Browsing by Subject "Medical Cell Biology"

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17Beta-Estradiol Suppresses Hydrogen Peroxide-Induced Nuclear Factor Kappa B Activation in HT22 Cells
(2008-05-01) Kim, Pil J.; Simpkins; Singh; Yang, Shaohua
Kim, Pil J., 17beta-estradiol suppresses hydrogen peroxide-induced nuclear factor κappa B activation in HT22 cells. Master of Science (Biomedical Sciences), May, 2008, 78pp., 20 illustrations, 66 titles. Reactive oxygen species (ROS) are natural byproducts of normal cellular reactions. They are oxygen ions, free (non)radicals, and peroxides that are highly reactive with normal macromolecules, such as lipids, DNA, and proteins. Cells are normally able to defend against the damages of ROS via enzymes that neutralize them into water. However, when cells are not able to cope with the accumulation of ROS, distributions in signaling pathways and gene transcription will occur, which will ultimately lead to cell death. It is now widely accepted that increased oxidative stress-induced damage in the brain is a major cause of neurodegenerative diseases, such as Alzheimer’s disease (AD). Nuclear factor κappa-B (NFκB) is not only a ubiquitously expressed transcription factor but also a signaling protein that is activated by ROS-induced oxidative stress. Our laboratory has demonstrated the neuroprotective effects of 17β-estradiol (E2) are elicited via an anti-oxidant effect. The purpose of this project was to determine the role of NFκB activation in E2-mediated neuroprotection against hydrogen peroxide (H2O2)-induced oxidative stress. HT-22, a murine immortalized hippocampal neuronal cell line, was utilized to determine whether NFκB is activated by hydrogen peroxide-induced oxidative stress and whether E2 suppresses H2O2-induced NFκB activation. We observed that H2O2 activated NFκB by phosphorylation of IκBα (pIκBα), one of the NFκB inhibitor proteins, reduction of total IκBα, and induction of NFκB (p65) nuclear translocation. In contrast, E2 suppressed H2O2-induced NFκB activation by dramatic reducing pIκBα, increasing total IκBα, and inhibiting p65 nuclear translocation. Our results show that one of the mechanisms by which estrogens are neuroprotective against oxidative stress is through the attenuation of H2O2-induced NFκB activation.
[3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors
(2000-05-01) Yagle, Monica A.; Dillon, Glenn; Martin, Michael; de Fiebre, Christopher
Yagle, Monica A., [3H] Ethynylbicycloorthobenzoate ([3H] EBOB) Binding in Native and Recombinant GABAA Receptors. Master of Science (Pharmacology), May 2000, 59 pp., 3 tables, 7 illustrations, bibliography, 75 titles. Modulation of the GABAA receptor has been studied with noncompetitive convulsant ligands such as tert-butylbicyclophosphorothionate (TBPS) and picrotoxin (PTX). EBOB is a more recently developed ligand that appears to bind in the same region of the channel at TBPS, but with a higher affinity. While only a few studies have examined the binding of EBOB to vertebrate brain tissue and insect preparations, none have examined potential subunit-dependent binding of EBOB. We have thus examined [3H] EBOB binding in rat cerebellum and HEK293 cells stably expressing human α1β2γ2, human α2β2γ2, and rat α6β2γ2 GABAA receptors. For comparison, [35S] TBPS binding was also examined in α1β2γ2 receptors. Saturation and Scatchard analyses revealed saturable [3H] EBOB binding at one site in all tissue preparations with Kd values ranging from 3 to 9nM. [3H] EBOB binding, like [35S] TBPS binding was inhibited by the CNS convulsants dieldrin, lindane, tert-butylbicyclophosphorothionate (TBOB), PTX, TBPS, and pentylenetetrazole (PTZ) at one site in a concentration dependent fashion. Affinities were in the high nM to low μM range for all compounds except PTZ (low mM range). GABA modulated [3H] EBOB binding in a biphasic manner in α1β2γ2 receptors with a 100-fold difference between stimulatory and inhibitory affinities. Inhibition of GABA-mediated current by TBOB in α1β2γ2 receptors resulted in a functional IC50 of 0.2 μM, in agreement with binding study results. Differences seen in binding between the different receptor subtypes examined suggest that some characteristics of EBOB binding are subunit dependent. In addition, we have shown that [3H] EBOB is a useful ligand in the study of recombinant GABAA receptors and that results obtained with [3H] EBOB are comparable to those obtained with [35S] TBPS.
