RESUMO
This study tested the hypothesis that women with higher prolactin feel more hostility, anger and aggression. A total of 66 women with moderate fertility problems were grouped into the 50% who had the highest and the 50% who had the lowest levels of prolactin. Levels of hostility, aggression and anger were compared. Women with higher prolactin levels did not report significantly increased hostility. After Bonferroni correction, women with lower prolactin showed non-significantly increased scores on two measures of state anger, and on a measure of trait temper. When comparing those with the highest and lowest 20% of prolactin levels, those with lower prolactin had non-significantly higher scores on trait temper and outward expression of anger, and non-significantly lower scores for control of anger. Although non-significant, these findings run counter to those of earlier studies on this topic. Implications for future research and patient care are discussed.
Assuntos
Agressão , Ira , Hostilidade , Infertilidade Feminina/sangue , Prolactina/sangue , Adulto , Estudos Transversais , Feminino , Humanos , Infertilidade Feminina/psicologia , Adulto JovemAssuntos
Janus Quinase 2/genética , Mutação , Policitemia Vera/genética , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Estudos de Casos e Controles , Éxons , Proteínas de Fusão bcr-abl/genética , Humanos , Índia , Dados de Sequência Molecular , Policitemia Vera/diagnóstico , Policitemia Vera/patologia , Análise de Sequência de DNARESUMO
STUDY QUESTION: Are women with polycystic ovary syndrome (PCOS) better at three-dimensional mental rotation than other women? SUMMARY ANSWER: Women with PCOS scored significantly higher on a mental rotation task than a female control group. WHAT IS KNOWN ALREADY: PCOS is a condition characterized by elevated testosterone levels. Some researches have found that three-dimensional mental rotation task performance is positively correlated with testosterone levels. STUDY DESIGN, SIZE, DURATION: This cross-sectional study was conducted between June 2006 and January 2009. The participants were 69 women with PCOS and 41 controls recruited from five gynaecology clinics in London. The control group consisted of non-PCOS women of comparable subfertility to PCOS group. These groups sizes gave roughly 80% power to detect moderate effect sizes for the main statistical test. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were recruited at London gynaecology clinics. The women were aged between 18 and 43. PCOS was diagnosed based on the Rotterdam criteria. Controls were women who experienced some degree of subfertility. Blood samples from participants were frozen for up to 4 months until being assayed by direct electrochemiluminescence. The mental rotation task was undertaken electronically. Some questionnaires and other tasks were completed as control measures. MAIN RESULTS AND THE ROLE OF CHANCE: Women with PCOS scored significantly higher than controls: median (range) 3.00 (0-9) and 2.00 (0-8), respectively (U = 1147.500, N1 = 69, N2 = 41, P < 0.047). Within the PCOS group, circulating levels of testosterone were significantly positively correlated with three-dimensional scoring (rs = 0.376, n = 56, P < 0.002), whereas estradiol was significantly negatively correlated with three-dimensional scoring (rs = -0.473, n = 29, P < 0.010). In the control group, the relationship between sex hormones and mental rotation was non-significant. Other factors, including general intelligence and social class, did not account for these findings. A subgroup analysis comparing hyperandrogenic PCOS cases, non-hyperandrogenic PCOS cases and controls, in which age and body mass index were controlled for using ANCOVA, found a non-significant difference in three-dimensional scoring between the three groups (F = 1.062, d.f. = 1, 73, P < 0.351). LIMITATIONS, REASONS FOR CAUTION: The small number of women in the control group meant that correlations were underpowered in this group. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first to find a benefit of PCOS in visuospatial cognition, and the first to find a link between visuospatial cognition and sex hormones in PCOS. The fact that the correlations went in the opposite direction in the PCOS group compared with the controls might suggest the influence of increased prenatal exposure to androgen in PCOS. STUDY FUNDING/COMPETING INTEREST(S): The assays for this study were funded by the Department of Psychology, City University London. All authors report no conflicts of interest.
Assuntos
Androgênios/fisiologia , Cognição , Síndrome do Ovário Policístico/psicologia , Percepção Visual , Adolescente , Adulto , Estudos Transversais , Estrogênios/sangue , Feminino , Humanos , Testosterona/sangue , Mulheres/psicologiaRESUMO
3-(4-Chlorophenyl)-4-substituted pyrazole derivatives were synthesised and tested for their in vitro antifungal activity. Some compounds showed very good antifungal activity against four pathogenic strains of fungi. The same compounds exhibited an interesting activity against the tested strain of Mycobacterium tuberculosis H37Rv. The results suggest that 1,3,4-oxadiazoles and 5-pyrazolinones bearing a core pyrazole scaffold may be promising antifungal and antitubercular agents.
