Browsing by Subject "gene expression"
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Item Effects of the Pleiotropic Gene csrA on Glycogen Metabolism in Escherichia Coli(1995-06-01) Yang, Honghui; Ming-Chi Wu; Wayne NicholsonYang, Honghui, Effects of the pleiotropic gene csrA on glycogen metabolism in Escherichia coli. Master of Science (Biomedical Sciences), June, 1995, 78 pp., 3 tables, 17 illustrations, bibliography, 59 titles. The csrA gene negatively regulates the expression of four genes glgB, glgC, glgA and glgS involved in glycogen synthesis. It also negatively regulates glgY, which encodes the enzyme glycogen phoshorylase involved in glycogen degradation, but no effect was observed on the glycogen debranching enzyme in this pathway. In addition, csrA exhibits a positive effect on the glycolytic enzyme triosephosphate isomerase. No significant effects were observed on the expression of two genes (zwf & gnd) participating in the pentose phosphate pathway. In vitro expression of glgB, glgC and glgA was specifically inhibited by cell extracts containing the csrA gene product (CsrA). This study provides evidence that csrA encodes an important regulator of intermediary carbon metabolism in Escherichia coli.Item Identification of Actin Binding Proteins Associated with Cross-Linked Actin Networks(2006-12-01) Mills, Christy E.; Clark, Abbot F.; Yorio, Thomas; Wordinger, Robert J.Mills, Christy E., Identification of Actin Binding Proteins Associated with Cross-Lined Actin Networks. Master of Science (Pharmacology and Neuroscience), December 2006, 95 pp., 9 tables, 16 figures, references, 122 titles. Glucocorticoid therapy can leady to ocular hypertension and glaucoma. The purpose of this study is to examine mechanisms contributing to increased intraocular pressure using tissue culture models of steroid-induced ocular hypertension through identification of specific actin-binding proteins associated with cross-linked actin network (CLANs). Human trabecular meshwork ™ cells were cultured to confluence and treated with dexamethasone or vehicle for 14 days. Total RNA was extracted for gene expression analysis to confirm steroid-induced expression of actin binding proteins in human TM cells. Western blots confirmed expression of actin binding proteins and demonstrated the specificity of selected antibodies. Fluorescence microscopy of treated TM cells showed cytoskeleton rearrangements from linear actin stress fibers to cross-linked actin networks and the position of candidate proteins in relation to CLANs. Dexamethasone treatment of TM cells altered the expression of actin-associated proteins that may be important in the formation of CLANs and increased outflow resistance.Item Intercellular Nef transfer and HIV-1 infection of astrocytes(2015-05-01) Luo, Xiaoyu; He, Johnny J.; Ghorpade, Anuja; Wordinger, Robert J.Acquired immune deficiency syndrome (AIDS) is a pandemic caused by human immunodeficiency virus type 1 (HIV-1). It is a major health issue in many parts of the world ever since its discovery in 1981. The most devastating effect of HIV-1 infection is the graduate loss of CD4+ T cells, which eventually leads to the dysfunction of the immune system, susceptibility to opportunistic infections and cancer. HIV-1 Nef protein is long known as an essential pathogenic factor for HIV-1/AIDS pathogenesis. A few recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, the underlying mechanism of intercellular Nef transfer is in dispute. In the first part of our study, we characterized two potential underlying mechanisms for intercellular Nef transfer: direct cell-cell contact and exosomes using several complementary strategies and a panel of exosomal markers. First, we showed that Nef was transferred from Nef-expressing or HIV-infected CD4+ T lymphocytes to CD4+ T lymphocytes and astrocytes, and that the transfer was mainly associated with tunneling nanotube formation. Then we determined that Nef enhanced virological synapse formation and induced cytoskeleton re-arrangement and cell surface protrusions, suggesting that Nef promotes the establishment of intercellular connection and communication between infected cells and uninfected cells. Thirdly, we examined the possibility of Nef transfer through exosomes. In the exosome uptake assay, Nef transfer was undetectable while exosome marker CD81 transferred rapidly. In contrast, Nef was detected in crude exosomes collected from Nef-transfected 293T. In addition, two different populations of exosomes were successfully separated by OptiPrep gradient fractionation and determined as AChE+/CD81low/TSG101low exosomes and AChE- /CD81high/TSG101high exosomes. We determined that Nef was selectively secreted into the AChE+/CD81low/TSG101low population. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out the cell. Taken together, this study shows that Nef transfer requires direct cell-cell contact such as tunneling nanotubes, not cell-free exosomes. In addition, this study reveals existence of two types of exosomes: AChE+/CD81low/TSG101low exosomes and AChE/CD81high/TSG101high exosomes. In the second part, we characterized HIV-1 infection of astrocytes. Astrocytes are the most abundant cells in the central nervous system (CNS) and play important roles in HIV-1/neuroAIDS. Detection of HIV-1 proviral DNA, RNA and early gene products but not late structural gene products in astrocytes in vivo and in vitro indicates that astrocytes are susceptible to HIV-1 infection albeit in a restricted manner. We, as well as others have shown that cell-free HIV-1 is capable of entering CD4- astrocytes through human mannose receptor-mediated endocytosis. In this study, we took advantage of several newly developed fluorescence protein-based HIV-1 reporter viruses and further characterized HIV-1 interaction with astrocytes. First, we found that HIV-1 was successfully transferred to astrocytes from HIV-infected CD4+ T cells in a cell-cell contact- and gp120-dependent manner. In addition, we demonstrated that compared to endocytosis-mediated cell-free HIV-1 entry and subsequent degradation of endocytosed virions, cell-cell contact between astrocytes and HIV-infected CD4+ T cells led to robust HIV-1 infection of astrocytes but retained the restricted nature of viral gene expression. Furthermore, we showed that HIV-1 latency was established in astrocytes. Lastly, we demonstrated that infectious progeny HIV-1 was readily recovered from latently infected astrocytes in a cell-cell contact-mediated manner. Taken together, our studies point to the importance of the cell-cell contact-mediated HIV-1 interaction with astrocytes and provide direct evidence to support the notion that astrocytes are HIV-1 latent reservoirs in the CNS.Item YY1 Mediated Competitve Regulation: A Governing Principle Behind Phenotype-Specific Gene Expression in Vascular Smooth Muscle Cells(2005-07-01) Roberts, Leslie Don; Stephen R. Grant; Neeraj Agarwal; Peter B. RavenLeslie Don Roberts, YYI Mediated Competitive Regulation: A governing principle behind phenotype-specific gene expression in vascular smooth muscle cells. Doctor of Philosophy (Biomedical Science), July 2005. The vascular smooth muscle cell (VSMC) lacks the typical phenotypic restriction that limit most cell-types to expressing a single phenotype, as a result, these cells are uniquely suited to wound repair, as well as, exacerbating several vascular disease-states. While much is known regarding the specific transcription factors that drive phenotype-specific gene expression the mechanisms that regulate the transition between phenotype-specific gene programs remain poorly defined. To further explore these mechanisms, we sought to better understand how VSMCs stably express their default contractile-specific gene program despite the inherent instabilities of their environment. This study explored the regulatory implications of a yet undescribed regulatory domain, that resides with a high-frequency in the promoters of the most contractile-specific gene. These domains, which we term dual regulatory domains (DRD), orient the core binding site for the transcriptional repressor Ying Yang-1 in close proximity to, or overlapping with, the core binding site for a variety of transcriptional activators. This study specifically examines the regulatory implications at two DRD where YY1 competes with the transcriptional activators C/EBPβ (C/CAAT-enhancer binding protein beta). Our findings demonstrate: i.) YY1 acts as a dominant, negative, regulator of the smooth muscle myosin heavy chain (SM-MHC) gene promoter; ii.) YY1 binds to, and repressing from, multiple sites within the regulatory context of this promoter; and iii.) The transactivation potential of C/EBPβ completes with transrepressive potential of YY1 for regulatory control over SM-MHC promoter activity and does so in a stoichiometric-dependent fashion. These findings argue that the relative concentrations of YY1 define the effective dose required of specific transcriptional activators to complete with and override the repressive effect of YY1, and by doing so, directly dictate which genes will be expressed.