Browsing by Subject "Proteins"
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Item A comparison of proteins synthesized and secreted by peri-implantation embryos of rats and mice(Texas Tech University, 1992-08) Liu, Shao-tungPsychoyos (1973) has defined embryo implantation in rodents as the result of coordinated interactions between a uterus that is "receptive" and an embryo that has reached the "blastocyst stage" of development. In rats and mice, uterine "receptivity" is vaguely understood as the ability of the endometrium to undergo decidualization and formation of the maternal plancenta; it is achieved only after exposure to ovarian hormones secreted in a specific sequence (Psychoyos 1976) . The process of decidualization involves profound changes in both morphological and biocherical characteristics of the endometrial stroma including a localized increase in vascular permeability and tissue edema, increased rates of cell proliferation, differentiation of stromal cells into so-called "decidual cells," and increases in synthesis of DNA, RNA and protein (De Feo 1967 and Finn 1977). Because decidualization only occurs in areas adjacent to the blastocysts and can be observed even before their attachment to the uterus, it is believed that a soluble factor from the embryo must exist which acts as a signal to trigger the reaction. Although the nature of the soluble factor has not been determined, it has been proposed at one time or another that steroids, histamine, prostaglandins or proteins from the embryos are responsible (see Kennedy 1983; Weitlauf 1988, for reviews).Item A study of the influence of dietary protein on resistance of the albino rat to whole body irradiation at multiple sublethal doses(Texas Tech University, 1958-08) Rivers, Jerry MargaretNot availableItem Amino acid nutrition and ideal protein for reproductive sows(Texas Tech University, 2004-12) Ji, FeiThe purpose of raising sows is to produce healthy weaning pigs. Sows should be maintained in healthy, strong, and good condition. High-prolific lean type sows have large litter size and high milk production, but low appetite during lactation, and therefore, high culling rate. Providing accurate amounts and profiles of amino acids to gestating and lactating sows will allow more efficient sow production and reduce feed cost and N excretion. Currently, there are some problems in sow nutrition: 1) current available amino acid requirements for gestating sows were adapted from growing pig data; 2) dietary amino acid ratios for the efficient protein utilization are not known for sows; and 3) current amino acid requirements may not provide accurate estimations for gestating and lactating sows. The current feeding system for gestating sows (one drop, single diet) is not sufficiently flexible to adjust the nutrient allowance according to the nutritional status of sows. Therefore, the amino acid requirements and ideal dietary amino acid ratios for gestating and lactating sows need to be better characterized. This dissertation covers the areas of: 1) changes of body weight, backfat thickness, and chemical contents in various maternal and fetal tissues in pregnant gilts and estimation of protein needs in different gestation stages of gilts; 2) growth of mammary glands of gilts during gestation; 3) estimation of ideal protein for pregnant gilts; 4) validation of ideal protein for pregnant gilts; and 5) validation of ideal protein for lactating sows. In the first study, 35 gilts were randomly allotted to seven slaughter groups: d 0, 45, 60, 75, 90, 102, and 112 of gestation. Gilts were bred and fed 2 kg/d gestation diet (as fed basis) during gestation. The gestation diet contained 3.1 Mcal/kg ME and 0.56% lysine (as fed basis). Maternal tissues and organs and fetuses were separated and weighed. Compositions of maternal and fetal tissues were analyzed. The protein accretion rates in various tissues increased (P < 0.05) after d 70 of gestation. Considering the needs of maintenance and maternal and fetal gains, we suggest that the diet of pregnant gilts should provide 6.8 and 15.3 g/d true ileal digestible lysine, respectively, before and after d 70 of gestation. Feeding gestating gilts based on a two-phase feeding program reflects the real protein needs for tissue protein accretion that may improve protein utilization. In the second study, individual mammary glands were separated and weighed. The first two pairs of glands were pooled as "anterior." The 3rd, 4th, and 5th pairs of glands were pooled as "middle." The 6th, 7th, and 8th pairs of glands were pooled as "posterior." The CP content in middle accreted faster than that in anterior and posterior. At d 112 of gestation, the CP content in individual mammary glands in middle was heavier (P < 0.05) than that in anterior and posterior. This result was different from the generally recognized concept that anterior mammary glands are larger and produce more milk than middle and posterior mammary glands. In the third study, based on changes of CP in various maternal and fetal tissues during gestation, ideal amino acid ratios for protein accretion and protein