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Biomedical subjects

M J Bailey

Publications and source records attributed to M J Bailey.

At least 37 records · Page 2Linked to original sources
PubMed2001

Increased human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes after lung transplantation correlates with HCMV pneumonitis.

BACKGROUND: Human cytomegalovirus (HCMV) reactivation and disease remain relatively common in lung transplant recipients (LTR) despite the use of ganciclovir prophylaxis protocols for all HCMV at-risk patients. The specific aims of this study were to (1) describe the HCMV DNA viral load in the peripheral blood leukocytes (PBL) of a cohort of LTR during the first 6 months after lung transplantation; (2) prospectively determine whether HCMV DNA viral load predicts episodes of HCMV pneumonitis in LTR; and (3) study the effect of ganciclovir on HCMV viral load. METHODS: Competitive polymerase chain reaction using an internal standard and fluorometric detection were used to quantitate HCMV DNA in the PBL of a cohort of 26 LTR monthly for the first 6 months after transplantation (145 samples). All patients were treated with standard triple immunosuppression, and ganciclovir prophylaxis was given to all at-risk LTR (donor or recipient HCMV seropositive) for at least 8 weeks after transplantation. RESULTS: Thirteen episodes of histopathologically proven HCMV pneumonitis in nine subjects occurred during follow-up with a wide intra- and intersubject variation in the HCMV DNA PBL levels. HCMV detection had a sensitivity of 92% and specificity of 76% for HCMV pneumonitis (negative likelihood ratio, 9.5), whereas greater than 10-fold increases in HCMV DNA load had a specificity of 93% and sensitivity of 67% (positive likelihood ratio, 11). HCMV DNA detection had an adjusted odds ratio for HCMV pneumonitis of 107 (95% confidence interval, 14-821; P<0.005). In those with detectable HCMV DNA in PBL (n=44), HCMV DNA levels were 4.4 (95% confidence interval, 1.2-16.8) times higher in those with HCMV pneumonitis than in those without HCMV pneumonitis. Although ganciclovir treatment was very effective in treating HCMV pneumonitis and suppressing HCMV DNA levels, thrice weekly ganciclovir prophylaxis only partially controlled HCMV DNA levels and did not eliminate HCMV pneumonitis risk as three patients developed HCMV pneumonitis while on this regimen. CONCLUSIONS: HCMV DNA detection, absolute levels, and relative change from baseline in the PBL of LTR correlate with HCMV pneumonitis episodes and may be a useful intermediate outcome measure of the efficacy of ganciclovir prophylaxis and treatment strategies.

Adult↗
PubMed2001

Ecological and physiological analyses of Pseudomonad species within a phenol remediation system.

A diverse collection of 700 bacteria obtained from an operational phenolic remediating industrial treatment plant was made to select potential strains as microbial biosensors. Pseudomonads were the most abundant group, of which 48 selected from the liquor or suspended solids were assessed for their physiological response to phenolic pollutant loading and niche specialisation. By FAME-MIS identification the Pseudomonads were clustered into six major species groups. Those isolates able to utilise phenol as a sole carbon source predominantly belonged to a non-clonal Pseudomonas pseudoalcaligenes cluster determined by REP-PCR genotyping. Rapid microtitre based respiration assays were developed to contrast activity in response to increasing concentrations of phenol. A considerable range in response for both phenol degrader and non-degrader strains was observed. This natural phenotypic and physiological heterogeneity could facilitate the selection of isolates for the development of a suite of ecologically relevant, custom designed sensors with predictable toxicity susceptibilities to monitor process efficacy.

Biodegradation, Environmental↗
PubMed2001

Strain characterization and 16S-23S probe development for differentiating geographically dispersed isolates of the phytopathogen Ralstonia solanacearum.

