• Characterization of the Thioredoxin System in the Diabetic Retina

      Lamoke, Folami; Department of Cellular Biology and Anatomy (2013-07)
      Diabetes is a group of diseases, which are characterized by high blood glucose levels that are a consequence of the inability to produce and/or utilize insulin. Type 1 diabetes (T1D, previously referred to as juvenile diabetes) is typically diagnosed in children and young adults. In this form, the body does not produce insulin primarily due to autoimmune-mediated destruction of pancreatic - cells, leading to insulin deficiency. Type 2 diabetes (T2D, adult onset or noninsulin dependent diabetes) is a chronic condition where the body either resists the effects or does not produce sufficient insulin. This form is more common in African Americans, Latinos, Native Americans, Asian Americans, Pacific Islanders, as well as the aged population.
    • Creating a Selective Advantage for Stem Cells: A Strategy for Gene Therapy

      Menezes, Kareena M.; Department of Cellular Biology and Anatomy (1999-10)
      (Statement of the Problem) Various approaches are used to treat the many known genetic diseases. The treatments are often incompletely effective, and they sometimes have undesirable side effects. Somatic cell gene therapy might provide truly effective permanent cures. Gene therapy, however, is still in the experimental stages, and much needs to be learned about stem cell biology before gene therapy becomes routine clinical practice. Moreover, inferences made from experiments in vitro do not necessarily model the in vitro setting. If treatments designed and tested in vitro can also be made workable and proven to be therapeutic in vivo, a major contribution to clinical gene therapy would be achieved. The described research, which attempts to encourage the stem cells to proliferate rather than divide down the hematopoietic cascade, could be significant in terms of increasing in number those hematopoietic cells that have been successfully modified by therapeutic vectors. The long-term goal of this research is to find a way to provide modified stem cells with a selective advantage in repopulating the marrow of a patient with a genetic disease. Ultimately it will be necessary to confer the selective advantage on somatic cells by introducing DNA into the patient’s defective bone marrow stem cells. However for purposes of preliminary laboratory analyses, a more reproducible system of testing a candidate genes’ potential for providing a selective advantage is necessary. In the present case, an Erythropoietin Receptor transgenic mouse line is used to provide stem cells, each of which already expresses the candidate selective-advantage gene.
    • DEFINING THE ROLE OF TROPOMYOSIN-1C IN CARGO TRANSPORT IN DROSOPHILA

      Boggupalli, Shankarappa Devi Prasad; Department of Cellular Biology and Anatomy (Augusta University, 2020-05)
      Cell polarity is the asymmetric organization of different organelles in a cell, including the plasma membrane and cytoskeleton. Such organization results from asymmetric sorting of proteins, either post-translationally or pre-translationally by messenger RNA localization. In Drosophila oocytes, posterior localization of oskar mRNA is required for germplasm assembly and establishing antero-posterior polarity. oskar mRNA is transported by Kinesin, however the adaptor that links Kinesin to oskar mRNA was not known. In Aim 1 of this thesis, we demonstrate that a novel isoform of Tropomyosin, namely Tm1C, binds directly to kinesin and functions as the adaptor in linking kinesin to oskar mRNA. Oskar expression is limited to female germline, however Tm1C is also expressed in male flies. This suggests that there might be additional cargoes for Tm1C. We attempted to identify novel cargoes of Tm1C by performing a proteomic assay in Drosophila S2 cells. Apart from Khc, we identified Supernumerary limbs (Slmb) as the main interacting partner. Our further investigation of Slmb suggests that it might not be a cargo. Instead, Slmb which is a component of E3 ubiquitin ligase, might regulate the expression of Tm1C. In Aim 2 of the thesis, we show that Slmb regulates the levels of Tm1C by ubiquitinating it and facilitating its degradation by the Proteasome.
    • DETERMINING THE MECHANISM OF EGALITARIAN-MEDIATED MRNA TRANSPORT IN DROSOPHILA

      Goldman, Chandler; Biomedical Sciences
      The establishment of cell polarity is critical for performing complex functions including division and migration. As such, the loss of polarity is implicated in many diseases including cancers. To establish polarity, many cell types rely on the asymmetric sorting of messenger RNAs or mRNAs. These mRNAs are held in a translationally-repressed state until reaching their destinations. Upon arrival, translation is allowed to commence, giving rise to spatially restricted proteins. Often, mRNAs are transported to their destinations along microtubules via linkage to one of the sub families of microtubule motors, Dynein or Kinesins. The mechanism by which mRNAs are linked to these motors is unknown for the vast majority of localizing mRNAs. Drosophila oocytes and embryos display a great number of mRNAs that are localized to specific regions. The protein Egalitarian (Egl) has been shown to directly bind several mRNAs and participates in their microtubule-based transport by promoting linkage to cytoplasmic Dynein. In Aim 1 of this thesis, we aim to determine the mechanism by which Egl and its interacting partners, Dynein light chain (Dlc/LC8), and Bicaudal D (BicD), tether mRNAs to the Dyenin motor for transport. Dlc is required for Egl homodimerization, which promotes binding to mRNAs. BicD then preferentially associates with mRNA-bound Egl and links the complex to the Dynein motor. In Aim 2, we further investigate Egl’s role in mRNA transport by determining the critical amino acid residues within its RNA binding domain required for mRNA association.
    • Diabetic Membrane Repair Deficiency and Repair Promotion By Vitamin E

