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H Webb

Publications and source records attributed to H Webb.

At least 19 recordsLinked to original sources
PubMed2003

Employment of broad-range 16S rRNA PCR to detect aetiological agents of infection from clinical specimens in patients with acute meningitis--rapid separation of 16S rRNA PCR amplicons without the need for cloning.

AIMS: The aim of this study was to develop a polyacrylamide gel electrophoresis (PAGE) method for the rapid separation of 16S rRNA PCR amplicons from aetiological agents of acute meningitis. METHODS AND RESULTS: Blood samples from 40 patients with suspected acute meningococcal meningitis were examined for the presence of causal agents, including Neisseria meningitidis employing two methods: (i) broad-range 16S rRNA PCR in conjunction with PAGE and automated sequencing and (ii) species-specific PCR employing ABI TaqMan technology for N. meningitidis. Analysis of clinical specimens employing 16S rRNA PCR yielded 33/40 (82.5%) positive for the presence of bacterial DNA. Species-specific PCR yielded 30/40 (75%) clinical specimens positive for N. meningitidis. Prior to separation by PAGE, only 6/33 (18.2%) amplicons were able to be identified by sequence analysis, the remaining amplicons (n=27) did not yield an identification due to the presence of mixed 16S rRNA PCR amplicons. Following separation, amplicons were re-amplified and sequenced, yielding 24/27 (88.9%) positive for N. meningitidis and three specimens positive for Acinetobacter sp., Staphylococcus aureus and Streptococcus pneumoniae. One specimen was positive for both N. meningitidis and Streptococcus spp. and another specimen was positive for N. meningitidis and Pseudomonas sp., by broad-range PCR. Seven clinical specimens were negative for N. meningitidis and other eubacteria using both detection techniques. CONCLUSIONS: Clinical specimens including blood and cerebrospinal fluid from patients with suspected acute bacterial meningitis, may become contaminated with commensal skin flora, resulting in difficulties in downstream sequencing of pathogen plus contaminant DNA. This study allows for the rapid separation of amplified pathogen from contaminant DNA. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrated the usefulness of the rapid separation of multiple 16S rRNA PCR amplicons using a combination of PAGE and automated sequencing, without the need of cloning. Adoption of this technique is therefore proposed when trying to rapidly identify pathogens in clinical specimens employing broad-range 16S rRNA PCR.

Acinetobacter↗
PubMed2001

The coatomer of Trypanosoma brucei.

Coatomer is a multisubunit complex involved in trafficking of vesicles between the endoplasmatic reticulum and the Golgi apparatus. From sequence homologies, all seven subunits, alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP, are encoded in the genome of Trypanosoma brucei. The complete predicted amino-acid sequences of beta-, beta'-, and zeta-COP show only 20-30% identity with higher eucaryotic homologues. The trypanosome coatomer complex was partially purified using a procedure similar to that used for bovine coatomer.

Amino Acid Sequence↗
PubMed1999

Identification of vitronectin as a novel insulin-like growth factor-II binding protein.

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(1-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/IGF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Amino Acid Sequence↗
PubMed1998

Conservation of genetic linkage between heat shock protein 100 and glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei and Trypanosoma cruzi.

The experiments described in this paper were designed to try and isolate a recombinant DNA clone encoding a Trypanosoma cruzi homologue of the Trypanosoma brucei glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) gene. Despite the ready biochemical detection of phospholipase C activities that hydrolyse GPI-anchors of cell surface proteins in T. cruzi, it did not prove possible to isolate any recombinant DNA clones using the T. brucei gpi-plc gene as a probe. On determining the DNA sequence to the 5' side of the gpi-plc gene it was found to be adjacent to a gene that encodes a 100 kDa heat shock protein (HSP100). To investigate whether this linkage between the hspl00 and gpi-plc genes was conserved in T. cruzi, a probe derived from the T. brucei hsp100 gene was used to isolate T. cruzi genomic clones. These were partially sequenced and shown to contain an hsp100 gene. Restriction enzyme fragments located to the 3' side of the T. cruzi hsp100 gene were then sequenced and found to contain a gene that encodes a polypeptide (TcPLC1) that has 46% amino acid sequence identity with the T. brucei GPI-PLC including most of the key residues involved in inositol binding and the catalytic histidine. A recombinant form of TcPLC1 was produced and shown to possess phospholipase C activity towards a GPI-substrate. Thus, the hsp100 and gpi-plc genes are adjacent in T. brucei and this linkage is conserved in T. cruzi. This observation has been used to facilitate the isolation of a clone encoding a T. cruzi phospholipase C gene.

