DNA topoisomerase II is an essential enzyme in eukaryotes and is required for many aspects of DNA metabolism including DNA replication, recombination, chromosome segregation and chromosome condensation. It is also a major component of the nuclear scaffold. Topoisomerase II from lower eukaryotes has been shown to be phosphorylated in vivo and this phosphorylation leads to a modulation of activity. However, unlike these lower eukaryotes, human topoisomerase II exists as two closely related, but genetically distinct, isozymes which have markedly different expression and localization patterns. Topoisomerase IIα is a 170kDa protein and topoisomerase IIβ is 180kDa. This study set out to analyse the phosphorylation of these specific isozymes and understand how this leads to the regulation of their distinct biological functions. In order to undertake this study, two polyclonal anti-topoisomerase II antibodies were generated and a series of other polyclonal and monoclonal antibodies characterized. Furthermore, the α isozyme of human topoisomerase II was purified to near homogeneity from cultured HeLa cells. A kinase activity with the biochemical characteristics of casein kinase II co-purified with and could phosphorylate the purified topoisomerase Hot protein. The α and β isozymes of human topoisomerase II were both shown to be phosphoproteins in vivo. The α isozyme is phosphorylated predominantly on serine residues but with a minor proportion of phosphothreonine. Both the α isozyme and a stable ISOkDa fragment of the β isozyme are phosphorylated in vitro by casein kinase II and the catalytic subunit of PKA (cAMP-dependent protein kinase). The α isozyme can also be phosphorylated in vitro by Ca²⁺-dependent and -independent isozymes of protein kinase C and the cell cycle-regulated p34^cdc2 kinase. Two-dimensional tryptic phosphopeptide mapping suggested that the pattern of phosphorylation of human topoisomerase Ha protein in vivo is complex with phosphorylation occurring on multiple residues. Comparison with in vitro maps suggested that casein kinase II and PKA could account for most of the phosphorylation seen in vivo. Using a one- dimensional phosphopeptide mapping approach, a major site of phosphorylation in vivo appeared to be within the C-terminal 20kDa, and that casein kinase II, PKA and PKC may all phosphorylate this region. Phosphorylation of human topoisomerase Hoc protein by casein kinase II, PKA and PKC all led to a stimulation of activity as measured by plasmid relaxation and decatenation. In contrast, dephosphorylation led to a marked decrease in activity of the enzyme. The dephosphorylated enzyme could be reactivated by casein kinase II but not PKA phosphorylation. These data suggest that phosphorylation plays a crucial role in the control of DNA tertiary structure in human cells via regulation of the activity of topoisomerase II proteins.

Glucose isomerase (E.C. 5.3.1.4) was extracted from Lactobacillus brevis N.CD.O 474 grown in xylose, containing medium with a yield of cells (dry weight) of 2.3 - 3.3gll of medium and 300-310 glucose isomerase units. Several methods for releasing the intracellular enzyme were investigated and the specific activity recovery was highest with the heat autolysis method. The crude extract preparation was further purified by nucleic acid precipitation with MnCl2, protein fractionation by ammonium sulphate and dialysis followed by chromatography on CM-cellulose, DEAE-cellulose and gel filtration on Sephadex G-200. A final purification of 24 fold was achieved with about 25% activity recovery in 4 purification steps as follows: enzyme extraction by heat autolyis, MnCl2 treatment (nucleic acid precipitation), ammonium sulphate (2-3.6M2pH 7.0) protein precipitation and CM-cellulose chromatography. A mol. wt. of approximately 120,000 was calculated for the purified enzyme by gel filtration (Sephadex G-200) which dissociated in small subunits with mol wt. of 54,000-42,600 calculated by electrophoresis on 5% polyacrylamide - 3% SDS-8M urea. The purified enzyme was immobilised with a PEI-derivative of nylon (polyethyleneimine) and the kinetic properties of both free and immobilized enzyme were investigated. Apparent Km values for the free purified enzyme were 7.4 x 10^-3M (D-xylose); 2.8M (D-glucose); 1.9M (D-fructose). The corresponding apparent V values were 0.45; 0.015 and 0.022 mumoles min-1. mug enzyme-1 respectively. Investigations were also carried out into several other possibilities of assaying glucose isomerase activity. Parameters for the coupled reaction assay system using sorbitol dehydrogenase -NADH were optimised.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 86-97).

No description available.

