RESUMO
OBJECTIVE: When a triploid pregnancy is diagnosed prenatally, gynaecologists have traditionally relied on the histopathological examination of the tissue from the terminated pregnancy to determine if the pregnancy is molar. However, reproducibility is poor and variability is high when diagnosing hydatidiform moles. Triploid pregnancies can have either the chromosomal constitution of two maternal and one paternal set, or two paternal and one maternal set, but only the conceptuses with two paternal sets have the potential to cause maternal complications. Therefore, it would be beneficial to introduce a method that gives the gynaecologist the parental origin of the genome of the triploid conceptus as early as possible, without delaying the process by first collecting parental samples. METHODS: Using methylation-specific multiplex ligation-dependent probe amplification, we measured methylation levels at different imprinted sites. RESULTS: We were able to correctly determine the parental origin of the genome in all 105 triploid pregnancies analysed. CONCLUSIONS: We present methylation-specific multiplex ligation-dependent probe amplification as a method capable of determining the parental origin of the genome of triploid conceptuses within 24 h; it is inexpensive, simple and easy to use, and parental samples are not needed.
Assuntos
Metilação de DNA/genética , Mola Hidatiforme/genética , Reação em Cadeia da Polimerase Multiplex , Pais , Diagnóstico Pré-Natal/métodos , Triploidia , Feminino , Humanos , Mola Hidatiforme/diagnóstico , Cariotipagem , Masculino , Gravidez , Complicações na Gravidez/genética , Reprodutibilidade dos Testes , Fatores de Risco , Neoplasias Uterinas/genéticaRESUMO
This study aims to evaluate the incidence of breast cancer after risk-reducing mastectomy (RRM) in healthy BRCA mutation carriers. This study is a long-term follow-up of 307 BRCA mutation carriers of whom 96 chose RRM. None of the study participants had a previous history of breast or ovarian cancer nor had they undergone RRM or risk-reducing bilateral salpingo-oophorectomy (BSO) prior to the time of BRCA testing. The annual incidence of post-mastectomy breast cancer was 0.8% compared with 1.7% in the non-operated group. Implications of these findings in relation to genetic counseling and future management are discussed.
Assuntos
Neoplasias da Mama/cirurgia , Mastectomia/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Feminino , Genes BRCA1 , Genes BRCA2 , Humanos , Pessoa de Meia-Idade , Mutação , Comportamento de Redução do Risco , Adulto JovemRESUMO
To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.
Assuntos
Granulócitos/citologia , Células-Tronco Hematopoéticas/citologia , Subpopulações de Linfócitos T/citologia , Telômero/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Envelhecimento/patologia , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Sequências Repetidas Terminais , Gêmeos Dizigóticos/genética , Gêmeos Monozigóticos/genéticaRESUMO
Once female carriers of a BRCA mutation are identified they have to make decisions on risk management. The aim of this study is to outline the uptake of risk-reducing surgery in the Danish population of BRCA mutation positive women and to search for factors affecting this decision. We analysed data from 306 healthy BRCA carriers with no personal history of ovarian or breast cancer. We found a 10-year uptake of 75% for risk-reducing salpingo-oophorectomy and 50% for risk-reducing mastectomy by time to event analysis. Age and childbirth influenced this decision. The uptake rate has not changed significantly over the last decade. Risk-reducing surgeries are widely acceptable among Danish BRCA mutation positive women and the uptake of prophylactic mastectomy is higher than in most other countries.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Tubas Uterinas/cirurgia , Heterozigoto , Mastectomia/métodos , Mutação/genética , Ovariectomia/métodos , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Fatores de Risco , Fatores de TempoRESUMO
OBJECTIVE: Deficiency of DNA mismatch repair (MMR) causes microsatellite instability (MSI) in a subset of colorectal cancers. Patients with these tumours have a better prognosis and may have an altered response to chemotherapy. Some of the tumours are caused by hereditary mutations (hereditary nonpolyposis colon cancer or Lynch syndrome), but most are epigenetic changes of sporadic origin. The aim of this study was to define a robust and inexpensive strategy for such classification in clinical practice. METHOD: Tumours and blood samples from 262 successive patients with colorectal adenocarcinomas were collected. Expression of the MMR proteins MLH1, MSH2, and MSH6 by immunohistochemistry (IHC) was compared with MSI DNA analysis. Methylation analysis of MLH1 and mutation analysis for BRAF V600E were compared in samples with MSI and/or lack of MLH1 expression to determine if the tumour was likely to be sporadic. RESULTS: Thirty-nine (14.9%) of the tumours showed MMR deficiency by IHC or by microsatellite analysis. Sporadic inactivation by methylation of MLH1 promoter was found in 35 patients whereby the BRAF activating V600E mutation, indicating sporadic origin, was found in 32 tumours. On the basis of molecular characteristics we found 223 patients with intact MMR, 35 patients with sporadic MMR deficiency, and four patients who were likely to have hereditary MMR deficiency. CONCLUSION: To obtain the maximal benefit for patients and clinicians, MMR testing should be supplemented with MLH1 methylation or BRAF mutation analysis to distinguish sporadic patients from likely hereditary ones. MMR deficient patients with sporadic disease can be reassured of the better prognosis and the likely hereditary cases should receive genetic counselling.