      Howard, Amber Cyran; Department of Cellular Biology and Anatomy (5/30/2014)
      Myopathy, characterized by muscle necrosis and atrophy, is a diabetic complication. The myopathy of at least one muscular dystrophy is linked to defective membrane repair. We hypothesized that defective membrane repair is also associated with diabetic myopathy. To test this hypothesis, we monitored repair in intact muscle from diabetic type 1 (INS2Akita+/-) and type 2 (db/db) mouse models. Myocytes were laser injured in the presence of a membrane impermeant dye, and cellular dye uptake through the disruption site was monitored. Dye influx of diabetic myocytes was significantly increased, compared to controls, indicating repair deficiency. This defect was mimicked in cultured cell models by high (30 mM) glucose exposure. Inhibiting the high glucose formation of advanced glycation endproducts (AGE) prevented this repair defect, but was induced in the absence of high glucose exposure by enhanced AGE receptor (RAGE) binding. We conclude that high glucose exposure leads to defective membrane repair in skeletal muscle, and that AGE/RAGE interactions underlie this defect. AGE/RAGE binding also induces generation of reactive oxygen species (ROS), which is increased in diabetes. ROS are also produced in skeletal muscle during eccentric contracts, an act that creates muscle membrane disruptions. Using a potent antioxidant, vitamin E (α-tocopherol), we were able to reverse the high glucose exposure repair defect. Interestingly, diets deficient in vitamin E results in a lethal muscular dystrophy. α-Tocopherol partitions into membrane bilayers where it is thought to act as a membrane stabilizer and/or as an antioxidant. We hypothesize that one important biological role of vitamin E is to promote muscle membrane repair. To test this hypothesis, cultured muscle cells were loaded with α-tocopherol and repair assessed with the laser assay. α-Tocopherol loading significantly decreased cellular dye influx, indicating that repair had been promoted. Strikingly, the HeLa cell, a non-muscle cell that normally displays unrestricted dye influx after laser disruption, e.g. not capable of repair via this form of injury, became repair competent after loading with α-tocopherol. Vitamin C, another antioxidant that can be loaded into cells, also significantly decreased dye influx after laser injury. However, horseradish peroxidase, an antioxidant that lacks transport across the plasma membrane was found to be ineffective in promoting repair. Cells injured in the presence of H2O2, displayed significantly more dye influx than controls injured in physiological saline lacking this oxidant. If however cells were loaded with vitamin E the H2O2 did not affect repair. We further tested H2O2 exposure in intact mouse skeletal muscle, and found repair to be significantly impaired. However, comparable to vitamin E loading in the cell model, Trolox (a water soluble analog of vitamin E) pretreatment prevented the H2O2 muscle membrane repair defect. We conclude that vitamin E promotes plasma membrane repair, and that its capacity as an anti-oxidant is crucial in this role.
    • Differntial Agonist-induced Signal Transduction Cascades and their Correlation with MARCKS Phosphorylation, StAR Phosphorylation, StAR Protein Synthesis, and Aldosterone Secretion in Cultured Bovine Adrenal Glomerulosa Cells

      Betancourt-Calle, Soraya V.; Department of Cellular Biology and Anatomy (1998-05)
      Aldosterone is a steroid hormone secreted by the cells of the zona glomerulosa of teh adrenal gland in response to increases in serum potassium (K+) concentrations, angiotensin II (AngII), and adrenocorticotropic hormone (ACTH). Although all of these agonists stimulate Ca2+ entry, which is required but not sufficient for aldosterone secretion, they generate other intracellular signals that are unique to each agent. in the first part of this study we addressed the possible involvement of Protein Kinase C (PKC) in the actions of these agonists, as measured by the phosphorylation of a specific endogenous substrate of PKC: the myristoylated alanine-rich C-kinase substrate (MARCKS).Both AngII and K+ induced an increase in MARCKS phosphorylation, while ACTH inhibited this response. We conclude that PKC activation is involved in aldosterone secretion stimulated by either AngII or K+ but not by ACTH. Although these three agonists act via different signaling pathways, it seems li9kely that at some point, the transducing events should converge. The transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane is the limiting step in steroidogenesis. the steroidogenic acut regulatory (StAR) protein is through to be a principal mediator of this transfer, with its acute synthesis and phosphorylation thought to be required for steroid production. Thus, StAR activation should be common to the actions of all three agonists. The second part of this study determined 1) the effect of these agonists on StAR protein synthesis and protein phosphorylation, and 2) how these events relate to the secretory response. Stimulation with AngII significantly increased StAR protein synthesis and StAR protein phosphorylation whereas stimulation with K+ significantly increased StAR protein phosphorylation but did not affect StAR protein synthesis. Finally, ACTH significantly increased in both events but the increase in StAR protein phosphorylation was less than that for AngII or K+. We conclude that these agonists differently regulate StAR protein synthesis and protein phosphorylation in cultured bovine adrenal glomerulosa cells. In addition, there is no simple correlation between these events and aldosterone production. These results suggest that StAR may not be the only factor regulating intramitochrondial cholesterol transport and steroid synthesis.
    • DNA METHYLATION REGULATION IN ACUTE KIDNEY INJURY

