• A Novel RNA Aptamer-Based Targeted Therapy for Ovarian Cancer with PRKCI Gene Amplification

      Rehmani, Hina Salam; Biomedical Sciences
      Ranked fifth in cancer death among women, ovarian cancer cure rates have remained low over the past two decades due to unsuccessful detection of early-stage disease, stagnant methodologies of treatment, and high relapse rates. Stage IV invasive epithelial ovarian cancer patients have a meager 17% relative 5-year survival rate and around 70% of patients relapse within the first two years of diagnosis. Accounting for more deaths than any other cancer of the female reproductive system, it is paramount to strategically offer ovarian cancer patients targeted therapies to improve outcome. Ovarian cancer contains a host of copy number aberrations (CNA) that can lead to the silencing or amplification of tumor suppressor genes, oncogenes, or non-coding RNAs that modify the expression of genes. Clinically, gene amplifications have prognostic and diagnostic usefulness as they can be a mechanism to promote tumorigenesis and/or attainment of drug resistance. Protein kinase C iota (PKCiota), a cytoplasmic serine-threonine protein kinase, is highly amplified and overexpressed within the 3q26 amplicon in ovarian cancer and is identified as an oncogene in multiple cancers. However, selectively targeting the iota isoform with small molecule inhibitors is challenging since it shares a 72% overall homology with another atypical PKC isoform, zeta. We found that small-molecule inhibitors currently being investigated do not offer selective inhibition nor specificity in ovarian cancer cell lines. We hypothesize that PRKCI amplification offers a unique opportunity to stratify patients into different risk categories and that specifically targeting PRKCI using a siRNA-aptamer can offer therapeutic benefits by impairing ovarian cancer cell tumorigenesis. We expect this approach to provide potent and selective anti-tumor activity compared to targeting using other pharmaceutical approaches. To test this hypothesis, we identified ovarian cancer cell lines with PRKCI gene amplification, identified a pattern of onco-addiction specificity in cell lines with amplification, and created an RNA-based aptamer that efficiently becomes internalized in ovarian cancer cells and mediates PRKCI mRNA silencing. In conclusion, we have identified a rationale to target, specifically, the PKCiota gene amplification in ovarian cancer and our RNA-based aptamer prevents ovarian tumorigenesis both in vitro and in vivo, opening a door for future therapies.
    • Activation of Arginase and the Endothelin System in Models of Ischemic Retinopathy

      Patel, Chintan; Department of Biochemistry and Molecular Biology; Department of Biochemistry and Molecular Biology (2014-07)
      Ischemic retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP) are characterized by microvascular degeneration, followed by an abnormal hypoxia-induced neovascularization (NV). Although the triggering insult varies among the diseases, they share a common end result of capillary loss due to increased oxidative stress, cellular inflammation and vascular injury and dysfunction. We have linked activation of the urea hydrolase enzyme arginase to the latter complications in models of DR. Both arginase and nitric-oxide synthase (NOS) enzymes utilize L-arginine as substrate. NOS dysfunction due to limitations in L-arginine availability has been implicated in the pathogenesis of diabetic complications. Our studies in streptozotocin-induced diabetic mice and high glucose treated retinal ECs have demonstrated signs of retinal vascular activation and injury. These were associated with increased arginase activity and expression, decreased bioavailable nitric oxide (NO), increased superoxide formation and increased leukostasis. Blockade of the arginase pathway prevented these alterations, suggesting a primary role of arginase in retinal vascular dysfunction and injury. Our studies have also shown that endothelium-dependent retinal vasorelaxation was impaired in diabetic mice, however, deletion of arginase improved retinal vessel function and improved blood flow. During ischemic retinopathies, disturbances in retinal blood flow can result in vasoconstriction, ischemia, tissue hypoxia and formation of neovascularization (NV). Such alterations have been linked to development of ROP, a blinding disease that adversely affects premature infants due to oxygen-induced damage of the immature retinal vasculature resulting in pathological NV. Our studies using a mouse model of ROP, the oxygen-induced retinopathy (OIR) model indicate that a potent vasoactive and angiogenic factor endothelin (EDN) is responsible for pathological NV. Our analysis revealed significant increases in EDN1, EDN2 and endothelin A receptor (EDNRA) mRNA and EDN2 protein expression during ischemia. EDN2 was localized to endothelial cells and retinal glia in OIR retinas. Treatment of OIR mice with EDNRA blocker, BQ-123, significantly increased vessel sprouting resulting in enhancement of physiological angiogenesis and decreased pathological NV. OIR triggered a significant increase in STAT3 activation and VEGFA production and increased mRNA expression of angiogenic and inflammatory mediators, which were all reduced by BQ-123 treatment. These studies suggest that EDNRA activation during OIR promotes vessel degeneration and pathological NV. Collectively, both arginase and endothelins are increased in models of ischemic retinopathies. These two pathways could be interrelated through an unknown cross-talk mechanism that needs to be elucidated.
    • Activities and Perceived Outcomes of Nurse Case Managers: Building a Case Management Model for Rural Hospitals

