• Recombinant Bone Morphogenetic Protein-2 Induces Up-Regulation of Angiogenesis and Inflammatory Transcripts: Role of Reactive Oxygen Species

      Akeel, Sara K; Department of Oral Biology (2012-12)
      Large bone defects in the oral and maxillofacial region are mostly secondary to tumor resection, gunshot wounds or craniofacial anomalies. Reconstruction of large bone defects remains a clinical challenge despite the ability of bone to regenerate itself after fracture, mainly because bone regeneration requires recruitment of new cells as well as development of new bone tissue in order to restore anatomical and mechanical functions. Several biological and mechanical factors regulate bone formation. Early vascularization plays a critical role in skeletal bone development and bone fracture repair, and without a vascular supply, osteogenesis is impaired (Glowacki 1998; Akeno, Czyzyk-Krzeska et al. 2001; Carano and Filvaroff 2003). Furthermore, in the treatment of bone defects, vascularized bone grafts show less bone remodeling when compared to non-vascularized bone grafts (Cutting and McCarthy 1983; Wang, Yamazaki et al. 1996). This highlights the importance of angiogenesis in bone formation and remodeling. The close proximity of osteoblasts and osteoclasts to endothelial cells during bone formation suggests there is a cross-talk between these cells. Osteogenesis-inducing growth factors, such as bone morphogenetic proteins (BMPs), especially BMP-2 and -7, and vascular endothelial growth factor (VEGF) which is known to have a major role in angiogenesis, have been reported in many studies to play a major role in osteoblast-endothelial cell communication (Mayer, Bertram et al. 2005). The general goal of this study is to understand some of the molecular events that occur at the site of bone healing and the interaction of local growth factors produced by resident cells such as osteoprogenitor and endothelial cells. This will help us in developing techniques that enhance bone formation and provide better integration of bone grafts in the recipient site. Specifically, the aim of this study is to understand the mechanism by which recombinant bone morphogenetic protein-2 (rhBMP-2) induces bone formation and whether or not the effect of rhBMP-2 is through enhancing angiogenesis and inflammation.
    • Recombination and genetic polymorphism at the mouse alpha-globin locus

      Lewis, Jill B; Department of Cell and Molecular Biology (1988-12-08)
      Unusual genetic phenomena are often responsible for dramatic evolutionary changes at many mammalian loci. Among the possible genetic mechanisms involved in these changes are point mutation, gene-conversion, and homologous or nonhomologous recombination. Rarely can such evolutionarily significant events be studied as recent occurrences in mammalian systems. In most case.s it can only be postulated that such events occurred ·in the distant past. In the case analyzed in this research, however, the genetic rearrangement has occurred within the last fifteen years since the origination of a certa~n inbred mouse strain known as AKXL-7. The AKXL-7 recombinant inbred strain is the product of inbred parental strains AKR and C57L. AKR.has the Hba "f" genetic type which specifies only alpha-globin chain 5. C57L has the Hba "a" genetic type that specifies only alpha-globin chain 1. Chains 1 and 5 are identical except for a gly --> ala substitution at position 78. Although one would predict that any recombinant inbred strain resulting from thes,e parents would be homozygous for one of these two alpha-globin types, this is not the case for the AKXL-7 strain. These mice express both alpha-globin chains, with chain 1 present in greater amounts than chain 5. The type of genetic reassortment that has occurred has been ascertained through the use of DNA probes to flanking regions of the two non-allelic or "tandem" alpha-globin 1 genes. Southern blot analysis has revealed that the left and right AKXL-7 alphaglobin gene flanking regions are homologous to regions from different parents. This result indicates that the novel AKXL-7 genotype is the result of a reciprocal recombination event. Further analysis using an intergenic region probe narrowed the region of crossover to approximately 5.2 kb. Using the most distal flanking region probes, chromosome walking was performed to recover probes useful for characterization of three different induced mouse alphathalassemia mutations. Results indicate that in all three cases the deletion spans. at least 45 kb, including both alphaglobin genes and the-embryonic alpha-like x gene.
    • Reduced binding to type I collagen of rat tibiae by transposon-mutated staphylococcus aureus

      Buxton, Thomas Brooks; Department of Immunology and Microbiolgy (1992-03)
    • Regional Variability of Dentin Permeability

      Andringa, Hans-Jan; Department of Oral Biology (1987-08)
    • Regulation and Function of the Major Stress-Induced HSP70 Molecular Chaperone in vivo: Analysis of Mice with Targeted Gene Disruption of the HSP70.1 or HSP70A1