Thesis (Ph. D. in Biochemistry)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

Made available in DSpace on 2016-04-27T12:28:41Z (GMT). No. of bitstreams: 2 maria_oliveira_ioc_mest_2015.pdf: 3167015 bytes, checksum: 4bc54f5dd3253314f23a2aa12a3fea12 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Embora existam diversos tratamentos disponíveis atualmente, o câncer ainda é uma das principais causas de morbidade e mortalidade no mundo e a leucemia é responsável por muitas delas. Além dos fármacos atuais apresentarem diversos efeitos adversos, já foram relatadas diversas ocorrências de resistência celular a eles. Dessa forma, é imprescindível a busca por novos compostos com atividade anticancerígena eficaz, mas que detenham melhor perfil toxicológico. Nesse âmbito, destacam-se os inibidores projetados contra alvos com mutação ou superexpressos em células cancerosas, devido ao fato de permitirem maior seletividade. As topoisomerases são enzimas essenciais para o desenvolvimento celular e estão superexpressas em células de câncer. Portanto, elas são alvos importantes na quimioterapia antitumoral. O lapachol, uma naftoquinona natural, e seus derivados parecem ser promissores para a oncoterapia e têm sido bastante investigados nos últimos tempos. Novos compostos derivados de \03B1 e \03B2- lapachona inibidores de topoisomerases (PNQs) foram avaliados quanto ao seu potencial efeito citotóxico em células de leucemia através de ensaios de viabilidade, apoptose e ciclo celular. As linhagens utilizadas apresentam mutações que permitem investigar a participação da via de morte mediada pelo Fas: a A3 é a linhagem selvagem, sensível à apoptose via Fas; a I2.1 é derivada da A3, sendo deficiente em FADD; e a I9.2, também derivada da A3, é deficiente em caspase-8. Tanto o FADD quanto a caspase-8 estão diretamente envolvidos com a via clássica de ativação do Fas Também foi utilizada uma linhagem celular normal (WSS-1) para que fosse analisada a seletividade das PNQs. A maioria dos compostos mostrou-se eficaz contra as células leucêmicas e a comparação dos resultados obtidos entre elas indicou uma dependência parcial da ativação da via Fas para a atuação dessas PNQs. De acordo com os dados obtidos para a linhagem I9.2 no ensaio de apoptose, essa ativação do Fas não é necessariamente dependente de caspase-8. Além disso, a partir da administração dos compostos, foram observadas alterações no ciclo celular com predomínio de células na fase G2/M em relação ao controle. Esse efeito foi mais acentuado na linhagem A3, sugerindo que a presença da via completa do Fas possa estar relacionada a regulações do ciclo celular após o tratamento com os compostos. Embora menos seletivas que seus precursores, algumas PNQs foram mais potentes. Esse achado favorece o estudo futuro de modificações estruturais alternativas para essas moléculas, a fim de manter sua potência anticancerígena, mas que contribuíram para aumentar sua seletividade. Juntos, os resultados encontrados permitem iniciar a elucidação da atuação intracelular desses novos derivados do lapachol / Even though there are diverse types of treatment currently available, cancer still is one of the main causes of morbidity and mortality in the world, and leukemia is responsible for many of them. In addition to presenting various side effects, it has been already reported many occurrences of cellular resistance to them. Therefore, the search for new compounds that present effective anticancer activity with a better toxicological profile is indispensable. In this context, stand out the inhibitors designed against mutated or overexpressed targets in cancer cells because they allow for more selectivity. Topoisomerases are essential enzymes to the cellular development and are overexpressed in cancer cells. Therefore, they are important targets on antitumor chemotherapy. Lapachol, a natural naphthoquinone, and its derivatives seem to be promising to oncotherapy, and have been widely investigated recently. New topoisomerase inhibitors derived from α and β-lapachone (PNQs) were evaluated as to their potential cytotoxic effect on leukemia cells through viability, apoptosis and cellular cycle asssays. The cell lines that were used present mutations that allow investigating the participation of the Fas death pathway: A3 is a wild-type cell line, sensible to apoptosis by Fas; I2.1 is derived from A3, being deficient in FADD; and I9.2, also derived from A3, is deficient in caspase-8. Both FADD and caspase-8 are directly involved with the classical Fas activation pathway. A normal cell line (WSS-1) was also used in order to analyse the selectivity of the PNQs. The majority of the compounds have been shown to be effective against leukemia cells and the comparison of the results obtained among them indicated a partial dependency of the Fas pathway activation for the action of these PNQs. According to the data obtained from the I9.2 cell line on the apoptosis test, this Fas activation is not necessarily dependent on caspase-8. Besides, from the administration of the compounds, alterations in the cell cycle were observed, triggering a G2/M phase arrest. That effect was more marked on the A3 cell line, suggesting that the presence of the complete Fas pathway may be related to the regulations of the cell cycle after the treatment with the compounds. Although they were less selective than their predecessors, some PNQs were more potent. That finding stimulates future structural modification studies for these molecules, in order to keep their anticancer potency while increasing selectivity. Together, the results contribute to start elucidating the intercellular action of these lapachol derivatives. / 2016-11-24

Thesis (Ph. D. in Biochemistry)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Pharmaceutical Science." Includes bibliographical references (p. 99-107).