A Calcium-Dependent Nuclear Signaling Pathway Transcriptionally Silences Atrial Natriuretic Factor Gene Expression
(1995-08-01) Zeng, Hong; Stephen R. Grant; Walter McConathy; Richard Easom
Zeng, Hong, A Calcium-Dependent Nuclear Signaling Pathway Transcriptionally Silences Atrial Natriuretic Factor Gene Expression. Master of Science (Biomedical Science), August, 1995, 85 pp., 2 tables, 20 illustrations, bibliography, 90 titles. A cultured myocardial cell model was used to examine a potential role of calcium-dependent protein kinases and phosphatases in regulating the induction of the atrial natriuretic factor (ANF) gene mediated through adrenoreceptor signaling. In primary culture, rat neonate cardiomyocytes supplemented with phenylephrine (PE) following transfection (24 h) with a full length ANF promoter-reporter construct, showed elevated levels of promoter activity when compared to transfected cardiomyocytes cultured in the absence of PE. Prazosin, a dedicated α1-antagonist, completely blocked the transcriptional induction mediated through PE stimulation. Two different calcium mobilizing agents, BAY K8644 and gramicidin D, significantly reduced PE-stimulated ANF promoter activity. The over-expression of co-transfected exogenous CaM kinase II isoforms resulted in transcriptional silencing of PE-induced promoter activity for cardiac ANF. Transfection of a constitutively active, mutant form of the calcium-dependent phosphatase 2B, calcineurin, gene also transcriptionally silenced ANF gene expression. Exposure of PE-induced cardiomyocytes to either FK-506-treated cells in the absence of PE exposure suggesting that transcriptional silencing may be mediated through a transcriptional repression mechanism. Taken together, these results suggest that the activation of a Ca2+-dependent nuclear signaling pathway mediated through either CaM kinase II or calcineurin leads to complete transcriptional silencing of the embryonic ANF gene expression.
A Clinical Research Study Involving the Use of Erythropoietin in Perioperative Patients Undergoing Surgery for Gynecologic Cancer
(2002-07-01) Larson, Sharon Beth; Richardson, Barbara; Martin, Michael
The purpose of this internship practicum report is to analyze the pathophysiology and impact of anemia in low-income gynecologic cancer patients. The report also assesses the impact of erythropoietin on hemoglobin levels prior to gynecologic cancer surgery. This report is based on a clinical research study to determine whether or not erythropoietin will mitigate the suppression of bone marrow inherent to the gynecologic cancer population and alleviate some of the symptoms and side effects of the anemia.
A DNA-Based Multiplex Screening Tool for Separation of Fragmented and Commingled Skeletal Remains
(2007-12-01) Ambers, Angie; Joseph Warren; John Planz; Arthur Eisenberg
Ambers, Angie, A DNA-based Multiplex Screening Tool for Separation of Fragmented and Commingled Skeletal Remains. Master of Science (Forensic Genetics), December, 2007, 63 pages, 13 tables, 19 figures, references, 38 titles. In mass death scenarios, human remains are often fragmented, scattered, and commingled. Ascertaining the number of victims and determining the victims’ identities in such scenarios is a challenging task. A DNA-based screening tool used early in the investigation of mass disasters or mass graves would provide a relatively quick way to initially assess casualty numbers and separate remains for further analysis. Such a tool would promote the most efficient allocation of resources and speed the identification process. The multiplex designed here incorporates a few genetic loci that show high variability in the human population, giving it sufficient discriminatory power for separation of commingled remains. Specifically, the multiplex includes the amelogenin sex-determining locus, D3S1358, and a 3’ (CA)n dinucleotide repeat in the mitochondrial D-loop. Further optimization/validation studies need to be conducted, and a fourth locus (D5S818) may need to be considered to increase the tool’s power of discrimination.
A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichia Coli
(2002-12-01) Weilbacher, Thomas; Jerry Simecka
Weilbacher, Thomas S., A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichi coli. Master of Science (Microbiology & Immunology), December, 2002, 57 pp., 2 tables, 12 illustrations, bibliography, 44 titles. Small untranslated RNAs (sRNAs) perform a variety of important functions in bacterial systems. The 245 nt sRNA of Escherichia coli K-12, CsrC, was uncovered using a genetic screen for genes that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post-transcriptionally regulates central carbon flux, biofilm formation, and motility in E. coli. CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both of these sRNAs possess similar imperfect repeat sequences (18 in CsrB, 9 in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is activated by CsrA and the response regulator UvrY. Complementation and in vitro transcription-translation experiments reveal that CsrA effects on csrC are mediated indirectly, through UvrY. Because CsrB and CsrC antagonize the activity of CsrA and are dependent on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. An updated model for the signaling circuitry of the Csr system is discussed.
A Study of Some Aspects of the Role of Mast Cells in Experimental Autoimmune Uveitis
(1994-06-01) Lee, Carol Hamberlin; Orr, Edward; Gracy, Robert; Lang, Laura S.