The causative agent of potato brown rot and bacterial wilt, Ralstonia solanacearum, results in serious world-wide economic losses, particularly in the tropics. In the last decade, however, the incidence of bacterial wilt in potatoes grown in Northern Europe has increased, presenting an interesting epidemiological puzzle. Its occurrence may be as a result of changes in agricultural practice or the emergence of a novel bacterial variety, better adapted to cooler conditions. To understand the distribution and genetic diversity of this phytopathogen, we have analysed a collection of 82 isolates from Europe and tropical regions. Both phenotypic [SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) profiling, FAME (fatty acid methyl esters) analysis, growth profiles and EPS (exopolysaccharide) production] and genotypic [16S rRNA RFLP (restriction fragment length polymorphism), ARDRA (amplified ribosomal DNA restriction analysis) and sequence analysis of 16S-23S rRNA ITS and flanking regions] methods were compared. Principal component analysis of FAME profiles clustered isolates into three groups and ARDRA of a 0.85 kb amplified fragment from the 16S-23S ITS region differentiated isolates into four groups. Using sequence analysis, specific primers were designed within the variable region 147-170 of the 23S rRNA. These primers, RsolT2 and RsolT3, respectively, differentiated isolates into two distinct clusters as described previously by Wullings and colleagues (Wullings et al., 1998). The European strains (Biovar 2, race 3) analysed in this study specifically hybridized with RsolT3, and showed considerable genetic homogeneity when compared with strains of other races from 'the rest of the world'. These data indicate the possible selection and proliferation of a 'European'-adapted variant.

Betaproteobacteria↗
PubMed2001

A dose-escalation study to assess the efficacy and safety of sildenafil citrate in men with erectile dysfunction.

OBJECTIVE: To assess the efficacy and safety of sildenafil citrate (Viagra, Pfizer Inc., USA) in a double-blind, placebo-controlled, dose-escalation study over a period of 26 weeks in men with erectile dysfunction of a broad spectrum of aetiology. PATIENTS AND METHODS: In all, 315 patients from five countries were randomized to receive treatment with placebo (156 men) or sildenafil (159 men). Significant concomitant medical conditions were hypertension (20%), a history of pelvic surgery (19%), diabetes mellitus (15%), and ischaemic heart disease (10%). Patients randomized to treatment received a starting dose of 25 mg of sildenafil or matching placebo, which could be increased to 50 mg and then to 100 mg of sildenafil, based on efficacy and tolerability. Assessments of efficacy comprised the 15-item International Index of Erectile Function (IIEF), including question three (ability to achieve an erection) and question four (ability to maintain an erection), a partner questionnaire, an overall efficacy question, and event-log data. RESULTS: After 12 weeks of treatment, 26%, 32% and 42% of patients were taking 25, 50 and 100 mg of sildenafil, respectively. A similar distribution of doses was reported after 26 weeks of treatment. Treatment with sildenafil significantly improved the patients' abilities to achieve and maintain an erection compared with treatment with placebo (P < 0.001). Scores for four of the five sexual function domains of the IIEF (erectile function, orgasmic function, intercourse satisfaction and overall satisfaction) also improved significantly (P < 0.001). There was a significant improvement in the mean score for the erectile function domain, regardless of the aetiology of erectile dysfunction (P < 0.001). After 12 weeks and 26 weeks of treatment, 82% and 79% of patients receiving sildenafil reported improved erections, compared with 24% and 23% of patients receiving placebo, respectively (P < 0.001). Treatment-related adverse events were mild to moderate and occurred in 27% of patients receiving sildenafil, compared with 8% of patients receiving placebo. CONCLUSION: Sildenafil is an effective and well-tolerated treatment for men with erectile dysfunction of a broad spectrum of aetiology.

Administration, Oral↗
PubMed2001

The usefulness of competitive PCR: airway gene expression of IL-5, IL-4, IL-4delta2, IL-2, and IFNgamma in asthma.

BACKGROUND: Asthma has been described as an eosinophilic bronchitis driven by interleukin(IL)-4 and IL-5. The quantification of cytokine mRNA levels in airway samples has been confounded by housekeeping gene expression which differs between and within asthmatics and controls. METHODS: The usefulness of competitive reverse transcriptase-polymerase chain reaction (RT-PCR) that is independent of housekeeping gene expression for quantitating the mRNA for interferon (IFN)gamma, IL-2, IL-5, IL-4 and its receptor antagonist encoding splicing variant IL-4delta2 was determined in a cross sectional study of 45 normal control subjects and 111 with asthma. RESULTS: Atopic controls and atopic asthmatic subjects expressed more IL-5 than non-atopic controls (p<0.02) in bronchoalveolar lavage (BAL) cells, but not in biopsy specimens. IL-5 mRNA expression in BAL cells from asthmatic subjects using inhaled corticosteroids (ICS) was significantly lower than those not receiving ICS (p=0.04). IL-2 mRNA levels differed with steroid use in biopsy specimens but not in BAL cells. IFNgamma, IL-4, and IL-4delta2 mRNA levels did not differ between any groups and were not affected by steroid use. IL-4 and IL-4delta2 mRNA levels were positively correlated (p<0.0001), suggesting coordinated transcription. CONCLUSIONS: While the signal differentiation of competitive PCR in asthma may rival that of in situ hybridisation and immunohistochemistry, the method is expensive and wasteful of material.