accretion plus maintenance for pregnant gilts were estimated. Amino acid needs of pregnant gilts for maintenance and amino acid accretion rates in maternal and fetal tissues changed during gestation; therefore, lysine-based amino acid ratios also changed. The ideal amino acid ratios were different in the early and late gestation. In the fourth study, thirty-four pregnant gilts at d 30 of gestation were used in two experiments. In Exp. 1, 14 pregnant gilts were randomly allotted to a control group (Cl, seven gilts) and an ideal protein group (IPl, seven gilts). In Exp. 2, 20 pregnant gilts were randomly allotted to a control group (C2, ten gilts) and an ideal protein group (IP2, ten gilts). Pregnant gilts had two meals of gestation diet (2.0 kg/d, as fed). The IPl was formulated to provide an ideal dietary amino acid ratio for maternal and fetal tissue gains, whereas the IP2 diet for both tissue gains and maternal maintenance. In Exp. 1, performance of gestation and subsequent lactation did not differ between the IPl and Cl. In Exp. 2, body weight gain of the IP2 was higher (P < 0.01) than that of the C2 fi'om d 30 to 109 of gestation. Serum urea level of the IP2 was lower (P < 0.05) than that of the C2 at d 90 and 109 of gestation. Litter mortality of the IP2 was lower (P < 0.05) than that of the C2 during lactation. Performance of the IPl was not improved, suggesting that the ideal amino acid ratios for protein accretion were not balanced for pregnant gilts. The IP2 had higher body weight gain (P < 0.01) from d 30 to 109 of gestation and lower serum urea level at d 109 of gestation (P < 0.05), suggesting that ideal amino acid ratios in the IP2 decreased the oxidation of amino acids and increased maternal protein accretion. In the fifth study, 51 sows were grouped according to their body weight at farrowing and parities. Within each group, sows were randomly allotted to four dietary treatments: 1) low-protein control (17.5% CP; LPC); 2) low protein with ideal protein (LPI); 3) high-protein control (19.5% CP; HPC); and 4) high protein with ideal protein (HPI). Ideal protein for lactating sows was obtained from the TTU-Sow Model (Kim et al., 2002). Feed intake and body weight loss of sows did not differ among four dietary treatments. Sows fed ideal protein had increased (P < 0.05) litter weight gain overall three parity at the low-protein diets. Sows fed ideal protein had decreased (P < 0.01) serum urea concentration at the high protein level. Sows fed ideal protein had increased (P < 0.05) litter weight gain of the first parity sows at the low-protein level. Sows fed ideal protein improved litter weight gain in the low-protein diet during lactation, indicating that consideration of ideal dietary amino acid ratio in lactation diet may enhance milk production. The benefits may be maximal for the first-parity sows because they have lower voluntary feed intakes and limited body tissue reserves. Current available estimation of amino acid needs of gestating and lactating sows for maintenance was based on the data of non-pregnant gilts. Whether pregnancy and lactation affect amino acid needs of gestating and lactating sows for maintenance is unknown. Further research is needed in these areas of nutritional physiology. In addition, we assumed that the metabolic rates of various amino acids in the small intestinal mucosa were similar between nonpregnant and pregnant pigs or between d 1 and d 21 of lactation, but some studies form other species suggested that the rates of amino acid synthesis and/or catabolism in enterocytes may vary with physiological status.Item Aromatic donor-acceptor interactions : bridging abiotic and peptide folding(2008-05) Bradford, Valerie Jean, 1980-; Iverson, Brent L.Aromatic donor-acceptor interactions have been utilized by the Iverson group in the development of abiotic molecules, called aedamers, that achieve new folding motifs, intermolecular association in heteroduplexes, and new material properties. These molecules exploit the interaction between the electron-rich 1,5-dialkoxynapthalene (DAN) and electron-deficient 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) units in a face-centered stacking geometry in aqueous solution. This dissertation describes the use of DAN-NDI interactions in the realm of peptides and proteins to expand the scope for applications of this interaction. This work specifically focuses on three areas of aromatic donor-acceptor interactions: achieving protein behavior with abiotic molecules, introducing the interaction into natural peptides, and utilizing the interaction in the intermolecular association of an abiotic molecule and a natural peptide. Chapter 2 refines the model of aggregation of an amphiphilic aedamer, which forms a hydrogel upon heating. The aedamer behaves similarly to proteins called amlyoids, which form fibrils and plaques in vivo which have been implicated in a variety of diseases, including Alzheimer's. Chapter 3 describes the synthesis of [alpha]-amino acids with DAN- and NDI-containing side chains. These amino acids can be used in a peptide model of [beta]-hairpin secondary structure. The