Bacterial toxins have been shown to modify animal cell proteins in vivo with ADPR. Animal cells also contain endogenous enzymes that can modify proteins. Indirect evidence for the existence in vivo of rat liver proteins modified by ADPR on arginine residues has been reported previously. Presented here is direct evidence for the existence of ADP-ribosylarginine in rat liver proteins. Proteins were subjected to exhaustive protease digestion and ADP-ribosyl amino acids were isolated by boronate chromatography.

The pathology of Alzheimer's disease (AD) remains elusive. Competing evidence links amylois \U+fffd\-peptide (A\U+fffd\) amyloid formation to the phenotype of AD (1). The mechanism of amyloid fibril formation has been an ongoing investigation for many years. A\U+fffd\10-23 peptide, a fragment of A\U+fffd\1-42 peptide, contained crucial hydrophobic core residues (2). In this study, an investigation was launched to study the aggreagation process of A\U+fffd\1023 peptide and its ability to form amyloid fibrils. Furthermore, the presence of its hydrophobic core showed importance for its ability to aggregate and form amyloid fibrils. Thereafter, the inhibition of A\U+fffd\1-42 peptide aggregation was studied by using pyrimidine-based compounds. A\U+fffd\1-42 peptides, known to be neurotoxic, aggregate to form amyloid fibrils (3). This investigation may provide insight into the development of novel small molecular candidates to treat AD. / by Ahmad Alex Hijazi. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

