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Biomedical subjects

A J Howard

Publications and source records attributed to A J Howard.

At least 37 records · Page 2Linked to original sources
PubMed1996

Susceptibility testing of Haemophilus influenzae--an international collaborative study in quality assessment.

In order to compare the prevalence of antibiotic resistance in different geographical areas, it is necessary to ensure that agreement is achieved between laboratories on the assignment of strains to 'susceptible' and 'resistant' categories. An international quality assessment study, involving 15 laboratories in eight countries, was performed to investigate the standard of performance of the susceptibility testing of Haemophilus influenzae. One hundred and fifty strains of H. influenzae were distributed from the London Hospital Medical College (LHMC) to all laboratories who were asked to test the susceptibility of the strains to ampicillin, chloramphenicol, tetracycline, trimethoprim, cephalosporins and ciprofloxacin. Laboratories were also asked to provide the details of methodology to test the susceptibility. Significant discrepancy between the LHMC and the participating laboratories appeared in the detection of resistance to ampicillin (especially beta-lactamase-negative strains resistant to ampicillin) as well as the assignment of susceptibility and resistance to chloramphenicol, tetracycline and trimethoprim. Often these reflected the use of inappropriate breakpoints which led to erroneous assignment of susceptibility. Other variations including disc content, medium and supplement, inoculum as well as failure to measure zone sizes properly also led to some repeating anomalies.

Ampicillin↗
PubMed1995

Epidemiology of invasive Haemophilus influenzae infections in England and Wales in the pre-vaccination era (1990-2).

This survey defined the pattern of invasive Haemophilus influenzae infections during 1990-2 in six regions in England and Wales during the pre-vaccination era providing a baseline against which any changes in patterns of disease due to the introduction of the Haemophilus influenzae type b vaccination programme can be monitored. A total of 946 cases of invasive Haemophilus influenzae were recorded during the survey period of which almost 90% were due to type b and most of the remainder were non-typeable. Type b infections occurred predominantly in children less than 5 years of age (88%) with the highest attack rate in male infants in the 6-11 month age group. Diagnostic category varied with both age and serotype; meningitis was the commonest presentation overall but pneumonia and bacteraemia were more common in adults and non-typeable isolates. Mortality was highest in neonates and the elderly (over 65 years of age) who were more likely to have an underlying predisposing condition than older children and adults. Children under 5 years of age had a higher case fatality rate for non-typeable than for type b infections. Ampicillin resistance was 15% and there were no cefotaxime resistant type b isolates.

Adolescent↗
PubMed1995

Chaos under canvas: a Salmonella enteritidis PT 6B outbreak.

An outbreak of Salmonella enteritidis PT 6B food poisoning, the first reported of this recently described phage type, resulted in illness among 46 of 49 members of a camping group in North Wales, 33 of whom were hospitalized. Epidemiological evidence (P < 0.0001) indicated that a lemon meringue pie was the vehicle of infection. Fresh shell eggs, stored after purchase at ambient temperature, appear to be the most likely source of infection, with multiplication during preparation and subsequent storage of the pie a significant contributory factor. Campers may be at greater risk than others and should consider the use of cold boxes for the transport and storage of eggs, and avoid the preparation of lightly cooked egg products under these basic conditions.

Abdominal Pain↗
PubMed1995

1.85 A structure of anti-fluorescein 4-4-20 Fab.

The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti-fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1.

Amino Acid Sequence↗
PubMed1995

Measurements of 220Rn emanation from rocks.

Alpha-particle and gamma-ray measurements are employed to determine the individual emanation and production rates associated with the thorium radioactive series for thirty-seven rock specimens of approximately 200 cm3 individual volume representing igneous, sedimentary, and metamorphic types. These results are combined to establish 220Rn emanating powers for each of these specimens, and a wide range of values is observed. The generally large 220Rn emanation observed for these specimens strongly indicates that non-tortuous diffusion paths are commonly present in the associated structures, which gives more evidence for the existence of well-connected pore networks at the nanometer level in many of the specimens studied. While the results are qualitatively explainable, they are not predictable in terms of the current macroscopic observables. Since 220Rn and 222Rn emanation rates from rock specimens have previously been found to be comparable in magnitude, the relatively fast determination of 220Rn emanation rates described herein (measurements involving 1-h duration) is in reasonable probability indicative of a comparable 222Rn emanation rate: The employment of 220Rn as a convenient screening tool for potentially high 222Rn emanation sources is indicated.

Alpha Particles↗
PubMed1994

A multicentre study of the in-vitro activity of cefotaxime, cefuroxime, ceftazidime, ofloxacin and ciprofloxacin against blood and urinary pathogens.

