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Biomedical subjects

J Day

Publications and source records attributed to J Day.

At least 73 records · Page 4Linked to original sources
PubMed1995

Structure of an anti-idiotypic Fab against feline peritonitis virus-neutralizing antibody and a comparison with the complexed Fab.

The crystal structure of anti-idiotopic Fab 409.5.3, made against an E2 specific feline infectious peritonitis virus-neutralizing antibody 730.1.4, has been determined in its free from, at 2.9 A resolution by molecular replacement. This antibody, used as an immmunogen, elicits the production of anti-anti-idiotypic antibodies that in turn neutralize the virus. The structure of the uncomplexed Fab was refined using constrained-restrained least squares minimization and simulated annealing in combination with conjugate gradient techniques to a crystallographic R of 0.22 based on 16,482 unique reflections between 20.0 and 2.9 A. The free antiidiotypic Fab shows, when compared to its complexed form, a 5 degrees rotation of its variable light with respect to its variable heavy domain and rearrangement of complementarity determining region loops, which permits optimization of the stereocomplementarity between interacting molecules. This finding supports the induced fit hypothesis for antibody antigen interaction.

Amino Acid Sequence↗
PubMed1995

Developing an immunisation protocol for the primary health care team.

Health visitors based at the Ely Bridge fundholding practice in Cardiff were concerned about the low uptake in child immunisations. The health visitor team describe how they developed a joint immunisation protocol in collaboration with primary health care team colleagues to enable improved uptake in line with the Health of the Nation and GP contract targets.

Child, Preschool↗
PubMed1994

Structures of three crystal forms of the sweet protein thaumatin.

Three crystal forms of the sweet-tasting protein thaumatin from the African berry Thaumatococcus daniellii have been grown. These include two naturally occurring isoforms, A and B, that differ by a single amino acid, and a recombinant form of isoform B expressed in yeast. The crystals are of space groups C2 with a = 117.7, b = 44.9, c = 38.0 A, and beta = 94.0 degrees, P2(1)2(1)2(1) with a = 44.3, b = 63.7 and c = 72.7 A, and a tetragonal form P4(1)2(1)2 with a = b = 58.6 and c = 151.8 A. The structures of all three crystals have been solved by molecular replacement and subsequently refined to R factors of 0.184 for the monoclinic at 2.6 A, 0.165 for the orthorhombic at 1.75 A, and 0.181 for the tetragonal, also at 1.75 A resolution. No solvent was included in the monoclinic crystal while 123 and 105 water molecules were included in the higher resolution orthorhombic and tetragonal structures, respectively. A bound tartrate molecule was also clearly visible in the tetragonal structure. The r.m.s. deviations between molecular structures in the three crystals range from 0.6 to 0.7 A for Calpha atoms, and 1.1 to 1.3 A for all atoms. This is comparable to the r.m.s. deviation between the three structures and the starting model. Nevertheless, several peptide loops show particularly large variations from the initial model.

Journal Article↗
PubMed1994

Characterization of crystals of satellite panicum mosaic virus.

Satellite panicum mosaic virus (SPMV) has been purified from pearl millet and obtained in a variety of different crystal forms, at least two of which appear suitable for high resolution X-ray diffraction analysis. The first is of cubic space group P4(2)32 with a = b = c = 183.1 A and two virus particles in the unit cell. The second is of a primitive orthorhombic space group with a = 166.1 A, b = 266.7 A and c = 269.1 A, with four virus particles in the unit cell. While the cubic crystal has as its asymmetric unit one twelfth of the icosahedron, or five capsid protein subunits, the asymmetric unit of the orthorhombic crystals is an entire particle. The cubic crystals diffract to at least 2.8 A resolution. We have also succeeded in crystallizing, but not yet characterizing the master virus, PMV.

Crystallization↗
PubMed1994

Crystal structure of an idiotype-anti-idiotype Fab complex.

