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M J Bailey

Publications and source records attributed to M J Bailey.

At least 73 records · Page 4Linked to original sources
PubMed1993

Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly.

A transposon mutant of Escherichia coli 5K was isolated which reduced 10- to 50-fold the secreted extracellular haemolytic activity of cells carrying the complete hlyCABD operon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA. The transposon insertion was identified within the rfaP gene (required for attachment of phosphate-containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restored in trans by the intact rfaP gene. The loss in cytolytic activity of the secreted HlyA protein was not related to the HlyC-directed acylation of the protoxin. Activity of the secreted toxin was restored by chaotropic agents and during rate-zonal centrifugation the mutant-secreted HlyA migrated as a larger species than the wild type. The results indicate that the rfaP mutation affects the aggregation behaviour of the active toxin during or following the signal peptide-independent secretion process.

Bacterial Proteins↗
PubMed1993

Hydrolytic properties of two cellulases of Trichoderma reesei expressed in yeast.

Two cellulases of the filamentous fungus Trichoderma reesei, cellobiohydrolase II (CBHII, EC 3.2.1.91) and endoglucanase I (EGI, EC 3.2.1.4), produced in recombinant strains of the yeast Saccharomyces cerevisiae, were tested in the hydrolysis of cellulose, xylan and other polymeric substrates. Both enzymes were active against unsubstituted, insoluble cellulose. CBHII had greater activity than EGI against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence for synergism was obtained when mixtures of the two enzymes were used with a constant total protein dosage. The EGI was also active against soluble substituted cellulose derivatives, whereas the activity of CBHII against these substrates was insignificant. Both enzymes were active against barley (1-->3,1-->4)-beta-glucan, but were inactive against (1-->3,1-->6)-beta-glucan (laminarin). An apparent low mannan-degrading activity of EGI against locust-bean (Ceratonia siliqua) gum galactomannan was not confirmed when homopolymeric mannan was used as substrate in a prolonged hydrolysis test. EGI exhibited considerably greater activity against insoluble, unsubstituted hardwood xylan than against amorphous cellulose. Soluble 4-O-methyl-glucuronoxylan was also attacked by EGI, although to a somewhat lesser extent than the unsubstituted xylan. By comparison with two purified xylanases of T. reesei, EGI produced xylo-oligosaccharides with a longer mean chain length when acting on both substituted and unsubstituted xylan substrates. CBHII was inactive against xylan.

Cellulose↗
PubMed1992

Escherichia coli HlyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes.

Synthesis and secretion of the 110kDa haemolysin toxin of Escherichia coli and other pathogenic Gram-negative bacteria are governed by the four genes of the hly operon. We have identified, by transposon mutagenesis, an E. coli cellular locus, hlyT, required for the synthesis and secretion of haemolysin encoded in trans by intact hly operons carrying the hly upstream regulatory region. Mutation of the hlyT locus specifically reduced the level of hlyA structural gene transcript 20-100-fold and thus markedly lowered both intracellular and extracellular levels of the HlyA protein. Genetic and structural analysis of the hlyT locus mapped it at co-ordinate 3680 kbp (minute 87) on the chromosome adjacent to the fadBA operon, and identified it specifically as the rfaH (sfrB) locus which is required for transcription of the genes encoding synthesis of the sex pilus and also the lipopolysaccharide core for attachment of the O-antigen of E. coli and Salmonella. Expression of the hly operon in the E. coli hlyT mutant was restored in trans by both the hlyT and rfaH genes, suggesting that the rfaH gene is an important activator of regulon structures that are central to the fertility and virulence of these pathogenic bacteria. DNA sequencing of the hlyT locus identifies the HlyT/RfaH transcriptional activator as a protein of 162 amino acids (Mr 18325) which shows no identity to characterized transcription factors.

Amino Acid Sequence↗
PubMed1991

Pilot scale production of a Trichoderma reesei endo-beta-glucanase by brewer's yeast.

Endo-beta-glucanase I (EGI) of Trichoderma reesei was produced in laboratory and pilot scale using recombinant strains of "bottom-fermenting" Saccharomyces cerevisiae. The gene eg/1 was integrated in the chromosome or an expression cassette was inserted on a multicopy plasmid. Expression levels were compared in a laboratory scale bioreactor. The best EGI-producing strain was cultivated in pilot scale. Adsorbent treatment was used to remove endogenous yeast proteins and other impurities from the culture filtrate during concentration. Effective pilot scale one-step purification of the EGI protein was obtained using DEAE-Sepharose, on which EGI was weakly bound. The purified enzyme reacted with antibodies prepared against T. reesei EGI and catalyzed the hydrolysis of both insoluble and soluble substrates.

Fermentation↗
PubMed1991

Proliferation in human bladder carcinoma measured by Ki-67 antibody labelling: its potential clinical importance.