The in-vitro susceptibilities of aerobic bacteria isolated from 1804 blood and 4529 urine specimens collected at nine hospitals in the UK were examined. An agar dilution method was used to determine the MICs of each isolate to three cephalosporins, cefotaxime, cefuroxime and ceftazidime, and to two fluoroquinolones, ofloxacin and ciprofloxacin. Sensitivities were then calculated using British Society for Antimicrobial Chemotherapy recommended breakpoints. Of the cephalosporins tested cefotaxime was the most active against the Enterobacteriaceae. All the systemic staphylococcus isolates collected were sensitive to both cefotaxime and cefuroxime. As expected, ceftazidime was the only cephalosporin active against the Pseudomonas isolates. Both quinolones were highly active against the Enterobacteriaceae and Pseudomonas spp. They also demonstrated good Gram-positive activity, particularly against Staphylococcus aureus and Enterococcus spp.

Anti-Infective Agents↗
PubMed1994

Hepatitis B immunization in UK renal units: failure to put policy into practice.

A postal questionnaire survey of renal units, and an analytical study of surveillance data on acute hepatitis B were undertaken to discover the numbers of renal units (a) routinely immunizing staff and patients against hepatitis B and (b) with recent experience of managing patients with hepatitis B. Seventy-three (86%) of 85 units returned completed questionnaires. Most (63, 84%) of the responding units routinely immunized staff but only 4 (5%) routinely immunized patients. A third (25) of the units had treated at least one HBsAg-positive patient between 1987 and 1991, and 11 units had cared for at least one patient with acute hepatitis B. Units with recent experience of treating an HBsAg-positive patient were no more likely to immunize staff or patients than those without. Guidance on hepatitis B immunization of staff has generally been implemented by UK renal units; that on immunization of patients has not. Renal unit patients remain largely susceptible to hepatitis B and the potential for outbreaks remains. Renal units need further encouragement to implement fully guidance on immunization.

Health Policy↗
PubMed1993

Crystallization of single-chain Fv proteins.

Single-chain Fv (sFv) proteins consist of the variable heavy chain (VH) and variable light chain (VL) domains of an antibody, covalently joined by an engineered polypeptide linker. We report the crystallization of single-chain Fv's with specificities for fluorescein (4-4-20 sFv) and the TAG-72 pan-carcinoma glycoprotein antigen (CC49 sFv). Concentration of these proteins, preliminary to crystallization, results in a monomer-multimer equilibrium, causing aggregation which interferes with crystallization. Aggregation has been observed to depend primarily on an intact linker between VL and VH domains, although other factors are likely to modulate this phenomenon as well, including the specific identity of Fv and ligand, presence or absence of the ligand, linker length and possibly sequence. We have found two methods to overcome sFv aggregation, both of which yield X-ray diffraction quality crystals. The first, discovered serendipitously, is by introducing a proteolytic clip into the linker region (effectively yielding an Fv fragment). The second is the purification of the sFv dimer form, with linker regions intact, from an equilibrium mixture of aggregates. The sFv molecular association in a dimer is believed to be unusual in that each VL/VH interface may not be formed by the two linker-connected VL and VH domains, but rather by interaction of VL and VH domains from two distinct sFv monomers. Structure determination of the CC49 sFv dimer, with the 14-residue linker designated 212, is underway to test this model. Increasing linker length, to relieve steric strain on the monomer, and inclusion of the appropriate antigen, to slow transitions between monomeric and multimeric forms, may prove valuable strategies with sFv proteins less amenable to crystallization.

Amino Acid Sequence↗
PubMed1992

1.67-A X-ray structure of the B2 immunoglobulin-binding domain of streptococcal protein G and comparison to the NMR structure of the B1 domain.

The structure of the B2 immunoglobulin-binding domain of streptococcal protein G has been determined at 1.67-A resolution using a combination of single isomorphous replacement (SIR) phasing and manual fitting of the coordinates of the NMR structure of B1 domain of streptococcal protein G [Gronenborn, A. M., et al. (1991) Science 253, 657-661]. The final R value was 0.191 for data between 8.0 and 1.67 A. The structure described here has 13 residues preceding the 57-residue Ig-binding domain and 13 additional residues following it, for a total of 83 residues. The 57-residue binding domain is well-determined in the structure, having an average B factor of 18.0. Only residues 8-77 could be located in the electron density maps, with the ends of the structure fading into disorder. Like the B1 domain, the B2 domain consists of four beta-strands and a single helix lying diagonally across the beta-sheet, with a -1, +3 chi, -1 topology. This small structure is extensively hydrogen-bonded and has a relatively large hydrophobic core. These structural observations may account for the exceptional stability of protein G. A comparison of the B2 domain X-ray structure and the B1 domain NMR structure showed minor differences in the turn between strands and two and a slight displacement of the helix relative to the sheet. Hydrogen bonds between crystallographically related molecules account for most of these differences.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of the rat glutathione peroxidase gene.