      Guo, Chunyuan; Department of Cellular Biology and Anatomy (4/26/2018)
      DNA methylation is a critical epigenetic mechanism, which is heritable during cell division, but does not involve the change of DNA sequence. It plays an essential role in regulating gene transcription in physiological and disease conditions. However, little is known about DNA methylation in renal diseases, especially in acute kidney injury (AKI). In this study, the role of DNA methylation in AKI was determined in both cell culture and mouse models. In cell culture, 5-aza-2’-deoxycytidine (5-aza), a pharmacological DNA methylation inhibitor, was used to inhibit DNA methylation. Interestingly, 5-aza increased both cisplatin- and hypoxia-induced apoptosis. These results suggest pharmacologic blockade of DNA methylation by 5-aza sensitizes renal tubular cells to apoptosis, supporting a cytoprotective role of DNA methylation in AKI. To determine the role of DNA methylation in vivo, we first successfully established conditional knockout mice that were deficient in DNMT1, DNMT3a or both exclusively in proximal tubules. In cisplatin-induced AKI, consistent with the effects of 5-aza in the cell culture, ablation of DNMT1 from proximal tubules exacerbated cisplatin-induced AKI in mice, and primary proximal tubular cells from PT-DNMT1-KO mice were more sensitive to cisplatin-induced apoptosis than wild-type cells. In sharp contrast, PT-DNMT1/3a-DK mice attenuated cisplatin-induced AKI, and primary proximal tubular cells from PT-DNMT1/3a-DK mice were more resistant to cisplatin-induced apoptosis. However, PT-DNMT3a-KO mice and PT-DNMT3a-WT mice showed similar AKI following cisplatin treatment. These results suggest different DNMTs play different roles in cisplatin-induced AKI. In ischemic AKI, none of the conditional knockout models showed differences in response to ischemia-reperfusion injury. Nevertheless, although ablation of both DNMT1 and DNMT3a in proximal tubular cells did not affect ischemia-reperfusion injury, it, indeed, suppressed renal fibroblast activation and ameliorated renal fibrosis. Furthermore, we found that Irf8 was regulated by DNA methylation during cisplatin treatment and knockdown of Irf8 in RPTC cells inhibited cisplatin-induced apoptosis, supporting a pro-death role of Irf8 in renal tubular cells. In ischemic AKI, although Bcl6 is hypermethylated and repressed in mice, overexpression of Bcl6 in RPTC cells had no impact on hypoxia-induced apoptosis. Collectively, these results suggest an important role of DNA methylation in AKI by regulating specific genes expression.
    • DRP-1 and BIF-1 Regulations in Mitochondrial Dynamics During Apoptosis

      Cho, Sung Gyu; Department of Cellular Biology and Anatomy (2013-06)
      Recent studies have revealed that mitochondrial fragmentation is a critical event in apoptosis. Mitochondria become fragmented and notably, the fragmentation causes the permeabilization o f the mitochondrial outer membrane and consequently contributes to mitochondrial dysfunction and apoptotic cell death. In apoptosis, mitochondrial fragmentation involves the activations of D rpl, a key fission protein, and Bif-1, a protein originally identified to interact with Bax. However, the molecular mechanisms by which Drpl and Bif-1 regulate mitochondrial dynamics during apoptosis remain unclear. In the first study o f my thesis work, I investigated Drpl regulation and its role in apoptosis o f rat proximal tubular cell (RPTC) following ATP depletion. During ATP depletion, Drpl was shown to be dephosphorylated at serine-637. The dephosphorylation could be suppressed by cyclosporine A and FK506, two calcineurin inhibitors, which also prevented mitochondrial fragmentation, Bax accumulation, cytochrome c release and apoptosis in RPTC. The results suggest that Drpl is activated by calcineurin-mediated dephosphorylation at serin-637. Upon activation, Drpl stimulates mitochondrial fragmentation and the permeabilization of outer membrane, resulting in the release o f apoptogenic factors and apoptosis. In the second study, I detected Bif-1 translocation to mitochondria during apoptosis o f RPTC. Notably, apoptotic events including mitochondrial fragmentation, Bax insertion and oligomerization, and cytochrome c release were all suppressed in Bif-1 deficient cells. Mechanistically, we showed that during apoptosis, Bif-1 bound to prohibitin-2 (PHB2), a mitochondrial protein implicated in mitochondrial inner membrane regulation. Furthermore, PHB2 was shown to form hetero-oligomeric complex with prohibitin-l (PHB1) in control cells and the complex broke down upon apoptosis, which was accompanied by the proteolysis of optic atrophy 1 (OPA1), the mitochondrial inner membrane fusion protein. In Bif-1 deficient cells, the breakdown o f PHB complexes and OPA1 proteolysis were both inhibited, supporting a critical role o f Bif-1 in mitochondrial inner membrane fragmentation by regulating PHB2 and OPA1. Our studies have shed new light on the critical molecular mechanisms responsible for the alteration o f mitochondrial dynamics upon cell stress, resulting in mitochondrial fragmentation, injury and apoptosis.
    • Dynamic Bayesian Network Analysis Reveals Unique and Conserved Elements of Genetic Circuitry Governing Two Different Cell-Specific Regenerative Paradigms in the Retina

