• THE ROLE OF KYNURENINE, A TRYPTOPHAN METABOLITE THAT INCREASES WITH AGE, IN MUSCLE ATROPHY AND LIPID PEROXIDATION)

      Kaiser, Helen E.; Department of Cellular Biology and Anatomy (Augusta University, 2020-05)
      Loss of mobility and independence are risk factors for falls and mortality, and drastically reduce the quality of life among older adults. The cellular and molecular mechanisms underlying loss of muscle mass and strength with age (sarcopenia) are not well-understood; however, heterochronic parabiosis experiments show that circulating factors are likely to play a role. Kynurenine (KYN) is a circulating tryptophan metabolite that is known to increase with age and is implicated in several age-related pathologies. Here I tested the hypothesis that KYN contributes directly to muscle loss with aging. Results indicate that that KYN treatment of mouse and human myoblasts increased levels of reactive oxygen species (ROS) two-fold, and significantly increased lipid peroxidation enzymes. Small-molecule inhibition of the Aryl hydrocarbon receptor (Ahr), an endogenous KYN receptor, in vitro did not prevent KYN-induced increases in ROS, and homozygous Ahr knockout in vivo did not protect mice from KYN-induced stress, suggesting that KYN can directly increase ROS independent of Ahr activation. In vivo, wild-type mice treated with KYN had reduced skeletal muscle strength, size, and increased oxidative stress and lipid peroxidation. Old wild-type mice treated with 1MT, a small molecule that suppresses KYN production by IDO1, showed an increase in muscle fiber size, peak muscle strength, and oxidative stress. Protein analysis identified mitochondrial lipid peroxidation as a downstream mechanism that is increased upon KYN treatment. Lipid peroxidation enzymes increased with KYN have been shown to produce H2O2 outside of the electron transport chain. Our data suggest that IDO inhibition may represent a novel therapeutic approach for the attenuation of sarcopenia and possibly other age-associated conditions associated with KYN accumulation such as bone loss and neurodegeneration.
    • THE ROLE OF NEDDYLATION IN EARLY CARDIAC DEVELOPMENT

      Littlejohn, Rodney; Department of Cellular Biology and Anatomy (Augusta University, 2020-07)
      Background. Early cardiac development is a tightly regulated process, involving spatiotemporal coordination of multiple signaling pathways and heterogenous cell populations, both generated de novo and externally sourced. While the roles of transcription, environmental, and epigenetic factors have all been studied extensively in the context of heart development, the roles of post-translational protein modification in regulating this process remain to be elucidated. NEDD8 (neural precursor cell expressed developmentally downregulated 8) is a novel ubiquitin-like protein modifier. Conjugation of NEDD8 to protein targets, a process termed neddylation, has been shown to regulate cell proliferation, cell signaling, and protein homeostasis, and play important roles in multiple physiological and pathological events. We have previously shown that neddylation is developmentally downregulated in the developing heart and is essential for mid-to-late gestational ventricular chamber maturation. However, whether and how neddylation regulates early cardiogenic events remains unknown. Methods and results. Mice with constitutive, cardiac progenitor cell-specific, cardiomyocyte- and vascular smooth muscle cell-specific deletion of NAE1, a regulatory subunit of the NEDD8-specific E1 activating enzyme, were created. Constitutive deletion of NAE1 led to early embryonic lethality before E9.5. Nkx2.5Cre-mediated deletion of NAE1 decreased neddylated proteins in the heart, disrupted normal cardiogenesis and resulted in embryonic lethality by embryonic day (E) 12.5 due to heart failure. Similarly, SM22αCre-driven deletion of NAE1 also caused cardiac failure and embryonic lethality by E13.5. The striking cardiac phenotypes were associated with myocardial hypoplasia, ventricular hypo-trabeculation, and pronounced endocardial and/or epicardial defects in both models. Unbiased transcriptomic analysis revealed dysregulated expression of genes associated with cardiomyocyte differentiation, proliferation, and maturation in NAE1-deficient hearts. Indeed, inhibition of neddylation disturbed cardiomyocyte proliferation, and myofibril assembly in vitro and in vivo. Moreover, defects in cardiomyocyte differentiation and maturation were linked to downregulation of Nkx2.5 and Mef2c, two key transcription factors regulating early cardiogenesis. Conclusion. Collectively, our findings demonstrate that neddylation in cardiac progenitor cells and cardiomyocytes is essential in the regulation of cardiogenesis in transgenic mouse models. Our results uncover a previously unknown role of post-translational modification in the regulation of cardiac development via potential roles in mediating cardiomyocyte proliferation, differentiation, and maturation.
    • THE ROLE OF THE DUFFY ANTIGEN RECEPTOR FOR CHEMOKINES (DARC) IN ETHNIC SUSCEPTIBILITY TO CARDIOMETABOLIC DISEASE