      Anderson-Loftin, Wanda; Department of Physiological and Technological Nursing (1996-12)
      The primary purpose of this study was to describe the activities and perceived outcomes of nurse case managers in a rural hospital setting and the relationship of nurse case managers' education, experience, and age to activities and perceived outcomes. Results of the study will be used to further develop and refine a portion of the investigator-developed model of Nursing Case Management for Rural Hospitals which served as the study framework. Nurse case managers in non-federal, rural hospitals listed in the American Hospital Association's (1995) guide to U. S. hospitals were surveyed using an investigatordeveloped instrument. Psychometric qualities of the instrument were determined as part of the study. The sample (N = 302) consisted primarily of white, middle-aged females. The majority were ADN or BSN nurses who averaged two to three years experience in case management and 16 years experience in nursing. Descriptive, correlational, and multivariate statistics were used to analyze the data. Results indicated that individual advocacy was the most frequent activity, and comments suggested that nurse case managers were becoming aware of the centrality of advocacy to their practice and job satisfaction. The second most frequent activity was teaching, with clinical practice third. The most frequent pattern of activities reflected assessment and coordination of community resources through an advocacy role for the client while performing managed care/quality assurance activities. The top ten perceived outcomes were: (a) increased patient satisfaction, (b) reduced fragmentation of care, (c) reduced length of stay, (d) increased job satisfaction, (e) increased job enjoyment, (f) increased quality of life, (g) increased functional health, (h) increased self-care, (i) increased autonomy, and (j) attainment of goals within the time-frame for reimbursement. Two-thirds of the nurse case managers thought their activities prevented delays in care and provided clients with a regular source of care, thus increasing access to care. Other significant (pi .05) findings indicated: (a) ADN, BSN, and MSN nurse case managers engaged in more teaching than nurse case managers with diplomas, (b) system advocacy was higher for MSN than for diploma, ADN, or BSN case managers, and (c) experienced nurse case managers engaged in more clinical practice than inexperienced nurse case managers.
    • Activity-Dependent Regulation of the Dopamine Transporter is Mediated by Cam Kinase II Signaling

      Padmanabhan, Shalini; Department of Pharmacology (2009-01)
      Dopamine signaling in the brain governs a variety of functions such as locomotor activity, reward, attention and working memory. The dopamine transporter (DAT) plays a crucial role in the clearance of extracellular dopamine and thus helps terminate dopamine neurotransmission. DAT is also the target for psychostimulant drugs of abuse and therapeutic agents. Changes in DAT expression occur in neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD) and chronic psychostimulant use, and variability in DAT abundance is associated with differences in working memory. However, mechanisms regulating DAT expression are poorly understood. We tested the hypothesis that neuronal activity is one of the non-genetic determinants of DAT abundance. Chronic perturbations in neuronal firing, caused by pharmacological agents, significantly altered DAT expression and function in primary cultures of mesencephalic neurons. Pharmacological experiments showed that calcium entry through L-type voltage-gated calcium channels and calcium/calmodulindependent protein kinase II (CaMKII) activity played a role in activity-dependent changes in DAT expression. In order to further evaluate the role of CaMKII in DAT regulation, the effect of sustained depolarization, a stimulus often used to study activity-dependent changes in gene expression, on DAT expression was tested. Surprisingly, chronic KCI-induced depolarization decreased DAT expression and function. Measurement of CaMKII activity in dopamine neurons showed that chronic depolarization led to a decrease in CaMKII activity, even in the presence of elevated intracellular calcium, due to activation of the serine/threonine protein phosphatase 2A. Moreover, increasing CaMKII activity in dopamine neurons by introducing a constitutively active CaMKII mutant caused a significant increase in DAT abundance while inhibiting CaMKII activity in dopamine neurons using a dominant-negative CaMKII mutant decreased DAT abundance suggesting that CaMKII activity is both sufficient and required to cause changes in DAT expression in a cell autonomous fashion. Taken together, our data demonstrate that CaMKII activity can govern DAT expression and may play an important role in dopamine neurotransmission in the brain.
    • Adoption of AACN Verification of Feeding Tube Placement Practice Alert by Critical Care Nurses

