• 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.
    • Analysis of Folate Transport Proteins in the Mammalian Retina and Retinal Pigment Epithelium: Characterization and Localization of Folate Receptor Alpha and Reduced-Folate Transporter-1

      Bridges, Christy C.; Department of Cellular Biology and Anatomy (2000-11)
      The purpose of these studies was to determine the cellular and molecular mechanisms of folate transport in the retinal pigment epithelium (RPE).
    • Androgenic Maintenance of Rat Penile Erection

      Reilly, Christopher M.; Department of Cellular Biology and Anatomy (1997-06)
      Prior studies from this laboratory, using untreated-castrated rats (CASTRATE) and testosterone-treated castrated rats (TESTO), have shown that the magnitude of the intracavemosal pressure increase during erection is androgen dependent. Studies from this and other laboratories have also presented evidence suggesting that penile erection is mediated principally by nitric oxide (NO). The present report was designed to confirm that androgens maintain the availability of cavemosal NO, and to determine if this androgenic action is exerted at the genomic level modulating the expression of the neuronal form of nitric oxide synthase (nNOS) gene. The results showed that administration of supplemental L-arginine failed to augment the erectile response in either group, suggesting that substrate availability is not a cause o f the reduced response in CASTRATE animals. Inhibition of NO synthesis with a nitro-arginine competitive inhibitor of NOS resulted in strong inhibition of erection in both TESTO and CASTRATE rats. When given in conjunction with ganglionic stimulation to induce erection, the NO releasing drug, sodium nitroprusside (SNP), increased intracavemosal pressure in CASTRATE but not in TESTO rats suggesting a deficiency of the available NO in CASTRATE animals. Finally, reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mRNA levels for the enzyme nNOS in the penis were greater in TESTO animals than in CASTRATE rats. These results support the hypothesis that androgens mediate the erectile response in the rat penis by stimulating the expression of the neuronal isoform of nitric oxide synthase, thus maintaining an adequate supply of NO.
    • 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.
    • 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).
    • Characterization of the Biphasic Effect of 17 B-Estradiol on Fish Responsiveness in Rat Granulosa Cells

      Cannady, William E.; Department of Cellular Biology and Anatomy (1998-07)
      It has been well documented that the duration of estrogen exposure regulates the release of gonadotropins from the hypothalamic-anterior pituitary axis. However, there are conflicting data as to the effects of estrogen in the ovary. Within the ovary, cyclic exposure to estrogen initiates follicle growth and development, which ultimately results in the formation of follicles capable of ovulating. However, some studies have shown that prolonged estrogen exposure may inhibit follicle development. Therefore characterizing this biphasic action of estrogen on ovarian follicle development will provide an explanation for these previously conflicting results. The following hypothesis is proposed: Short durations of estradiol exposure synergizes with follicle stimulating hormone by inducing FSH receptor; longer durations of estradiol not only decrease circulating levels of FSH but also suppress the responsiveness of granulosa cells to FSH.
    • Characterization of the Retinal Phenotype In Methylene Tetrahydrofolate Reductase (Mthfr) Deficient Mice, A Model Of Mild Hyperhomocysteinemia

      Markand, Shanu; Department of Cellular Biology and Anatomy (2015-05)
      Homocysteine (hcy), a sulfur containing amino acid, is an integral part of methionine metabolism. Elevated plasma level of hcy (Hhcy) is identified as a risk factor for cardiovascular disorders and implicated in various retinal diseases such diabetic retinopathy, glaucoma, age related macular degeneration and central retinal vein occlusion. Cystathionine β-synthase (CBS) and methylene tetrahydrofolate reductase (MTHFR) are key enzymes of hcy metabolism. CBS catalyzes the transsulfuration pathway yielding beneficial downstream products such as taurine, H2S and glutathione (GSH). MTHFR is required for methylation of hcy. Mutations in MTHFR are the most common genetic cause for Hhcy. Murine models of CBS and MTHFR are an invaluable tools to understand Hhcy pathophysiology in humans. Our lab has reported the retinal phenotype of CBS mutant mice. Depending upon the loss of one or both alleles, mild to marked retinal neurovascular and functional alterations are observed. The data from CBS mutant mice raise an important question: is the retinal neurovasculopathy observed in absence/deficiency of CBS attributed to excess hcy levels or is it due to decline in availability of taurine, H 2S and GSH? This can be addressed by studying the retinal phenotype of MTHFR mutant mice which have an intact CBS pathway. No information is available is currently available about the retinal expression of MTHFR and current data regarding CBS in the mouse retina is contentious. This thesis work tested the hypothesis that CBS and MTHFR are expressed in the mouse retina at gene and protein levels and that Hhcy would induce retinal functional and neurovascular alterations in MTHFR-deficient mice. For gene and protein expression studies, RNA and protein were isolated from retinas for analysis of Cbs and Mthfr gene expression by RT-PCR and protein expression by Western blotting. Eyes were harvested from C57BL6 mice and used for immunodetection of CBS and MTHFR in the retina. RT-PCR revealed robust Cbs and Mthfr expression in retina. Western blotting detected CBS and MTHFR protein in mouse retina. In immunohistochemical studies of the intact retina, CBS was present most abundantly in the ganglion cell layer of WT retina while MTHFR showed widespread retinal expression. Our immunofluorescence studies revealed presence of CBS and MTHFR in retinal ganglion, Müller and RPE cells. Taken together, we have compelling molecular evidence that CBS and MTHFR are expressed in mouse retina at gene and protein levels. These data indicate the underlying importance of hcy metabolism in the retina. For characterization of the retinal phenotype in MTHFR deficient mice, we employed tools such as ERG, Fundus and FA, OCT, HPLC, morphometric, immunohistochemistry (IHC) and PCR arrays. ERG revealed a significant decrease in positive scotopic threshold response in retinas of Mthfr+/- mice at 24 wks. FA revealed areas of focal vascular leakage in 20% of Mthfr+/- mice at 12-16 wks and 60% by 24 wks suggesting potential vascular damage mediated by Hhcy. SD-OCT revealed a significant decrease in NFL thickness at 24 wks in Mthfr+/- compared to Mthfr+/+ mice. There was a 2-fold elevation in retinal hcy at 24 wks in Mthfr+/- mice by HPLC and IHC. Morphometric analysis revealed ∼20% reduction in cells in the ganglion cell layer of Mthfr+/- mice at 24 wks. IHC indicated significantly-increased GFAP labeling suggestive of Müller cell activation. The similar loss of ganglion cells, focal vascular leakage, 2-fold increase in retinal hcy, gliosis and functional abnormities were reported in Cbs+/- mice. Taken together, these data support our hypothesis that Hhcy induces retinal neurovascular and functional alterations in MTHFR deficient mice. In addition, we explored retinal mitochondrial gene alteration as a possible mechanism of Hhcy mediated retinal alterations. PCR array data analysis revealed upregulation of pro-apoptotic genes and downregulation of genes associated with normal mitochondrial transport function. Future studies will validate these results at protein and functional levels. To conclude, our data support the hypothesis that Hhcy may be causative in certain retinal neurovasculopathies. These data contribute to our understanding of the potential effects of Hhcy on the retina and may prove useful in other disease model systems of Hhcy.
    • 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.