Adult↗
PubMed2001

Are asthma medications and management related to deaths from asthma?

There is controversy about the role of beta-agonists in asthma mortality, and the impact of asthma management plans remains unclear. We compared blood beta-agonist levels in patients dying from asthma with those in controls, and estimated the risks associated with specific classes of medication and patterns of management. We identified 89 asthma deaths and recruited 322 patients presenting to hospitals with acute asthma. A questionnaire was administered to the next of kin in 51 cases, and to 202 controls. Blood drawn from 35 cases and 229 controls was assayed for salbutamol. Smoking, drinking, and family problems were significantly more likely among the cases of asthma death than among the controls. The two groups were reasonably well matched with regard to markers of chronic asthma severity. Cases of asthma death were significantly less likely than controls to use a peak flow meter. Written action plans were associated with a 70% reduction in the risk of death. Use of nebulized bronchodilators or oral steroids was significantly more likely in cases of asthma death. Mean blood salbutamol concentrations were 2.5 times higher in cases of asthma. The use of oral steroids for an attack of asthma reduced the risk of death by 90%. More widespread adoption of written asthma management plans, with less reliance on beta-agonists and closer medical supervision, should reduce asthma mortality.

Adrenergic beta-Agonists↗
PubMed2000

In vitro recruitment of the RfaH regulatory protein into a specialised transcription complex, directed by the nucleic acid ops element.

An unusual regulatory mechanism that controls transcription elongation in long fertility and virulence operons in bacteria is effected by two specialised components, the RfaH protein and the nucleic acid ops element. Without direct interaction, ops acts to reduce the concentration of RfaH required to stimulate distal gene transcription, and we have proposed that ops recruits RfaH to the transcription machinery. To provide direct experimental evidence for this view, we used gel fitration to identify potential RfaH complexes assembled in Escherichia coli cell extracts that carry out RfaH-dependent transcription. This novel molecular weight shift assay revealed that RfaH-dependent transcription elongation occurs concomitantly with recruitment of RfaH into a high molecular weight transcription complex, and that this recruitment is specifically directed by the ops element. Assembly of this complex required RNA polymerase and nucleotide hydrolysis, but not processive transcription. Neither assembly of the complex nor RfaH-dependent transcription was observed in in vitro reactions containing only ops, RfaH and purified core (alphabetabeta') RNA polymerase; both processes required the combination of subcellular fractions containing the RNA polymerase complex, the cytoplasmic membrane and ribosomes. The data confirm that the ops element directs recruitment of RfaH into a multi-component RNA polymerase complex that resists transcription termination.

Adenosine Triphosphate↗
PubMed2000

Immuno-capture differential display method (IDDM) for the detection of environmentally induced promoters in rhizobacteria.

A rapid immunological method for trapping and selection of functionally regulated prokaryotic promoters is described. The method is based on application of a novel mini-Tn5 derived promoter probe (pUTTKZY-promoterless lacZY as a reporter and kanamycin resistance) to mutagenise a plant growth promoting fluorescent pseudomonad, Pseudomonas fluorescens 54/96. The transposon allows selection of operon fusion mutants (lacZY(+)) directly on media containing lactose as a sole carbon source as well as selection for kanamycin and lacZ (beta-galactosidase) expression on X-gal indicator media. We have extended the technique to target the surface expression of the induced lactose permease gene (lacY) from mutagenised libraries and the immuno-capture of bacteria with magnetic beads and anti-LacY monospecifc antisera. The benefits of the lacZY reporter are that a library can be rapidly generated and screened in vitro to isolate non-expressed mutants for further in situ screening. Here we demonstrate the development and utility of the technique and its potential as a differential display method for the isolation of promoters that direct regulated gene expression in the phytosphere, or under other imposed conditions.

DNA Transposable Elements↗
PubMed2000

Identification of conserved traits in fluorescent pseudomonads with antifungal activity.