      Walker, Steven L.; Department of Cellular Biology and Anatomy (2014-03)
      Regeneration—the capacity to replace lost body parts—has fascinated scientists since the time of Aristotle. Regenerative phenomena became popular with observationalists during the 18th and 19th centuries and a fertile ground for experimentation. In fact, early regenerative biology practitioners such as Abraham Trembley (1710-1784) and his colleagues have been credited with establishing the foundations of modem experimental biology. However, interest faded during the 20th century in part due to the rise of Genetics and later reinforced by the dominance of mammalian model species which have a limited capacity for tissue replacement. At the start of the 21st century, a confluence of “stem cell promise” and new genetic manipulation techniques applicable to a broad range of species has rekindled the field. (Rosenthal, 2003) One aspect, however, remains a consistent and somewhat limiting theme: the emphasis on large-scale injury paradigms (e.g., limb loss). Conversely, the diseases most often cited as potential therapeutic beneficiaries of stem cell/regenerative research advances are typically linked to the loss of specific cell types (e.g., Parkinson’s disease, Type 1 diabetes). We and others have begun to explore cell-specific regenerative paradigms in order to increase understanding of how the loss of individual cell types is detected, how the response to cell loss is regulated, and ultimately how cell-type specific regeneration can be promoted. The goal of this project is to identify genetic networks that regulate the 12 regeneration of individual retinal neuron subtypes. Visual impairment is cited as the second most feared disease following cancer (Office, 2004). While currently considered ‘irreversible’ in mammals, including humans, we posit that retinal cell loss can be ‘cured’ by stimulating dormant regenerative capacities of adult neural stem cells located in the eye (Das et al., 2006); i.e., that reparative therapeutic strategies can be developed that restore visual function to patients by replacing cells lost to degenerative disease or ocular trauma. We developed a system for studying cell-specific loss and replacement in the zebrafish retina, a highly regenerative species (Poss, Wilson, & Keating, 2002), as a means of understanding how the regenerative potential retinal stem cells is regulated. Mammals, including humans, have a limited innate capacity for retinal regeneration (Das et al., 2006). However, recent data suggests that the potential for regeneration is retained in mammals; treatments with discrete molecular factors can enhance retinal cell replacement in mammalian disease models and human retinal stem cells can give rise to new neurons in cell culture. In addition, key cellular and molecular mechanisms governing retinal regeneration appear to be conserved between fish and mammals (e.g., Muller glia acting as injury-induced retinal stem cells) (Reh & Fischer, 2006). Accordingly, we sought to identify genes and genetic networks which regulate the regeneration of individual retinal cell subtypes using temporally resolved differential expression assays combined with cutting-edge statistical methods for establishing connectivity patterns in genetic circuits. By defining pathways which stimulate retinal stem cells to respond to cell losses in a regenerative manner, we aspire to further the development of novel therapies aimed at reversing vision loss in humans.
    • Early Events in the Periovulatory Interval: Steroidogenesis and Proliferation in Macaque granulosa cells

      Fru, Karenne N; Department of Cellular Biology and Anatomy (2006-06)
      The periovulatory interval is defined as the period of time between the ovulatory stimulus and ovulation of the ovarian follicle. It is initiated by a midmenstrual cycle release of luteinizing hormone (LH) from the pituitary and initiates a cascade of events that eventually lead to extrusion of a fertilizable oocyte as well as remodeling of the follicle into the corpus luteum. Previous experiments looking beyond 12hr after the ovulatory stimulus have identified multiple changes to the preovulatory follicle while little is known of the early periovulatory interval. In spite of the paucity of information available about this time period, it was hypothesized that multiple unknown changes occur early in the interval that are critical to normal ovulation and luteinization. Two endpoints were examined in the periovulatory interval; steroidogenic changes as well as mural granulosa cell proliferation. The novel observation of CYP 21 induction was made as well as identification of 11-deoxycorticosterone (DOC) synthesis in response to hCG both in vivo and in vitro. Additionally, mineralocoritoid receptor (MR) is expressed by granulosa cells thus establishing their potential for corticosteroid sensitivity. Antagonism of MR ablates the normal synthesis of progesterone in response to hCG although the mechanism remains unclear. It was also concluded that even though mural granulosa cells are less likely to proliferate in response to exogenous stimulus in the form of epidermal growth factor (EGF) after hCG, proliferation can be enforced in even luteinizing granulosa cells using insulin. Moreover, mural granulosa cells express EGF family members in response to hCG and express EGF receptor constitutionally. However, more work needs to be done to elucidate the absence of EGF driven proliferation in luteinizing but not non-luteinized granulosa cells.
    • The Effect of Blood Flow Rate on PMN Adherence and Protection Against Injury in the Isolated Blood Perfused Canine Lung Lobe Stimulated with PMA