Amino Acid Sequence↗
PubMed1998

The properties and function of the glycosylphosphatidylinositol-phospholipase C in Trypanosoma brucei.

The purpose of this review is to consider recent results obtained concerning the properties and function of the glycosylphosphatidylinositol-phospholipase C (GPI-PLC) in Trypanosoma brucei. A mutagenesis study that provides evidence that the GPI-PLC is more closely related to bacterial PI-PLCs than previously realised is described. The variant specific glycoprotein (VSG), which dominates the surface of the mammalian stages of the trypanosome, is almost certainly the major substrate of the GPI-PLC. The hydrolysis of the GPI-anchor of the VSG under stress conditions and hypotonic lysis is well established. To investigate whether this hydrolysis of the GPI-anchor plays any role during the life cycle a GPI-PLC null mutant has been made. The phenotype indicates that the gene is non-essential, but its absence alters the course of infection in mice.

Amino Acid Sequence↗
PubMed1997

Mutagenesis study of the glycosylphosphatidylinositol phospholipase C of Trypanosoma brucei.

The glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei is particularly effective in hydrolysing the GPI-anchors of some proteins. The enzyme is inhibited by Zn2+ and p-chloromercurylphenylsulphonic acid, both of which can act as sulphydryl reagents, suggesting that a cysteine residue may be important in catalysis. Single cysteine to serine mutants have been produced for all eight cysteines in GPI-PLC; all the mutants were fully active in vitro and were still susceptible to p-chloromercurylphenylsulphonic acid inhibition. In contrast, a single histidine 34 to glutamine mutation totally inactivated GPI-PLC. The histidine was chosen after a sequence alignment with the Bacillus cereus phosphatidylinositol phospholipase C (PI-PLC) suggested a conservation of active site residues, including histidine 34 which is central to the proposed reaction mechanism (Heinz D.W., Ryan M., Bullock T.L., Griffith O.H. EMBO J 1995;14:3855-3863). The results suggest that the GPI-PLC and bacterial PI-PLCs have conserved active sites and that the inhibition of GPI-PLC by sulphydryl reagents can occur through more than one residue.

4-Chloromercuribenzenesulfonate↗
PubMed1997

The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.

Animals↗
PubMed1996

Simultaneous but independent activation of adenylate cyclase and glycosylphosphatidylinositol-phospholipase C under stress conditions in Trypanosoma brucei.

Previous observations suggested a concomitant relationship between the release of the variant surface glycoprotein (VSG) and the activation of adenylate cyclase in the bloodstream form of the parasitic protozoan Trypanosoma brucei. In order to evaluate this hypothesis, adenylate cyclase activity was measured in live trypanosomes subjected to different treatments known to induce the shedding of the VSG coat, namely low pH and trypsin digestion. In both cases adenylate cyclase activation occurred in parallel with the release of the VSG. The latter was found to be mediated by the glycosylphosphatidylinositol-specific phospholipase C that cleaves the glycosylphosphatidylinositol anchor of the protein (VSG lipase). Furthermore, both adenylate cyclase and VSG release were activated by the incubation of trypanosomes with specific inhibitors of protein kinase C, suggesting a repressive role for protein kinase C on both VSG lipase and adenylate cyclase activities. Significantly, in mutant trypanosomes lacking VSG lipase, adenylate cyclase was activated under conditions where VSG release did not occur. Moreover,VSG release was also found to occur in the absence of activation of the cyclase, as observed in the presence of low concentration of the thiol modifying reagent p-chloromercuriphenylsulfonic acid. These observations provide the first demonstration that release of the VSG in response to cellular stress is mediated by the VSG lipase and that while both release of the VSG and activation of adenylate cyclase occur in response to the same stimuli they are not obligatorily coupled.