      Huang, Lei; Georgia Cancer Center (6/3/2002)
      (First Paragraph) The cellular response to stress, including exposure to environmental (UV radiation, heat shock, heavy metals), pathological (infection, fever, inflammation, malignancy, ischemia) or physiological (growth factor, hormonal stimulation, tissue development) stimuli is represented at the molecular level by synthesis of groups of protein named heat shock proteins [hsp(s)] (Benjamin 1998; Feder and others 1992; Jolly and Morimoto 2000; Li and Mivechi 1986; Lindquist 1986; Smith 1998). The presence of hsp(s) protect host cells from the damage caused by thermal stress, and after induction of hsp expression, cells are protected well from higher temperatures than they can normally tolerate. This phenomenon is defined as themiotoleranee (Gemer 1975; Li and Mivechi 1986). The protective role of hsp(s) is attributed to several functional properties, including active participation in maintaining proteins in their native correctly folded states, promoting degradation and refolding of misfolded proteins, and minimizing aggregation and incorrect interactions between proteins (Agashe and Hartl 2000; Gething and Sambrook 1992). In addition, hsp(s) can function in cellular protection by modulating the engagement and progression of apoptosis induced by a variety of stress stimuli (Beere and Green 2001). Besides the recognition of the cytoprotective function of hsp(s) under stress conditions, widespread clinical interests exist in their chaperone function during a range of human pathologies, including neurodegenerative conditions, such as amyloidosis, prion disease, and Alzheimer's disease, and cardiovascular diseases, such as myocardial ischemia, cardiac hypertrophy, stroke, and blood vessel injury (Benjamin 1998; Planas and others 1997; Smith 1998).
    • The regulation of aldosterone secretion in the rat

      Lin, Cheng-Hsiung; Department of Endocrinology (1973-06)
    • Regulation of androgen receptor expression by testosterone

      Mora, Gloria R.; School of Graduate Studies (1996-03)
    • Regulation of Cytochrome Oxidase and F1Fo ATP Synthase by Protein Kinase C Isozymes: Implications for Cardiac Preconditioning and Ischemia / Reperfusion Injury

      Nguyen, Tiffany Tuyen M.; Department of Pharmacology and Toxicology (2010-03)
      Despite decades of intense research, heart disease associated with myocardial ischemia/reperfusion (IR) injury remains the most frequent cause of lethality worldwide. It has been known for over 2 decades that the mammalian heart can be protected from IR injury if exposed to a paradoxical response known as cardiac ischemic preconditioning (PC). Of interest, mitochondrial protein kinase C (PKC) isozymes have emerged as central players in both PC and IR mechanisms. Mitochondrial oxidative phosphorylation (OXPHOS) complexes are responsible for greater than 90% of myocardial ATP synthesis, and ATP levels decline substantially during myocardial IR injury. Therefore, we determined if direct protein-protein interactions occurred between individual PKC isozyme and specific subunits of each of these complexes during cardioprotective and cardiac IR responses. First, we have utilized an in situ rat coronary ligation model to establish an PKC-cytochrome oxidase subunit IV (COIV) coimmunoprecipitation (co- IP) in myocardium exposed to PC. This co-IP correlated with a 2.8-fold increase in mitochondrial PKC autophosphorylation (activation) and a 2-fold enhancement of cytochrome-c oxidase activity. In a second line of study, we demonstrated that following prolonged 4- phorbol 12-myristate-13-acetate (PMA) and hypoxia (Hx), PKC interacts with the “d” subunit of F1Fo ATP synthase (dF1Fo) to inhibit F1Fo activity in neonatal cardiac myocytes (NCMs). We next developed cell-permeable, mitochondrial-targeted peptide modulators (derived from the amino acid sequence of dF1Fo) based on the PKCdF1Fo interaction. In vitro binding assays and co-IP experiments using NCMs revealed a facilitator [NH2-YGRKKRRQRRMLATRALSLIGKRAISTSVCRVREYEKQLEKIKNMIDYKDDDK- COOH] and an inhibitor peptide [NH2-YGRKKRRQRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK- COOH] of the PKC-dF1Fo interaction. The inhibitor peptide reduced PMA/Hx-induced inhibition of F1Fo activity or PMA-Hx-induced PKC-dF1Fo co-IP in NCMs while the facilitator peptide has opposite effects. Administration of the inhibitor peptide to isolated rat hearts immediately after a 20 min global ischemia exposure, and just prior to a 90 min reperfusion, decreased infarct size and released of cardiac troponin I compared to rat hearts receiving a scrambled-sequence (inactive) peptide prior to IR exposures alone. Collectively our studies have revealed two key mitochondrial OXPHOS regulatory events involving PKC-enhancement and protection of cytochrome oxidase by ischemic PC and PKC suppression of F1Fo ATP synthase during IR injury. Further, our work suggests that the PKC-dF1Fo inhibitor peptide may have potential as a therapeutic compound targeting myocardial ischemia in humans.
    • Regulation of Embryonic Stem Cell Pluripotency and Differentiation by Notch Signaling

