• The c-MYC oncogene deregulates global DNA methylation and hydroxymethylation to control genome-wide gene expression for tumor maintenance in leukemia/lymphoma

      Poole, Candace Jean; Biomedical Sciences (Augusta University, 2019-05)
      Aberrant DNA methylation is a characteristic feature of tumor cells. However, our knowledge of how DNA methylation patterns are established and maintained to contribute to tumorigenesis is limited. Inactivation of the c-MYC oncogene triggers tumor regression in T-cell acute lymphoblastic leukemia (T-ALL) resulting in dramatic changes to the chromatin landscape including DNA methylation. In this study, I investigated how MYC regulates DNA methylation and hydroxymethylation patterns to contribute to gene expression programs important for tumor maintenance in T-ALL and Burkitt lymphoma. I report that MYC maintains 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) patterns by regulating the DNA methylation machinery, which is important for gene expression in T-ALL. DNA methyltransferases (DNMTs) initiate 5mC marks, while Ten-eleven translocation methylcytosine dioxygenases (TETs) oxidize 5mC to produce 5hmC as an intermediate modification, ultimately leading to active DNA de-methylation. I demonstrated that DNMT1 and DNMT3B are MYC target genes and that their expression is dependent on high MYC levels. Knockdown of DNMT3B in T-ALL reduced cell proliferation through cell cycle arrest and caused the reactivation of gene transcription through reversing promoter/CpG island methylation. Furthermore, I demonstrated that TET1 and TET2 expression is MYC-dependent, as high TET1 and low TET2 levels depend on oncogenic MYC. Knockdown of TET1 in T-ALL reduced cell proliferation through cell cycle arrest and caused genome-wide changes in 5mC and 5hmC corresponding to changes in gene programs important for ribosomal biosynthesis and protein synthesis. In contrast, ectopic expression of TET2 reduced tumor cell proliferation through apoptosis/necrosis and caused genome-wide changes in 5mC and 5hmC corresponding to changes in transcriptional regulatory gene programs. My finding that a coordinated interplay between components of the DNA methylation machinery is necessary for MYC-driven tumor maintenance highlights the potential of targeting specific DNMT or TET proteins for therapeutic strategies.
    • Calpain-2 Activates Akt via the TGF~ 1-mTORC2 Pathway in Pulmonary Artery Smooth Muscle Cells

      Abeyrathna, Prasanna; Deparment of Pharmacology and Toxicology (8/23/2016)
      Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to the AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibition of calpain-2 using the calpain inhibitor MDL28170 and calpain-2 siRNA attenuated Akt phosphorylation at serine-473 (S473) and threonine-308 (T308) as well as collagen synthesis and cell proliferation ofPASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylation at S4 73 and T308. Moreover, knockout of calpain attenuated Akt phosphorylation at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable specific TGF~ receptor inhibitor SB431542 attenuated Akt phosphorylation at both S473 and T308 induced by PDGF and overexpressed calpain- 2 in PASMCs. Moreover, SB-431452 and knock down of ALK5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF~l using a cell-impermeable TGF~l neutralizing antibody did not affect PDGF-induced Akt phosphorylation at S473 and T308. Further, inhibition of mTORC2 by knocking down its component protein Rictor prevented Akt phosphorylation at S473 and T308 induced by PDGF and overexpressed calpain-2. These data provide the first evidence supporting that calpain-2 up-regulates PDGF-induced Akt phosphorylation via an intracrine TGF~ 1/mTORC2 mechanism.
    • CaMKIIβ association with F-actin in developing cortical neurons