Assuntos
Adenocarcinoma/classificação , Adenocarcinoma/genética , Neoplasias Colorretais/classificação , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Adulto , Idoso , Idoso de 80 Anos ou mais , Metilação de DNA , Proteínas de Ligação a DNA/análise , Feminino , Humanos , Imuno-Histoquímica , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Proteína 2 Homóloga a MutS/análise , Proteína 3 Homóloga a MutS , MutaçãoRESUMO
This paper characterizes the distribution of telomere length on individual chromosome arms in humans. By fluorescent in situ hybridization (FISH), followed by computer-assisted analysis of digital images, it is shown that the distribution of telomere length on individual chromosome arms is not random, but that humans have a common telomere profile. This profile exists in lymphocytes, amniocytes and fibroblasts, and seems to be conserved during life. A closer look at the overall pattern of the profile shows that the length of the telomeres in general follows the total chromosome length. In addition to the common profile, it is found that each person has specific characteristics, which are also conserved throughout life. Studying both twins and families we have obtained indications that these individual characteristics are at least partly inherited. Altogether, our results suggest that the length of individual telomeres might occasionally play a role in the heritability of life span.
Assuntos
Envelhecimento/genética , Cromossomos Humanos/genética , Variação Genética , Longevidade/genética , Telômero/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/fisiologia , Âmnio/citologia , Fibroblastos/fisiologia , Humanos , Hibridização in Situ Fluorescente , Linfócitos/fisiologia , Pessoa de Meia-Idade , Gêmeos Dizigóticos , Gêmeos MonozigóticosRESUMO
The degradation of medium-chained dicarboxylic (DC) acids was investigated on purified mitochondria and peroxisomes. Intact organelles were incubated with dodecanedioic acid (DC12), suberic acid (DC8) and adipic acid (DC6), and the production of lower-chained DC-acids and of acetyl-CoA + acetyl-carnitine was monitored. It was shown, that intact peroxisomes could beta-oxidize DC12, DC10, and DC8 at least as far as DC6, while intact mitochondria readily beta-oxidized DC12, and DC10 as far as succinic acid. DC8 and DC6 were not oxidized by intact mitochondria when these two acids were presented externally to the intact organelle. When they were formed intramitochondrially from DC12 and DC10, both DC8 and DC6 were, however, to a great extent beta-oxidized as far as succinic acid. The major reason for this difference between mitochondrial oxidation of externally and internally located DC8 and DC6 seems to be an inability to transport these two acids through the mitochondrial membrane. For DC12 and DC10, the mitochondrial transport systems, which were indicated to be identical to the systems used by the corresponding monocarboxylic acids, were found to be rate-limiting in the beta-oxidation of these acids. A contributing factor to the undetectable beta-oxidation of externally located DC8 and DC6 may also be, that the Km values of DC8-CoA (460 +/- 70 mumol/l) and DC6-CoA (980 +/- 90 mumol/l) towards the acyl-CoA dehydrogenases are very high. These results imply that very high concentrations of intermediates are created intramitochondrially during beta-oxidation, concentrations which are probably only formed through formation of DC8-CoA and DC6-CoA from longer DC-acids and not by transport from outside the mitochondria. The data presented thus for the first time give evidence to a pathway for medium-chained monocarboxylic acids (especially lauric acid and decanoic acid) through cytosolic omega-oxidation followed by activation, transport over the mitochondrial membrane and beta-oxidation to succinic acid.