      Noggle, Scott A; Institute of Molecular Medicine and Genetics (2004-05)
      (First Paragraph) Mammalian development prior to implantation is primarily involved with establishing the support tissues needed for interaction with the mother’s uterine tissue and blood stream as the uterine tissue prepares to receive the embryo. Development of the embryo proceeds slowly while initiating differentiation of the first cell lineages, trophectoderm and the primitive endoderm, that are necessary for interactions with the mother’s uterine tissue. In mice, development from fertilization to the initiation of implantation of the blastocyst into the uterine wall takes about four and a half days (Fig. 1) whereas this period in human development requires about 7 days. This is in contrast to the much-accelerated development, for example, of amphibian species that produce embryos that develop in isolation of maternal support after fertilization.
    • Regulation of embryonic stem cell pluripotency and differentiation by notch signaling

      Noggle, Scott A.; School of Graduate Studies (2004-05)
      Mammalian development prior to implantation is involved with establishing the support tissues needed for.interaction with the maternal blood stream. Molecular studies of these processes are in early stages. Embryonic stem cells (ESCs) derived from the inner cell mass of preimplantation embryos offer a window into the molecular biology· of preimplantation development. While many studies have explored the potential for differentiation into germ layer derivatives, there are few examples that have explored the ability of ESCs to differentiate into extraembryonic lineages. I have used two ESC model systems to study the molecular basis of extraembryonic lineage development. Human ESC (HESC) lines derived from human blastocysts seem to require cell-cell interactions to be maintained in an undifferentiated state. However, the passaging techniques currently in use can result in heterogeneous cultures as assayed by pluripotency markers. Here I show that there is spontaneous differentiation of extraembryonic cell types resembling primitive endoderm in HESC cultures. The Notch signaling pathway has been shown to control many developmental processes. The Notch pathway is present and can be activated in undifferentiated HESCs. However, the pathway may not be activated in undifferentiated HESCs and may instead be involved in the differentiation of primitive endoderm. Notch expression is regulated upon differentiation but constitutive signaling could not induce differentiation. Notch is activated in the primitive endoderm population of HESC-derived embryoid bodies. Inhibition of y-secretase reduces the proportion of differentiating HESCs. Thus, inhibition of y-secretase mediated Notch signaling may improve the homogeneity of HESCs and suggests a molecular mechanism that controls early human development. In the mouse ESC system, primitive endoderm can be efficiently removed from mouse embryoid bodies by hypotonic lysis to isolate primitive ectoderm-like cells. Lysed embryoid bodies maintain a primitive ectoderm marker in a defined serum-free medium and under defined conditions may form neurectoderm. This technique may allow isolation of an alternate pluripotent cell type in vitro and has implications for homogeneous differentiation of embryonic cell types for cell therapy applications.
    • Regulation of Endothelial Nitric Oxide Synthase by Subcellular Localization and

