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Biomedical subjects

I Findlay

Publications and source records attributed to I Findlay.

At least 37 records · Page 2Linked to original sources
PubMed1998

Comparison of FISH PRINS, and conventional and fluorescent PCR for single-cell sexing: suitability for preimplantation genetic diagnosis.

PURPOSE: Although conventional polymerase chain reaction (PCR) was the first method used for sexing in preimplantation genetic diagnosis, fluorescent in situ hybridization (FISH) has become the method of choice. Recently two new techniques, primed in situ synthesis (PRINS) and fluorescent PCR, have been developed. This study compares the reliability and accuracy of these four techniques in single cells. RESULTS: In buccal cells, fluorescent PCR and FISH had similar reliability (94 and 93%) and accuracy (97 and 96%) rates. The reliability and accuracy of PRINS (91 and 25%) and conventional PCR (79 and 89%) were lower. In human blastomeres, FISH and fluorescent PCR had similar reliability (100%, 717; 95%, 190/201) rates. Accuracy rates were 71% (517) and 99% (188/190) for FISH and fluorescent PCR, respectively, however, too few blastomeres were analyzed by FISH for meaningful comparison. However, when these data are compared with published data, the method of choice for blastomere sexing appears to be fluorescent PCR. CONCLUSIONS: Flouroscent PCR has major implications for PGD.

Biotin↗
PubMed1998

Rapid determination of trisomy 18 parental origin using fluorescent PCR and small tandem repeat markers: case reports.

Trisomy 18 is the second most common genetic defect after trisomy 21, almost 90% of which are due to additional chromosome from the mother. The parental origin of the additional chromosome can, if required, be determined by two methods: karyotyping, which takes several weeks; or, more recently, by polymerase chain reaction (PCR) which is often problematic. Fluorescent PCR of small tandem repeats (STRs) can determine the parental origin in the majority of cases within 5 h. Although the incidence of paternal origin is known for both trisomy 21 and trisomy 18, this technique can rapidly determine the parental origin in cases where there is insufficient samples to perform conventional tests. Determining parental origin by these methods may also have clinical significance in the diagnosis of chromosomal translocations or in the diagnosis of genetic disease using linkage analysis.

Chromosomes, Human, Pair 18↗
PubMed1998

Prenatal detection of trisomy 21 and 18 from amniotic fluid by quantitative fluorescent polymerase chain reaction.

Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis on amniotic fluid. This requires lengthy laboratory procedures and high costs, and is unsuitable for large scale screening of pregnant women. An alternative method, which is both rapid and inexpensive and suitable for diagnosing trisomies even from single fetal cells, is the fluorescent polymerase chain reaction using polymorphic small tandem repeats (STRs). In this paper we present the preliminary results of a larger study comparing parallel prenatal diagnoses of trisomies 21 and 18 using cytogenetics with quantitative fluorescent polymerase chain reaction using STR markers. The results obtained by the two techniques were concordant in all cases. This is the first study reporting significant numbers of prenatal diagnoses using the quantitative fluorescent polymerase chain reaction. We believe that further studies on greater numbers of samples will determine the absolute reliability of this technique. These results also provide a model for diagnosis of trisomy from single fetal cells isolated from maternal blood.

Amniocentesis↗
PubMed1998

Same day diagnosis of Down's syndrome and sex in single cells using multiplex fluorescent PCR.

The major reason for prenatal diagnosis lies in the detection of trisomies, particularly trisomy 21 (Down's syndrome). Current techniques require lengthy laboratory procedures and high costs. Furthermore, diagnosis is often not possible if the sample is of small size or is contaminated. An alternative method, quantitative fluorescent polymerase chain reaction (PCR) of short tandem repeats (STRs), can also be used to diagnose trisomies and it has the advantage that a result is obtained within five to eight hours. However, this method is currently limited to relatively large amounts of sample, which restricts diagnostic confidence and value. Recently, genetic diagnosis using fluorescent PCR has been applied at the single cell level but is limited to sex or single gene defect diagnosis. This study, using quantitative multiplex fluorescent PCR, provides for the first time simultaneous diagnosis and confirmation of sex and trisomy in single cells. Two markers for chromosome 21 increase diagnostic confidence, informativeness, and confirmation. This system is rapid (five hours), reliable, and accurate and we believe that it will be more cost effective than alternative methods. The technique has direct application to preimplantation genetic diagnosis, early prenatal diagnosis, and other diagnostic systems where sample size is limited.