      Lin, Yu-Chih; Department of Pharmacology and Toxicology (2008-08)
      Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is best known for its role in synaptic plasticity and memory .. Although multiple roles of CaMKII have been identified in the hippocampus, its role in the developing cerebral cortex is less well understood. Immunostaining showed Ca~KII~, but not CaMKIIa was expressed in embryonic day 18 (E 18) cortical neurons at 4 days in vitro (DIV) and localized to a F-actin rich cytoskeletal structure we termed "micro spike". Further characterization of micro spikes revealed that micro spikes were composed of bundled actin, and were stable over time. Besides CaMKII~, several actin binding proteins, such as Arp3, cortactiti"and ~1-integrin were also colocalized in microspikes. Fluorescence recovery after photo bleaching (FRAP) analyses showed different dynamics of actin and CaMKII~ in microspikes compared to dendrite spines. The colocalization of CaMKII~ and F-actin in microspikes was dependent on the F-actin binding domain and the oligomerization domain. FRAP analyses confirmed the association of CaMKIIP with F-actin in microspikes was via the F-actin binding domain. This association was altered by the co-expression of CaMKIIa. FRAP analyses with stabilized F-actin using jasplakinolide or cytochalasin-D further indicated CaMKIIP, but not CaMKIIa, had a strong interaction with stable F-actin. Inhibiting calmodulin binding on CaMKII using a CaMKII inhibitor, KN93, dissociated CaMKIIP from stable F-actin. Increasing CaMKIIP activity with KCl or an active form of CaMKIIP, CaMKIIPT287D, also dissociated CaMKIIP from stable F-actin. A calmodulin binding mutant, CaMKIIPA303R, or a kinase dead mutant, CaMKIIPK43R, however, did not recover differently from wildtype CaMKIIp. The differential binding of CaMKIIP with F-actin shown in FRAP analyses correlated with CaMKIIP enrichment in microspikes and the prominence of microspikes. While overexpressed CaMKIIP increased the number of cells with microspikes, knockdown of CaMKIIP with shRNA reduced it. Taken together, these data suggested that CaMKIIP is associated with F-actin in cortical neurons, and this association is regulated by CaMKIIa and calcium signals · contributing to the stability of micro spikes.
    • Can novel Apo A-I polymorphisms be responsible for low HDL in South Asian immigrants?

      Dodani, Sunita; Dong, Yanbin; Zhu, Haidong; George, Varghese; Department of Medicine (2010-03-19)
      Coronary artery disease (CAD) is the leading cause of death in the world. Even though its rates have decreased worldwide over the past 30 years, event rates are still high in South Asians. South Asians are known to have low high-density lipoprotein (HDL) levels. The objective of this study was to identify Apolipoprotein A-I (Apo A-I) polymorphisms, the main protein component of HDL and explore its association with low HDL levels in South Asians. A pilot study on 30 South Asians was conducted and 12-h fasting samples for C-reactive protein, total cholesterol, HDL, low-density lipoprotein (LDL), triglycerides, Lipoprotein (a), Insulin, glucose levels, DNA extraction, and sequencing of Apo A-I gene were done. DNA sequencing revealed six novel Apo A-I single nucleotide polymorphisms (SNPs) in South Asians, one of which (rs 35293760, C938T) was significantly associated with low (<40 mg/dl) HDL levels (P = 0.004). The association was also seen with total cholesterol (P = 0.026) and LDL levels (P = 0.032). This pilot work has highlighted some of the gene-environment associations that could be responsible for low HDL and may be excess CAD in South Asians. Further larger studies are required to explore and uncover these associations that could be responsible for excess CAD risk in South Asians.
    • Cancer Stressors and Protective Factors: Predictors of Stress Experienced During Treatment for Childhood Cancer