      McCloud, Laryssa; Department of Cellular Biology and Anatomy (1998-05)
      In the lung neutrophil (PMN)-endothelial interactions contribute to the endothelial damage that occurs in many disease states, such as the adult respiratory distress syndrome (ARDS). Current literature states that PMN adherence is greater at low blood flow rates. How high blood flow rates affect PMN-mediated injury in the lung has not been investigated. This study was designed to determine the effects of increased blood flow on the ability of phorbol myristate acetate (PMA) to cause lung injury in the isolated canine lung lobe and on the ability of agents to protect against this injury. Injury was assessed by examining luminal endothelial bound angiotensin converting enzyme (ACE) activity, pulmonary vascular resistance (PVR), pulmonary artery pressure (Pa), double vascular occlusion pressure (Pdo), and the capillary filtration coefficient (Kf). PMN sequestration was measured using circulating white blood cell counts [WBC] and differentials and 51Cr labeled PMN retention by the lung. Lung lobes were perfused at low flow (LF, 0.599±0.001 L/min) or high flow (HF, 1.185±0.004 L/min) and divided into four groups. Group I, LF PMA, Group II, LF Control, Group III, HF PMA, and Group IV, HF Control. Groups I and III received PMA (10* M) while Groups II and IV were treated with the PMA vehicle. PMA decreased ACE activity and [WBC] at both flows while Pa, PVR and Kf were increased. PMA caused lung injury independent of blood flow rate. Isoproterenol (ISO) has been shown to protect against some forms of lung injury. To study the effect of flow rate on the ability of ISO (10*SM) to protect against PMAinduced injury, lobes were perfused at either 0.603±0.003 or 2.015±.0.064 L/min and were pretreated with either saline (Group I, LF Vehicle + PMA) and (Group II, HF Vehicle + PMA) or ISO (Group III, LF ISO + PMA) and (Group IV, HF ISO + PMA) for 20 min before PMA. After PMA Group I and II lobes showed significant decreases in ACE activity and increases in Pa and PVR. Kf measurements after injury could be completed in only three of the six lobes in Group II due to severe edema. Pa and PVR increased after injury in Group III lobes. In Group IV lobes ISO protected against the increases in Pa and PVR and decreases in ACE activity but caused an increase in Kf that was further increased after PMA. Thus, ISO protected against endothelial ectoenzyme dysfunction and partially protected against hemodynamic changes after PMA in lungs perfused at high blood flow rate. Lobes perfused at a low flow rate were not protected from the hemodynamic effects of PMA by ISO pretreatment. Pentoxifylline (PTX) is another agent reported to provide protection against various forms of lung injury. To study the ability of PTX (10'3M) to protect against PMA-induced injury, lobes were perfused at low flow (LF, 0.601±0.002 L/min) or high flow (HF, 1.170±0.005 L/min) and divided into four groups. Group I, LF PTX Control, Group II, LF PTX + PMA, Group III, HF PTX Control, and Group IV, HF PTX + PMA. Lobes were treated with PTX 30 min before PMA or vehicle. [WBC] and blood smear differentials were performed. PTX increased [WBC] in all groups but did not change any other measured parameters. In the presence of PTX, PMA resulted in no changes in ACE activity, Kf or hemodynamic parameters. PMA decreased [WBC] (P<0.05) in both th epresence and absence of PTX. PTX provided protection against PMA-induced lung injury at both flow rates. The injury to PMA was found to occur in lung lobes perfused at both high and low flow. PMA increased Pa, PVR and the Kf while decreasing circulating WBC counts, circulating PMN counts, A ^ /K ^ , and % metabolism of 3H-BPAP. Although the injury to PMA was found to occur independently of flow rate, the ability of ISO to protect against PMA-induced injury was found to be greatest during high flow perfusion. At high flow, ISO completely protected against increases in Pa, Pdo and PVR while attenuating the increase in the Kf. Plasma cAMP levels were also significantly increased by ISO pretreatment and were not altered by PMA in the high flow group. At low flow ISO did not prevent PMA-induced increases in Pa, Pdo or PVR. ISO did however protect against increases in the Kf and tended to increase plasma cAMP levels. Unlike ISO, PTX provided protection against PMA-induced lung injury independently of flow rate. During both high and low flow perfusion PTX protected against PMA-induced increases in Pa, PVR and the Kf while protecting against decreases in ACE enzyme activity. PTX caused the release of WBC from the lung significantly increasing both total WBC and PMN counts. PTX did not prevent the sequestration of PMN or the release of superoxide in response to PMA.
    • Effect of Phorbol Esters on the Regulation of Rat Decidual Cell Regression

      George, Philip; Department of Cellular Biology and Anatomy (1997-12)
      Specific Aims: 1. To determine the decidual stromal cell cycle by analysis of mitotic figures, PCNA expression and flow cytometry and examine its correlation with PKC enzyme activity in stromal cells at 8, 10, 12, 14, and 17 days of pregnancy. 2. To determine the changes induced by the administration of phorbol esters on mitotic figures, PCNA expression, flow cytometry and PKC enzyme activity in stromal cells and examine the correlation between cell cycle changes with PKC enzyme activity. 3. To determine the effects of phorbol esters on ER and PR mRNAs and progesterone binding sites at day 10 and day 14 of pregnancy, the time of decidual stromal cell proliferation and regression respectively. 4. To determine the effect of antiprogestin (RU 486) on PKC enxyme activity in stromal cells at day 10 of pregnancy.
    • The Effects of Dental Resin Polymerization Initiators on Cell Lipid Metabolism

      Datar, Rahul A.; Department of Cellular Biology and Anatomy (2003-04)
      Benzoyl peroxide and camphorquinone, initiators of heat and light polymerized dental resins, are considered cytotoxic and the mechanism of cytotoxicity suggested is lipid peroxidation-induced membrane damage. The mechanism of such damage is not clear. The objectives of our current study were I) To study the effects of the various concentrations of initiators benzoyl peroxide and camphorquinone on cell lipid metabolism, 2) To study the effects of peroxidation-inducing concentrations of benzoyl peroxide on turnover of major lipids, 3) To study the effects of the materials on the lipid second messenger ceramide and on apoptotic responses in cells. Methods. Lipid metabolism i.e. synthesis as well as turnover, was measured using l4C acetate in HCP and THP-l cells. The lipids were extracted using the Bligh & Dyer method of lipid extraction and separated using one and two-dimensional thin-layer chromatography. The lipid peroxidation was measured using thio-barbituric acid reactive substance (T-BARS) produced in response to benzoyl peroxide combined with ferric chloride and camphorquinone with, or without activation with light, when combined with an enhancer dimethylaminoethyl ethyl methacrylate (DMAEMA). Ceramides were detected by extracting neutral lipids using chloroform/methanol extraction and separated by high performance thin-layered chromatography (HPTLC). DNA fragmentation assay was used to detect apoptosis. Results. Benzoyl peroxide and camphorquinone at minimally inhibitory concentrations induced similar changes in neutral lipids such as increased triglycerides and decreased cholesterol synthesis. Sphingomyelin changes were specific to HCP cells exposed to camphorquinone. The changes were mostly related to altered synthesis rather than turnover. The changes were also cell-type specific. Toxic concentrations induced peroxidation as measured by T-BARS in a time and dose dependent manner only in HCP cells while THP-1 showed different responses. Major lipid profiles were unaltered at peroxidation-inducing concentrations. Sub-toxic concentrations of benzoyl peroxide induced ceramide elevation at 24 hours, after an initial inhibition at 10 minutes, in both cell types. DNA fragmentation was, however, evident only in THP-l cells at sub-toxic concentration. Conclusion. Both initiators, benzoyl peroxide and camphorquinone, induced changes in neutral lipids. Their mechanism of peroxidation-inducing membrane damage was not dependent on the quantitative alteration in major polar lipids. Benzoyl peroxide induced changes in ceramides in both HCP and THP-l cells. Induction of apoptosis was clearly seen only in THP-l cells in response to benzoyl peroxide while HCP cells lacked this response.
    • The Effects of pp60v-src Expression on the Development of the Chicken Optic Tectum