      Benson, Tyler W; Biomedical Sciences
      Cardiometabolic disease is a cluster of pathologies relating to obesity and cardiovascular diseases with many shared risk factors and are hallmarked by chronic inflammation mediated by the aberrant production of cytokines and chemokines. Cardiometabolic disease risk is highly stratified by ethnicity. African Americans have a higher prevalence of metabolic syndrome, vascular stiffness and hypertension compared to Caucasians. Concomitantly, African Americans typically have elevated levels of circulating IL-6 and c reactive protein, both of which have been implicated in the progression of cardiometabolic disease. Conversely, the incidence of abdominal aortic aneurysms (AAA) and coronary artery calcification, pathologies that are also mechanistically linked to inflammation, are markedly reduced in African Americans compared to Caucasians. These disparate findings in cardiometabolic disease rates among ethnic populations suggest that the relationship between obesity, cardiovascular disease, and inflammation is complex and is likely not explained solely on the basis of conventional risk factors. Interestingly, mutations in the Duffy antigen receptor for chemokines (DARC) are highly prevalent in African American, Asian, and Hispanic populations as a result of negative selection pressure imposed by P. vivax malaria. Indeed, up to 80% of African Americans harbor a mutation in the DARC resulting in complete loss of expression on erythrocytes. DARC is prominently expressed on the surface of erythrocytes and is a promiscuous non-signaling chemokine receptor whose function has been described as a chemokine ‘buffer-sink’. Loss of functional DARC is associated with dysregulation of its ligands, including MCP-1. In the present study, we investigate the role of DARC in inflammatory driven cardiometabolic diseases employing a mouse that is genetically deficient in DARC. We found that mice deficient in DARC are more prone to the development of metabolic syndrome in the setting of diet induced obesity, reduced incidence of AAA, but increased hypertension. Our findings in mice deficient in DARC mirror disparities in cardiometabolic disease observed in human populations expressing allelic variants of DARC suggesting a role for DARC in ethnic disparities of cardiometabolic disease.
    • The therapeutic alliance, awareness, and medication compliance in the treatment of schizophrenia

      Chadwick, Nanenia Brannen; School of Graduate Studies (2001-04)
      Schizophrenia is one of the most debilitating and costly of the major illnesses in the UJ?ited States. Individuals diagnosed with schizophrenia are at-risk for impaired awareness, poor therapeutic alliance, and, failure to comply with medication regimens. Although awareness has been related to medication compliance, and the therapeutic alliance has been suggested as an explanation of medication compliance when individuals are not aware of their illness· symptoms, treatment response or social consequences of · their mental illness, findings of previous studies have been unclear concerning the influence of the therapeutic alliance on the relation between awareness and medication compliance. This cross-sectional correlational study was designed to test a hypothesized model in which the therapeutic alliance inflμences the relation between awareness and medication compliance in the treatment of a sample of individuals diagnosed with schizophrenia. The.settings for the study were two day treatment programs. Adults diagnosed with o!le of the types of schizophrenia and their primary therapists were invited to participate. Seventy-six patients and 12 primary therapists were offered the opportunity to participate, with 70 patients and 10 therapists who completed the·study. It was hypothesized that the therapeutic alliance would influence the relation . between awareness and medication compliance. Data were analyzed using hierarchical multiple regression anaiyses. The hypothesis was partially supported. The therapeutic alliance and awareness had significant main effects on medication compliance, but their interaction was not significant. In this sample, both the therapeutic alliance and awareness hc;!d additive effects on medication compliance, but their interaction did not significantly add to the prediction of medication compliance. The study suggests that primary therapists should spend time on both developing the therapeutic alliance and on improving patients' awareness of illness symptoms, treatment response, and social consequences of their mental illness in order to have the greatest effect on medication compliance for individuals diagnosed with schizophrenia.
    • Therapeutic induction of fetal hemoglobin in sickle cell disease: development of a novel prodrug AN-233

