• From Adipokines to Atherosclerosis: The Role of Adipose Tissue in Inflammation and Etiology of Vascular Disease

      Bundy, Vanessa; Vascular Biology Center (2007-04)
      The prevalence of overweight and obese has steadily increased over the years among males and females of all ages, all racial and ethnic groups, and all educational levels. Recent studies have established adipose tissue as a dynamic, endocrine organ with the capacity to secrete a number of adipokines which may act directly upon the vasculature to stimulate adhesion molecule expression and exacerbate vascular disease. Our aim was to elucidate the associations of vasoactive pro- and anti- inflammatory factors, including adhesion molecules, with adiposity, blood pressure and endothelial function, and to distinguish race and sex variations in these relationships. To accomplish this, we expanded upon existing measurements within a Georgia Prevention Institute cross-sectional study entitled Lifestyle, Adiposity & Cardiovascular Health in Youths (LACHY) by adding two cardiovascular disease risk factor domains: inflammation and vascular adhesion. Our model included measurements of adiposity, adiponectin, C-reactive protein, leptin, insulin, resistin, tumor necrosis factor-a, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, blood pressure and endothelial-dependent arterial dilation. Our findings include numerous race and sex differences in the concentration of circulating risk factors along with significant interactions between them and measurements of adiposity. However, we did not find circulating cardiovascular disease risk factors or their concentration differences to be significantly associated with blood pressure or endothelial function. We believe this to be largely due to the fact that our subjects were young and apparently healthy at time of measurement. Overall, our findings provide insight into the relationships between adiposity, inflammation and cardiovascular outcomes in black and white, male and female adolescents. Future studies are needed to further elucidate these relationships and how they may change over time.
    • The Function of NELF and ESR1 in Puberty Development and Fertility

      Quaynor, Samuel Nii Djangmah; Department of Obstetrics and Gynecology (2013-07)
      Reproductive disorders affect 10-15% of the population in the United States and result in debilitating social and economic issues. Reproductive dysfunction may be displayed as delayed puberty or infertility due to defects in the hypothalamic-pituitary gonadal (HPG) axis. This axis is controlled by gonadotropin releasing hormone (GnRH) through the release of GnRH peptide. An important critical barrier to understanding normal puberty is the lack of known specific genes that regulate the development, migration and function of hypothalamic GnRH neurons, which synthesize and secrete GnRH neuropeptide in a pulsatile pattern into the portal blood. Several human diseases occur as the result of impaired GnRH function. Idiopathic hypogonadotropic hypogonadism (IHH) patients present with decreased gonadotropins and sex steroids leading to permanent delayed of puberty and infertility. Kallmann syndrome (KS) combines IHH phenotypes in addition to anosmia/hyposmia as there is impairment in migration o f GnRH neurons and olfactory neurons/axons. To date, the molecular basis has only been identified for approximately 40% o f all IHH/KS patients. The purpose of this study was to understand the role and function o f genes, such as NELF (nasal embryonic LHRH factor), in pubertal development and fertility. NELF, a nuclear protein isolated from migratory GnRH neurons, has been shown to be involved in the pathogenesis of IHH/KS, and knockdown impairs GnRH neuron migration in vitro. The mechanism o f NELF’s role relevant to GnRH migration/signaling and its effects on pubertal development and fertility are unclear. NELF function has been studied in GnRH cell lines, but not in the whole organism. The overall goals of this study are to characterize the phenotypic features of /Ve/^knockout (KO) mice, classify the prevalence of the different variants of NELF reported, and the inheritance patterns of IHH/KS genes including NELF, and finally examine the phenotype and hormonal profile o f an estrogen receptor-a (ESR1) mutation in a woman. Methods: Following breeding of heterozygous mice to generate homozygous N elf knockout mice, pubertal onset was assessed by daily monitoring of weight, vaginal opening (females), and anogenital distance (males). In addition we determined fertility of the mice by doing a continuous 90 day breeding study. Immunocytochemical labeling o f GnRH neurons was performed and analyzed for migration and count; hematoxylin and eosin stains were performed to visualize gonadal morphology. Three additional studies were also performed: 1) NELF splice variants were studied in immortalized human and mouse GnRH neurons; 2) the prevalence of digenic disease in IHH/KS was determined when the 13 most common genes were sequenced in 48 patients; and 3) the first woman with an estrogen receptor-a (ESR1) mutation was characterized clinically and by in vitro analysis. Results: Female NelfK O mice had delayed puberty, small uteri, and a decrease in GnRH neuron distribution distance, but male KO mice had normal puberty and GnRH neuron distribution distance. Both male and female KO mice had decreased litter size. Furthermore, NELF variant 2 was the dominant variant in both mouse and human GnRH neurons. Both nuclear and non-nuclear splice variants were expressed at the RNA and protein levels. DNA sequencing of 13 genes in 48 IHH/KS patients demonstrated that -90% of the cases are monogenic. Finally, the ESR1 mutation in the female patient led to absent pubertal development as ESR1 signaling is profoundly defective.
    • Function of TNF Death Receptors in Apoptosis and Cancer Immune Surveillance

