Now showing items 21-40 of 79

    • Bisphosphonate-Related Osteonecrosis of the Jaw: From Mechanism to Treatment

      Howie, Rebecca; Department of Cellular Biology and Anatomy (2015-04-20)
      With 55 million prescriptions issued each year, bisphosphonates are the second most prescribed class of drug in the United States. They are widely used to treat diseases with excessive osteoclastic resorption, including post-menopausal osteoporosis, Paget’s disease, and tumor metastasis to bone. Unfortunately, with long term intravenous administration of nitrogen-containing bisphosphonates some patients develop bisphosphonate-related osteonecrosis of the jaw (BRONJ). This debilitating disease has limited treatment options once it has manifested and no mechanism for its development has been elucidated. This dissertation explores the novel concept that bisphosphonates cause osteonecrosis of the jaw by impairing osteocyte-induced osteoclastogenesis and, through the physical accumulation of bisphosphonates in bone, impairing the ability of recruited osteoclasts to attach thereby arresting bone healing. Furthermore, it explores the possibility that chelating agents can be used for the removal of bisphosphonate attachment from bone systemically and locally during extractions, potentially leading to a future preventive treatment. It was found that 13 weeks of 80µg/kg intravenous tail vein injections of Zoledronate followed by two mandibular molar extractions caused the clinical presentation of BRONJ as analyzed by the gross, radiographic, and histological methods. Bone dynamic parameters and TRAP staining suggested an impaired ability for the bone to remodel and defective osteoclast attachment in treated groups that persisted eight weeks after the cessation of treatment. Additionally, it was found through the use of a fluorescently tagged bisphosphonate, that the decalcifying agents cadmium, EDTA, and citric acid all had the ability to cause the significant release of bound bisphosphonate from bone. Finally, this dissertation showed that the migration of monocytes treated with low doses of Zoledronate had increased migration, while their migration to conditioned media of osteocytes treated with Zoledronate was impaired. Collectively, these data suggest that invasive trauma by itself consistently precipitated massive bone necrosis in Zoledronate treated animals, possibly through a bisphosphonate driven alteration of monocyte migration and that the use of decalcifying agents could acutely remove bisphosphonate from bone both systemically and locally. This study establishes and effective rodent model for BRONJ and a possible preventive strategy for the side-effects of bisphosphonates during high-risk situations.
    • Characterization of Cardiac L-Type and T-Type Calcium Channels During Normal and Defective Chick Heart Development

      Nichols, Carol A.; Department of Cellular Biology and Anatomy (2000-03)
      (First Paragraph) The human heart is vital for survival from early in embryonic development throughout life. It begins developing around the third week of gestation from a pair of endocardial tubes that fuse to form a single primitive heart tube. The single-lumen heart tube develops a series of expanded areas and infoldings that divide it into four presumptive chambers. As the embryo grows, the heart begins looping. This looping process serves to bring the four presumptive chambers into the appropriate orientation for septation. The developing heart remodels itself into four separated chambers (two atria or holding chambers, two ventricles or pumping chambers) which provide for separate systemic and pulmonary circulation at birth. In most mammals, oxygenated blood enters the left atrium through four pulmonary veins. The blood is forced into the left ventricle when the left atrium contracts. When the left ventricle contracts, blood is pumped through the aorta and carried throughout the body. Deoxygenated blood returns to the right atrium via the superior and inferior vena cavae. Blood is forced into the right ventricle by contraction of the right atrium. Blood is then pumped through the pulmonary trunk and arteries to the lungs to be re-oxygenated. The four- chambered heart is formed by the eighth week o f gestation. (Larsen, 1997; de la Cruz & Markwald, 1998).
    • Regulation of Reduced-Folate Transporter-1 in Retinal Pigment Epithelium