      Zhang, Qian; Department of Pharmacology and Toxicology (2007-04)
      Endothelial nitric oxide synthase (eNOS) is regulated by post-translational modifications that target eNOS to the plasma membrane (PM) and the perinuclear/Golgi region. It has been shown in COS-7 cells that targeting of eNOS to the Golgi or PM regulates the mechanism and degree of eNOS activation. However, little is known about the functional significance of eNOS targeting in endothelial cells (ECs). Our first goal was to isolate these two pools of enzyme in ECs and determine their functional significance in response to agonist stimulation and manipulation of membrane cholesterol levels. Using an RNAi strategy, we generated stable populations of EC that had greater than 90% inhibition of eNOS expression and lacked the ability to produce NO. Reconstitution of these eNOS “knockdown” EC with Golgi and PM targeted eNOS restored the ability of EC to produce NO. This approach can be broadly applied to endothelial cells from a number of different species and from different vascular beds and should have broad utility. Using these cells we found that the PM is the optimal location within the cell to produce NO, but it is also the most vulnerable to changes in cholesterol and oxidized LDL. Calcium-dependent agonists were the more efficient stimulus for the PM-restricted eNOS in EC. In contrast, Golgi eNOS was less responsive to both calcium and Akt-dependent agonists. The functional significance of the increased NO produced by the PM eNOS is reflected in the greater ability to elicit endothelium-dependent relaxation, greater suppression of vWF secretion, a key regulator of platelet aggregation, and inhibition of endothelial cell proliferation. Mechanistically, PM eNOS induces more nitrosylation of proteins such as NSF, but this is related to the amount of NO being produced, rather than its intracellular location. Increased superoxide formation in endothelial cells (ECs) has been identified as a causative factor in endothelial dysfunction by reducing nitric oxide (NO) bioavailability, uncoupling eNOS. A major source of intracellular superoxide is the NADPH oxidase (Nox) family of enzymes. In experiments to address the effect of superoxide on local eNOS activity, we found that Nox5 increased eNOS activity paradoxically in both cotransfected COS-7 cells and transduced bovine aortic ECs determined by chemiluminescence to measure the NO metabolite. Nox5 also activated eNOS in human aortic ECs as detected by a cGMP reporter assay that measured the release of biologically functional NO from cells in the presence of superoxide dismutase (SOD). To establish the functional significance of this observation in blood vessels, the endothelium of mouse aorta was tranduced with Nox5 or control adenoviruses. Nox5 potently inhibited Achinduced relaxation, potentiated contractile responses to phenylephrine. In precontracted blood vessels, acute exposure to SOD induced significant vascular relaxation in vessels exposed to Nox5 versus control and unmasked the ability of Nox5 to activate eNOS in blood vessel endothelium. These results are in contrast to a number of described mechanisms for eNOS inhibition and provide valuable clues that in complex diseases such as diabetes and hypertension that ROS production is not the sole cause of endothelial cell dysfunction.
    • Regulation of GluN2C-Containing N-methyl-D-aspartate (NMDA) Receptors

      Chung, Connie; Department of Neuroscience and Regenerative Medicine (6/3/2016)
      NMDA receptors (NMDARs) play a major role in the pathological events following excitotoxicity. Post-ischemic activation of NMDARs has been linked to opposing signaling that mediates pro-survival or pro-death activity. This dichotomy is largely due to distinct GluN2 subunit compositions governing important receptor functions including channel properties, receptor trafficking, and synaptic localization. Compared to GluN2A- and GluN2B-containing NMDARs, the trafficking of GluN2C in non-cerebellar granule neurons is less well understood. Moreover, the role of GluN2C following cerebral ischemia remains unknown. Here, we report 14-3-3 isoform-specific binding and regulation of GluN2C. Our findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission. Next, we sought to investigate the role of GluN2C following cerebral ischemia. We show that GluN2C expression promotes neuronal survival as a homeostatic mechanism by which intracellular Ca2+ levels are maintained by upregulation of GluN2C. Through such a mechanism, not only the intracellular Ca2+ level but also NMDAR signaling can be maintained at equilibrium.
    • Regulation of Granulosa Cell Proliferation in the Rat

