• 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.
    • Mechanisms of Homocysteine-Induced Retinal Ganglion Cell Death

      Ganapathy, Preethi S.; Department of Cellular Biology and Anatomy (2010-12)
      The purpose of these studies was to determine the effect of excess homocysteine on retinal ganglion cell viability. An overview of homocysteine metabolism and the literature concerning homocysteine-induced neurotoxicity is given below, followed by detailed descriptions of the eye, the retina, and retinal ganglion cells.
    • 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.
    • 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.
    • Murine CD19+ Plasmacytoid Dendritic Cells Expressing Indoleamine 2,3 Dioxygenase

      Kahler, David J.; Department of Cellular Biology and Anatomy (2008-10)
      Indoleamine 2,3 Dioxygenase (IDO) is a potent immunomodulatory enzyme whose role has been described in diverse physiologic states including pregnancy, cancer, tissue transplants, autoimmune disease, chronic inflammation, and depression. IDO suppresses antigen specific T cell proliferation via mechanisms including tryptophan degradation and the production of toxic metabolites, and the activation of resting regulatory T cells (Tregs). IDO expression is tightly regulated in the murine spleen, as only rare dendritic cell (DC) subsets are competent to express IDO. Therefore, an accurate phenotype by which to identify IDO competent DCs in tissues is important when ascribing the role of IDO competent DCs in disease models. Here we show that IDO competent CD19+ pDCs (CD19+ pDCs) express high levels of costimulatory receptors (CD80 / CD86) under homeostatic conditions indicating a mature or activated phenotype and uniquely express the Class I MHC-like molecule CD1d, and the chemokine receptor CCR6. IDO competent pDCs do not share the same lineage as other murine splenic DCs as they were the only DC subset to express Pax5, and were present in reduced numbers in murine models of B cell development indicating that they develop from B cell precursors. Distinct signaling requirements regulate IDO induction in IDO competent pDCs as MyD88 was required for IDO induction and function in inflamed skin draining lymph nodes following phorbol myristate acetate application but not for IDO transcript expression or STAT1 or STAT2 protein phosphorylation following treatment with recombinant cytokines. CD19+ pDCs from WT mice but not mice genetically deficient for the IDO1 gene formed stress granules (SG) following treatment with IFNγ, which were not prevented by inhibitors of IDO activity indicating that SG formation was not IDO dependent. We hypothesize that IDO competent murine splenic pDCs uniquely expressing CD19 are phenotypically and functionally distinct from other splenic DC subsets and respond to inflammatory signals by expressing IDO. We further hypothesize that activated IDO causes distinct yet undefined biochemical changes within IDO competent pDCs following induction most probably by activating the integrated stress response and the eif2a kinases GCN2, PKR, and PERK.
    • NEUROVASCULAR DEGENERATION FOLLOWING RETINAL ISCHEMIA REPERFUSION INJURY: ROLE OF ARGINASE 2

      Shosha, Esraa; Department of Cellular Biology and Anatomy (2017)
      Ischemic retinopathies such as retinopathy of prematurity, central retinal artery occlusion and diabetic retinopathy are leading causes of visual impairment and blindness. These pathologies share common features of oxidative stress, activation of inflammatory pathways and neurovascular damage. There is no clinically effective treatment for these conditions because the underlying mechanisms are still not fully understood. In the current study, we used a mouse model of retinal ischemia reperfusion (I/R) insult to explore the underlying mechanisms of neurovascular degeneration in ischemic retinopathies. The arginase enzyme utilizes the L-arginine amino acid for the production of L-ornithine and urea. Here, we investigated the role of the mitochondrial arginase isoform, arginase 2 (A2) in retinal I/R induced neurovascular injury. We found that retinal I/R induced neurovascular degeneration, superoxide and nitrotyrosine formation, glial activation, cell death by necroptosis and impairment of inner retinal function in wild type (WT) mice. A2 homozygous deletion (A2-/-) significantly protected against the neurovascular degeneration after retinal I/R. That was attributed to decreased oxidative stress and glial activation. A2 deletion protected against I/R induced retinal function impairment. Using Optical coherence tomography (OCT), we evaluated the retinal structure in live animals and found that A2-/- retinas showed a more preserved structure and less retinal detachment. To investigate the underlying mechanisms of A2 induced vascular damage after I/R, we used an in vitro model of oxygen glucose deprivation/ reperfusion (OGD/R) in bovine retinal endothelial cells (BRECs). Analysis of oxidative metabolism showed impaired mitochondrial function. We also found an increase in dynamin elated protein 1 (Drp1), a mitochondrial fission marker. Mitochondria labeling studies showed fragmented mitochondria after OGD/R. Arginase inhibition reduced mitochondrial fragmentation in OGD/R insult. This dissertation presents A2 as a new therapeutic target in reducing neurovascular damage in ischemic retinopathies.
    • Oxidation of Dietary Amino Acids Disrupts their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells

      El Refaey, Mona M.; Department of Cellular Biology and Anatomy (2016-07)
      Age-dependent bone loss has been well documented in both human and animal models. Since it has been proposed that aging is associated with an increase in the generation of damaging reactive oxygen species (ROS), our hypothesis was that the oxidized products of dietary amino acids could play a role in age-induced bone loss by altering osteoprogenitor cell differentiation and function or activating osteoclastic activity. We first examined the effects of the oxidized nutrients on the bone marrow-derived mesenchymal stem cells and our data showed a decrease in the protein and gene expression of osteogenic markers normally stimulated by nutrients. Aromatic amino acids activated signaling pathways involved in protein synthesis in vitro, and thus, in contrast, the oxidized metabolites of these aromatic amino acids had no effect on the activation of these anabolic pathways. We then examined the bone marrow concentration of the oxidized aromatic amino acids in mature (12 months) vs. aged (24 months) C57BL/6 mice and found that kynurenine, the oxidized product of the aromatic amino acid tryptophan, was found in the highest concentration in 12 months mice. Thus, we tested the effects of kynurenine, fed as a dietary supplement, on the bone mass of twelve-month-old C57BL/6 mice compared to a normal protein diet to see if the oxidized amino acid would induce a pattern consistent with age-related bone loss. Twelve-month-old, male C57BL/6 mice were fed one of four diets; 18% protein diet (normal protein diet); 8% protein diet + tryptophan; 8% protein diet + kynurenine (50 μM) and 8% protein diet + kynurenine (100 μM) for 8 wks. Bone densitometry and micro-CT analyses demonstrated bone loss following the kynurenine diet. Histological and histomorphometric studies showed a decreased bone formation and an increased MONA M. EL REFAEY Oxidation of Dietary Amino Acids Disrupts Their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells (Under the direction of DR. CARLOS M. ISALES) osteoclastic activity in the kynurenine groups; these animals also exhibited an increase in serum pyridinoline, a marker of bone breakdown. Thus, these data demonstrate that feeding an oxidized product of an essential amino acid induces bone loss in a pattern consistent with accelerated aging, and we propose that one of the mechanisms involved in age-induced bone loss may be from alterations of dietary nutrients by the increased generation of ROS associated with aging.
    • PKC and ATR Mediated Regulation of Cisplatin-Induced Renal Tubular Cell Apoptosis

      Pabla, Navjotsingh; Department of Cellular Biology and Anatomy (2009-03)
      Cisplatin is one of the most widely used anti-cancer drug. However, its use and efficacy is limited due to nephrotoxicity. One fourth of patients treated with cisplatin develop varying degree of renal impairment, frequently resulting in acute kidney injury. Due to high mortality associated with acute kidney injury, effort has been made to understand the molecular basis of cisplatin nephrotoxicity and develop effective renoprotective strategies. In kidneys, cisplatin is accumulated in tubular cells; however the uptake mechanism that is responsible for high accumulation of cisplatin in renal cells is unclear. In tubular cell, cisplatin accumulation induces cell death by apoptosis. Mechanistically, our laboratory has demonstrated a critical role of p53 in tubular cell apoptosis during cisplatin nephrotoxicity. However, the proximal events that contribute to p53 activation and related signaling are unknown. The focus of my work was to decipher these early events during cisplatin nephrotoxicity. Firstly, my results suggest that the copper transporter Ctr1 is highly expressed in renal tubular cells and is responsible for renal uptake of cisplatin. Secondly, I show that DNA damage response involving ATR-Chk2 is responsible for p53 activation and consequent apoptosis during cisplatin-induced kidney injury and nephrotoxicity. Thirdly, I have identified that PKCd is a novel regulator of cisplatin nephrotoxicity. During cisplatin treatment PKCd is activated in a Src dependent manner and is responsible for activation of MAPKs, contributing to renal cell death. Most importantly, my results suggest that pharmacological inhibition of PKCd ameliorates renal injury without affecting the anticancer efficacy of cisplatin. These results have not only provided new insights into the 3 molecular mechanism of cisplatin nephrotoxicity, but have also identified a novel strategy to mitigate the side effects of cisplatin in normal renal tissues.
    • 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.
    • Rapamycin, an evolving role in up-regulation of autophagy to improve stroke outcome and increase neuronal survival to stroke type injuries

      Buckley, Kathleen; Department of Cellular Biology and Anatomy (2015-10)
      Rapamycin was shown to reduce infarct size in a non-reperfusion and a slow reperfusion model of murine stroke; it also improved neurological score and survival in the slow-reperfusion model. The rapamycin improvement was 50 percent greater than that observed with chloroquine. In HT22 mouse hippocampal neurons, rapamycin was shown to improve survival to an oxidative/reperfusion injury with H2O2 and a hypoxic/ischemic injury with oxygen and glucose deprivation to a larger degree than chloroquine. Rapamycin treatment increased punctate microtubule light chain associated protein 3, LC3, in the HT22 neurons in an uninjured and oxygen and glucose deprivation injured HT22 neurons compared to untreated neurons. Finally, genetic knockdown of autophagy with shRNA to autophagy protein 5, ATG5, abrogated the rapamycin’s positive effect on survival to injury.