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Biomedical subjects

A C Boyd

Publications and source records attributed to A C Boyd.

At least 19 recordsLinked to original sources
PubMed2004

Percutaneous pulmonary vein isolation for treatment of atrial fibrillation.

BACKGROUND: Transvenous catheter ablation for the treatment of atrial fibrillation is an evolving technique. AIM: The purpose of this study was to identify subgroups of patients most likely to benefit from pulmonary vein electrical isolation. METHODS: Patients with symptomatic atrial fibrillation resistant to pharmacological therapy were studied. Mapping-guided segmental application of radio-frequency energy was used to electrically isolate the pulmonary veins in 74 patients. Ischaemic or dilated cardiomyopathy was present in 34% of patients. Atrial fibrillation had been present for a mean time (+/- standard deviation) of 6.6 +/- 6.1 years. It was paroxysmal in 53 patients (72%). RESULTS: The mean number of procedures was 1.6/patient. After 6 +/- 6 months, 73% of patients (54/74) were in sinus rhythm. Thirteen of those in sinus rhythm were using anti-arrhythmic medications (25%). Recurrence of atrial fibrillation soon after pulmonary vein isolation occurred in 50%. Patients with persistent/permanent atrial fibrillation were less likely to be in sinus rhythm at follow up (11/21 (52%) vs 43/53 (81%); P = 0.01). However, the rate of early recurrence was similar in the intermittent and the persistent/permanent groups (26/53 (49%) vs 11/21(52%), respectively; P-value not significant). Patients with persistent atrial fibrillation were more likely to experience a recurrence of atrial fibril-lation (89%; P = 0.04). No other baseline factors predicted procedural success. Cardiac tamponade occurred in two patients and moderate pulmonary vein stenosis (>50% diameter narrowing) occurred in three patients. CONCLUSIONS: Pulmonary vein isolation is an effective curative treatment for a broad group of patients with atrial fibrillation. However, the procedure is only suitable for patients with problematic atrial fibrillation resistant to other therapies because of the small risk of serious complications.

Adult↗
PubMed2001

Sodium bicarbonate improves the chance of resuscitation after 10 minutes of cardiac arrest in dogs.

The likelihood of successful defibrillation and resuscitation decreases as the duration of cardiac arrest increases. Prolonged cardiac arrest is also associated with the development of acidosis. These experiments were designed to determine whether administration of sodium bicarbonate and/or adrenaline in combination with a brief period of cardiopulmonary resuscitation (CPR) prior to defibrillation would improve the outcome of prolonged cardiac arrest in dogs. Ventricular fibrillation (VF) was induced by a.c. shock in anaesthetised dogs. After 10 min of VF, animals received either immediate defibrillation (followed by treatment with bicarbonate or control) or immediate treatment with bicarbonate or saline (followed by defibrillation). Treatment with bicarbonate was associated with increased rates of restoration of spontaneous circulation. This was achieved with fewer shocks and in a shorter time. Coronary perfusion pressure was significantly higher in NaHCO3-treated animals than in control animals. There were smaller decreases in venous pH in NaHCO3-treated animals than in controls. The best outcome in this study was achieved when defibrillation was delayed for approximately 2 min, during which time NaHCO3 and adrenaline were administered with CPR. The results of the present study indicate that in prolonged arrests bicarbonate therapy and a period of perfusion prior to defibrillation may increase survival.

Animals↗
PubMed2000

Conformationally constrained anesthetic steroids that modulate GABA(A) receptors.

Various cyclic ether and other 3 alpha-hydroxyandrostane derivatives bearing a conformationally constrained hydrogen-bonding moiety were prepared. Their anesthetic potency and their binding affinity for GABA(A) receptors, measured by intravenous administration to mice and inhibition of [(35)S]TBPS binding to rat whole brain membranes, were compared with that of known anesthetic 3 alpha-hydroxypregnan-20-ones. Synthetic steroids with similar in vitro and in vivo activities to the endogenous 3 alpha-hydroxypregnan-20-ones all had an ether oxygen on the beta-face of the steroid D-ring. These results suggest that for optimal GABA(A) receptor modulation, the hydrogen bond-accepting substituent should be near perpendicular to the plane of the D-ring on the beta-face of the steroid.

Androstanols↗
PubMed2000

Impaired ability of glycated insulin to regulate plasma glucose and stimulate glucose transport and metabolism in mouse abdominal muscle.

