Browsing by Subject "mRNA"
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Item 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, RustinA 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.Item 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-HouVopat, 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.Item Characterization and Optimization of Nanoparticles for Polynucleotide delivery(2015-05-01) Conjeevaram Nagarajan, Bhavani Saranya; Lacko, Andras G.; Cistola, David P.; Krishnamoorthy, Raghu R.Nucleic acid therapeutics involves the use of polynucleotides (DNA, RNA) as novel therapeutic agents for the treatment of a wide range of diseases including cancer and several metabolic and genetic disorders. However, the highly unstable nature of RNA molecules necessitates the use of drug carriers to prevent them from nuclease degradation and facilitate targeted delivery in vivo. Hence, this study was conducted to optimize the preparation of nanoparticle carriers in order to improve the stability of the polynucleotides (siRNA and mRNA). Additionally, as heterogeneity and stability of nanoparticle formulations are major issues preventing the clinical approval of therapeutic formulations this study was also focused on improving the homogeneity and the stability of the nanoparticles. In the siRNA study, reconstituted high density lipoprotein (rHDL) nanoparticles were used as the delivery vector. Optimization of siRNA-rHDL formulation was attempted with respect to homogeneity, size of the nanoparticle and entrapment efficiency of siRNA. The results showed that the inclusion of the lyophilization step in the preparation of nanoparticles resulted in a marginal increase in the homogeneity. The size analysis of siRNA rHDL nanoparticles using AFM and TEM imaging revealed the presence of spherical nanoparticles in the range of 10-16nm. Optimization studies with mRNA peptide nanoparticle formulation were conducted using a combination of cationic detergents and peptides at varied concentrations. The particle size analysis via Dynamic Light Scattering (DLS) detector revealed the presence of 268 nm diameter particles as the major component of the mRNA nanoparticle formulation that involved the combination of DOTAP (neutralizer) and Myr-5A (Apo A-I mimetic peptide). Further optimization of this formulation will be required to improve the homogeneity of the nanoparticles.Item Exercise-Evoked Metabolic Adaptations in Canine Myocardium(1999-12-01) Stuewe, Steven Richard; Robert Mallet; Neeraj Agarwal; Patricia GwirtzStuewe, Steven Richard, Exercise-Evoked Metabolic Adaptations in Canine Myocardium. Doctor of Philosophy (Biomedical Sciences), November 1999; 128 pp; 4 tables; 17 figures; bibliography, 130 titles. Aerobic exercise training evokes adaptations in the myocardial contractile machinery that enhance cardiac functional capabilities, and the myocardium’s capacity to consume energy. Despite considerable investigative effort, the effects of exercise training on myocardial intermediary metabolism, the source of energy for cardiac function, have not been defined. The investigations described herein were undertaken to delineate the effects of aerobic exercise training on key rate-controlling enzymes of myocardial intermediary metabolism and energy transport, and to characterize the effects of acute exercise on cardiac messenger RNA transcripts encoding metabolic enzymes. To address these questions, dogs were conditioned by a 9 wk treadmill running program or cage rested for 4 wk. Exercise conditioning was documented by a significant decrease in heart rate at rest and during submaximal exercise. A panel of glycolytic and oxidative enzymes was measured in myocardial extracts. It was demonstrated that aerobic exercise training of dogs selectively increased capacities of key rate-controlling enzymes of each of the major pathways of intermediary metabolism in ventricular myocardium. In addition, it appeared that the training-evoked increases in enzyme activities were due to increased enzyme contents, not to changes in substrate affinity. The same training program was implemented to investigate the effects of aerobic exercise training on the myocardium’s energy shuttling system. Total creatine kinase (CK) activity and content of the CKMB isoenzyme were measured in canine myocardial extracts. It was demonstrated that aerobic exercise training increased total myocardial CK activity and CKMB content, although the CKMB isoenzyme remained minor component of the myocardial CK system. A third investigation was conducted to examine the effects of aerobic exercise on the abundance of messenger RNA (mRNA) encoding key enzymes involved in myocardial energy production and transport. Left ventricular myocardium was sampled 30 min after an exercise bout, and messenger RNA transcripts were analyzed by reverse transcriptase polymerase chain reaction. Exercise increased in the myocardial abundance of mRNA transcripts encoding glyceraldeheyde 3-phosphate dehydrogenase, citrate synthase, and the CK-M subunit. These mRNA enhancements could be responsible, at least in part, for exercise-evoked adaptations in myocardial metabolic enzymes demonstrated in the first two investigations.Item Properties of a Human Metastatic Variant Lung Cancer Model(2003-05-01) Poirot, Julie E.; Mart Hart; Robert Wordinger; Rick KitsonPoirot, J. Properties of a Human Metastatic Variant Lung Cancer Model. Master of Science (Molecular Biology and Immunology). May 2003. 44 pp., 11 illustrations, 1 table, 39 bibliography titles. A model of non-small cell lung cancer (NSCLC) has been developed for screening and preclinical drug evaluation by implanting the A549 lung cancer cell line orthotopically into immunocompromised (SCID) mice. Aggressive metastatic sublines were then derived from metastases from the primary implant. The purpose of this project is to elucidate some of the cellular properties involved in the tumor aggressiveness of the metastatic variant cell lines. In vitro migration and invasion assays produced data showing no significant differences between the rates of migration or invasion of parental and metastatic sublines. In vivo tumor burden experiments, however, produced data showing significant differences in the numbers and sizes of metastatic tumors formed when the three cell lines were compared in SCID mice. RT-PCR analysis has indicated that there are differences in the mRNA levels of certain matrix metalloproteinases. The A549 parental cells have matrix metalloproteinase-2 (MMP-2) but not MMP-9, while both metastatic variants show MMP-9 mRNA but no MMP-2. Western blots and gelatin zymographies also confirm these findings. RT-PCR analysis and casein zymography experiments have also shown no differences in the message or activity of urokinase plasminogen activator *uPA0 among the cell lines. Multidrug resistance studies were done on the tumor cell lines in order to compare their resistance to various classes of antineoplastic drugs. These studies indicate that there is no significant difference in the resistance to doxorubicin or paclitaxel, but the parental cell line is substantially more resistant to cisplatin than either of the metastatic sublines.