Protein arginine methylation is a posttranslational modification catalyzed by the family of proteins known as the protein arginine methyltransferases (PRMTs). Thousands of methylated arginines have been found in mammalian cells. Many targets of arginine regulation are involved in important cellular processes like transcription, RNA transport and processing, translation, cellular signaling, and DNA repair. Since PRMT dysregulation has been linked to a variety of disease states, understanding how the activity of the PRMTs is regulated is of paramount importance. PRMT1 is the predominant PRMT, responsible for about 85% of all arginine methylation in cells, but very little is known about how PRMT1 is regulated. Although a few methods to regulate PRMT1 activity have been reported, the details of interaction and regulatory mechanisms remain largely unknown. To better understand how PRMT1 is able to bind its substrates and how PRMT1 activity is regulated, we followed a mechanistic and structural biology approach to better understand how PRMT1 interacts with its substrates and protein regulators. In this study the regulation of Hmt1 methyltransferase activity by the Air1 and Air2 proteins was analyzed and only one was determined to affect Hmt1 activity. The posttranslational phosphorylation of Hmt1 had also been reported to affect Hmt1 activity in vivo and our preliminary studies suggest that additional factors may help influence the regulatory effect of phosphorylation. Lastly, we report a new method of PRMT regulation through the reversible oxidation of key PRMT1 cysteine residues. We are also able to show that this regulation occurs in cells and affects several PRMT isoforms.

Indiana University-Purdue University Indianapolis (IUPUI) / Streptococcus mutans, an inhabitant of oral biofilm or dental plaque, adheres to the tooth surface via protein antigen I/II (PA I/II). Pathologic lesions of atherosclerosis (AT) and infective endocarditis (IE) harbor S. mutans. Serotypes f and k strains with collagen binding protein genes cbm and cnm are uncommon in the mouth, but these are the most prevalent S. mutans strains in AT and IE tissues and can invade endothelial cells (EC) in vitro. Tobacco use increases the risk for cardiovascular and oral diseases. Oral S. mutans encounter many substances including nicotine. Arginine is present in saliva and the EC glycocalyx that coats and protects ECs from shear forces of blood flow. Prior studies demonstrated arginine alters S. mutans biofilm. This work characterizes S. mutans serotype k strains and serotype c strains, the most prevalent in the mouth. The effects of nicotine and arginine on biofilm mass, metabolic activity and EC invasion were investigated. Biofilm production by serotypes c and k strains did not differ; there were no differences in responses to nicotine and arginine between these serotypes. Increased production of biofilm was associated with the cbm and cnm genes. Nicotine increased biofilm for all strains whereas arginine plus nicotine reduced bacteria and the extracellular polymeric substances. Previous EC invasion studies were performed with planktonic cultures of S. mutans; therefore, EC invasion by biofilm was evaluated. Significant factors for EC invasion by S. mutans are presence of the cbm gene and lack of PA I/II expression on the bacterial cell surface. Presence of the cnm gene increased EC invasion by biofilm but not planktonic cells. Planktonic cells of six strains invaded better than biofilm, whereas four strains showed increased invasion by biofilm cells. Neither nicotine nor arginine significantly altered the ability of S. mutans biofilm cells to invade ECs. Not all strains with cbm or cnm and no PA I/II expression invaded EC. A strain with PA I/II expression and without cbm and cnm genes invaded EC. While cbm, cnm and PA I/II expression are predictors of EC invasion, additional mechanisms for EC invasion by S. mutans remain to be revealed. / 2021-08-21

The objective of this study was to evaluate the effects of dietary lysine on plasma concentrations of amino acid (AAs) and other metabolites of finishing pigs. Eighteen crossbred late-stage finishing pigs (9 barrows and 9 gilts) were assigned to three dietary treatments, Diets I (lysine-deficient), II (lysineequate), and III (lysine-excess), according to a randomized complete block design with gender as block. After 4 weeks on trial, jugular vein blood were collected and centrifuged for plasma samples, which were analyzed for concentrations of 24 AAs using HPLC method and 6 selected metabolites related to major nutrient metabolism using ACE Alera Clinical Chemistry System. Dietary lysine had a significant effect on plasma concentrations of 13 AAs and 3 metabolites (albumin, cholesterol, and urea nitrogen). The patterns of AA and metabolite concentrations alteration indicated complex metabolic interactions between lysine and other AAs, which subsequently had effects on other metabolites and animal growth performance.

Studies that have investigated the effects of L-arginine supplementation on blood flow to the uterus have not determined the optimal time of supplementation. No research has been conducted on nitrite concentration in the placenta and blood of mares supplemented with L-arginine. L-arginine is a precursor of NO and therefore, should increase nitrite production both in blood as well as in expelled placenta. Presence of nitrites would also be indicative of increased angiogenesis. No studies have addressed placental efficiency in mares supplemented L-arginine. Placental efficiency is vital for production of a healthy foal. Therefore, to address these short-comings the objectives of this study were to supplement pregnant mares with L-arginine on effects of supplementation at differing time points on blood flow to the uterus, nitrite concentration in blood and placenta, and placental efficiency.

