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Biomedical subjects

T Atkinson

Publications and source records attributed to T Atkinson.

At least 91 records · Page 5Linked to original sources
PubMed1990

Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B.

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.

Amino Acid Sequence↗
PubMed1990

The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene.

A 26-mer oligonucleotide probe was synthesized (based on the determined amino acid sequence of the N-terminus of the Clostridium botulinum type A neurotoxin, BoNT/A) and used in Southern blot analysis to construct a restriction map of the region of the clostridial genome encompassing BoNT/A. The detailed information obtained enabled the cloning of the structural gene as three distinct fragments, none of which were capable of directing the expression of a toxic molecule. The central portion was cloned as a 2-kb PvuII-TaqI fragment and the remaining regions of the light chain and heavy chain as a 2.4-kb ScaI-TaqI fragment and a 3.4-kb HpaI-PvuII fragment, respectively. The nucleotide sequence of all three fragments was determined and an open reading frame identified, composed of 1296 codons corresponding to a polypeptide of 149 502 Da. The deduced amino acid sequence exhibited 33% similarity to tetanus toxin, with the most highly conserved regions occurring between the N-termini of the respective heavy chains. Conservation of Cys residues flanking the position at which the toxins are cleaved to yield the heavy chain and light chain allowed the tentative identification of those residues which probably form the disulphide bridges linking the two toxin subfragments.

Amino Acid Sequence↗
PubMed1990

Expression and purification of a truncated recombinant streptococcal protein G.

The gene for Protein G from Streptococcus strain G148 was cloned and expressed in Escherichia coli. The regions on the gene corresponding to the albumin-binding domains and the Fab-binding region were then deleted by site-directed mutagenesis. The translation of regions corresponding to the cell-wall- and membrane-binding domains was prevented by introduction of stop codons upstream of these domains. This recombinant DNA sequence codes for a protein (G') that contains repetitive regions and that binds only the Fc portion of IgG, analogously to Protein A. Translation of the sequence produces a protein with an Mr of about 20,000. The nucleotide sequence differs from those published previously [Guss, Eliasson, Olsson, Uhlén, Frej, Jornvall, Flock & Lindberg (1986) EMBO J. 5, 1567-1575; Olsson, Eliasson, Guss, Nilsson, Hellman, Lindberg & Uhlén (1987) Eur. J. Biochem. 168, 319-324]. The protein can be substantially purified on a large scale by chromatography on IgG-Sepharose 4B. Homogeneous Protein G' can be prepared by anion-exchange f.p.l.c. on Mono Q HR. This Protein G' has a pI of 4.19 and SDS/PAGE gives an apparent anomalous Mr of 35,000.

Amino Acid Sequence↗
PubMed1990

Development and validation of an automated enzyme assay for paracetamol (acetaminophen).

A rapid, enzymatic assay for serum or plasma paracetamol has been developed with the potential for adaptation to a wide range of clinical analysers. The method involves the action of an amidase enzyme to produce 4-aminophenol from paracetamol, which in turn reacts with 8-hydroxyquinoline in the presence of manganese ions to form a blue dye. Two stable reagents are used and excellent precision is achieved over the drug concentration range 0-2.5 mmol/l. The method, which is complete within 6 min, has been validated using a Monarch centrifugal analyser and shows no significant interference from endogenous serum compounds, drugs or paracetamol metabolites.

Acetaminophen↗
PubMed1990

Identification of a reversible structural transition in the metal-depleted glycerol dehydrogenase from Bacillus stearothermophilus.

Evidence is presented to demonstrate that the Zn2+ -depleted, inactive form of the glycerol dehydrogenase from Bacillus stearothermophilus exists in one of two possible conformations in equilibrium, the position of which is temperature sensitive. The conformation of the metal-depleted enzyme favoured by higher temperatures (20-40 degrees C) is able to bind Zn2+ and regain catalytic activity, whereas that favoured at lower temperatures (0-10 degrees C) is unable to bind metal ions and is thus inactive. This equilibrium is also pH dependent with a pK of 6.6. At pH 6.0, the equilibrium lies in favour of the form of the enzyme able to bind metal ions and exhibit activity.

Geobacillus stearothermophilus↗
PubMed1990

Recognition of pertussis toxin by antibodies to synthetic peptides.