DNA supercoiling is an important determinant of variety of molecular processes because of its influence on substrate properties of DNA. Many proteins that bind DNA interact in a negatively supercoil dependent manner. The higher free energy associated with negative supercoiling is utilized for binding and subsequent transactions. Protein components of DNA replication, segregation, transcription and recombination machinery interact with DNA in topology dependent manner. Topoisomerases, thus play essential role in DNA transaction processes. Owing to their influence on diverse cellular functions, the enzymes are considered as global regulators. Movement of macromolecular assemblies along the DNA helix generates internal torsional strain during replication and transcription. This strain manifests itself as local domains of supercoils ahead and behind the helix tracking machinery. If left unchecked, accumulation of these supercoils could severely affect the survival of the cells. Cellular systems thus employ more than one topoisomerase, which act in concert to regulate DNA topology and maintain a steady-state level of supercoiling necessary for integrity and functionality of DNA molecules.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia, Florianópolis, 2010 / Made available in DSpace on 2012-10-25T00:26:53Z (GMT). No. of bitstreams: 1 277022.pdf: 4321942 bytes, checksum: 61f110123aa6e16130d34cb59a61133a (MD5) / DNA topoisomerases são enzimas que participam de diversos processos celulares tais como: replicação, transcrição, recombinação e segregação dos cromossomos. Atuando na clivagem transitória de uma fita (tipo I) ou ambas as fitas (tipo II) da molécula de DNA, as topoisomerases são alvos de agentes bactericidas e de drogas antitumorais e podem ser um importante alvo para quimioterapia de doenças causadas por parasitos. Neste estudo, descrevemos e caracterizamos os genes que codificam as topoisomerases tipo II de Trypanosoma rangeli (TrTop2). Os genes TrTop2 apresentaram um quadro aberto de leitura com 3.696 (TrTop2mt) e 4.368 pares de bases (TrTop2?), codificando polipeptídios preditos de 1.232 (138,8 kDa) e 1.456 (164,1 kDa) aminoácidos, respectivamente. Ambos os genes apresentaram uma alta similaridade da sequência protéica com topoisomerases ortólogas de outros tripanosomatídeos, sobretudo com T. cruzi (96% e 85%). Entre os dois genes a similaridade a nível de aminoácidos foi de 56%, sendo ambos de cópia única no genoma do T. rangeli. Anticorpos dirigidos a fragmentos protéicos de ambas as proteínas foram utilizados em ensaios de western blot e revelaram bandas de aproximadamente 130 kDa em extratos protéicos totais de T. rangeli. Embora com tamanho similar, foram identificadas proteínas distintas quando avaliadas por eletroforese 2D (pI 6,4 e 7,6). Através de géis de poliacrilamida em condições não desnaturantes foi possível determinar que ambas as proteínas nativas formam um complexo protéico de alto peso molecular indicando uma possível interação entre proteínas ou subunidades. Ensaios de imunolocalização com os distintos anticorpos contra DNA topoisomerases II apontaram diferentes padrões de localização celular, com reconhecimento no núcleo ou no cinetoplasto em formas epimastigotas e em alguns casos dispersa no citoplasma de formas tripomastigotas. A novobiocina, inibidor de topoisomerases tipo II procarióticas, foi ativo in vitro contra o T. rangeli. Acima de 300 ?g/ml observa-se uma redução no crescimento dos parasitos com alterações morfológicas e estruturais a nível nuclear e do cinetoplasto. Acima de 150 ?g/ml observa-se completa inibição da diferenciação celular in vitro. Utilizando as proteínas mtHSP70, DHLADH e a DNA topoisomerase II mitocondrial (TrTop2mt) como marcadores biológicos, foi observado redução da expressão das mesmas durante a diferenciação do T. rangeli, assim como nos tratamentos com novobiocina. Conclui-se que eventos relacionados as DNA topoisomerases II podem ser essenciais na redução do crescimento e da diferenciação celular do T. rangeli.