Previous studies have shown that glycated insulin is secreted from pancreatic beta-cells under conditions of hyperglycaemia. This study has investigated the effects of monoglycated insulin on plasma glucose homeostasis and in vitro cellular glucose transport and metabolism by isolated abdominal muscle of mice. Monoglycated insulin was prepared under hyperglycaemic reducing conditions, purified by RP-HPLC and identified by electrospray ionisation mass spectrometry (5971.1 Da). When administered to mice at an intraperitoneal dose of 7 nmoles/kg body weight, insulin (non-glycated) decreased plasma glucose concentrations and substantially reduced the glycaemic excursion induced by conjoint intraperitoneal injection of 2 g glucose/kg body weight. In comparison, the same dose of monoglycated insulin decreased plasma glucose concentrations to a lesser extent (P < 0.05), corresponding to an approx. 20% reduction of glucose lowering potency. Using isolated abdominal muscle, insulin (10(-9)-10(-7) M) stimulated dose-dependent increases in cellular 2-deoxy-D-[1-3H]glucose uptake, D-[U-14C]glucose oxidation and glycogen production. Monoglycated insulin was approx. 20% less effective than native insulin in stimulating glucose uptake and both indices of metabolism, generally requiring 10-fold greater concentrations to achieve significant stimulatory effects. These data indicate that the impaired biological activity of glycated insulin may contribute to glucose intolerance of diabetes.

Abdominal Muscles↗
PubMed2000

Structure, antihyperglycemic activity and cellular actions of a novel diglycated human insulin.

Human insulin was glycated under hyperglycemic reducing conditions and a novel diglycated form (M(r) 6135.1 Da) was purified by RP-HPLC. Endoproteinase Glu-C digestion combined with mass spectrometry and automated Edman degradation localized glycation to Gly(1) and Phe(1) of the insulin A- and B-chains, respectively. Intraperitoneal (i.p.) administration of diglycated insulin to mice alone or in combination with glucose (7 nmol/kg) resulted in a 43-61% and 11-34% reduction in glucose lowering activity, respectively, compared with native insulin. Consistent with these findings, diglycated insulin (10(-9) to 10(-7) mol/liter) was 22-38% less effective (P < 0.001) than native insulin in stimulating glucose uptake, glucose oxidation and glycogen production in isolated mouse abdominal muscle.

Amino Acids↗
PubMed2000

Genomic sequence analysis of Fugu rubripes CFTR and flanking genes in a 60 kb region conserving synteny with 800 kb of human chromosome 7.

To define control elements that regulate tissue-specific expression of the cystic fibrosis transmembrane regulator (CFTR), we have sequenced 60 kb of genomic DNA from the puffer fish Fugu rubripes (Fugu) that includes the CFTR gene. This region of the Fugu genome shows conservation of synteny with 800-kb sequence of the human genome encompassing the WNT2, CFTR, Z43555, and CBP90 genes. Additionally, the genomic structure of each gene is conserved. In a multiple sequence alignment of human, mouse, and Fugu, the putative WNT2 promoter sequence is shown to contain highly conserved elements that may be transcription factor or other regulatory binding sites. We have found two putative ankyrin repeat-containing genes that flank the CFTR gene. Overall sequence analysis suggests conservation of intron/exon boundaries between Fugu and human CFTR and revealed extensive homology between functional protein domains. However, the immediate 5' regions of human and Fugu CFTR are highly divergent with few conserved sequences apart from those resembling diminished cAMP response elements (CRE) and CAAT box elements. Interestingly, the polymorphic polyT tract located upstream of exon 9 is present in human and Fugu but absent in mouse. Similarly, an intron 1 and intron 9 element common to human and Fugu is absent in mouse. The euryhaline killifish CFTR coding sequence is highly homologous to the Fugu sequence, suggesting that upregulation of CFTR in that species in response to salinity may be regulated transcriptionally.

Amino Acid Sequence↗
PubMed2000

Production and characterization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues.