model system can determine whether aromatic donor-acceptor interactions are useful in stabilizing peptide and protein structure. Chapter 4 describes the study of the Anchored Periplasmic Expression System (APEx) for use in screening random peptide libraries. A random peptide library is used to determine the sequence of a natural peptide, potentially containing electron-rich aromatic residues, which could bind an NDI oligomer with high affinity for use as a protein expression tag. Chapter 5 describes work toward the use of cyclic NDI bisintercalators for binding both the major and minor grooves of a specific sequence of DNA simultaneously, in addition to the use of cyclic NDI and DAN molecules for the further study of NDI-DAN interactions in abiotic intermolecular assembiles. Overall, this work has advanced the application of aromatic donor-acceptor interactions in peptides and should serve as a foundation for the future study of this interaction in protein folding and behavior in biological systems.Item Biochemical Characterizations of the Steroidogenic Acute Regulatory (StAR) Protein(Texas Tech University, 1998-05) King, Steven RobertWhile the first step in the production of steroid hormones in the body is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage system (CSCC), the rate-limiting step is the delivery of substrate cholesterol from the outer to the inner mitochondrial membrane and the CSCC. Although it has been known for over three decades that this requires the de novo synthesis of a protein(s) in response to trophic hormone stimulation, the identity of this factor has remained unknown. Our laboratory has recently cloned and characterized a candidate mitochondrial protein that can induce steroid production in the absence of stimulation in transfected MA-10 mouse Leydig tumor cells. As a result, it was named the Steroidogenic Acute Regulatory (StAR) protein. In this work, we showed that all previously described forms of StAR can be generated from a single mRNA, the 37 kDa form of StAR is the precursor form, and that upon stimulation, transcription of StAR is upregulated. StAR was demonstrated to be sufficient to induce steroidogenesis in isolated MA-10 cell mitochondria, in a specific and time- and dose-dependent manner. Together with other lines of evidence, we conclude that StAR is the long-sought acute regulator of steroidogenesis. We have further investigated requirements for StAR function. StAR-induced cholesterol transfer was determined to require ATP hydrolysis and a mitochondrial electrochemical gradient in vitro. Consistent with the former requirement, phosphorylation of StAR at serine 194 in mouse and 195 in human was found to be critical for maximal activity. This phosphorylation appears to occur as the precursor form of StAR is processed by the mitochondria to die 32 kDa intermediate. Disruption of either the electrochemical gradient or the potential across the inner mitochondrial membrane in whole cells resulted in inhibition of steroidogenesis induced by StAR as well as mitochondrial import. Therefore, either import of StAR or another factor. such as mitochondrial calcium, is requisite for stimulation of intermembrane cholesterol delivery to the CSCC. Since we find that StAR can induce cholesterol transfer in nonsteroidogenic cells, the mechanism by which StAR acts is probably conserved through different cell types.Item Cardiomyocyte Autophagy is Induced by Protein Aggregation in Heart Disease(2009-06-19) Tannous, Paul; Hill, Joseph A.Autophagy is associated with diverse forms of myocardial stress. When I initiated my studies activators of this pathway had not been identified in the heart, nor was it clear weather autophagy is an adaptive or maladaptive response in the stressed myocardium. My initial research focused on autophagy in hypertension-induced heart failure, the most common cardiovascular disease in Western nations. Early evidence demonstrated generation of reactive oxygen species, protein damage, and protein aggregation in the acute period of pressure overload. Given the simultaneous presence of autophagosomes and aggregates, and autophagy's role in bulk degradation, I postulated these events were mechanistically linked. I designed experiments to test the hypotheses that protein aggregates are activators of autophagy in the heart, and that autophagy functions in aggregate clearance. Here I report novel findings that link pressure overload-induced protein aggregation to increased cardiomyocyte autophagy. Specifically, in the pressure-stressed ventricle 1) generation of reactive oxygen species is an early pathological event, 2) there is extensive protein aggregation with higher-order processing into aggresomes, 3) protein aggregation induces cardiomyocyte autophagy, and 4) in this setting autophagy functions in its role as a mechanism of bulk protein degradation. These findings are the first to demonstrate proteinopathy of non-genetic etiology contributes to hypertension-induced heart failure and that protein aggregates are robust activators of cardiomyocyte autophagy. To directly address the role of autophagy in cardiomyocyte