AGE) prevented this repair defect, but was induced in the absence of high glucose exposure by enhanced AGE receptor (RAGE) binding. We conclude that high glucose exposure leads to defective membrane repair in skeletal muscle, and that AGE/RAGE interactions underlie this defect. AGE/RAGE binding also induces ~eneration of reactive oxygen species (ROS), which is increased in diabetes. RCDS are also produced in skeletal muscle during eccentric contracts, an act that qrlates muscle membrane disruptions. Using a potent antioxidant, vitamin E (a-tlcopherol), we were able to reverse the high glucose exposure repair defect. f erestingly, diets deficient in vitamin E results in a lethal muscular dystrophy. arocopherol partitions into membrane bilayers where it is thought to act as a membrane stabilizer and/or as an antioxidant. We hypothesize that one important biological role of vitamin E is to promote muscle membrane repair. To test this hypothesis, cultured muscle cells were loaded with a-tocopherol and repair assessed with the laser assay. a-Tocopherol loading significantly decreased cellular dye influx, indicating that repair had been promoted. Strikingly, the Hela cell, a non-muscle cell that normally displays unrestricted dye influx after laser disruption, e.g. not capable of repair via this form of injury, became repair competent after loading with a-tocopherol. Vitamin C, another antioxidant that can be loaded into cells, also significantly decreased dye influx after laser injury. Howeve~, horseradish peroxidase, an antioxidant that lacks transport across the plasma membrane was found to be ineffective in promoting repair. Cells injured in the presence of H202, displayed significantly more dye influx than controls injured in physiological saline lacking this oxidant. If however cells were loaded with vitamin E the H202 did not affect repair. We I further tested H202 exposure in in~act mouse skeletal muscle, and found repair to be significantly impaired. However, comparable to vitamin E loading in the cell model, Trolox (a water soluble analog of vitamin E) pretreatment prevented the H202 muscle membrane repair defect. We conclude that vitamin E promotes plasma membrane repair, and that its capacity as an anti-oxidant is crucial in this role.
    • Differential agonist-induced signal transduction cascades and their correlation with MARCKS phosphorylation, StAR phosphorylation, StAR protein synthesis, and aldosterone secretion in cultured bovine adrenal glomerulosa cells

      Betancourt-Calle, Soraya V.; School of Graduate Studies (1998-05)
      Aldosterone is a steroid hormone secreted by the cells of the _zona glomerulosa of the adrenal gland in response to increases in serum potassium (K+) concentrations, angiotensin II (Angll), and adrenocorticotropic hormone (ACTH). Although all of these agonists stimulate Ca2+ entry, which is required but not sufficient for aldosterone secretion, they generate other intracellular signals that are unique to each agent. In the first part of this study we addressed the possible involvement of Protein Kinase C (PKC) in the actions of these agonists, as measured by the phosphorylation of a specific endogenous ~ substrate of PKC: the myristoylated alanine-rich C-kinase substrate (MARCKS). Both Angll and K+ induced an increase in, MARCKS phosphorylation, while ACTH inhibited this response. We conclude that PKC activation is involved in aldosterone secretion stimulated by either Angll or K+, but not by ACTH .. Although these three agonists act via different signaling pathways, it seems likely that at some point, the transducing events should converge. The _ transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane - is the limiting step in steroidogenesis. The steroidogenic· g_cute .regulatory (StAR) protein is thought to be a principal mediator of this transfer, with its acute synthesis and phosphorylation thought to be required for steroid production. Thus, StAR activation should be common to the actions of all three agonists. The second part- of this study determined 1) the effect of these agonists on StAR protein synthesis and protein phosphorylation, and 2) how these events relate to the secretory. response. Stimulation with Angll significantly increased StAR protein synthesis and StAR protein phosphorylation whereas stimulation with K+ significantly increased StAR ' ' protein phosphorylation but did not affect StAR protein synthesis. Finally, ACTH significantly increased . both events but the increase in StAR protein phosphorylation was less than that for Angll or K+. We conclude that these agonists differentially regulate StAR protein synthesis and protein phosphorylation in cultured bovine adrenal glomerulosa cells. In addition, there is no simple correlation between these events and aldosterone production. These results suggest that StAR may not be the only factor regulating intramitochondrial cholesterol transport and steroid synthesis.
    • The Differential Roles of PI3K P110 Isoforms in Regulating CD4 T Cell Subset Polarization