Lee, Carol Hamberlin, A Study of Some Aspects of the Role of Mast Cells in Experimental Autoimmune Uveitis. Doctor of Philosophy (Biomedical Sciences), June 1994, 141 pp., 6 tables, 29 illustrations, bibliography, 115 titles. Choroidal mast cells have been implicated in experimental autoimmune uveitis (EAU), an ocular inflammatory disease induced by S-antigen (Sag). Activation of ocular mast cells in Lewis rats was evaluated by determining changes in numbers of mast cells, levels of histamine, and wet weights of ocular tissues. A decrease in choroidal mast cells was confirmed statistically, and limbal mast cells were found to be activated earlier than choroidal mast cells. The ocular histamine distribution was altered during EAU, decreasing in the anterior eye, and increasing in the posterior eye. Retinal histamine levels increased when EAU symptoms occurred, but decreased while the disease was still intense. Levels of histamine methyltransferase, which degrades histamine, increased significiantly in retinal tissue when histamine levels fell. Signficant weight increases indicated edema, which can result from mast cell mediator action. Leflunomide, an immunomodulating drug that is known to affect mast cells in vitro, prevented induction of EAU. Leflunomide also suppressed changes in the mast cell-related parameters, histamine levels and wet weights. Mechanisms for activation of ocular mast cells in EAU were investigated. Results suggest that mast cell activation does not occur through mast cell surface IgE-antigen crosslinking. The adjuvant used, complete Freund’s adjuvant, is not conducive to IgE production. Histamine releasing factors, HRFs, are produced by various immune system cellular components. Preliminary efforts did not demonstrate HRF activity. Mast cell numbers, histamine levels, and wet weights were also evaluated in a milder form of EAU induced by M-peptide (Mpep), a peptide fragment of Sag. Mpep/EAU produces few disease symptoms in the anterior eye, but destroys the same retinal area as Sag/EAU—photoreceptor cells and their outer segments. Inflammation is less intense, restricted primarily to the target area. Mast cell numbers did not change, but histamine levels and wet weights changed significantly, suggesting that mast cells are also involved in Mpep/EAU. Overall, the results of this study add to evidence that mast cells are involved in pathogenesis of EAU. The results also point to topics of further investigation into the role of mast cells in EAU and in normal function in ocular tissues.
A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis
(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, Rustin
A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.
Alterations in mRNA Levels of Selected Gene Products During Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells
(2001-08-01) Vopat, Kelly S.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-Hou
Vopat, K., Alterations in mRNA Levels of Selected Gene Products during Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells. Master of Science (Biomedical Science), August 2001. 54 pp., 2 tables, 10 illustrations, bibliography, 105 titles. In order to explore the mechanisms involved in the signal transduction pathways of ischemia-induced apoptosis of RGCs in glaucoma, an in vitro ischmia model of transformed rat retinal ganglion cells (RGC-5) was utilized. RGC-5 cells were exposed to hypoglycemia, hypoxia, and ischemia for six hours. Hypoxia and ischemia resulted in apoptosis of RGC-5 cells as determined by TUNEL assay. The bax mRNA levels increased significantly in cells exposed to hypoxia. The mRNA levels of hemoxygenase, c-fos HSP 70, and BDNF showed a trend of increase in both the hypoxic and ischemic conditions. These results demonstrate that retinal ganglion cells undergo apoptosis in hypoxic conditions likely via an increase in bax/bcl-2. The up-regulation of BDNF and some stress proteins may be part of a cellular rescue effort trying to overcome the damage created by hypoxic and ischemic stresses.
Alzheimer's Fibroblasts are More Susceptible to Oxidative Stress
(2001-05-01) Marshall, Pamela L.; Neeraj Agarwal; Robert Gracy
Marshall, Pamela L., Alzheimer’s Fibroblasts Are More Susceptible to Oxidative Stress. Master’s of Science (Biomedical Sciences). May 2001. Recent evidence indicates that oxidative stress contributes to neuronal death in Alzheimer’s disease (AD). In addition, it has been suggested that AD is a systemic illness in which the development of the disease is only visible in the brain. The aim of this research is to develop experimental procedures using a simple cell model, the fibroblast, to determine if proteins derived from AD skin fibroblasts are more sensitive to oxidation by reactive oxygen species than non-AD cells, and to assess the ability of antioxidants to prevent this oxidative damage in AD fibroblasts. Preliminary findings suggest that changes in sensitivity are already detectable in fibroblasts from AD patients, probably as a consequence of genetic component as well as other risk factors. Therefore, this biochemical marker might have the potential for identifying individuals at risk for AD.
Amplification of Mitochondrial DNA Regions HVI and HVII in its Entirety and Reducing Cycle Sequencing
(2004-08-01) Ariyo, Bolanle; Joseph Warren; John Planz; Arthur Eisenberg
Ariyo, Bolanle. Amplification of Mitochondrial DNA Regions HVI and HVII in its Entirety and Reducing Cycle Sequencing Reactions. Master of Science (Forensic Genetics), August 2004, 46 pages, 10 figures, 7 tables, 18 references. Mitochondrial DNA is widely used in the forensic community because of its high copy number in cells, location, and mode of inheritance. Yet this method of analysis is expensive, time consuming, and labor intensive, therefore labs should take steps to improve the procedure of mtDNA analysis. This study is performed to validate the use of amplifying HVI and HVII region in its entirety (2 primer sets) for use in reference samples. Amplification performed using primers F15989-R16410 (HVI) and F73-R340 (HVII). The current method of amplification is 4 primer sets at full cycle sequencing reactions. The cost of Cycle Sequencing Kit is also expensive, therefore performing half and quarter reactions would be beneficial in reducing the amount of kit consumed. To validate the use of reducing cycle sequencing reactions, half and quarter cycle reactions were performed using 2 and 4 primer sets. Results demonstrate that sequence data for reducing cycle sequence data is consistent with the sequence data using the current method. Results also show that sequence data obtained using two primer sets was consistent with sequence data amplified by the current method with the exception of two samples at length heteroplasmy polyctosine regions.