      Mogan, John C.; Department of Biology and Anatomy (1999-03)
      The chicken optic tectum (OT) develops from the dorsal mesencephalon (midbrain) and processes crossed input from each retina. Previous experiments using a replicationdeficient retrovirus that contained the marker gene lacZ have demonstrated the normal pattern of development for the OT. Clonal cohorts derived from a single neuroepithelial stem cell migrate both radially and tangentially and differentiate into many types of neurons and at least three types of glia (radial glia and two types o f astrocytes). The goal of our laboratory is to identify important proteins involved in tectal development by: (1) directly altering expression of endogenous proteins through senseor antisense-containing retroviruses, or (2) indirectly altering endogenous protein expression or function by retroviral expression of an exogenous protein. These two approaches will allow us to determine which proteins are important in the normal and abnormal development of tectal clones. Many processes are involved in the development of the OT: proliferation, migration, differentiation, and synapse formation. Four non-receptor tyrosine kinases of the Src family (c-src, c-src+, fyn, and yes) are expressed in a spatially and temporally regulated manner in the nervous system. Their expression patterns in neural cells in vivo and in vitro have implicated these Src family members in all four of the major developmental processes mentioned above. Knockout mice of these three Src family members individually (c-src, fyn, or yes), however, show few or no overt neural developmental abnormalities. These unexpected results indicated that other members of the Src family can assume the roles of the missing kinase. Knockout mice for the major known negative regulator of Src family kinases, Csk (c-src kinase), however, show severe developmental abnormalities and defects in neural tube closure. These mice died around E9-E10 and showed elevated kinase activity for at least three Src family members (c-src, fyn, and Iyn). This fact makes it impossible to conclude that the overactivity of any one Src family tyrosine kinase is responsible for the developmental defects observed, and the early death of these embryos prevents the study of neural cell lineage, migration and differentiation in vivo. Given these results, the use of antisense to reduce expression of c-src would yield little information about the role of this kinase in neural development due to functional redundancy among Src family kinases. I decided to express in tectal clones an unregulated member of the Src family, pp60“'src, to determine how its expression alters normal tectal development. The v-src oncogene of Rous sarcoma virus was the first member of the Src family to be discovered, v-src encodes an activated tyrosine kinase (pp60,'"irc) which has lost a critical regulatory tyrosine present in the carboxy terminus of all other Src family members. Consequently, pp60'fcsrc expression affects the proliferation, migration and differentiation of many cell types in vitro and in vivo, but the effects of its expression on neural development in vivo are not well characterized. Expression of this kinase in tectal clones will provide an excellent system to study how a single unregulated Src kinase influences development of the nervous system. Expression o f a protein (pp60v'src) known to affect many different processes (proliferation, migration/cell adhesion and differentiation) in tectal clones will allow us to answer many questions of biological significance: (1) Is the proliferative potential of neuroepithelial stem cells restricted in vivo, or can stem cells generate clones of larger size?, (2) If multiple cell adhesion systems are presumptively inactivated in pp60*N,rc - expressing tectal neuroblasts, then how will clonal migration patterns differ from the norm?, (3) Is clonal differentiation in the OT controlled by only extracellular influence (e.g., growth factors, gradients) or can the developmental fate of stem cell progeny be altered by expression of pp60*’'src. In the first set of experiments I wanted to determine how wild-type pp60w'5rc expression alters the development of tectal clones in vivo. I used a replication-deficient retrovirus (LZIS), which efficiently coexpresses both LacZ and pp60*fc*rc, to determine the effects of pp60,,'*rc expression on several clonal parameters: cell number, migration pattern, and differentiation. In the next set of experiments I constructed and tested retroviral vectors which efficiently coexpress LacZ and mutated pp60w‘src proteins with deleted SH2 or SH3 domains (LZISASH2 and LZISASH3). These domains normally allow the pp60u'*rc tyrosine kinase to associate with certain cellular proteins which contain phosphotyrosines or a proline-rich stretch of amino acids, respectively. Mutation or deletion of these domains alters the biochemical and biological function of pp60^rc. I hoped to determine if the SIC or SID domains of pp60v'src are necessary for the wild-type pp60ltsrc phenotype, and to determine if they afford a unique but altered clonal phenotype compared to wild-type pp60v^rc. These experiments are novel in that they demonstrate that the overexpression of activated forms of Src family kinases influences development of the vertebrate brain. I conclude from my results that: (1) the proliferative potential of neuroepithelial stem cells in the OT is not restricted, (2) tangential migration of neuroblasts in the developing OT appears enhanced with pp60*fc*rc expression, and (3) the proper differentiation of radial glia is hindered but not prevented by pp60lHirc
    • Emotional and Physical Health Impacts of Intergenerational Caregiving for the Cognitively and/or Functionally Impaired Elderly in Korea