Assuntos
Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Candida/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazóis/farmacologia , Antifúngicos/síntese química , Antifúngicos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
PURPOSE OF THE STUDY: The overall aim of the present work was to elucidate the effects of iontophoresis on assisting permeation/deposition of peptide dendrimers across/within human skin. PROCEDURES: A series of peptide dendrimers containing arginine and histidine as terminal acids were synthesized and characterized. These dendrimers were subjected to passive and iontophoretic permeation studies across human epidermis. RESULTS: The synthesized peptide dendrimers were found to be stable in epidermal, dermal and skin extracts up to 6 h. Passive diffusion studies revealed that none of the synthesized peptide dendrimers permeated human epidermis up to 6 h, although minute concentrations of low molecular weight dendrimers were detected in receptor medium at the end of 24 h. Application of iontophoresis significantly increased the permeation of all the tested peptide dendrimers across human skin in a molecular weight-dependent manner compared to simple passive diffusion. Electromigration was found to be the dominant mechanism behind the iontophoretic permeation of peptide dendrimers across human skin. CONCLUSIONS: The present study demonstrates that iontophoresis is an effective technique in enhancing the transdermal permeation of peptide dendrimers. MESSAGE OF THE PAPER: This study foresees the possibility of applying peptide dendrimers in iontophoretic delivery of drugs and macromolecules across/within the skin.
Assuntos
Dendrímeros/metabolismo , Peptídeos/metabolismo , Pele/metabolismo , Adulto , Difusão , Feminino , Humanos , Técnicas In Vitro , Iontoforese , Absorção CutâneaRESUMO
As a part of our research to develop novel antitubercular and antimicrobial agents, a series of 3-(4-chlorophenyl)-4-substituted pyrazoles have been synthesised. These compounds were tested for antitubercular activity in vitro against Mycobacterium tuberculosis H37Rv strain using the BACTEC 460 radiometric system, antifungal activity against a pathogenic strain of fungi and antibacterial activity against gram-positive and gram-negative organisms. Among them tested, many compounds showed good to excellent antimicrobial and antitubercular activity. The results suggest that hydrazones, 2-azetidinones and 4-thiazolidinones bearing a core pyrazole scaffold would be potent antimicrobial and antitubercular agents.
Assuntos
Anti-Infecciosos/síntese química , Antituberculosos/síntese química , Pirazóis/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antituberculosos/química , Antituberculosos/farmacologia , Fungos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazóis/síntese química , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
Chromatographic fingerprinting has been widely accepted as a crucial method for qualitative and quantitative analyses of bioactives within traditional Chinese medicine. A fingerprint provides detailed information, specific for any given herb, thus facilitating the quality control measures of a given traditional Chinese medicine. In this article, quality assessment of Radix Scrophulariae was achieved by using high performance liquid chromatography combining diode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS). Eight batches of sample obtained from different origins in China were used to establish the fingerprint and quantitative analyses. By comparing the retention times, UV and MS spectral data with reference standards, four characteristic peaks in the chromatograms were confirmed as corresponding to acetoside, angoroside C, cinnamic acid, and harpagoside. In addition, other two characteristic peaks were tentatively identified, following the literature interpretation of HPLC-ESI-MS and LC-MS/MS (affording structural information) to be sibirioside A and scrophuloside B(4), respectively. The results indicated that the newly developed HPLC-DAD-MS fingerprint method would be suitable for quality control of Radix Scrophulariae.