La petite protéine de choc thermique Hsp27 chez Drosophila melanogaster est localisée au noyau. Dans le but d'identifier la séquence peptidique responsable de cette localisation, la mutagenèse dirigée d'un signal potentiel de localisation nucléaire bipartite situé entre les acides aminés 42 et 59 a été effectuée, et les vecteurs obtenus ont été transfectés dans des cellules de mammifères HeLa. Il s'est avéré que la mutation des arginines 54, 55 et 56 de la protéine occasionne une diminution importante de la localisation nucléaire, bien qu'on observe toujours des résidus nucléaires localisés au niveau de structures granulaires. La localisation au niveau de structures granulaires est également observée avec la protéine sauvage. Des analyses avec des marqueurs de corps nucléaires ont donc été effectuées afin de déterminer si ces granules contenant Hsp27 correspondaient à des corps nucléaires connus. Il a été observé que la protéine colocalise avec le facteur d'épissage SC35 au niveau des "speckles" nucléaires. Ces observations s'avéreront utiles dans l'identification de fonctions pour la protéine Hsp27.

Les récepteurs couplés aux protéines G (RCPG) représentent la plus grande famille de protéines membranaires. Ils sont activés par une grande variété d'hormones, de neurotransmetteurs, et par des stimuli sensoriels. Ces récepteurs jouent un rôle central dans le contrôle de la grande majorité des fonctions physiologiques et constituent une cible thérapeutique majeure. Plusieurs études supportent l'existence de plusieurs états conformationnels de ces récepteurs stabilisés par les ligands. Le caractère dynamique des RCPG est essentiel dans leur fonctionnement. Une question majeure est de déterminer comment un ligand modifie la structure et la fonction de son récepteur. Pour cela, nous avons analysé les changements conformationnels d'un récepteur prototype de la famille des récepteurs à ligands peptidiques, le sous-type V2 de la vasopressine (V2R). Le V2R présente un large éventail de ligands ayant des efficacités différentes (agoniste partiels et complets, agonistes inverses et antagonistes) ainsi que des agonistes biaisés vis-à-vis des voies de signalisation dépendantes de Gs et des arrestines. Afin de déterminer les bases structurales de l'efficacité (amplitude de la réponse biologique) et de la sélectivité fonctionnelle (la capacité d'un RCPG à activer ou à inactiver préférentiellement une voie de signalisation parmi l'ensemble des voies de transduction auxquelles il est couplé), nous avons purifié et stabilisé le V2R par reconstitution en amphipols neutres (Napols). La fonctionnalité du récepteur a été vérifiée par mesure de son interaction directe avec la protéine Gs et les arrestines purifiés. Les profils d'efficacité des ligands vis-à-vis des deux voies de signalisation sont cohérents avec ceux décrits dans des cellules vivantes. Afin d'aborder directement les changements conformationnels dépendants des ligands, nous avons développé deux approches de fluorescence, la fluorescence intrinsèque des tryptophanes et le LRET (Lanthanide Resonance Energy Transfer). La liaison des ligands ayant des efficacités opposées pour la voie Gs ont induit des variations opposées de la fluorescence intrinsèque des tryptophanes, suggèrant l'existence d'états conformationnels distincts. En parallèle, l'analyse des changements des signaux de LRET entre deux domaines fonctionnels du récepteur marqués par deux fluorophores compatibles, le domaine transmembranaire 6 (TM6) côté cytoplasme et l'extrémité C-terminale distale, a permis de calculer une distance moyenne de 33 Å. En accord avec les variations de fluorescence intrinsèque des tryptophanes, les ligands ayant des efficacités opposées pour la voie Gs ont induit un mouvement opposé de ces deux domaines. Les agonistes complets entraînent un éloignement de la boucle i3 et de l'extrémité C-terminale (+2.4 Å) alors qu'un rapprochement des deux domaines est associé à la liaison de l'agoniste inverse (-0.9 Å). Nos résultats démontrent qu'un récepteur à ligands peptidiques répond à la liaison de ses ligands spécifiques par des changements conformationnels dynamiques. Chaque ligand est caractérisé par un ou plusieurs états conformationnels distincts. De plus, les changements conformationnels du V2R jouant un rôle dans le couplage à Gs sont différents de ceux impliqués dans le recrutement des arrestines. Ces données apportent des éléments essentiels de compréhension des mécanismes moléculaires et structuraux de l'activation des RCPG. A plus long terme, une étude plus extensive de la dynamique des RCPG devrait guider le développement de molécules thérapeutiques possédant des propriétés de sélectivité fonctionnelle. / G protein-coupled receptors (GPCR) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Several studies support the existence of multiple ligand-specific conformational states of GPCR. The dynamic character of GPCR is likely to be essential for their functioning, and a better understanding of this molecular plasticity might facilitate structure-based drug discovery. A major question is to determine how ligands modify receptor structure and function. To this end, we have been studying the structural dynamics of the human vasopressin type 2 receptor (V2R), a prototypical peptide-activated class A GPCR. The V2R is coupled to Gs protein and to β-arrestins, and it has been well characterized pharmacologically using a large panel of ligands with different efficacies. Several display functional selectivity (Gs activation and concomitant β-arrestin inhibition). To demonstrate that ligand efficacy and functional selectivity are achieved through the stabilization of multiple conformational states, we have purified and reconstituted the V2R in amphipathic polymers (amphipol) and developed fluorescence-based approaches. The functionality of the V2R was monitored by direct activation of the purified Gs protein and interaction with purified β-arrestin 1. In these two assays, the effect of ligands correlated well with their known efficacy in cellular systems. Binding of V2R ligands with opposite efficacies toward Gs pathway led to opposite variations in the tryptophan intrinsic fluorescence of the receptor, suggesting the presence of different conformational states of the receptor. In parallel, we used Lanthanide-based resonance energy transfer (LRET) to directly analyze dynamics of the V2R and more particularly conformational changes between fluorophore-labeled extreme C-terminus and transmembrane domain 6. We calculated a basal mean distance of 33 Å between these domains. Interestingly, ligands with different efficacies towards Gs protein elicited opposite LRET changes as for tryptophan fluorescence spectroscopy. Indeed, the two labeled domains moved away upon full agonist binding (+2.4 Å), and closer in presence of inverse agonist (-0.9 Å). These data provide the first evidence of ligand-specific conformational changes in a peptide-activated receptor, and demonstrate that receptor conformational changes involved in Gs coupling are different from those responsible for arrestin recruitment. The results shed some light into the molecular and structural mechanisms of GPCR activation that may be relevant to the design of signaling pathway-selective drugs.