Eight synthetic peptides, selected from the amino acid sequence of pertussis toxin (PT) subunits S1, S2, S3 and S4, were assessed for their ability to induce protein-recognizing and neutralizing antibodies. Seven of these peptides, prepared as conjugates of either keyhole limpet haemocyanin or tetanus toxoid, induced significant levels of antibody, all of which reacted with SDS-denatured PT on Western blots. Six of the antibodies bound to PT-coated ELISA plates; this binding was inhibited by homologous peptide antigen. However, none of the antibodies, including those directed against the N-terminus of subunit S1, were able to attenuate in vivo or in vitro toxin-dependent activity. Further investigation revealed that only one antibody, specific for the C-terminus of S1 (peptide Slc, 237-255), could recognize the conformation of native PT in solution. The other five antipeptide antibodies which reacted with PT-coated ELISA plates did not recognize PT when captured onto ELISA plates via either a monoclonal antibody or fetuin, unless the conformation of the toxin had been relaxed by reduction with dithiothreitol. Conversely, the native PT-recognizing response of peptide Slc did not bind the conformationally relaxed PT molecule. From this study, it appears likely that a peptide capable of inducing PT-neutralizing antibody must closely resemble the conformation of the cognate sequence in the native protein.

Amino Acid Sequence↗
PubMed1990

Effect of hirudin on tissue plasminogen activator induced clot lysis.

The effect of recombinant hirudin in the in vitro tPA fibrinolytic and thrombolytic activity was investigated. The activity was evaluated by following lysis of radiolabelled fibrin or plasma clot formed in the presence of tPA alone or with hirudin. The results obtained indicate that increasing concentrations of hirudin had a potentiating effect, with faster clot lysis rates and reduced time to complete lysis. However, when radiolabelled plasma or whole-blood clots were immersed in autologous plasma in the presence of tPA and hirudin, no significant difference in the lysis rates and time to complete lysis was observed. The findings suggest that hirudin or hirudin-thrombin complex interferes with the forming fibrin, thereby making clots more susceptible to lysis, while the presence of hirudin in the surrounding medium during lysis of formed clots helps to rapidly neutralize active thrombin released during clot lysis, thereby preventing further activation of coagulation. Thus, use of hirudin as an anticoagulant during thrombolytic therapy may prove to be helpful in reducing the incidence of reocclusion.

Fibrinolysis↗
PubMed1990

Colorimetric glucose assay using thermostable glucokinase.

A method for assaying glucose in serum or plasma samples using a thermostable glucokinase was developed. Glucokinase from Bacillus Stearothermophilus was coupled with glucose-6-phosphate dehydrogenase to produce NADPH, which reduced the tetrazolium dye MTT to its formazan. Detection of the product at 660 nm allowed samples containing up to 30 mmol/L glucose to be assayed with an endpoint method. Use of the optimal wavelength for formazan detection, 570 nm, increased sensitivity for NADPH detection by over threefold compared to UV detection. The stability of glucokinase assay mixtures was extensively studied, with variation in buffers, salt and enzyme stabilizers. Maximal half life for reagent stability at room temperature was approximately 30 days, with storage of assay mixtures in two solutions. Various drugs and metabolites were tested for interference in the method and no significant interferences were found.

Blood Glucose↗
PubMed1990

Recombinant methionyl bovine prolactin: loss of bioactivity after single amino acid deletions from putative helical regions.

We have previously described a method for producing recombinant methionyl bovine PRL (Met-bPRL), which is as bioactive as the authentic hormone in the Nb2 cell lactogen bioassay; in contrast, a Met-bPRL variant lacking tyrosine 28 was essentially devoid of bioactivity. In the present study we have investigated this loss of bioactivity at the molecular level by determining the bioactivities of a number of Met-bPRL variants engineered to contain specific changes in their primary structures. It was found that the presence of tyrosine per se at the 28 position in Met-bPRL was not essential for high bioactivity, since Met-bPRL variants prepared by replacing tyrosine 28 with other amino acids (arginine, phenylalanine, alanine, and histidine) still had substantial bioactivity (40-74% that of Met-bPRL). Neither was the loss of bioactivity related to a shift in the relative positions of conserved histidines 27 and 30; in fact, histidine 27 was found not to be essential for the bioactivity of the hormone. The loss of bioactivity after deletion of tyrosine 28 from Met-bPRL appears to be related to the removal of an amino acid from the middle of a putative helix (no. 1) rather than to the loss of a residue specific to lactogen function. This suggestion is supported by the finding that Met-bPRL variants obtained by deletion of selected single amino acids from center domains of putative helix 2, 3, or 4 were also essentially devoid of bioactivity. It is speculated that this lack of bioactivity reflects an inability of the proteins to assume a native conformation.

Amino Acid Sequence↗
PubMed1990

Dopamine-beta-hydroxylase deficiency in humans.

