Mycobacterial aetiology of sarcoidosis.
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Base Sequence↗
Biomedical subjects
M T Moss
Publications and source records attributed to M T Moss.
Typing by DNA probe of mycobacterial species isolated from patients with AIDS.
Restriction fragment length polymorphism (RFLP) types of Mycobacterium avium intracellulare and Mycobacterium kansasii isolated from patients with AIDS were examined. We demonstrate that one RFLP type is much more common than others which confirms previous findings. Carriage of individual RFLP types is constant over long periods of time. In addition, we document a disseminated infection in a patient with M. avium intracellulare of three RFLP types.
AIDS-Related Opportunistic Infections↗
IS902, an insertion element of the chronic-enteritis-causing Mycobacterium avium subsp. silvaticum.
An insertion sequence element of Mycobacterium avium subsp. silvaticum was isolated and its complete nucleotide sequence determined. IS902 is 1470 bp in size and is repeated 10-12 times per genome. An open reading frame of 1200 bp was identified, encoding a protein product of Mr 43932. This protein is highly similar to the predicted proteins of IS900 of Mycobacterium paratuberculosis, IS116 of Streptomyces clavuligerus and IS110 of Streptomyces coelicolor. IS902 lacks terminal inverted repeats and flanking direct repeats but displays insertion site specificity.
Amino Acid Sequence↗
p43, the protein product of the atypical insertion sequence IS900, is expressed in Mycobacterium paratuberculosis.
The novel mycobacterial insertion sequence IS900 was analysed by coupled transcription-translation, of both strands independently, in a cell-free E. coli extract using an exogenous promoter. This revealed only one protein product, p43, as predicted from the nucleotide sequence. The protein was readily translated in recombinant E. coli, using the tac promoter, though it did not appear as a major product by SDS-PAGE analysis. A synthetic peptide was used to generate and affinity-purify a specific anti-p43 antibody, which clearly identified the protein in recombinant E. coli. p43 was relatively stable in exponential phase and stationery phase bacteria, though a 28 kDa processed form was seen to accumulate over a period of hours. Both forms appeared in the soluble fraction of the bacterial lysate. The anti-p43 antibody also identified p43, as a 28 kDa processed product, in Western blots of protein extracts from Mycobacterium paratuberculosis, indicating a level of expression which would be unusually high for a classical transposase. These data have important implications for the relationship between IS900 and its host.
Amino Acid Sequence↗
Mycobacterium paratuberculosis DNA in Crohn's disease tissue.
Crohn's disease has long been suspected of having a mycobacterial cause. Mycobacterium paratuberculosis is a known cause of chronic enteritis in animals, including primates, but may be very difficult to detect by culture. IS900 is a multicopy genomic DNA insertion element highly specific for M paratuberculosis. A polymerase chain reaction (PCR) based on the 5' region of IS900 and capable of the specific detection of a single M paratuberculosis genome was developed. This was applied to DNA extracts of full thickness samples of intestine removed at surgery from 40 patients with Crohn's disease, 23 patients with ulcerative colitis, and 40 control patients without inflammatory bowel disease. Stringent precautions were taken that excluded contamination artefact. M paratuberculosis was identified in 26 of 40 (65%) Crohn's disease, in 1 of 23 (4.3%) ulcerative colitis, and in 5 of 40 (12.5%) control tissues. Positive samples from Crohn's disease were from both the small intestine and colon, those from control tissues were from the colon those from control tissues were from the colon only. All PCR internal control reactions were negative. The presence of M paratuberculosis in a small proportion of apparently normal colonic samples is consistent with a previously unsuspected alimentary prevalence in humans. The presence in two thirds of Crohn's disease tissues but in less than 5% of ulcerative colitis tissues is consistent with an aetiological role for M paratuberculosis in Crohn's disease.
Adult↗
Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum in long term cultures from Crohn's disease and control tissues.