Amplified Fragment Length Polymorphism Analysis of White Oak Tree Leaves
(2005-07-01) Patel, Kaajal Devendra; John Planz; Joseph Warren; Arthur Eisenberg
The AFLP technique at first seems to be a remarkable new technology that can be applied to the growing area of non-human DNA testing. The ability to identify organisms without prior genetic knowledge would be an asset to a field such as non-human DNA testing since not enough research in the area is being conducted. With any new technique or theory in science, intense scrutiny must be used to examine the applicability of the new technology. In the area of forensic science, the severe consequences of a false result extend far beyond the realm of scientific error. Errors make in forensic casework could result in life changing occurrences for the families of not only the victim, but the defendant as well. From this study it can be seen that AFLP as a technique may not stand up to the high expectations of reliability, and reproducibility required for a technique to be adopted into the field of forensic science. Several problems occurred through this study that may prevent this technology from becoming a widely accepted technique in non-human DNA testing. The initial problems with the technique were associated with reproducible results. The first several attempts were conducted under the same conditions, by the same analyst but yielded results that were no comparable. The RFUs of each experiment were inconsistent, not only between samples examined at different times, but samples examined within the same tiral as well. AFLP as a technique is supposedly insensitive to template concentrations however, it has been previously shown to produce differences in the electropherogram when the template is excessively diluted (26). Vos et al. (1995) determined that high dilutions yielding template DNA concentrations below 1 pg could result in irreproducible fingerprints. In this study 27.5 ng of template DNA was added to each digestion-ligation reaction, yet the resulting quantity of amplified fragments varied. These variations in quantities of amplified product could be due to PCR inefficiencies when comparing samples from different trials, but it does not explain instances where duplicate trials were inconsistent with each other (10, 22). When new ligase was introduced the resulting electropherograms did produce considerably higher RFUs for each peak, but the lack of interpretable peaks observed previously may not have been solely due to inefficient ligase. In an inter-laboratory study, Jones et al. (1997) noted that several laboratories encountered problems in obtaining complete AFLP profiles. For several groups, up to 50% of the bands were missing during the preliminary testing. Though this problem subsided with successive attempts, this approach to achieving successful results may not be feasible in a forensic setting. Often the evidence received from a crime scene may be insufficient to allow for multiple testing. In addition, multiple attempts to obtain results may open up areas for scrutiny and attack by the defense counsel. Repetitive testing may appear to be a biased search for condemning evidence against the questioned party, rather than the production of reliable results. Repetitive testing may also not be possible since laboratory reagents and time involved in the production of these results may not be within the constraints of a crime laboratory. In this study, capillary electrophoresis was used to visualize the fluorescent dyes attached to each fragment however, laboratories could use radioisotopes and polyacrylamide gels instead. This method of visualizing AFLP fingerprints is not only costly, but time consuming as well. Conducting repetitive tests in order to obtain a sample with sufficiently intense bands for analysis may not be feasible. These limitations may therefore restricts the use of the AFLP technique from only being conducted in laboratories with sufficient time and funds to conduct repetitive testing as is needed (10). Despite the potential cost in time and funds, the technique was able to produce AFLP fingerprints that were consistent with each other when the electropherograms were compared. The major source of error resulted from the method used to determine the presence of peaks within the designated categories. Since not all peaks crossed the 50 RFU detection threshold, they were not identified by the Genotyper macros. However, when the actual electropherograms were compared, these peaks were present. It has been suggested that to verify whether each peak is present in the pre-designated categories a scan of the electropherogram should be done and any peaks that were not called by the macro should be manually entered into the binary table or should be reanalyzed (Heather Coyle, personal communications). Although this method could potentially aid in the correct genotyping of each sample, it requires a considerable amount of user intervention. A considerable amount of time is needed to examine each electropherogram for the presence of peaks that are below the 50 RFU threshold. Without a redefined interpretation threshold, the analysis of each electropherogram can be highly subjective. Peaks that are relatively low need to be distinguished from peaks that may be associated with background noise. Therefore, in order to eliminate analyst bias a peak detection threshold must be established. Generally the interpretation threshold is established by a validation study of the analysis technique. In this study the lower threshold was previously established at 50 RFU for the instrument being used, but this threshold was insufficient for the recognition of all peaks present during the AFLP analysis. The question then becomes to what extend the peaks can or should be called in order to correctly identify each organism without errors. The exclusion of some peaks could lead to discrepancies, such as those observed during the blind study, which could result in an initial false match or exclusion. The interlaboratory study by Jones et al. found only one scoring difference associated with the absence of one band out of a total of 172 in the AFLP profiles. This error was later associated with experimental errors that incurred during