      Bourgault, Annette M.; Department of Nursing; Georgia Regents University (2012-04)
      The intent of clinical practice guidelines is to help bridge gaps between evidence and practice, yet there is no correlation between availability of guidelines and changes in practice. Little is known about how critical care nurses adopt guidelines, since few studies have sampled nurses exclusively. This descriptive, exploratory study examined factors influencing adoption of the American Association of Critical-Care Nurses (AACN) Verification of Feeding Tube Placement Practice Alert and four clinical practices recommended by this guideline.
    • Adrenal Zona Glomerulosa Targeting in Transgenic Mice

      Parmar, Jeniel; Department of Physiology (2009-12)
      The final step in the production of aldosterone is performed by the enzyme aldosterone synthase (CYP11B2). CYP11B2 is primarily expressed in the zona glomerulosa (ZG) of the adrenal cortex. Adrenocortical expression of CYP11B2 is primarily regulated by circulating levels of angiotensin II (Ang II) and K+, but the molecular mechanisms that control its ZG-specific expression are not clearly defined. Considerable in vitro analyses have been performed towards defining the mechanisms that control CYP11B2 expression. Previous studies from our laboratory and others have identified several c/s-regulatory elements on the 5' flanking promoter region (at -71/64, -129/114, -351/343 and -773/766) that regulate basal expression as well as maximal stimulation of CYP11B2 gene transcription. Moreover, key transcription factors that bind these c/s-regulatory regions including NGFIB, NURR1, SF-1 and COUP-TF have also been identified. Hence, through several in vitro analyses, a considerable evidence exists supporting the contention that these regulatory elements found within the 5' flanking promoter region may control ZG-specific expression of CYP11B2 gene. However, thus far, all evidence is based on in vitro analyses of transcriptional regulation, which does not always depict in vivo occurrences. To initiate our in vivo assessment of CYP11B2 promoter, we began by comparing the DNA sequences between human, mouse, and rat CYP11B2 genes, which interestingly revealed high sequence similarity in the 5' flanking promoter region of the CYP11B2 gene. This result suggested that the cisregulatory regions identified by in vitro analyses likely plays an important role in CYP11B2 ZG-specific gene expression. Therefore, we generated transgenic mouse lines by pronuclear injection of a Transgenic (Tg) DNA construct containing 985 base pairs (bp) of the mouse Cyp11b2 promoter driving expression of a LacZ reporter gene. Importantly, 4 founder Tg mouse lines revealed LacZ expression exclusively in the adrenal ZG. Mice fed a normal sodium diet (0.3 %) and a low sodium diet (0.03 %) showed LacZ mRNA expression exclusively in adrenal tissue. Furthermore, (3-galactosidase protein (the product of LacZ) was localized solely in the ZG of the Tg mice. Hence, the role of the proximal promoter region of the Cyp11 b2 gene was confirmed, in vivo, as this region allowed induction of LacZ exclusively in the adrenal ZG of Tg mice. Moreover, with the expression of LacZ properly restricted to adrenal ZG, we concluded that regions required for Cyp11b2 gene repression in the adjacent inner two zones of the adrenal cortex were also confined within the 985 bp promoter. This regulatory fragment will be an invaluable tool for adrenal ZG targeting of genes believed to play a role in adrenocortical diseases and aldosterone dysregulation. While developing Tg mice, we also focused on characterization and development of novel adrenocortical cell lines. As aforementioned, in vitro culture models have allowed a multitude of studies that have broadened our understanding of normal adrenocortical endocrine function. Primary cultures of adrenocortical cells have been an excellent source for in vitro studies. However, the eventual onset of senescence in primary cultures of cells creates a recurring need for the costly and difficult isolations of fresh adrenocortical cells. Hence, the use of primary cultures has been increasingly supplemented by immortalized cell lines. We utilized an adrenocortical carcinoma to develop a human adrenocortical cell line. We entitled it the human adrenocortical carcinoma cell line clone 15 (HAC15). HAC15 represents only the second human adrenocortical cell line available that exhibits physiological hormonal responses, steroidogenesis, and expression of steroid-metabolizing enzymes. The ability of HAC15 to respond to Ang II, K+, and ACTH makes it the first adrenal cell line capable of responding to the three main physiologic regulators of the adrenal cortex.
    • AKAP350 Targets to the Golgi Apparatus Where it Interacts with CLIC5B and CIP4/S