      Hu, Xiaolin; Department of Biochemistry and Molecular Biology (2012)
      Tumor necrosis factor receptor (TNFR) is a group of death receptors that can mediate extrinsic apoptosis in target cells upon specific ligand engagement. Members of the TNFR super family play a pivotal role in cytotoxic T cell-mediated immune surveillance and immune regulation. However, cancer cells can acquire apoptosis resistance through down-regulating surface TNFR level or alteration of key mediators in the TNFR signaling pathway so that cancer cells can evade TNFR-mediated and immune cell-based cytotoxicity. Therefore, understanding the underlying mechanism of cancer resistance to TNFR-mediated apoptosis will provide the basis for identifying specific molecular targets and effecient cancer therapy. In this study, we discovered that chronic myelogeneous leukemia (CML) cells use DNA methylation to down-regulate IRF8, a tumor suppressor gene, to acquire apoptosis resistance to Fas-mediated apoptosis. More importantly, we showed that acid ceramidase is a transcription target of IRF8. Restoration of IRF8 or inhibition of acid ceramidase can re-sensitize CML cells to FasL-induced apoptosis in vitro. Furthermore, such manipulation can prolong mouse survival in vivo. While targeting Fas-mediated apoptosis is one strategy to restore immune cell-mediated surveillance, targeting other TNFR members sheds light on monoclonal antibody (mAb) based therapy. We demonstrated that LTβR, as a member of the death receptor super family, could function in cancer immune surveillance through mediating tumor cell apoptosis in sarcoma, colon and mammary carcinoma in a caspase dependent way. Because LTR is selectively up-regulated on cancer cells, it could potentially be a good target for cancer-selective killing. Targeting the TNFR pathway might not only eliminate tumors, but it may also emerge as a promising target to eliminate myeloid derived suppressor cells that accumulate in the peripheral blood under neoplastic conditions. We demonstrated that tumor induced myeloid derived suppressor cells (MDSC) were more resistant to apoptosis than the cells with the same phenotype in tumor free mice. We also identified that up-regulation of Bcl-xL is one of the mechanisms responsible for apoptosis resistance in tumor induced MDSC and that inhibiting Bcl-xL by BH3 mimetics could greately sensitize MDSC to FasL induced apoptosis.
    • Functional analysis of a hematopoietic-specific gene identified using high-throughput tissue-specific cDNA screening in zebrafish

      Marty, Scott D.; School of Graduate Studies (2001-06)
      Zebrafish hematopoiesis begins in an intra-embryonic tissue called the intermediate cell mass (ICM), which specifically expresses the erythroid-specific transcription factor GATA-1. Using a cDNA library constructed from embryonic zebrafish GATA-1- positive cells, we identified a prothymosin alpha-like gene, ProTaL-1. RNA whole mount in situ hybridization showed that ProT~-1 was specifically expressed in the ICM. Microinjection of a construct encoding ProTa.L-1 fused to the green fluorescent protein revealed nuclear localization of ProTa.L-1. Oye~-expression of ProTaL-1 mRNA increased gata-1 expression, whereas knockdown ofProTaL-1 using an antisense, morpholino oligonucleotide resulted in a specific decrease of gata-1 expression in whole embryos. Fluorescence activated cell sorting analy~is of green fluorescent proteinlabeled GATA-I-positive cells from transgenic zebrafish indicated that ProTa.L-1 acted primarily to increase the total number· of ~mbryonic hematopoietic cells. s·ince prothymosin alpha is conserved among species, ProTa.L-1 may play a role in promoting , ' proliferation ofhematopoietic-ptogenitor cells in,higher vertebrates.
    • Functional Characterization of the P53 Family Protein P63 and the EPHA2 Receptor Tyrosine Kinase, a Novel P53 Family Target Gene