Ki-67 is a monoclonal antibody which recognises a human nuclear antigen expressed in proliferating cells. The antibody was used to assess proliferation in primary human bladder tumours from 64 patients. Ki-67 index (the number of Ki-67 positive tumour cells divided by the total number of tumour cells %) was derived from 59 tumours. A wide range of Ki-67 indices were recorded, range 3.0-65.8%, mean 20.2%. The Ki-67 index correlated with known prognostic factors: T stage (P = 0.002) and histological grade (P less than 0.001), early stage disease and more differentiated tumours having lower Ki-67 indices. Patients with invasive disease (21 patients) had significantly higher Ki-67 indices than those with non-invasive disease (P = 0.01). Patients with metastatic disease at presentation (four cases) all had a Ki-67 index of greater than or equal to 29%. Ki-67 antibody staining is a simple technique for assessing the proliferation fraction than can be performed on a small amount of tissue taken at routine biopsy without prior injection of thymidine analogues.

Aged↗
PubMed1991

Mobile extracorporeal shockwave lithotripsy.

During the last 18 months, extracorporeal shockwave lithotripsy (ESWL) has been provided at Epsom District Hospital using a mobile unit containing a Dornier HM4 lithotriptor. Patients with upper ureteric and renal stones were selected for treatment, which was performed without anaesthesia or sedation as a day-case procedure; 83 patients were treated, 5 of them with bilateral stones. Seventy patients required 1 treatment session, 17 required 2 and 1 patient required 3. There were no serious complications but 3 patients needed ureteroscopy to remove obstructing stones. The overall success rate was 86%. The cost to treat each NHS patient was 253 pounds. Mobile lithotripsy as a day-case procedure is a safe and cost-effective means of treating urolithiasis and can be performed in a District General Hospital.

Ambulatory Care↗
PubMed1990

Evaluation of two new assays for tumor-associated antigens, CASA and OSA, found in the serum of patients with epithelial ovarian carcinoma--comparison with CA125.

Two new assays have been developed to measure tumor-associated antigens designated ovarian serum antigen (OSA) and cancer-associated serum antigen (CASA). Both assays are dual epitope ELISAs using the same capture monoclonal antibody (BC2); the second antibodies in the OSA and CASA assays are OM-1 and BC3, respectively. Using arbitrary cutoffs of 2.5 and 3.0 units/ml, 82 and 76% of 80 serum samples from ovarian cancer patients were positive for OSA and CASA, respectively, compared with 5 and 2.5% of samples from a control population of 40 women. A strong correlation was found between the two assays (r = 0.80, P less than 0.001). CA125 levels were obtained from 49 of the 80 samples; 82% of these samples were positive for CA125 (greater than 35 U/ml), 82% for OSA and 73% for CASA. Of the 9 samples negative for CA125, 3 were positive for OSA and 3 were positive for CASA. Serum OSA, CASA, and CA125 levels were determined in serial samples from 20 ovarian carcinoma patients throughout the course of their treatment. Clinical course was accurately reflected by CA125 levels in 85% of patients, by CASA in 65%, and by OSA in 75%. In 4 patients, a rise in CASA levels and, in 2 patients, a rise in OSA levels significantly predated rising CA125 levels to predict recurrence. Six of 7 serum samples obtained prior to positive second-look laparotomy were negative for CA125, while 4 were positive for OSA and 6 were positive for CASA. These results indicate that the OSA and CASA assays could be superior to CA125 for detection of small volume occult ovarian carcinoma.

Antigens, Neoplasm↗
PubMed1990

Pilot scale production of a heterologous Trichoderma reesei cellulase by Saccharomyces cerevisiae.

Cellobiohydrolase II of Trichoderma reesei was produced in laboratory and pilot scale using a transformant strain of Saccharomyces cerevisiae harbouring a multicopy expression plasmid. Different strategies were compared for concentration and partial purification of the enzyme produced in a 200 1 pilot cultivation. After efficient separation of biomass and sub-cellular particulate matter, a combination of ultrafiltration and adsorbent treatment for removal of protein impurities was used to provide a concentrate for chromatographic purification. Effective purification of the CBH II protein was obtained by passing the concentrate through a column of DEAE Sepharose, on which almost all the yeast proteins were adsorbed. The purified enzyme reacted with antibodies prepared against T. reesei CBH II and catalyzed partial solubilization of crystalline cellulose to soluble sugars.

Biotechnology↗
PubMed1989

Glycosylation is not required for the fusion activity of the G protein of vesicular stomatitis virus in insect cells.

