Biomedical subjects
Publications and source records attributed to D Barber.
A patient with a very rare benign bronchial tumour who presented in a most unusual manner is described. An endobrachial hamartoma was expectorated 24 hours after bronchoscopic manipulation for a suspected malignant tumour.
Smaller chromosomes have higher rates of meiotic reciprocal recombination (centimorgans per kilobase pair) than larger chromosomes. This report demonstrates that decreasing the size of Saccharomyces cerevisiae chromosomal DNA molecules increases rates of meiotic recombination and increasing chromosome size decreases recombination rates. These results indicate that chromosome size directly affects meiotic reciprocal recombination.
A 16 kDa protein, designated CM16*, which strongly binds IgE from baker's-asthma patients has been identified as a glycosylated form of the previously reported WTAI-CM16, which is a subunit of the wheat tetrameric alpha-amylase inhibitor. A glycosylated form (CMb*) of BTAI-CMb, the equivalent inhibitor subunit from barley, has been also found to have significantly enhanced IgE-binding capacity. In all, 14 purified members of the alpha-amylase/trypsin-inhibitor family showed very different IgE-binding capacities when tested by a dot-blot assay. The glycosylated components CM16*, CMb* and the previously described non-glycosylated 14.5 kDa allergen from barley (renamed BMAI-1) were found to be the strongest allergens.
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Several monoclonal antibodies (MAbs) were raised against Olea europaea pollen-extract components. Two of these antibodies, named OL 2 and OL 7, recognize two nonoverlapping, nonrepeating epitopes on the olive-allergen Ole e I, as demonstrated by different techniques. The allergen was purified in a single step by MAb-based affinity chromatography, and the allergen revealed a band at molecular weight 20 kd as well as a minor band at 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The contribution of allergen Ole e I to the allergenic activity of O. europaea pollen extracts was determined from the effect of allergen depletion by affinity chromatography on skin reactivity and a histamine-release test. The removal of allergen caused a large reduction in the activity of the preparation in 25 monospecific olive-allergic patients. In agreement, the affinity-purified allergen demonstrated a similar response when it was compared with the whole extract in these assays. The results indicated that Ole e I is by far the most important olive-pollen allergen. A two-site solid-phase radioimmunoassay was developed for the quantitation of the allergen Ole e I in mass units. The assay was based on the MAbs, OL 2 and OL 7, and had a detection limit in the nanogram range. A good correlation was found between allergenic activity, as determined by RAST inhibition, and allergen content in 18 olive-pollen extracts. This result indicates that the assay can be a good alternative to RAST inhibition for the standardization of O. europaea extracts.
Inhalant food allergy has been described many times in literature, but double-blind clinical trials to support successful hyposensitization to these allergens has seldom been reported. Some authors have suspected that certain adverse reactions after immunotherapy may be mediated by immune complexes. Furthermore, the FDA does not recommend injection therapy with food extracts. We present a study on the detection of adverse effects after immunotherapy with an inhalant food (wheat flour) in a double-blind clinical trial in 26 patients with bakers' asthma. We investigated the presence of circulating immune complexes (CICs) after 2 years of treatment with hyposensitization to wheat flour.
Occupational allergy due to hypersensitivity to cereal flours is relatively common among bakers and grain-store workers. Storage mites can contaminate wheat flour and could be an important cause of allergic symptoms due to inhalation. Forty-three patients with criteria for allergic sensitization to wheat flour (skin tests, specific IgE to wheat flour and positive challenge tests) were included in a study to investigate the prevalence of cosensitization to Lepidoglyphus destructor (Ld). This mite was the predominant species in the wheat flour samples supplied by our patients. We found that 30% of the patients had IgE-mediated hypersensitivity of Ld. Of these, 23% did not have a relationship with any bakery or agriculture. We conclude that the prevalence of sensitization to Ld in patients sensitized to wheat flour is important.
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We have identified the major antigens or IgE binding components from wheat flour. Thirty-five sera from patients with baker's asthma were used to analyze the reaction with wheat salt-soluble proteins. We found a 15 kDa SDS-PAGE band which reacted with all sera tested. Purified members of the alpha-amylase inhibitor family, which are the main components of the 15 kDa band, were recognized by specific IgE when tested with a pool of reactive sera. Immunodetection after two-dimensional electrophoretic fractionation of crude inhibitor preparations from wheat endosperms also detected several inhibitor subunits as major low-molecular-weight allergens.
One hundred thirty-nine bakers and pastry cooks were included in a prevalence study of IgE-mediated hypersensitivity to wheat flour demonstrated by skin tests, specific IgE to wheat flour (RAST), and inhalation challenge. From the sensitized workers, we selected 30 asthmatic patients. Twenty patients were treated with a standardized wheat flour extract, and ten with a placebo in a double-blind clinical trial. Before and after immunotherapy we performed tests in vivo (skin tests with wheat flour and methacholine tests), and in vitro (total IgE and specific IgE to wheat flour). We found substantial prevalence of wheat flour allergy (25.17% of workers), and a significant decrease (P less than .001) in hyperresponsiveness to methacholine, skin sensitivity (P = .002), and specific IgE (P less than .005) to wheat flour after 20 months of immunotherapy. There was also significant subjective improvement (P less than .001). The placebo group showed no changes in these variables.
Marked in vitro allergenic potency variations were observed among six different olive pollen batches as determined by RAST inhibition, in direct correlation with the content of a multimeric component of 55,000 D as analyzed by size-exclusion high performance liquid chromotography. Electrophoretical patterns displayed differences in a heterogeneous band of 20,000 D and isoelectric point around 5, which probably represents the monomeric form of the major allergen.