      Oseghale, Aluya; Biomedical Sciences (Augusta University, 2019-05)
      The reactivation of fetal hemoglobin (HbF) in sickle cell disease (SCD) ameliorates the clinical severity of the illness and improves patient's survival. Pharmacological induction of HbF has been a major strategy for SCD treatment and several research studies have focused on a wide variety of agents for their potential to induce HbF. However, hydroxyurea (HU) remains the only Food and Drug Administration (FDA)-approved drug proven to elevate HbF in about 50% of adults with SCD. Efficacy of HU has been very limited due to many side effects including bone marrow suppression, susceptibility to infections and long-term infertility. Our group reported potent HbF induction by sodium butyrate in erythroid cells through p38 MAPK activation. Nevertheless, oral administration of a butyrate (BA) derivative to SCD patients was ineffective due to rapid metabolic inactivation by the liver. Therefore, the need for better therapies exists. This project investigated a novel prodrug conjugate of BA and δ-aminolevulinate (ALA) denoted as AN-233. As an ester, AN-233 [1-(butyryloxy) ethyl-5-amino-4-oxopentanoate] undergoes cellular hydrolysis in an esterase dependent manner to yield two active drugs BA and ALA. In prior studies, oral administration of AN-233 to mice increased total hemoglobin but the effect on HbF was unknown. We proposed the hypothesis; that AN-233 upregulates γ-globin gene expression and elevates HbF synthesis via transcriptional and post-transcriptional mechanisms. We investigated AN-233 using in-vitro, in-vivo and ex-vivo model systems including K562 cell lines, sickle progenitors and β-YAC mice. Treatment of K562 cells showed AN-233 significantly increased mRNA levels. Flow cytometry analyses show the prodrug significantly increased HbF protein expression and Western blotting of whole cell lysates confirmed increased synthesis of HbF. Treatment of CD34+ stem cell-derived primary erythroid cells increased early stage (basophilic) erythroblasts by 2.4fold and decreased late stage (orthochromatophilic) erythroblasts by 2.5 fold. In sickle progenitors, AN-233 again elevated F-cell% by 1.5fold and HbF protein by over 2.6fold. Mechanistic studies in K562 cells show AN-233 significantly elevated heme biosynthesis, decreased phosphorylation of HRI and eIF2α thereby promoting the protein synthesis of globin chains. Additionally, AN-233 enhanced histone acetylation at the γ-globin promoter and LCR DNase hypersensitive site 2 (LCR HS2). Treatment of sickle progenitors with AN-233 decreased %sickled cells by up to 50%. The transcription factor BACH1 was reduced while NRF2 was increased in AN-233 treated K562 cells. In vivo, AN-233 increased F-cell% and F-cell MFI of treated β-YAC mice within 4-weeks. Our data support AN-233 as a potent HbF inducer in erythroid progenitors and in mice. The prodrug represents a drug candidate, which can be developed for the treatment of SCD patients.
    • Therapeutic Targeting of P2X7 After Traumatic Brain Injury