clearance of toxic protein species, I turned my attention to CryABR120G-induced desmin-related cardiomyopathy (DRCM), an aggregate-associated disease with autosomal dominant inheritance. My studies demonstrated that 1) autophagy is activated by CryABR120G-induced protein aggregation, 2) aggregate formation is inversely proportional to the degree of autophagic activity and 3) blunting autophagy accelerates pathological myocardial remodeling and the onset of heart failure. Extending this work to clinical medicine, we observed increased autophagy in the skeletal muscle from patients with desmin-related skeletal myopathy. Cumulatively these data are the first to demonstrate autophagy is induced in DRCM and functions as a protective cellular response. These findings suggest autophagy is a pathway amenable to therapeutic intervention in patients suffering from myofibrillar myopathy, a disease class for which there are limited therapeutic options.Item Characterization of Herc5: the major ligase for ISG15, an antiviral ubiquitin-like protein(2007-08) Dastur, Anahita R., 1975-; Huibregtse, Jon M.Human ISG15 is a 17 kDa ubiquitin-like protein (Ubl) that is induced by type I interferons (interferons [alpha] and [beta]) and plays a role in antiviral responses. ISG15 is conjugated via its C-terminus to more than 150 cellular proteins, and like ubiquitin, an E1-E2-E3 enzymatic cascade is required for conjugation. Ube1L and UbcH8 were previously identified as the E1 and E2 enzymes for this pathway. My experiments identified Herc5, a HECT domain E3, as the major ligase for ISG15. Like ISG15, Ube1L, and UbcH8, expression of Herc5 is transcriptionally induced by type I interferons. siRNAs against Herc5 abrogated ISG15 conjugation to the vast majority of target proteins in interferon-treated cells. Wild type Herc5, but not the catalytically inactive C994A mutant, supported conjugation of ISG15 in non-interferon-treated cells co-transfected with Ube1L, UbcH8 and ISG15. IQGAP1, a scaffold protein, was identified as another essential component of the ISG15 system. IQGAP1 was discovered to interact with Herc5, and this interaction was mediated by the C-terminal domain of IQGAP1 and the N-terminal RCC1-like repeats of Herc5. IQGAP1 was required for auto-conjugation of ISG15 to Herc5, and I propose a model where IQGAP1 functions, at least in part, by relieving an auto-inhibitory conformation of Herc5. Thus, I have identified two factors that are critical for ISG15 conjugation and my discoveries have increased our understanding of the ISG15 pathway. Identification and characterization of the conjugation apparatus will aid in establishing an in vitro biochemical system for ISG15 conjugation, which in turn, will be important to decipher the biological function of ISG15 modification.Item Computational modeling of transport through polymer membranes and globular proteins(2012-08) Jiang, Yingying, doctor of chemical engineering; Sanchez, Isaac C., 1941-; Paul, Donald R.; Freeman, Benny D.; Truskett, Thomas M.; Elber, RonWithin a polymer thin film, free-volume elements have a wide range of size and topology. This broad range of free-volume element sizes determines the ability for a polymer to perform molecular separations. Herein, the free volume and transport properties (diffusion, permeability, and selectivity) in both rubbery and glassy polymers were simulated using fully atomistic models. Extension of the computational tool to study the void structure in proteins is also included in this thesis. Six permeable thermally rearranged (TR) polymers and their precursors were studied. Using atomistic models, cavity size (free volume) distributions determined by a combination of molecular dynamics and Monte Carlo methods were consistent with experimental observation that TR polymers are more permeable than their precursors. The cavity size distributions determined by simulation were also consistent with free volume distributions determined by positron annihilation lifetime spectroscopy. The diffusion, solubility and permeation of gases in TR polymers and their precursors were also simulated at 308 K, with results that agree qualitatively with experimental data. A new hybrid Monte Carlo/Molecular Dynamics method is developed for estimating the slow diffusion processes of light gases transporting in glassy polymers. Diffusion coefficients, as small as 10⁻⁵ to 10⁻⁹ cm²/s are estimated for penetrants in four different polymers at 298 K. In all cases, agreement between literature experimental data and values obtained from the fast hybrid molecular dynamics method ranges from good to excellent. A new technique is developed using Monte Carlo methods to characterize the cavity size distribution and surface atoms in globular proteins. New statistical metrics have been defined for the structural characterization of globular proteins. Some of these metrics include volume, surface area, asymmetry ratio, interior cavity size distribution, and the identification of percolation channels. Wild-type (WT) myoglobin (Mb) and 5 Mb mutants have been studied in this research as examples. An analysis of cavity