      Shanks, Ryan; Department of Biochemistry and Molecular Biology (2002-03)
      A-kinase anchoring proteins (AKAPs) are defined by their ability to scaffold PKA, but their function depends upon their targeting of PKA and other scaffolded signaling proteins to specific subcellular compartments. We have investigated one AKAP, AKAP350, which can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. The AKAP350 gene is multiply spliced to create three carboxyl terminal splice variants which we have designated AKAP350A, AKAP350B and AKAP350C. Immunocytochemistry in HCA-7 cells demonstrated that AKAP350A was localized specifically to the Golgi apparatus. GFP-fusion proteins representing the carboxyl terminus of AKAP350A identified a carboxyl terminal region responsible for the Golgi apparatus targeting of AKAP350A. Yeast two-hybrid analysis was utilized to screen a rabbit gastric parietal cell library with a 3.2kb segment of AKAP350 (nucleotides 3611-6813) which is weakly homologous to pericentrin. This screen yielded two positive clones representing rabbit chloride intracellular channel 1 (CLIC1) and rabbit Cdc42 interacting protein 5 (CIP5). Further yeast-two hybrid binary analysis determined that CLIC1 and CIP5 bound to AKAP350 through adjacent domains located with in the PHR. CLIC1 belongs to a family of proteins which all contain a high degree of homology in their carboxyl termini, and this conserved domain is responsible for several CLIC family member’s ability to bind AKAP350. We isolated the human homologue of bovine p64, CLIC5B, from an HCA-7 colonic adenocarcinoma cell cDNA. A splice variant of CLIC5, the predicted molecular weight of CLIC5B corresponds to the molecular weight of a major CLIC immunoreactive protein in HCA-7 cells. Immunocytochemistry determined that CLIC5B colocalized with AKAP350 at the Golgi apparatus. Yeast-two hybrid binary analysis determined that the final 120 amino acids of CLIC5B interacted with AKAP350. Furthermore, expression of a GFP-fusion protein containing the final 120 amino acids targeted to the Golgi apparatus in HCA-7 cells. CIP5 contains high homology to the human protein Cdc42 interacting protein 4 (CIP4). Yeast-two hybrid binary analysis determined that the first 117 amino acids of both human CIP4 and CIP5 interacted with AKAP350. Immunocytochemistry in HCA-7 cells determined with an antibody recognizing CIP4 and CIP5 localized to the Golgi apparatus. These results suggest that AKAP350 associates with CLIC proteins and CIP4/5, and these proteins interact with the AKAP35A splice variant at the Golgi apparatus.
    • AKAP350: A Centrosome Associated Scaffold Protein

