PART I - The base compositions of deoxyribonucleic acid, isolated from whole cells, nuclei, and mitochondria of rat intestinal mucosa were compared. DNA from whole cells or nuclei was fractionated by column chromatography on methylated albumin kieselguhr, MAK. The guanine plus cytosine content of these DNA fractions was determined mainly by the method of heat denaturation, although the methods of acid hydrolysis and equilibrium centrifugation in cesium chloride solutions were also used. The mole % G+C for the DNA fractions from whole cellular extracts ranged between 34.39 and 52.92, while the nuclear DNA fractions showed a range between 37.31 and 50.97. Although the main DNA band which was eluted with 0.6 M NaCl solution had separated into two or three peaks, the detection of a major base compositional class of DNA was not evident. Unfractionated DNA from whole cells or nuclei has a G+C content of 42.2 which corresponds to a midpoint of thermal denaturation, Tm of 86.6° C. DNA isolated from the mitochondria of intestinal mucosa was observed to have a Tm of approximately 85.0° C and a density of 1.702 g/cm³. This density corresponded to the value determined for unfractionated DNA from the whole mucosa cells. PART II - In initial experiments, DNA polymerase from Escherichia coli was isolated and partially purified by treatment with streptomycin, ammonium sulfate, and by chromatography on DEAE-cellulose. After these introductory experiments, DNA polymerases from the small intestinal mucosa of the rat were studied. Using suitable assay systems with ¹⁴C-2-dTTP or ¹⁴C-8-dATP, both a replicative and a terminal DNA nucleotidyl-transferase were detected in extracts of nuclei. The replicative enzyme incorporated a labeled precursor into a native or heat denatured DNA primer in the presence of all four complementary triphosphates. The terminal enzyme preferentially incorporated single deoxyribonucleoside triphosphates onto the terminal position of heat denatured DNA primers. Treatment of the DNA products formed in the terminal addition reaction with snake venom phosphodiesterase indicated that the labeled precursors were added to 3'-hydroxy terminal positions of the chains. A heterogeneous nature of the DNA polymerases from rat intestinal mucosa was indicated by the appearance of three fractions of enzyme activity following DEAE-cellulose chromatography. A distinct peak of terminal-addition enzyme activity was detected by rechromatography on DEAE-cellulose. Gel filtration through Sephadex G-150 or G-200 and sucrose density gradient centrifugation showed that these DNA polymerases varied in molecular sizes. The molecular weights of the DNA polymerase fractions were estimated to be between 2.5 x 10⁴ and 3 x 10⁵ by comparisons with marker proteins. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 108-126).

Thesis (Ph.D)--Boston University / Characteristics of deoxyribonuclease II from calf thymus and spleen were described and verified by the following experimental procedures. Nuclei from calf thymus were prepared free of cytoplasm both by washing homogenized tissue in sucrose solutions, and by density gradient centrifugation in organic solvents. The action of the enzyme from nuclei and from a thymus homogenate was found to be similar when measured by light scattering and hyperchromic shift. Deoxyribonuclease II from calf spleen was purified by ammonium sulfate precipitation. Further purification of the enzyme on column chromatography resulted in fractions similar to those reported in the literature. The effect of sodium sulfate on the activity of splenic DNase II, purified by ammonium sulfate precipitation, was measured by the production of acid-soluble nucleotides from deoxyribonucleic acid during hydrolysis. The effect of the sulfate ion was also measured by viscometry, as was the effect of magnesium chloride, manganese chloride, sodium citrate and iodoacetic acid. The substances were all found to be inhibitory, which is in agreement with the results reported in the literature. Deoxyribonucleases active at an acid pH were extracted from representatives of four invertebrate phyla (Echinodermata, Mollusca, Arthropoda and Annelida) and compared with vertebrate deoxyribonuclease II with respect to ionic requirements and pH optima. [TRUNCATED]