On rare occasions, reproducible exercise-induced anaphylactic reactions (EIA) occur in some patients only after certain foods have been eaten before exercise, yet eating these foods alone or exercising alone causes no symptoms. This special response has been evident sometimes with shellfish, nuts, and wheat. We describe a patient in whom grain flour was a triggering factor for EIA. Skin tests and RAST were positive for grain flours. Normally, the patient tolerated grain flours without symptoms and IgE mechanisms had not been suspected. Testing for food hypersensitivity may be important in patients with EIA.
Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of epsilon(ox.) (410)=282x10(3) litre.mol(-1).cm(-1) was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d(1) components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d(1) bound NO. Titration experiments on the reduced protein over the pH range 5-7, with nitrite as a precursor of NO, showed that the haem d(1) had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.
The magnetic properties of the haem groups of Pseudomonas cytochrome oxidase and its cyanide-bound derivatives were studied in both the oxidized and reduced states by means of m.c.d. (magnetic circular dichroism) at low temperatures. In addition, the oxidized forms of the enzyme were also investigated by e.p.r. (electron-paramagnetic-resonance) spectroscopy, and a parallel study, using both e.p.r. and m.c.d., was made on Pseudomonas cytochrome c-551 to aid spectral assignments. For ascorbate-reduced Pseudomonas cytochrome oxidase, the temperature-independence of those features in the m.c.d. spectrum corresponding to the haem c, and the temperature-dependence of those signals corresponding to the haem d1, showed the former to be low-spin and the latter to be high-spin (s = 2). However, addition of cyanide to the reduced enzyme gave a form of the protein that was completely low-spin. The e.p.r. and m.c.d. sectra of oxidized Pseudomonas cytochrome oxidase and its cyanide derivative were consistent with the haem c and d1 components being low-spin in both cases. Pseudomonas cytochrome c-551 was found to be low-spin in both its oxidized and reduced redox states.
Techniques for computer-assisted diagnosis have been largely confined to off-line use, and there has been little examination of their potential role directly in the clinical setting. It is argued that the potential benefits of on-line diagnostic aids are considerable. A major barrier to such developments is uncertainty that the practical use of an on-line system would lead to the improvements in diagnostic effectiveness that more conventional off-line systems have already demonstrated. A series of studies in which clinicians took the histories of computer-simulated 'patients' presenting with dyspepsia, and formed diagnoses on the basis of these histories with and without computer assistance, suggested that diagnostic effectiveness may be greatly improved with on-line computer assistance. Some reasons for this improvement are discussed and it is concluded that the clinician and the on-line computer bring different but complementary types of knowledge to the diagnostic task. Though many practical questions remain to be answered the findings establish the principle of on-line symptom processing.
The binding of cyanide to both oxidized and ascorbate-reduced forms of Pseudomonas cytochrome c-551 oxidase was investigated. Spectral studies on the oxidized enzyme and its apoprotein showed that the ligand can bind to both the c and d, haem components of the molecule, and kinetic observations indicated that both chromophores reacted, under a variety of conditions, with very similar rates. Cyanide combination velocities were dependent on ligand concentration, and increasing the pH also accelerated the reaction; the second-order rate constant was estimated as approx. 0.2M-1 . s-1 at pH 7.0. The binding of cyanide to the protein was observed to have a considerable influence on reduction of the enzyme by ascorbate. Spectral and kinetic observations have revealed that the species haem d13+-cyanide and any unbound haem c may react relatively rapidly with the reductant, but the behaviour of cyanide-bound haem c indicates that it may not be reduced without prior dissociation of the ligand, which occurs relatively slowly. The reaction of reduced Pseudomonas cytochrome oxidase with cyanide is radically different from that of the oxidized protein. In this case the ligand only binds to the haem d1 component and reacts much more rapidly. Stopped-flow kinetic measurements showed the binding to be biphasic in form. Both the rates of these processes were dependent on cyanide concentration, with the fast phase having a second-order rate constant of 9.3 X 10(5) M-1 . s-1 and the slow phase one of 2.3 X 10(5) M-1 . s-1. The relative proportions of the two phases also showed a dependency on cyanide concentration, the slower phase increasing as the cyanide concentration decreased. Computer simulations indicate that a reaction scheme originally proposed for the reaction of the enzyme with CO is capable of providing a reasonable explanation of the experimental results. Static-titration data of the reduced enzyme with with cyanide indicated that the binding was non-stoicheiometric, the ligand-binding curve being sigmoidal in shape. A Hill plot of the results yielded a Hill coefficient of 2.6.
Stopped-flow kinetics were made of the reaction between ascorbate-reduced Pseudomonas cytochrome oxidase and potassium ferricyanide under both N2 and CO atmospheres. Under N2 three kinetic processes were observed, two being dependent on ferricyanide concentration, with second-order rate constants of 9.6 X 10(4)M-1.s-1 and 1.5 X 10(4)M-1.s-1, whereas the other was concentration-independent, with a first-order rate constant of 0.17 +/- 0.03s-1. Measurements of their kinetic difference spectra have allowed the fastest and second-fastest phases of the reaction to be assigned to direct bimolecular reactions of ferricyanide with the haem c and haem d, moieties of the enzyme respectively. Under CO, the second-order rate constant for the reaction of the haem c was, at 1.3 X 10(5)M-1.s-1, slightly enhanced over the rate in a N2 atmosphere, but the reaction velocity of the haem d1 component was greatly decreased, being apparently limited to that of the rates of CO dissociation from the molecule (0.15s-1 and 0.03s-1). The results are compared with those obtained during a previous study of the reaction of reduced Pseudomonas cytochrome oxidase with oxidized azurin.