Biomedical subjects

J W Dale

Publications and source records attributed to J W Dale.

At least 19 recordsLinked to original sources

PubMed1992

Simplified procedures for detection of amplified DNA using fluorescent label incorporation and reverse probing.

Conventional methods of detecting polymerase chain reaction (PCR) products require equipment and expertise which may not be available in diagnostic bacteriology laboratories, especially in developing countries. To this end we have examined other methods of product detection, including fluorescein-12-dUTP incorporation during PCR amplification, and reverse probing, where the PCR product is used as the probe in a scaled down hybridization with a fixed capture probe consisting of a fragment entirely internal to the sequence of the PCR product. These techniques have shown sensitivities of 20 fg of purified mycobacterial DNA, which corresponds to approximately five cells.

Base Sequence↗

PubMed1992

Control of acyl-CoA carboxylase activity in mycobacteria.

Acyl-CoA carboxylase activity in four pathogenic mycobacteria and Mycobacterium smegmatis was shown with both acetyl-CoA and propionyl-CoA substrates. Only very low activity was detected in mycobacteria grown in host tissues or on egg-based media rich in lecithin and avidin. This appeared to be a result of severe depression of activity, as strains which could be grown both in host tissue and egg-based media, and in the relatively simple Dubos or Sauton's media showed 8 to 120-fold higher activity in the simpler media.

Animals↗

PubMed1992

Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains.

Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.

BCG Vaccine↗

PubMed1991

A modified broth dilution assay for antibiotic sensitivity testing of Mycobacterium avium-intracellulare using paraffin slide cultures.

Mycobacterium avium-intracellulare (MAI) can utilize paraffin wax as the sole carbon source in basal media. Paraffin slide culture (Para SL/C) has been employed for isolation and speciation of MAI derived from clinical sources. We have evaluated an adaptation of this method for antimicrobial sensitivity testing. Sixteen clinical isolates of MAI were tested against ciprofloxacin amikacin, and azithromycin by Para SL/C and compared with sensitivities obtained with a conventional broth microtiter procedure. The system can be performed rapidly over a median time interval of 6-8 days. The MIC was defined as the lowest concentration of antimicrobial agent necessary to inhibit growth on paraffin wax coated slides. With Para SL/C, the MIC values were determined at the time when the corresponding control tubes showed confluent growth. The procedure was reproducible with all of the agents tested. The MIC50 and MIC90 values obtained from the Para SL/C assay and from serial broth microtiter dilutions correlated well for ciprofloxacin and amikacin. However, results of the MIC50 and MIC90 for azithromycin did not correlate.

Amikacin↗

PubMed1991

Plasmid analysis of Mycobacterium avium-intracellulare (MAI) isolated in the United Kingdom from patients with and without AIDS.

One hundred and forty-seven isolates (128 strains) of Mycobacterium avium-intracellulare (MAI) were screened by agarose gel electrophoresis for the presence of plasmids. Plasmids were characterised according to size and by Southern hybridisation analysis of intact and restriction endonuclease-digested DNA. Two cloned MAI plasmids, pLR7 and pLR20, were used as probes. There was no significant difference in the rate of plasmid carriage in MAI strains isolated from patients with the acquired immuno-deficiency syndrome (AIDS) and from non-AIDS patients in the UK, but a higher rate of plasmid carriage was observed in a panel of American strains from AIDS patients. Plasmids were grouped into two broad categories: small (mostly 14-30 kb) and large (greater than 150 kb). Southern blot analysis identified two distinct groups of small plasmids, the majority of which showed homology with pLR7. Plasmids from this group were significantly more common in strains of serotypes 4 and 8 which are particularly associated with AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains.

Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like element, and these strains are highly polymorphic with regard to the site of integration in the chromosome. In contrast, Mycobacterium bovis BCG contains a single copy of the insertion element, and in all strains this copy is integrated at the same site in the chromosome. In this study, we determined the sequence of the single-copy insertion element from M. bovis BCG, IS987, and its flanking regions. The analysis of IS987 revealed that this element was virtually identical to the sequence of IS986 from M. tuberculosis. IS987 is located in a region containing direct repeats (DRs). The cloned flanking regions contained 20 virtually identical DRs of 36 bp, each separated by 35 to 41 bp of spacer DNA. Analysis of chromosomal DNA by the polymerase chain reaction revealed the presence of a cluster of 49 DRs, and IS987 is inserted in the 30th DR. Furthermore, the DR sequences were found to occur only in species of the M. tuberculosis complex and not in nine other mycobacterial species tested. Analysis of 14 M. tuberculosis strains revealed the presence of one insertion sequence element in the DR-containing region of eight strains, two insertion sequence elements were located in the DR region of five strains, and one strain did not contain an insertion sequence element in this region. Additionally, the DR-containing regions of these 14 M. tuberculosis strains were polymorphic in length and composition. We conclude that the DR cluster is a specific, hot-spot region for integration of insertion elements in the chromosome of M. tuberculosis complex strains.