Anti-idiotypic monoclonal antibody 409.5.3 is raised against an antibody that neutralizes feline infectious peritonitis virus. This antibody, used as an immunogen, elicits the production of anti-anti-idiotypic antibodies that in turn neutralize the virus. The crystal structure of the complex between anti-idiotypic Fab 409.5.3 and idiotypic Fab fragment of virus-neutralizing antibody has been solved by molecular replacement using real-space Patterson search and filtering by Patterson correlation-coefficient refinement. The structure has been refined to an R value of 0.21 based on 21,310 unique reflections between 40.0 and 2.9 A. The three-dimensional structure reveals extensive, specific interactions that involve 118 van der Waals contacts and at least 9 probable hydrogen bonds. The two Fabs are rotated 61 degrees with respect to each other around the approximate long axis of the complex and are within 26 degrees being aligned along their major axes.

Amino Acid Sequence↗
PubMed1994

Refined molecular structure of pig pancreatic alpha-amylase at 2.1 A resolution.

The structure of pig pancreatic alpha-amylase has been determined by X-ray diffraction analysis using multiple isomorphous replacement in a crystal of space group P2(1)2(1)2(1) (a = 70.6 A, b = 114.8 A, c = 118.8 A) containing nearly 75% solvent. The structure was refined by simulated annealing and Powell minimization, as monitored by 2Fo-Fc difference Fourier syntheses, to a conventional R of 0.168 at 2.1 A resolution. The final model consists of all 496 amino acid residues, a chloride and a calcium ion, 145 water molecules and an endogenous disaccharide molecule that contiguously links protein molecules related by the 2(1) crystallographic operator along x. The protein is composed of a large domain (amino acid residues 1 to 403) featuring a central alpha ta-barrel of eight parallel strands and connecting helices with a prominent excursion between strand beta 3 and helix alpha 3 (amino acid residues 100 to 168). The final 93 amino acid residues at the carboxyl terminus form a second small domain consisting of a compact Greek key beta-barrel. The domains are tightly associated through hydrophobic interfaces. The beta 3/alpha 3 excursion and portions of the central alpha/beta-barrel provide four protein ligands to the tightly bound Ca ion; three water molecules complete the coordination. The Cl- ion is bound within one end of the alpha/beta-barrel by two arginine residues in a manner suggesting a plausible mechanism for its allosteric activation of the enzyme. A crystalline complex of the pancreatic alpha-amylase with alpha-cyclodextrin, a cyclic substrate analog of six glucose residues, reveals, in difference Fourier maps, three unique binding sites. One of the alpha-cyclodextrin sites is near the center of the long polysaccharide binding cleft that traverses one end of the alpha/beta-barrel, another is at the extreme of this cleft. By symmetry this can also be considered as two half sites located at the extremes of the active site cleft. This latter alpha-cyclodextrin displaces the endogenous disaccharide when it binds and, along with the first sugar ring, delineates the extended starch binding site. The third alpha-cyclodextrin binds at an "accessory site" near the edge of the protein and is quite distant from the polysaccharide binding cleft. Its presence explains the multivalency of alpha-amylase binding to dextrins in solution. The extended active site cleft is formed by large, sweeping, connecting loops at one end of the alpha/beta-barrel. These include three sequence segments that are highly conserved among alpha-amylases.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Effects of breathing a normoxic helium mixture on exercise tolerance of patients with cystic fibrosis.

Breathing helium-oxygen (He-O2) mixtures of 20.9% O2/79.1% He has been shown to increase exercise ventilation and peak oxygen uptake in healthy subjects. The improved exercise performance is thought to be due to the reduced density of He-O2 compared to air and the resulting increases in ventilation. Patients with cystic fibrosis (CF) frequently have abnormal pulmonary function test results, low exercise ventilations and diminished exercise tolerance. This led to the hypothesis that in CF the exercise tolerance of patients might improve when breathing He-O2. To test this hypothesis, 11 patients with CF or mild to severe airway obstruction performed spirometry and progressive maximal exercise tests while breathing air or He-O2. The He-O2 mixture significantly increased (P < 0.05) forced expiratory volume in 1 sec (FEV1) by 8.2%, peak expired flow by 39%, and maximal voluntary ventilation (MVV) by 17.9% compared to air, while forced vital capacity (FVC) and forced mid-expiratory flow rate (FEF25-75%) were unchanged by breathing He-O2. Ventilation and oxygen uptake at matched submaximal power outputs were not increased while breathing He-O2, nor were peak exercise ventilation (VEpeak) or peak exercise oxygen uptake (VO2peak). Estimated hemoglobin saturation and total exercise time were also unchanged during He-O2 breathing. However, there was a trend for the subjects with the better FEV1 to increase VO2peak. Increases in VO2peak when breathing He-O2 and air were correlated (r = 0.67, P < 0.05) with the percent of predicted FEV1 values. Still, in the 11 patients as a group, breathing He-O2 did not significantly improve VO2peak, VEpeak, or exercise tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Giant infectious intracavernous carotid artery aneurysm presenting as intractable epistaxis.