statistics provides an efficient method to quantify local properties such as packing density and transport pathways. The average cavity sizes of WT Mb and its mutants are around 4.0-5.0 Å.Item Conformational dynamics of an unfolded biopolymer : theory and simulation(2012-12) Cheng, Ryan; Makarov, Dmitrii E.; Florin, Ernst-Ludwig; Elber, Ron; Henkelman, Graeme; Keatinge-Clay, Adrian T.The conformational dynamics of an unfolded biopolymer such as a polypeptide or DNA has attracted a significant amount of attention in the context of protein folding and the design of biomimetic technologies. To this end, recent advances in single-molecule experiments have allowed for biomolecules to be probed with an unprecedented level of detail, shedding light on their dynamics. Motivated by the need to interpret experimental data and to help guide future studies, we use concepts from polymer physics, computer simulations, and experimental data to study the timescales in which an unfolded biopolymer undergoes conformational rearrangement. First, we examine the end-to-end loop formation time in the experimentally relevant scenario where the dynamics are probed using a fluorescence probe and quencher. We show that the loop formation time in the experimentally relevant case is quantitatively dissimilar from the predictions of previous theoretical studies that neglect the quenching kinetics, which are often used to interpret experimental data. We additionally find that the loop formation times can be re-casted in a simple, universal dependence that is characteristic of random-coils. Furthermore, deviations from this universal dependence can be used as a sensitive tool for detecting structural order in unfolded biopolymers. We also consider a surface-tethered polymer chain and investigate the rate of a reaction between the free end and the surface. We explore this rate in the reaction-controlled limit and the diffusion-controlled limit, providing evidence for near-universal dependences of the rate in the respective limits. Next, we examine the transit time of end-to-end loop formation in a case study. We find that approximating the end-to-end dynamics as diffusion in a 1D potential of mean force fails dramatically to describe the transit time. Furthermore, we find that the transit time is uninfluenced by the average entropic force imposed by the polymer chain and is well described by a simple free-diffusion model. Finally, we explore the role of internal friction in the dynamics of an unfolded protein. Using simple polymer models that incorporate internal friction as an adjustable free parameter, we mimic typical single-molecule experiments that probe the unfolded state dynamics and make several experimentally verifiable predictions.Item Determining roles of the SUN domain proteins klaroid and Dspag4 in Drosophila development(2008-08) Kracklauer, Martin, 1971-; Fischer, Janice AnnIn eukaryotes, the process of nuclear migration is critical in fusion of haploid pronuclei after fertilization, in separation of daughter nuclei during mitosis, and in nuclear positioning in interphase cells. Experiments in several organisms have identified the basic protein requirements for nuclear migration and positioning: molecular motors that provide motive force; the cytoskeleton along which motors move nuclei, or to which the nuclei are anchored; and proteins of the outer and inner nuclear envelopes. These nuclear membrane proteins interact with the motors, the nuclear lamina and each other to effect nuclear migration and positioning. Proteins containing a SUN domain, which were first characterized in S. pombe Sad1 and C. elegans UNC-84, are inner nuclear envelope linkers of the nucleus to the cytoskeleton. In fungi, C. elegans, D. discoideum and vertebrates, these proteins are required not only for nuclear positioning, but also for maintaining the connection of the nucleus to the MTOC, for centrosomal duplication, for homologous pairing of chromosomes in meiosis, for distribution of nuclear pore complexes and for connecting the centrosome to chromatin to ensure genomic stability. The D. melanogaster genome has two genes, CG18584 and CG6589, which encode SUN domain proteins. The specific aims of my dissertation research were to generate null mutants in these genes, to characterize their null phenotypes, and to analyze where the genes are expressed. CG18584 = klaroid mutants are grossly normal, but adult eyes are mildly rough due to a defect in nuclear positioning that occurs during larval eye development. Klaroid protein is perinuclear in every cell of the eye, and functions by localizing the MTOC connector Klarsicht to the outer nuclear envelope. CG6589 = dspag4 null mutants are male sterile. In mature sperm, Dspag4 protein localizes rostrally to the sperm centriole. In the absence of Dspag4, most steps of gametogenesis occur normally, however, prior to the final steps of sperm maturation, the sperm nucleus dissociates from its centriole. Klaroid and Dspag4 thus have cellular roles typical for SUN domain proteins, and Dspag4 is unique in that its function is to attach nuclei to centrioles exclusively