      Schmidt, Hank; Department of Biochemistry and Molecular Biology (2000-06)
      A-kinase anchoring proteins (AKAPs) are recognized as key components of compartmentalization and transduction in intracellular cAMP signaling. They allow localization of the Type II c AMP-dependent protein kinase to specific subcellular domains, effectively positioning the enzyme near its substrate to await activation by cAMP. The role of AKAPs as protein scaffolds allows binding of multiple enzymes, regulatory molecules, and structural elements, functioning as a virtual platform for modulation of specific cellular events (i.e. membrane channel activity, receptor clustering). We have cloned a novel 350 kDa AKAP (AKAP350) from human gastric cDNA, and identified partial clones in human lung and rabbit parietal cells. The genomic region containing AKAP350, found on chromosome 7q21, is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, an NMDA receptorassociated protein. We identified three unique AKAP350 C-termini (AKAP350A, -B, and -C) resulting from alternative splicing of the 3' end of the gene. AKAP350 is associated with centrosomes, as well as with the cleavage furrow during anaphase and telophase by immunocytochemistry. Polyclonal antibodies to individual AKAP350 Cterminal splice variants demonstrate tissue dependent combinations of centrosomal and non-centrosomal distribution. In the polarized HCA-7 colon cell line AKAP350A is purely non-centrosomal while AKAP350B and -C are centrosomal. Anti-AKAP350C is limited to mitotic cells, suggesting that this isoform may be expressed only at entry into M phase. A yeast two-hybrid screen of a rabbit parietal cell library identified a novel TACC (Transforming Acidic Coiled coil Containing) protein family member as a ligand of the final pair of arginine residues in the AKAP350A splice variant. A GFP fusion with the novel AKAP interacting protein verified co-localization with AKAP350 at the centrosome exclusively during mitosis. Microinjection of dividing sea urchin embryos with GST fused to the AKAP interacting protein arrested cell division. Therefore, the AKAP350 protein scaffold may function as a large docking station, providing kinase / phosphatase signals for coordination of cytoskeletal dynamics as well as cell division.
    • Alpha-Tocopherol as an Ergogenic Factor in the Guinea Pig

      Allen, Hugh Clement; Department of Anatomy (1968-06)
    • Alterations in Articular Cartilage of the Rabbit Mandibular Condyle Following Surgical Induction of Anterior Disc Displacement: Light and Electron Microscopic Immunocytochemistry Using Colloidal Gold Conjugates

      Choi, Won-Seok; Department of Oral Biology (1996-05)
      The purpose of this study was to test the hypothesis that surgical induction of anterior disc displacement (ADD) in the rabbit craniomandibular joints (CMJ) will lead to degenerative osteoarthritic changes detectable a t the molecular, subcellular and cellular levels in the articular cartilage of the rabbit mandibular condyle. Ultrastructural features of the normal rabbit mandibular condyle were compared to those of experimental condyles a t two weeks following induction of ADD. The quantities of type-VI and -IX collagens, as well as the components of proteoglycans, such as chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), k e ra tan sulfate (KS) and link protein (LP) were measured using immunogold labeling technique at the light and the electron microscopic levels. The right joint of each of 20 rabbits was exposed surgically, and all discal attachments were severed except for th e posterior attachment. The disc was then displaced anteriorly and sutured to the zygomatic arch. The left joint served as a sham -operated control. Ten additional joints were used as non­ operated controls. Deeply anesthetized rabbits were perfused with 2% buffered formalin two weeks after surgery. The mandibular condyles were excised and decalcified in ethylenediam inetetraacetic acid (EDTA). Paraffin embedded tissues were sectioned a t 5 (im for light microscopic study, while water-soluble plastic embedded sections were used for electron microscopy. Sections were incubated in monoclonal antibodies directed against C4S, C6S, KS and LP, and in polyclonal antibodies against type-VI and -IX collagens. After incubation in the appropriate colloidal gold conjugated secondary antibodies, tissue sections were studied with light and electron microscopes. In addition, immunostaining for proliferating cell nuclear antigen (PCNA) was performed using paraffin sections, and the PCNA indices of control and experimental condyles were determined. Pathological alterations were obvious in the experimental condyles, and appeared to be characteristic osteoarthritic changes. These include cartilage neovascularization, chondrocyte clustering, vacuolation, loss of extracellular matrix next to the membranes of chondrocytes, and an increase in num ber of apoptotic chondrocytes. Increased num bers of PCNA-positive cells in the osteoarthritic cartilage of the experimental group indicated a n active chondrocytic proliferation. Ultrastructural changes in injured chondrocytes included increased amounts of RER and Golgi, suggesting an increase in the synthesis and secretion of possibly degradative enzymes with a decrease in the normal secretory products. The results of th e immunocytochemistry using colloidal gold conjugates both a t the light and electron microscopic levels showed statistically significant depletion of C4S, C6S, KS, LP, type-VI collagen and type-IX collagen in the osteoarthritic cartilage (P < 0.05). The reduction of binding molecules such as LP, type-VI and type-IX collagens suggest a possible mechanism for the observed loss of integrity of the extracellular matrix. It is concluded that surgical induction of ADD in the rabbit CMJ leads to molecular, cellular and extracellular alterations in the articular cartilage of the mandibular condyle similar to those described previously in hum an ADD and in osteoarthritis of other synovial joints. The results of this study provide evidence that the loss of the shock absorber function of the disc, and the exposure of the condyles to overloading may cause the injured chondrocytes to secrete degenerative cytokines as indicated by the loss of proteoglycans, binding collagens and LP. These molecular changes are expressed a t the subcellular and cellular levels as osteoarthritis or degenerative joint disease.
    • Amyloid Peptide-a7 Nicotinic Acetylcholine Receptor Interactions: Implications For Cytoprotection In Vitro

