Browsing by Subject "Proteins -- Synthesis"
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Item Estradiol regulation of uterine protein synthesis at the translational level by transfer RNAs(Texas Tech University, 1988-05) Rasmussen, Kathleen GoertzA study of the mechanism(s) of the estrogen-mediated increase in the specific amino acid acceptor activity of uterine tRNAs from ovariectomized rats was undertaken. tRNA isolated from uteri of estradiol (E2)-deprived rats has been found to contain a defective 3'[CCA] terminus. tRNA nucleotidyltransferase activity (3H-CMP incorporation into the 3'-terminus of tRNApC*) and 3'- exoribonuclease activity (degradation of 3'-terminal trinucleotide sequence of labeled tRNApCpCpA*) in control and E^-treated rats were assayed and results demonstrated that the activity of neither enzyme was found to be significantly modified by the hormone. Experiments were carried out to determine if substrate levels of CTP or ATP could regulate the rate of synthesis of the 3'[CCA] terminus. Results indicated that E2 has a dramatic effect on uterine ATP, GTP, CTP, and UTP pools as determined by HPLC analysis. Experiments were carried out to ascertain if E^-administration had an effect on the source of these expanded nucleotide pools in the E^- treated uterus by examining the salvage of nucleic acid catabolites [measured by the coupled reactions of nucleoside diphosphate kinase and tRNA nucleotidyltransferase activities (incorporation of [3H]CDP or [3H]ADP into tRNA)] and the de novo biosynthetic pathway (measured CTP synthetase which converts UTP into CTP in the presence of glutamine). A 3-fold increase in CTP synthetase activity was observed after a 12 h E2 stimulation and the glutamine antagonist, azaserine, blocked this E^-induced increase in activity. In addition, azaserine suppressed the E^-induced stimulation of leucine acceptor activity of uterine tRNAs suggesting that E2 increases CTP levels which in turn facilitates the repair of the 3'[CCA] terminus. The salvage pathway enzyme was not stimulated by E2-treatment. Studies were carried out to evaluate the relationship between the estrogen-induced repair/synthesis of the 3'[CCA] terminus of tRNApro and tRNAg'v (the most stimulated tRNAs by E^-treatment) and synthesis of E^-induced proteins with clusters of these amino acids in their sequences. The E^-induced increase in the estrogen receptor, progesterone receptor, and glucose-6-phosphate dehydrogenase, were preceded by and correlated with stimulation of tRNA acceptor activity for proline and glycine and these responses were specifically and simultaneously inhibited by prior azaserine treatment, which inhibited the E^-induced repair/synthesis of the 3'[CCA] acceptor terminus of tRNAs. Estrogen regulation of protein synthesis at the level of tRNA in the rat pituitary was examined by measuring the specific amino acid acceptor activity of pituitary tRNAs from ovariectomized animals treated with E2 for 1 or 6 h. E2(1 h)-treatment enhanced the specific amino acid acceptor activity of specific types of pituitary tRNAs, with aspartic acid, glutamic acid, tyrosine and proline the most affected. Other tRNAs were more chargable when purified from control pituitaries versus tRNAs collected from £2(1 h)- treated animals. At 6 h after E^-treatment, the shift in increased specific amino acid acceptor activity for the above tRNAs returned to control levels. In summary, E2 effects on the intactness of the 3'[CCA] terminus and the availability of certain tRNAs to be charged with their cognate amino acid could regulate the amount of time required to translate E^-induced mRNAs and thus alter the amounts of proteins synthesized.Item Functional characterization of transcriptional inhibitory domains in the C/EBP-epsilon basic region/leucine zipper transcription factor(Texas Tech University, 2003-08) Li, YaminC/EBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors and is primarily expressed in neutrophils. Multiple functional domains were previously identified in the C/EBPe polypeptide including a regulatory domain (RD or RDI) that negatively regulates the activity of C/EBPe. Within the RD a five amino acid motif (the regulatory domain motif (RDM)) is conserved in three other C/EBP family members and is critical for the inhibitory function of the RD. Interestingly the RDM is similar to the recognition sequence for the small ubiquitin-like modifier protein (SUMO) and attachment of SUMO to the RDM can activate C/EBPe. The goal of this study was to explore the molecular mechanisms by which the RD controls C/EBPe activity. These experiments may provide important information about general transcriptional regulatory mechanisms as RD-like elements, and SUMO attachment sites, exist in inhibitory domains