Assuntos
Inibidores da Angiogênese/análise , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/análise , Raízes de Plantas/química , Scrophularia/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/normas , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/normas , Mapeamento de Peptídeos/métodos , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
The aim of the present work was to develop and validate a simple RP-HPLC method with UV detection to quantify peptide dendrimers in skin permeation experiments. Six dendrimers of varying positive charges (4(+), 8(+) and 16(+)) containing either histidine or arginine as terminal aminoacids were prepared by solid phase peptide synthesis. Mobile phase containing 0.02% (v/v) heptafluorobutyric acid in 90% acetonitrile-water was capable of separating all dendrimers from interfering peaks of receptor fluid. For the calibration of each dendrimer, a different dendrimer from the same class was selected as the internal standard. The results of preliminary human skin permeation studies showed that the developed analytical method can be successfully used for the quantification of cationic poly(aminoacid)-based dendrimers in skin permeation experiments.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dendrímeros/análise , Peptídeos/análise , Pele/metabolismo , Dendrímeros/metabolismo , Humanos , Estrutura Molecular , Peptídeos/metabolismo , Reprodutibilidade dos TestesRESUMO
An 80-year-old man presented with the characteristic triad of yellow nail syndrome (chronic respiratory disorders, primary lymphedema and yellow nails) in association with coronary artery bypass graft surgery. Treatment with mechanical pleurodesis and vitamin E resulted in near complete resolution of the yellow nails, pleural effusions, and lower extremity edema. The etiology of the yellow nail syndrome has been described as an anatomical or functional lymphatic abnormality. Several conditions have previously been described as associated with this disease. This is the first report of the association of this syndrome with thoracic surgery.
Assuntos
Ponte de Artéria Coronária , Complicações Pós-Operatórias , Síndrome das Unhas Amareladas/etiologia , Idoso de 80 Anos ou mais , Humanos , Masculino , Pleurodese/métodos , Cirurgia Torácica Vídeoassistida/métodos , Resultado do Tratamento , Vitamina E/uso terapêutico , Vitaminas/uso terapêutico , Síndrome das Unhas Amareladas/diagnóstico , Síndrome das Unhas Amareladas/terapiaRESUMO
The quest towards achieving a better understanding of underlying mechanisms by which genetic factors contribute to human disease has gathered considerable momentum, most notably due to the drafting of the complete human genome sequence. This has in turn accelerated research into identifying genes responsible for a plethora of genetic, infectious and metabolic diseases with the vision that therapies can then be developed. Although achieving a therapeutic intervention by gene delivery is perfectly feasible, the practical approach to achieving such a goal, at least in vivo, has proved far more challenging. Employing viruses as gene vectors has to-date proven to be the most effective method of delivery however concerns have emerged about both the short and long-term risks they pose. These fears being confirmed by incidents which led to the tragic deaths of subjects believed to have been triggered by adeno- & retroviral vectors used in clinical trials. This prompted many in the field to turn their research focus towards developing non-viral vectors deemed not only to be safer (non-immunogenic) than their viral counterparts but with a greater gene loading capacity. Polycationic dendrimers (PCDs) as vectors for this purpose have attracted significant interest due to their ease of synthesis, versatility and tolerability. This review will explore the physicochemical parameters crucial to PCD-mediated gene delivery and highlight some innovative strategies designed to maximise transfection efficacy and facilitate tissue-targeting of these elaborate macromolecules.
Assuntos
Dendrímeros/administração & dosagem , Sistemas de Liberação de Medicamentos/tendências , Técnicas de Transferência de Genes/tendências , Animais , Sistemas de Liberação de Medicamentos/métodos , Terapia Genética/métodos , Terapia Genética/tendências , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Transfecção/métodos , Transfecção/tendênciasRESUMO
The ongoing shortage of donors for cardiac transplantation has led to a trend toward acceptance of donor hearts with some structural abnormalities including left ventricular hypertrophy. To evaluate the outcome in recipients of donor hearts with increased left ventricular wall thickness (LVWT), we retrospectively analyzed data for 157 cardiac donors and respective recipients from January 2001 to December 2004. There were 47 recipients of donor heart with increased LVWT >or=1.2 cm, which constituted the study group and 110 recipients of a donor heart with normal LVWT < 1.2 cm that formed the control group. At 3 +/- 1.5 years, recipient survival was lower (50% vs. 82%, p = 0.0053) and incidence of allograft vasculopathy was higher (50% vs. 22%, p = 0.05) in recipients of donor heart with LVWT > 1.4 cm as compared to LVWT
Assuntos
Transplante de Coração/patologia , Transplante de Coração/fisiologia , Ventrículos do Coração/patologia , Miocárdio/patologia , Doadores de Tecidos/estatística & dados numéricos , Disfunção Ventricular Esquerda/patologia , Adulto , Anti-Hipertensivos/uso terapêutico , Biópsia , Angiografia Coronária , Ecocardiografia , Feminino , Transplante de Coração/imunologia , Transplante de Coração/mortalidade , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Estudos Retrospectivos , Análise de Sobrevida , Doadores de Tecidos/provisão & distribuição , Coleta de Tecidos e Órgãos/métodos , Resultado do Tratamento , Disfunção Ventricular Esquerda/diagnóstico por imagemRESUMO
Idiopathic hypertrophic pachymeningitis is an extremely rare entity. It usually affects cranial meninges. The spinal form is further uncommon and presents as a chronic progressive disease. We describe a 42 year old female with isolated idiopathic hypertrophic cervical pachymeningitis who had a relapsing remitting course under observation for five years. Laminectomy and immunosuppressive therapy produced temporary and partial relief. The long term course and relevant literature is reviewed.