by Scott P. Kelly. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 168-190). / Acknowledgements --- p.iv / Chapter Chapter 1 : --- Introduction --- p.1 / Chapter Chapter 2 : --- Review of the literature / Part A : Nutritional requirements of fish and performance assessment / Chapter A2.1 --- Introduction --- p.5 / Chapter A2.2 --- Dietary protein requirement --- p.6 / Chapter A2.2.1 --- Factors influencing dietary protein requirement / Chapter A2.2.1.1 --- Protein quality / Chapter A2.2.1.2 --- Nonpotein energy / Chapter A2.2.1.3 --- Protein energy to total energy ratio / Chapter A2.2.1.4 --- Fish size/age / Chapter A2.2.1.5 --- Feeding rate / Chapter A2.2.1.6 --- Natural food / Chapter A2.2.1.7 --- Economics / Chapter A2.2.1.8 --- Environmental factors / Chapter A2.3 --- Dietary lipid --- p.14 / Chapter A2.3.1 --- Dietary lipid requirement / Chapter A2.4 --- Dietary carbohydrate --- p.16 / Chapter A2.4.1 --- Dietary carbohydrate requirement / Chapter A2.5 --- Dietary vitamins / Chapter A2.5.1 --- Dietary vitamin requirements / Chapter A2.6 --- Dietary minerals --- p.19 / Chapter A2.6.1 --- Dietary mineral requirements / Chapter A2.7 --- Evaluation criteria --- p.21 / Chapter A2.7.1 --- Introduction / Chapter A2.7.2 --- Growth and conversion efficiencies / Chapter A2.8 --- Digestion --- p.22 / Chapter A2.9 --- Metabolism in relation to nutritional status --- p.23 / Chapter A2.9.1 --- Nitrogen excretion / Chapter A2.9.2 --- Metabolic rate (Oxygen consumption) / Chapter A2.10 --- Biochemical indices of metabolic performance --- p.25 / Chapter A2.10.1 --- Tissue composition / Chapter A2.10.1.1 --- Proximate composition and organ indices / Chapter A2.10.1.2 --- Essential amino acid (EAA) profile / Chapter A2.10.2.3 --- Lipid/essential fatty acid (EFA) profile / Chapter A2.10.2.4 --- Tissue RNA/DNA ratio / Chapter A2.11 --- Haematological characteristics --- p.27 / Chapter A2.12 --- Enzymes --- p.28 / Chapter A2.12.1 --- Enzymes of the intermediate metabolism / Chapter A2.12.2 --- Intestinal enzymes / Chapter A2.13 --- Thyroid hormones --- p.30 / Part B : Teleostean salinity adaptation / Chapter B2.1 --- Introduction --- p.31 / Chapter B2.2 --- Influence of salinity on growth --- p.32 / Chapter B2.3 --- Influence of salinity on weight loss during starvation --- p.37 / Chapter B2.4 --- Metabolic rate (oxygen consumption) and salinity adaptation --- p.38 / Chapter B2.5 --- Biochemical indices of performance during salinity adaptation --- p.41 / Chapter B2.5.1 --- Carcass and tissue composition / Chapter B2.5.2 --- Haematological characteristics / Chapter B2.6 --- Influence of salinity on protein requirements of fish --- p.46 / Chapter B2.7 --- Effect of salinity on digestion and digestive enzymes --- p.48 / Chapter B2.8 --- Cortisol and osmotic adjustment --- p.50 / Chapter B2.9 --- Conclusion --- p.52 / Chapter Chapter 3: --- Materials and methods / Chapter 3.1 --- Culture conditions --- p.54 / Chapter 3.2 --- Composition of experimental diets --- p.55 / Chapter 3.2.1 --- Dietary protein / Chapter 3.2.2 --- Dietary carbohydrate / Chapter 3.2.3 --- Dietary lipid / Chapter 3.2.4 --- Dietary vitamins / Chapter 3.2.5 --- Dietary minerals / Chapter 3.2.6 --- Chromic oxide / Chapter 3.2.7 --- Binder / Chapter 3.3 --- Proximate analysis of experimental diets --- p.61 / Chapter 3.3.1 --- Moisture / Chapter 3.3.2 --- Lipid / Chapter 3.3.3 --- Protein / Chapter 3.3.4 --- Ash / Chapter 3.3.5 --- Nitrogen free extractives / Chapter 3.3.6 --- Metabolizable energy / Chapter 3.4 --- Dietary evaluation criteria --- p.65 / Chapter 3.4.1 --- Growth rate / Chapter 3.4.2 --- Protein efficiency ratio / Chapter 3.4.3 --- Food conversion ratio / Chapter 3.4.4 --- Feed conversion efficiency / Chapter 3.5 --- Digestibility --- p.66 / Chapter 3.6 --- Metabolism --- p.67 / Chapter 3.6.1 --- Ammonia excretion / Chapter 3.6.2 --- Oxygen consumption / Chapter 3.7 --- Biochemical analysis / Chapter 3.7.1 --- Organ indices / Chapter 3.7.2 --- Serum metabolites / Chapter 3.7.2.1 --- Collection of serum / Chapter 3.7.2.2 --- Serum ions / Chapter 3.7.2.3 --- Serum protein / Chapter 3.7.2.4 --- Serum α-amino acids / Chapter 3.7.2.5 --- Serum ammonia / Chapter 3.7.2.6 --- Serum glucose / Chapter 3.7.2.7 --- Serum lipids / Chapter 3.7.2.8 --- Serum hormones / Chapter 3.7.3 --- Proximate analysis of white muscle --- p.74 / Chapter 3.7.4 --- Analysis of hepatic tissue --- p.75 / Chapter 3.7.4.1 --- Proximate analysis / Chapter 3.7.4.2 --- Liver glycogen / Chapter 3.7.4.3 --- Hepatic enzymes / Chapter 3.7.4.3. --- a Glucose-6-phosphatase / Chapter 3.7.4.3. --- b Glucose-6-phosphate dehydrogenase / Chapter 3.7.4.3. --- C Hexokinase / Chapter 3.7.5 --- Intestinal enzymes --- p.80 / Chapter 3.7.5.1 --- γ-Glutamyltranspeptidase / Chapter 3.7.5.2 --- Trypsin / Chapter 3.7.5.3 --- α-Amylase / Chapter 3.7.6 --- Statistical analysis --- p.83 / Chapter Chapter 4: --- Results / Chapter 4.1 --- Growth and conversion efficiencies --- p.84 / Chapter 4.2 --- Metabolism --- p.90 / Chapter 4.2.1 --- Ammonia excretion / Chapter 4.2.2 --- Oxygen consumption / Chapter 4.3 --- Biochemical analysis --- p.93 / Chapter 4.3.1 --- Organ indices --- p.93 / Chapter 4.3.2 --- Serum metabolites --- p.98 / Chapter 4.3.2.1 --- Serum ions / Chapter 4.3.2.2 --- "Serum protein, α-amino acids and ammonia" / Chapter 4.3.2.3 --- Serum glucose / Chapter 4.3.2.4 --- Serum lipids / Chapter 4.3.2.5 --- Serum hormones / Chapter 4.3.3 --- Proximate composition of white muscle --- p.113 / Chapter 4.3.4 --- Composition of hepatic tissue --- p.117 / Chapter 4.3.4.1 --- Proximate composition and liver glycogen / Chapter 4.3.4.2 --- Hepatic enzymes / Chapter 4.3.5 --- Total digestibility --- p.126 / Chapter 4.3.6 --- Intestinal enzymes --- p.126 / Chapter Chapter 5: --- Discussion / Chapter 5.1 --- Growth and conversion efficiencies --- p.132 / Chapter 5.2 --- Metabolism --- p.135 / Chapter 5.2.1 --- Ammonia excretion / Chapter 5.2.2 --- Oxygen consumption / Chapter 5.3 --- Organ indices --- p.137 / Chapter 5.4 --- Serum metabolites --- p.140 / Chapter 5.4.1 --- Serum ions / Chapter 5.4.2 --- "Serum protein, α-amino acids and ammonia" / Chapter 5.4.3 --- Serum glucose / Chapter 5.4.4 --- Serum lipids / Chapter 5.4.5 --- Serum hormones / Chapter 5.5 --- Proximate composition of white muscle --- p.150 / Chapter 5.6 --- Proximate composition of liver --- p.152 / Chapter 5.7 --- Hepatic enzymes / Chapter 5.7.1 --- Glucose-6-phosphate dehydrogenase (G6P-DH) / Chapter 5.7.2 --- Hexokinase / Chapter 5.7.3 --- Glucose-6-phosphatase (G-6-Pase) / Chapter 5.8 --- Total digestibility --- p.159 / Chapter 5.9 --- Intestinal enzymes / Chapter 5.9.1 --- Trypsin / Chapter 5.9.2 --- γ-Glutamyltranspeptidase (γ-GT) / Chapter 5.9.3 --- α-Amylase / Chapter Chapter 6: --- Conclusion --- p.164 / References --- p.168