Assuntos
Caprilatos , Ácidos Dicarboxílicos/metabolismo , Fígado/metabolismo , Acetilcoenzima A/metabolismo , Acetilcarnitina/metabolismo , Adipatos/metabolismo , Animais , Ácidos Decanoicos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Feminino , Cinética , Fígado/ultraestrutura , Microcorpos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Modelos Biológicos , Oxirredução , Ratos , Ratos EndogâmicosRESUMO
The beta-oxidation rate of dodecanedioic acid in rat liver homogenates (600 X g supernatant fraction) was determined by simultaneous measurements of the C6-C12-dicarboxylic acids, i.e., adipic, suberic, sebacic and dodecanedioic acids, in relation to time in assays incubated with dodecanedioic acid. Measurements were performed by a combined gas chromatographic-mass spectrometric technique, i.e., selected ion-monitoring. The beta-oxidation rate was registered as the consumption rate of dodecanedioic acid and as the initial rise in the concentrations of C6-C10-dicarboxylic acids. The beta-oxidation rate of C8-C12-dicarboxylic acids was increased many times in homogenates from clofibrate-treated rats. Moreover, it was unexpectedly found that 2.0 mM cyanide was unable to inhibit the beta-oxidation rate of the dicarboxylic acids in vitro, but in fact caused a minor increase in the rate of beta-oxidation in homogenates from both normal and clofibrate-treated rats. It was concluded that the present results strongly indicate the existence of a peroxisomal beta-oxidation of dicarboxylic acids.
Assuntos
Clofibrato/farmacologia , Cianetos/farmacologia , Ácidos Dicarboxílicos/metabolismo , Microcorpos/metabolismo , Organoides/metabolismo , Cianeto de Potássio/farmacologia , Animais , Feminino , Cinética , Oxirredução , Ratos , Ratos Endogâmicos , Relação Estrutura-AtividadeRESUMO
An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild-type MCAD with respect to solubility, subcellular location, mature protein size and tetrameric structure. In immunoblot comparisons, the MCAD protein was present in normal fibroblasts, but essentially undetectable in patient fibroblasts homozygous for the prevalent mutation. We suggest that the MCAD protein carrying this mutation has an impaired ability to form correct tetramers, leading to instability and subsequent degradation of the enzyme. This finding is discussed in relation to the results from expression of human MCAD in Escherichia coli, where preliminary results show that production of mutant MCAD leads to the formation of aggregates.
Assuntos
Acil-CoA Desidrogenases/genética , Mutação , Acil-CoA Desidrogenase , Animais , Northern Blotting , Western Blotting , Linhagem Celular Transformada , Clonagem Molecular , DNA , Células Eucarióticas , Vetores Genéticos , Herpesvirus Humano 4/genética , Homozigoto , Humanos , Regiões Promotoras Genéticas , RNA/genética , Vírus 40 dos Símios/genética , TransfecçãoRESUMO
The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism underlying MCAD deficiency caused by the prevalent K304E mutation. Depending on which of the three amino acids--lysine (wild-type), glutamic acid (K304E) or glutamine (K304Q) are present at position 304 of the mature polypeptide, three different patterns were observed in our assay system: (i) solubility, tetramer formation and yield of enzyme activity of wild-type MCAD is largely independent of GroESL co-overexpression; (ii) the larger part of the K304Q mutant is insoluble without and solubility is enhanced with GroESL co-overexpression; solubility correlates with the amount of tetramer detected and the enzyme activity measured as observed for the wild-type protein. (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected in Western blotting. In a first attempt to estimate the specific activity, we show that tetrameric K304E and K304Q mutant MCAD display a specific activity in the range of the wild-type enzyme. Taken together, our results strongly suggest, that the K304E mutation primarily impairs the rate of folding and subunit assembly. Based on the data presented, we propose that lysine-304 is important for the folding pathway and that an exchange of this amino acid both to glutamine or glutamic acid leads to an increased tendency to misfold/aggregate. Furthermore, exchange of lysine-304 with an amino acid with negative charge at position 304 (glutamic acid) but not with a neutral charge (glutamine) negatively affects conversion to active tetramers. A possible explanation for this latter effect--charge repulsion upon subunit docking--is discussed.