      Dohn, Michael Robert; Department of Biochemistry and Molecular Biology (2001-10)
      A cell’s ability to survive as part of a multicellular organism often depends on how well it communicates with other cells in its surrounding environment. The need for coordinated and complementary cellular activities requires a means o f sending and receiving signals to and from neighboring cells. This is achieved by the presence of an assortment of proteins both linked to and within the plasma membrane. The plasma membrane acts as a barrier between the intracellular and extracellular milieu, and plasma membrane proteins allow for much o f the communication between these two environments. An important class o f proteins found on the plasma membrane is the superfamily of receptor protein tyrosine kinases (RPTKs). RPTKs are membrane-spanning proteins, that contain domains on both sides of the plasma membrane, joined by a single transmembrane domain (1). Binding of a growth factor or cytokine to the extracellular domain of an RPTK results in receptor dimerization and autophosphorylation o f specific tyrosine residues on the cytosolic domains (1). These phosphotyrosine residues then act as docking sites for cytosolic proteins containing modular structures such as Srchomology- 2 (SH2) and phosphotyrosine binding (PTB) domains (2). Binding of SH2- and PTB-containing proteins initiates an intracellular signaling cascade that often results in the regulation of transcription factors in the nucleus (3-5). It is through these RPTKmediated cascades that many extracellular signals are received by a cell.
    • Functions of NF-κB in the Tumor Microenvironment

      Redd, Priscilla Simon; Department of Biochemistry and Molecular Biology (11/7/2017)
      NF-κB is a master transcription factor whose signaling pathway regulates the expression of genes involved in a substantial number of pathways including immunity, cell survival, cell death, inflammation, and proliferation. NF-κB has been shown to promote and suppress tumor development, however, the molecular mechanism underlying this contrasting role of NF-κB is unknown. Our central hypothesis is that the relative functions of NF-κB in immune cells and tumor cells control the dynamic interactions between immune and tumor cells in the tumor microenvironment to dictate tumor progression or regression. To test this hypothesis, we studied NF-κB expression and function in myeloid cells, T cells, and tumor cells. Here we provide evidence for the specific roles of NF-κB in 1) iNOS expression in myeloid and tumor cells and 2) radiation-induced TNFα and Fas expression in tumor cells. In the first publication, we found that iNOS is expressed in colon carcinoma cells and tumor-infiltrating immune cells. We not only show that NF-κB is specifically binding to the nos2 promoter in both human colon carcinoma cells and murine myeloid cells, but specifically a p65/p65 and p50/p50 homodimer is binding to the nos2 promoter in myeloid cells. In the second publication, we found that radiation induces rapid activation of NF-κB in human soft tissue sarcoma. Specifically, radiation is activating the p65/p50 heterodimer and p50/p50 homodimer that is then binding to TNFα and FAS promoter sequences. Using a syngeneic mouse model, irradiated tumors had a significant reduction in tumor growth and induced a Th1/Tc1 T cell response, shown by an increase in signature genes of T cells in which NF-κB has been reported to be a transcriptional activator. NF-κB therefore acts as a molecular link between tumor cells and immune cells in the tumor microenvironment in irradiated tumors. In summary, our data indicate that the different NF-κB dimers regulate the expression of both tumor suppressors such as Fas and tumor promoters such as iNOS. Therefore, the cellular context-dependent NF-κB dimer composition might underlie the contrasting NF-κB functions in tumor promotion or suppression through regulating the expression of genes with opposite functions during tumor development.
    • Gamma-diketone neuropathy : evaluation of the energy hypothesis

      Pearson, Jerry K; Department of Anatomy (1988-12)
    • [Gamma]-glutamyl transferase from hog kidney

      Pace, Judith Lee; Department of Cell and Molecular Biology (1970-05)
    • A Gata Ten Motif Found in the b-Globin LCR Possesses Insulator and Silencer Properties