      Cannon, Jennifer D.; Department of Biological Sciences (2006-08)
      The growth and maturation of the ovarian follicle is central to reproductive cyclicity in females and is under the endocrine control of the pituitary gonadotropins FSH and LH, as well as locally produced steroids and growth factors. A fundamental aspect of follicle development is proliferation and differentiation of granulosa cells, although the precise temporal dynamics and regulation remain largely unknown. FSH acts through its receptor to stimulate estradiol synthesis, which together with FSH induces proliferation of the granulosa cells in preantral and small antral follicles (95). In contrast, the LH surge initiates ovulation and formation of the corpus luteum (luteinization) in preovulatory follicles. Luteinization has been considered the terminal differentiation of granulosa to luteal cells and has been associated with a rapid reduction in granulosa cell proliferation (36, 114). However, more recent evidence suggests that (a) cell cycle control and luteinization are not functionally and/or mechanistically linked (113, 137), and (b) luteinization is associated with additional granulosa cell proliferation (100, 124, 126-128). The data presented herein challenge the dogma that granulosa cell proliferation is strictly associated with follicle growth and exit from the cell cycle is strictly associated with the LH surge and luteinization. The role of granulosa cell proliferation and differentiation in follicular development and luteinization is only just now beginning to be elucidated. Several ovarian pathologies are associated with defects in these processes, including luteinized unruptured follicle syndrome and polycystic ovarian syndrome (PCOS). PCOS, in particular, is a serious public health issue, affecting anywhere from 3.4% to 6.8% of women (138). Among females undergoing assisted reproductive technology (ART) cycles in the United States in 2003, 6% had been diagnosed with ovulatory dysfunction (139). Thus, pathologies associated with follicular development and luteinization have the potential to afflict large numbers of reproductive-age women. Further, the use of controlled ovarian stimulation (COS) cycles to generate large numbers of oocytes for use in ART procedures is fraught with problems, such as a shortened luteal phase as well as a heterogeneous population of antral follicles likely differing in size, health, and oocyte quality (140) all affecting the rate of pregnancy from these procedures. For example, of the 122,872 ART cycles performed in 2003, only 35, 785 (-30%) resulted in a live birth (139). Finally, a better understanding of the processes that govern granulosa cell proliferation has important ramifications for ovarian cancer, especially rare juvenile granulosa cell tumors that are nevertheless associated with a high mortality (141).
    • Regulation of Granulosa Cell Proliferation in the Rat

      Cannon, Jennifer D.; School of Graduate Studies (2006-08)
      The growth and maturation of the ovarian follicle is central to reproductive cyclicity in females. A_ fundamental aspect of follicle development is proliferation and differentiation of granulosa cells although the precise temporal dynamics and regulatory mechanisms remain largely unknown. Using the PMSG-primed immature rat as a model to study follicle growth, we show a decline in mural granulosa cell proliferation of large antral follicles prior to an ovulatory stimulus, although cumulus granulosa cells continue proliferating up to 10 h after an ovulatory bolus of hCG. Expression and activity of CCNE1-CDK2 correlates with granulosa cell cycle progression suggesting that regulators of this complex are likely to be key mediators of granulosa cell proliferation. Because of the spatial and· temporal organization of granulosa cell proliferation and differentiation within the follicle, we examined the relationship betw~en these two processes using both an in vivo and in vitro approach. By forcing proliferation in both of these models, we were unable to prevent an increase in markers of differentiation or in hCG/ forskolininduced markers of luteinization suggesting the uncoupling of granulosa cell proliferation and differentiation and/ or luteinization. The findings from these studies could have major implications hi contraceptive development, infertility treatment, and understanding and treatment of granulosa cell tumors.
    • Regulation of Pharyngeal Region Patterning and Organogenesis by Sonic Hedgehog

      Moore-Scott, Billie A.; Department of Biochemistry and Molecular Biology (2005-03)
      Patterning of the pharyngeal region determines the proper development of multiple organs in addition to structures of the face, head and neck. The thymus and parathyroid originate from the third pharyngeal pouch and regulate the development of the immune system and calcium homeostasis, respectively. Although functionally these organs are in no way similar, they are derived from a common endodermal primordium, which develops around E l 1.0-El 1.5 in the mouse. At this stage it is apparent that the rudiment has regionalized into the parathyroid and thymus specific domains. Sonic hedgehog (Shh) is a secreted molecule that contributes to the patterning and regionalization of multiple tissues throughout embryonic development. As a morphogen, Shh initially specifies heterogeneous cell types within a field of cells, regionalizing the target tissue into functional domains. Shh activity can also regulate the proliferation and/or survival of those same cells continuing to contribute to the development of the tissue both morphologically and functionally. Since Shh expression is present in the pharyngeal endoderm, it was a promising candidate for patterning of the pharyngeal region as well as the regionalization of the common parathyroid/thymus primordium. Since die pharyngeal region is a specialized vertebrate structure from which several endocrine organs are derived, correct patterning of the pharyngeal region is important for these organs to initiate and develop properly. Our analysis of the Shh'A mutant has revealed multiple organ phenotypes which are die result of die necessity for Shh signaling at multiple stages of development in the pharyngeal region. These include loss of pharyngeal pouch identity, atrophy of the first pharyngeal arch, parathyroid agenesis, dysmorphic and ectopic growth of the thyroid; and thymic hypoplasia and improper differentiation of the thymic epithelial cell microenvironment. This dissertation is a description of our analysis of the early patterning and organogenesis phenotypes of the Shh'A mutant. Based on these data we propose a model describing the Shh-dependent mechanisms that regulate these events in the mid-gestation staged mouse embryo.