Photocopy of typescript. / Digitized by Kansas Correctional Industries

The first goal of this study was to understand the role of u-calpain in skeletal muscle protein degradation in cultured muscle cells. Several strategies were developed to down-regulate endogenous u-calpain activity and m-calpain activity in rat myotubes. These included over-expression of antisense u-calpain (AnsL), dominant negative u-calpain (DN-u-CL), antisense 30K subunit (AnsS) and fused antisense u-calpain/30K (AnsLS, i.e., 80K/30K). The ability to regulate calpain activity was confirmed by fodrin degradation (an index of calpain activity). Our data supported the contention that u-calpain contributes significantly to total protein degradation in myotubes. Specifically, over-expressing DN-u-calpain reduced total protein degradation by 7.9% (P<0.01) at 24 hr time point and by 10.6% (P<0.01) at a 48 hr time point. Similarly, over-expression of antisense u-CL and the 30K subunit reduced total protein degradation significantly at the 24 hr time point (P<0.05). However, over-expression of the fused antisense (80K/30K) did not affect (P>0.05) the total protein degradation. In addition to this we determined that desmin was a calpain substrate and that calpain could not degrade tropomyosin. The second goal of this study was to evaluate the relationships among u- and m-calpain and the 30KD subunit. The rationale for this study was that our earlier work indicated coordinated regulation of the calpain subunits. Our data demonstrated for the first time that the transcription and translation of u-calpain and 30K, and m-calpain and 30K are coordinately regulated, respectively. However, the expression of u-calpain did not affect the expression of m-calpain The third goal of this study was to develop a skeletal muscle-specific inducible expression system that may be used in transgenic animal research. A skeletal muscle a-actin promoter was used to replace the cytomegalovirus immediate-early promoter (pCMV) in the ecdysone inducible mammalian expression system. LacZ was used as a reporter gene. A beta-galactosidase staining assay and high-sensitivity B-gal activity assay indicated that the skeletal muscle-specific expression system functioned in myotubes. After 48 hr of administration of ponasterone A (inducer), the treated cells had 15-fold higher B-gal activity than the control cells. / Graduation date: 2002

No description available.

