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Biomedical subjects

T McNally

Publications and source records attributed to T McNally.

34 records · Page 2Linked to original sources
PubMed1994

The Met repressor-operator complex: DNA recognition by beta-strands.

The crystal structure of the E. coli met repressor in complex with a synthetic 19-base pair oligonucleotide reveals two dimeric repressor molecules bound to adjacent sites on the DNA. The oligonucleotide contains two adjacent repeats of an 8-mer known as a met-box, which represents the consensus of the met operator sites. Each met repressor dimer is centered on a met box and interacts with the adjacent dimer through antiparallel alpha-helices, which explained the observed cooperative nature of the binding. DNA binding takes place through the insertion of a beta-ribbon into the major groove of B-form DNA, representing a novel DNA binding motif. Sequence specificity arises from direct interactions between side chains of the beta-strands and the edges of the bases in the major groove. The local DNA conformation confers additional specificity through interactions between protein and the phosphate backbone. The repressor is activated through binding of S-adenosyl methionine (SAM), the corepressor, to the face opposite to that used for DNA binding. The lack of significant conformational change upon SAM binding, together with electrostatic calculations, suggests that DNA binding enhancement occurs through long-range electrostatic interactions.

Bacterial Proteins↗
PubMed1994

Heparin clearance during continuous veno-venous haemofiltration.

OBJECTIVE: To determine whether premature clotting of haemofiltration circuits could be related to heparin removal across the filter membrane into the ultrafiltrate. DESIGN: Randomised study using either unfractionated (n = 8) or low molecular weight (n = 7) heparin for anticoagulation of the haemofiltration circuit at 1000 and 600 U/h respectively. Samples were drawn at 1 and 2 h from arterial and venous limbs of the haemofilter circuit for measurement of plasma heparin (as anti-Factor Xa activity), antithrombin III and haematocrit. Ultrafiltrate samples were collected at the same time for measurement of anti-Xa activity. SETTING: Intensive care unit. PATIENTS: Patients in acute renal failure requiring haemofiltration. RESULTS: Both unfractionated and low molecular weight heparin plasma levels were within the range required for therapeutic anticoagulation in all but one patient at 2 h. Ultrafiltrate anti-Xa levels were insignificant. Antithrombin III levels in these critically ill patients were subnormal in 11 of the 15 studies. CONCLUSIONS: Despite their small sizes, neither unfractionated nor low molecular weight heparins cross the haemofilter membrane into the ultrafiltrate in any measurable quantity. Both heparins were present in plasma at a level suitable for therapeutic anticoagulation. Subnormal levels of antithrombin III may be an important factor in determining filter longevity.

Acute Kidney Injury↗
PubMed1994

Beta 2 glycoprotein-I antigen is increased in primary hyperlipidaemia.

In order to determine whether elevated levels of beta 2 glycoprotein-I (beta 2GPI) are associated with increased plasma lipids, we measured plasma beta 2GPI antigen levels in 47 patients with primary hyperlipidaemia (20 severe hypercholesterolaemia, nine severe hypertriglyceridaemia, and 18 mixed hyperlipidaemia) and 34 normal healthy subjects. Mean beta 2GPI levels were significantly increased in each patient group (302.3, 272.9 and 299.1 mg/l, respectively) compared to controls (199.6 mg/l) (P < 0.01). Significant correlations were demonstrated between beta 2GPI levels and triglyceride and total cholesterol levels in the control group (r = 0.387, r = 0.559; P < 0.05), but were not observed in all patient groups. These results indicate that beta 2GPI is increased in hyperlipidaemia and that its distribution between plasma lipid fractions is perturbed. Plasma lipid levels should therefore be considered when interpreting results of beta 2GPI antigen assays.

Adult↗
PubMed1994

The Taipan snake venom time: a new test for lupus anticoagulant.

AIMS: To develop a specific test for lupus anticoagulant activity with reduced sensitivity to coagulation factor deficiency that would be suitable for analysis of plasmas from patients receiving oral anticoagulants. METHODS: A coagulation test based on the Taipan snake venom time (TSVT) with a platelet neutralisation procedure (PNP) was developed and compared with dilute Russell's viper venom time (DRVVT). The TSVT was used to test plasmas from patients receiving oral anticoagulant or heparin with mild liver dysfunction and with documented lupus anticoagulant. RESULTS: The optimised conditions for the TSVT were established and a reference range was determined in normal healthy subjects. Results were considered positive for lupus anticoagulant if the ratio was > or = 1.1 and was reduced by > or = 10% or to < 1.1 in the PNP. In 43 samples from patients receiving oral anticoagulants there was no correlation between level of anticoagulation and TSVT, and only seven samples had increased TSVTs. Of these, five corrected on mixing with normal plasma and two gave equivocal results. The patients with mild liver dysfunction all had normal TSVTs. The TSVT in plasmas from patients receiving heparin correlated with the heparin concentrations (as measured by the APTT, r2 = 0.81). Some anticoagulated plasmas showed correction in the PNP and were regarded as false positive. Fourteen of 17 patients known to have lupus anticoagulant (on the basis of DRVVT results) were also positive by the TSVT; two of the remaining three were borderline and one was negative. CONCLUSIONS: The TSVT showed satisfactory intra-assay precision and reasonable sensitivity to lupus anticoagulant, compared with the DRVVT. The TSVT was influenced by the presence of heparin but was not sensitive to the effects of oral anticoagulant. Like other lupus anticoagulant tests, it does not seem to have a 100% detection rate, but this may be due to the presence of lupus anticoagulant subtypes with distinct activities or the requirement of cofactors other than prothrombin or beta 2 glycoprotein-I.