      Kimbler, Donald E.; Department of Neurosurgery (2012-02)
      Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Cerebral edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP) and is a common life-threatening neurological complication following TBI. Unfortunately, neurosurgical approaches to alleviate increased ICP remain controversial and medical therapies are lacking due in part, to the absence of viable drug targets. In the present study, genetic inhibition (P2X7-/- mice) of the purinergic P2X7 receptor attenuated the expression of the pro-inflammatory cytokine, interleukin-iP (IL-ip) and reduced cerebral edema following controlled cortical impact, as compared to wild-type mice. Similarly, the clinically useful P2X7 inhibitor, brilliant blue G (BBG), inhibited the expression of IL-ip, limited edemic development and prevented the development of post-traumatic depression and anxiety. The beneficial effects of BBG were observed following either prophylactic administration via the drinking water for one week prior to injury or via an intravenous bolus administration up to four hours after TBI, suggesting a clinically-implementable therapeutic window. Notably, P2X7 localized within astrocytic end feet and administration of BBG decreased the expression of glial fibrillary acidic protein (GFAP), a reactive astrocyte marker, and reduced the expression of aquaporin-4 (AQP4), an astrocytic water channel that promotes cellular edema. Together, these data implicate P2X7 as a novel therapeutic target to prevent secondary neurological injury after TBI, a finding that warrants further investigation.
    • Therapeutic Targeting of P2X7 after Traumatic Brain Injury

      Kimbler Jr, Donald E; Department of Neuroscience and Regenerative Medicine (Georgia Health Sciences University, 2012-02)
      Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Cerebral edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP) and is a common life-threatening neurological complication following TBI. Unfortunately, neurosurgical approaches to alleviate increased ICP remain controversial and medical therapies are lacking due in part, to the absence of viable drug targets. In the present study, genetic inhibition (P2X7-/- mice) of the purinergic P2x7 receptor attenuated the expression of the pro-inflammatory cytokine, interleukin-I~ (IL-1~) and reduced cerebral edema following controlled cortical impact, as compared to wild-type mice. Similarly, the clinically useful P2X7 inhibitor, brilliant blue G (BBG), inhibited the expression of IL-1~, limited edemic development and prevented the development of post-traumatic depression and anxiety. The beneficial effects of BBG were observed following either prophylactic administration via the drinking water for one week prior to injury or via an intravenous bolus administration up to four hours after TBI, suggesting a clinically-implementable therapeutic window. Notably, P2X7 localized within astrocytic end feet and administration of BBG decreased the expression of glial fibrillary acidic protein (GFAP), a reactive astrocyte marker, and reduced the expression of aquaporin-4 (AQP4), an astrocytic water channel that promotes cellular edema. Together, these data implicate P2X7 as a novel therapeutic target to prevent secondary neurological injury after TBI, a finding that warrants further investigation.
    • This retrospective study attempted to identity differences between student groups occurring before and after the restructuring of the Army occupational therapy assistant (OTA) program. Records of 168 students were reviewed (104 records of students in the pre-restructuring group, 64 in the post-restructuring group). Several areas were examined: (a) demographic data, (b) course attrition rates, (c) performance on level II fieldwork, and (d) national certification exam pass rates. Analyses of demographic data revealed that students in the post-restructuring group were significantly older (p<.OOl) and of higher rank (p<.001) than students in the pre-restructuring group. Analysis using a chi-square test revealed a significant decrease in the attrition rate (p<.001) after the restructuring. At-test revealed that students in the post-restructuring group performed significantly better on their level II fieldwork (p=.030). National certification exam pass rates for the groups could not be determined due to the confidential nature in which scores are reported

      Harrison-Weaver, Sandra; Medical College of Georgia (Augusta University, 1997-03)
      This retrospective study attempted to identity differences between student groups occurring before and after the restructuring of the Army occupational therapy assistant (OTA) program. Records of 168 students were reviewed (104 records of students in the pre-restructuring group, 64 in the post-restructuring group). Several areas were examined: (a) demographic data, (b) course attrition rates, (c) performance on level II fieldwork, and (d) national certification exam pass rates. Analyses of demographic data revealed that students in the post-restructuring group were significantly older (p<.OOl) and of higher rank (p<.001) than students in the pre-restructuring group. Analysis using a chi-square test revealed a significant decrease in the attrition rate (p<.001) after the restructuring. At-test revealed that students in the post-restructuring group performed significantly better on their level II fieldwork (p=.030). National certification exam pass rates for the groups could not be determined due to the confidential nature in which scores are reported
    • Tissue culture study of clinical specimens from an outbreak of rubella-like illness