      Webb, Mason James; Department of Biochemistry and Molecular Biology (5/12/2017)
      Class IA phosphatidylinositol-4,5-bisphosphate 3-kinases, or PI3K’s, are one of the earliest bottlenecks for T cell receptor signaling transduction, without which phosphorylated phosphatidylinositides cannot be generated and the T cell activation cascade becomes impaired. Of the catalytic class IA PI3K subunits, there are three isoforms designated as p110α, p110β, and p110δ. The Khleif laboratory has discovered that these catalytic subunits display unique roles in T regulatory cells and non-polarized activated CD4+ T cells. This thesis aims to determine what differential control these p110 isoforms have upon distinct polarized CD4+ T cell subsets.
    • Differntial Agonist-induced Signal Transduction Cascades and their Correlation with MARCKS Phosphorylation, StAR Phosphorylation, StAR Protein Synthesis, and Aldosterone Secretion in Cultured Bovine Adrenal Glomerulosa Cells

      Betancourt-Calle, Soraya V.; Department of Cellular Biology and Anatomy (1998-05)
      Aldosterone is a steroid hormone secreted by the cells of the zona glomerulosa of teh adrenal gland in response to increases in serum potassium (K+) concentrations, angiotensin II (AngII), and adrenocorticotropic hormone (ACTH). Although all of these agonists stimulate Ca2+ entry, which is required but not sufficient for aldosterone secretion, they generate other intracellular signals that are unique to each agent. in the first part of this study we addressed the possible involvement of Protein Kinase C (PKC) in the actions of these agonists, as measured by the phosphorylation of a specific endogenous substrate of PKC: the myristoylated alanine-rich C-kinase substrate (MARCKS).Both AngII and K+ induced an increase in MARCKS phosphorylation, while ACTH inhibited this response. We conclude that PKC activation is involved in aldosterone secretion stimulated by either AngII or K+ but not by ACTH. Although these three agonists act via different signaling pathways, it seems li9kely that at some point, the transducing events should converge. The transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane is the limiting step in steroidogenesis. the steroidogenic acut regulatory (StAR) protein is through to be a principal mediator of this transfer, with its acute synthesis and phosphorylation thought to be required for steroid production. Thus, StAR activation should be common to the actions of all three agonists. The second part of this study determined 1) the effect of these agonists on StAR protein synthesis and protein phosphorylation, and 2) how these events relate to the secretory response. Stimulation with AngII significantly increased StAR protein synthesis and StAR protein phosphorylation whereas stimulation with K+ significantly increased StAR protein phosphorylation but did not affect StAR protein synthesis. Finally, ACTH significantly increased in both events but the increase in StAR protein phosphorylation was less than that for AngII or K+. We conclude that these agonists differently regulate StAR protein synthesis and protein phosphorylation in cultured bovine adrenal glomerulosa cells. In addition, there is no simple correlation between these events and aldosterone production. These results suggest that StAR may not be the only factor regulating intramitochrondial cholesterol transport and steroid synthesis.
    • Dimensional change of demineralized dentin matrix produced by monomer/alcohol primer saturation and solvent evaporation

      Becker, Thomas D.; School of Graduate Studies (2005-05)
      The purpose of this work was to evaluate the use o_f ethanol ~nd methanol as ·, ' solvents for resin monomers and more specifically to evaluate their 'e'ffects on the expansion and modulus of elasticity of infiltrated demineralized dentin matrices, and the _ subsequent matrix shrinkage following solvent evap·oration. Twenty-eight dentin specimens approximately 200 J..tm thick were demineralized with thirty-seven percent phosphoric acid for four hours. The linear variable differential transformer portion of four thermomechanical analyzers were used to measure changes iti demineralized disc thickness upon solvent application, model-primer saturation, and solvent evaporation. Dentin matrix modulus of elasti~ity was measured while saturated with watervs. model primers. One-way analysis of variance and Students-Newman-Kuels post-hoc tests demonstrated a significant (p < 0.05) correlation between the solvent's Hoy's solubility parameter for hydrogen bonding, 8h, and its ability to expand the matrix. Solvents with higher 8h were able to expanded the matrix at a higher rate and extent. Regression analysis and analysis of covariance showed a significant (p < 0.05) correlation between the model primer concentration and matrix collapse upon solvent evaporation. Model primers with higher monomer concentrations demonstrated less matrix collapse upon solvent evaporation than more dilute primers.
    • DISCOVERY AND VALIDATION OF A NOVEL NEUTROPHIL ACTIVATION MARKER ASSOCIATED WITH OBESITY