Down Syndrome↗
PubMed1998

Prenatal detection of trisomy 13 from amniotic fluid by quantitative fluorescent polymerase chain reaction.

Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. However, this requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. An alternative method, which is rapid, inexpensive and suitable for diagnosing trisomies, even from single fetal cells, is the fluorescent polymerase chain reaction (PCR) using polymorphic small tandem repeats (STRs). In this paper, we present the method of rapid prenatal detection of trisomy 13 from amniotic fluid using fluorescent PCR and two highly polymorphic STRs (D13S258 and D13S631). The results obtained by quantitative fluorescent PCR amplification of fetal DNA were concordant with amniocyte karyotyping results in all cases. Two cases of trisomy 13 were detected from 212 amniotic fluids and the results obtained from D13S631 and D13S258 amplification are presented. In the first trisomy 13 case, a triallelic pattern was detected by both markers, and in the second case, D13 markers showed a characteristic 2:1 dosage allele ratio, both of which demonstrate trisomy 13 status. All other heterozygous disomic samples showed an allele intensity ratio of 1:1.

Amniocentesis↗
PubMed1997

Contrasting effects of intracellular redox couples on the regulation of maxi-K channels in isolated myocytes from rabbit pulmonary artery.

1. The effects of intracellular redox couples were investigated on the activation by voltage, Ca2+ and NS 1619 of maxi-K channels in enzymatically isolated smooth muscle cells from large pulmonary arteries of rabbits. 2. In inside-out membrane patches, maxi-K channels were characterized by a single-channel conductance of 266 pS in symmetrical 140 mM KCl solutions. The relationship between the open-state probability (Po) and the membrane potential could be fitted to the Boltzmann equation. The activating action of intracellular Ca2+ was reversible, concentration dependent, and was manifested as the reduction in the voltage necessary to half-activate the channel (V1/2) with no change in the slope factor. NS 1619 also predisposed the maxi-K channel to open at more hyperpolarized membrane potentials. 3. The oxidizing agent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB, 1 mM) activated maxi-K channels by inducing a negative shift of the activity-voltage curve, while the reducing agent 2-hydroxy-1-ethanethiol (beta-mercaptoethanol) (BME, 1 mM) had no effect. DTNB increased the efficacy of Ca2+ in activating maxi-K channels. The action of DTNB was not reversible upon wash-out, but could be counteracted by BME. 4. Maxi-K channel activity was unaffected by other oxidizing agents, such as NAD (2 mM) and glutathione disulphide (GSSG, 5 mM), or by their reduced forms (NADH and GSH). Mg-ATP (0.1 and 1 mM) increased the channel activity in a dose-dependent manner, while guanine nucleotides (100 microM GTP gamma S, 500 microM GDP and 200 microM GDP beta S) had no effect. 5. Our data suggest that a change in the intracellular redox state, which would be expected during acute hypoxia, does not alter the activity of maxi-K channels of large pulmonary artery smooth muscle cells. The sulfhydryl-specific redox reagents (DTNB and BME) must act through another regulatory mechanism.

Animals↗
PubMed1996

Fluorescent PCR: a new technique for PGD of sex and single-gene defects.

BACKGROUND: For single-cell diagnosis, particularly preimplantation genetic diagnosis to be successful four main criteria must be achieved: sensitivity, reliability, accuracy, and identification/elimination of contamination. METHODS AND RESULTS: Fluorescent PCR achieves all four necessary criteria and, in addition, currently allows genes on up to nine chromosomes to be simultaneously investigated. Fluorescent PCR has high sensitivity (approximately 1000 x conventional analysis systems), high reliability (97%), and high accuracy (97%) rates for both sex and CF diagnosis in single somatic cells. The low detection threshold allows allelic dropout (one of the main causes of misdiagnosis) to be easily distinguished from PCR phenomena such as preferential amplification. High reliability (90%) and accuracy (97-100%) have been achieved in sex and CF diagnosis in human blastomeres. Fluorescent PCR can also be used to DNA fingerprint (STR profiling) single cells to identify the source/origin of the cell and determine if contamination has occurred. CONCLUSIONS: Fluorescent PCR is therefore a suitable method for PGD.