in maturing spermatids in the male germline.Item Development and Application of Proteomic Technologies for the Analysis of Post-Translational Modifications(2007-08-08) Sprung, Robert William, Jr; Zhao, YingmingPost-translational modifications represent a rapid and dynamic means for diversifying the chemistry of the ~20 ribosomally coded amino acids. As such, they provide an ideal mechanism for promoting cellular adaptability by facilitating the tuning of protein interactions and functions in response to changing environmental conditions. Despite their fundamental importance in regulating cellular functions and their wide implications in physiology, efficient means for the detection, enrichment and identification of proteins bearing specific modifications are lacking for most modifications. The availability of such methods would constitute invaluable tools supporting efforts to better understand the essential regulatory roles of modifications and the means by which aberrant modifications result in the onset and progression of disease. Towards this end, my dissertation describes the development and application of novel methods for the proteomic analysis of proteins bearing known modifications, including O-GlcNAc, lysine acetylation and methyl esterification. The identification of known targets of the modifications support some of the current ideas regarding their potential impact and serve as a means of validating the methods. More importantly, the identification of novel targets for the modifications challenges some currently held concepts, in particular regarding the relatively limited regulatory roles associated with lysine acetylation. In addition, the unparalleled power of proteomics as a screening strategy is demonstrated through compelling evidence of the existence of novel lysine acylations in vivo with respect to propionylation and butyrylation. Together, the methods described in this dissertation and the datasets generated embody powerful platforms and rich resources for the ongoing exploration of the fundamental contributions of post-translational modifications to the regulation of biological processes.Item Dispersibility indices, emulsion capacities, and electrophoretic comparisons of protein extracted from defatted soy flake suspensions at pH 4.5, pH 3.0 and pH 3.0 in the presence of calcium chloride(Texas Tech University, 1980-05) Hayslip, Jack LeonNot availableItem Effects of the proportion of supplemental dietary crude protein supplied by urea on performance and carcass characteristics of finishing beef cattle fed steam-flaked corn-based diets with Sweet Bran® wet corn gluten feed(Texas Tech University, 2004-05) Richeson, John TAn experiment was conducted to examine the effects of urea level in steam-flaked corn-based diets containing 25%) (DM basis) Sweet Bran® wet corn gluten feed (WCGF) on performance and carcass characteristics of beef steers. British x Continental steers were blocked by BW (average initial BW = 402.76 kg ± 10.75; n = 240) and assigned to one of three dietary treatments, which consisted of three different ratios (N basis) of urea:cottonseed meal provided in the supplemental CP: (1) 33%)urea:67%) cottonseed meal (33%); (2) 67%) urea:33% cottonseed meal (67%); and (3) 100%) urea:0%) cottonseed meal (100%). Eight pens per treatment were arranged in a randomized complete block design. Performance and carcass data were analyzed using mixed model procedures of S AS (SAS Institute, Cary, NC), with pen designated as the experimental unit and block as the random effect. There was a quadratic (P = 0.06) effect of the proportion of urea in supplemental CP on ADG from d 0 to 56, as steers fed the 33% diet gained less than cattle fed either the 67 or 100%) treatment. From d 0 to 112, ADG increased linearly (P = 0.09) with increasing proportion of urea provided in the supplement. For the overall feeding period, but especially early in the feeding period, ADG was numerically greatest in the steers fed the diet with 67%) urea:33%) cottonseed meal. Average daily DM intake (DMI) was affected linearly (P = 0.001), by urea level, as cattle fed the 33%) treatment consumed less than those fed the 67 or 100%> treatments from d 0 to 28. For the entire feeding period, DMI tended (P = 0.14) to increase linearly with increasing proportion of urea. There was a quadratic effect on gain:feed ratio from d 0 to end; steers fed the diet containing 67% urea:33%. cottonseed meal gained more efficiently (P = 0.09) than those fed the 33%) diet, whereas gain:feed by steers fed the 100%) treatment did not differ from that of steers in the other two treatments. Furthermore, there was a tendency for a quadratic effect (P = 0.14) of urea level relative to hot carcass weight (HCW). Average HCW was 393.0 kg for the 67% treatment, whereas the 33%) treatment averaged 384.3 kg, with an intermediate value of 390.5 kg for the 100%) treatment. Percentage of internal fat was least (P = 0.10, linear effect of urea level) for the 33%) diet. There were no treatment effects for yield grade, dressing percent, percentage of cattle grading USD A Choice, marbling score, backfat