The properties of the deoxyribonuclease activity in the intestinal mucosa of rat have been studied. It was found that the activity was extractable from a whole cell preparation of the tissue with either Krebs Ringer phosphate buffer (pH 7.8) or physiological saline. The former was a better extracting medium, the buffered extract being more stable to storage at -20°C. Two active proteins were precipitated on fractionation of the Krebs Ringer phosphate buffer extract with ammonium sulfate. One fraction precipitated at 20% saturation of (NH₄)₂SO₄ was identified as DNase I by its optimum pH, ionic requirements and by its reaction to known DNase I inhibitors such as EDTA, citrate and arsenate. Ion-exchange chromatography on DEAE-cellulose (chloride form) established that the products of reaction ranged from mononucleotides through to oligonucleotides with a degree of polymerization larger than 7. These products were shown to carry a phosphomonoester linkage on the 5’-carbon. This fraction was designated as the 20%P enzyme and represented a 20 fold purification of the crude deoxyribonuclease extract. The supernatant fraction obtained after the 20%P enzyme was removed by centrifugatlon was found to still contain - i i - considerable DNase activity. This residual activity disappeared from the solution when the ammonium sulfate concentration was increased to 30% saturation, but all attempts to recover the DNase activity from the protein precipitated at this salt concentration were unsuccessful. Consequently, studies of this residual DNase activity were carried out using the crude extract. This second deoxyrlbonuclease was shown to be qualitatively different from the 20% enzyme. It did not require activation by Mg⁺⁺, and was not inhibited by EDTA, citrate or arsenate. Some of the products formed by the crude enzyme extract were shown to terminate in 3’-phosphoryl groups. These products were not formed by the 20%P enzyme and therefore must be due to the residual deoxyrlbonuclease. In all likelihood then, this second deoxyribonuclease activity was of the DNase II type. The intracellular distribution of the intestinal mucosal deoxyribonuclease was studied by differential centrifugation of the tissue homogenate. DNase I activity was found to occur in the mitochondria whereas DNase II was associated with some lighter subcellular particles. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The properties of the deoxyribonuclease activity in the intestinal mucosa of the rat have been studied. Two DNases were found in a cell-free extract prepared by homogenizing the mucosal tissue in Krebs-Ringer phosphate buffer and then centrifuging the homogenate at 105,000 x g for 60 mins. Resolution and partial purification of these two enzymes were achieved by ion-exchange chromatography on DEAE-cellulose and partition on hydroxylapatite of the cell-free extract or an acetone powder preparation of the enzymes. One of the enzymes was identified as DNase I by its optimum pH (6.5 to 6.8), its requirements for bivalent metals and by its reaction to known DNase I inhibitors such as EDTA, citrate and arsenate. It was also found to be active toward native DNA, and, to a lesser degree, toward heated DNA. The second DNase was shown to be qualitatively different from DNase I. It has an acidic optimum pH (3.5-4.0), does not require activation by bivalent cations and is not inhibited by EDTA, citrate or arsenate. This second DNase activity is therefore of the DNase II type. The linkage specificity of the two enzymes was studied by isolating the products of the reaction and examining them with respect to chain length, base composition of the mononucleotides, relative frequencies of the dinucleotides and base frequencies at the ends of the oligonucleotides. The hydrolysis reaction was carried out under a variety of conditions. When Mg++ was used as the activating ion and native DNA as substrate, DNase I was found to show a preference for the linkages pApC, pApT and pGpT. But when Mn++ was the activator, or when heated DNA was used as substrate in the presence of Mg++, DNase I did not show any significant order of specificity. With regard to DNase II, the enzyme was found to attack native DNA preferentially at the ApCp, GpCp and GpTp bonds. The mechanism of metal activation of intestinal DNase I was also studied. Preliminary experiments showed that the enzyme was inhibited by high concentrations of both metals and DNA. In addition, optimal activation was found to depend on the molar ratio of metal ion to DNA phosphorus. These experiments suggested that the metal-DNA complex is probably the true substrate. Consequently, a rate equation in terms of the metallosubstrate concentration was developed based on the assumption that intestinal DNase I can combine individually and simultaneously with free metal, free DNA and the metal-DNA complex. Data obtained from initial velocity studies carried out at varying concentrations of metal and DNA fitted this rate equation well. On this basis, it was suggested that the enzyme, the metal activator and the DNA substrate combine to form a ternary complex which then dissociates to give the products. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