      Hockenberry-Eaton, Marilyn; Department of Physiological and Technological Nursing (1992-05)
      The purpose of this study was to evaluate cancer stressors and protective factors as predictors of stress experienced during treatment for childhood cancer. The conceptual framework evolved from the stress and coping literature and childhood cancer research. A convenience sample of 44 children between 6 ½ and 13 ½ years of age receiving treatment for cancer were evaluated during two clinic visits. Protective factors included the child’s self-perception, coping strategies, perceived social support, and family environment. Cancer stressors include acute stressors represented by the type of treatment received during two clinical visits. Chronic stressors were evaluated by the child’s perception of stressors related to the cancer experience. Responses to stressors were assessed by physiologic and psychologic indicators of stress. Physiologic measures include epinephrine, norepinephrine, and cortisol measures of urine and psychologic measures of state and trait anxiety. No significant differences were found in the physiologic or psychologic response to stressors in relation to the type of treatment received during either clinic visit. Epinephrine and norepinephrine were elevated for children during both clinic visits. Stepwise multiple regression analyses revealed that family expressiveness and the child’s perceived global self-worth were the best predictors of epinephrine levels. Family activities and recreation and family intellectual cultural orientation were the best predictors of state anxiety. The intensity of chronic cancer stressors, family activities and recreation, family intellectual cultural orientation, the child’s perception of physical appearance, and presence of family conflict had the greatest effect of trait anxiety. This study is the first to examine the child’s perception of chronic cancer stressors and protective factors associated with treatment for cancer. The findings provide insight into the importance of the interactions among the nature of the stressor, perceptual meaning of the stressor, and physiologic and psychologic responses to stressors that may affect long-term adjustment to childhood cancer.
    • Cannabidiol protects retinal neurons by preserving glutamine synthetase activity in diabetes.

      El-Remessy, Azza B.; Khalifa, Yousef; Ola, S; Ibrahim, Ahmed S.; Liou, Gregory I.; Department of Ophthalmology; Vision Discovery Institute (2010-08-31)
      PURPOSE: We have previously shown that non-psychotropic cannabidiol (CBD) protects retinal neurons in diabetic rats by inhibiting reactive oxygen species and blocking tyrosine nitration. Tyrosine nitration may inhibit glutamine synthetase (GS), causing glutamate accumulation and leading to further neuronal cell death. We propose to test the hypothesis that diabetes-induced glutamate accumulation in the retina is associated with tyrosine nitration of GS and that CBD treatment inhibits this process. METHODS: Sprague Dawley rats were made diabetic by streptozotocin injection and received either vehicle or CBD (10 mg/kg/2 days). After eight weeks, retinal cell death, M?�ller cell activation, GS tyrosine nitration, and GS activity were determined. RESULTS: Diabetes causes significant increases in retinal oxidative and nitrative stress compared with controls. These effects were associated with M?�ller cell activation and dysfunction as well as with impaired GS activity and tyrosine nitration of GS. Cannabidiol treatment reversed these effects. Retinal neuronal death was indicated by numerous terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)-labeled cells in diabetic rats compared with untreated controls or CBD-treated rats. CONCLUSIONS: These results suggest that diabetes-induced tyrosine nitration impairs GS activity and that CBD preserves GS activity and retinal neurons by blocking tyrosine nitration.
    • Canonical Wnt Signaling in Antigen Presenting Cells Regulates Microbiota-Induced Inflammation and Immune Cell Homeostasis in the Colon

      Swafford, Daniel Joseph; Department of Biochemistry and Molecular Biology / Cancer Center (8/3/2018)
      Aberrant Wnt/β-catenin-signaling occurs in several inflammatory diseases including inflammatory bowel disease (IBD) and IBD-associated colon carcinogenesis. However, its role in shaping mucosal immune responses to commensals in the gut remains unknown. Here, we investigated the importance of canonical Wnt signaling in CD11c+ antigen presenting cells (APCs) in controlling intestinal inflammation. Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt-signaling in intestinal CD11c+ antigen presenting cells (APCs) controls intestinal inflammation by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt co-receptors, low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6) in CD11c+ APCs in mice (LRP5/6ΔCD11c mice) resulted in enhanced intestinal inflammation with increased histopathological severity of colonic tissue. This was due to microbiota-dependent increased production of pro-inflammatory cytokines and decreased expression of immune regulatory factors such as IL-10, retinoic acid (RA), and IDO. In addition, loss of LRP5/6-mediated signaling in CD11c+ APCs resulted in altered microflora and T cell homeostasis, which led to a loss of systemic tolerance to oral antigen. Furthermore, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice resulted in reduced acute intestinal inflammation with decreased histopathological severity of colonic tissue. Loss of canonical Wnt signaling in CD11c+ APCs also results in an increase in colonic polyp formation and exacerbation of chronic inflammation/injury. This was also heavily dependent on the presence and composition of the gut microbiota, as fecal transfers from LRP5/6ΔCD11c mice to floxed control (LRP5/6FL/FL) mice that were administered an antibiotic cocktail produces a polyp load and weight loss similar to that of LRP5/6ΔCD11c mice without treatment. Additionally, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice reduces severity of inflammation-associated colon carcinogenesis in these mice. Furthermore, we show that treatment of LRP5/6ΔCD11c mice with either RA or IL-10 reduces severity of inflammation-associated colon carcinogenesis. Mechanistically, RA and IL-10 may independently reduce key inflammatory factors at the acute phase of colitis. These results ultimately reveal a mechanism by which intestinal APCs control intestinal inflammation and immune homeostasis via the canonical Wnt signaling pathway, which may serve as a promising target for chronic inflammatory disorders.
    • Carcinomatous Meningitis: The Natural History of Successfully Treated Metastatic Bladder Cancer