the AFLP procedure. Discrepancies such as this can lead to an erroneous identification of samples that could have severe consequences in a criminal case. At this time, the utilization of AFLP technique for further testing of other organisms such as Cannabis sativa does not seem feasible. A variety of adjustments in the technique need to be addressed before this technology should be further applied to organisms in forensic casework. In order for AFLP typing to be used for forensic casework, major improvements in the technique need to be made. Consistency in obtaining reliable electropherograms with peaks well above the RFU detection threshold must be resolved in order to allow for accurate sample interpretation. This will not only allow for greater consistency between replicates, but will also help in establishing new databases for organisms that are being tested. As with any type of forensic DNA analysis, a database must be established for each organism being tested. Without a reliable database, accurate identification of crime scene evidence cannot be established. A major improvement that is required for the utilization of AFLP typing is the process by which genotypes are identified. Utilizing the macros to identify control and variable peaks to create the binary table was a quick and easy method, however it was not always able to identify the correct genotype. The overlapping of electropherograms in GeneScan ultimately was the best method for accurate identification of the blind samples, but in a real case scenario it would not be feasible to compare each evidentiary electropherogram with those in a database. Advancements in technology will continually introduce new techniques and procedures that could be applicable to the field of forensic science. As with any new technique, the methods and theories must be validated in order to determine whether they can be used in a criminal case. The field of non-human DNA testing is growing and with the advent of new technology such as AFLP, the possibility for establishing a non-human DNA identification method may be on the horizon.
Analysis of a Tn917 Transposon Mutant and Preliminary Characterization of NonHemolytic, Catalase-Deficient Variants of Staphylococcus aureus
(1999-06-01) Crum, Russell M.
Crum, Russell M., Analysis of a Tn917 Transposon Mutant and Preliminary Characterization of Nonhemolytic, Catalase-Deficient Variants of Staphylococcus aureus. Masters of Science (Microbiology). June 1999. Pages-101. Tables-15. Figures-10. A Tn917 transposon mutant of Staphylococcus aureus S6C was isolated and analyzed due to its deficiency in hemolysin and lipase activities. The transposon insertion did not occur in any of the known genetic regulators, which suggested the insertion occurred in a novel regulator of at least, hemolysin and lipase activities. One end of the region where the insertion occurred was isolated, sequenced, and compared with known DNA databases. Sequence comparisons revealed the insertion occurred in one of six rRNA DNA operons, which was confirmed by Southern analysis. Transduction of the transposon insertion back into the parental strain did not result in a mutant phenotype thereby indicating that the transposon insertion into a rRNA DNA operon was not responsible for the observed mutant phenotype. Further analysis of the parent strain, S. aureus S6C, revealed a population of four relatively stable variants differing in their hemolysin and catalase activities. These data suggest that the Tn917 mutant was one of these four S6C variants.
Anatomical and Biochemical Characterization of the Porcine Spinal Arachnoid
(1994-06-01) Taylor, Martin T.; Turner, James; Yorio, Thomas; Wordinger, Robert J.
Taylor, Martin T., Anatomical and Biochemical Characterization of the Porcine Spinal Arachnoid. Doctor of Philosophy (Biomedical Sciences), June, 1994. Mast cell products modulate many biologic barrier systems. In the central nervous system (CNS) two such barriers are the blood-brain barrier (B-B-B) and its analogous cerebrospinal fluid-blood barrier (CSF-B-B). Published research has demonstrated that mast cell products increase the permeability of the BBB, but no comparable research has been described for the CSF-B-B. The main goal of this dissertation was to begin to assess the role of mast cell products on the chief component of the CSF-B-B, the arachnoid mater. Specifically, the hypothesis that mast cell products modulate arachnoid function through receptor mediated second messenger system regulation was postulated. Initially, the light and electron microscopic features of the porcine spinal meninges generally and the arachnoid mater specifically were characterized and found to be similar to those of other species and CNS regions. In addition, mast cells were found to be present in the meninges where their product could affect the arachnoid mater. To study the effects of the selected mast products on the arachnoid mater, arachnoid cells were isolated and cultured. Morphologic, immunohistochemical, and physiological studies confirmed the cultured cells were arachnid cells and that they were capable of developing attributes of a barrier membrane in vitro, (e.g. tight junctions, increased transcellular resistance). The effects of mast cell products on arachnoid cells were then assessed biochemically. Arachnoid cells were found to produce cyclic adenosine monophosphate (cAMP) in response to forskolin and prostaglandin D2 (PGD2). Histamine inhibited both forskolin and PGD2 stimulated production of cAMP. Additionally, arachnoid cells produced inositol phosphates (IP) in response to carbachol and histamine via muscarinic and H1-histamine receptors respectively. Since histamine and PGD2 are produced and released by activated mast cells, and since cAMP and IP levels are known to modulate cellular barrier systems, it is concluded that meningeal mast cells and their products may regulate or modulate permeability of the CSF-B-B. An understanding of the specific biochemical actions of mast cell products on the arachnoid may ultimately aid in the understanding of many physiologic and pathologic processes involving the arachnoid such as hydrocephalus, subarachnoid and subdural hemorrhages, cerebral edema, meningitis, and meningiomas.