L'anévrysme de l'aorte abdominale (AAA) est une pathologie vasculaire caractérisée par une augmentation du diamètre de l'aorte de plus de 1, 5 fois le diamètre de référence et une perte du parallélisme de la paroi au niveau infra rénal. Les AAA affectent de manière prépondérante les homes âgés avec une prévalence de 5%. La rupture d'un AAA est responsable de 1à 4% de la mortalité chez les hommes de plus de 65 ans. Dans le cas d'une rupture, la mortalité est supérieure à 70-95%. C'est pourquoi les AAA sont une des causes majeures de mortalité dans les pays industrialisés avec une population vieillissante. L'age, le sexe masculin, la consommation de tabac, la susceptibilité génétique et la présence d'une autre localisation d'athérosclérose sont des facteurs de risque connus pour le développement d'un AAA. La rupture peut être prévenue par une chirurgie vasculaire qui permet de diminuer la mortalité chez les patients atteints d'AAA. Néanmoins, la plupart des patients atteints d'AAA sont asymptomatiques et la majorité ne sont pas diagnostiqués avant la rupture. Au cours de ce travail de thèse, nous avons utilisé des techniques de protéomique différentielle dans le but d'analyser et comparer des échantillons provenant de plasma et de cellules (cellules musculaires lisses, monocytes différenciés en macrophages) de patients présentant ou non un AAA. Ces analyses ont été réalisé avec comme but les objectifs suivants : a) Identifier et évaluer les marqueurs biologiques potentiels pour un dépistage des AAA, qui permettrait un diagnostique précoce et donc un traitement précoce de cette pathologie. b) De permettre une meilleure connaissance des mécanismes physiopathologiques impliqués dans l'évolution des AAA par le biais des résultats obtenus lors de l'analyse protéomique différentielle. En ce qui concerne l'analyse protéomique des cellules, l'utilisation de la technique DIGE par saturation associée avec l'utilisation du logiciel SameSpots, a permis l'identification de 2 profils protéiques différents dans l'ensemble des types cellulaires étudiés, indépendamment du statut du patient. Ces deux protéomes différents sont le résultat d'un biais technique du à l'utilisation d'un traitement à la DNase I des échantillons dans le but d'éliminer les acides nucléiques présents. Nous avons montré que le traitement à la DNase I produisait des changements de profil protéique de ces deux types cellulaires. Les protéines principalement affectées par ce traitement sont celles impliquées dans le mouvement cellulaire, la réorganisation du cytosquelette d'actine, et l'apoptose. Du fait de la présence de ce biais technique, la comparaison entre les échantillons de cellules obtenues de patients présentant ou non un AAA n'a pas permis de résultats concluants. L'analyse protéomique du plasma a été effectué en collaboration avec le laboratoire du Pr Goodlett à Seattle (USA). Deux approches différentes de spectrométrie de masse ont été utilisées pour comparer les échantillons plasmatiques de patients présentant ou non un AAA. La première approche combine l'utilisation de l'analyse MS PAcIFIC avec le calcul spectral et la seconde approche combine l'analyse des données MS de manière indépendante avec un marquage TMT isobarique. Après validation par western blot, quatre protéines ont été validées comme différentiellement exprimées dans le plasma de patients présentant ou non un AAA : l'adiponectine, la superoxide dismutate extracellulaire, la kallistatine et la carboxipeptidase B2. Ces protéines sont impliquées dans la pathologie anévrysmale et peuvent être des potentiels marqueurs biologiques pour le diagnostique de AAA. Des études dans de grandes cohortes seront nécessaires pour valider leur utilisation pour le dépistage des AAA dans la population à risque. Cela permettrait une détection précoce de la maladie et une diminution de la mortalité de la population âgée dans les pays industrialisés.

3′ repair exonuclease 1 (TREX1) is a known DNA exonuclease involved in autoimmune disorders and the antiviral response. In this work, we show that TREX1 is also a RNA exonuclease. Purified TREX1 displays robust exoribonuclease activity that degrades single-stranded, but not double-stranded, RNA. TREX1-D200N, an Aicardi-Goutieres syndrome disease-causing mutant, is defective in degrading RNA. TREX1 activity is strongly inhibited by a stretch of pyrimidine residues as is a bacterial homolog, RNase T. Kinetic measurements indicate that the apparent Km of TREX1 for RNA is higher than that for DNA. Like RNase T, human TREX1 is active in degrading native tRNA substrates. Previously reported TREX1 crystal structures have revealed that the substrate binding sites are open enough to accommodate the extra hydroxyl group in RNA, further supporting our conclusion that TREX1 acts on RNA. These findings indicate that its RNase activity needs to be taken into account when evaluating the physiological role of TREX1.