Assuntos
Acil-CoA Desidrogenases/genética , Proteínas de Bactérias/metabolismo , Chaperoninas/metabolismo , Escherichia coli/genética , Proteínas/metabolismo , Acil-CoA Desidrogenase , Acil-CoA Desidrogenases/química , Acil-CoA Desidrogenases/metabolismo , Sequência de Bases , Expressão Gênica , Glutamatos , Ácido Glutâmico , Glutamina , Humanos , Lisina , Dados de Sequência Molecular , Mutação , Plasmídeos , Dobramento de ProteínaRESUMO
To try epidermis as a target for somatic gene therapy we studied transfected primary human keratinocytes grown in culture and grafted onto athymic mice. We have developed a novel technique for grafting cultured epidermal sheets onto mice. First, the graft is placed on the dorsal muscle fascia underneath the mouse skin using the latter as a bandage. Secondly, the mouse skin above the graft is removed, which exposes the grafted skin to open air and thus stimulates terminal differentiation. A novel method for the discrimination between murine and human epidermal cells is also presented, employing in situ hybridization with human Alu repeated DNA sequences. During monolayer culture the keratinocytes were lipofected with the gene for human growth hormone in an Epstein-Barr virus-based expression vector. The cells were allowed to develop a multilayered tissue for 5 d, secreting human growth hormone into the medium at a daily rate of at least 50 ng/cm2 of tissue. The transfected tissues were then grafted onto mice. We detected human growth hormone at levels of up to 2.6 ng/ml in mouse serum for 4 d, but later no human growth hormone could be found, although the transplants survived for months. To investigate the fate of the transfected cells in the transplanted tissue, we labeled them with the beta-galactosidase reporter gene. The cells staining positive for X-gal were found exclusively in the most superficial differentiated layers at 7 d after transplantation. This may be the main reason why no human growth hormone is found in the mouse circulation at this time.
Assuntos
Transplante de Células , Células Epidérmicas , Epiderme/transplante , Técnicas de Transferência de Genes , Tolerância Imunológica , Animais , Células Cultivadas , Feminino , Genes Reporter , Humanos , Queratinócitos/fisiologia , Queratinócitos/transplante , Camundongos , Camundongos Nus , Transfecção , beta-Galactosidase/genéticaRESUMO
The function of a series of LDL receptor GFP fusion proteins with different, flexible, unstructured spacer regions was analysed. An optimised version of the fusion protein was used to analyse the effect of an LDL receptor mutation (W556S) found in FH patients and characterised as transport defective. In cultured liver cells this mutation was found to inhibit the transport of LDL receptor GFP fusion protein to the cell surface, thus leading to impaired internalisation of fluorescent labelled LDL. Co-localisation studies confirmed the retention of the mutant protein in the endoplasmic reticulum. Wild type (WT) and W556S LDL receptor GFP fusion proteins were expressed in mouse liver by means of hydrodynamic delivery of naked DNA. Two days after injection liver samples were analysed for GFP fluorescence. The WT LDL receptor GFP protein was located on the cell surface whereas the W556S LDL receptor GFP protein was retained in intracellular compartments. Thus, the GFP-tagged LDL receptor protein allows both detailed time lapse analysis and evaluations in animals for the physiological modelling of mutations. This method should be generally applicable in functional testing of gene products for aberrant processing.
Assuntos
Receptores de LDL/fisiologia , Animais , Transporte Biológico , Linhagem Celular , Endocitose , Genótipo , Proteínas de Fluorescência Verde , Humanos , Hiperlipoproteinemia Tipo II/genética , Lipoproteínas LDL/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Microscopia Confocal , Mutação , Receptores de LDL/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
In a group of unrelated Danish patients with familial hypercholesterolemia (FH) we recently reported two common low-density lipoprotein (LDL) receptor mutations, W23X and W66G, accounting for 30% of the cases. In this study, we describe another common LDL receptor mutation, a G to C transition at cDNA position 1730 in exon 12, causing a tryptophan to serine substitution in amino acid position 556 (W556S). In the Danish patients, the W556S mutation was present in 12% of 65 possible mutant alleles. The pathogenicity of the W556S mutation, which is located in one of the five conserved motifs Tyr-Trp-Thr-Asp in the epidermal growth factor homology region, was studied in transfected COS-7 cells expressing normal and mutant LDL receptor cDNAs. Results obtained by immunofluorescence flow cytometry and confocal microscopy, as well as by immunoprecipitation, were compatible with complete retention of the mutant protein in the endoplasmic reticulum. The transport-defective W556S mutation and the W23X and W66G mutations seem to account for about 40% of the LDL receptor defects in Danish families with FH.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Mutação Puntual , Receptores de LDL/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Sequência Conservada , Dinamarca , Éxons , Citometria de Fluxo , Imunofluorescência , Humanos , Microscopia Confocal , Sequências Repetitivas de Ácido Nucleico , Serina , Transfecção , TriptofanoRESUMO
PRimed IN Situ labeling (PRINS) is a fast and sensitive alternative to fluorescence in situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.
