Mycobacterium tuberculosis proteins. Structure, function, and immunological relevance.
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Antibody Formation↗
Biomedical subjects
A B Andersen
Publications and source records attributed to A B Andersen.
A Latex agglutination test for rapid serodiagnosis of leprosy in the field.
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Enzyme-Linked Immunosorbent Assay↗
Cloning of the lysA gene from Mycobacterium tuberculosis.
The lysA and proC genes of Mycobacterium tuberculosis were cloned by screening of a recombinant lambda gt11 M. tuberculosis DNA library for phages able to complement lysA and proC Escherichia coli mutants. The lysA gene encodes diaminopimelic acid decarboxylase which catalyzes the conversion of diaminopimelic acid (DAP) to lysine. The lysA gene from M. tuberculosis encodes a 44-kDa protein, as determined by maxicell experiments. The nucleotide sequence of the structural gene was established. The deduced amino acid sequence was found to exhibit significant homology (from 55% to 73% similarity, and from 27% to 53% identity) to DAP decarboxylase sequences from other bacterial species.
Amino Acid Sequence↗
Polymerase chain reaction for detection of Mycobacterium tuberculosis in sputum.
The polymerase chain reaction (PCR) was evaluated in a trial which, with respect to the positive-to-negative ratio, approximated the situation of a diagnostic laboratory in a tuberculosis-endemic area. Three hundred sputum samples were included in the study, of which one-third were known to contain mycobacteria as judged by direct microscopy. The repetitive insertion sequence IS6110/IS986 of Mycobacterium tuberculosis was used as a target. The samples were spiked with DNA from a modified IS6110/IS986 sequence, which gives rise to PCR products easily distinguished from PCR products amplified from chromosomal Mycobacterium tuberculosis DNA. This allowed identification of samples that contained substances inhibitory to the Taq polymerase. The detection limit of the assay was 0.05 pg to 0.5 pg of purified Mycobacterium tuberculosis DNA, corresponding to 10 to 100 organisms. The sensitivity and specificity of the PCR was compared with that of conventional microscopy and culture. It was concluded that this method is fast and sensitive, but that culture currently is crucial for assessing viability and thus infectivity.
Base Sequence↗
Role of cathepsin D in the degradation of human serum albumin by peritoneal macrophages and veiled cells in antigen presentation.
Murine peritoneal macrophages (PMO) and veiled cells (VC) isolated from the thoracic duct of irradiated lymphadenectomized (MNLX) mice presented intact human serum albumin (HSA) to stimulated T lymphocytes, but VC were not as effective as PMO in presenting the antigen. Pepstatin A significantly inhibited the presentation of HSA by VC. Lysates prepared from PMO degraded [125I]HSA at pH 4.0 to peptides as demonstrated by SDS-polyacrylamide-gel electrophoresis and autoradiography. Degradation was inhibited by pepstatin A, suggesting that cathepsin D might be responsible for processing the antigen. In contrast, lysates prepared from VC did not degrade [125I]HSA. The localization of cathepsin D, by light microscopy, was examined on cytospins of PMO and VC by means of a peroxidase antiperoxidase technique (PAP). Cathepsin D was found in vacuoles in the cytoplasm of PMO and, in some cases, appeared to be bound to some areas of the cell surface, but the enzyme could not be detected in VC.
Animals↗
Detection of antibodies in serum against M. tuberculosis using western blot technique. Comparison between sarcoidosis patients and healthy subjects.
Antibodies in serum of IgG and IgA class against Mycobacterium tuberculosis were detected by Western blot technique in 32 patients with pulmonary sarcoidosis and in 10 BCG and 10 non-BCG vaccinated healthy individuals. All three groups demonstrated multiple IgG and IgA antibodies against Mycobacterium tuberculosis protein antigens. Preabsorption of the sera with an Escherichia coli antigen did not influence the results. There were no consistent differences in the antibody profiles in patients with sarcoidosis and healthy subjects.
Adult↗
The Mycobacterium tuberculosis 38-kDa antigen: overproduction in Escherichia coli, purification and characterization.
