Biomedical subjects

J W Dale

Publications and source records attributed to J W Dale.

At least 37 records · Page 2Linked to original sources

PubMed1994

Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M. tuberculosis.

The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance. Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated. Genes from M. leprae and M. tuberculosis encoding a biotinylated protein have been cloned and sequenced. Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins.

Acetyl-CoA Carboxylase↗

PubMed1994

Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories.

PCR is, in principle, a simple and rapid test for use in the detection of Mycobacterium tuberculosis. However, virtually no data are available on the reliability and reproducibility of the method. In order to assess the validity of PCR for the detection of mycobacteria in clinical samples, seven laboratories participated in a blinded study of 200 sputum, saliva, and water samples containing either known numbers of Mycobacterium bovis BCG cells or no added organisms. Each laboratory used its own protocol for pretreatment, DNA extraction, and detection of the amplification product. Insertion sequence IS6110 was the target for DNA amplification. Several participating laboratories reported high levels of false-positive PCR results, with rates ranging from 3 to 20% and with one extreme value of 77%. The levels of sensitivity also ranged widely among the different participants. A positive PCR result was reported for 2 to 90% of the samples with 10(3) mycobacteria. Although most participants did include control tests to check the sensitivity and specificity of the PCR, the sequence of operations from sample pretreatment to purification of DNA from bacteria was not always monitored adequately. During these procedures cross-contaminating DNA was introduced and/or bacterial DNA was lost. The results of the study show that the implementation of an effective system for monitoring sensitivity and specificity is required before the PCR can be used reliably in the diagnosis of tuberculosis.

Base Sequence↗

PubMed1993

Transpositional activity of IS986 in Mycobacterium smegmatis.

The insertion sequence IS986 (also known as IS6110) has been widely used for typing Mycobacterium tuberculosis isolates, due to the extensive multiple polymorphism shown using this probe. Although this polymorphism is presumed to be due to the mobility of IS986, transposition of this element has not previously been demonstrated in the laboratory. Using artificial composite transposons constructed in a vector unable to replicate in mycobacteria, we have succeeded in demonstrating the mobility of IS986 in M. smegmatis, apparently through cointegrate formation and transposition. The apparently random nature of IS986 insertions in M. tuberculosis is advantageous for transposon mutagenesis, although its efficient use will require more effective delivery systems.

Base Sequence↗

PubMed1993

Restriction fragment length polymorphism analysis of epidemiologically related Mycobacterium tuberculosis isolates.

Restriction fragment length polymorphism (RFLP) analysis of a large number of Japanese isolates of Mycobacterium tuberculosis, containing isolates from small outbreaks of M. tuberculosis infection, and clinical isolates of M. bovis BCG, was carried out using a DNA probe derived from the insertion sequence IS986. Clinical isolates of M. tuberculosis had a high degree of RFLP. The occurrences of the IS element varied from 1 to 19, the majority of isolates having 8 to 15 copies. Very similar fingerprints, however, were seen among strains isolated in the Kanto district. In particular, 3 strains were of the same pattern with or without an additional band. Similarity of the banding patterns of strains isolated in the same district was observed in other areas. Six groups of strains, each group arising from a suspected common source of infection, were analyzed. Of these, 5 showed identical fingerprints within each group, but one showed different fingerprints. RFLP patterns of three strains isolated from individuals with lymphadenitis developed about two months after BCG vaccination, and one strain isolated from a bladder cancer patient with BCG instillation therapy were identical to those of BCG-Tokyo which had been used for the vaccination and therapy. These results confirm that RFLP analysis using IS986 is a suitable tool for epidemiology of tuberculosis.

DNA Fingerprinting↗

PubMed1993

Construction and use of integrative vectors to express foreign genes in mycobacteria.

We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin-resistance gene into a 'suicide' vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18 kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot-and-mouth disease virus VP1 140-160 epitope were successfully cloned into the 18 kDa gene and expression in M. smegmatis was obtained.

Amino Acid Sequence↗

PubMed1993

Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology.

DNA fingerprinting of Mycobacterium tuberculosis has been shown to be a powerful epidemiologic tool. We propose a standardized technique which exploits variability in both the number and genomic position of IS6110 to generate strain-specific patterns. General use of this technique will permit comparison of results between different laboratories. Such comparisons will facilitate investigations into the international transmission of tuberculosis and may identify specific strains with unique properties such as high infectivity, virulence, or drug resistance.

Base Sequence↗

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

Differential induction of peroxisomal and microsomal fatty-acid-oxidising enzymes by peroxisome proliferators in rat liver and kidney. Characterisation of a renal cytochrome P-450 and implications for peroxisome proliferation.

The induction of renal fatty-acid-oxidising enzymes has been investigated following short-term exposure to a group of structurally diverse peroxisome proliferators and compared to the more extensively documented hepatic responses in the rat. There was a marked compound dependence on induction of both cytochrome P-450-IVA1-dependent omega-hydroxylation of lauric acid and enzymes of the peroxisomal fatty acid beta-oxidation pathway (measured as cyanide-insensitive palmitoyl-CoA oxidation and enoyl-CoA hydratase). Cytochrome P-450 IVA1 (or a very closely related isoenzyme in the same gene family) was a major constitutive haemoprotein in rat kidney microsomes and actively supported the omega-hydroxylation of lauric acid. This activity was induced 2-3-fold by peroxisome proliferators such as clofibrate, di-(2-ethylhexyl)phthalate, bezafibrate and nafenopin. By using a cDNA probe to the cytochrome P-450 IVA1 gene in Northern blot analysis, we have shown that increased renal and hepatic omega-hydroxylation of lauric acid, after treatment with peroxisome proliferators is a consequences of a substantial increase in the mRNA coding for this haemoprotein. In addition, programming of an in vitro rabbit reticulocyte translation system with both renal and hepatic RNA resulted in the synthesis of similar (if not identical) cytochrome-P-450-IVA1-related polypeptides. Furthermore, we have provided Western blot evidence that both rat liver and kidney microsomes contain two closely related cytochrome P-450 IVA1 polypeptides, the major one characterised by a monomeric molecular mass of 51.5 kDa (identical to authentic, purified hepatic cytochrome P-450 IVA1) and a minor one of 52 kDa. The kidney-supported fatty acid omega-hydroxylase activity was refractory to inhibition by a polyclonal antibody to liver cytochrome P-450 IVA1, which may be related to the existence of two closely related (but immunochemically distinct) fatty acid hydroxylases in this tissue. Our studies have also demonstrated that certain of the compounds tested (including clofibrate, bezafibrate and nafenopin) induced renal fatty acid beta-oxidation, mirroring the increased omega-hydroxylase activity in the endoplasmic reticulum. Our studies have also indicated that the kidney was more refractory to induction of the endoplasmic reticulum and peroxisomal fatty-acid-oxidising enzymes than the liver. Taken collectively, our data is strongly suggestive of a possible linkage of the renal fatty acid oxidative enzymes in these two organelles, a situation that also occurs in the liver. In addition, our studies have provided a possible conceptual framework that may rationalise the decreased susceptibility of the k

Amino Acid Sequence↗