      Tadepalli, S.; Coleman, Teresa; Hackett, Ladawn A.; Liles, G.B.; Department of Medicine; Department of Pathology (2011-08-24)
      Carcinomatous meningitis due to bladder cancer is a rare entity reported only in case reports. Optimal therapy is thus poorly defined with earlier cases reporting an unsuccessful outcome. Here we report a case of late carcinomatous meningitis secondary to transitional cell carcinoma (TCC) of the bladder occurring in a patient in complete remission. He was successfully treated with intrathecal methotrexate and whole brain irradiation and experienced prolonged survival after treatment. With modern chemotherapy increasing complete remissions and survival rates in patients with TCC, more and more patients are being reported with carcinomatous meningitis. We raise the question of whether central nervous system prophylaxis should be considered in patients with TCC achieving a complete remission to chemotherapy in the metastatic setting.
    • Case Selection Criteria for use with Resin-Infiltrative Treatment of Enamel Decalcification

      Raley, N; Clayton, Ashley; Fortson, WM; Deleon, E; Rueggeberg, FA; Department of Orthodontics, Department of Restorative Sciences (Augusta University, 2019)
      Although one of the primary aims for many orthodontic patients is to achieve improvement in their dental esthetic condition, a high percentage of these patients develop unesthetic, white spot lesions (WSL) during the course of treatment. These lesions develop due to enamel decalcification resulting from bacterial plaque accumulation around difficult to clean brackets and overlying wires and ligation devices. Acids produced locally in this retained plaque will decalcify enamel along the peripheral border of the bonded bracket. Quite often, despite repeated admonishment by the clinician to the patient to take extra care in cleansing these susceptible locations, patients return with large plaque deposits around the brackets, and evidence of the early stages of enamel decalcification: the so-called “white spot lesion” (WSL). The problem becomes obvious at the time of bracket removal, when, although the teeth may now be arranged in near-to-perfect alignment and occlusion, large, white areas of enamel decalcification are prominently displayed, denoting the exact location of where the bonded bracket used to be.
    • Case Study: Legalize It All

      Wilder, Corneshia S.; Department of Sociology, Criminal Justice & Social Work (2017-03)
      Currently, there are over 20 states in the United States that made using marijuana legal for medical purposes. With nearly half of the states legalized marijuana, some believe that stronger illegal drugs, such as cocaine and meth, should be legalized. Dan Baum, a writer, wrote an article in Harper’s Magazine that it is time to “legalize it all”. Other do not believe that harder drugs should be legalized. Mark Kleiman, a public policy expert and professor, predicts that alcohol and cocaine addiction will rise if harder drugs become legal. Is it ethically permissible to legalize harder drugs in the United States? In my presentation, I am going to explain the case in detail. It will include information about the stakeholder and how each stakeholder will be affected. I will also explain my viewpoint of the case by using theories and statistics to explain my position in the case.
    • Cash Flow Pattern Analysis of Fraud and Non-Fraud Firms: A Comparison and Contrast

