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The mechanism of Formyl-Coenzyme A transferase, a Family III CoA transferase, from Oxalobacter formigenesJonsson, Stefan. January 2004 (has links)
Thesis (Ph. D.)--University of Florida, 2004. / Title from title page of source document. Document formatted into pages; contains 79 pages. Includes vita. Includes bibliographical references.
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The role of carnitine acetyltransferases in the metabolism of Saccharomyces cerevisiaeKroppenstedt, Sven 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: L-carnitine is a compound with a long history in biochemistry. It plays an important
role in mammals, where many functions have been attributed to it. Those functions
include the p-oxidation of long-chain fatty acids, the regulation of the free CoASH/
Acyl-CoA ratio and the translocation of acetyl units into mitochondria. Carnitine is
also found in lower eukaryotic organisms. However, in contrast to the multiple roles it
plays in mammalian cells, its action appears to be restricted to the transport of
activated acyl residues across intracellular membranes in the lower eukaryotes. In
the yeast Saccharomyces cere visiae , the role of carnitine consists mainly of the
transfer of activated acetyl residues from the peroxisome and cytoplasm to the
mitochondria. This process is referred to as the carnitine shuttle. This system
involves the transfer of the acetyl moiety of acetyl-CoA, which cannot cross
organellar membranes, to a molecule of carnitine. Subsequently, the acetylcarnitine
is transported across membranes into the mitochondria, where the reverse transfer
of the acetyl group to a molecule of free CoA occurs for further metabolism. Carnitine
acetyl transferases (CATs) are the enzymes responsible for catalysing the transfer of
the activated acetyl group of acetyl-CoA to carnitine as well as for the reverse
reaction.
In the yeast S. cerevisiae, three CAT enzymes, encoded by the genes CAT2,
YAT1 and YAT2, have been identified. Genetic data suggest, that despite the high
sequence similarity, each of the genes encodes for a highly specific activity that is
part of the carnitine shuttle. So far, the specific function of any of the three CAT
enzymes has been elucidated only partially.
The literature review focuses mainly on the importance of the carnitine system in
mammals. After discussing the discovery and biosyntheses of carnitine, the
enzymatic background of and molecular studies on the carnitine acyltransferases are
described.
The experimental section focuses on elucidating the physiological roles and
cellular localisation of the three carnitine acetyltransferase of S. cere visia e. We
developed a novel enzymatic assay to study CAT activity in vivo. By C-terminal
tagging with a green fluorescent protein, we localised the three CAT enzymes.
However, all our genetic attempts to reveal specific roles for and functions of
these enzymes were unsuccessful. The overexpression of any of the CAT genes
could not cross-complement the growth defect of other CAT mutant strains. No
phenotypical difference could be observed between strains carrying single, double
and triple deletions of the CAT genes. Furthermore, the expression of the
Schizosaccharomyces pombe dicarboxylic acid transporter can complement the
deletion of the peroxisomal citrate synthase, but has no effect on the carnitine shuttle
per se. Our data nevertheless suggest that Cat2p is the enzyme mainly responsible
for the forward reaction, e.g. the formation of acetylcarnitine and free CoA-SH from acetyl-CoA and carnitine, whereas Yat1 pand Yat2p may be required mainly for the
reverse reaction. / AFRIKAANSE OPSOMMING: L-karnitien is 'n verbinding met 'n lang geskiedenis in die biochemie-veld. Dit speel 'n
belangrike rol in soogdiere, waar verskeie funksies daaraan toegeskryf word. Dié
funksies sluit in die p-oksidasie van lang-ketting-vetsure, die regulering van die vrye
KoA-SH-tot-asiel-KoA-verhouding en die oordrag van asetieleenhede na die
mitochondria. Karnitien word ook in laer eukariotiese organismes gevind. In
teenstelling met die verskeidenheid rolle wat dit in soogdierselle vervul, is die funksie
in laer eukariote tot die transport van geaktiveerde asetielderivate oor intrasellulêre
membrane beperk. In die gis Saccharomyces cerevisiae is die funksie van karnitien
meestal beperk tot die vervoer van geaktiveerde asetielresidu's vanaf die sitoplasma
en piroksisome na mitochondria, 'n proses wat as die "karnitiensiklus" bekend staan.