      Kim, Jin-Sun; Department of Physiological and Technological Nursing (2000-05)
      The purpose of this study was to examine the emotional and physical health of daughter and daughter-in-law caregivers who cared for cognitively and/or functionally impaired parents or parents-in-law in Korea and to identify factors that explain the emotional and physical health of Korean daughter and daughter-in-law caregivers. The study was guided by Riegel’s (1975,1979) and Lemer’s (1985, 1986, 1991) human developmental theories with emphasis on cultural factors and social network interactions. A purposive sample of 120 daughter and daughter-in-law caregivers who cared for cognitively and/or functionally impaired parents or parents-in-law was selected for this study. Care-recipients were predominantly female, widowed and less educated. Levels of cognitive and functional impairment were relatively low compared to Western studies. Caregivers were predominantly daughters-in-law and married. Most provided caregiving due to a general sense of obligation and responsibility rather than affectional motives. Caregivers in this study reported relatively poor emotional and physical health. Hierarchical regression analyses revealed that poor emotional health of caregivers was predicted by lower family income, the presence of dementia in carerecipients, and higher social conflict. Poor physical health of caregivers was predicted by older age, fewer competing roles, and poor emotional health. Among cultural variables, only social conflict was a significant predictor of caregivers ’ emotional health, while competing roles were significant predictors of caregivers ’ physical health. In addition to regression analyses, path analysis was used to test an overall conceptual model of caregiver health. Social conflict emerged as an important mediating variable for caregiver emotional health; furthermore, social conflict and the caregivers * emotional health were mediators for caregiver physical health. This study confirmed the importance o f a comprehensive understanding of social network interactions. Social conflict, especially intrafamily conflict was a powerful predictor of caregivers ’ negative health outcomes. Interventions to relieve negative social network interaction may prevent or relieve the negative health outcomes of caregivers.
    • Impact of Genetic Predisposition and Environmental Stress on Measures of Preclinical Essential Hypertension

      Poole, Joseph C.; Department of Cellular Biology and Anatomy (2006-06)
      The main objective of this project was to determine the impact of genetic risk and chronic environmental stress on measures of preclinical essential hypertension (EH) (e.g., exaggerated cardiovascular reactivity, increased resting hemodynamics and increased left ventricular mass [LVM]). A secondary objective was to evaluate the moderating and interactive effects of ethnicity, gender, body mass index [BMI] and anger expression on EH risk indices. Two genes with relevance for blood pressure (BP) control (e.g., beta-2 adrenergic receptor [ADRB2] gene and serotonin transporter [5-HTT] gene) were used to define genetic risk. Chronic environmental stress was assessed by socioeconomic status (SES) and subjective social status (SSS). The project consisted of three sequential studies on a large, multiethnic cohort of young adults (N>500). The first two studies were cross-sectional and based on the analysis of cardiovascular reactivity, resting hemodynamics and LVM data collected at a single visit. The third study was longitudinal and involved the tracking of BP and LVM over a 15-year span from childhood to early adulthood. In the first study, ADRB2 haplotype significantly interacted with anger suppression in African Americans such that high anger suppressing carriers had the highest resting SBP (p<.05) and TPR reactivity to a cold pressor task (p<.01). In European Americans, ADRB2 haplotype significantly interacted with BMI to predict resting hemodynamics, such that carriers who were high in BMI showed the highest SBP (p<.05). In the second study, a significant interaction between the 5-HTT promoter region polymorphism (5-HTTLPR) and social status was found for cardiovascular reactivity, such that S allele homozygotes who were low in SES and high in SSS exhibited the greatest BP and TPR reactivity to the video game stressor (p-values<.05). No significant interaction was found between 5- HTTLPR and social status in the longitudinal study, however a significant 5- HTTLPR by BMI interaction was determined for LVM, such that obese LL homozygotes had the greatest LVM over time (p<.001). Results from this project expand what is currently known with regard to EH etiology and carry implications for the prevention of EH through the early consideration of genetic, environmental and demographic risk factors.
    • In search of genetic mutations for familial keratoconus

      Khaled, Mariam Lotfy; Department of Cellular Biology and Anatomy (Augusta University, 2019-05)
      Keratoconus (KC) is the most common corneal degenerative disorder and a leading cause of corneal transplantation in developed countries. KC is a multi-factorial disease with involvement of genetic, environmental, and hormonal factors. Although KC has been widely studied, the main cause of the disease and the molecular mechanism remain unknown. We aimed to study the molecular genetics of KC via utilizing next-generation sequencing technology including RNA-Seq, whole exome sequencing, and whole genome sequencing. We used RNA-Seq to study the KC-affected corneal transcriptome. We identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas with a |fold change| ≥ 2 and a false discovery rate ≤ 0.05. Pathway analysis, using WebGestalt, indicated the enrichment of the genes involved in the extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Co-expression analysis revealed 296 pairs of genes with significant KC-specific correlations. The RNA-Seq data analysis highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis. Next, we used whole genome and exome sequencing to figure out the causal mutation(s) in a four-generation KC family with a linkage locus on Chr5q14.3-q21.1. We found a missense mutation in the phosphatase domain of PPIP5K2 (c.1255T>G, p.Ser419Ala). We found another missense mutation in the same domain of PPIP5K2 (c.2528A>G, p.Asn843Ser) in a second KC family. PPIP5K2 is a bifunctional enzyme involved in the inositol phosphate metabolic pathway. In vitro functional assays indicated the impact of the identified mutations on the enzymatic activity of PPIP5K2. PPIP5K2 expresses at a higher level than its homolog PPIP5K1 in both human and mouse corneas. A transgenic mouse model with the loss of phosphatase activity and elevated kinase activity of Ppip5k2 exhibited corneal structural abnormalities emphasizing the important role of PPIP5K2 in the homeostasis of corneal integrity. This study advances our knowledge of KC genetic etiology and helps in identifying a potential therapeutic target for KC.
    • IN VITRO AND IN VIVO STUDIES DEMONSTRATE A ROLE FOR SH3PX1 IN LAMELLIPODIA FORMATION.

