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Biomedical subjects

I Findlay

Publications and source records attributed to I Findlay.

At least 19 recordsLinked to original sources
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↗
PubMed1994

Interactive regulation of the ATP-sensitive potassium channel of cardiac muscle.

A variety of ligands, including intracellular nucleotides, pharmaceutical products, and neurohormones, influence the adenosine triphosphate (ATP)-sensitive potassium channel of cardiac muscle. In this review it is emphasized that these ligands act not only on the channel-gating mechanism but also influence each others' effects or effectiveness in a quite complex series of interactions. Thus, ATP, which directly closes the channel-gating mechanism, also enhances the efficacy of sulfonylurea drugs and is necessary for the action of certain potassium-channel-opening drugs and neurohormones. Intracellular nucleoside diphosphates that, depending on the conditions, either act on the channel-gating mechanism or influence the effectiveness of ATP, also facilitate and reduce the effectiveness of potassium-channel opening and sulfonylurea drugs, respectively. Our knowledge of the mechanisms that underlie the interactions between these different ligands is limited. The cloning of the channel and the characterization of the different ligand-binding sites will be a great step forward.

Adenosine↗
PubMed1993

Molecular cloning, functional expression and localization of an inward rectifier potassium channel in the mouse brain.

We have cloned an inward-rectifier potassium channel from a mouse brain cDNA library, studied its distribution in the brain by in situ hybridization and determined the chromosomal localization of the gene. A mouse brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. Two duplicate clones of approximately 5.5 kb were obtained. Xenopus ococytes injected with cRNA derived from the clone expressed a potassium channel with inwardly rectifying channel characteristics. The amino acid sequence of the clone was identical to that of IRK1 recently cloned from a mouse macrophage cell line. In situ hybridization study showed the mouse brain IRK1 to be generally distributed throughout the brain, but in particular subsets of neurons at high levels. The gene was placed in the distal region of mouse chromosome 11, which contains several uncloned neurological mutations. These results provide the first demonstration of the cloning and distribution of an inward rectifier potassium channel from the nervous system.

Animals↗
PubMed1993

YM934, a novel K+ channel opener, activates ATP-sensitive K+ channels in cardiac myocytes.

2-(3,4-Dihydro-2,2-dimethyl-6-nitro-2H-1,4-benzoxazin-4-yl)pyridin e N-oxide (YM934) is a newly synthesized benzoxazin. The effects of YM934 on ATP-sensitive K+ (KATP) channels in guinea pig cardiac ventricular myocytes and in an insulin-secreting cell line, HIT T15 beta-cells, were examined using the gigaohm-seal patch-clamp techniques. Under the whole-cell clamp condition, YM934 induced in ventricular myocytes a time-independent, glibenclamide-sensitive K+ current in a concentration-dependent fashion (EC50 = approximately 3 microM). On formation of inside-out patches in ATP-free solution, the KATP channel current abruptly appeared and then ran down. YM934 was applied to inside-out patches before, during and after channel "run-down." Because nucleoside diphosphates, such as uridine diphosphate (UDP), can induce channel openings after complete run-down, the effects of YM934 on the UDP-induced channel openings were also examined. Before run-down, YM934 enhanced KATP channel activity by decreasing the sensitivity of channels to intracellular ATP. YM934 also enhanced the partially run-down channel, even in the absence of ATP. After run-down, YM934 had no effect but could enhance the UDP-induced KATP channel openings. These effects of YM934 on cardiac KATP channels were similar to those of pinacidil and lemakalim. In HIT T15 beta-cells, 100 microM YM934 was ineffective in both cell-attached and inside-out patch configurations, suggesting the tissue-specific nature of the action of this novel K+ channel opener.

Adenosine Triphosphate↗
PubMed1993

Sulphonylurea drugs no longer inhibit ATP-sensitive K+ channels during metabolic stress in cardiac muscle.