Die proses behels die oordrag van die asetielgedeelte van asetiel-KoA, wat nie oor
organelmembrane kan beweeg nie, na 'n molekuul van karnitien. Gevolglik word die
asetielkarnitien oor die membraan na die mitochondria vervoer, waar - met die oog
op verdere metabolisme - die omgekeerde oordrag van die asetielgroep na 'n vrye
molekuul van KoA plaasvind. Karnitienasetiel-transferases (KAT's) is die ensieme
wat verantwoordelik is vir die katalisering van die oordrag van die geaktiveerde
asetielgroepe van asetiel-KoA na karnitien, sowel as vir die omgekeerde reaksie.
In die gis S. cerevisiae is drie KAT-ensieme geïdentifiseer wat deur die gene
CAT2, YAT1 en YAT2 gekodeer word. Genetiese data dui daarop dat, ten spyte van
die hoë mate van homologie van die DNA-volgordes, elke geen vir 'n hoogs
spesifieke aktiwiteit, wat deel van die karnitiensiklus is, kodeer. Tot dusver is die
spesifieke funksie van die drie individuele KAT-ensieme net gedeeltelik ontrafel.
Die literatuurstudie fokus hoofsaaklik op die belangrikheid van karnitiensisteme
in soogdiere. Na 'n bespreking van die ontdekking en biosintese van karnitien, word
die ensimatiese agtergrond en molekulêre studies van KAT's beskryf.
Die eksperimentele deel konsentreer op die ontrafelling van die fisiologiese rol
en intrasellulêre lokalisering van die drie KAT-ensieme van S. cerevisiae. Eerstens is
'n nuwe ensimatiese toets ontwikkel om KAT-aktiwiteit in vivo te bestudeer. Deur
C-terminale aanhegting van 'n groen fluoreserende proteïen kon die drie KATensieme
gelokaliseer word.
Daar kon egter nie met behulp van genetiese studies verder lig gewerp word op
die spesifieke rolle en funksies van hierdie KAT-ensieme nie. Die ooruitdrukking van
enige van die KAT-gene kon nie die groeidefek van ander KAT-mutantrasse
kruiskomplementeer nie. Geen fenotipiese verskil tussen rasse wat 'n enkel, dubbel
of trippel delesie van die KAT-gene bevat, kon waargeneem word nie. Verder kon die
uitdrukking van Schizosaccharomyces pombe se dikarboksielsuurtransporter die
delesie van die peroksisomale sitraatsintetase komplementeer, maar het dit as sulks
geen effek op die karnitiensiklus gehad nie. Die data wat deur hierdie studie verkry is, dui nogtans daarop dat Cat2p die ensiem is wat hoofsaaklik verantwoordelik is vir
die voorwaartse reaksie, met ander woorde die vorming van asetielkarnitien en vrye
KoH-SH van asetiel-KoA en karnitien, terwyl Yat1 p en Yat2p hoofsaaklik vir die
omgekeerde reaksie benodig word.
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Rat liver UDP-glucuronosyl transferase phospholipid dependence, purification, and biochemical characterization /Gorski, Jeffrey P. January 1975 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1975. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
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Site Directed Mutagenesis of β-Ketoadipate Succinyl-Coenzyme A Transferase II from Acinetobacter CalcoaceticusSheng, Mei 08 1900 (has links)
The role of specific amino acid residues in β-ketoadipate succinyl-coenzyme A transferase II from Acinetobacter calcoaceticus was investigated. A 1412 base pair BamiHI-EcoRI fragment carrying the catIJ genes was amplified by polymerase chain reaction and inserted into pUCl9 to generate the plasmid pCATEl9. Escherichia coli DH5α (pCATEl9) carrying only the catlJ genes expressed 3-fold higher enzyme activity than the parent strain. Two mutants were constructed by site directed mutagenesis so that glutamate was replaced by a glutamine at positions Gln155 and Gln193 in the ß subunit of the primary amino acid sequence of the CoA transferase. Both mutants produced transferase that was catalytically active suggesting that Glu155 and Glu193 do not participate directly in catalysis.