Previous studies have shown that glycation of insulin occurs in pancreatic beta-cells under conditions of hyperglycaemia and that the site of glycation is the N-terminal Phe(1) of the insulin B-chain. To enable evaluation of glycated insulin in diabetes, specific antibodies were raised in rabbits and guinea-pigs by using two synthetic peptides (A: Phe-Val-Asn-Gln-His-Leu-Cys-Tyr, and B: Phe-Val-Asn-Gln-His-Leu-Tyr-Lys) modified by N-terminal glycation and corresponding closely to the N-terminal sequence of the glycated human insulin B-chain. For immunization, the glycated peptides were conjugated either to keyhole limpet haemocyanin or ovalbumin using glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester or 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. Antibody titration curves, obtained using I(125)-tyrosylated tracer prepared from glycated peptide A, revealed high-titre antisera in five groups of animals immunized for 8-28 weeks. The highest titres were observed in rabbits and guinea-pigs immunized with peptide B coupled to ovalbumin using glutaraldehyde. Under radioimmunoassay conditions, these antisera exhibited effective dose (median) (ED(50)) values for glycated insulin of 0.3-15 ng/ml and 0.9-2.5 ng/ml respectively, with negligible cross-reactivity against insulin or other islet peptides. The degree of cross-reaction with glycated proinsulin was approximately 50%. Glycated insulin in plasma of control and hydrocortisone-treated diabetic rats measured using rabbit 3 antiserum (1:10 000 dilution; sensitivity <19 pg/ml) was 0. 08+/-0.01 and 1.5+/-0.6 ng/ml (P<0.01), corresponding to 4 and 16% of total circulating insulin concentration respectively. Immunocytochemistry studies of the pancreas of streptozotocin-treated diabetic rats using a 1:1000 dilution of guinea-pig 2 antiserum revealed clusters of fluorescent positively stained cells in islets. These studies document the successful production of polyclonal antisera specific for glycated insulin and their usefulness in radioimmunoassays and immunocytochemistry. The demonstration of glycated insulin in plasma and islets of animal models of diabetes supports the view that glycation of insulin is involved in the pathogenesis of this disease.

Animals↗
PubMed1999

Elimination of contaminant Escherichia coli chromosomal DNA from preparations of P1 artificial chromosome recombinants facilitates directed subcloning.

The subcloning of large inserts (>50 kbp) from P1-derived artificial chromosomes (PACs) was found to be hindered by the presence of contaminating Escherichia coli chromosomal fragments which, because of their smaller median size, are recovered preferentially as unwanted subclones. A significant fraction of contaminating DNA was seen to persist after conventional plasmid purification methods. We describe a rigorous protocol for eliminating the bulk of contamination that involves plasmid isolation on commercially available silica-based columns followed by three pulsed field gel electrophoresis steps. Using this, we were able to subclone 55, 85 and 90 kbp PAC inserts but failed to subclone a 195 kbp PAC insert. After surveying a range of DNA purification methods, we devised an optimised protocol that allowed us to subclone the 195 kbp insert. The optimised protocol, which reliably yields DNA with essentially no contaminating material, consists of plasmid isolation on silica-based columns followed by treatment with highly purified DNaseI and retrieval by electroelution of restriction-digested DNA electrophoresed on a single pulsed field gel. By inference it is applicable to the purification of large inserts from other single-copy plasmid vectors such as bacterial artificial chromosomes (BACs).

Chromosomes, Bacterial↗
PubMed1999

pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis.

A modified bacterial artificial chromosome (BAC) vector, pSURF-2, adapted for the selective subcloning of yeast artificial chromosome (YAC) sequences was constructed. DH10B-U, a pyrF derivative of the highly transformable E. coli strain DH10B was also constructed and used for the detection of Ura+ recombinants carrying DNA linked to YAC right arms. The vector's properties were illustrated in two main ways. (i) An intact 25-kb YAC containing a mouse tyrosinase minigene was cloned into pSURF-2. Appropriately spliced tyrosinase RNA was detected by reverse transcription (RT)-PCR in extracts of cells transiently lipofected with the cloned YAC. (ii) Cells expressing human cystic fibrosis transmembrane conductance regulator (CFTR) from an integrated pSURF-2 recombinant containing a cDNA expression cassette were selected using the hygromycin-resistance (HyTK) marker of the vector and characterized by RT-PCR and immunoprecipitation. The unique I-SceI site and HyTK marker of pSURF-2 are designed to facilitate subsequent functional studies of cloned DNA.

Animals↗
PubMed1997

Efficient Cre-lox linearisation of BACs: applications to physical mapping and generation of transgenic animals.

Due to the size of BAC, PAC and P1 clones, it is often difficult to construct detailed restriction maps, with large number of restriction fragments leading to ambiguity of mapping data. We report the use of Cre recombinase to linearise and asymmetrically introduce label at the unique loxP site of large loxP-containing clones. Subsequent partial digestion allows the direct ordering of restriction fragments. Additionally, BAC DNA linearised using the Cre-lox system has been used successfully to generate transgenic animals.