      Reddick, Rhoda Anne; Department of Cell and Molecular Biology (1968-06)
    • Tissue culture study of clinical specimens from an outbreak of rubella-like illness

      Reddick, Rhoda Anne; Department of Cell and Molecular Biology (1965-06)
    • Tissue metabolism of thyroid hormones in hypothyroid and growth hormone treated hypothyroid mothers, their fetuses and their progenies

      Berdecia-Rodriguez, Joseph; Department of Physiology and Endocrinology (1990-05)
      Studies of Man et al. have shown that the children of hypothyroxinemic women have an .increased rate of mental and .motor retardation. A rat model which presents the ~etabolic and motor defects of these children developed by Hendrich et al. was used for these studies. The metabolism of T4 was studied i~ the progenies of Tx-only and GH-treated Tx dams utilizing a radioimmunoassay for T4 detection and equations based on those of Inglebleek .~d Malvaux. In the above mentioned study, the hypothyroid dams showed an impaired reproductive performance that included lower body weight gain, smaller liter size and greater mortality for their pups. Furthermore, maternal hypothyroidism had a detrimental effect on their offsprings body and tissue weights. The parameters of metabolism for T4 showed that the offsprings of hypothyroid mothers have a clear derangement in the utilization of this hormone at the ages studied (i.e., 5, 30, 60 days of age). The next step in our investigation wa~ to measure the iodothyronine concentrations intracellularly to determine how these J molecules were utilized. We developed a new technique for the extraction of iodothyronines based on HPLC fluorescence. The extracted iodothyronines were derivatized with dansyl chloride and then rap in our chromatographic system. The iodothyronine concentrations ·in the tissues {i.e., brain and liver) and plasma for the progenies of hypothyroid mothers at all ages {i.e,. 22 day, 10, 30, 60 day-olds} were consistently abnormal confirming what we saw with the metabolic studies of T4 done initially. The hypothyroid mothers had iodothyronine concentrations consistently abnormal in all tissues studies. The progenies of hypothyroid mothers appear to suffer an insult that causes a severe impairment in the metabolism of T4. This insult not only affects the metabolism of T4 but also affects the concentrations' of other iodothyronines in liver, brain and plasma
    • Toll-like receptor 2 contributes to cerebrovascular dysfunction and cognitive impairment in diabetes

      Hardigan, Trevor; Department of Physiology (2016-03)
      The risk of cognitive decline in diabetes (Type 1 and Type 2) is significantly greater compared to normoglycemic patients, and the risk of developing dementia in diabetic patients is doubled. The etiology for this is likely multifactorial, but one mechanism that has gained increasing attention is decreased cerebral blood flow (CBF) as a result of cerebrovascular dysfunction. The innate immune system has been shown to play a role in diabetic vascular complications, notably through Toll-like receptor (TLR) stimulated release of proinflammatory cytokines and chemokines that leads to vascular damage. TLR2 has been implicated in the development of diabetic microvascular complications such as nephropathy, and thus we hypothesized that TLR2-mediated cerebrovascular dysfunction leads to decreased CBF and cognitive impairment in diabetes. Vascular TLR2 expression was increased and local TLR2 antagonism improved cerebrovascular function in diabetes. While the anti-hyperglycemic dipeptidylpeptidase-IV (DPP-IV) inhibitor linagliptin prevented TLR2 expression in brain microvascular endothelial cells (BMVEC) when applied locally, chronic in vivo treatment did not decrease vascular smooth muscle TLR2 expression. Treatment with linagliptin restored CBF in diabetes independent of effects on blood glucose levels, and this increase in CBF was correlated with decreased endothelin-1 (ET-1)-mediated vasoconstriction, decreased pathological remodeling, and increased endothelium-dependent relaxation. Knockout of TLR2 conferred protection from impaired CBF in early-stage diabetes and from hyperperfusion in long-term diabetes, prevented the development of endothelium dependent vascular dysfunction in diabetes, created a hyperactive and anxiolytic phenotype, and protected against diabetes induced impairment of long term hippocampal- and prefrontal cortex- mediated fear learning. In conclusion, these findings support the involvement of TLR2 in the pathogenesis of diabetic vascular disease and cognitive impairment.
    • Toll-like receptor 9 contributes to vascular dysfunction in hypertension