thickness, or longissimus muscle area. Incidence of liver abscess did not differ (P = 0.30) among the three treatments; however, the 33% treatment had a numerically higher rate (12.25%) than the 67% (8.26%)) and the 100% (7.50%) treatments. Results indicate that when feeding a finishing diet based on steam-flaked corn that contains 25%o (DM basis) Sweet Bran® WCGF, providing supplemental CP with a ratio of at least 67%) urea:33%o cottonseed meal improves ADG and feed efficiency compared with 33%) urea:67%> cottonseed meal.Item Expanding the genetic code in mammalian cells(2011-08) Xiang, Liang; Zhang, Zhiwen Jonathan; Georgiou, George; Roy, Krishnendu; Ren, Pengyu; Yin, WhitneyProteins are diverse polymers of covalently linked amino acids. They play a role in almost every biological process that occurs within an organism. Twenty different amino acids are genetically encoded by mammalian cells to build proteins. The sequence of these amino acids determines the protein’s final shape, structure, and function. Modern molecular cloning techniques allow for the genetic encoding and expression of mutant proteins that have one or more amino acids replaced with one of the others. The roles of individual amino acids in a protein can therefore be studied. Proteins with novel functions have also been designed or evolved using this technology. However, the genetic code is limited to the twenty natural amino acids. Nonnatural amino acids have unique side groups that not found on any of the twenty natural amino acids. They can be site-specifically incorporated using a mutant orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. Each pair only allows for one type of nonnatural amino acid to be genetically encoded. This technology has resulted in the incorporation of over fifty different types of nonnatural amino acids into proteins in prokaryotic and eukaryotic cells. Unfortunately, most of these pairs are not orthogonal outside of prokaryotic systems and only a few have been developed for mammalian cells. To create more mammalian pairs a nonnatural aaRS has to be evolved and screened in a cumbersome process. In this dissertation an approach is outlined that can be used to change the orthogonality of existing nonnatural suppressor tRNA/aaRS pairs. As a result of the orthogonality change many previously unavailable pairs can be shuttled into mammalian cells. The ability to genetically encode a 21st amino acid is a powerful tool in the study and engineering of proteins.Item Extraction of protein from defatted soy flake suspensions at pH 4.5, pH 3.0, and pH 3.0 in the presence of calcium chloride(Texas Tech University, 1978-05) Walker, David JosephNot availableItem Factors determining the pKa values of the ionizable groups in proteins: their intrinsic pKas and the effects of hydrogen bonding on buried carboxyl groups(Texas A&M University, 2007-04-25) Thurlkill, Richard LeeA goal of the modern protein chemist is the design of novel proteins with specific activities or functions. One hurdle to overcome is the ability to accurately predict the pKas of ionizable groups upon their burial in the interior of a protein, where they are typically perturbed from their intrinsic pKas. Most discussion of intrinsic pKas is based on model compound data collected prior to the 1960's. We present here a new set of intrinsic pKas based on model peptides, which we think are more applicable than the model compound values. We observe some differences with the model compound values, and discuss these by critically examining the compounds originally used for the dataset. One interaction affecting the pKas of ionizable groups in proteins that is not well understood is the effect of hydrogen bonds. The side chain carboxyl of Asp33 in RNase Sa is buried, forms 3 intramolecular hydrogen bonds, and has a pKa of 2.4 in the folded protein. One of these hydrogen bonds is to the side chain hydroxyl of Thr56. We mutated Thr56 to alanine and valine and observed that the mutations relieves the perturbation on the carboxyl group and elevates its pKa by 1.5 and 2 units, respectively. The side chain carboxyl of Asp76 in RNase T1 is completely buried, forms 3 intramolecular hydrogen bonds to other side chain groups, and has a pKa of 0.5 in the folded protein. Mutating any of the hydrogen bonding groups to the carboxyl affects its pKa differently, depending on the group mutated. Mutating all of the hydrogen bonding groups, creating a triple mutant of RNase T1, reverses the perturbation on the pKa and elevates it to about 6.4, very near the observed pKa of other carboxyl groups buried in hydrophobic environments. We compared these experimental results with predicted results from theoretical models based on the Solvent Accessibility Corrected Tanford- Kirkwood Equation and the finite difference solution to the linearized Poisson- Boltzmann Equation. The comparisons revealed that these models, most often used by theoreticians, are flawed when typically applied, and some possible improvements are proposed.Item Functional characterization of the role of Bruno protein in translational regulation and germ line development