Animals↗
PubMed1997

Anesthetic activity of novel water-soluble 2 beta-morpholinyl steroids and their modulatory effects at GABAA receptors.

(3 alpha,5 alpha)-3-Hydroxypregnan-20-ones and (3 alpha,5 alpha)-3-hydroxypregnane-11,20-diones bearing a 2 beta-morpholinyl substituent were synthesized, and the utility of these steroids as anesthetic agents was evaluated through determination of their potency and duration of hypnotic activity in mice after intravenous administration. Alkylation of the morpholinyl substituent or chlorination at C-21 afforded the novel amino steroids (2 beta,3 alpha,5 alpha)-3-hydroxy-2-(2,2-dimethyl-4-morpholinyl)-pregnane-11,20-dione (19) and (2 beta,3 alpha,5 alpha)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one (37) that were more potent and advantageously produced shorter sleep times than related compounds which were previously reported. Furthermore, salts of these and other amino steroids generally retained good aqueous solubility. In a radioligand binding assay the compounds inhibited the specific binding of [35S]-tert-butyl bicyclophosphorothionate to rat whole brain membranes, and in an electrophysiological assay they potentiated GABAA receptor-mediated currents recorded from voltage-clamped bovine chromaffin cells. These in vitro results are consistent with the anesthetic activity of the amino steroids being related to their modulatory effects at GABAA receptors.

Anesthesia↗
PubMed1997

Glycation of insulin results in reduced biological activity in mice.

Bovine insulin was glycated by in vitro incubation with 20-220 mM D-glucose for 1-48 h. The percentage of glycation was dependent on time, glucose concentration, temperature and pH, attaining values up to 28%. Glucose-lowering activities of glycated and control (non-glycated) insulin preparations were assessed in mice by intraperitoneal injection in a 39% (w/v) glucose solution (2 g/kg body weight) at doses of 0.05 and 0.25 units/kg body weight. Injection of glucose alone significantly (P < 0.001) increased plasma glucose concentrations at 30 min. Simultaneous administration of non-glycated insulin with glucose significantly decreased the 30-min glycaemic excursion (P < 0.001) in a dose-dependent manner. Glycated insulin exhibited a significant reduction (P < 0.001) in glucose-lowering activity under these conditions. The relationship between the extent of insulin glycation and glucose-lowering activity at 0.25 units/kg was assessed using five different insulin preparations glycated between 6%-28%. The insulin-induced decrease in plasma glucose at 30 min was inversely related to the extent of glycation (r = 0.99). Glycated insulin (10(-8) and 10(-6) M) also exhibited a significantly reduced (P < 0.05) ability to stimulate glucose oxidation in isolated mouse diaphragm muscle compared with non-glycated insulin. These data indicate that glycated insulin exhibits impaired biological activity which may contribute to glucose intolerance in diabetes. Further studies are required to determine if glycation of insulin occurs in man and if this process contributes to the pathogenesis of diabetes.

Animals↗
PubMed1995

The pCLIP plasmids: versatile cloning vectors based on the bacteriophage lambda origin of replication.

A series of general-purpose plasmid vectors based on the phage lambda origin of replication (ori) has been constructed. Each vector consists of a backbone plasmid encoding chloramphenicol resistance (CmR) and containing a unique HaeII site into which the lacZ alpha-complementing multiple cloning site (MCS) region of an established vector was inserted. To increase the cloning potential of the inserted MCS, superfluous restriction sites in the backbone were removed by a variety of techniques. The vectors, designated pCLIP (for CmR lambda ori integration proficient) plasmids, are of medium copy number and are compatible with most other vectors in common use. A total of 17 unique restriction sites in pCLIP8, pCLIP9, pCLIP18, pCLIP19 and pCLIP23 are available for cloning. As well as possessing the usual properties of vectors, the pCLIP plasmids are able to integrate reversibly into lambda prophage by homologous recombination. Thus, cloned DNA can be maintained in single or multiple copy at will. By integrating recombinant plasmids into appropriate deletion prophages followed by induction, phage::plasmid hybrids are produced which can be manipulated as phage. These properties are demonstrated using a test recombinant plasmid, pCLIPLEU2. The pCLIP vectors are therefore useful for routine plasmid cloning, complementation analysis and applications where the ability to manipulate recombinants in plasmid, phage or prophage forms is advantageous.

Amino Acid Sequence↗