Anionic Ligand-Gated Ion Channels: The Convulsive Site and Mechanism of Action
(2001-08-01) Dibas, Mohammed I.; Das, Hriday; Yorio, Thomas; Agarwal, Neeraj
Dibas, Mohammed, Anionic Ligand-Gated Ion channels: The Convulsive Site And Mechanism of Action. Doctor of Philosophy (Biomedical Sciences), August 2001, pp153, 1 table, 24 illustrations, 76 titles. Picrotoxin, a CNS convulsant inhibits all anionic ligand gated ion channels. The mechanism and the binding site for picrotoxin and its related ligands are still undefined. The second transmembrane (TMII) domain of these ligand gated ion channels is found to play a key role in the mechanism of block by picrotoxin. It has been shown that the incorporation of a phenylalanine residue in place of threonine at position 6’ within the TMII domain of B2 subunit conferred high resistance toward picrotoxin in GABAA a3B2(T6’F)y2 receptors. Mediating their blocking effect through the PTX-site, PTZ, TBPS, and U-93631 lost their inhibitory effects due to the same mutation B2(T6’F). Interestingly, this mutation uncovered a low affinity, highly efficacious stimulatory site for PTZ. PTZ seems to mediate its stimulatory effect through a novel distinct site different from that for benzodiazepine. The effect of varying subunit configuration of GABAA receptors dramatically affected the ability of the mutation B2(T6’F) to abolish the inhibitory effect of picrotoxin. While picrotoxin failed to block the current induced by GABA in a3B2(T6’F)y2 receptors, picrotoxin partially blocked the current in a3B2(T6’F)y2 receptors. In B2(T6’F)y2 receptors, picrotoxin restores its full efficacy. When phenylalanine was incorporated at position 6’ in the a1 subunit, picrotoxin completely blocked the current induced by GABA in a1(T6’F)B2y2 receptors. The combined results showed that the ability of (T6’F) mutation to regulate the inhibitory mechanism of picrotoxin as dependent on the subunit configurations and at which subunit is mutated. In addition, picrotoxin is known to inhibit GABAA receptors in use-facilitated mechanism, while it inhibits the glycine receptor in a non-use facilitated fashion. The molecular determinant behind the use-facilitated mechanism was modulated by the nature of the amino acid at position 15’ within the second transmembrane domain. The mutation of serine 15’ to either glutamine or asparagine in the glycine a1 receptors converted picrotoxin from a non-use facilitated blocker to a use-facilitated one. The latter finding suggested that this residue might residue within the PTX binding site or play a key role in the transduction pathway for picrotoxin mechanism. The overall results further support the fact that TMII domain plays a key role in the picrotoxin mechanism.
Approaches to Cloning and Identification of the Ligand for Natural Cytotoxicity Receptor NKp44
(2008-07-01) Horton, Nathan C.; Jones, Harlan; Stevens, Stanley; Krishnamoorthy, Raghu
Horton, Nathan C., Approaches to Cloning and Identification of the Ligand for the Natural Cytotoxicity Receptor, NKp44. Masters of Science (Microbiology & Immunology), July 2008, 64 pp., 22 illustrations, 37 titles. Natural Killer (NK) cells represent a specialized lymphoid population that mediate innate immune responses against tumor or virally infected cells. NK cell cytotoxicity is regulated by inhibitory and activating receptors. Activating receptors include the Natural Cytotoxicity Receptors (NCRs), 2B4, and NKG2D. The NCRs play a key role in recognition and killing of tumor cells and include the receptors NKp30, NKp46, and NKp44. The ligands for the NCRs are not yet known. NKp44 is of particular interest because it is only expressed on activated NK cells, and is implicated in increased cytotoxicity and HIV infection. To identify and clone the ligand for NKp44, a recombinant fusion protein containing the extracellular domain of NKp44 was constructed and used to identify a cell line, DB, expressing a ligand for NKp44. A directional complimentary DNA (cDNA) library was constructed from this cell line and screened by mammalian expression cloning, resulting in the isolation of several putative cDNA clones of NKp44 ligands.
Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB
(2001-11-01) Chang, Woo-Jin; Alvarez, Rafael; Mathew, Porunelloor A.; Goldfarb, Ronald H.