The effects of the sulphonlyurea drugs glibenclamide and tolbutamide were tested upon ATP-sensitive K+ channels activated by dinitrophenol or carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) in ventricular myocytes isolated from rat hearts. In whole-cell current recording, 1 microM glibenclamide or 1 mM tolbutamide totally but only transiently inhibited the K+ current activated by dinitrophenol or FCCP. In cell-attached membrane patches, 1 to 100 microM glibenclamide initially inhibited the activation of ATP-sensitive K+ channels induced by dinitrophenol or FCCP, but failed to prevent the activation of these channels during contracture. Myocyte contracture induced by caffeine or Ca++ entry during K+ depolarization did not activate ATP-sensitive K+ channels. In excised membrane patches, 1 to 10 microM glibenclamide did not block completely opening of ATP-sensitive K+ channels. Neither intracellular ADP nor phosphorylable substrate were able to reliably influence the effect of glibenclamide. It is concluded that sulphonylurea drugs, otherwise highly effective blockers of this type of ion channel, were no longer able to inhibit the opening of ATP-sensitive K+ channels during the final stages of metabolic stress. These channels could therefore be responsible for both the glibenclamide-sensitive and glibenclamide-insensitive phases of K+ loss during cardiac ischemia.

Adenosine Triphosphate↗
PubMed1992

Preimplantation diagnosis of a human beta-globin transgene in biopsied trophectoderm cells and blastomeres of the mouse embryo.

The preimplantation diagnosis of a HbSA-globin transgene in biopsied trophectoderm cells and blastomeres in embryos using a transgenic mouse model for the trait of human sickle-cell anaemia has been undertaken. A sensitive procedure was developed for the amplification of the human beta-globin gene sequence flanking the sickle mutation. Polymerase chain reaction (PCR) assays were undertaken on one to five biopsied trophectoderm cells and isolated blastomeres of the preimplantation mouse embryo. After biopsy the blastocysts were cultured whilst the cells were analysed for the presence of the transgene, and a high proportion (82-91%) were viable as assessed by the presence of a blastocoele cavity within a 5-h period. The majority of the biopsied cultured blastocysts were frozen and used to confirm the diagnosis; 90 biopsied cultured blastocysts were transferred to pseudopregnant recipients and 34% established pregnancy. Material from day 13.5 post-coitum fetuses was also used to confirm the original diagnosis. The time (4-5 h) required to carry out the analysis obviates a need for extended culture or cryopreservation of the biopsied embryo. In individual experiments under optimal conditions, the presence of the transgene in biopsied cells was detected with 100% accuracy, and the PCR analysis was sensitive at the 1-cell level. The overall success rate of diagnosis and confirmation of the presence or absence of the human beta-globin sequence in the biopsied embryo was 70%. Over the entire experimental period (14 months) DNA contamination from a variety of sources did occasionally occur; the methods used to overcome this problem are discussed.

Anemia, Sickle Cell↗
PubMed1992

An interactive videodisc 'cancer patients and their families at home', designed for education in primary health care.

This article describes the development of an interactive videodisc program to support professional education in the field of cancer for the primary health care team. Based on a survey of needs, it concentrates on common symptoms and their control, and problems in communication. The educational approach adopted was chosen on the basis of an extensive consideration of the needs, and what was felt most appropriate for adult learners. Plans were carefully reviewed at each stage, with external advisers, and the prototype system independently evaluated in four sites before completing the package. With financial backing from the 'Europe against Cancer' initiative of the European Commission, and close cooperation with national practitioners, Dutch, German, Greek and Portuguese versions of the package have been developed. Local differences in practice are taken into account and translations of key aspects are provided.

Home Care Services↗
PubMed1992

Inhibition of ATP-sensitive K+ channels in cardiac muscle by the sulphonylurea drug glibenclamide.

The effects of the sulphonylurea drug glibenclamide were tested upon ATP-sensitive K+ (KATP) channels activated by 20 microM SR 44866 in isolated ventricular myocytes from guinea pig hearts. Glibenclamide inhibited the openings of KATP channels in both cell-attached and excised inside-out membrane patches. Glibenclamide inhibited the whole-cell KATP current (I-KATP) with an EC50 of 6 nM and Hill Coefficient of 1.26. The kinetics of the onset of inhibition of I-KATP were complex and dose-dependent. The recovery of I-KATP from inhibition was largely dose-independent. The application of glibenclamide for short periods of time was followed by the continued development of inhibition. These results suggest that the cell was loaded with the drug before it interacted with the KATP channel. It is proposed that the sulphonylurea receptor site is to be found in the lipid phase of the membrane. In consequence critical factors involved in the sulphonylurea drug-induced inhibition of KATP channels will include the rates of drug absorption into and exit from the membrane lipid. That sulphonylureas will be concentrated in the membrane lipid suggests that the EC50 value for channel inhibition does not represent the true affinity of the drugs for their receptor.

Adenosine Triphosphate↗