The 38-kDa protein (Ag38) of the Gram+ bacterium, Mycobacterium tuberculosis H37Rv, is an immunodominant antigen of potential utility for diagnosis and vaccine development. Assessment of this potential requires large amounts of the purified protein that would be difficult, if not impossible, to obtain from M. tuberculosis itself. The gene coding for Ag38 had been previously cloned and in the present study was expressed as an unfused protein in Escherichia coli under the control of strong transcriptional (bacteriophage lambda pLpR) and translational (atpE) signals. Fermentation of the recombinant E. coli K-12 strain CAG629[pMS9-2], which is deficient in Lon protease and the heat-shock response, produced recombinant Ag38 (reAg38) at high levels (about 10% of total cellular protein). The reAg38, which accumulated as inclusion bodies, was completely solubilized in 6 M guanidine.HCl, refolded and purified to apparent homogeneity. The product showed the expected amino acid composition and M(r), and had similar reactivities as the native protein with three different mAb. Polyclonal antibodies raised against reAg38 reacted strongly with the native antigen in enzyme-linked immunosorbent assay. These results demonstrate that reAg38, which cannot be distinguished antigenically from the native protein of M. tuberculosis, can be prepared in quantity from E. coli.
Amino Acid Sequence↗
Structure and function of a 40,000-molecular-weight protein antigen of Mycobacterium tuberculosis.
A gene encoding a protein antigen from Mycobacterium tuberculosis with a molecular weight of 40,000 has been sequenced. On the basis of sequence homology and functional analyses, we demonstrated that the protein is an L-alanine dehydrogenase (EC 1.4.1.1). The enzyme was demonstrated in M. tuberculosis and Mycobacterium marinum but not in Mycobacterium bovis BCG. The enzyme may play a role in cell wall synthesis because L-alanine is an important constituent of the peptidoglycan layer. Although no consensus signal sequence was identified, we found evidence which suggests that the enzyme is secreted across the cell membrane. The enzyme was characterized and purified by chromatography, thus enabling further studies of its role in virulence and interaction with the immune system of M. tuberculosis-infected individuals.
Alanine Dehydrogenase↗
Results of the third immunology of leprosy/immunology of tuberculosis antimycobacterial monoclonal antibody workshop.
An international workshop was sponsored by the World Health organization to screen new antimycobacterial monoclonal antibodies and to identify antibodies which could be recommended as standard reagents giving consistent results under differing assay conditions. Fifty-eight antibodies were submitted to the workshop by eight independent laboratories. Nineteen of the antibodies recognized antigens distinct from those identified in earlier workshops, defining at least 10 new protein antigens. Monoclonal antibodies characterized in the workshop provide a set of convenient reagents for further characterization of mycobacterial antigens.
Animals↗
The W ileal reservoir: long-term assessment after proctocolectomy for ulcerative colitis and familial polyposis.
BACKGROUND: This report examines the viability of the W reservoir as a reliable option for the treatment of ulcerative colitis and familial polyposis and studies W reservoir adaptation as reflected by changes in compliance and stool frequency. METHODS: Since 1984, 109 patients have undergone proctocolectomy with W reservoir reconstruction. Ileal reservoir static compliance was measured in 70 and 57 patients at 2 and 12 months after ileostomy takedown and in 25 patients at 3 years. Compliance was calculated as the change in volume over change in pressure. RESULTS: Twenty-four-hour stool frequency decreased from 7.3 +/- 0.2 at 2 months to 4.9 +/- 0.2 at 1 year for patients with ulcerative colitis and from 6.3 +/- 0.4 to 3.4 +/- 0.4 for patients with familial polyposis (p less than or equal to 0.05). Compliance increased from 12.7 +/- 0.6 ml/mm Hg to 14.3 +/- 0.6 ml/mm Hg between 2 months and 1 year. No significant increase in compliance occurred after 1 year. Ninety-six percent of patients were continent during the day at 12 months although 10% experienced occasional minor leakage at night. Average postoperative morbidity (for example, small-bowel obstruction, anastomotic complications) was 35%. No operative deaths, pelvic sepsis, or reservoir loss occurred. CONCLUSIONS: We conclude that W ileal reservoirs (1) are an excellent option for ileal reservoir reconstruction, (2) have optimal functional and compliance properties versus lower capacity designs and straight ileoanal pull-through procedures, and (3) maintain stable compliance characteristics and functional reservoir volume after the initial year of adaptation.