The gene encoding the complete glycoprotein of vesicular stomatitis virus (VSV, Indiana serotype G protein) with potential asparagine-linked glycans at amino acid residues 179 and 338 was inserted into a baculovirus transfer vector pAcYM1, derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). The gene was placed under the control of the AcNPV polyhedrin promotor and expressed by the derived recombinant viruses to high levels in Spodoptera frugiperda cell lines. The principal product was the glycosylated version of the G protein, although some alternative (including probable degradation) forms of the protein were also observed. Similar recombinant viruses were prepared with deletion of one, the other, or both glycosylation sites of the VSV G protein. All forms expressed VSV G protein derivatives and mediated cell fusion and the production of syncytia at low pH. The fusogenic properties of the VSV G protein expressed on the surface of insect cells was prevented using anti-VSV sera, or by elevating the pH above 6.2. A reduction of the pH to 5.5, or 5.0, accelerated the rate of syncytia formation.

Animals↗
PubMed1989

Comparative antigenic analysis of Treponema pallidum laboratory and street strains.

The polypeptide and antigenic profiles of Treponema pallidum Nichols strain and two other more recently isolated 'street' strains of T. pallidum have been compared. PAGE and immunoblotting identified a 34.5 kDa polypeptide present in the Nichols strain which was absent from one of the other street strains. This polypeptide was shown to be associated with the axial filament in T. pallidum. Three other axial-filament-associated polypeptides of 37, 33 and 30 kDa were present in all strains examined. Axial filaments of all three strains were morphologically identical and all three strains were equally motile.

Antigens, Bacterial↗
PubMed1989

Cloning and expression of Treponema pallidum antigens in Escherichia coli.

A library of Treponema pallidum genomic DNA fragments produced by partial Sau3A digestion was established in Escherichia coli K12 using the plasmid vector pAT153. The library was screened using immune syphilitic rabbit serum and six recombinant phenotypes expressing eight treponemal polypeptides were detected. With two exceptions, all the recombinant gene products were the same size as polypeptides detected on Western immunoblots of T. pallidum. The genes encoding three novel gene products, with molecular masses in SDS-PAGE of 42, 17 and 15.5 kDa, which had not been cloned previously from T. pallidum were also identified. Monoclonal antibodies which reacted with four of the eight recombinant polypeptides were generated.

Antibodies, Monoclonal↗
PubMed1987

Extracorporeal shockwave lithotripsy: first 1000 cases at the London Stone Clinic.

One thousand patients underwent extracorporeal shockwave lithotripsy for renal and ureteric calculi at this clinic. An overall success rate of 91.8% was achieved (stone free or less than 2 mm fragments at three months) and for stones measuring 1 cm 96.3%. Lithotripsy produced extremely low morbidity, and no deaths have occurred at the clinic. Patients who had lithotripsy alone had a mean hospital stay of three days and in most instances were able to perform their full range of activities on discharge. Planned combination of lithotripsy with minimally invasive endourological procedures such as percutaneous nephrolithotomy and ureterorenoscopy has allowed us to extend the range of treatable cases to include large stones. Prophylactic use of Double-J ureteric stents in selected cases has reduced the incidence of obstruction by stone fragments after lithotripsy, thereby decreasing morbidity and hospital stay.

Adolescent↗
PubMed1987

Analysis of sheath and core structures of the axial filament of Treponema pallidum.

Electron microscopy and SDS-PAGE have been used to analyse the polypeptide and antigenic composition of the sheath and core components of the axial filament of Treponema pallidum. The sheath contains a major 37 kDa polypeptide which was solubilized by a combination of trypsin and urea treatments with concurrent loss of binding of anti-37 kDa monoclonal antibody. These studies also indicated some antigenic heterogeneity within the axial filament population. Trypsin treatment alone removed a number of antigenic determinants from the axial filament but left others intact, suggesting differences in their location within the sheath structure. A second 31.5 kDa polypeptide may also be associated with the sheath. The axial filament core comprises at least two components, an antigenically dominant 33.5 kDa polypeptide and a second of 34 kDa. The structure of the axial filament in T. pallidum and Treponema phagedenis biotype Reiterii was similar, but antigenic cross-reactivity of sheath and core components was incomplete.

Antigens, Bacterial↗
PubMed1987

Monoclonal antibodies directed against surface-associated polypeptides of Treponema pallidum define a biologically active antigen.

Murine monoclonal antibodies (Mabs) were raised against two outer-membrane-associated polypeptides of Treponema pallidum (47 and 44 kDa). Three Mabs against each polypeptide were investigated further and only those directed against the 44 kDa polypeptide were demonstrated to have immobilizing activity. The specificity of the Mabs for T. pallidum was determined by Western blotting procedures and the surface association of the antigens was inferred by immunogold electron microscopy. The clear distinction between these two polypeptides in their biological activity could help to explain the pathobiology of syphilis infections as the 47 kDa antigen has been shown to be associated with the outer membrane of this organism. Inactivity of such a surface-located protein in antibody-mediated anti-treponemal mechanisms could account for the observed ability of this organism to survive in the face of strong antibody responses in infection.

Animals↗