in Drosophila melanogaster(2006-05) Yan, Nan, 1979-; Macdonald, Paul M.Both body axes of the Drosophila egg are determined by localization of several mRNAs to specific regions within the oocyte. One of these mRNAs, oskar (osk), is required for posterior body patterning. Localization and translational control are both crucial for the correct deployment of osk. Bruno (Bru) binds specifically to the 3’UTR of the osk mRNA and represses osk translation. In this dissertation, I first describe a genetic screen looking for dominant modifiers of the arrest (aret) mutant phenotype (aret encodes Bru). Two modifiers suggested additional targets for Bru action. One is Star, a gene that contributes to provision of Gurken activity. The second suggested target is a gene acting in the Delta signaling pathway. A final modifier, Lk6, encodes a protein kinase predicted to regulate eIF4E. I also took a biochemical approach trying to understand how Bru regulates osk translation. Bru protein contains three RNA Recognition Motifs, but the remainder of the protein had no known function. I identified a domain, which is required for interaction to Bru itself, Cup and Apontic. Subsequent analysis of mutant forms of Bruno defective in these interactions led us to an unexpected discovery that Bru also acts as an activator of osk translation. Parallel analysis of Bru binding sites in osk 3’UTR fully support the notion that Bru has a dual role. There are two clusters of Bru Recognition Elements in either end of osk 3’UTR. Point mutations in one cluster cause overproduction of Osk protein while point mutations in the other cluster largely prevent translation of the message. To understand the molecular basis of the opposing roles of Bru, I used quantitative methods to better define and compare the binding of Bru to the different regulatory elements: those that either repress or activate osk mRNA translation. Using purified components I found that Bru binds to two clusters of binding sites in the osk 3’UTR differently, in terms of affinity, cooperativity and apparent compaction of the RNA. This work raises the possibility that the details of how Bru binds its substrate may determine whether it acts as a repressor or an activator.Item Genotypic variability in protein content and amino acid composition of pearl millet (Pennisetum Typhoideum)(Texas Tech University, 1978-05) Han, Ruth Tan-PiNot availableItem Influence of substrate on the amino acid profile and macromolecular composition of a Brevibacterium species(Texas Tech University, 1978-08) Miller, Leslie T.Not availableItem ISGylation and phosphorylation : two protein posttranslational modifications that play important roles in influenza A virus replication(2008-08) Hsiang, Tien-ying, 1976-; Krug, Robert M.Two posttranslational modifications, ISGylation and phosphorylation, impact the replication of influenza A virus, a human pathogen responsible for high mortality pandemics. The ubiquitin-like ISG15 protein is induced by type 1 interferon (IFN) and is conjugated to many cellular proteins by three enzymes that are also induced by IFN. Experiments using ISG15-knockout (ISG15-/-) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus, but inhibition was not detected in mouse embryo fibroblasts in tissue culture. The present study is focused on the effect of ISG15 and/or its conjugation on the replication of influenza A virus in human cells in tissue culture. IFN-induced antiviral activity against influenza A virus in human cells was significantly alleviated by blocking ISG15 conjugation using small interfering RNAs (siRNAs) against ISG15 conjugating enzymes. IFN-induced antiviral activity against influenza A virus gene expression and replication was reduced 10-20-fold by suppressing ISG15 conjugation. Unconjugated ISG15 does not contribute to this antiviral activity. Consequently human tissue culture cells can be used to delineate how ISG15 conjugation inhibits influenza A virus replication. SiRNA knockdowns were also used to demonstrate that other IFN-induced proteins, specifically p56, MxA and phospholipid scramblase 1, also inhibit influenza A virus gene expression in human cells. The research on phosphorylation focused on the viral NS1A protein, a multifunctional virulence factor. Although threonine phosphorylation of the NS1A protein was discovered 30 years ago, the sites of phosphorylation and its function had not been identified. A recombinant influenza A virus encoding an epitope-tagged NS1A protein was generated, enabling the purification of NS1A protein from infected cell extracts. Mass spectrometry identified phosphorylation at T49 and T215. A recombinant virus in which phosphorylation at 215 was abolished by replacing T with A is attenuated, and an apparently aberrant NS1A protein is produced. Attenuation did not occur when T was changed to E to mimic a constitutively phosphorylated state, or surprisingly when T was changed to P to mimic avian NS1A proteins. These results suggest that T215 phosphorylation in human viruses and P215 in avian viruses can support analogous functions.
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