Chang, Woo-Jin, Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB, Doctor of Philosophy (Microbiology and Immunology), November, 2001, 92 Pages, 20 figures, 3 schemes, and bibliography. Poly(ADP-ribose) polymerase (PARP-1, E.C. 2.4.2.30) is a constitutively expressed nuclear enzyme. It comprises about 1% of the total nuclear protein and in phylogenetically well conserved in most eukaryotes, with a notable exception in yeast. PARP-1 post transitionally modifies DNA-binding proteins by transferring the ADP-ribose moiety from BNAD+. Although the exact biological function of poly(ADP-ribosyl)ation has not been clearly elucidated, the process is thought to be involved in DNA repair, replication, and gene expression. Previous studies have indicated that PARP-1 participates in eukaryotic gene expression including the genes under the control of nuclear factor-kB (NF-kB). It has been demonstrated that PARP-1 deficient mice are more resistant to lipopolysaccharide-induced endotoxic shock than isogenic wild-type mice due to the inactivation of NP-kB in the mutants. In order to further analyze the interactions between PARP-1, NF-kB, and its consensus DNA in a cell-free system, we co-incubated recombinant PARP-1 protein and the p50-subunit of NF-kB (NF-kB-p50) in the absence of DNA strand-breaks. Electrophoretic mobility shift assays (EMSA) showed that sequence-specific DNA-binding of NF-kB-p50 was dependent on autopoly(ADP-ribosyl)ation of PARP-1. The NF-kB-p50 DNA-binding was inhibitied when PARP-1 was not auto-poly(ADP-ribosyl)ated either in the absence of BNAD+ or in the presence of 3-aminobenzamide, an enzymatic inhibitor of PARP-1. Coimmunoprecipation and immunoblot analysis demonstrated that NF-kB-p50 formed a heterodimer with PARP-1 when PARP-1 was not auto-poly(ADP-ribosyl)ated. In addition, poly(ADP-ribosyl)ation assays showed that NF-kB-p50 protein was not susceptible to poly(ADP-ribosyl)ation under normal incubation conditions. Those in vitro observations described above were confirmed by experiments utilizing HeLa nuclear extracts. EMSA showed that NF-kB DNA-binding was inhibited in 3-AB-pre-treated HeLa cells. To our knowledge, this is the first report demonstrating that auto-poly(ADP-ribosyl)ation reaction by PARP-1 reversibly regulates the function of a transcription factor by inhibiting the formation of heterodimer between PARP-1 and a transcription factor.
Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways
(2003-05-01) Taylor, Sara A.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-Hou
Taylor, Sara A., Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways. Master of Science (Biomedical Sciences), January, 2003, 112 pp., 4 tables, 39 illustrations, bibliography, 68 titles. Purpose: Glutamate has been implicated in many pathologies affecting the Central Nervous System including those in the retina, but the exact nature of the role of glutamate in neuronal degeneration remains unclear. In the retina. Müller cells are resistant to glutamate insults that are normally toxic to other cells of the retina, however the molecular and biochemical mechanisms that control their death or survival are not well understood. We used a series of pharmacological inhibitors and molecular biology agents on cultured Müller cells to dissect two key signaling pathways normally involved in cell survival, the Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) pathway and the Phosphatidylinositide 3 Kinase (PI3K) pathway. Since preliminary data in our laboratory showed that Müller cells upregulate their secretion of neurotrophins including Brain Derived Growth Factor (BDNF) in response to glutamate treatment, we also examined the effect of BDNF on the activation of these two signaling pathways. Methods: Early passaged Müller cells were treated with various concentrations (5 nM -50 μM) of inhibitions of the MAPK(ERK) pathway (GW5074, U0126, and PD98059) or with various concentrations (1-50 μM) of inhibitors of the PI3K pathway (LY294002 or Akt inhibitor) in the presence and absence of 50 ng/ml of BDNF for 24 hours. These experiments were repeated in Müller cells transfected with either NFκB or Bc12 DNA. Cell cultures were then analyzed for surviving cells with an MTS/PMS assay, a colorametric method for determining the number of viable cells in a proliferation assay. Results: The MAPK (ERK) inhibitors PD98059 and GW5074 both resulted in decrease in Müller cell survival. PD98059 did not decrease Müller cell survival until concentrations were high enough to suppress ERK2 phosphorylation. Müller cells transfected with NFκB or Bc12 DNA were able to resist treatment with concentrations of PD98059 that reduced cell number in untransfected cells. The PI3K inhibitor LY294002 also resulted in significant decreases in Müller cell survival in both untransfected cells and cells transfected with NFκB or Bc12 DNA. Treatment with an inhibitor farther down in the PI3K pathway, Akt inhibitor, did not significantly decrease Müller cell survival. Finally, BDNF was not able to increase cell survival in Müller cells treated with PD98059 or U0126, although it did increase the survival of cells treated wit GW5074. BDNF was also able to reverse the decrease in cell survival caused by LY294002 in both untransfected Müller cells or Müller cells transfected with NFκB or Bc12 DNA. Conclusions: Our data shows that Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) and Phosphatidylinositide 3 Kinase (PI3K) are both essential for Müller cell survival. There is modulation between the pathways and they may interconnected far upstream at a protein previously associated with only the MAPK(ERK) pathway. These results are consistent with a role for both pathways in Müller cell survival.