      Pan, Yue; Biomedical Sciences
      Obesity and its related comorbidities such as cardiovascular disease (CVD) have imposed a huge burden on public health worldwide. Identification of the mechanistic pathways by which obesity impacts cardiovascular health is urgently needed to provide new targets for prevention of obesity and its associated CVDs. Low-grade systemic inflammation accompanies obesity and etiologically contributes to obesity-induced CVD. Neutrophils represent the most abundant type of leukocytes in humans and neutrophil activation is a fundamental process in the inflammatory response. Growing evidence supports that neutrophils are most likely to be the target peripheral leukocyte subtype initiating the adipose tissue inflammatory cascade in response to obesity. As depleting neutrophils is not a choice in humans, identification of obesity induced neutrophil activation markers becomes a prerequisite to develop targeted treatment. Therefore, our central hypothesis is that there are neutrophil activation markers that can specifically respond to obesity status and mediate obesity’s effect on CVD risks. The goal of this study is to identify these markers and their roles on CVD risk using a step-wise approach including an unbiased omic step (i.e. the discovery phase) and a target step (i.e. the validation phase). To achieve this goal, we used the biological samples of 688 subjects from multiple cohorts that generally have neutrophils, white blood cells, and plasma stored. CVD risk factors including blood pressure, insulin resistance, lipid profile, and pulse wave velocity have been measured. In the discovery phase, genome wide DNA methylation, RNA-sequencing and quantitative proteomics were obtained from the purified neutrophils. A significant difference was found for one gene, ALPL, across 3 omics platforms. In the validation phase, ALPL expression and the cellular protein levels were found to be higher in obese compared with lean subjects. Within the obese population, we observed ALPL expression level positively associated with CVD risk factors including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), carotid intima–media thickness (IMT), triglycerides (TG), and fasting insulin. This study identified one novel marker of neutrophil activation in response to obesity and provided evidence that obesity induced changes in ALPL expression were associated with CVD risk factors.
    • Dissecting the Roles of Reactive Oxygen Species in Cardiovascular Disease

      Wang, Yusi; Vascular Biology Center (2015-09)
      Cardiovascular disease remains the leading cause of death in the USA. While much has been learned about the root causes, the underlying mechanisms remain incompletely understood. In particular, elevated levels of reactive oxygen species (ROS) have been observed in the vasculature of blood vessels from animal models and humans with hypertension, atherosclerosis and diabetes. The importance of ROS to cardiovascular disease and the mechanisms by which it alters the function of cells of the cardiovascular system are the goals of this dissertation.
    • The distribution of arylamidase in some selected bacteria

      Lumpkin, Elizabeth Bell; Department of Cell and Molecular Biology (1967-05)
    • DNA METHYLATION AS A KEY PLAYER IN INFLAMMATION-MEDIATED COLON TUMORIGENESIS

      Ibrahim, Mohammed Mahmoud Labib; Biomedical Sciences (Augusta University, 2019-05)
      A causal link between chronic inflammation and tumorigenesis is now well established in the literature with a great deal of supporting evidences from genetic, epigenetic, pharmacological and epidemiological perspectives. In particular, inflammatory bowel diseases represent an important risk factor for colon cancer development. Moreover, it seems that even sporadic colon cancers that do not develop as a complication of chronic colitis are also driven by inflammation. However, the molecular mechanisms behind inflammation-mediated colon tumorigenesis have remained largely unknown. Colitis associated cancer development is thought to be multifaceted due to a combination of genetic and epigenetic aberrations. Recently, epigenetic alterations -particularly aberrant DNA methylation- have gained great attention in cancer biology and have been observed to play a key role in the pathogenesis of inflammation-associated tumors; especially in colitis-associated cancer. IRF8, a key transcription factor originally identified in myeloid cells, has been reported to play a crucial role in myeloid cells differentiation and immune response regulation. IRF8 deficiency is associated with deregulation of myeloid cell differentiation and accumulation of immature myeloid subsets phenotypically and functionally resemble MDSCs (Myeloid Derived Suppressor Cells). IRF8 is thought to function as a tumor suppressor and was found to be silenced in different types of cancers including colon cancer. Myeloid derived-IRF8 has been extensively studied. However, the role of epithelial-derived IRF8 in colon inflammation and colon cancer initiation remains a point to be addressed. In this study, we generated conditional Irf8cKO mice in which IRF8 is specifically deleted in colon epithelium. Irf8cKO mice exhibit a more aggressive pattern of colitis associated cancer with higher tumor incidence and severe loss of body weight. Additionally, we provide evidence that chronic inflammation promotes the accumulation and infiltration of CD11b+Gr1+ MDSCs, which plentifully secrete IL10 in colon tissue. IL10 then induces STAT3 phosphorylation and nuclear translocation to bind to Dnmt1 and Dnmt3b promoters to upregulate their expression, leading to DNA hyper-methylation at the Irf8 promoter to silence IRF8 expression in colonic epithelial cells and promote colon tumorigenesis. Collectively, our data pinpoint the MDSC-IL10-STAT3-DNMT3b-IRF8 axis as a novel bridge between chronic inflammation and colon cancer formation.
    • DNA METHYLATION REGULATION IN ACUTE KIDNEY INJURY