Amino Acid Sequence↗

PubMed1990

The use of paraffin wax metabolism in the speciation of Mycobacterium avium-intracellulare.

Paraffin-wax utilisation or baiting of Mycobacterium avium-intracellulare (MAI) complex organisms and other 'atypical mycobacteria' and the inability of Mycobacterium tuberculosis to utilise paraffin are known and useful if forgotten facts. Strains of possible AIDS-related MAI have been introduced into Czapek broth devoid of any carbon source other than paraffin-wax coated slides. Replicate slides showing 'in situ' growth were subjected to the following battery of tests: acid alcohol fast staining and microscopic examination of 'in situ' growth, tellurite reduction in 3 days, absence of urea hydrolysis, inability to reduce nitrates and inability to hydrolyse Tween 80. The system has been utilised to isolate and identify MAI organisms in blood from AIDS patients. The simplicity, low cost, and reduced risk of contamination make the system especially suitable for small rural laboratories and field stations as well as laboratories in developing countries.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Does Mycobacterium tuberculosis have plasmids?

Evidence of the presence of plasmids in Mycobacterium tuberculosis is lacking, whereas they are widespread in some other mycobacterial species. We examined, by agarose gel electrophoresis, a total of 197 clinical isolates of M. tuberculosis, mostly resistant to one or more antibiotics, and were able to detect bands of apparently extrachromosomal DNA at a low level in some isolates. These presumptive plasmids could not be isolated by CsCl/ethidium bromide gradient ultracentrifugation, and may consist of unusual forms of DNA. The possible existence of single stranded plasmid DNA is discussed.

Antitubercular Agents↗

PubMed1990

Cloning and characterization of the gene for the '19 kDa' antigen of Mycobacterium bovis.

Monoclonal antibody CMA134.1 reacted with a protein antigen of apparent molecular mass 22 kDa from Mycobacterium bovis and Mycobacterium tuberculosis, and with an apparently 24 kDa antigen of Mycobacterium kansasii, but not with other mycobacteria or related species. This antibody was used to screen a gene library of M. bovis in lambda gt11 and identified a recombinant clone that expressed a protein with an apparent molecular mass of 19-20 kDa. Gene expression occurred from the lac promoter in lambda gt11, but used an unidentified vector promoter, possibly that of the replication primer RNA, in the final plasmid construct. The sequence of an 840 bp fragment was determined and shown to code for a product of 15 kDa. This sequence is identical to that, independently determined, of a gene from M. tuberculosis, usually referred to as the 19 kDa antigen. The reasons for the apparent size discrepancies are discussed.

Amino Acid Sequence↗

PubMed1990

Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family.

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).

Amino Acid Sequence↗

PubMed1990

Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis.

IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis.

Animals↗

PubMed1989

Oxacillin-hydrolysing beta-lactamases. A comparative analysis at nucleotide and amino acid sequence levels.

We have extended the sequence of the OXA-2 beta-lactamase which together with S1 mapping has enabled us to identify the promoter site for this gene. This lies in a region that is found upstream from a variety of resistance genes on different plasmids; each gene appears to have been inserted at the same specific site and to be expressed from the same promoter. The ancestral plasmid thus appears to function as a natural expression vector. The sequence of the recombination site at the 5' end of the OXA-2 gene shows a marked similarity with the attP sequence of lambda. DNA-probe analysis confirmed that the OXA-2 and OXA-3 beta-lactamases are related, and indicated no similarity with other beta-lactamase genes. However, a comparison of amino acid sequences demonstrates that the OXA-2, OXA-1 and PSE-2 beta-lactamases show some similarities to the typical class A enzymes, especially in the central helical domain of the latter, which is largely responsible for forming the active site of the enzyme. The three oxacillinases also show marked amino acid sequence similarity with the product of a regulatory gene, blaR1, required for beta-lactamase induction in Bacillus licheniformis.