Adenomatous Polyposis Coli↗
Neurological correlates of unilateral and bilateral "strokes" of the middle cerebral artery in the rat.
Rats with unilateral lesions of the middle cerebral artery (MCA) were tested for the ability to detect (touch) and remove a square of adhesive tape from each forepaw, and for performance on a number of neurological tests (e.g., placing and hopping reflexes, activity). Rats with MCA damage showed deficits in both touching and removing the tape from the paw contralateral to the damage, but not ipsilateral to the damage, while performing within normal limits on the other tests. After scores of the MCA rats dropped into the control group range on the adhesive tape test, they sustained damage to the opposite MCA. This did not reinstate the original deficit, suggesting that the recovery seen after the unilateral lesion was not mediated by the opposite cortex. The second lesion, however, caused a deficit in removing the adhesive tape from the limb opposite the new stroke. Some of the rats that originally had sham operations received bilateral MCA lesions at this time. These animals showed much more severe deficits on the adhesive tape test than the rats with sequential strokes. Rats with bilateral MCA damage (simultaneous or sequential) also slipped on a long narrow plank more often than control animals. Nimodipine did not enhance recovery on any of the behavioral measures.
Animals↗
Recording single motor unit activity of human nasal muscles with surface electrodes: applications for respiration and speech.
A method is presented for recording nasal single motor unit (SMU) potentials from the skin surface using a 3-pole 'branched' bipolar electrode. Stable, high-quality recordings of single motor unit activity were obtained for up to 3 h. Branched electrode arrays were capable of locating an SMU's maximal voltage point within 5 mm. We examined nasal SMU discharge patterns in relation to respiration in 9 adult humans. The majority of SMUs which discharged during quiet breathing began firing late in expiration and ceased firing in mid-inspiration, other SMUs discharged only during expiration, and a few fired continually with frequency modulation during breath cycles. With increased ventilation, new SMUs were recruited, and previously active SMUs increased the frequency and duration of their discharge. We examined the discharge of 13 units (5 adults) which discharged during speech but were never active during quiet or moderately increased breathing. Some of these SMUs fired during production of nasal consonants, and others were active for articulations involving facial movements (bilabial stops, labio-dental fricatives, and vowels produced with lip movement). By providing information about motor neuron recruitment which cannot be obtained from gross EMG recordings, surface recording of unit potentials may be useful in studying the central nervous control of the nasal upper airway, face, and neck for respiration and speech.
Electrodes↗
MPB 64 possesses 'tuberculosis-complex'-specific B- and T-cell epitopes.
We have developed monoclonal antibodies (MoAb) reactive with a protein from Mycobacterium tuberculosis of apparent molecular mass 24 kDa. This protein was shown to be identical with MPB 64 (Harboe et al.,) MoAb bound to four different epitopes of which two were restricted to the 'tuberculosis complex' and two were also found in mycobacteria not belonging to the 'tuberculosis complex'. The cross-reactive MoAb demonstrate that MPB 64 is present in more mycobacterial species than previously assumed. MPB 64 was shown to induce strong delayed type hypersensitivity (Dth) reactions in outbred guinea pigs immunized with M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). No reaction was observed in animals immunized with mycobacteria not belonging to the 'tuberculosis complex'. The Dth-inducing capacity of MPB64 was compared with that of another 24 kDa protein purified from M. tuberculosis and of the previously described 38 kDa protein. The Dth responses to these three antigens were further analysed in four inbred guinea pig strains. A genetic restriction of the ability of the animals to respond to MPB 64 as well as to the 38 kDa protein was observed.
Animals↗
Lipopolysaccharide-instructed, cryopreserved, human monocytes sequentially convert plasma fibrinogen to fibrin and lyse the fibrin formed.