Assuntos
Vértebras Cervicais , Dura-Máter/patologia , Meningite/patologia , Adulto , Anti-Inflamatórios/uso terapêutico , Azatioprina/uso terapêutico , Feminino , Seguimentos , Humanos , Hipertrofia , Imunossupressores/uso terapêutico , Índia , Imageamento por Ressonância Magnética , Meningite/tratamento farmacológico , Prednisolona/uso terapêuticoRESUMO
Condensation of 5-cyano-2-hydrazino-3-N-methyl-6-phenyl/p-chlorophenyl-3,4-dihydropyrimidin-4-one (3a and 3b) with 2,4-bisalkyl/arylamino-6-chloro-s-triazine (4) gave the corresponding 2,4-bisalkyl/arylamino-6-[5'-cyano-3'-N-methyl]-6'-phenyl/pchlorophenyl-3',4'-dihydropyrimidin-4'-one-2'-yl-hydrazino-s-triazines (5a-n and 6a-n). The compounds 4 have been prepared by the condensation of cyanuric chloride and different alkyl/aryl amines. The reaction between 5-cyano-3-N-methyl-2-methylthio-6-phenyl/p-chlorophenyl-3,4-dihydropyrimidin-4-one (2a and 2b) with hydrazine hydrate furnished 3a and 3b, respectively. The condensation of 6-phenyl/p-chlorophenyl/5-cyano-2-mercapto-3,4-dihydropyrimidin-4-one (1a and 1b) with methyl iodide yielded 2a and 2b, respectively. All the products have been evaluated in vitro for their antimicrobial activity against several microbes and antitubercular activity against Mycobacterium tuberculosis H37 Rv.
Assuntos
Antituberculosos/síntese química , Pirimidinonas/síntese química , Antituberculosos/química , Antituberculosos/farmacologia , Testes de Sensibilidade Microbiana , Pirimidinonas/química , Pirimidinonas/farmacologia , Relação Estrutura-AtividadeRESUMO
Treatment of ovarian carcinomas with the antimitotic antitumor drug paclitaxel is highly efficacious. However, development of drug resistance presents a major obstacle. The common cellular phenotypes associated with paclitaxel resistance are an increased expression of the drug transport protein P-glycoprotein (P-gp), an alteration in the levels of beta-tubulin isotypes, and/or changes in the drug binding affinity of the microtubules. We established two paclitaxel-resistant human ovarian carcinoma cell lines. The 2008/17/4 cells exhibited a "classic" multidrug-resistant phenotype (overexpression of P-gp associated with cross-resistance to natural product drugs), whereas the 2008/13/4 cells were an atypical multidrug-resistant subline (no overexpression of P-gp). In addition to being paclitaxel resistant (250-fold), the 2008/13/4 cells were also cross-resistant to etoposide (39-fold) and vincristine (460-fold). To identify the alterations in the gene expression profile associated with the development of atypical paclitaxel resistance, we used the Clontech Atlas Human Cancer cDNA Microarray (spotted with 588 genes). The expression of retinoic acid receptor (RAR)-gamma was significantly higher in the paclitaxel-resistant (2008/13/4 and 2008/17/4) cells than in the parental (2008) cells. Northern blotting analysis demonstrated that the expression of RAR-gamma was 7-fold higher in the 2008/13/4 and 2008/17/4 cells than in the 2008 cells, whereas the expression of RAR-alpha and RAR-beta was not observed in any cell line. Whereas the 2008, 2008/13/4, and 2008/17/4 cells were found to resist the antiproliferative effects of all-trans-retinoic acid, the paclitaxel-resistant cells were 6- to 7-fold cross-resistant to the antiproliferative effects of CD437 (a synthetic RAR-gamma-selective agonist; 6-[-(1-admantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid) compared with the sensitivity of the parental cells. To further understand the association of paclitaxel and CD437 resistance with the observed RAR-gamma overexpression, we transfected the 2008 cells with a full-length RAR-gamma cDNA construct. Two transfectants with increased expression of the RAR-gamma mRNA and protein were isolated and subjected to growth inhibition assays in the presence of various concentrations of paclitaxel, etoposide, vincristine, and CD437. The sensitivity of the 2008 transfected clones (displaying increased expression of RAR-gamma) to the cytotoxic effects of paclitaxel, etoposide, vincristine, and CD437 was similar to that observed in the parental 2008 cells. These results suggest that the overexpression of RAR-gamma (observed in the 2008/13/4 and 2008/17/4 cells) by itself is not capable of inducing paclitaxel and CD437 resistance (or resistance to etoposide and vincristine).