A single plaque-pure lambda clone designated λBA84 that hybridized to a ˆ32P-labeled bovine arginine tRNA was isolated from a bovine genomic library harbored in a lambda bacteriophage vector. A 2.3-kilobase segment of this clone was found to contain an arginine transfer RNAccg gene by Southern blot hybridization analysis and dideoxyribonucleotide DNA sequencing. This gene contains the characteristic RNA polymerase III split promoter sequence found in all eukaryotic tRNAs and a potential RNA polymerase III termination site, consisting of four consecutive thymine residues, in the 3'-flanking region. Several possible cis-acting promoter elements were found within the 5'-flanking region of the sequenced gene. The function of these elements, if any, is unknown.

Pancreatic cancer is one of the most devastating human cancers with the lowest survival rate among 24 commonly diagnosed cancers. It is the seventh and the sixth leading cause of cancer-related deaths in the world and Hong Kong respectively. The current pancreatic cancer treatment options, have limited efficacy and undesirable side effects. Because of the high mortality rate and unsatisfactory treatment outcome, it is necessary to develop new strategies for pancreatic cancer therapy. / In human, an abundant arginine reserve is known to be crucial for tumor cell proliferation. Arginine is a semi-essential amino acid because most of the somatic cells can re-synthesize it from other metabolites like citrulline in urea cycle. However, arginine auxotrophy is observed in certain tumors, such as hepatocarcinoma, melanoma and sarcoma, where restriction or depletion of arginine will lead to tumor death. Further studies have found that deficiency in either argininosuccinate synthetase 1 (ASS1) or ornithine transcarbamylase (OTC) expression contributes to arginine auxotrophy in these tumors. These findings implicated the potential of using arginine deprivation as a novel pancreatic cancer treatment strategy. / PEG-BCT-100 is a pegylated recombinant human arginase that metabolizes arginine into urea and ornithine. This study examined the preclinical anti-tumor efficacy of PEG-BCT-100 and the underlying mechanism in pancreatic cancer. Six pancreatic cancer cell lines AsPC-1, BxPC-3, CFPAC-1, Capan-2, MIA PaCa-2 and Panc10.05 were used as in vitro cell model. Cell growth was either completely stopped or dramatically reduced in arginine-free medium, suggesting pancreatic cancer cells were arginine auxotrophic. The protein and mRNA expression levels of the ASS1, OTC and argininosuccinate lyase (ASL), which are enzymes involved in arginine, were studied. The