Adult↗
PubMed1994

The interaction of beta 2 glycoprotein-I and heparin and its effect on beta 2 glycoprotein-I antiphospholipid antibody cofactor function in plasma.

beta 2 glycoprotein-I (beta 2GPI), a cofactor for antiphospholipid antibody (aPA) binding, binds to many anionic macromolecules including heparin. The nature of this interaction with heparin is not well understood and its effect on the purported biological functions of beta 2GPI is unknown. We have examined the interactions of dermatan sulphate (DS) and different pharmaceutical preparations of heparin with beta 2GPI by crossed immunoelectrophoresis (CIE) and investigated the effect of these agents on plasma levels of beta 2GPI antigen (beta 2GPI:Ag) by a standardised enzyme linked immunosorbent assay (ELISA). beta 2GPI aPA cofactor activity (beta 2GPI:Cof) was also measured using a modified solid phase anti-phosphatidylserine (aPS) ELISA. CIE results confirmed a heparin-beta 2GPI interaction with unfractionated (UF) heparin. beta 2GPI:Ag levels were unaffected by any of the preparations investigated. There were no significant differences in beta 2GPI:Cof activities of the samples containing LMW heparins or DS but levels of beta 2GPI:Cof were increased in samples containing UF sodium and calcium heparin preparations (0.5 IU/ml Monoparin, p < 0.05, and 10 IU/ml Liquemin and Calciparine, p < 0.05).

Antibodies, Antiphospholipid↗
PubMed1993

Immunoelectrophoresis and ELISA techniques for assay of plasma beta 2 glycoprotein-1 and the influence of plasma lipids.

Beta 2 glycoprotein-1 (beta 2GP1) has been identified as a cofactor for the binding of some antiphospholipid antibodies to anionic phospholipids and has been demonstrated to possess anticoagulant properties in vitro. We have investigated Laurell rocket immunoelectrophoresis (IEP) and ELISA techniques for measurement of beta 2GP1. Western blotting and crossed immunoelectrophoresis (CIE) of plasma demonstrated free beta 2GP1 and beta 2GP1 complexed with unidentified plasma constituents. The free and complexed forms were not distinguished in immunoelectrophoresis assays, allowing measurement of total beta 2GP1. Standard and detergent modified IEP and ELISA techniques were compared: significant correlation was demonstrated between unmodified and detergent modified IEP, detergent modified IEP and ELISA and unmodified IEP and ELISA. The intra-assay co-efficients of variation (CVs) of the unmodified and modified IEPs and ELISA were 7.5, 3.7 and 7.9%. Inter-assay CVs determined for the modified IEP and ELISA were 5.8 and 9.1% respectively. The purified beta 2GP1 used to standardise the assays was shown to have subfraction selectivity and different calibration values were obtained for pooled normal plasma by the unmodified IEP (242 mg/l) and modified IEP and ELISA (201 mg/l). We have also investigated the influence of some pre-test variables on beta 2GP1 levels and shown that heparin, citrate or EDTA plasma and serum samples are suitable for assay of this glycoprotein and that levels are unaffected by repeated freeze/thawing. The influence of plasma lipids on beta 2GP1 measurement was also examined and we demonstrated no significant differences between pre and postprandial samples, which suggests that fasting status is not an important consideration for assay of beta 2GP1 in healthy subjects.

Apolipoproteins↗
PubMed1993

Postoperative multiple organ dysfunction syndrome associated with gut mucosal hypoperfusion, increased neutrophil degranulation and C1-esterase inhibitor depletion.

We have examined the relationship between gut mucosal perfusion, as determined by gastric intramucosal pH (pHi), changes in plasma neutrophil elastase concentrations and components of the contact system during elective major surgery and related these findings to patient outcome. Of the 26 patients studied, 16 developed gut mucosal hypoperfusion (pHi < 7.32) by the end of surgery; four of these developed multiple organ dysfunction syndrome; three of these died. In this group there was a significant increase in neutrophil elastase (P < 0.005) and significant reductions in plasma components of the contact system from immediately before surgery to 24 h later. Ten patients maintained gut mucosal perfusion (pHi > or = 7.32); none of these developed life threatening complications. In this group there was no significant increase in neutrophil elastase and, although there were significant reductions in some plasma components of the contact system, concentrations of C1-esterase inhibitor (the main inhibitor of the contact system) were not significantly reduced. We conclude that gut mucosal hypoperfusion, neutrophil degranulation and activation of the contact system to the extent that C1-esterase inhibitor becomes depleted are associated with a poor outcome after major surgery.

Adult↗
PubMed1992

Probing met repressor-operator recognition in solution.