      Guo, Chunyuan; Department of Cellular Biology and Anatomy (4/26/2018)
      DNA methylation is a critical epigenetic mechanism, which is heritable during cell division, but does not involve the change of DNA sequence. It plays an essential role in regulating gene transcription in physiological and disease conditions. However, little is known about DNA methylation in renal diseases, especially in acute kidney injury (AKI). In this study, the role of DNA methylation in AKI was determined in both cell culture and mouse models. In cell culture, 5-aza-2’-deoxycytidine (5-aza), a pharmacological DNA methylation inhibitor, was used to inhibit DNA methylation. Interestingly, 5-aza increased both cisplatin- and hypoxia-induced apoptosis. These results suggest pharmacologic blockade of DNA methylation by 5-aza sensitizes renal tubular cells to apoptosis, supporting a cytoprotective role of DNA methylation in AKI. To determine the role of DNA methylation in vivo, we first successfully established conditional knockout mice that were deficient in DNMT1, DNMT3a or both exclusively in proximal tubules. In cisplatin-induced AKI, consistent with the effects of 5-aza in the cell culture, ablation of DNMT1 from proximal tubules exacerbated cisplatin-induced AKI in mice, and primary proximal tubular cells from PT-DNMT1-KO mice were more sensitive to cisplatin-induced apoptosis than wild-type cells. In sharp contrast, PT-DNMT1/3a-DK mice attenuated cisplatin-induced AKI, and primary proximal tubular cells from PT-DNMT1/3a-DK mice were more resistant to cisplatin-induced apoptosis. However, PT-DNMT3a-KO mice and PT-DNMT3a-WT mice showed similar AKI following cisplatin treatment. These results suggest different DNMTs play different roles in cisplatin-induced AKI. In ischemic AKI, none of the conditional knockout models showed differences in response to ischemia-reperfusion injury. Nevertheless, although ablation of both DNMT1 and DNMT3a in proximal tubular cells did not affect ischemia-reperfusion injury, it, indeed, suppressed renal fibroblast activation and ameliorated renal fibrosis. Furthermore, we found that Irf8 was regulated by DNA methylation during cisplatin treatment and knockdown of Irf8 in RPTC cells inhibited cisplatin-induced apoptosis, supporting a pro-death role of Irf8 in renal tubular cells. In ischemic AKI, although Bcl6 is hypermethylated and repressed in mice, overexpression of Bcl6 in RPTC cells had no impact on hypoxia-induced apoptosis. Collectively, these results suggest an important role of DNA methylation in AKI by regulating specific genes expression.
    • DNA Probes for the Identification of Nocardia Asteroides

      Belcher, Keith E; Department of Cell and Molecular Biology (1988-07)
    • DNA sensing via STING regulates immunity

      Mohamed, Eslam; Department of Neuroscience and Regenerative Medicine (2016)
      The stimulator of interferon genes (STING) is an adaptor protein downstream of an array of cytosolic DNA sensors such as cyclic GMP-AMP synthase (cGAS). STING activation by the second messengers, cyclic dinucleotides (CDNs) induces interferon type I (IFN-I). STING/IFN-I signaling incites autoimmunity in mice lacking the DNA catabolizing enzyme Trex-1. Paradoxically, we find the DNA sensing to activate STING/IFN-I signaling induces dendritic cells (DCs) to express indoleamine 2,3 dioxygenase (IDO), which activates regulatory T cells (Tregs). Thus treatment with DNA nanoparticles (DNPs) or CDNs to activate STING attenuated experimental autoimmune encephalomyelitis (EAE) and therapeutic responses were dependent on STING/IFN-I signaling to induce IDO. DNP and CDNs treatments were also effective in slowing type I diabetes (T1D) progression in susceptible female non-obese diabetic (NOD) mice. Recent reports revealed that DNA sensing to activate STING in DCs that engulfed dying tumor cells impeded growth of immunogenic tumors and potentiated responses to therapy. Consistent with these findings synthetic STING agonists enhanced tumor regression. Paradoxically, lewis lung carcinoma (LLC), grew slower in STING-deficient mice, revealing that STING is required for optimal LLC growth. Mechanistically, STING ablation abolished IDO upregulation in DCs located in tumor draining lymph nodes during LLC growth. Consequently, expression of the regulatory cytokine IL-10 and infiltration of myeloid derived suppressor cells (MDSCs) into the tumor microenvironment (TME) were diminished in mice lacking STING. In contrast, STING was not required for optimal growth of LLC tumors expressing neo-antigens, revealing a pivotal role for tumor antigenicity in influencing responses to DNA in the TME. Thus, DNA from dying cells are sensed in the TME to activate STING, which induces dominant regulatory responses via IDO when tumor antigenicity is low and dominant immunogenic responses when tumor antigenicity is enhanced. Collectively, these findings support the hypothesis that DNP cargo DNA and DNA from dying tumor cells is sensed to activate STING\IFN-I in regulatory DCs that suppresses T cell immunity and autoimmunity at sites of chronic inflammation associated with autoimmunity and tumor growth.