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Phosphate Analogues as Probes of the Catalytic Mechanisms of MurA and AroA, Two Carboxyvinyl TransferasesZhang, Fuzhong 08 1900 (has links)
<p> The two carboxyvinyl transferases MurA and AroA are essential for bacterial survival, and are proven or potential antibiotic targets. The reactions they catalyze are chemically challenging, involving protonation of an ethylene group in the first step, and deprotonation of a methyl in the second step. In order to probe how the enzymes promote these reactions, the reverse reactions from enolpyruvyl compounds (EP-OR) plus phosphate to phosphoenolpyruvate (PEP) plus R-OH were investigated, and compared with EP-OR hydrolysis reactions catalyzed by phosphate analogues. </p> <p> Thirteen phosphate analogues were used to study EP-OR hydrolysis. Among these phosphate analogues, many could bind to the free enzymes, but only three could promote hydrolysis. The products were pyruvate and the corresponding alcohol (S3P in AroA/EPSP reaction and UDP-GicNAc in MurA/EP-UDP-GicNAc reaction). The most effective analogue was arsenate. The mechanism of the arsenate-promoted reaction was examined in detail. The hydrolysis reaction proceeded though an arseno-tetrahedral intermediate with AroA, a similar reaction pathway to the natural reaction. This arseno-tetrahedral intermediate was converted to arsenoenolpyruvate and hydrolyzed spontaneously. MurA also likely catalyzed arseno-tetrahedral intermediate formation, and appeared to catalyze arsenoenolpyruvate breakdown, though it is possible that it was a bystander in the reaction, with the tetrahedral intermediate being formed by water attack on C2 of EP-UDP-GicNAc. There was a fast solvent exchange step before EP OR was converted to arseno-THI by AroA or MurA. This strongly indicated an oxacarbenium ion like intermediate before the arseno-tetrahedral intermediate. </p> <p> The catalytic machinery for stabilizing such an unstable oxacarbenium ion like intermediate was investigated by studying ligand binding. Based on information from all the phosphate analogues, there were evidence that the enzyme undergoes an conformational change upon binding with phosphate, by which EPSP was distorted into an oxacarbenium ion like intermediate. </p> / Thesis / Master of Science (MSc)
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Estudo da Expressão dos Genes DNMT1, DNMT3A, DNMT3B, MGMT e Efeitos da Zebularina em Glioblastoma / Estudo da Expressão dos Genes DNMT1, DNMT3A, DNMT3B, MGMT e Efeitos da Zebularina em GlioblastomaMoreno, Daniel Antunes 24 August 2012 (has links)
Os gliomas são tumores que surgem a partir de células da glia e são considerados os mais comuns do sistema nervoso central. São subdivididos em quatro grupos: astrocitoma pilocítico (grau I), astrocitoma difuso (grau II), astrocitoma anaplásico (grau III) e glioblastoma (grau IV ou GBM). Entre esses, o GBM é o tumor mais agressivo e mais freqüente. Apesar de ser encontrado em qualquer faixa etária, esse tumor é raro em crianças. Atualmente a cirurgia seguida de radioterapia e quimioterapia com temozolomida (TMZ) tem sido utilizado como protocolo de tratamento padrão para a maioria dos pacientes e mesmo assim a sobrevida se mantem extremamente baixa. Além disso, grande parte dos pacientes não respondem ao tratamento com TMZ indicando a necessidade de agentes quimioterápicos alternativos. A zebularina (ZB) é um agente inibidor de DNA metiltransferases (iDNMTs) estável, pouco tóxico, que promove radiosensibilização e tem mostrado efeitos promissores em diversos tipos de neoplasias, entretanto pouco se sabe a respeito dos efeitos da ZB em glioblastoma. Os objetivos deste trabalho foram analisar a expressão dos genes DNMT1, DNMT3A, DNMT3B, MGMT em 5 amostras de substâncias brancas (SB), 6 linhagens de GBM e 33 amostras de gliomas (13 grau I, 2 grau II e 18 grau IV), correlacionar a expressão desses genes com os diferentes graus de gliomas e analisar os efeitos da ZB combinada ou não com TMZ em linhagens de