Adenylyl Cyclases↗
PubMed1996

Characterization of serum-derived and recombinant rat IGF-I and their use for measuring true concentrations of IGF-I in rat plasma.

While numerous researchers have used rat models to investigate the in vivo actions of IGF-I, interpretation of the results in terms of true concentrations of rat IGF-I (rIGF-I) in plasma has been hampered by the absence of homologous reference standards. In order to overcome this we have produced recombinant rIGF-I (rrIGF-I) from Escherichia coli using procedures similar to those we have previously described for the production of other recombinant IGFs. The rrIGF-I is indistinguishable from serum-derived rIGF-I when characterized in a number of in vitro assays including ability to stimulate protein synthesis and inhibit protein degradation in cultured rat cells, as well as in interactions with the rat type-1 IGF receptor and with rat IGF-binding proteins. Moreover, both the serum-derived and the recombinant rat proteins are similar to recombinant human IGF-I (rhIGF-I) in these assays. However, differences between the human and rat IGFs are apparent when tested in immunoassays using some antibodies raised against rhIGF-I. Furthermore, the differences between rhIGF-I and rrIGF-I are even greater when rhIGF-I is used as the competing radiolabel in these assays, a situation that can lead to a two- to threefold underestimation of the actual concentration of IGF-I in rat plasma. These results indicate that, while immunoassays employing antibodies raised against rhIGF-I and rhIGF-I reference standards reliably indicate trends in IGF-I concentrations in rat plasma, the true amounts of rIGF-I present can only be assured in an assay using homologous tracer and reference peptides.

Animals↗
PubMed1995

Risk factors for hearing disorders: epidemiologic evidence of change over time in the UK.

The prevalence** of all bilateral sensorineural hearing impairments of at least 40 dB HL was assessed by a retrospective ascertainment study to be about 1.2 children per 1000 births per annum, over the period 1983-1988. This prevalence was highly variable over year and region of the study. For all profound (at least 95 dB HL) impairments, the prevalence was 1 in 2703 children per birth cohort. The contribution of acquired impairments and inter-region transfers after birth was not trivial, being over 25 percent for the profound impairments. The proportion of the bilateral moderately profoundly hearing impaired who might have been in the neonatal intensive care unit, have a family history of childhood deafness, or craniofacial anomaly recognizable prior to maternity discharge was 64 percent. Changes in the relative distribution of the etiology of hearing impairment seem to have taken place since the EC 1969 birth cohort study. These changes should have considerable impact on the planning of services for hearing-impaired children and child health surveillance in general.

Child, Preschool↗
PubMed1994

The role of GPI-PLC in Trypanosoma brucei.

The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG). However the evidence that it is involved in VSG metabolism in the living trypanosome is circumstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotonic lysis. Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease. To try to determine the role of the GPI-PLC, a homozygous null mutant T. brucei has been constructed. The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes. The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype. The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be.

Animals↗
PubMed1993

Influence of selective decontamination of the digestive tract on microbial biofilm formation on endotracheal tubes from artificially ventilated patients.