We report a 42-year-old man with dopamine-beta-hydroxylase deficiency, an autonomic disorder characterized by lifelong severe orthostatic hypotension, ptosis, nasal stuffiness, hyperextensible joints, and retrograde ejaculation. There is isolated deficiency of norepinephrine in both central and peripheral neurons, which contain and release dopamine instead. Dopamine-beta-hydroxylase deficiency should be suspected also in infants presenting with delayed eye opening, hypoglycemia, hypothermia, or hypotension. It can be diagnosed definitively by assay of plasma norepinephrine and dopamine.

Adult↗
PubMed1990

Development and validation of an automated, enzyme-mediated colorimetric assay of salicylate in serum.

This salicylate-specific assay can be adapted for use with most discrete analyzers, for rapid emergency or routine testing with small serum or plasma sample volumes and a single calibration. The basis of this method is as follows: salicylate monooxygenase (EC 1.14.13.1) converts salicylate to catechol in the presence of NADH; the catechol then reacts with 4-aminophenazone under alkaline conditions, catalyzed by manganese ions, to produce a red dye. Incorporation of an NADH-regenerating system, involving glucose and glucose dehydrogenase, into the enzyme reagent ensures that the working reagent is stable for more than two weeks. The standard curve is linear over the drug concentration range 0 to 5 mmol/L. The CV was less than 4% over 20 days. Results correlated well with those by the Trinder colorimetric method and an HPLC method. We saw no interference by any of 80 drugs we tested at therapeutic concentrations or by endogenous compounds in serum.

Autoanalysis↗
PubMed1989

Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD.

A reactive bis-dichloro derivative of the Procion dye Green HE-4BD was shown to inactivate irreversibly methionyl-tRNA synthetase (MTS) from Escherichia coli and also tryptophyl-tRNA synthetase (WTS) and tyrosyl-tRNA synthetase (YTS) from Bacillus stearothermophilus at pH 8.5 and 37 degrees C. At a 5-fold excess of reactive dye over enzyme subunit concentration MTS was quantitatively inactivated within 20 min in the ATP/pyrophosphate exchange assay, whereas WTS and YTS show an 80% loss of activity over the same time period. The inactivation is affected by the addition of substrates, which either protect (WTS and YTS) or promote (YTS with tyrosine) the dye-mediated enzyme inactivation. Green HE-4BD-OH was shown to be a competitive inhibitor of MTS with respect to MgATP, methionine and tRNA substrates.

Adenosine Triphosphate↗
PubMed1989

Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus.

A protocol for the rapid purification of the glycerol dehydrogenase (glycerol: NAD+ 2-oxidoreductase, EC 1.1.1.6) from the thermophile Bacillus stearothermophilus has been developed using a combination of chromatographic techniques including affinity chromatography on a Sepharose-immobilised triazine dye (Procion red, HE3B, ICI). Substrate specificity has been examined and Km values determined. The protein has been shown to have an oligomeric Mr of approx. 180,000 and consists of four identical subunits of Mr 42,000. Exposure to chelating agents (e.g., EDTA) leads to total loss of activity; the EDTA-inactivated enzyme can be reactivated by Zn2+ and requires 1 mol equivalent of zinc per subunit for full catalytic activity. Other divalent cations such as Cd2+ and Co2+ will reactivate the apo-enzyme but yields an enzyme of lower specific activity. The enzyme binds 1 equivalent of NADH per subunit and during catalysis transfers the 4-pro-R hydride from the nicotinamide ring of the reduced-coenzyme to the substrate. Glycerol increases the dissociation constant for the interaction between NADH and Zn-metallo-glycerol dehydrogenase (ZnGDH) but has no effect on the equilibrium between NADH and metal-depleted enzyme.

Amino Acids↗
PubMed1989

A comparison of the effects of various stimulatory agents on t-PA secretion by normal and malignant cell lines.

Succinyl con A and acetyl con A both stimulated epithelial cells to produce similar yields of tissue plasminogen activator (t-PA) to those previously obtained with native con A. However, unlike con A, the derivatized lectins did not adversely affect cell morphology and viability, and cells treated with succinyl con A could secrete t-PA for a prolonged period. Con A and the two derivatives produced similar morphological effects in Bowes melanoma cells, but t-PA production was not increased. Elevated cyclic nucleotide concentrations did not affect t-PA production from epithelial cells, but calcium ionophore treatment generated t-PA yields similar to those obtained with lectins. Azacytidine, which enhanced t-PA production from epithelial cells, did not increase yields from Bowes melanoma cells, and also sodium butyrate, reported to increase t-PA yields from human endothelial cells, had no effect on either cell line.

Azacitidine↗