Infectious intracavernous carotid artery aneurysms usually present with ophthalmoplegia and/or signs of cavernous sinus thrombosis. We report an unusual case in which a patient with AIDS presented with intractable epistaxis secondary to rupture of a giant infectious intracavernous carotid artery aneurysm. Culture of the aneurysm grew mycobacterium avium intracellulare (MAI). The patient was treated successfully by excision of the aneurysm and reconstruction of the internal carotid artery with a saphenous vein interposition graft.

AIDS-Related Opportunistic Infections↗
PubMed1994

Screening for non-insulin-dependent diabetes mellitus (NIDDM): how often should it be performed?

OBJECTIVES: To examine whether a screening programme for diabetes repeated after an interval of 30 months is worthwhile both in terms of yield of new cases and continued high response rate. METHODS: Self testing for postprandial glycosuria was used as it has been shown to have a good response rate, a good yield of cases of diabetes, and a sensitivity and specificity which compares favourably with more expensive and invasive screening methods. A total of 3231 subjects aged 45-70 years in one practice were screened on two occasions 30 months apart. Eighty seven subjects known to have diabetes were excluded. This number included five new patients who had presented since the initial screen; two of whom had moved into the practice, one who screened positive at the initial screen but failed to attend for the oral glucose tolerance test (OGTT), and two subjects who had screened negative, the first presenting 24 months after screening. RESULTS: At repeat screening the return rate was lower than at the first screening (72.5% v 79.2%, P < 0.0001). Glycosuria was detected in 52 subjects (2.3%), at the repeat screening, similar to that at the initial screen. Attendance for the OGTT after a positive screening test was 93.2%. Only six subjects of the 24 with glycosuria but normal glucose tolerance at initial screen were found to have glycosuria again; repeat OGTTs were all normal. Of the remaining 46 subjects, 10 had non-insulin-dependent diabetes mellitus (NIDDM) and five impaired glucose tolerance (IGT). The number of subjects with diabetes was not significantly different from that at the initial screening (0.44% v 0.72%, P = 0.2). CONCLUSIONS: Repeat screening after 30 months has a high response rate, similar rate of detection of glycosuria, and a further yield of 0.44% of newly diagnosed cases of diabetes. A screening programme detecting postprandial glycosuria identifies additional diabetic subjects 30 months after a previous screening programme.

Age Factors↗
PubMed1994

State-dependent release of acetylcholine in rat thalamus measured by in vivo microdialysis.

Mesopontine cholinergic neurons have long been thought to play a key role in behavioral state control. In particular, they have been implicated in the process of EEG desynchrony and in the generation of rapid eye movement (REM) sleep. However, the behavioral profile of identified mesopontine cholinergic neurons has not been unequivocally demonstrated. In an attempt to address this issue, in vivo microdialysis was used to monitor acetylcholine (ACh) release across behavioral state in the rat thalamus, a major projection site of mesopontine cholinergic neurons. Because REM periods in rats are of short duration, a method was developed to collect and accumulate sufficiently large samples from each of the individual states of wake, slow-wave sleep, and REM sleep to permit off-line analysis via (HPLC-ECD). Probe placement and the source of cholinergic innervation to the vicinity of the microdialysis probe were verified using retrograde tracing combined with ChAT immunohistochemistry. Finally, the sodium and calcium dependence of ACh measured in the thalamus were tested using TTX and calcium-free dialysates. The results showed that (1) extracellular ACh concentrations in the thalamus are high during both wake and REM sleep and significantly lower during slow-wave sleep, (2) the majority of cholinergic projections to the vicinity of the dialysis probes originate in the mesopontine tegmentum, and (3) ACh release in the thalamus is due to sodium- and calcium-dependent mechanisms. In contrast to predictions of some previous hypotheses, these results demonstrate that mesopontine cholinergic neurons are active during both wake and REM sleep.