      Ramchandran, Ramani; Department of Biochemistry and Molecular Biology (1997-08)
      In gene transfer experiments, transgene expression has been reported to be influenced by host sequences proximal to the integration sites. Such influences have been referred to as position effect of gene expression or position effect variegation (PEV) (Milot et al., 1996). The position effect (PEV) poses significant problems in the study of transgene regulation. However, studies on the human P-globin gene locus have shown that a 20 kb Locus Control Region (LCR) shields a as-linked transgene from position effects and thus confers position-independent expression of the transgene in transgenic mice (Grosveld et al., 1987). The LCR regulates the expression of the far downstream ay-linked P-like globin genes during erythroid development (Townes et al., 1985; Behringer et al., 1990; Enver et al., 1990). The 20 kb P-LCR is defined by four erythroid specific, DNase I hypersensitive sites, HS1, 2, 3, 4 and a unique HS5 site (Tuan et al., 1985; Forrester et al., 1987; Grosveld et al., 1987; Dhar et al., 1990). The unique HS5 site is located upstream of the HS 1-4 sites, at the apparent 5' border of the LCR (Tuan et al., 1985; Dhar et al., 1990). Like the boundary element of the chicken lysozyme gene (Steif et al., 1989), the HS5 site is located in a span of DNA found to be associated with the nuclear scaffold (Jarman et al., 1988). More recently, a 3 kb DNA fragment containing the HS5 site has been reported to possess insulator activity (Li et al., 1994; Yu et al., 1994). Insulators are DNA sequence elements that block activation of a promoter by an enhancer, but only when placed between them (Eissenberg et al., 1991). When placed upstream of an enhancer, an insulator shields transgene expression from position effects. HS5 fits this definition. When used to flank 5' or 3' of a transgene in transfected plasmids, the 3 kb HS5 fragment was capable of shielding the expression of the transgene from interference by host DNA proximal to the integration sites. Furthermore, when interposed between the promoter and enhancer of the transgene, the HS5 fragment was found to block enhancer-promoter communication, thus repressing the activity of the m-linked enhancer (Chung et al., 1993; Li et al., 1994). The properties of the HS5 fragment are similar to the insulator activity of the scs (Kellum et al., 1991; 1992) and gypsy (Cai and Levine, 1995; Roseman et al., 1993) elements of Drosophila and the A element of the chicken lysozyme gene locus (Steif et al., 1989). DNA Sequencing of the region between HS5 and HS4 has revealed a 40 bp DNA which consists of ten tandem GATA motifs found within 1 kb 3' of HS5 in the human genome (Figure 1). The (GATA)10 motif is unique in the 73 kb P-like globin gene domain, using a computer-aided analysis designed by Hardison et al. (1994). This (GATA)10 motif was named 5a. The question under study is whether or not the 5a sequence located proximal to HS5 contributes to the insulator function of HS5. In this study, we sought to characterize the function of 5a in recombinant plasmids, using transient and stable transfection assays. The experimental results show that: 1. When 5a was spliced either upstream or downstream of the HS2 enhancer in recombinant CAT plasmids, it repressed enhancer activity in erythroid K562 and HEL cells. 2. In non-erythroid N-Tera cells, 5a repressed HS2 enhancer activity when it was spliced upstream of the HS2 enhancer; however, 5a activated the HS2 enhancer when it was spliced downstream of the enhancer. 3. Further, the 5a sequence also possessed silencer activity (Brand et al., 1985). When spliced upstream of the promoter of the housekeeping phosphoglycerate kinase gene (Adra et al., 1987) in the enhancer-less plasmid, 5a did not activate but repressed promoter activity in both erythroid and non-erythroid cells. 4. Electrophoretic mobility shift assays using both K562 and N-Tera nuclear extracts showed that the 5a sequence is bound by the transcription factor GATA-1 but not detectably by GATA-2. 5. Also, mutation of the GATA motifs in the 5a sequence from a Head-Tail to a Head-Head orientation did not alter the function of the 5a sequence but affected the binding affinity for the GATA-1 transcription factor. The results suggest that in erythroid K562 and HEL cells the 5a sequence possesses both insulator and silencer activities and that transcription factor GATA-1 bound at 5a is a repressor of the cfr-linked promoter and the HS2 enhancer. From these findings we conclude that the 5a/GATA-l complex represses the activities of a proximal promoter and enhancer and contributes to the observed insulator activity of the HS5 fragment.
    • Gene regulation by the putative Campylobacter jejuni diguanylate cyclase CbrR