      Runger, Shannon; Knox School of Accountancy (Augusta University, 2016-05)
      Companies may exhibit one of eight possible cash flow patterns on their Statement of Cash Flows. By pair-matching 30 firms that were known to have issued fraudulent financial statements with 30 non-fraud firms of similar size and industry, a comparison and contrast of the cash flow patterns can be made and the results analyzed. In my research, I examine and analyze the cash flow patterns of fraud and non-fraud firms as reported on the Statement of Cash Flows to determine whether or not the patterns provide some indication of fraudulent activity. I hypothesize that the fraud firms would be more likely to show a cash flow pattern during the year prior to fraud that indicated the firm was struggling and that alternatively, the pattern during the fraud year would be one that indicates a firm is stable and profitable. My findings not only do not support this hypothesis, they also indicate that this method of cash flow pattern analysis does not provide a reliable indication or prediction of fraudulent activity.
    • Caspase-14: A novel caspase in the retina with a potential role in diabetic retinopathy

      Al-Shabrawey, Mohamed; Ahmad, Saif; Megyerdi, Sylvia; Othman, Amira; Baban, Babak; Palenski, Tammy L.; Shin, Eui Seok; Gurel, Zafer; Hsu, Stephen; Sheibani, Nader; et al. (2012-07-14)
      Purpose: The purpose of this study was to evaluate caspase-14 expression in the retina under normal and diabetic conditions, and to determine whether caspase-14 contributes to retinal microvascular cell death under high glucose conditions.
    • Cassandra Radical Feminist Nurses Network: Feminism, Nursing, and a History for the Present

      Dillard-Wright, Jessica Susan; Nursing (Augusta University, 2020-12)
      As the last light of the Equal Rights Amendment (ERA) faded in 1982, a group of radical feminist nurses coalesced around their shared outrage at nursing’s disciplinary failure to engage deeply with feminist causes. The 1982 American Nurses Convention coincided with this last gasp of the ERA, held in a hotel in Washington, D.C. where thousands of nurses, overwhelmingly women, converged for professional development and camaraderie. And although the city outside the hotel roiled in protest, the Convention unfurled with nary a mention of the constitutional amendment that would secure legal equality irrespective of gender. Incensed by this omission, and with nursing’s general resistance to political engagement, these radical nurses descended on the hotel bar and began organizing what would become Cassandra Radical Feminist Nurses Network. Cassandra Radical Feminist Nurses Network (“CASSANDRA” hereafter, in the convention established by the organization in their Newsjournal) was an activist network active from 1982 until 1991. This study used historical research methods to document CASSANDRA’s legacy while unpacking the complex interrelationship between nursing and feminism. This includes examining the influences of race, gender, and sexuality, influences that shapes normative understandings of nursing from its Victorian origins to the present. CASSANDRA was unusual in its overt affiliation as a nurses’ organization with a radical feminist allegiance during an era when feminism and nursing were frequently at odds. As a decentralized, radical feminist “web,” the aim of CASSANDRA was to “create and develop a group that would truly provide an open forum for feminist nurses from all walks of life and how to avoid the usual male-oriented hierarchy and rigidity of most national organizations” (LaGodna, 1982, p. 1). In unfurling the nuances of gender and sexuality that CASSANDRA navigated, it is clear that the work of CASSANDRA envisioned a radical space for collective resistance and connection, reflecting the normative expectations in nursing that stemmed from nursing’s Victorian imaginary. Even while CASSANDRA’s work around gender and sexuality was bold and transgressive, their engagement with race was poorly articulated. Because of this, the organization’s work reinforced white normativity. Ultimately, like mythological Cassandra, CASSNADRA would eventually quiet to a whisper. What understanding the thrums of CASSANDRA, of nursing’s rich and complex history can do is provide a clear view of nursing’s disciplinary history. This is a fundamental prerequisite for a more just, equitable nursing future.
    • Catching the Asthma: Family Caring for School-aged Asthmatic Children