Cryopreserved human monocytes have been examined for their procoagulant and profibrinolytic capacities on lipopolysaccharide (LPS) stimulation, when given the possibility to act upon fibrinogen in heparinized plasma. As shown by the appearance of fibrinopeptide A, indicating thrombin action on the fibrinogen molecule, and by the appearance of D-Dimer, indicating the action of plasmin on fibrin, it is apparent that LPS-instructed monocytes give rise to fibrin which is subsequently lysed. Thus, delineation (fibrin formation) appears to be followed by fibrin removal (restitution).
Blood Coagulation Factors↗
Sensorimotor cortical lesion effects and treatment with nimodipine.
Rats with unilateral ablations of the sensorimotor cortex and others with control operations were tested for their ability to touch and remove adhesive tape applied to both forelimbs. Half of each group was administered a calcium channel antagonist (nimodipine) for two weeks following the lesions and the other half received vehicle. The rats with lesions showed a bias to remove the ipsilateral stimulus first and exhibited contralateral deficits relative to control animals. Nimodipine was shown to reduce the contralateral stimulus removal time when the animals began testing two weeks after surgery, but not when testing began 1 day after surgery and overlapped the period of drug administration. Lesion effects also appeared on tests for neurologic impairment and activity, but nimodipine did not reduce these deficits. These findings indicate that nimodipine has the potential to reduce some deficits after sensorimotor cortical lesions, but that the effects of this drug may be task specific.
Animals↗
Evidence that protein antigen b of Mycobacterium tuberculosis is involved in phosphate metabolism.
Protein antigen b (Pab) of Mycobacterium tuberculosis has previously attracted interest because of its immunological and diagnostic relevance. In this study we present evidence that Pab possesses a signal sequence and is secreted from the cytoplasm of M. tuberculosis. The synthesis of Pab is enhanced under phosphate starvation indicating that the protein is involved in phosphate metabolism in M. tuberculosis.
Amino Acid Sequence↗
Comparison of the immunological activity of five defined antigens from Mycobacterium tuberculosis in seven inbred guinea pig strains. The 38-kDa antigen is immunodominant.
We have examined the immunological activity of five affinity-purified protein antigens from Mycobacterium tuberculosis in seven inbred and one outbred guinea pig strains. The test systems were measurements of delayed-type hypersensitivity (Dth) responses, lymphocyte stimulation assays (LS), and antibody response measurements. The results showed significant differences in the immunogenicity of the single-protein antigens and, when the antigens were considered separately, highly significant guinea pig strain differences. The outbred guinea pig strain behaved as a Dth high responder to all antigens studied. The order of magnitude of the Dth responses was not usually correlated with that of the corresponding antibody responses for the individual guinea pig strain-antigen combinations. In particular, when compared with the other strains, strain 2 guinea pigs generally gave the lowest Dth, but the highest antibody responses. A 38,000 molecular weight protein, possessing M. tuberculosis complex-specific B-cell determinants, appeared immunodominant in 5 out of 7 strains. Our Dth data in the inbred strains further suggest the presence of an M. tuberculosis-specific T-cell epitope. A T-cell line, 11D9, derived from the high-responder guinea pig strain 13 reactive to this protein, was shown to be able to confer a tuberculin-like skin reaction in vivo. LS assays with recombinant 38-kDa protein and truncated versions of the protein mapped the 11D9-defined T-cell epitope to the middle part of the molecule.
Amino Acid Sequence↗
Polymerase chain reaction for detection of Mycobacterium tuberculosis.
A polymerase chain reaction for the specific detection of mycobacteria belonging to the Mycobacterium tuberculosis complex was developed. Using a single primer pair derived from the nucleotide sequence of protein antigen b of M. tuberculosis, we achieved specific amplification of a 419-base-pair DNA fragment in M. tuberculosis and M. bovis. After DNA was extracted from mycobacteria by using a simple, safe lysis procedure, we detected the 419-base-pair sequence in samples containing few mycobacteria. Preliminary data suggested that this technique could be applied to clinical specimens for early and specific diagnosis of tuberculosis.
Base Sequence↗