      McCarthy, Cameron; Department of Physiology (2016-03)
      Inappropriate immune system activation is common in hypertension; however, the exact mechanisms by which this occurs are not well understood. Innate immune system recognition and response to damage-associated molecular patterns (DAMPs) is becoming an increasingly accepted mechanism. Mitochondrial DNA (mtDNA) is a DAMP that is recognized by Toll-like receptor (TLR)9, and it is elevated in the circulation of spontaneously hypertensive rats (SHR). Therefore, we hypothesized that (1) inhibition of TLR9 in SHR with a TLR9 antagonist (ODN2088) or TLR9 inhibitor (chloroquine) would lower blood pressure and improve vascular function and that (2) treatment of normotensive rats with a TLR9 agonist (ODN2395) would cause vascular dysfunction and increase blood pressure. Both ODN2088 and chloroquine lowered high blood pressure in SHR and treatment with chloroquine also improved cyclooxygenase-dependent endothelial function and prevented the full recruitment of the adaptive immune system in SHR. On the other hand, treatment of normotensive rats with ODN2395 increased blood pressure and rendered their arteries less sensitive to acetylcholine-induced relaxation and more sensitive to norepinephrine-induced contraction. This dysfunctional vasoreactivity was due to cyclooxygenase activation, increased reactive oxygen species generation, and reduced nitric oxide bioavailability. In conclusion, these findings support the involvement of the innate immune system pattern recognition receptor TLR9 in the pathogenesis and maintenance of hypertension. Specifically, circulating mtDNA may activate TLR9 and contribute to high blood pressure and endothelial dysfunction in SHR.
    • Toothbrush abrasion of a novel porcelain esthetic characterization technique

      Chi, Woo Jun Amos; Medical College of Georgia (Augusta University, 2008-07)
    • Toxicity of visible light-cured denture resins

      Barron, Dara Jewell; Department of Oral Biology (1992-04)
      In this study three commercial formulations of visible light-cured (VLC) denture resins have been analyzed. The products used are those suggested for the reline, repair and fabrication of dentures to improve their fit. The biocompatibility of these resins was investigated by measuring RNA and DNA synthesis of oral epithelial cells in vitro. The extent to which oral cells recover from toxic resin exposure, the conversion of monomer into polymer, the presence of inorganic filler, and resin leacha~ility have also been studied. It was shown that VLC denture resins inhibit the synthesis of RNA and· DNA relative to a heat-cured resin control (p~0.05). Although epithelial cells appeared to recover from toxic resin exposure, this recovery was inconsistent among experiments. Infrared spectroscopy illustrated chemical group differences that occurred before and after photo-polymerization. Using these differences, the conversion of monomer into polymer ranged from 77% to 97%. This conversion was significantly affected (p< .003) by the type of curing unit, duration of photo-polymerization, and surface exposed to visible light. Soluble substances in cured and uncured resin products were analogous using HPLC. The range of inorganic filler present was 0-15%. These investigations suggest that visible light-cured denture resins may impair the replication of oral epithelial cells. This effect may be related to the leachability of unpolymerized resin constituents, the presence or absence of filler particles, or polymerization by-products.
    • Transcriptional Coactivator and Oncoprotein CoAA