      Li, Xinyu D.; Department of Cellular Biology and Anatomy (2006-11)
      Brain deposition of (3-amyloid peptide 1-42 (A(31 -42)-containing senile plaques has been a consistent finding in Alzheimer’s disease (AD). However, the link between Apl-42 and neuronal degeneration remains unclear. It has been reported that AP peptides bind with selectivity to a l nicotinic acetylcholine receptors (a7nAChRs), in both healthy and Alzheimer’s Diseased brain tissues. The goal of this study was to demonstrate the functional inhibition of oc7nAChRs induced by Api-42, both in systems in vitro and in vivo. Initially, differentiated PC-12 cells were preloaded with fura 2-AM and intracellular free Ca2+ levels were determined by fluorescent imaging. Nicotine-induced Ca2+ signals were inhibited by pretreatment with the a7nAChR-selective antagonists, abungarotoxin (BTX) and methyllycaconitine (MLA). Nicotine induced Ca2+ influx was also blocked by pretreatment with 100 nM Api-42. In the same model, nicotine produced a concentration-dependent increase in cell viability in differentiated PC-12 cells that underwent nerve growth factor (NGF) withdrawal for 24 hr. The cytoprotective action of nicotine was efficiently antagonized by co-treatment with a7nAChR antagonists. A concentration-dependent inhibition of the cytoprotective action of nicotine also was produced by co-treatment with Apl-42 (1-100 nM). Also in differentiated PC-12 cells, nicotine induced a concentration-dependent increase in cell surface Trk A receptor expression. This increase was almost completely reversed by a7receptor-selective antagonists, and by co-treatment with Api-42. In in vivo studies with rats, intracerebroventricular (icv) injection of choline, a selective a7nAChR agonist, produced transient, but dose-dependent pressor responses and prolonged decreases in heart rate. Icv pretreatment with BTX and MLA significantly inhibited the cardiovascular responses to subsequent injection of choline. Pretreatment with the Api-42 also significantly inhibited the choline-induced cardiovascular changes suggesting that the peptide can block an oc7nAChR-mediate response in vivo. Nicotine also was administered to rats by direct injection into a lateral cerebral ventricle. Estimation of Trk A expression in necropsied brain tissues revealed significant increases in hippocampus and entorhinal cortex. These increases were significantly inhibited in rats co-treated with a-bungarotoxin or with Api-42. The data derived from these in vitro and in vivo experiments support the hypothesis that low physiological concentrations of AP peptides inhibit the function of a7nAChRs, thereby contributing to the loss in neuronal viability that accompanies Alzheimer’s disease.
    • An Analysis of Diabetes Predictors and Diagnostic Tests in a Sample of African Americans at Risk for Diabetes

