RESUMO
Quadruplexes are involved in the regulation of gene expression and are part of telomeres at the ends of chromosomes. In addition, they are useful in therapeutic and biotechnological applications, including nucleic acid diagnostics. In the presence of K(+) ions, two 15-mer sequences d(GGTTGGTGTGGTTGG) (thrombin binding aptamer) and d(GGGTGGGTGGGTGGG) (G3T) fold into antiparallel and parallel quadruplexes, respectively. In the present study, we measured the fluorescence intensity of one or more 2-aminopurine or 6-methylisoxanthopterin base analogs incorporated at loop-positions of quadruplex forming sequences to develop a detection method for DNA sequences in solution. Before quadruplex formation, the fluorescence is efficiently quenched in all cases. Remarkably, G3T quadruplex formation results in emission of fluorescence equal to that of a free base in all three positions. In the case of thrombin binding aptamer, the emission intensity depends on the location of the fluorescent nucleotides. Circular dichroism studies demonstrate that the modifications do not change the overall secondary structure, whereas thermal unfolding experiments revealed that fluorescent analogs significantly destabilize the quadruplexes. Overall, these studies suggest that quadruplexes containing fluorescent nucleotide analogs are useful tools in the development of novel DNA detection methodologies.
Assuntos
2-Aminopurina/química , Corantes Fluorescentes/química , Quadruplex G , Xantopterina/análogos & derivados , Aptâmeros de Nucleotídeos/química , Dicroísmo Circular , Desnaturação de Ácido Nucleico , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Xantopterina/químicaRESUMO
INTRODUCTION: The presence of more than one musculoskeletal disease has been found to impair quality of life (QoL). The influence of low bone mineral density (BMD) on symptoms and function in those with fibromyalgia syndrome (FMS) is unknown. METHODOLOGY: A cross sectional study was carried out on 158 patients attending camps in rural South India. BMD was determined using quantitative ultrasound of the distal radius. Symptoms and function were assessed using a visual analogue scale (VAS) and the Fibromyalgia Impact Questionnaire (FIQ). RESULTS: Low BMD was seen in 81.6% (129/158) of the persons screened. FMS was seen in 37/158 persons, of which 31/37 (83.7%) had low BMD. CONCLUSION: FMS with low bone density leads to higher levels of pain and a poorer QoL compared to those without FMS. Coexisting musculoskeletal problems could also contribute to this. Therefore, medical practitioners and rehabilitation specialists should consider screening for bone density among those with FMS and should use this information to decide appropriate therapies to reduce pain and improve QoL.
Assuntos
Densidade Óssea , Fibromialgia/fisiopatologia , Fibromialgia/psicologia , Osteoporose/fisiopatologia , Osteoporose/psicologia , Qualidade de Vida , Rádio (Anatomia)/fisiopatologia , Saúde da População Rural , Adulto , Idoso , Idoso de 80 Anos ou mais , Comorbidade , Efeitos Psicossociais da Doença , Estudos Transversais , Feminino , Fibromialgia/diagnóstico , Fibromialgia/epidemiologia , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Osteoporose/diagnóstico por imagem , Osteoporose/epidemiologia , Medição da Dor , Rádio (Anatomia)/diagnóstico por imagem , Fatores de Risco , Inquéritos e Questionários , Ultrassonografia , Adulto JovemAssuntos
Doenças Ósseas Metabólicas/epidemiologia , Osteoporose/epidemiologia , Absorciometria de Fóton , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Ósseas Metabólicas/diagnóstico , Doenças Ósseas Metabólicas/tratamento farmacológico , Feminino , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Osteoporose/diagnóstico , Osteoporose/tratamento farmacológico , Prevalência , Adulto JovemRESUMO
We previously developed a method, known as quadruplex priming amplification (QPA), which permits isothermal amplification of DNA. The assay is based on a DNA quadruplex formed by the GGGTGGGTGGGTGGG (G3T) sequence. G3T has three unique properties that are fundamental for QPA; (i) G3T forms a quadruplex with significantly more favorable thermodynamics than the corresponding DNA duplexes; (ii) removal of guanines at the 3'-end inhibits quadruplex formation; and (iii) incorporated fluorescent nucleotides, such as 2-aminopurine (2AP) or 6-methylisoxanthopterin (6MI), which are quenched by neighboring nucleotides, regain maximum emission upon quadruplex formation. New model studies carried out here with primers missing one, two and three guanines reveal that the driving force for QPA comes from the difference in thermal stability between the primer/template and the product complexes. Primers missing one and two guanines are able to self-dissociate from the template upon elongation, whereas QPA is not observed when the primer lacks three 3'-nucleotides. QPA reaches its maximum rate at temperatures slightly higher than the T(m) of the primer/template complex and is more efficient in the presence of only dGTP. QPA-based assays also revealed that Taq is able to incorporate thymidines opposite template 2AP, while no significant incorporation was observed opposite template 6MI.
Assuntos
Primers do DNA/genética , DNA/genética , Quadruplex G , Técnicas de Amplificação de Ácido Nucleico/métodos , 2-Aminopurina/química , Sequência de Bases , DNA/química , Primers do DNA/química , Corantes Fluorescentes/química , Guanina/química , Espectrometria de Fluorescência , Temperatura , Moldes Genéticos , Termodinâmica , Xantopterina/análogos & derivados , Xantopterina/químicaRESUMO
OBJECTIVE: Ghrelin acylation by ghrelin O-acyltransferase (GOAT) has recently been reported to be essential for the prevention of hypoglycemia during prolonged negative energy balance. Using a unique set of four different genetic loss-of-function models for the GOAT/ghrelin/growth hormone secretagogue receptor (GHSR) system, we thoroughly tested the hypothesis that lack-of-ghrelin activation or signaling would lead to hypoglycemia during caloric deprivation. METHODOLOGY: Male and female knockout (KO) mice for GOAT, ghrelin, GHSR, or both ghrelin and GHSR (dKO) were subjected to prolonged calorie restriction (40% of ad libitum chow intake). Body weight, fat mass, and glucose levels were recorded daily and compared to wildtype (WT) controls. Forty-eight hour blood glucose profiles were generated for each individual mouse when 2% or less body fat mass was reached. Blood samples were obtained for analysis of circulating levels of acyl- and desacyl-ghrelin, IGF-1, and insulin. PRINCIPAL FINDINGS: Chronic calorie restriction progressively decreased body weight and body fat mass in all mice regardless of genotype. When fat mass was depleted to 2% or less of body weight for 2 consecutive days, random hypoglycemic events occurred in some mice across all genotypes. There was no increase in the incidence of hypoglycemia in any of the four loss-of-function models for ghrelin signaling including GOAT KO mice. Furthermore, no differences in insulin or IGF-1 levels were observed between genotypes. CONCLUSION: The endogenous GOAT-ghrelin-GHSR system is not essential for the maintenance of euglycemia during prolonged calorie restriction.