Assuntos
Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos/fisiologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Receptores do Ácido Retinoico/biossíntese , Retinoides/farmacologia , Northern Blotting , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Receptores do Ácido Retinoico/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Receptor gama de Ácido RetinoicoRESUMO
Neurenteric cysts are very rare, particularly in adults. These are congenital intraspinal cysts of endodermal origin. A 67 years old man, presenting with backache and paraesthesiae of one and half years' duration, followed by subacute flaccid paraplegia, developing in a week is described. MRI revealed intramedullary cyst at T7. He underwent emergency thoracic laminectomy and complete excision of the cyst. Histopathology confirmed a neurenteric cyst. In view of their rarity, peculiarity in terms of age, location and presentation, we report this case.
Assuntos
Bulbo , Defeitos do Tubo Neural/diagnóstico , Defeitos do Tubo Neural/cirurgia , Medula Espinal/anormalidades , Idoso , Humanos , Imageamento por Ressonância Magnética , Masculino , Defeitos do Tubo Neural/patologia , Medula Espinal/patologiaRESUMO
Potential mechanisms accounting for the high cardiovascular death rates observed in Asian-Indians are dyslipidemia and insulin resistance. Polymorphisms in the APOC3 promoter (-455 T/C and -482 C/T) were frequently encountered in young Asian-Indians and they correlated with reduced concentrations of apolipoprotein A-I.
Assuntos
Apolipoproteínas C/genética , Doença das Coronárias/etnologia , Doença das Coronárias/genética , Polimorfismo Genético , População Branca/genética , Adulto , Feminino , Humanos , Índia/etnologia , Masculino , Pessoa de Meia-Idade , Prevalência , Estados UnidosRESUMO
Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2'-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.
Assuntos
Reparo do DNA/genética , DNA Recombinante/metabolismo , DNA de Cadeia Simples/metabolismo , Conversão Gênica/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Plantas , RNA/metabolismo , Animais , Sequência de Bases , Extratos Celulares , Linhagem Celular , Sistema Livre de Células , DNA Recombinante/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Eletroporação , Resistência a Canamicina/genética , Camundongos , Proteína 2 Homóloga a MutS , Proteína 3 Homóloga a MutS , Mutação/genética , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Células Vegetais , Plantas/genética , Plasmídeos/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA/genética , Resistência a Tetraciclina/genética , Transformação BacterianaRESUMO
Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.
Assuntos
Reparo do DNA/genética , Mutação da Fase de Leitura , Oligonucleotídeos/farmacologia , Mutação Puntual , Zea mays/genética , Sequência de Bases , Sistema Livre de Células , Hibridização de Ácido Nucleico , Oligonucleotídeos/genéticaRESUMO
Self-complementary chimeric oligonucleotides that consist of DNA and 2'-O-methyl RNA nucleotides arranged in a double-hairpin configuration can elicit a point mutation when targeted to a gene sequence. We have used a series of structurally diverse chimeric oligonucleotides to correct a mutant neomycin phosphotransferase gene in a human cell-free extract. Analysis of structure-activity relationships demonstrates that the DNA strand of the chimeric oligonucleotide acts as a template for high-fidelity gene correction when one of its bases is mismatched to the targeted gene. By contrast, the chimeric strand of the oligonucleotide does not function as a template for gene repair. Instead, it appears to augment the frequency of gene correction by facilitating complex formation with the target. In the presence of RecA protein, each strand of a chimeric oligonucleotide can hybridize with double-stranded DNA to form a complement-stabilized D-loop. This reaction, which may take place by reciprocal four-strand exchange, is not observed with oligonucleotides that lack 2'-O-methyl RNA segments. Preliminary sequencing data suggest that complement-stabilized D-loops may be weakly mutagenic. If so, a low level of random mutagenesis in the vicinity of the chimera binding site may accompany gene repair.