      Fulmer, Claudia; Department of Biochemistry and Cancer Biology (Augusta University, 2020-05)
      As a leading cause of bacterial gastroenteritis, Campylobacter jejuni incurs health care costs estimated at $290 million a year in the US and up to 40,000 deaths in children aged 5 and younger worldwide. As such, determining those proteins that regulate C. jejuni virulence factors are prime targets to possibly develop a prophylactic therapy, that as of yet does not exist. CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), the class of enzyme that catalyzes the synthesis of cyclic di-GMP, a universal bacterial second message molecule, from GTP. In C. jejuni strain DRH212, an unmarked deletion mutant, cbrR-, and complemented mutant, cbrR+, were constructed. Soft agar motility tests, biofilm formation assays, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were performed. Site-directed mutagenesis was performed on cbrR to make alanine substitutions in both the autoinhibitory site (I-site) and active site and differential radial capillary of action ligand (DRaCALA) assays were performed to determine nucleotide binding by wild-type CbrR and the CbrR point mutants. Soft agar motility assays indicated a hyper-motile phenotype associated with the C. jejuni cbrR- mutant, whereas motility was all but negated in the cbrR+ complement. Biofilm assays and SEM demonstrated similar formation and robustness of biofilms between wild type and cbrR- mutant, however cbrR+ was unable to form significant biofilms in 72 hours. TEM images showed similar cellular morphology between cbrR-, wild type, and cbrR+, however cbrR+ cells had fewer flagella. DRaCALA assays showed wild-type CbrR and the active site mutant were both able to bind GTP and cyclic di-GMP, whereas the I-site mutant lost the ability to bind cyclic di-GMP, indicating the product binding site on CbrR. The highly conserved diguanylate cyclase CbrR is the only annotated DGC in the C. jejuni genome. Though the active site sequence is highly variant when compared to the consensus sequence, this protein is able to bind both substrate and product of the chemical synthesis of cyclic di-GMP and has now been shown to be a negative regulator of motility, a critical virulence factor in C. jejuni pathogenesis.
    • Gene-Environment Interaction Modulates Schizophrenic Endophenotypes in Heterozygous Reeler Mice

      Howell, Kristy R.; Department of Psychiatry and Health Behavior (2013-07)
      Aim 1: To determine the effect of chronic stress on the VEGF signaling pathway. Aim 2: To determine the effects of prenatal hypoxia on VEGF signaling, behavioral activities, blood flow, and brain volume in heterozygous reeler mice during early adulthood. Aim 3: To evaluate long lasting effects of prenatal hypoxia on VEGF signaling, behavioral activities, blood flow, and brain volume in heterozygous reeler mice. Aim 4: To determine the correlation between serum VEGF levels and brain volumes in schizophrenia subjects.
    • Genetic labeling reveals novel cellular targets of schizophrenia susceptibility gene ERBB4 and neuregulin-1 – ERBB4 signaling in monoamine neurons

      Bean, Jonathan C; Department of Neuroscience and Regenerative Medicine (2015)
      Neuregulin 1 (NRG1) and its receptor ErbB4 are schizophrenia risk genes. NRG1-ErbB4 signaling plays a critical role in neural development and regulates neurotransmission and synaptic plasticity. Nevertheless, its cellular targets remain controversial. ErbB4 was thought to be expressed in excitatory neurons although recent studies have disputed this view. Utilizing mice that express a fluorescent protein under the promoter of the ErbB4 gene, I determined in what cells ErbB4 is expressed and their identity. ErbB4 was widely expressed in the mouse brain, being highest in amygdala and cortex. Almost all ErbB4-positive cells were GABAergic in cortex, hippocampus, basal ganglia, and most of amygdala in neonatal and adult mice, suggesting GABAergic transmission as a major target of NRG1-ErbB4 signaling in these regions. Non-GABAergic, ErbB4-positive cells were present in thalamus, hypothalamus, midbrain and hindbrain. In particular, ErbB4 was expressed in both dopamine neurons in the substantia nigra and ventral tegmental area and in serotoninergic neurons of raphe nuclei, but not in norepinephrinergic neurons of the locus coeruleus. In hypothalamus, ErbB4 was present in neurons that express oxytocin. ErbB4 was expressed in a group of cells in the subcortical areas that are positive for S100β. These results identify novel cellular targets of NRG1-ErbB4 signaling. Finally, perfusion of NRG1 into the medial prefrontal cortex enhanced both dopamine and serotonin release but with differing time courses.
    • Genetic Modeling and Pathophysiological Analysis of FAM109A, a Putative Human Disease Gene