GBM irradiadas e não irradiadas. Para análise da expressão gênica foi realizada a técnica de PCR em tempo Real. Os ensaios de proliferação celular, clonogênico, radiação e apoptose foram realizados em 3 linhagens de GBM (U251, SF188 e T98G) e uma de fibroblastos (MRC5). Também foi realizado o ensaio de proliferação celular em 5 culturas primárias de GBM tratadas com zebularina. Os genes DNMT3A e MGMT mostraram expressão maior nas amostras de SB comparando-se com gliomas e linhagens de GBM. O gene DNMT3B foi mais expresso nas linhagens de GBM comparando-se com as SB. O gene DNMT1 não mostrou diferenças significativas entre as amostras analisadas. Os ensaios de proliferação celular mostraram diminuição na proliferação com doses a partir de 50-100µM de ZB e de 250-500µM de TMZ nas linhagens e a partir de 50µM de ZB para as culturas primárias de GBM. As combinações de ZB com TMZ não mostraram sinergia na grande maioria das doses testadas. A ZB aumenta a apoptose nas 3 linhagens com doses a partir de 100µM. A ZB e TMZ mostraram diminuição na formação de colônias com as doses de 100µM e 10µM nas linhagens U251 e SF188 não irradiadas e irradiadas com 2, 4 e 6 Gy. A linhagem T98G expressa o gene MGMT, mostrou resistência a 10µM de TMZ e respondeu ao tratamento com 100µM de ZB. Também foi observado que 10µM de TMZ é mais citotóxico do que 100µM de ZB em fibroblastos não irradiados e irradiados (2Gy). Os resultados obtidos neste estudo mostram que a ZB pode representar um alvo terapêutico interessante para o estudo em glioblastoma. / Gliomas arise from glial cells and are the most common central nervous system tumors. They are divided in four groups: pilocytic astrocytoma (grade I), difuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma (grade IV or GBM). GBM is the most frequent and aggressive glioma. This type of tumor can occur in any age but its rare in children. Actually, surgery, radiotherapy and temozolomide (TMZ) adjuvant/concomitant chemotherapy has been the standard treatment protocol but the survival is extremely poor. In addition most patients do not respond to TMZ indicating the need for alternative chemotherapeutic agents. Zebularine (ZB) is a DNA metiltransferase inhibitor (DNMTi) stable, slight toxic that has been showed promise effects in cancer including radiosensitivity but little is known about ZB in glioblastoma. The objectives of this study were analyze DNMT1, DNMT3A, DNMT3B, MGMT gene expression profile in 5 samples of normal brain, 6 GBM cell lines and 33 glioma samples (13 grade I, 2 grade II e 18 grade IV), correlate with different gliomas grades and analyze the effects of ZB isolate and in combination with TMZ in irradiated and non irradiated GBM cell lines. Gene expression assays was made using Real Time PCR. Proliferation, clonogenic, radiation and apoptosis assays were realized in three GBM cell lines (U251, SF88, T98G) and one fibroblast cell line (MRC5). We also made proliferation assays in 5 primary cultures of samples of GMB. MGMT and DNMT3A genes showed higher expression in normal brain compared to gliomas and GBM cell lines. DNMT3B gene showed higher expression in GBM cell lines compared with normal brain and DNMT1 showed no significant differences among samples analyzed. We observed decrease of cell proliferation from 50-100µM of ZB and 250-500µM of TMZ on GBM cell lines and from 50µM of ZB for primary GBM samples. It was not observed synergy in the most combinations doses of ZB and TMZ (Calcusyn software). It was observed that 100µM of ZB and 10µM of TMZ decrease colony formation on U251 and SF188 cell lines non irradiated and irradiated with 2, 4, and 6Gy. T98G that express MGMT, did not respond to TMZ but showed response to ZB. It was also observed that 10µM of TMZ is more cytotoxic than 100µM of ZB in fibroblast cell line non irradiated and irradiated with 2Gy. ZB increase apoptosis from 100µM on the three GBM cell lines. Results obtained in this study can indicate that ZB may be an interestig therapeutic target for future studies in glioblastoma.