      Horner, Sharon D; Department of Physiological and Technological Nursing (1992-04)
      The purpose of this study was to explore the process of family caring in families that had a school-aged child with asthma. One focus of this study was to explore the impact of chronicity on the family, specifically looking beyond illness-management issues. Another focus of this study was to uncover the evolution of family caring within the context of school-aged developmental changes. Grounded theory was the research methodology used to discover the strategies, goals, and dimensions of family caring. The research questions used to begin this exploration were: "What is (are) the experience(s) of family caring?" "How does chronicity impact family caring?" These questions presented a number of avenues for exploration. The impact of chronicity on family caring has not previously been explored in depth. Management of the needs of the family member who has a chronic illness (care-taking) has been studied extensively. Various studies have identified the problems families experience related to care-taking tasks, meeting family members' needs, financial burden, stress, role overload, as well as other dynamics of family functioning. None of these studies have explored the emotive and commitment dimensions of caring (caring for and caring about). An exploration of illness-management in day-to-day living was certainly included in the interviewing process; however, this study extended the exploration of chronicity into all aspects of family life to uncover the dimensions of family caring. Specifically, "How do family members care for and care about each other, while taking care of self and others?"
    • CBD Analysis in Oils and Foods

      Foley, Joanna; Department of Chemistry and Physics (Augusta University, 2020-05)
      Cannabidiol (CBD) has become a very prominent topic in the medical community and popular marketplace because of its widespread consumer use. Tetrahydrocannabinol (THC) and other similar molecules can be present in commercial CBD products, so testing is necessary to determine the presence of the CBD. Existing methods of analysis for CBD oils are only known on GC-FID (gas chromatography – flame ionization detector) and these methods are not optimal for the wide variety of commercial CBD products available. Thus, a GC-MS (mass spectroscopy) method, based on a published GC-FID method, was created to optimize the detection of CBD because not only separation but also identification can be obtained. This method can be applied to a wide variety of foods, gummies, and other items that may contain CBD and similar molecules. The method has been optimized by varying GC column temperature, and sample preparation, to find a balance between analysis time, analyte detection, and resolution for the various types of cannabinoid molecules present in commercial CBD oil samples. The optimized method was able to determine that a 1:3 ratio of oil to solvent gave optimal signal of all CBD oils tested. The optimized method was then tested on a variety of commercial and self-prepared CBD edibles to determine that CBD was still present and was not degraded into THC.
    • Cell Membrane Disruption Stimulates NO/PKG Signaling and Potentiates Cell Membrane Repair in Neighboring Cells

      Togo, Tatsuru; McNeil, Paul L.; Department of Cellular Biology and Anatomy (2012-08-7)
      Resealing of a disrupted plasma membrane at the micron-diameter range requires Ca2+-regulated exocytosis. Repeated membrane disruptions reseal more quickly than the initial wound, and this potentiation of membrane resealing persists for at least 24 hours after the initial wound. Long-term potentiation of membrane resealing requires CREB-dependent gene expression, which is activated by the PKC- and p38 MAPK-dependent pathway in a wounded cell. The present study demonstrates that membrane resealing is potentiated in both wounded and neighboring cells in MDCK cells. Wounding of cells expressing CREB133, a mutant variant of CREB, does not show the potentiated response of cell membrane resealing in either wounded or neighboring cells. Furthermore, wounding of cells induces CREB phosphorylation, not only in wounded cells, but also in neighboring cells. Inhibition of the nitric oxide/PKG signaling pathway suppresses CREB phosphorylation in neighboring cells, but not in wounded cells. The potentiation of membrane resealing in neighboring cells is suppressed if the nitric oxide/PKG pathway is inhibited during the initial wound. Together, these results suggest that the nitric oxide/PKG pathway stimulates CREB phosphorylation in neighboring cells so that subsequent cell membrane disruptions of the neighboring cells reseal more quickly.
    • Cell-Type Specific Expression of a Dominant Negative PKA Mutation in Mice

      Willis, Brandon S.; Niswender, Colleen M.; Su, Thomas; Amieux, Paul S.; McKnight, G. Stanley; Mei, Lin; Department of Neurology (2011-04-12)
      We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any celltype that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIa regulatory subunit (RIaB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIaB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIaB demonstrated a reduction in PKA activity and inhibition of cAMPresponsive gene expression. Mice expressing RIaB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIaB allele in vivo provides a novel system for the analysis of PKA function in physiology.
    • Cellular and Immunocytochemical Response to Mandibular Distraction Using an Implanted Lengthening Device