The increased activity of glutathione peroxidase (GSHPx) in rat lungs is associated with the development of tolerance of the animals to hyperoxia. To understand further the regulation of expression of this enzyme, the molecular structure of the corresponding rat gene was characterized. The rat GSHPx gene consists of two exons interrupted by a single intron of 217 base pairs. The same initiation sites for transcription were found to be utilized in both lung and liver. The promoter of the GSHPx gene contains neither a 'TATA' box nor a 'CAAT' box. Instead, it comprises two copies of Sp1 binding motif and one copy of AP-2 binding motif. These features of the promoter may offer a clue to the mechanisms by which the expression of this gene is controlled.

Amino Acid Sequence↗
PubMed1992

A survey of invasive Haemophilus influenzae infections.

A survey of invasive H. influenzae infections has been underway in six regions of England and Wales since September 1990. In the first year, there were 433 cases of which 362 (84%) were due to H. influenzae type b (Hib). The majority of Hib infections were in children aged less than 5 years; there being an annual incidence of 26.4/100,000 in this age group. Meningitis occurred in 56% of cases of Hib infection. The results confirm previous evidence of the need to incorporate Hib vaccination into the childhood immunisation schedule. The ongoing survey data will provide useful information to assess the impact of an Hib immunisation programme.

Adolescent↗
PubMed1991

Epidemiology of Haemophilus influenzae type b invasive disease in Wales.

OBJECTIVE: To investigate the epidemiology of invasive disease due to Haemophilus influenzae type b, the clones responsible, and the antibiotic resistance of the isolates. DESIGN: Prospective population based analysis of clinical and epidemiological data collected for Gwynedd during 1980-90 and in the whole of Wales during 1988-90. SETTING: 19 hospitals in Wales; all medical microbiology laboratories in Wales participated. PATIENTS: 82 patients with confirmed invasive infections caused by H influenzae type b in Gwynedd during 1980-90 and 207 in Wales during 1988-90. MAIN OUTCOME MEASURES: Clinical and epidemiological measures; analysis of the clonal types of the isolates based on the electrophoretic mobilities of 17 metabolic enzymes; and antibiotic resistance. RESULTS: The annual incidence of H influenzae type b infections in Gwynedd was 3.2 cases/100,000 and in Wales was 2.5 cases/100,000. Most cases occurred in children aged under 5 years, the highest annual incidence being in those aged under 1 (84.6/100,000 and 56.9/100,000 in Wales). The cumulative risk of acquiring H influenzae type b disease by the fifth birthday was one in 456 in Gwynedd and one in 578 in Wales. Fifteen per cent of cases in Gwynedd and 7% of those in Wales occurred in adults. Predominant clinical conditions were meningitis in children and pneumonia in adults. In Gwynedd 2/70 (3%) children and 5/12 (42%) adults died. Long term neurological sequelae occurred in 8% (4/48) of children who survived haemophilus meningitis. Children presenting with infection were usually the youngest members of their family. No secondary household cases were identified. 100 of 128 (78%) strains were of a single clone, electrophoretic type 12.5, and 4/207 (1.9%) isolates from Wales were resistant to both ampicillin and chloramphenicol. CONCLUSIONS: The annual rate of infection in children aged under 5 in four Welsh counties was 12-44% higher than that previously published for the United Kingdom. The study emphasises the potential value of a vaccine effective in early infancy and provides baseline data to assess its efficacy after its introduction. Alternatives to ampicillin and chloramphenicol should be used as first line, empirical treatment for severe infections that might be caused by H influenzae type b in Wales.

Adolescent↗
PubMed1991

A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose.

The crystal structure of recombinant Streptomyces rubiginosus D-xylose isomerase (D-xylose keto-isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 A resolution. As observed in an earlier study at 4.0 A (Carrell et al., J. Biol. Chem. 259: 3230-3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight-stranded parallel beta-barrel surrounded by eight helices with an extended C-terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions. The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D-xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 A, R = 0.15 at 1.71 A, R = 0.15 at 1.60 A, and R = 0.14 at 1.60 A, respectively. Both the ring oxygen of the cyclic alpha-D-xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His-54 in the structure crystallized in the presence of D-xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D-xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains. A detailed mechanism for D-xylose isomerase is proposed. Upon binding of cyclic alpha-D-xylose to xylose isomerase, His-54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D-xylose, involving two divalent cations, in an extended conformation. The isomerization of D-xylose to D-xylulose involves a metal-mediated 1,2-hydride shift. The final step in the mechanism is a ring closure to produce alpha-D-xylulose. The ring closing is the reverse of the ring opening step. This mechanism accounts for the majority of xylose isomerase's biochemical properties, including (1) the lack of solvent exchange between the 2-position of D-xylose and the 1-pro-R position of D-xylulose, (2) the chemical modification of histidine and lysine, (3) the pH vs. activity profile, and (4) the requirement for two divalent cations in the mechanism.

Aldose-Ketose Isomerases↗