Assuntos
Ácidos Nucleicos/química , Cromossomos Humanos , Primers do DNA , Sondas de DNA , DNA Satélite , Feminino , Técnicas Genéticas , Humanos , Hibridização in Situ FluorescenteRESUMO
Five urine samples were collected in clinically quiet periods over a period of one year from a patient suffering from D-glyceric acidemia, and investigated for presence or absence of glycine-conjugates. The findings of isovalerylglycine, 2-methylbutyrylglycine, isobutyrylglycine, and tiglylglycine are interpreted as indications of intracelluar accumulations of isovaleryl-CoA, 2-methylbutyryl-CoA and isobutyryl-CoA. Similarly, the findings of elevated amounts of butyric acid and hexanoic acid together with butyrylglycine, hexanoylglycine, and suberic acid suggest intracellular accumulations of straight-chain acyl-CoA's. It is therefore suggested that this child has a common derangement in his acyl-CoA dehydrogenase (in addition to his primary defect). As possible secondary consequences of this, two points can be mentioned: firstly hyperglycinemia, from which the patient suffered, and secondly, diminished tendency to ketosis, a condition from which the child never suffered, not even in connection with severe intercurrent disease.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/urina , Ácidos Glicéricos/sangue , Glicina/análogos & derivados , Butiratos/urina , Caproatos/urina , Caprilatos/urina , Pré-Escolar , Glicina/urina , Humanos , Hidrólise , Modelos QuímicosRESUMO
Five children, who received calcium levulinate (calcium 4-oxopentanoate) intravenously in pharmacological doses excreted in the following 24-h period both 4-oxopentanoic acid (3.5--11.0 mg/24 h) and 4-hydroxypentanoic acid (4.5--10.4 mg/24 h). Attention is drawn to the fact that in gas chromatographic-mass spectrometric systems these compounds closely resemble the two acids found in children with beta-ketothiolase deficiency.
Assuntos
Ácidos Carboxílicos/urina , Ácidos Levulínicos/uso terapêutico , Erros Inatos do Metabolismo Lipídico/diagnóstico , Acetil-CoA C-Aciltransferase/deficiência , Cálcio , Cromatografia Gasosa/métodos , Erros de Diagnóstico , Estudos de Avaliação como Assunto , Humanos , Recém-Nascido , Ácidos Levulínicos/urina , Espectrometria de Massas/métodosRESUMO
Urine samples from 18 individuals with various types of dicarboxylic acidurias have been investigated by mass fragmentography for N-dicarboxyl-mono-glycines (dicarboxylglycines). One patient with methylmalonic acidemia excreted 14-20 microgram methylmalonylglycine/mg creatinine, three patients with glutaric aciduria excreted 20-60 microgram glutarylglycine/creatinine, and one patient with C6-C10-dicarboxylic aciduria excreted 120-365 microgram succinylglycine/mg creatinine. Excretion of C6-C10-dicarboxylic acids in patients with ketosis and glycogenosis and in neonates were not accompanied by excretion of C8-C10-dicarboxylglycines in measurable amounts (greater than 1 microgram/mg creatinine). Nor did patients with succinic aciduria excrete succinylglycine in amounts larger than 1 microgram/mg creatinine. On the basis of these data it is argued that production of short- and medium-chain dicarboxylglycines is not a metabolic pathway of biological significance for the elimination of short- and medium-chain dicarboxylic acids from individuals with dicarboxylic acidurias.
