Identification and Structural Analysis of the Androgen Receptor From Normal and Androgen Insensitive Human Fibroblasts

Resistance to androgens in utero and at puberty causes a dysfunction of male sexual differentiation, and results in a form of male pseudohermaphroditism. From whole cell binding studies, using cultured human genital skin fibroblasts (GSF) a number of defects in the androgen receptor have been discovered, these have been classified as: absent, deficient, thermolabile, defective activation to the DNA/nuclear binding form, and finally, failure to "up-regulate" the basal binding level in response to prolonged (i.e. 24h) incubation of cells with hormone. There is, however, a need to study the receptor protein directly, without relying on the reversible binding of 3H ligand. Therefore, in the present study the androgen receptor from human GSF was extracted using 0.5M-KCl, and partially purified by 35% ammonium sulphate preciptitation prior to further studies

Role of Redox-Active Trace Metal (RATM) Oxidants in the Regulation of Toll-Like Receptor 4 (TLR4) Signaling-Mediated Inflammatory Phenotype in Synovial Fibroblasts

Title from PDF of title page viewed December 18, 2019Dissertation advisor: Orisa J IgweVitaIncludes bibliographical references (pages 207-246)Thesis (Ph.D.)--School of Pharmacy. University of Missouri--Kansas City, 2018While the involvement of oxidant stress in rheumatoid arthritis (RA) has been suspected, the role of redox-active trace metals (RATM) as exogenous pro-oxidants in the pathogenesis of RA has not yet been investigated. Evidence suggests that oxidant-induced Toll-like receptor 4 (TLR4) activation plays a significant role in initiating “sterile” inflammation. Here, we investigate for the first time the role of RATM-induced oxidant stress in the molecular mechanism of the pathogenesis of RA. Potassium peroxychromate (PPC) (Cr⁺⁵), cuprous chloride (Cu⁺), and ferrous chloride (Fe⁺²) RATM agents were used as exogenous sources of reactive species. LPS-EK as a TLR4 specific agonist was used as a positive control for TLR4 activation. Given the importance of synovial fibroblasts in the development of RA, HIG-82, a rabbit fibroblast like-synoviocytes (FLS) cell line, was used as a model system in the studies proposed in this dissertation research. The expression of TLR4 in HIG-82 was confirmed by quantitative PCR (RT-PCR) and Western blots. Intracellular reactive oxygen species (iROS) production was visualized and quantified by fluorescence imaging microscopy and flow cytometry (FC), respectively. Activation of TLR4 signaling pathway was determined by measuring the expression of TLR4 and the downstream signaling proteins. Either ELISA kits or FC quantified levels of TNF-α, interleukin (IL-1β), and HMGB1 (as pro-inflammatory agents), and IL-10 (as an anti-inflammatory mediator) released into the culture medium. Proliferation index of FLS and examination of the effects of RATM on apoptosis and autophagy-related protein levels were quantified by FC and Western blots. We found that (1) RATM induced iROS production, which was attenuated by pretreatment.with antioxidants (2) Similar to TLR4 specific agonist LPS-EK, RATM significantly increased the activity of TLR4, which was blocked by pretreatment with TLR4 signaling inhibitor (CLI 095). (3) To our surprise, RATM increased proliferation of FLS and protected cells against apoptosis through activation of autophagy which is in agreement with the pathophysiological changes that occur in active RA. (4) RATM exogenous RS activate TLR4-mediated different down-stream signaling cascades that lead to an increased production of pro- and anti inflammatory mediators in FLS, and (5) Further studies reveal that RATM exogenous RS treatment increased the expression of all three major MAPK; (Extracellular signal-regulated protein kinase (ERK), the c-Jun N-terminal Kinase (JNK), and P38 MAPK pathways). Moreover, RATM concurrently increased the expression of AP-1 nuclear protein through TLR4 stimulation. Taken together, our findings indicate that TLR4 has mediated RATM induced inflammatory phenotype through AP-1 pathway activation in synovial fibroblasts. Therefore, oxidant stress through TLR4 activation may initiate and propagate inflammatory processes that maintain many chronic diseases. The design of dual-functioning antioxidants possessing both metal chelating and oxidative stress scavenging properties will be an essential milestone in pharmacotherapy and could help us live free of many chronic diseases. For the first time, we present evidence that supports a connection between exogenous and endogenous reactive species in enhancing inflammatory phenotype in synovial fibroblasts which is likely responsible for the initiation, propagation, and maintenance of RA.Introduction -- Review of literature and background -- Central hypothesis and specific aims -- General materials and methods -- Redox-active trace metals (RATM) as pro-oxidants -- Effect of RATM on HMGB1 release and markers of inflamatory phenotypes -- Effect of RATM on cell proliferation and cell death -- Effect of RATM pro-oxidants on TLR4-coupled AP-1 signaling pathway -- Research summary, conclusions, and future directions -- Appendi

Mitochondria: From Physiology to Pathology

Mitochondria play an increasingly central role in the context of cellular physiology. These organelles possess their own genome (mtDNA), which is functionally coordinated with the nuclear genome. Mitochondrial gene expression is mediated by molecular processes (replication, transcription, translation, and assembly of respiratory chain complexes) that all take place within the mitochondria. Several aspects of mtDNA expression have already been well characterized, but many more either are under debate or have yet to be discovered. Understanding the molecular processes occurring in mitochondria also has clinical relevance. Dysfunctions affecting these important metabolic ‘hubs’ are associated with a whole range of severe disorders, known as mitochondrial diseases. In recent years, significant progress has been made to understand the pathogenic mechanisms underlying mitochondrial dysfunction; however, to date, mitochondrial diseases are complex genetic disorders without any effective therapy. Current therapeutic strategies and clinical trials are aimed at mitigating clinical manifestations and slowing the disease progression to improve the quality of life of patients. The goal of the Special Issue ‘Mitochondria: from Physiology to Pathology’ published in Life (ISSN: 2075-1729) was to collect research and review articles covering the physiological and pathological aspects related to mtDNA maintenance and gene expression, mitochondrial biogenesis, protein import, organelle metabolism, and quality control