Alleles↗
PubMed1996

The Total Ischaemic Burden European Trial (TIBET). Effects of atenolol, nifedipine SR and their combination on the exercise test and the total ischaemic burden in 608 patients with stable angina. The TIBET Study Group.

OBJECTIVES: To determine the effects of atenolol, nifedipine and their combination on exercise parameters and ambulatory ischaemic activity in patients with mild chronic stable angina. SETTING: Multicentre, multinational study involving 608 patients from 69 centres in nine countries. DESIGN: Placebo washout followed by double-blind parallel-group study comparing atenolol 50 mg bd, nifedipine SR 20 mg bd, and their combination. Patients underwent maximal exercise testing using either a bicycle (n = 289) or treadmill (n = 319) and 48 h of ambulatory ST segment monitoring outside the hospital environment at the end of the placebo washout period and after 6 weeks of active therapy. RESULTS: Both medications alone and in combination caused significant improvements in exercise parameters and significant reductions in ischaemic activity during daily activities, when compared with placebo. There were, however, no significant differences between groups, for any of the measured ischaemic parameters although combination therapy resulted in a greater fall in resting systolic and diastolic blood pressure than either treatment alone. CONCLUSIONS: In the management of mild chronic stable angina there appears to be little advantage gained from using combination therapy for ischaemia reduction.

Adrenergic beta-Antagonists↗
PubMed1996

Rapid genetic diagnosis at 7-9 weeks gestation: diagnosis of sex, single gene defects and DNA fingerprint from coelomic samples.

Prenatal diagnosis (chorionic villus sampling (CVS) or amniocentesis) is performed at a relatively late stage of pregnancy (11-18 weeks). Such tests have significant disadvantages including increased risk of miscarriage and delay before results are known. Earlier prenatal diagnosis (< 11 weeks) has been discontinued because of the risk of fetal abnormalities. Recently fetal cells have been recovered from the coelomic cavity at 7-12 weeks gestation (coelocentesis). This study has established that highly sensitive fluorescent polymerase chain reaction (PCR) can provide rapid (4-5 h), reliable and accurate multiple genetic diagnoses (sexing and single-gene diagnosis) from coelomic cells. As prenatal diagnosis has a significant risk of contamination, we have also shown that coelomic cells can be simultaneously DNA fingerprinted to determine that contamination has not occurred. This earlier method of prenatal diagnosis would be very valuable, as it may overcome some problems of later conventional prenatal diagnosis and allow reassurance/treatment to be undertaken at a much earlier stage. Successful application of these techniques may supersede alternative methods of prenatal diagnosis. Although these techniques appear very promising, extensive clinical trials must be undertaken to determine safety of coelocentesis, diagnostic reliability and accuracy in a clinical setting.

Adult↗
PubMed1995

Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis.

Previously the diagnosis of sex and cystic fibrosis status has been studied on single cells using the polymerase chain reaction (PCR). It has been suggested that allelic drop-out (PCR failure of one allele) and/or preferential amplification (hypo-amplification of one allele) may contribute to poor reliability and misdiagnosis, although this remains controversial as some reports suggest that allelic drop-out does not occur. We investigated an improved method of diagnosing sex and cystic fibrosis in single cells using a new technology (fluorescent PCR) to determine the base level of PCR artefacts (allelic drop-out and preferential amplification) which, in combination with improved sensitivity, should improve PCR reliability and accuracy. Fluorescent PCR gives high reliability (approximately 97%) and accuracy rates (approximately 97%) in somatic cells for both sex and cystic fibrosis diagnosis and its lower detection threshold allows allelic drop-out and preferential amplification to be easily distinguished. We also achieved high reliability and accuracy in diagnosing cystic fibrosis in human blastomeres. This study confirms earlier reports of both allelic drop-out and preferential amplification in single cell analysis. We demonstrate that both allelic drop-out and preferential amplification occur in somatic cells and suggest these are separate phenomena. Preferential amplification appeared common in single cell PCR while allelic drop-out apparently occurred at random in each allele. Preferential amplification was mainly amplification of the larger allele. We suggest that some inaccuracy/misdiagnosis may be due to both preferential amplification as well as allelic drop-out. Other findings were variability in drop-out between PCR and that amplification of signals from human blastomeres may be linked to embryo quality. We suggest that allelic drop-out is dependent on the number of cells within the sample.