Cellular and Molecular Mechanisms that Distinguish the Effects of Progestorone and Medroxyprogesterone Acetate on Neuroprotection
(2006-07-28) Kaur, Paramjit; Goldfarb, Ronald; Singh, Meharvan; Agarwal, Neeraj
Kaur, Paramjit. Cellular and Molecular Mechanisms That Distinguish the Effects of Progesterone and Medroxyprogesterone Acetate on Neuroprotection., Doctor of Philosophy, (Pharmacology and Neuroscience), July, 2006, 203 pp., 5 illustrations, 20 figures and bibliography. Women have a higher prevalence for Alzheimer’s disease (AD) than men, suggesting that the precipitous decline in gonadal hormone levels following the menopause may contribute to the risk of developing AD. However, principal results from the Women’s Health Initiative concluded that women taking conjugated equine estrogens combined with medroxyprogesterone acetate (MPA, tradename: Prempro) incurred more harmful than beneficial outcomes versus the placebo group (Rossouw et al., 2002). This dissertation was aimed at determining if the discrepancy between basic science reports and these clinical studies could have been due to the synthetic progestin, MPA. I hypothesized that P4 and MPA differed in their ability to protect against the excitotoxic/oxidative insult, glutamate. Further, I proposed that this difference in neuroprotective potential would be reflected in the difference in the ability of these hormones to elicit key effectors of two neuroprotection-associated signaling pathways, the ERK/MAPK and P13-Kinase pathways. Finally, studies were initiated to evaluate the potential importance of BDNF (brain-derived neurotrophic factor) in mediating the protective effects of P4. I used organotypic explants of the cerebral cortex, and found that both P4 and MPA elicit the phosphorylation of ERK and Akt, two signaling pathways implicated in neuroprotection, with maximal phosphorylation occurring at a concentration of 100 nM. Interestingly, P4 protected against glutamate- induced toxicity however, while an equimolar concentration of MPA (100nM) did not. Further, P4 resulted in an increase in BDNF, while MPA did not. Our data bring into question the relevance of using MPA as a component of hormone therapies in postmenopausal women, and instead, argue that the relevant progestin for use in treating brain-related disorders is progesterone. Collectively, the data presented here suggest that P4 is protective via multiple, and potentially related mechanism, and importantly, its neurobiology is different from the clinically used progestin, MPA.
Cellular Mechanisms in the Ocular Actions of Endothelin
(1996-12-01) White, Karen A.; Yorio, Thomas; Pang, Iok-Hou; Dobbs, Richard
White, Karen A., Cellular Mechanisms in the Ocular Actions of Endothelin. Doctor of Philosophy (Biomedical Sciences/Pharmacology), December, 1996, 151 pp., 25 tables, 23 figures, references, 111 titles. Endothelins are a family of regulatory peptides which could have important implications in this regulation of aqueous humor outflow and intraocular pressure (IOP). The objectives of this dissertation were to investigate the cellular mechanism of endothelin (ET) receptor interactions in ocular tissues focusing on their effect on second messengers such as phospholipase C (PLC) and calcium, and their interactions with phospholipase A2 (PLA2) in ciliary muscle cells. The hypothesis was that in human ciliary muscle (HCM) cells, endothelin-1 (ET-1), via the ETA receptor and a pertussis toxin sensitive G-protein, activates PLC, which in turn stimulates calcium mobilization. Independent of this pathway, ET-1 also activates PLA2 and increases the release of prostaglandins. These two pathways provide a cellular second messenger balance that influences ciliary smooth muscle contraction. The current study demonstrated that ET-1 and endothelin-2 (ET-2) stimulate calcium mobilization in HCM cells via an ETA receptor subtype. It appears that the increase in intracellular calcium ([Ca2+]i) is the result of ET coupled to PLC via a pertussis toxin sensitive G-protein. A biphasic calcium response is elicited with ET stimulation consisting of a transient increase in [Ca2+]I which appears to be primarily due to release of intracellular stores, followed by a lower sustained phase which appears to be dependent on the influx of extracellular calcium. Endothelin-1 also appears to stimulate an increase in prostaglandin E2 (PGE2) formation through activation of PLA2. Furthermore, it appears that the effects of ET-1 on PLC and calcium are independent of the ET-1 effects on PGE2 production, such that the ET-1 induced increase in [Ca2+]I are coupled to the PLC signaling pathway, whereas increase in PGE2 production appears to be the result of an ETA receptor coupled PLA2. Whether there are different subtypes of ETA receptors or the receptor is coupled through different G-proteins is uncertain. Endothelin-1 and Big ET-1 immunoreactivity was also observed in both HCM and human nonpigmented ciliary epithelial (HNPE) cells. This is the first time that ET-1 and Big ET-1 immunoreactivity has been detected in the HCM cells, suggesting that these cells have the capability to synthesize both peptides. Furthermore, the increase in ET-1 and Big ET-1 immunoreactivity upon stimulation with TNF-α suggests that cytokines may be important regulators of ET synthesis and release. The findings of this research aid in the understanding of the mechanism of action whereby ETs regulate aqueous humor dynamics and IOP. Through a better understanding of the cellular actions of ET, insight is gained into the development of new ocular selective agents acting at the ET receptor.