A collection of 29 fluorescent pseudomonads, some with known biological control activity against a range of phytopathogenic fungi, were characterized phenotypically and genotypically by comparing carbon source utilization patterns, suppression of Pythium ultimum both in planta and in vitro and the potential to produce known secondary metabolites. Fatty acid profiling and restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA operon (ribotyping) were used to determine the diversity of isolates. A small group of genetically related Pseudomonas spp. with similar properties was identified; each isolate produced a diffusible bioactive product in vitro and was active against Pythium ultimum in planta. However, other isolates that were able to suppress damping off disease but did not inhibit hyphal extension in vitro clustered outside this group. Phenotypic analyses revealed that the accumulation of C17:0 cyclopropane fatty acid (17CFA) and the production of hydrogen cyanide correlated significantly with biological control activity and with the antagonism of fungal development. The potential of 17CFA as a marker for the selection of fluorescent pseudomonads with biocontrol agent (BCA) potential was demonstrated by the isolation of a novel active strain. This was selected after the screening of 13 clonal groups of fluorescent pseudomonads identified from 500 isolates from the phytosphere of sugar beet. Levels of 17CFA synthesis possibly reflect the efficacy of the rpoS allele in particular strains.

Cyclopropanes↗
PubMed2000

Chromosomal insertion of phenazine-1-carboxylic acid biosynthetic pathway enhances efficacy of damping-off disease control by Pseudomonas fluorescens.

A disarmed Tn5 vector (pUT::Ptac-phzABCDEFG) was used to introduce a single copy of the genes responsible for phenazine-1-carboxylic acid (PCA) biosynthesis into the chromosome of a plant-growth-promoting rhizobacterium Pseudomonas fluorescens. The PCA gene cluster was modified for expression under a constitutive Ptac promoter and lacked the phzIR regulators. PCA-producing variants significantly improved the ability of the wild-type P. fluorescens to reduce damping-off disease of pea seedlings caused by Pythium ultimum, even under conditions of heavy soil infestation. Under conditions of oxygen limitation that are typical of the rhizosphere, PCA production per cell in vitro was greater than that recorded in fast-growing, nutrient-rich cultures. Similarly, when the in vitro nutrient supply was limited, P fluorescens::phz variants that produced the most PCA effectively competed against P. ultimum by suppressing mycelial development. Soil-based bioassays confirmed that the level of PCA biosynthesis correlated directly with the efficacy of biological control and the persistence of inocula in soil microcosms. They also showed that soil pretreatment with bacteria provides a suitable method for plant protection by reducing infection, effectively decontaminating the soil. These data demonstrate that the insertion of a single chromosomal copy of the genes for a novel antifungal compound, PCA, enhances the ecological fitness of a natural isolate already adapted to the rhizosphere and capable of suppressing fungal disease.

DNA Transposable Elements↗
PubMed2000

Seasonal population dynamics and interactions of competing bacteriophages and their host in the rhizosphere.

We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium. Further, we provide evidence of in situ competition between these phages. This is the first in situ demonstration of such microbial interactions in soil. To achieve this, sugar beet seeds were inoculated with Serratia liquefaciens CP6RS or its lysogen, CP6RS-ly-phi 1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages. Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. A Podoviridae phage, identified as phi CP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, the Siphoviridae phage phi CP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some phi CP6-1 phage up to day 137, yet hardly any phi CP6-4 phages were found, and this was consistent with previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model.

Chenopodiaceae↗
PubMed2000

Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition.

A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.

Bacteria↗
PubMed2000

Bacterial community structure and physiological state within an industrial phenol bioremediation system.

The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater. Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor. Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the alpha, beta and gamma subclasses of the class Proteobacteria [alpha-, beta-, and gamma-Proteobacteria, respectively], the Cytophaga-Flavobacterium group, and the Pseudomonas group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting. At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to the Cytophaga-Flavobacterium group and to the gamma-Proteobacteria. The alpha subclass Proteobacteria were of low relative abundance throughout the treatment system whilst the beta subclass of the Proteobacteria exhibited local dominance in several of the processing compartments. Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA. For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content. The gamma subclass of the Proteobacteria had a ribosome content which correlated positively with total phenolics and thiocyanate. While members of the Cytophaga-Flavobacterium group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical parameters. The potential importance of the gamma-Proteobacteria and the Cytophaga-Flavobacteria during this bioremediation process was highlighted.

Aged↗
PubMed2000

A clinical trial of the Buteyko Breathing Technique in asthma as taught by a video.