Acetylcholine↗
PubMed1993

Preliminary crystallographic study of a complex between an Fab of a monoclonal feline peritonitis virus neutralizing antibody and its anti-idiotypic Fab.

A complex between an Fab fragment of an E2 specific feline infectious peritonitis virus neutralizing antibody 730.1.4 and Fab fragment from anti-idiotypic monoclonal antibody 409.5.3, was crystallized from ammonium sulfate using vapor diffusion methods. The complex crystals diffract to about 2.9 A resolution and are of orthorhombic space group P2(1)22(1) with a = 75.2 A, b = 80.6 A and c = 187.6 A. There are two Fab molecules, or one idiotype-anti-idiotype complex, comprising the asymmetric unit. The long 187.6 A c axis suggests that the long axis of the complex might lie along this direction. Although small and radiation-sensitive, crystals are suitable for three-dimensional structural analysis.

Animals↗
PubMed1993

Determination of three crystal structures of canavalin by molecular replacement.

Canavalin, the major reserve protein of the jack bean, was obtained in four different crystal forms. From the structure determined by multiple isomorphous replacement in a hexagonal unit cell, the structures of three other crystals were determined by molecular replacement. In two cases, the rhombohedral and cubic crystals, placement was facilitated by coincidence of threefold molecular symmetry with crystallographic operators. In the orthorhombic crystal the canavalin trimer was the asymmetric unit. The rhombohedral, orthorhombic and cubic crystal structures were subsequently refined using a combination of several approaches with resulting R factors of 0.194, 0.185 and 0.211 at resolutions of 2.6, 2.6 and 2.3 A, respectively. Variation in the conformation of the molecule from crystal to crystal was small with an r.m.s. deviation in Calpha positions of 0.89 A. Packing is quite different among crystal forms but lattice interactions appear to play little role in the conformation of the molecule. Greatest variations in mean position are for those residues that also exhibit the greatest thermal motion. Crystal contacts in all crystals are mediated almost exclusively by hydrophilic side chains, and three to six intermolecular salt bridges per protein subunit are present in each case.

Journal Article↗
PubMed1993

Three-dimensional structure of satellite tobacco mosaic virus at 2.9 A resolution.

The crystal structure of satellite tobacco mosaic virus (STMV) has been solved by a combination of multiple isomorphous replacement and molecular replacement methods and refined at 2.9 A resolution to a conventional R-factor of 0.215. STMV, a T = 1 icosahedral virus, is the smallest whose structure has been determined. The coat protein is an eight-stranded "Swiss roll" beta-barrel with an amino-terminal strand that extends away from the beta-barrel by more than 60 A. This strand is primarily responsible for quaternary interactions within the capsid. The most arresting feature of the virus structure is the intimate association of each capsid protein dimer with a Watson-Crick base-paired segment of RNA double helix on the interior of the virion. The icosahedral 2-fold axis of each dimer pair is coincident with that of the central base-pair of each helical RNA segment whose helical axis is along the edge of the icosahedron. The helical RNA segments are seven base-pairs in length with a stacked base at each 3' end so that a total of 16 nucleotides is clearly visible. The character of the RNA helix is somewhat different than any of the canonical forms. Assuming full occupancy, then approximately 45% of the total RNA genome is present in the electron density map. The close association of capsid with highly structured nucleic acid suggests that assembly of STMV is likely to be a highly co-operative process involving both protein and RNA. The nucleic acid is distributed within the virion with a high degree of order. The capsid protein is a true double helical RNA binding protein and a number of prominent interactions between protein and RNA can be clearly seen.

Capsid↗
PubMed1993

Double-helical RNA in satellite tobacco mosaic virus.