A specific method has been developed for the extraction and measurement of staphylococcal nuclease in cheese in which Staphylococcus aureus has grown. Ten grams of cheese sample were homogenized with ninety milliliters of pH ten buffer for three minutes. Ammonium sulfate fractionation was used and a forty to eighty percent fraction was collected and concentrated using ultrafilters. The nuclease activity was determined using a toluidine blue deoxyribonucleic acid agar slide method and a spectophotometric method. The DNA agar slide method was used to compare staphylococcal growth with nuclease production in cheese under varying conditions. When Staphylococcus aureus plate counts indicated populations of three to four thousand per milliliter, it was possible to detect nuclease in the cheese sample. A method has also been developed to detect Staphylococcus aureus colonies using DNase agar and toluidine blue, utilizing the heat stability of Staphylococcus aureus nuclease.

INTRODUÇÃO: A síndrome de Sjögren primária (SSp) é uma doença autoimune inflamatória crônica que afeta principalmente as glândulas exócrinas, levando aos sintomas de síndrome sicca. O olho seco é uma das características mais importantes dessa síndrome e um estudo recente relatou redução da atividade da DNase I em lágrimas de pacientes com olho seco de várias etiologias. Portanto, postulamos que pacientes com SSp possam ter anticorpos direcionados à DNase I. MÉTODOS: Avaliamos em um estudo de corte transversal 85 pacientes com SSp (conforme os critérios de classificação do American European Consensus Group Criteria, 2002), 50 pacientes com artrite reumatoide (AR) (American College of Rheumatology Criteria/ 1987) sem sintomas de síndrome sicca e 88 voluntários saudáveis. A reatividade IgG anti-DNase I foi detectada por ELISA utilizando a enzima de pâncreas bovino como antígeno e confirmada por Imunoblotting. RESULTADOS: A idade e sexo foram comparáveis nos três grupos (p > 0,05). A anti-DNase I foi detectada em 43,5% dos pacientes com SSp, conforme determinado por ELISA. Em contraste, essa reatividade estava ausente em todos os pacientes com AR (p= 0,0001). Comparações adicionais dos pacientes com SSp com (n= 37) e sem (n= 48) anti-DNase I revelaram que o primeiro grupo tinha níveis séricos de IgG mais altos (2293,2 ± 666,2 vs. 1483,9 ± 384,6 mg/dL, p= 0,0001) e uma frequência maior de leucopenia não induzida por drogas (43% vs. 19%, p= 0,02). A análise de regressão logística multivariada mostrou que apenas os níveis de IgG foram independentemente associados com o anti-DNase I. CONCLUSÃO: Descrevemos uma alta frequência de anticorpos anti-DNase I em pacientes com SSp associados a níveis séricos de IgG mais elevados. A falta dessa reatividade em pacientes com AR sem sintomas de sicca sugere que esse anticorpo pode ser útil no diagnóstico diferencial dessas doenças / INTRODUCTION: Primary Sjögren\'s syndrome (pSS) is a chronic inflammatory autoimmune disease that mainly affects exocrine glands. Dry eye is one of the most important features of this syndrome and a recent study reported reduced DNase I activity in tears of patients with dry eye of various etiologies. We therefore postulated that patients with pSS may have antibodies targeting DNase I. METHODS: We have evaluated in a cross-sectional study 85 pSS patients (American European Consensus Group Criteria/ 2002), 50 rheumatoid arthritis (RA) patients (American College of Rheumatology Criteria/ 1987) without sicca symptoms and 88 healthy volunteers. The IgG anti-DNase I reactivity was detected by ELISA using bovine pancreas enzyme as antigen and confirmed by Immunoblotting. RESULTS: Age/ gender were comparable in the three groups (p > 0.05). Anti-DNase I was detected in 43.5% of the pSS patients as determined by ELISA. In contrast, this reactivity was absent in all RA patients (p= 0.0001). Further comparison of pSS patients with (n= 37) and without (n= 48) anti-DNase I revealed that the former group had higher IgG serum levels (2293.2 ± 666.2 vs. 1483.9 ± 384.6 mg/dL, p= 0.0001) and a higher frequency of non-drug induced leukopenia (43% vs. 19%, p= 0.02). A multivariate logistic regression analysis identified that only IgG levels were independently associated with anti-DNase I. CONCLUSION: We describe a high frequency of anti-DNase I antibodies in pSS patients associated with higher serum IgG levels. The lack of this reactivity in RA patients without sicca symptoms suggests that this antibody may be helpful in the differential diagnosis of these diseases