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The molecular basis of glutamate formiminotransferase deficiency /Hilton, John Frederick. January 2001 (has links)
Glutamate formiminotransferase deficiency (OMIM 229100) is an autosomal recessive disorder marked by clinical heterogeneity. The severe phenotype, first identified in patients of Japanese descent, includes high levels of formiminoglutamate (FIGLU) in the urine in response to histidine loading, megaloblastic anemia, and mental retardation. The mild phenotype is marked by high levels of FIGLU in the urine in the absence of histidine loading, mild developmental delay and no hematological abnormalities. The gene for human glutamate formiminotransferase-cyclodeaminase consists of 15 exons and is located at 21q22.3. The protein consists of a tetramer of dimers, with dimerization essential for both formiminotransferase and cyclodeaminase activity. / Genomic DNA extracted from cell lines from three patients with suspected glutamate formiminotransferase deficiency was analyzed by PCR and sequencing of individual exons. Cell lines WG 1758 and WG 1759 are from two siblings of Germanic descent. Both siblings are heterozygous for the mutations c457 C → T and c940 G → C. The c457 C → T changes a conserved arginine to a cysteine in a loop involved in the binding of formiminotetrahydrofolate to the enzyme. The c940 G → C mutation converts an arginine to a proline in an alpha-helix essential for the dimerization of the formiminotransferase domain. Cell line WG 1795 is from a patient of Danish descent. The patient appears to be hemizygous for a c1033 insG mutation. Quantitative PCR suggests the presence of a deletion on the other chromosome, which minimally encompasses exon 9. All of the FTCD gene changes were absent in 100 control individuals (200 alleles).
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The Salmonella enterica virulence plasmid : its role in bacterial adaptation to mammalian and protozoan cells /Tezcan-Merdol, Dilek, January 2004 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.
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Estudo da Expressão dos Genes DNMT1, DNMT3A, DNMT3B, MGMT e Efeitos da Zebularina em Glioblastoma / Estudo da Expressão dos Genes DNMT1, DNMT3A, DNMT3B, MGMT e Efeitos da Zebularina em GlioblastomaDaniel Antunes Moreno 24 August 2012 (has links)
Os gliomas são tumores que surgem a partir de células da glia e são considerados os mais comuns do sistema nervoso central. São subdivididos em quatro grupos: astrocitoma pilocítico (grau I), astrocitoma difuso (grau II), astrocitoma anaplásico (grau III) e glioblastoma (grau IV ou GBM). Entre esses, o GBM é o tumor mais agressivo e mais freqüente. Apesar de ser encontrado em qualquer faixa etária, esse tumor é raro em crianças. Atualmente a cirurgia seguida de radioterapia e quimioterapia com temozolomida (TMZ) tem sido utilizado como protocolo de tratamento padrão para a maioria dos pacientes e mesmo assim a sobrevida se mantem extremamente baixa. Além disso, grande parte dos pacientes não respondem ao tratamento com TMZ indicando a necessidade de agentes quimioterápicos alternativos. A zebularina (ZB) é um agente inibidor de DNA metiltransferases (iDNMTs) estável, pouco tóxico, que promove radiosensibilização e tem mostrado efeitos promissores em diversos tipos de neoplasias, entretanto pouco se sabe a respeito dos efeitos da ZB em glioblastoma. Os objetivos deste trabalho foram analisar a expressão dos genes DNMT1, DNMT3A, DNMT3B, MGMT em 5 amostras de substâncias brancas (SB), 6 linhagens de GBM e 33 amostras de gliomas (13 grau I, 2 grau II e 18 grau IV), correlacionar a expressão desses genes com os diferentes graus de gliomas e analisar os efeitos da ZB combinada ou não com TMZ em linhagens de GBM irradiadas e não irradiadas. Para análise da expressão gênica foi realizada a técnica de PCR em tempo Real. Os ensaios de proliferação celular, clonogênico, radiação e apoptose foram realizados em 3 linhagens de GBM (U251, SF188 e T98G) e