Thirty one cultures were established in MG3 medium from the intestinal tissues of 29 patients, including 18 with Crohn's disease, five with ulcerative colitis, and six non-inflammatory bowel disease controls. All cultures grew either acid fast bacilli or uncharacterized spheroplasts. Pellets from these cultures were coded and assayed blind for M paratuberculosis and M avium subsp silvaticum using IS900- and IS902-PCR (polymerase chain reaction) assays, respectively. IS900 and IS902 are multicopy DNA insertion elements specific for these two organisms. Six Crohn's disease cultures and a single non-inflammatory bowel disease control were positive for M paratuberculosis. A further six cultures were positive for M avium subsp silvaticum, of which two each were from Crohn's disease, ulcerative colitis, and non-inflammatory bowel disease controls. The intensity of the IS900-PCR signals indicated very low numbers of M paratuberculosis organisms and bore no relation to visible spheroplastic or bacillary mycobacterial growth. The results suggest that M paratuberculosis isolated from man exists in a form which hardly replicates if at all when cultured in MG3 medium in vitro, and are consistent with the involvement of this known animal enteric pathogen in a proportion of chronic enteritis in man.
Base Sequence↗
OR supply storage takes on a new look.
As with any system, gridwall does not solve all the supply problems encountered in the OR; however, when properly applied, it is an excellent tool for improving performance and eliminating some of the problems inherent in traditional storage methods. Operating rooms that use case cart storage systems could benefit from a gridwall. Because supplies in these areas are minimal and are chosen irregularly, inventory control and locating items could be simplified greatly. Gridwall also can solve storage problems in other areas of the hospital. Depending on the volume of supplies and the quantities kept on site, the gridwall system can be used effectively in sterile supply storage areas, labor and delivery areas, emergency rooms, and laboratories, and on patient floors. Instrument set and small equipment storage is another possible way gridwall could be used in the hospital. Instruments frequently are misplaced or unavailable. The gridwall system, with its enhanced supply visibility, could solve this problem as well.
Efficiency↗
Specific detection of Mycobacterium paratuberculosis by DNA hybridisation with a fragment of the insertion element IS900.
This paper describes the evaluation of a newly developed DNA probe for Mycobacterium paratuberculosis. DNA probe PCR278 is a 278 bp fragment obtained by polymerase chain reaction (PCR) amplification of the 5'-region of IS900, an insertion element contained in the genome of M paratuberculosis. This DNA probe can specifically distinguish M paratuberculosis from a wide range of other organisms, including members of the M avium-M intracellulare complex. When used in conjunction with the PCR amplification technique DNA probe PCR278 could detect as little as 10 fg (equivalent to two genomes) starting material of M paratuberculosis genomic DNA. Use of PCR amplification assays based on IS900, for the detection of M paratuberculosis, and homologous IS elements found in disease isolates of M avium should greatly help our understanding of the role of these organisms in Crohn's disease and other chronic inflammatory disorders.
Base Sequence↗
Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis.
The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.
Amino Acid Sequence↗
DNA probes demonstrate a single highly conserved strain of Mycobacterium avium infecting AIDS patients.
Strains of the Mycobacterium avium intracellulare complex (MAIC) have become important colonisers of patients with acquired immunodeficiency syndrome (AIDS). Restriction fragment length polymorphisms were used to study the DNA from 88 MAIC isolates, including 51 derived from 47 AIDS patients. MAIC isolates from 33 of 45 AIDS patients were identical at the molecular level and distinct from the mycobacteria isolated from the stools of healthy subjects. The study also showed that serotyping correlates poorly with the genetic identity of these organisms. Mycobacterium paratuberculosis, which has been implicated in Crohn's disease, was not identified in any of the cultures studied.
Acquired Immunodeficiency Syndrome↗
Insertion elements in mycobacteria.
We have isolated and characterised a repetitive element from the genome of Mycobacterium paratuberculosis. This repetitive element shows many features characteristic of a bacterial insertion element.
Cloning, Molecular↗
Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans".
The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.
Cloning, Molecular↗
Out of the frying pan into the fire?
During the last 10 years or so, many departments of therapeutic radiology have lost their medical and fiscal independence by administrative incorporation into "departments of oncology." The author feels that the aims of oncology centers, viz., consolidation of cancer-related activities, promotion of the image of a functioning cancer center, and the multidiscipline care of cancer patients, do not justify the radiotherapist's loss of identity within such a complex.
Hospital Departments↗