      Brooks, Yang Sui; Department of Pathology (2008)
      CoAA contains two copies of RNA recognition motifs (RRM) and an intrinsic transactivation domain rich in repetitive tyrosines and glutamines (YxxQ domain). Previously, CoAA has been shown to be a transcriptional coactivator that stimulates transcriptional activation and regulates alternative splicing. A pattern and profile search revealed that the YxxQ domain in CoAA shared significant pattern homology with the oncogenic EWS activation domains (EAD) in TET family proteins, including, TLS/FUS, EWS and TAFII 68. It was further demonstrated that CoAA’s YxxQ domain and EWS’ EAD also shared functional similarities. Based on these findings, this work investigated the aberration of CoAA in cancers and its pathophysiological significance. The results showed that the CoAA gene was amplified in a high percentage of inflammation-related human cancers with recurrent loss of the 5’ regulatory element upstream of its promoter. This genomic aberration resulted in CoAA protein overexpression, which in turn, induced the transformation of NIH3T3 cells. Subsequently, it was shown that the lost 5’ regulatory element could modulate the alternative splicing of the CoAA gene during stem cell differentiation and that the unbalanced expression of CoAA and its splice variant, CoAM could potentially impact the cell differentiation process. To further characterize the regulation of CoAA alternative splicing, two conserved trans-splicing events between CoAA and its downstream RBM4 were identified. These events yield a novel zinc finger- containing coactivator, CoAZ, and a non-coding splice variant, ncCoAZ. Both variants regulated their parental genes’ mRNA expression as well as activities, suggesting a linked control between CoAA and RBM4. Moreover, the expression patterns of CoAA, RBM4 and their trans-splicing variants switched during neural stem cell differentiation, resulting in lineage-specific expression of each variant. Our phylogenetic analysis suggests that mammalian CoAA and RBM4 share a common ancestor with the Drosophila melanogaster gene, Lark. In this regard, the trans-splicing events between CoAA and RBM4 represent a functional regulation preserved during evolution. This study established the connection between CoAA and human cancer and provides evidence for CoAA’s involvement in the regulation of cell differentiation. Moreover, this study is the first to report a functional trans-splicing variant in mammalian cells.
    • Transcriptional coactivator and oncoprotein CoAA

      Brooks, Yang Sui; Medical College of Georgia (Augusta University, 2008-08)
      CoAA contains two coptes of RNA recognition motifs (RRM) at).d an intrinsic transactivation domain rich in repetitive tyrosines and glutamines (YxxQ domain). Previously, CoAA has been shown to be a transcriptional coactivator that stimulates transcriptional activation and regulates alternative splicing. A pattern and profile search revealed that the Y xxQ domain in CoAA shared significant pattern homology with the oncogenic EWS activation domains (BAD) in TBT family proteins, including, TLS/FUS, 1!: EWS and TAFII 68. It was further demonstr~ted that CoAA's YxxQ domain and BWS' BAD also shared functional similarities. Based on these findings, this work investigated the aberration of CoAA in cancers and its pathophysiological significance. The results showed that the CoAA gene was amplified in a high percentage of inflammation-related human cancers with recurrent loss of the 5' regulatory element upstream of its promoter. This genomic aberration resulted in CoAA protein overexpression, which in tum, induced the transformation of NIH3T3 cells. Subsequently, it was shown that the lost 5' regulatory element could modulate the alternative splicing of the CoAA gene during stem cell differentiation and that the unbalanced expression of CoAA and its splice variant, CoAM could potentially impact the cell differentiation process. To further characterize the regulation of CoAA alternative splicing, two conserved trans-splicing events between CoAA and its downstream RBM4 were identified. These events yield a novel zinc finger containing coactivator, CoAZ, and a non-coding splice variant, ncCoAZ. Both variants regulated their parental genes' mRNA expression as well as activities, suggesting a linked control between CoAA and RBM4. Moreover, the expression patterns of CoAA, RBM4 and their trans-splicing variants switched during neural stem cell differentiation, resulting in lineage-specific expression of each variant. Our phylogenetic analysis suggests that mammalian CoAA and RBM4 share a common ancestor with the Drosophila melanogaster gene, Lark. In this regard, the trans-splicing events between CoAA and RBM4 represent a functional regulation preserved during evolution. This study established the connection between CoAA and human cancer and provides evidence for CoAA's involvement in the regulation of cell differentiation. Moreover, this study is the first to report a functional trans-splicing variant in mammalian cells.