      Williams, Lovoria B.; Department of Biobehavioral Nursing (2011-05)
      Recently the ADA and International Expert Committee (IEC) endorsed HbA1C for diagnosis of glucose states. Concerns exists regarding discordance between fasting plasma glucose (FPG) and HbA1C; the committees do not agree on the HbA1C cut-point for diagnosis of sub-diabetic states; and the HbA1C may be more sensitive in AAs. A secondary data analysis of the Fit Body and Soul (FBAS) sample (n = 393) was conducted. FPG and HbA1C values were classified by the current ADA and the IEC HbA1C criteria. A risk factor analysis was also conducted. Results indicate different subject classification based on choice of diagnostic test and criterion used. Subjects classified as normoglycemic based on ADA FPG, ADA HbA1C and IEC HbA1C criterion were (78.9%; 30.7%; 55%) of the sample, respectively. Sub-diabetic state was (18.1%; 55.9%; 31.5%), respectively. Diabetes was (3%; 13.4%; 13.4%), respectively. Moderate correlation exists between HbA1C and FPG (Pearson’s r = 0.63 p < 0.001); there is only slight to fair agreement between ADA HbA1C and ADA FPG classifications and IEC HbA1C and ADA FPG classifications; Cohen’s Kappa = 0.127; 0.234 (p < 0.001), respectively; McNemar’s Chi Square (χ23df = 182.8; 81.54 p < 0.001) respectively. Significant predictors of HbA1C by linear regression were waist circumference (WC) and age; FPG predictors were age, WC and family history of diabetes. The risk factor analysis indicated poor agreement with either diagnostic test.
    • Analysis of Folate Transport Proteins in the Mammalian Retina and Retinal Pigment Epithelium: Characterization and Localization of Folate Receptor Alpha and Reduced-Folate Transporter-1

      Bridges, Christy C.; Department of Cellular Biology and Anatomy (2000-11)
      The purpose of these studies was to determine the cellular and molecular mechanisms of folate transport in the retinal pigment epithelium (RPE).
    • Androgen Effects on Follicular Atresia and Ovulation

      Bagnell, Carrol A; Department of Endocrinology (1983-03)
    • Androgenic Maintenance of Rat Penile Erection

      Reilly, Christopher M.; Department of Cellular Biology and Anatomy (1997-06)
      Prior studies from this laboratory, using untreated-castrated rats (CASTRATE) and testosterone-treated castrated rats (TESTO), have shown that the magnitude of the intracavemosal pressure increase during erection is androgen dependent. Studies from this and other laboratories have also presented evidence suggesting that penile erection is mediated principally by nitric oxide (NO). The present report was designed to confirm that androgens maintain the availability of cavemosal NO, and to determine if this androgenic action is exerted at the genomic level modulating the expression of the neuronal form of nitric oxide synthase (nNOS) gene. The results showed that administration of supplemental L-arginine failed to augment the erectile response in either group, suggesting that substrate availability is not a cause o f the reduced response in CASTRATE animals. Inhibition of NO synthesis with a nitro-arginine competitive inhibitor of NOS resulted in strong inhibition of erection in both TESTO and CASTRATE rats. When given in conjunction with ganglionic stimulation to induce erection, the NO releasing drug, sodium nitroprusside (SNP), increased intracavemosal pressure in CASTRATE but not in TESTO rats suggesting a deficiency of the available NO in CASTRATE animals. Finally, reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mRNA levels for the enzyme nNOS in the penis were greater in TESTO animals than in CASTRATE rats. These results support the hypothesis that androgens mediate the erectile response in the rat penis by stimulating the expression of the neuronal isoform of nitric oxide synthase, thus maintaining an adequate supply of NO.
    • Angiotensin II Regulation of Aldosterone Synthase