The three-dimensional crystal structure of the Escherichia coli methionine repressor, MetJ, complexed with a DNA operator fragment is described in an accompanying article. The complex exhibits several novel features of DNA-protein interaction. DNA sequence recognition is achieved largely by hydrogen-bond contacts between the bases and amino-acid side chains located on a beta-ribbon, a mode of recognition previously hypothesized on the basis of modelling of idealized beta-strands and DNA, and mutagenesis of the Salmonella phage P22 repressors Arc and Mnt. The complex comprises a pair of MetJ repressor dimers which bind to adjacent met-box sites on the DNA, and contact each other by means of a pair of antiparallel alpha-helices. Here we assess the importance of these contacts, and also of contacts that would be made between the C-helices of the protein and DNA in a previous model of the complex, by studying mutations aimed at disrupting them. The role of the carboxy-terminal helix face in operator binding was unclear, but we demonstrate that recognition of operator sequences occurs through side chains in the beta-strand motif and that dimer-dimer interactions are required for effective repression.

Base Sequence↗
PubMed1991

Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase.

Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the convenient purification of 15-20 mg enzyme from 11 culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.

Chromatography, Gel↗
PubMed1989

The yeast pyruvate kinase gene does not contain a string of non-preferred codons: revised nucleotide sequence.

The sequence of the gene encoding pyruvate kinase from Saccharomyces cerevisiae was re-determined because of failures with oligonucleotide-directed mutagenesis experiments involving a region thought to contain a string of five contiguous non-preferred codons. This region was found to be difficult to sequence and was shown to have three extra bases when compared with the published sequence [(1983) J. Biol. Chem. 258, 2193-2201]. The revised sequence demonstrates that the yeast pyruvate kinase gene does not have a cluster of non-preferred codons, and that it therefore is not an example of the class of genes which possibly exhibit translational control by the presence of non-preferred codons.

Amino Acid Sequence↗
PubMed1987

Isolation of a repeated DNA sequence from Bordetella pertussis.

A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.

Autoradiography↗
PubMedDate not supplied

Accidental envenoming by a Gaboon viper (Bitis gabonica): the haemostatic disturbances observed and investigation of in vitro haemostatic properties of whole venom.

We report the successful treatment of envenoming by the Gaboon viper (Bitis gabonica) and include results of in vitro investigations of the haemostatic properties of the whole venom. The patient was admitted to casualty soon after the bite with chest tightness, dizziness, nausea and swelling at the site of the bite and was treated immediately with polyspecific antivenom, hydrocortisone, chlorpheniramine and antibiotics. Results of haemostatic investigations were essentially normal on admission but on day 3 the thrombin time became prolonged and was associated with significant hypofibrinogenaemia and elevated D-dimers. Factors V and VIII, antithrombin III and protein C levels and platelet number were not significantly reduced. The haemostatic disturbances persisted for more than 24 h despite treatment with blood products (16 units of cryoprecipitate, 2 units of fresh frozen plasma and 6 units of platelet concentrate). Resolution of the abnormalities occurred only after administration of a further dose of antivenom. The period of hypofibrinogenaemia occurred at a time when venom antigen was undetectable in plasma by enzyme-linked immunosorbent assay. Studies in vitro with whole venom and a panel of amidolytic substrates commonly employed for measurement of haemostatic proteins revealed significant activity of venom with substrates sensitive to kallikrein and plasmin. The venom inhibited washed platelet aggregation induced by collagen, thrombin, arachidonic acid and the calcium ionophore A23187 in a dose-dependent manner.

Adult↗
PubMedDate not supplied

Confusing extrication with immobilization: the inappropriate use of hard spine boards for interhospital transfers.

INTRODUCTION: To determine if air medical interhospital transport of patients with spinal injuries is done with techniques that minimize ischemic skin damage. METHODS: A formal telephone survey instrument was given to all U.S. flight services accredited by the Commission on Accreditation of Medical Transport Systems (CAMTS). RESULTS: Thirty-seven active services were listed by CAMTS; the author's service was excluded from the survey. One service did only scene responses; one was unreachable by phone; four were unwilling to complete the form, leaving 30 services for evaluation. Twenty-nine services used metal, plywood, or plastic "spine" boards for immobilization during interhospital transport. Eight services padded boards with blankets or cloth for patients immobilized for "extended periods." Eighteen services routinely reimmobilized all major trauma patients even if cleared by the sending physician, and four others reimmobilized patients not "cleared" by a radiologist. No service moved patients with known spinal injuries to softer, more conforming devices before transport. Only three services followed patients for complications throughout hospitalization. Two services reported cases of skin breakdown thought to be a result of prolonged immobilization. CONCLUSION: Air medical services often transport patients several hours after injury. Patients, particularly those unable to move because of their injuries, medication, or paralysis, are at risk for ischemic necroses of their skin. Decubitus ulcers are a major cause of morbidity and mortality, and preventing ulcers requires a very soft, conforming surface. Despite these facts, the highly select services surveyed continue to use hard, slippery boards designed for extrication at trauma scenes to immobilize patients for transport.

Accreditation↗