The effect of selective decontamination of the digestive tract on the nature and incidence of microbial biofilm formation on endotracheal tubes was assessed. Thirty endotracheal tubes were obtained post-extubation from patients in the intensive care unit who had been ventilated for a 1 to 15 day period and who did or did not receive the antibiotic regimen. Extensive biofilm formation was identified by scanning electron microscopy on 97% of tubes examined. Endotracheal tube biofilm in tubes obtained from patients who received selective decontamination of the digestive tract showed a high prevalence of colonization with yeast (4 of 15 tubes) and gram-positive bacteria (streptococci, staphylococci and diphtheroids) (14 of 15 tubes). Staphylococcus aureus was isolated only from this group. Pseudomonas spp. were isolated from 2 of 15 tubes in both patient groups. Enteric gram-negative organisms (coliforms, Klebsiella and Proteus spp.) were isolated only from tubes of patients who did not receive the antibiotic regimen (4 of 15 tubes). Yeasts, however, were not isolated from these tubes. Group D streptococcal isolates were resistant to tobramycin as were half of the Staphylococcus aureus isolates. For gram-negative bacteria, the MIC of tobramycin was in the range 1-64 micrograms/ml and the MIC of polymyxin in the range 0.5-16 micrograms/ml. Although a reduction was observed in the incidence of gram-negative microorganisms, this antibiotic regimen does not inhibit biofilm formation on the endotracheal tube by other pathogens associated with pneumonia in ventilated patients. This persistent nidus may be a factor in the pathogenesis of nosocomial pneumonia.

Adolescent↗
PubMed1992

The effects of captopril on training in patients with ischemic heart disease.

We studied a group of 30 patients to determine the effect of captopril on the exercise training response after a period of training in patients with ischemic heart disease but without cardiac failure. The study was a double-blind placebo-controlled comparison of captopril and placebo. The patients studied were 28 men and 2 women, mean age 53.6 +/- 6.9 years. All were 8 to 12 weeks postmyocardial infarction or coronary artery bypass surgery. These patients underwent an organized exercise training program consisting of exercise training sessions 3 times weekly for a period of 8 weeks. On commencement and completion of the program patients were assessed for exercise tolerance using submaximal exercise stress testing. Patients were assessed in the untreated state. Both groups showed a statistically significant training effect with increased exercise duration, decreased heart rate for equal workload, increased energy expenditure, and reduced functional aerobic impairment. There was no statistically significant change in systolic blood pressure, but the captopril group alone showed a significant reduction in diastolic blood pressure (p less than 0.001). The change in heart rate at rest over the 8-week period was not significant in both groups. In summary, this study shows that treatment with captopril does not affect the exercise training response in patients with ischemic heart disease undergoing an organized exercise training program.

Blood Pressure↗
PubMed1992

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae.

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.

Amino Acid Sequence↗
PubMed1992

The effects of verapamil on training in patients with ischemic heart disease.

Verapamil is a calcium-channel blocking agent with antianginal and antiarrhythmic properties that have been widely studied. Its myocardial depressant effect is well known. The purpose of this study was to examine the effects of verapamil on the training response in patients with ischemic heart disease. The study group consisted of 41 male patients with a mean age of 53.3 +/- 7.2 years who had suffered a myocardial infarction or had undergone coronary artery bypass surgery 8 to 12 weeks previously. They were chosen on a consecutive basis from eligible patients entering a cardiac rehabilitation program. With use of a double-blind technique, 21 patients were assigned to receive verapamil, 120 mg three times daily, while the other 20 were given an identical placebo. Each patient underwent exercise stress testing in the untreated state to permit comparison between tests performed on commencement and completion of training. The training effect was determined by comparing exercise response before and after the eight-week program. There was an increase in exercise duration (p less than 0.001) and a decrease in functional aerobic impairment (p less than 0.001), without difference between the two groups. Energy expenditure increased in both groups, but the highest level was achieved by those receiving active treatment (p less than 0.02). Heart rate for equal workload was significantly reduced after training (p less than 0.001), although this was lower in the placebo patients (p less than 0.001) and the patients who had a recent myocardial infarction (p less than 0.01). It appears that treatment with verapamil does not impair the development of a training effect in patients with ischemic heart disease who are undergoing organized training.

Anaerobic Threshold↗