Amino Acid Sequence↗

PubMed1989

Polymorphic repetitive DNA sequences in Mycobacterium tuberculosis detected with a gene probe from a Mycobacterium fortuitum plasmid.

Clinical isolates of Mycobacterium tuberculosis were shown by Southern blotting to contain DNA sequences hybridizing to a probe derived from a Mycobacterium fortuitum plasmid. Two such M. tuberculosis DNA fragments, isolated from a gene library, were used as probes to show restriction fragment length polymorphism in M. tuberculosis strains by detecting a repetitive sequence apparently located at different points on the chromosome. This could indicate the presence of a transposable element in M. tuberculosis which is partly homologous to a region of the M. fortuitum plasmid. The probes described can be used to fingerprint M. tuberculosis isolates, and in addition are capable of distinguishing M. tuberculosis from Mycobacterium bovis and BCG.

Blotting, Southern↗

PubMed1989

Transformation of Mycobacterium smegmatis with E. coli plasmids.

One limiting factor in the studies of tuberculosis and leprosy is the lack of a versatile system for genetic analysis and manipulation of mycobacteria. One strategy used in constructing a plasmid vector for transforming Mycobacterium smegmatis was to insert fragments of a mycobacterial plasmid into an Escherichia coli plasmid. We found that the parental E. coli plasmid is capable of self-replication in M. smegmatis yielding chloramphenicol-resistant colonies. Plasmids from different passages of one M. smegmatis transformant were recovered and characterised by restriction digest analysis. The plasmid from the earlier passage was found to be indistinguishable from the original plasmid by restriction analysis. Plasmids from later preparations, however, were found to have undergone modifications in the M. smegmatis host resulting in an apparent increase in transformation efficiency for M. smegmatis. These plasmids can be used as a shuttle vector for the genetic manipulation of mycobacterial species.

DNA, Bacterial↗

PubMed1979

The effect of a plasmid on growth and survival of E. coli.

We studied the growth characteristics of a pair of Escherichia coli strains, isogenic apart from the possession of a nonconjugative plasmid. There was no difference between the two strains when they were grown separately. In mixed culture, a second slow phase of growth that normally occurred following the end of rapid exponential growth, was absent from the plasmid-carrying strain. This resulted in a considerable decrease in the proportion of the cells that carried the plasmid after overnight incubation. The effect of different conditions of growth is reported. The plasmid-carrying strain survived extended incubation (150 days at 37 degrees C) as well as did the plasmid free strain separately. In a mixture, the proportion of plasmid-carrying cells declined rapidly, and none was detected after 100 days.

Cell Division↗

PubMed1978

High and low level tetracycline resistance in Shigella sonnei.

The results presented in this paper confirm the existence of two types of tetracycline resistance in Shigella sonnei. One group of strains had a high level of resistance to tetracycline and oxytetracyline, with a variable level of minocycline resistance. The second group had a lower level of tetracycline resistance and were sensitive to minocycline. After conjugation with E. coli K12 the selected E. coli transconjugants had the same levels of resistance as the parent Sh. sonnei strain, with one exception. Sh. sonnei 87 was resistant to a high level of tetracycline, but was able to transfer only low level resistance. It is suggested that Sh. sonnei 87 carriers two plasmids: pSU1, a conjugative plasmid conferring a low level of tetracycline resistance, and pSU2, a non-conjugative plasmid which confers a high level of resistance to tetracycline.

Microbial Sensitivity Tests↗

PubMed1977

Semiprecious alloys for cast restorations: a preliminary report.

A previous clinical evaluation indicated that semiprecious metals had a definite place in the construction of dowels and cores in endodontically treated teeth. 2 The results from the present in vitro study suggest that those alloys do have a place in the fabrication of single castings as type III and IV gold alloy substitutes. Although when compared with a type III gold alloy the behavior of the semiprecious alloys tested was less like that of a noble alloy, the semiprecious alloys appeared less active than the base-metal alloy. This suggests that the semiprecious alloys should be acceptably resistant to corrosion in the oral environment.

Crowns↗

PubMed1977

A clinical evaluation of semiprecious alloys for dowels and cores.

The semiprecious metals investigated and hard dental gold alloys may be manipulated in a similar manner. The semiprecious metals produced accurate and well-fitted dowels needing minimal adjustment. Being harder than type III gold alloy, they would be ideal for casting dowels and cores. Before considering these semiprecious alloys as final restorations, however, they must be further examined for resistance to tarnish and corrosion and their marginal adaptability and burnishability.

Crowns↗