      Naggar, Hany A.; Department of Cellular Biology and Anatomy (2003-04)
      (First Paragraph) The purpose of these studies was to analyze the regulation of the folate transport protein, reduced-folate transporter (RFT-1) in the retinal pigment epithelium (RPE) under conditions o f hyperglycemia, hyperhomocysteinemia and folate deficiency. A detailed description o f the retina, followed by information regarding folate and regulation o f RFT-1, is provided below.
    • 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
    • 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.
    • Use of Sigma Receptor Ligands to Prevent Retinal Ganglion Cell Apoptosis Characteristic of Diabetic Retinopathy

      Martin, Pamela M; Department of Cellular Biology and Anatomy (2003-04)
      (First Paragraph)Diabetic retinopathy is a major sight-threatening disease and is the leading cause of blindness among working-aged Americans, affecting approximately 10 to 12 million persons (Wu, 1995). Although retinal vasculature is particularly vulnerable to damage in diabetes, other retinal cells are at risk. Very recently, Barber et al. (1998) documented increased apoptosis of neural retinal cells in experimental diabetes in rats and diabetes mellitus in humans. Notably, retinal ganglion cells (RGCs) were found to be at particular risk. Ganglion cell death in diabetic retinopathy is thought to be mediated via overstimulation o f N-methyl-D-aspartate (NMDA) receptors by glutamate. oRl is a nonopiate and nonphencyclidine-binding site that has numerous pharmacological and physiological functions. In some studies, agonists for aR l have been shown to afford neuroprotection against overstimulation of the NMDA receptor. The purpose of these studies was to evaluate the potential use of aR ligands, particularly those that bind specifically to o R l, as neuroprotective agents in the treatment of RGC apoptosis characteristic of diabetic retinopathy. A detailed description of the retina, followed by information about diabetes and the mechanisms thought to be involved in the pathogenesis of diabetic retinopathy, particularly the apoptotic death of RGCs associated with diabetic retinopathy, is provided below.
    • 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.
    • Amyloid Peptide-a7 Nicotinic Acetylcholine Receptor Interactions: Implications For Cytoprotection In Vitro

      Li, Xinyu D.; Department of Cellular Biology and Anatomy (2006-11)
      Brain deposition of (3-amyloid peptide 1-42 (A(31 -42)-containing senile plaques has been a consistent finding in Alzheimer’s disease (AD). However, the link between Apl-42 and neuronal degeneration remains unclear. It has been reported that AP peptides bind with selectivity to a l nicotinic acetylcholine receptors (a7nAChRs), in both healthy and Alzheimer’s Diseased brain tissues. The goal of this study was to demonstrate the functional inhibition of oc7nAChRs induced by Api-42, both in systems in vitro and in vivo. Initially, differentiated PC-12 cells were preloaded with fura 2-AM and intracellular free Ca2+ levels were determined by fluorescent imaging. Nicotine-induced Ca2+ signals were inhibited by pretreatment with the a7nAChR-selective antagonists, abungarotoxin (BTX) and methyllycaconitine (MLA). Nicotine induced Ca2+ influx was also blocked by pretreatment with 100 nM Api-42. In the same model, nicotine produced a concentration-dependent increase in cell viability in differentiated PC-12 cells that underwent nerve growth factor (NGF) withdrawal for 24 hr. The cytoprotective action of nicotine was efficiently antagonized by co-treatment with a7nAChR antagonists. A concentration-dependent inhibition of the cytoprotective action of nicotine also was produced by co-treatment with Apl-42 (1-100 nM). Also in differentiated PC-12 cells, nicotine induced a concentration-dependent increase in cell surface Trk A receptor expression. This increase was almost completely reversed by a7receptor-selective antagonists, and by co-treatment with Api-42. In in vivo studies with rats, intracerebroventricular (icv) injection of choline, a selective a7nAChR agonist, produced transient, but dose-dependent pressor responses and prolonged decreases in heart rate. Icv pretreatment with BTX and MLA significantly inhibited the cardiovascular responses to subsequent injection of choline. Pretreatment with the Api-42 also significantly inhibited the choline-induced cardiovascular changes suggesting that the peptide can block an oc7nAChR-mediate response in vivo. Nicotine also was administered to rats by direct injection into a lateral cerebral ventricle. Estimation of Trk A expression in necropsied brain tissues revealed significant increases in hippocampus and entorhinal cortex. These increases were significantly inhibited in rats co-treated with a-bungarotoxin or with Api-42. The data derived from these in vitro and in vivo experiments support the hypothesis that low physiological concentrations of AP peptides inhibit the function of a7nAChRs, thereby contributing to the loss in neuronal viability that accompanies Alzheimer’s disease.
    • T-Type Calcium Current and Calcium-Induced Calcium-Release in Developing Chick Myocardium