uma de fibroblastos (MRC5). Também foi realizado o ensaio de proliferação celular em 5 culturas primárias de GBM tratadas com zebularina. Os genes DNMT3A e MGMT mostraram expressão maior nas amostras de SB comparando-se com gliomas e linhagens de GBM. O gene DNMT3B foi mais expresso nas linhagens de GBM comparando-se com as SB. O gene DNMT1 não mostrou diferenças significativas entre as amostras analisadas. Os ensaios de proliferação celular mostraram diminuição na proliferação com doses a partir de 50-100µM de ZB e de 250-500µM de TMZ nas linhagens e a partir de 50µM de ZB para as culturas primárias de GBM. As combinações de ZB com TMZ não mostraram sinergia na grande maioria das doses testadas. A ZB aumenta a apoptose nas 3 linhagens com doses a partir de 100µM. A ZB e TMZ mostraram diminuição na formação de colônias com as doses de 100µM e 10µM nas linhagens U251 e SF188 não irradiadas e irradiadas com 2, 4 e 6 Gy. A linhagem T98G expressa o gene MGMT, mostrou resistência a 10µM de TMZ e respondeu ao tratamento com 100µM de ZB. Também foi observado que 10µM de TMZ é mais citotóxico do que 100µM de ZB em fibroblastos não irradiados e irradiados (2Gy). Os resultados obtidos neste estudo mostram que a ZB pode representar um alvo terapêutico interessante para o estudo em glioblastoma. / Gliomas arise from glial cells and are the most common central nervous system tumors. They are divided in four groups: pilocytic astrocytoma (grade I), difuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma (grade IV or GBM). GBM is the most frequent and aggressive glioma. This type of tumor can occur in any age but its rare in children. Actually, surgery, radiotherapy and temozolomide (TMZ) adjuvant/concomitant chemotherapy has been the standard treatment protocol but the survival is extremely poor. In addition most patients do not respond to TMZ indicating the need for alternative chemotherapeutic agents. Zebularine (ZB) is a DNA metiltransferase inhibitor (DNMTi) stable, slight toxic that has been showed promise effects in cancer including radiosensitivity but little is known about ZB in glioblastoma. The objectives of this study were analyze DNMT1, DNMT3A, DNMT3B, MGMT gene expression profile in 5 samples of normal brain, 6 GBM cell lines and 33 glioma samples (13 grade I, 2 grade II e 18 grade IV), correlate with different gliomas grades and analyze the effects of ZB isolate and in combination with TMZ in irradiated and non irradiated GBM cell lines. Gene expression assays was made using Real Time PCR. Proliferation, clonogenic, radiation and apoptosis assays were realized in three GBM cell lines (U251, SF88, T98G) and one fibroblast cell line (MRC5). We also made proliferation assays in 5 primary cultures of samples of GMB. MGMT and DNMT3A genes showed higher expression in normal brain compared to gliomas and GBM cell lines. DNMT3B gene showed higher expression in GBM cell lines compared with normal brain and DNMT1 showed no significant differences among samples analyzed. We observed decrease of cell proliferation from 50-100µM of ZB and 250-500µM of TMZ on GBM cell lines and from 50µM of ZB for primary GBM samples. It was not observed synergy in the most combinations doses of ZB and TMZ (Calcusyn software). It was observed that 100µM of ZB and 10µM of TMZ decrease colony formation on U251 and SF188 cell lines non irradiated and irradiated with 2, 4, and 6Gy. T98G that express MGMT, did not respond to TMZ but showed response to ZB. It was also observed that 10µM of TMZ is more cytotoxic than 100µM of ZB in fibroblast cell line non irradiated and irradiated with 2Gy. ZB increase apoptosis from 100µM on the three GBM cell lines. Results obtained in this study can indicate that ZB may be an interestig therapeutic target for future studies in glioblastoma.
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The molecular basis of glutamate formiminotransferase deficiency /Hilton, John Frederick. January 2001 (has links)
No description available.
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