The Buteyko Breathing Technique (BBT) is promoted as a drug-free asthma therapy. It is based on the premise that raising blood PaCO2 through hypoventilation can treat asthma. Our study was designed to examine whether the Buteyko Breathing Technique, as taught by a video, is an efficacious asthma therapy. Thirty-six adult subjects with mild to moderate asthma were randomized to receive either a BBT or placebo video to watch at home twice per day for 4 weeks. Asthma-related quality of life, peak expiratory flow (PEF), symptoms, and asthma medication intake were assessed both before and after intervention. Our results demonstrated a significant improvement in quality of life among those assigned to the BBT compared with placebo (p = 0.043), as well as a significant reduction in inhaled bronchodilator intake (p = 0.008). We conclude that the BBT may be effective in improving the quality of life and reducing the intake of inhaled reliever medication in patients with asthma. These results warrant further investigation.

Adult↗
PubMed1999

Limitations of hepatocytes and liver homogenates in modelling in vivo formation of acyl glucuronide-derived drug-protein adducts.

The covalent binding of drugs or their metabolites to proteins is of increasing interest in the investigation of the toxicity of these compounds. Recent attention on biological consequences of protein adduct formation with carboxylate drugs, derived via their reactive acyl glucuronide metabolites, has focussed on liver tissue. Although the intact animal represents undisturbed hepatic physiology, other hepatic models can offer advantages, e.g., multiple experiments from a single liver. In this study we set out to compare the patterns of covalent binding of zomepirac (ZP) to proteins in the livers of intact rats, isolated rat hepatocytes (in culture or suspension), and in rat liver homogenates. Rats were dosed i.v. with 25 mg ZP/kg, and their livers were removed 3 h later. Isolated hepatocytes or liver homogenates were exposed to ZP at 100 microg/mL for 3 h at 37 degrees C. Liver homogenates were exposed to ZP and also zomepirac acyl glucuronide (ZAG) at 100 microg ZP equivalents/mL for 3 h at 37 degrees C. Covalent binding of ZP species was examined by SDS-PAGE and Western blotting with a polyclonal ZP antiserum. In livers from dosed animals, the strongest staining appeared at about 110120, 140, and 200 kDa. Few similarities existed with the results from isolated hepatocytes and, not surprisingly, liver homogenates. Only the 200-kDa band was common to all treatments. Many proteins seemed to be modified, at least to some extent. The differences in major bands are most likely caused by the loss of liver and hepatocyte architecture. The variability across different model systems in respect to covalent binding to hepatic proteins emphasizes the need for care in interpretation of results.

Animals↗
PubMed1998

Zomepirac acyl glucuronide covalently modifies tubulin in vitro and in vivo and inhibits its assembly in an in vitro system.

Drugs possessing a carboxylate functional group usually form acyl glucuronides as major metabolites. These electrophilic metabolites can undergo several spontaneous reactions, including covalent adduct formation with proteins. The present study examined whether covalent adducts were formed with microtubular protein (MTP, 85%, alpha/beta-tubulin) and whether this influenced its ability to assemble into microtubules. Bovine brain microtubular protein (MTP) was purified by assembly-disassembly cycles and incubated with the nonsteroidal anti-inflammatory drug (NSAID) zomepirac (ZP), its acyl glucuronide (ZAG) and rearrangement isomers (iso-ZAG) at various concentrations for 2 h at room temperature and pH 7.5. Assembly was monitored by change in turbidity (increase in absorbance at 340 nm). Both ZAG and iso-ZAG caused dose-dependent inhibition of assembly (50% inhibition at about 1 mM), while ZP caused modest inhibition (< 50% inhibition at 4 mM). In a slightly different system, incubation of performed microtubules with 4 mM ZAG caused about 35% inhibition of reassembly ability, while modification of MTP under similar conditions resulted in about 85% reduction of assembly ability. Immunoblotting with a ZP antiserum showed that ZAG and iso-ZAG covalently modified MTP in a dose-dependent manner, while ZP itself caused no modification. Tubulin and many minor proteins comprising MTP were modified. ZP-modified tubulin was shown to be present in the cytosol of livers from rats dosed twice daily for 3 days with ZP at 50 mg/kg, using a sandwich ELISA with ZP and tubulin antisera. Whether any perturbation of microtubule assembly occurs in vivo as a result of this in vivo modification is currently under investigation.

Animals↗