Alleles↗
PubMed1995

Simultaneous DNA 'fingerprinting', diagnosis of sex and single-gene defect status from single cells.

Sex and cystic fibrosis status have been previously diagnosed separately at the single cell level. We have developed a sensitive, reliable, accurate and rapid (within 5-6 h) system for the simultaneous diagnosis of sex, cystic fibrosis and a DNA 'fingerprint' within a single reaction from a variety of single cells. As contamination cannot be totally excluded, particularly at the single cell level, DNA 'fingerprinting' can be used to assess the risk of contamination. High sensitivity with single cells is combined with very high specificity (estimated matching probability of 10(-7)-10(-8)), allowing the source of the amplified cell to be identified with a very high degree of probability. Fluorescent primers were multiplexed for six tetranucleotide microsatellite sequences to determine the DNA fingerprint; the amelogenin gene was used to diagnose sex, and primers for the CFTR region were used to determine cystic fibrosis (CF) status. Analysis of the fluorescent product was undertaken using an automated DNA sequencer with Genescan software. This technique has many applications such as prenatal and preimplantation diagnosis, forensic identification of small or degraded samples, and detection of contamination sources. DNA fingerprints of single haploid spermatozoa and other cells can be assessed, so ensuring the detection of both diploid and haploid contamination during preimplantation diagnosis.

Cystic Fibrosis↗
PubMed1994

Molecular cloning and functional expression of cDNA encoding a second class of inward rectifier potassium channels in the mouse brain.

We have cloned a second class of inward rectifier potassium channels, designated MB-IRK2, from a mouse brain cDNA library. The amino acid sequence of this clone shares 70% identity with the mouse IRK1. Xenopus oocytes injected with cRNA derived from MB-IRK2 expressed a K+ current, which showed inward rectifying channel characteristics similar to the MB-IRK1 current. In contrast to the MB-IRK1 current, however, the MB-IRK2 current exhibited significant inactivation during hyperpolarizing pulses. In patch clamp experiments with 140 mM K+ in the pipette, the single channel conductance of MB-IRK2 was 34.2 +/- 2.1 picosiemens (n = 5), a value significantly larger than that of MB-IRK1 (22.2 +/- 3.0 picosiemens, n = 5). Consistent with the whole cell current, the steady-state open probability (Po) of the MB-IRK2 channel decreased with hyperpolarization, whereas that of the MB-IRK1 remained constant. Northern blot analysis revealed the mRNA for MB-IRK2 to be expressed in forebrain, cerebellum, heart, kidney, and skeletal muscle. In the brain, the abundance of mRNA for MB-IRK2 was much higher in cerebellum than in forebrain and vice versa in the case of MB-IRK1. These results demonstrate that the IRK family is composed of multiple genes, which may play heterogenous functional roles in various organs, including the central nervous system.

Amino Acid Sequence↗
PubMed1994

Dualistic behavior of ATP-sensitive K+ channels toward intracellular nucleoside diphosphates.

ATP-sensitive K+ (KATP) channels are intracellular ligand-gated channels which regulate diverse cellular functions. Intracellular nucleoside diphosphates (NDPs) are essential for the physiological opening of KATP channels which would otherwise be permanently closed by their overt sensitivity to intracellular ATP. We find that KATP channels exhibit dualistic behavior toward NDPs depending on their operative condition. When channels are in the spontaneous operative condition, NDPs antagonize channel inhibition by intracellular ATP. When channels have "run down", NDPs induce channel opening but no longer antagonize intracellular ATP. The switch of the KATP channel response to the same ligand, i.e., NDPs, is controlled by a Mg-ATP-dependent reaction. The condition of the target protein therefore determines the effect of the ligand. This property provides a novel basis to evaluate the dynamic regulation of ion channels by their ligands.

Adenosine Triphosphate↗