      Nogueira, Edson da F.; Department of Physiology (2009-07)
      Angiotensin II (Ang II) is the major physiological regulator of aldosterone production acting acutely to stimulate aldosterone biosynthesis and chronically to increase the capacity of the adrenals to produce aldosterone. Aldosterone is principally synthesized in the zona glomerulosa of the adrenal by a series of enzymatic reactions leading to the conversion of cholesterol to aldosterone. The major goal of our study was to define the Ang II-induced mechanisms regulating the expression of aldosterone synthase (CYP11B2) in adrenocortical cells. We approached the analysis of the protein synthesis-dependent regulation of this enzyme by defining, through microarray and real time PCR analysis, the transcription factors that are rapidly induced by Ang II incubation of adrenocortical cell models from three species (human, bovine, and rat). The gene list generated by this comparison included: ATF3, BTG2, NR4A1, NR4A2, NR4A3, EGR1, FOS, FOSB, and JUNB. Importantly, pretreatment of H295R cells with cycloheximide had no effect on Ang II induction of these genes, suggesting that they are direct targets of Ang II signaling. Co-transfection studies, used to investigate the role of these transcription factors in the regulation of CYP11B2, determined that out of the nine transcription factors listed above, only the NGFI-B family members (NGFI-B, NURR1, and NOR1) increased expression of CYP11B2. The importance of NGFI-B in the regulation of CYP11B2 was confirmed by the decrease in CYP11B2 expression in the presence of a dominant-negative (DN)- NGFI-B. A pharmacological approach used to characterize the Ang II pathways regulating transcription of NGFI-B family genes suggested that Ang II binding to the AT1R increases activity of protein kinase C (PKC), Ca -dependent calmodulin kinases (CaMK), and SRC kinase (SRC), which act to regulate the expression of the family of NGFI-B genes as well as CYP11B2. In the current study we also analyzed protein synthesis-independent mechanisms regulating CYP11B2 expression. We studied the role of the ATF/CREB family of transcription factors (ATF1, ATF2, CREB, and CREM), which may bind the cAMP response element (CRE) in the promoter region of the CYP11B2 gene. Importantly, analysis of these transcription factors in the human H295R adrenocortical cell line revealed very low expression of CREB in comparison to the other CRE-binding proteins herein studied. We investigated Ang II-induced phosphorylation of these transcription factors, their binding to the promoter region of CYP11B2, and their effect on CYP11B2 expression. Ang II time-dependently induced phosphorylation of ATF1, ATF2, and CREM in H295R cells. The association of these transcription factors with the CYP11B2 promoter region was induced by Ang II and K+. Transfection of siRNA for ATF1, ATF2, and CREM significantly reduced CYP11B2 expression in Ang II-stimulated conditions. Expression of NURR-1 alone or with constitutively active ATF1, ATF2, CREB, and CREM increased the promoter activity of CYP11B2 in H295R cells. In summary, Ang II rapidly induces expression of newly synthesized transcription factors as well as the phosphorylation of transcription factors already present in the adrenocortical cell. These events are followed by increased CYP11B2 expression and, therefore, represent important mechanisms to increase the adrenal capacity to produce aldosterone.
    • Angiotensin II Signaling Mechanisms Involved in the Elevation of Arginase Activity/Expression and Vascular Dysfunction

      Shatanawi, Alia; Department of Pharmacology and Toxicology (2011-11)
      Vascular endothelial dysfunction is a major cause of morbidity and mortality in patients with cardiovascular diseases such as hypertension, atherosclerosis and diabetes. Nitric oxide (NO) produced by endothelial nitric oxide synthase (NOS) is needed for normal vascular function. During hypertension, diabetes or atherosclerosis, elevated levels of arginase can compete with NOS for available L-arginine thus reducing vascular NO production. Elevated angiotensin II (Ang II) is a key participant of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. In this study we explored the signaling pathway leading to increased arginase expression/activity in responses to Ang II in bovine aortic endothelial cells (BAEC). Treatment of BAEC with Ang II (10-7 M, 24 hrs) caused a 40±6% increase in arginase activity. This was accompanied by 30±8% decrease in NO production. Our studies indicate involvement of the RhoA/ROCK-p38 mitogen activated protein kinase (MAPK) in Ang II-induced arginase upregulation and reduced NO production, as inhibitors of ROCK or p38 MAPK prevented the Ang II-induced increase in arginase activity. Our studies in mice also show involvement of p38 MAPK in Ang II-induced vascular dysfunction associated with elevated arginase activity and expression. Ang II (42 μg/kg/h) caused impaired EC-dependent vasorelaxation in mouse aorta (55±7% vs. 75±8% for control). This impairment was prevented by treatment with p38 inhibitor SB203580 (5 μg/kg/day). Ang II also caused a 6.2 fold increase in vascular arginase activity/expression that was completely prevented by p38 MAPK inhibition. Additionally, treatment of BAEC with Ang II causes phosphorylation of activating transcription factor-2 (ATF-2) and enhancement of the binding of ATF-2 to arginase promotor through an AP-1 site as evident from electrophoretic mobility shift assay experiments. Transfection of BAEC with ATF-2 siRNA prevents Ang II-induced increases in arginase activity/expression and maintains NO production. These results indicate that ATF-2 is necessary for enhanced expression of arginase by Ang II. Collectively, our results indicate that Ang II increases endothelial arginase activity/expression through a RhoA/ROCK-p38 MAPK-ATF-2 pathway leading to reduced NO production and endothelial dysfunction. Targeting these signaling steps might be therapeutic points for preventing vascular endothelial dysfunction associated with elevated arginase activity/expression.