      Kitchens, Susan A.; Department of Cellular Biology and Anatomy (2002-02)
      HYPOTHESES 1. The contribution of T-type calcium currents to the calcium transient are greater at young developmental ages, but decline with chick heart development. The decrease in contribution of T-type calcium current to the calcium transient mirrors the normal developmental reduction in magnitude of T-type current in the chick heart. 2. T-type calcium current plays a role in calcium-induced calcium-release during chick heart development. T-type current plays a significant role in the calcium-induced calcium-release process in younger embryos due to the greater magnitude of the current at earlier developmental stages. 3. More than one isoform of the T-type calcium channel is present in developing chick myocardium. The multiple isoforms will function concomitantly to provide sufficient T-type calcium current for proper development. 4. The expression of the T-type calcium channel in ventricle decreases with development. There is a concomitant decrease in T-type Ca2* current stimulation of CICR. SPECIFIC AIMS 1. To determine the contribution of T-type calcium current to the calcium transient during development in chick ventricular myocytes. The approach is to use a fluorescent calcium indicator to measure the transients from myocytes at embryonic day (ED) 5, EDI 1 andED15. 2. To determine the contribution of T-type calcium current to calcium-induced calciumrelease during chick heart development. The approach is to use pharmacological agents to quantify the contribution to the Ca3* transient from T-type Ca3* current stimulated CICR. 3. To determine which isoforms of the T-type calcium channel are likely to be present in chick myocardium. The approach is to use PCR methods to identify any T-type channel isoform mRNA expressed in chick ventricle. 4. To determine the level of expression of T-type calcium channel isoforms during the development of chick ventricle. The approach is to use molecular quantitation methods to examine the expression pattern of T-type channel isoforms in chick ventricle during development.
    • 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.
    • Artificial Chromosome Transgenesis Reveals Long-Distance Negative Regulation of ragl in Zebrafish

      Jessen, Jason R.; Department of Biochemistry and Molecular Biology (1999-11)
      Despite the essential roles played by the recombination activating genes (ragl and rag2) during V(D)J recombination, the mechanisms that restrict their expression to lymphoid cells are undefined. Using a novel approach to achieve artificial chromosome transgenesis in zebrafish, we demonstrate that distal regulatory elements are critical to suppress ragl expression in inappropriate tissues. In contrast to smaller reporter gene constructs, 125 and 75 kb artificial chromosomes containing the zebrafish rag genomic locus directed GFP expression in a pattern reflective of endogenous rag 1. Mapping experiments identified a positive element 5' of ragl that enhances GFP expression in both lymphoid and non-lymphoid tissues and a negative element 5' of ra g l that specifically suppresses GFP expression in the skeletal muscle. Our transgenic zebrafish also express GFP in olfactory neurons which we show represent an authentic ra g l expression site in zebrafish.
    • 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.
    • 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.
    • Modulation of a Conserved Cathepsin B-Like Extracellular Matrix Protein Impacts Wing and Egg Formation in Drosphila Melanogaster