      Elbokle, Nadar N; Department of Oral Biology (2004)
      Distraction osteogenesis (DO) is a biologic process that generates new bone between surfaces of bone segments, which are gradually separated by traction forces. It is a uniquely effective method with multiple applications in the craniofacial region. This concept has been the basis of all bone-lengthening operations; it involved an osteotomy of the shortened bone and an external/internal fixator device, which slowly elongates the bone to its new dimension while a bony callus is being formed at the side to distraction. The biology of DO is similar to callus fracture healing. The bony regenerate passes through the same phases: formation of a collagen fibril template, mineralization, bony union and finally remodeling. The mechanisms by which the mechanical stresses applied to the bone tissue cause the cells to proliferate and form new bone are not well understood. More studies are needed to understand the cellular events underlying DO and the effects of the strains applied during DO on cellular proliferation and mineral apposition.
    • Ceramide Compartments and Protein Interaction: Structure Meets Function

      Kong, JiNa; Department of Neuroscience and Regenerative Medicine (12/27/2016)
      Ceramide is a key sphingolipid, regulating a variety of critical cellular processes. Although exosomes and cilia are derivatives of the membrane, little is known about the role of lipids in their formation. Here we examined the novel role of ceramide in two ceramide-enriched, subcellular compartments: 1) secreted, extracellular vesicles (EVs) termed exosomes, and 2) cell membrane protrusions termed cilia. Firstly, we attempted to address the role of ceramide in exosome secretion and breast cancer. Breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP). We show that incubation of human breast cancer MDA-MB-231 cells with the farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, which is known to induce exosome secretion. Ceramide elevation by combined treatment with gug and bex induced BCRP secretion in exosomes and reduced cellular BCRP in cancer and cancer stem-like cells. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex treatment increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death. Our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy. Secondly, to investigate the role of ceramide in ciliogenesis, in particular motile cilia, we used Chlamydomonas reinhardtii (Chlamydomonas) and murine ependymal cells as models. Motile cilia are specialized organelles formed by cell membrane protrusions to function in movement of body fluids. We show for the first time that Chlamydomonas expresses serine palmitoyl transferase (SPT), the first enzyme in the sphingolipid biosynthetic pathway. Ceramide depletion, by the SPT inhibitor myriocin and a neutral sphingomyelinase deficiency (fro/fro mouse), led to glycogen synthase kinase-3 (GSK3) dephosphorylation and defective flagella and cilia, respectively. A novel activation mechanism for GSK3 by the sphingolipids phytoceramide and ceramide is shown to be critical for ciliogenesis in Chlamydomonas and ependymal cells, respectively. We conclude that ceramide promotes exosome secretion to reduce MDR in MDA-MB-231 cells and regulates GSK3-mediated ciliogenesis in Chlamydomonas and murine ependymal cells.
    • Challenge of a Difficult Airway and Anesthetic Management in a Patient with Still's Disease

      Donald, Ranita R.; Taylor, Emi; Gallen, Thomas; Department of Anesthesiology and Perioperative Medicine (American Society of Anesthesiologists, 2010-10)
      Adult-onset Still’s disease (AOSD) is a rare systemic inflammatory disorder of unknown etiology with articular and extra-articular (systemic) manifestations. The disorder owes its name to Sir George Frederick Still, who in 1897 described 22 children with symptoms consistent with what is currently known as systemic onset juvenile idiopathic arthritis. AOSD was established almost a century later in 1971, when Eric Bywaters encountered and described adult patients presenting with pediatric Still’s disease symptoms. Compared to rheumatoid arthritis in adults, AOSD runs a much more acute course, quite often affecting many parts of the body before settling in the various joints. Diagnosis of AOSD is difficult to establish due to the nonspecific clinical and laboratory findings. Tracheal intubation may become difficult due to impairment of cervical spine, temporomandibular joint and laryngeal involvement (crico-arytenoid arthritis). Patients with chronic articular disease have more disability and worse prognosis than patients with only systemic symptoms.