of a large number of transcriptional regulators. Two models were designed to explain the mechanism of the function of the RD. One model proposes that intra-molecular interactions exist between the RD and its linked activation domain (AD), thereby blocking access of the AD to components of the transcriptional machinery. In this model, the attachment of SUMO to the RDM releases the AD and activates C/EBPe. Physical interactions between the RD and AD were not detected in either a mammalian two-hybrid assay or a direct interaction assay. The validity of this first model was also assessed by examining the activation domain specificity of the RD. The C/EBPe RD was capable of inhibiting linked ADs from three different classes, suggesting that the RD is unlikely to function according to model one. The second model assumes the RD functions by recruiting as yet unknown inhibitory RDM binding factor(s) (RDM-BF). In this model, SUMO attachment would displace the RDM-BFs thereby releasing the inhibitory effect of the RD. The first candidate RDM-BF tested was the family of proteins with histone deacetylase (HDAC) activity as these proteins are classically associated with co-repressor activity. Although the activity of C/EBPe was increased in the presence of the general HDAC inhibitor Trichostatin A, this effect was not dependent on the integrity of the RDM. These results indicate that C/EBPe may recruit HDACs through a domain separate from the RDM. In conclusion, the AD and RD of C/EBPe appear not to directly interact, however, additional candidate RDM-BFs must be identified and examined to determine whether the RDM functions by recruiting accessory, inhibitory proteins.Item Insulin-Mediated Inhibition of Tyrosinase Activity and Protein Synthesis in Melanoma Cell Cultures(Texas Tech University, 1984-05) Ehlers, Susan ElizabethInsulin lowers basal levels of tyrosinase activity and inhibits the MSH-stimulated rise in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. These cultures are very sensitive to insulin. A concentration of insulin as low as 5 x 10 M insulin produces optimum inhibition. At maximum inhibition, tyrosinase activity is reduced to approximately 50% of control levels. Insulin inhibits cellular proliferation in melanoma cells; however, inhibition of tyrosinase activity precedes this effect. Insulin also inhibits the (Bu)2 cAMP and theophylline stimulated rise in enzyme activity. This finding suggests that insulin exerts its effects at a site distal to cAMP production. Insulin, in fact, does not lower cAMP levels in melanoma cells, nor does it alter the MSH-stimulated rise of cAMP. The inhibitory effect of insulin on tyrosinase activity could not be mimicked by either (Bu)2 cGMP or 8-bromo-cGMP, suggesting that insulin does not exert its effects by altering cellular levels of this nucleotide. Insulin decreases the incorporation of [3H]-leucine into trichloracetic acid insoluble material by 50%, an inhibition which corresponds well with the observed level of reduction of tyrosinase activity. This finding suggests that the inhibition of tyrosinase activity may be caused by a general reduction in protein synthesis due to insulin treatment.Item The Effect of the Suspensor and Gibberellic Acid on Phaseolus Vulgaris Embryo Protein Synthesis(Texas Tech University, 1982-12) Walthall, Ellen DeloyIn this thesis the role of the suspensor and gibberellic acid in Phaseolus vulgaris embryo protein content and synthesis was examined. The plant embryo exists in a very specialized environment and this environment must be maintained in tissue culture for continued normal embryonic development. Optimum sucrose concentrations for cult-ure of 0.2 mm and 0.5 mm embryos in Gamborg B5 medium were determined to be 12^ and 6^ respectively. Protein content and synthesis of various culture combinations of these embryos and their suspensors were examined by polyacrylamide electrophoresis. Two-tenths millimeter embryos required an attached suspensor for maximum protein content. Virtually all protein synthesis was dependent upon an attached suspensor. Maximum protein quantity and synthesis in 0.5 mm embryos were observed when the embryo was cultured attached to the suspensor. Protein levels decreased when it was cultured detached from or without _7 the suspensor. Gibberellic acid of 10 M elicited the same protein 35 diversity and greater S -methionine incorporation than the attached suspensor in 0.2 mm embryos. Five-tenths millimeter embryos did not appear to be differentially responsive to various gibberellins concentrations. All of the Gl storage protein subunits and the 32 and 3^ kD G2 storage protein subunits were observed in 0.2 mm embryos. The 0.5 mm embryos in gibberellin had all of the Gl and G2 storage protein subunits.