      Ates, Kristin Marie; Department of Neuroscience and Regenerative Medicine (Augusta University, 2019-05)
      A critical barrier in the treatment of endocytic diseases is the lack of information and understanding of the in vivo mechanisms of endocytosis. Part of this is due to the diverse array of endocytic adaptor proteins that have not yet been studied. We address this by investigating a key endocytic adaptor protein, FAM109A, which interacts with OCRL1, a causative gene for Lowe syndrome. Previous in vitro studies have identified FAM109A as a regulator for endosomal trafficking, particularly in the recycling of receptors in endosomes and sorting of cargo to lysosomes, based on knock-down studies. Here we conduct the first study into the developmental and physiological functions of FAM109A in vivo, utilizing the zebrafish model. We find that depletion of both zebrafish orthologs, zFAM109A and zFAM109B, in our maternal-zygotic homozygous mutant models (AB mutant) disrupts fluid-phase endocytosis and ciliogenesis in the pronephros. Partial knockdown of OCRL1 in the AB mutants exacerbates the endocytosis deficit, confirming that OCRL1 and FAM109 proteins are linked in a common endocytic pathway. In addition, we discover that zFAM109A/B mutant animals exhibit reduced jaw size and delay in chondrocyte maturation, indicating a novel role for zFAM109A and zFAM109B in craniofacial development. This is consistent with the phenotype in a patient within the NIH’s Undiagnosed Diseases Program (UDP). The UDP patient carries a de novo arginine (R) to cysteine (C) mutation (R6C) in FAM109A and presents with craniofacial abnormalities, developmental delay, auditory and vision impairments, and renal dysfunction. Expressing zFAM109A with the R6C mutation in zebrafish exacerbated craniofacial deficits, suggesting that the R6C allele acts in a dominant-negative manner. Together, these results show that FAM109A is involved in fluid-phase endocytosis and ciliogenesis in vivo. Moreover, we provide further insight into the potential pathogenesis of a UDP patient’s disease in association with a de novo mutation in FAM109A.
    • Genetic mutations cause primary aldosteronism

