RESUMO
Oxidative damage to mitochondrial DNA (mtDNA) increases with age in the brain and can induce G:C to T:A and T:A to G:C point mutations. Though rare at any particular site, multiple somatic mtDNA mutations induced by oxidative damage or by other mechanisms may accumulate with age in the brain and thus could play a role in aging and neurodegenerative diseases. However, no prior study has quantified the total burden of mtDNA point mutation subtypes in the brain. Using a highly sensitive cloning and sequencing strategy, we find that the aggregate levels of G:C to T:A and T:A to G:C transversions and of all point mutations increase with age in the frontal cortex (FCtx). In the substantia nigra (SN), the aggregate levels of point mutations in young controls are similar to the levels in the SN or FCtx of elderly subjects. Extrapolation from our data suggests an average of 2.7 (FCtx) to 3.2 (SN) somatic point mutations per mitochondrial genome in elderly subjects. There were no significant differences between Parkinson's disease (PD) patients and age-matched controls in somatic mutation levels. These results indicate that individually rare mtDNA point mutations reach a high aggregate burden in FCtx and SN of elderly subjects.
Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Osso Frontal/patologia , Doença de Parkinson/genética , Mutação Puntual , Substância Negra/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Linhagem Celular/metabolismo , Criança , Pré-Escolar , Análise Mutacional de DNA , DNA Mitocondrial/metabolismo , Feminino , Osso Frontal/metabolismo , Humanos , Lactente , Masculino , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Substância Negra/metabolismoRESUMO
We describe the physiochemical characterization and immunological evaluation of plasmid DNA vaccine formulations containing a nonionic triblock copolymer adjuvant (CRL1005) in the presence and absence of a cationic surfactant, benzalkonium chloride (BAK). CRL1005 forms particles of 1-10 microns upon warming above its phase-transition temperature (approximately 6-8 degrees C) and the physical properties of the particles are altered by BAK. DNA/CRL1005 vaccines formulated with and without BAK were evaluated in rhesus macaques to determine the effect of CRL1005 and BAK on the ability of plasmid DNA to induce a cellular immune response. Immunogenicity results indicate that the addition of CRL1005 to human immunodeficiency virus-1 gag plasmid DNA formulated in phosphate-buffered saline leads to an enhancement in the gag-specific cellular immune response. Moreover, the addition of BAK to human immunodeficiency virus-1 gag plasmid DNA/CRL1005 formulations produces an additional enhancement in gag-specific cellular immunity. In vitro characterization studies of DNA/CRL1005 formulations indicate no detectable binding of DNA to CRL1005 particles in the absence of BAK, suggesting that the enhancement of cellular immunity induced by DNA/CRL1005 formulations is not due to enhanced DNA delivery. In the presence of BAK, however, results indicate that BAK binds to CRL1005 particles, producing cationic microparticles that bind DNA through electrostatic interactions. If BAK is present at the phase-transition temperature, it reduces the particle size from approximately 2 microns to approximately 300 nm, presumably by binding to hydrophobic surfaces during particle formation. Zeta potential measurements indicate that the surface charge of CRL1005-BAK particles changes from positive to negative upon DNA binding, and DNA bound to the surface of CRL1005-BAK particles was visualized by fluorescence microscopy. These results indicate that the addition of BAK to DNA/CRL1005 formulations leads to the formation of approximately 300 nm CRL1005-BAK-DNA particles that enhance the cellular immune response in rhesus monkeys.
Assuntos
Adjuvantes Farmacêuticos/química , Microesferas , Plasmídeos/química , Vacinas de DNA/química , Adjuvantes Farmacêuticos/administração & dosagem , Animais , Bovinos , Química Farmacêutica , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Imunidade Celular/imunologia , Macaca mulatta , Tamanho da Partícula , Plasmídeos/administração & dosagem , Plasmídeos/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
Promoter escape efficiency of E. coli RNA polymerase is guided by both the core promoter and the initial transcribed sequence (ITS). Here, we quantitatively examined the escape properties of 43 random initial sequence variants of the phage T5 N25 promoter. The position for promoter escape on all N25-ITS variants occurred at the +15/+16 juncture, unlike the +11/+12 juncture for the wild type N25. These variants further exhibited a 25-fold difference in escape efficiency. ITS changes favoring promoter escape showed a compositional bias that is unrelated to nucleotide substrate binding affinity for the initial positions. Comparing all variants, the natural N25 promoter emerges as having evolved an ITS optimal for promoter escape, giving a high level of productive synthesis after undergoing the shortest abortive program. We supplemented GreB to transcription reactions to better understand abortive initiation and promoter escape in vivo. GreB supplementation elevated productive RNA synthesis 2-5-fold by altering the abortive RNA pattern, decreasing the abundance of the medium (6-10 nt) to long (11-15 nt) abortive RNAs without changing the levels of short (2-5 nt) and very long abortive RNAs (16-20 nt). The GreB-refractive nature of short abortive RNA production may reflect a minimum length requirement of 4-5 bp of the RNA-DNA hybrid for maintaining the stability of initial or backtracked complexes. That the very long abortive RNAs are unaffected by GreB suggests that they are unlikely to be products of polymerase backtracking. How the ITS might influence the course of early transcription is discussed within the structural context of an initial transcribing complex.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transcrição Gênica/fisiologia , Sequência de Bases , Escherichia coli/enzimologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Fagos T/genética , Sítio de Iniciação de Transcrição/fisiologiaRESUMO
The mitochondrial theory of aging proposes that mitochondrial DNA (mtDNA) accumulates mutations with age, and that these mutations contribute to physiological decline in aging and degenerative diseases. Although a great deal of indirect evidence supports this hypothesis, the aggregate burden of mtDNA mutations, particularly point mutations, has not been systematically quantified in aging or neurodegenerative disorders. Therefore, we directly assessed the aggregate burden of brain mtDNA point mutations in 17 subjects with Alzheimer's disease (AD), 10 elderly control subjects and 14 younger control subjects, using a PCR-cloning-sequencing strategy. We found that brain mtDNA from elderly subjects had a higher aggregate burden of mutations than brain mtDNA from younger subjects. The average aggregate mutational burden in elderly subjects was 2 x 10(-4) mutations/bp. The bulk of these mutations were individually rare point mutations, 60% of which changed an amino acid. Control experiments ensure that these results were not due to artifacts arising from PCR error, mistaken identification of nuclear pseudogenes or ex vivo oxidation. Cytochrome oxidase activity correlated negatively with increasing mutational burden. These findings significantly bolster the mitochondrial theory of aging.