results showed that ASL was highly expressed in all cell lines, suggesting it is not an essential regulator in arginine auxotrophy in pancreatic cancer. On the other hand, ASS1 was only detected in BxPC-3 and CFPAC-1, while OTC was undetectable in all cell lines in both mRNA and protein levels. The effect of PEG-BCT-100 was illustrated via cell cycle progression, cell proliferation and viability. Single drug effect combining PEG-BCT-100 with other anti-tumor drugs, such as 5-FU and gemcitabine, was further explored. Synergistic effect of PEG-BCT-100 and gemcitabine under combination of PEG-BCT-100 and gemcitabine was observed in CFPAC-1 and MIA PaCa-2. Overexpression of OTC and ASS1 decreased the sensitivity of towards PEG-BCT-100 significantly. Taken together, OTC deficiency is a potential indicative marker for the sensitivity of arginine depletion treatment in pancreatic cancer. / 胰腺癌是最具毀滅性的人類癌症之一，在二十四種常見的癌症中，它有着最低的存活率。儘管不在發病率最高的十種癌症中，胰腺癌仍舊是世界第七大致死癌症，以及香港第六大致死癌症。手術治療，放射治療，以及化學藥物治療是現今常用的胰腺癌治療手段，但是這些療法不是限制繁多，就是收效甚微，並常常伴有強烈的副作用。由於胰腺癌具有很高的致死率以及缺乏有效的治療方法，所以新的治療策略亟待開發。 / 於人類而言，精氨酸是一種半必需氨基酸，因爲它可以通過尿素循環中的其他代謝產物，如鳥氨酸以及瓜氨酸，重新合成。然而，精氨酸缺陷出現在多種腫瘤中，像肝癌，黑色素瘤，以及血癌。限制或者減少精氨酸的供應會導致這些腫瘤死亡。除此之外，腫瘤細胞的快速生長也依賴於充足的精氨酸。進一步的研究表明，在這些腫瘤中，精氨琥珀酸合成酶1（ASS1）或者鳥氨酸氨甲醯基轉移酶（OTC）的任意一個缺乏都會導致精氨酸缺陷。本文將探討將剝奪精氨酸作爲一種新策略來治療胰腺癌的可行性。 / PEG-BCT-100又名金氨素，是一種聚乙二醇化重組人精氨酸酶，它可以催化精氨酸分解爲尿素和鳥氨酸。我們研究了PEG-BCT-100在胰腺癌細胞中的抗癌效果以及探討了與其相關的作用機理。在我們的研究中，AsPC-1, BxPC-3, CFPAC-1, Capan-2, MIA PaCa-2以及Panc10.05這六個細胞株用作體外的細胞模型。爲了評估PEG-BCT-100治療胰腺癌的可行性，我們首先調查了精氨酸對胰腺癌細胞的重要性。通過將這些胰腺癌細胞培養在有精氨酸供應和沒有精氨酸供應的完全培養基中，我們發現剝奪精氨酸能完全停止或者極大地減少了胰腺癌細胞的生長。這說明了這些胰腺癌細胞也都是精氨酸營養缺陷型的細胞。通過蛋白印跡和實時定量聚合酶鏈式反應實驗，我們進一步研究了精氨酸代謝相關基因在這些胰腺癌細胞中的表達水平。結果表明，精氨琥珀酸裂解酶（ASL）在全部的六條細胞系中都有被檢測到。ASS1只出現在BxPC-3和CFPAC-1中。然而在全部的細胞中，無論是蛋白質水平還是mRNA水平，OTC都沒有被檢測到。緊接着，我們研究了PEG-BCT-100在胰腺癌細胞活力，細胞增殖，細胞週期以及細胞凋亡等方面的影響。結果表明，PEG-BCT-100可以從多個方面抑制胰腺癌細胞。我們還嘗試探索了PEG-BCT-100與其他胰腺癌治療藥物在胰腺癌細胞中的聯合使用效果。然後發現PEG-BCT-100與吉西他滨（gemcitabine）聯合使用具有協同效果。最後，我們構建了四種不同表達類型的MIA PaCa-2細胞模型：（ASS1-/OTC-）, (ASS1-/OTC+), (ASS1+/OTC-)以及(ASS1+/OTC+)。接着我們測試了PEG-BCT-100在這些細胞模型中的效果。結果表明，同時在MIA PaCa-2細胞中表達ASS1和OTC可以明顯地提高其對PEG-BCT-100的抗性，單表達其中一個基因對PEG-BCT-100的抗性也有些許提高，但效果不如雙表達明顯。 / 總而言之，對於胰腺癌細胞而言，精氨酸是必不可少的。PEG-BCT-100有很明顯的胰腺癌效果。在胰腺癌中，OTC的表達情況可以作爲預估PEG-BCT-100治療效果的重要生物標誌。 / Deng, Haohao. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 111-117). / Abstracts also in Chinese. / Title from PDF title page (viewed on 14, October, 2016). / Deng, Haohao. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成5年5月14日 雄山博文氏の博士論文として提出された

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains xii, 73 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 64-70).