      Dinkins, Michael B; Department of Cellular Biology and Anatomy (2011-03)
      Conserved in Bilaterian species, the tubulointerstitial nephritis antigen (TIN-ag) family of cathepsin B-like extracellular matrix proteins has been proposed to have roles in cell adhesion and regulation of basement membrane assembly based on in vitro studies of mammalian family members. Here we examined the single Drosophila ortholog, CG3074, and found conservation of its basement membrane localization as well as a role in cell adhesion. RNAi knockdown resulted in wing blistering and held-out wings following eclosion, consistent with defects in adhesion of wing epithelia and tendon cells to the underlying extracellular matrix, but no defects were detected during pupal development. We were unable to demonstrate a genetic or physical interaction with laminin and CG3074 but did detect genetic interactions with integrins and dystroglycan in the wing. A serine substitutes for cysteine in all TIN-ag family members at the 'active site' of the cathepsin B-like domain and is predicted to render the protein inactive as a protease. Overexpression of the mutant CG3074 S213C, in which the 'catalytic' cysteine of cathepsin is restored, resulted in gain-of-function defects in egg formation and larval development. We provide genetic and biochemical evidence that these defects arise from a neomorphic activity of the S213C protein that supports a role of this highly conserved domain in wildtype CG3074 function. These studies broaden our understanding of TINag family function and identify tissue and pathway models for future studies.
    • 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.
    • Progesterone Regulation of Proliferation and Regression of Rat Decidua Basalis

      Dai, Donghai; Department of Cellular Biology and Anatomy (1998-07)
      During implantation mesometrial cells o f the uterine stroma become decidualized under the coordinate actions of progesterone (P4 ) and estrogen (E) [1,2]. This process is characterized by transformation o f phenotype and stromal cell proliferation between Days 8-12 of gestation, resulting ultimately in the formation o f the decidua basalis (DB) [3,4], By Day 14. however, the DB begins to regress and a reduced layer o f stromal cells persists to the end of pregnancy [5.6]. The regression of DB is accompanied by development o f two other layers, namely junctional zone (JZ) and labyrinth zone (LZ), which are fetal parts of the placenta and morphologically become predominant at the end stages o f pregnancy. Although the morphological changes have been well documented and numerous functions have been revealed for DB [3-8], the mechanism and factors involved in the regulation of proliferation and regression o f DB have not been elucidated. The transition of DB from proliferation to regression occurs in such a delicate way that the morphological integrity and functional competence of the DB and placenta are maintained even though stromal cells are being lost. The objective o f this study was to identify the intracellular signals initially favoring proliferation and synthetic processes and those promoting remodeling and regression as pregnancy progresses.
    • 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.
    • Studies on NF-kB Activation During Reperfusion Injury Following Reversible Acute Ischemic Stroke

      Chen, Qiang; Department of Cellular Biology and Anatomy (1998-10)
      Specific Aim # 1. To investigate whether reoxvsenation o f hypoxic cells results in the activation o f NF-kB in the HBMEC model. I f NF-kB is activated, identify the Rel family proteins in the activated NF-kB. Specific Aim # 2: To investigate whether reperfusion o f the ischemic brain tissue results in the activation o f NF-kB in the rat stroke model. I f NF-kB is activated, identify the Rel family proteins in the activated NF-kB. Specific Aim # 3: To test the efficacy o f antioxidants /NAC. PDTC) on the inhibition o f NF-kB activation following TNF-a-treatment or kwoxiaJreperfusion in the HBMEC model.
    • 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.
    • The Mechanobiology of Cranial Sutures

      Byron, Craig D.; Department of Cellular Biology and Anatomy (2005-07)
      A central hypothesis that cranial suture growth and modeling vary with respect to the mechanical loading environment is tested in a mouse sagittal suture model using three Specific Aims. Experiments within these aims were designed to elucidate mechanisms of bone formation and bone resorption at the cellular level and to determine how these processes influence the morphology and performance of cranial suture connective tissues. It is argued that suture waveform complexity (measured using fractal analysis) is generated by the positive coupling of osteogenesis along convex bone margins and bone resorption along concave bone margins and that this turnover cycle is regulated in large part by mechanical forces acting on the suture bone-ligament interface. This suture formfunction relationship is believed to operate via mechanosensing mechanisms within skeletal connective tissues. Although mechanically-induced cell wounding appears to be involved in normal suture biology, it does not occur in the fashion predicted. Apoptosis is not directly implicated. Thus, it is predicted that bone resorption in cranial sutures does not localize according to regions of shear-induced cell death but rather to regions adjacent to osteoblastic activity. Tension rather than shear is most likely to be the driving force in this system.