Satellite tobacco mosaic virus (STMV) is the spherical satellite to an obligatory rod-shaped helper tobacco mosaic virus (TMV), which is required for replication. STMV has 60 protein subunits of M(r) 17,500 on a T = 1 icosahedral capsid containing a single-stranded RNA genome of 1,059 bases. STMV appears similar to another virus, STNV, but is approximately 20 per cent smaller. It shows no amino-acid homology or immunological cross-reactivity with either STNV or its host TMV. Here we report the X-ray crystal structure of STMV, which shows that the coat protein of STMV contains a 'Swiss roll' beta-barrel. An amino-terminal strand extends more than 60A and is primarily responsible for quaternary interactions. Each capsid dimer is associated with a segment of genomic RNA double helix comprising seven base pairs. The dyad of each protein dimer is coincident with that of the central base pair of the associated RNA segment whose helix axis is directed along an icosahedral edge. Protein-nucleic acid interactions are extensive. The RNA helices, which have additional stacked bases at their 3' termini, differ significantly from canonical nucleic acid helical forms.

Capsid↗
PubMed1993

Dopaminergic regulation of cortical acetylcholine release: effects of dopamine receptor agonists.

The regulation of the basal forebrain cholinergic system by D1 and D2 dopamine receptors was assessed in the rat using in vivo microdialysis of cortical acetylcholine. The D1 agonist CY 208-243 significantly increased cortical acetylcholine release; in contrast, the D2 agonists quinpirole and (+)-4-propyl-9-hydroxynaphthoxazine were without significant effects. Moreover, when administered in combination with CY 208-243, quinpirole failed to potentiate the D1 agonist-induced increases in cortical acetylcholine release. The non-selective dopamine receptor agonist apomorphine also increased cortical acetylcholine release, and this was completely blocked by the selective D1 receptor antagonist SCH 23390 and slightly, but not significantly attenuated by the D2 antagonist raclopride. The present results indicate that stimulation of D1 receptors activates cortically-projecting cholinergic neurons; however, a minor contribution of D2 receptors cannot be excluded.

Acetylcholine↗
PubMed1993

Evaluation of fluticasone propionate (500 micrograms day-1) administered either as dry powder via a Diskhaler inhaler or pressurized inhaler and compared with beclomethasone dipropionate (1000 micrograms day-1) administered by pressurized inhaler.

Five hundred and eighty-five patients with moderate asthma, currently receiving 400-1000 micrograms day-1 of an inhaled corticosteroid, were treated for 6 weeks in a double-blind, randomized, parallel group study with either 500 micrograms day-1 fluticasone propionate as a dry powder via a Diskhaler inhaler, 500 micrograms day-1 fluticasone propionate via a pressurized inhaler or 1000 micrograms day-1 beclomethasone dipropionate via a pressurized inhaler. For all three treatment groups, mean morning and evening peak expiratory flow rates (PEFRs) increased within 1 week of the start of treatment. There were also improvements in clinic lung function, daytime and night-time asthma symptoms and a reduction in daytime and night-time rescue bronchodilator medication in all three groups. There were no statistically significant differences between the two formulations of fluticasone propionate in any of the efficacy parameters. Fluticasone propionate via the Diskhaler was significantly more effective than beclomethasone dipropionate over the 6 week study period in reducing diurnal variation (mean difference--4 l min-1, 95% CI--8 to 0 l min-1: P = 0.03). Fluticasone propionate via the Diskhaler produced a statistically significant improvement in night-time symptoms when compared to beclomethasone dipropionate whereas, beclomethasone dipropionate 1000 micrograms day-1 was statistically significantly more effective than both formulations of fluticasone propionate in improving daytime symptoms (P < 0.05). However, these statistical differences must be viewed together with the fact that very few patients recorded a score of 2 or more for both daytime or night-time symptoms. There was a similarly low incidence of adverse events with all three treatments with no evidence of hypothalamic pituitary adrenal (HPA)-axis suppression. The results of the 6-week comparative study showed that 500 micrograms day-1 fluticasone propionate whether administered via pressurized inhaler or Diskhaler is as effective and as safe as 1000 micrograms day-1 beclomethasone dipropionate administered via a pressurized inhaler in the treatment of moderate asthma. Over 12 months fluticasone propionate 500 micrograms day-1 via a pressurized inhaler was at least as effective and as well tolerated as beclomethasone dipropionate 1000 micrograms day-1.

Adolescent↗