      Hicks, Lawrence Joseph; Department of Cellular Biology and Anatomy (5/22/2018)
      Actin remodeling and endocytosis are essential functions for most cells. Defects in these processes present in a variety of diseases. Sorting nexins are known to contribute to endocytic uptake, cytokinesis, the retromer complex, and autophagy. Sorting nexin 9 (Snx9) interacts with major endocytic factors and proteins involved in regulation of actin cytoskeleton dynamics. Nonetheless, Snx9’s exact in vivo roles in these basic cellular processes and disease mechanisms are not known. By examining the roles of Sh3px1, we can better understand the mechanism by which this protein contributes to endocytosis and actin remodeling in vivo. Two additional paralogs, Snx18 and Snx33, complicate studies in mammalian models due to potential redundant mechanisms. Utilizing the single ortholog in Drosophila, sh3px1, this report describes the function of Sh3px1 in membrane organization and actin dynamics. Drosophila S2 cells that are depleted of Sh3px1 fail to form lamellipodia, a process that is also dependent on the actin nucleation factor, Scar. In addition, over-expression of Sh3px1 in S2 cells results in the formation of tubules and also long membrane protrusions, atypical of a classical BAR domain protein. An intact PX-BAR domain is required for these overexpression phenotypes. sh3px1 null flies are viable; however, mutant females have significantly compromised fertility. Female sh3px1 null egg chambers show many morphological defects. The age-dependent degeneration of the null egg chamber is not likely due to compromised endocytosis. Additionally, collective border cell migration is attenuated in the absence of Sh3px1. These cells are known for their reliance on endocytosis and modulation of actin dynamics for migration. We have found that Sh3px1 is essential in efficient lamellipodia production at the start of border cell migration. Our findings also suggest that Scar directly interacts with Sh3px1 and is upregulated in sh3px1 nulls. Mutation of Scar enhances many reproductive defects in sh3px1 nulls. Thus, our work reveals a main in vivo function of Sh3px1 in actin regulation for the production of structures such as lamellipodia.
    • Increased Membrane Thiol Oxidation in Sickle Erythrocytes

      Hill, Benjamin Albert; Department of Cell and Molecular Biology (1988-06)
    • Infiltrating Cells, Interferon-gamma and Intraocular Spread of HSV-1 after Anterior Chamber Injection

      Cathcart, Heather M.; Department of Cellular Biology and Anatomy (2009-12)
      Following uniocular anterior chamber (AC) inoculation of HSV-1 (KOS), the anterior segment of the injected eye becomes inflamed and infected; however, virus does not spread from the anterior segment to infect the retina of the injected eye. The overall goal of this study was to identify interferons (IFNs) and early infiltrating cells which may play a role in protecting the retina of the ipsilateral (injected) eye. Female BALB/c, IFNy-/- and macrophage depleted (clodronate, CI2MBP treated) mice were injected in one AC with 3*104 - 6x104 PFU of HSV-1 (KOS). Mice were killed at various time points ranging from 12 to 120 hours post injection (p.i.). The injected eyes were enucleated, snap frozen and frozen sections were stained with antibodies specific for HSV-1, IFNy, Mac-1 (CD11b), Gr-1, CD49b, F4/80, CD4, CD8 and CD11c. The same antibodies were also used to stain freshly isolated single-cell suspensions from the eye or spleen for flow cytometry. Additionally, whole injected eyes were used to determine gene expression levels of IFNs and IFN associated genes. In the anterior segment of the injected eye, the ciliary body and iris were virus infected and inflamed, and infiltrating cells increased during the period of observation. Mac-1 + and F4/80+ cells colocalized with IFNy in the anterior segment and Mac-1 + cells increased in the injected eye beginning at 24 hours p.i. and continuing through 72 hours p.i. Although virus staining was increased in the ciliary body of macrophage depleted mice at 48 and 72 hours p.i., destructive retinitis was not observed in the injected eye of these animals. IFNy gene expression was up regulated in injected eyes of BALB/c mice from 48 to 120 hours p.i., and while HSV-1 infection of IFNy-/- mice resulted in increased virus staining in the ciliary body, destructive retinitis was rarely observed in IFNy-/- mice. Microglia and IFNy play important roles in the immune response to virus infection, but depletion of single cell types or cytokines did not result in early panretinal HSV-1 infection in the injected eye. Taken together, these findings support the idea that the timing and appearance of different cell types and cytokines is critical to protection of the retina of the injected eye from infection due to direct spread of virus; however, it is likely that during the innate immune response in the eye, other cell types and cytokines can compensate for the absence of a single cell type or of a single cytokine.