Assuntos
Aminoácidos Dicarboxílicos/urina , Glicina/análogos & derivados , Adolescente , Adulto , Criança , Pré-Escolar , Creatinina/urina , Cromatografia Gasosa-Espectrometria de Massas , Glicina/urina , Humanos , Lactente , Recém-Nascido , Malonatos/urinaRESUMO
Biotinylated DNA hybridization probes offers a stable, cheap and non-radioactive alternative to probes labelled with 32P. Insufficient sensitivity has, however, up till now, been prohibitive for the use of such probes in detecting unique sequences in Southern blots of human DNA. By optimizing the steps in the procedure we have improved the sensitivity enough for such use. We have showed (1) that long probes (greater than 500 nucleotides) perform unproportionally better than short probes; (2) that a simple affinity labelling with avidin alkaline phosphatase conjugate performs better than laborious immunochemical systems; (3) that use of 3% BSA as blocking agent at 37 degrees C and the presence of 0.5 mol/l NaCl together with 1% BSA during the affinity labelling nearly eliminate background staining; (4) that a dramatic gain in sensitivity is gained by affinity labelling at pH 9.0 instead of 7.5; (5) that biotin-labelling can be highly reproducibly performed on a preparative scale with cheap and easily synthesized bio-11-dUTP in a two step nick-translation and (6) that biotinylated probes and hybridization mixtures can be stored for months and reused. The study has resulted in the presentation of a fast procedure, which is generally applicable to routine DNA diagnostic work, also in parts of the world where it is difficult to get a regular supply of 32P.
Assuntos
DNA/análise , Sequência de Bases , Biotina , Colódio , DNA/imunologia , Humanos , Hibridização de Ácido Nucleico , Radioisótopos de Fósforo , Biossíntese de Proteínas , Uridina Trifosfato/análiseRESUMO
Two boys, who are not related, with hypoglycemia and C6-C10-dicarboxylic aciduria were investigated. Besides substantial amounts of adipic, suberic and sebacic acids, the urinary metabolic profile of organic acids contained 5-OH-caproic acid and caproylglycine. During acute attacks the concentrations of adipic, suberic and sebacic acids were 300--530, 160--200 and 35--200 micrograms/mg creatinine, respectively, and the excretions of 5-OH-caproic acid and caproylglycine were 75--330 and 41--260 micrograms/mg creatinine, respectively. It is argued that the biosynthesis of adipic acid passes through an omega-oxidation, that the production of 5-OH-caproic acid is caused by an omega-1-oxidation, and that caproylglycine formation passes through a glycine-N-acylase catalysed conjugation of accumulated caproic acid in the patients. Suberic acid and sebacic acid are in the same way omega-oxidation products of accumulated caprylic acid and capric acid, respectively. From the excretion pattern presented it is hypothesized that the patients suffer from a defect in the dehydrogenation of fatty acids in the beta-oxidation pathway. The biological significance of the findings is discussed.
Assuntos
Caprilatos , Ácidos Dicarboxílicos/urina , Hipoglicemia/urina , Erros Inatos do Metabolismo Lipídico/urina , Adipatos/urina , Caproatos/urina , Pré-Escolar , Cromatografia Gasosa , Ácidos Decanoicos/urina , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Glicina/análogos & derivados , Glicina/urina , Humanos , Hidroxiácidos/urina , Hidroxibutiratos/urina , Lactente , Masculino , Espectrometria de Massas , Valeratos/urinaRESUMO
Urinary analysis of the pattern of 23 organic acid metabolites derived from fatty acids in three patients with general (medium-chain) acyl-CoA dehydrogenase deficiency was performed. Although there exist quantitative differences in the excreted amounts of the different metabolites in the three patients the qualitative picture was the same. The excretion of adipic, suberic and sebacic acids was substantial, whereas that of dodecanedioic acid was within or just above control limit. The monounsaturated C6-C10-dicarboxylic acid excretion was only marginally or not increased. 5-OH-hexanoic acid and hexanoylglycine were excreted in excessive amounts, whereas 7-OH-octanoic acid, 9-OH-decanoic acid, octanoylglycine and decanoylglycine were excreted in limited amounts. The excreted amounts of 6-OH-hexanoic, 8-OH-octanoic and 10-OH-decanoic acids were not or only marginally elevated compared to controls. In one of the patients the excretion of ethylmalonic and methylsuccinic acids was enhanced, whereas the excretion of these two acids in the two other patients was comparable to that in controls. The urinary excretion of hexanoic, octanoic, decanoic and dodecanoic acids was just a little above the control limit, whereas the esterified hexanoic and octanoic acids were excreted in appreciable amounts. It is argued that the microsomal omega- and omega-1-oxidation systems are involved in the dicarboxylic and omega-1-OH-monocarboxylic acids formation at C10 and C12 level and that the C8-C6-dicarboxylic and omega-1-OH-monocarboxylic acids are formed from higher chained acids by beta-oxidation in both mitochondria and peroxisomes.