      Hattangady, Namita G; Department of Physiology (2014-10)
      The human adrenal glands are complex endocrine organs that are physiologically located above the kidney. The cortex of the adrenal gland may be considered as a combination of three different steroidogenic tissue-types which form concentric zones within each adrenal. The three cortical zones include zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR). Each zone, under independent regulation, produces unique steroid(s) which exhibit specific functions. The outermost ZG layer secretes the steroid, aldosterone due to ZG specific expression of aldosterone synthase (CYP11B2). Aldosterone regulates sodium reabsorption, and therefore, blood pressure. Aldosterone production is tightly regulated by the renin-angiotensin-aldosterone system. Thus, aldosterone levels are in direct proportion with renin levels. Other known physiological regulators of aldosterone production include serum K+ and adrenocorticotrophic hormone. A type of endocrine hypertension termed ‘Primary Aldosteronism’ (PA), is characterized by aldosterone secretion under suppressed renin levels. PA accounts for almost 10 % of hypertension. More recently, genetic mutations in an inward rectifying K+ channel (KCNJ5) that occur as both, somatic and germline cases, have been implicated in the pathology of PA. The goal of this dissertation is to define the role of KCNJ5 mutations in PA. In this dissertation, I will summarize my studies that describe the acute and chronic events involved in mutated KCNJ5 mediated aldosterone excess. In addition, I will define a novel mutation in KCNJ5 of germline nature identified at Georgia Regents University. Finally, I will also describe some interesting lessons we learnt from the expression of mutated KCNJ5 in primary cultures of human adrenals. The prevalence of a hereditary form of PA termed as Familial Hyperaldosteronism type III (FH III) is very rare. Thus far, only a few mutations in the KCNJ5 gene, including T158A, G151R, G151E and I157S, are confirmed as causing FH III, following Mendelian genetics. Perhaps the most interesting feature of this disease is the varied phenotype between the different mutations. T158A-affected patients present with massive hyperplasia and require bilateral adrenalectomy. In contrast, patients affected by the G151E mutation have more severe hypertension, although their adrenals are near normal in appearance. In this study we identify a new germline mutation (Y152C). The index case was a 61 year old woman who underwent unilateral adrenalectomy. The patient with the Y152C mutation exhibited a milder hypertension phenotype (like the G151E-affected patient) with extensive hyperplasia (as seen in the T158A-affected patient). In vitro analyses of the Y152C mutation indicated a pathology similar to other known mutations in KCNJ5, including change in conductance to Na+ ions and elevated calcium levels, and increase in CYP11B2 mRNA and aldosterone production. The inherent challenge presented by current studies utilizing constitutive expression of KCNJ5 mutations is the limitation in studying acute temporal events such as post translational modifications of steroidogenic enzymes and transcription factors. To address this issue, we generated a doxycycline inducible cell model system for the T158A harboring KCNJ5 transgene. Herein, we demonstrate a useful system that was amenable to the study of acute and chronic events involved in mutant-KCNJ5 mediated aldosterone excess. Our findings suggest that mutant KNCJ5 increases CYP11B2 expression through the activation of transcriptional activators of CYP11B2. Additionally, this is the first study to demonstrate that mutant KCNJ5 also activates steroidogenic acute regulatory protein (StAR) at the levels of translation and post translational phosphorylation. We also demonstrate calcium channel blocker, verapamil as an efficient blocker of mKCNJ5 mediated aldosterone production. Finally, one of the sharp advantages of our study was the use of primary cultures of human adrenal cells to confirm the effects of mutated KCNJ5. Interestingly, transduction of cells with constitutive viruses for mutant KCNJ5, confirmed an increase in KCNJ5 mRNA, although no change in CYP11B2 expression levels was observed. Pilot data including treatment of primary cells with calcium ionophores indicated that ZF/ZR cells may have a phenotype that is ‘muted’ for calcium mediated pathways. We could also speculate that this may disprove some current hypotheses that APA harboring KCNJ5 mutations may originate from the ZF. Overall, this study has improved our knowledge regarding the pathogenesis of PA caused by KCNJ5 mutations and has identified verapamil as a potentially effective therapeutic strategy in the inhibition of aldosterone excess in this type of PA.
    • The Genetics of the Hemoglobins of the Domestic Goat

      Ray, Adams H.; Department of Cell and Molecular Biology (1970-09)
    • Genomic and Functional Analysis of Vesicular Inhibitory Amino Acid Transporter During Mouse Embryogenesis

      Oh, Won-Jong; Institute of Molecular Medicine and Genetics (2006-01)
      The specification of particular neuronal phenotypes during embryonic development requires the appropriate activation and regulation of genes encoding the proteins required for neurotransmitter synthesis, vesicular packaging and re-uptake from the synaptic cleft. Each neurotransmitter is packaged into synaptic vesicles by its own distinct vesicular transporter. In addition, neurotransmitter packaging is well controlled by other co-factors (reviewed in Ahnert-Hilger et al., 2003). Components of GABAergic neurons GABAergic neurons are the principal inhibitory neurons in the mammalian central nervous system (CNS), where GABA is synthesized from glutamate by two glutamate decarboxylases (GAD), namely GAD65 (Gad2) and GAD67 (Gad1) (Erlander et al., 1991). GABA is then loaded into synaptic vesicles by the vesicular inhibitory amino acid transporter (VIAAT, also known as VGAT). Four GABA transporters (GAT 1-4) are responsible for the re-uptake of GABA from the synaptic cleft through the plasma membrane. Inhibitory GABAergic transmission is mediated by binding of GABA to its ionotropic receptors, GABAA and GABAC, which are ligand-gated chloride channels, and its metabotropic receptor, GABAB (Fig. 1).