Groucho/transducin-like Enhancer of split (Gro/TLE) family proteins are corepressors found as part of multiple transcriptional complexes that play significant roles during many developmental processes, including neurogenesis. This thesis sought to characterize the molecular mechanisms underlying the biological activity of Gro/TLE1. More specifically, the aim was to clarify the contribution of different transcriptional cofactors, as well as phosphorylation events induced by cofactor binding, to Gro/TLE1 ability to inhibit neuronal differentiation from proliferating neural progenitor cells. / By characterizing specific point mutations within the C-terminal domain of Gro/TLE1, we were able to selectively impair binding of Gro/TLE1 to different classes of DNA-binding proteins and then assess the effect of those mutations on Gro/TLE1 anti-neurogenic function. These studies showed that the inhibition of cerebral cortex (cortical) neuron differentiation by Gro/TLE1 requires interaction with transcription factors that use short tetrapeptide sequences, WRP(W/Y), to recruit Gro/TLE1. In contrast, interactions with proteins that either interact with the C-terminal domain of Gro/TLE1 using a different type of binding sequence, termed engrailed homology 1 (Eh1) motif, or bind to the N-terminal part of the protein, are not required for Gro/TLE1 anti-neurogenic function. / Using a similar strategy based on mutation analysis, we characterized point mutations that block the hyperphosphorylation of Gro/TLE1 induced by transcription cofactor binding ("cofactor-activated phosphorylation") without impairing cofactor binding and transcriptional corepression ability. These mutations map at phosphorylatable serine residues, Ser-286, Ser-289, and Ser298. Mutation of those residues to alanine blocks/reduces both cofactor-activated phosphorylation and anti-neurogenic activity of Gro/TLE1, demonstrating that cofactor-activated phosphorylation is required for that function. Tandem mass spectroscopy analysis showed further that Ser-286 is phosphorylated. Taken together, these findings characterize the role of cofactor-activated phosphorylation and identify residues important for this mechanism. / Our studies also showed that homeodomain-interacting protein kinase 2 (HIPK2) mediates phosphorylation of Gro/TLE1 when the latter is complexed with transcriptional partners of the WRP(W/Y) motif family. However, HIPK2 is not involved in Gro/TLE1 cofactor-activated phosphorylation. Rather, HIPK2--mediated phosphorylation is antagonistic to the latter and decreases the ability of Gro/TLE1 to interact and repress transcription with WRP(W/Y) motif proteins. / Taken together, these results improve significantly our understanding of the mechanisms underlying the anti-neurogenic function of Gro/TLE1. This information provides new insight into the regulation of mammalian neuronal development and, possibly, other developmental processes controlled by Gro/TLE proteins.

In the past three decades, various steps of the human immunodeficiency virus type 1 (HIV-1) life cycle have been thoroughly studied. Many of these steps, such as viral entry, reverse transcription and proteolysis have been targets of antiretroviral therapy. Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity and this process appears to be chaperoned by the nucleocapsid (NC) protein of HIV-1. In this dissertation, the role of NC in genome dimerization and other aspects of the viral life cycle have been thoroughly studied. Various positions of the NC protein have been mutated through site-directed mutagenesis and relevant and dispensable positions of NC have been identified through this method. 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The various mutations introduced into NC show the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal or distal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch (plateau) or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein (Pr55gag). In the second section of my work, I studied the effect of Pr55gag processing on gRNA dimerization by introducing rate alternating mutants into Pr55gag protein cleavage sites. I showed that Maturation ofNCp15 into NCp9 is essential for fast rates of genomic RNA dimerization and maturation of NCp9 into NCp7 has no incidence on genomic RNA dimerization but is essential for viral replication. In order to delineate the amount of viral protease activity needed to produce mature virus 48 hours post transfection, we also studied, by cotransfection studies, the effect of various ratios of wild-type (BH10) and protease-inactive (PR- ) plasmids and found that HIV-1 reaches its full genomic RNA dimerization despite 75% unprocessed Pr55gag polyproteins. We have also shown that wild type BH10 plasmid can rescue those mutations in NCp7 protein that have an effect on gRNA dimerization through rescue experiments. Overall, this thesis sheds light on the role of NC in HIV-1 genome dimerization and other aspects of the viral life cycle and identifies the importance of each component of NC during these processes.

Access to abstract permanently restricted. / Access to thesis permanently restricted. / Department of Biology

The present project investigated the effect of epidural blockade with local anesthetic on the catabolic stress response during and immediately after abdominal surgery in fasting patients and during infusion of glucose at 2 mg&middot;kg-1&middot;min-1. The kinetics of glucose and protein metabolism were assessed by the stable isotope tracers [6,6-2H2]glucose and L-[1-13C]-leucine. / Epidural blockade was associated with a lower plasma glucose concentration and glucose production when compared to control subjects in the fasted state. Whole body protein breakdown, amino acid oxidation and protein synthesis were suppressed during surgery, and epidural blockade had no modifying effect on perioperative protein metabolism. The suppression of endogenous glucose production by exogenous glucose was more pronounced in the presence of epidural blockade. Perioperative protein metabolism, however, was not influenced by epidural blockade during glucose infusion. / Although epidural blockade suppressed glucose metabolism both in the fasted state and during glucose administration, it failed to exert a modifying effect on perioperative protein metabolism.