Biomedical subjects

B T Cookson

Publications and source records attributed to B T Cookson.

25 records · Page 2Linked to original sources

PubMed1989

Biological activities and chemical composition of purified tracheal cytotoxin of Bordetella pertussis.

Specific destruction of ciliated epithelial cells lining the large airways is the primary respiratory tract cytopathology associated with human Bordetella pertussis infections. We have purified a single low-molecular-weight glycopeptide, tracheal cytotoxin (TCT), that appears to cause this pathology. By using a combination of solid-phase extraction and reversed-phase high-pressure liquid chromatography, about 700 nmol of biologically active peptide can be isolated from 1 liter of B. pertussis culture supernatant (approximately 60% yield). TCT at concentrations of 1 microM destroyed the ciliated cell population when incubated with respiratory epithelium in vitro. This concentration of TCT is similar to the concentrations found in the culture supernatant of growing B. pertussis. Purified TCT also inhibited DNA synthesis of hamster trachea epithelial cells in a quantitative, dose-dependent fashion. Endotoxin was not detected in the purified material, and neither B. pertussis nor Escherichia coli endotoxin could duplicate the biological activities of TCT. Amino acid and amino sugar analyses of purified TCT revealed the presence of glucosamine, muramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1. This suggests that TCT, the released ciliostatic principle of B. pertussis, is a disaccharide tetrapeptide subunit of peptidoglycan.

Amino Acids↗

PubMed1988

Dermonecrotic toxin and tracheal cytotoxin, putative virulence factors of Bordetella avium.

We examined Bordetella avium for virulence factors common to Bordetella pertussis, including pertussis toxin, filamentous hemagglutinin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin. B. avium produced a dermonecrotic toxin and a tracheal cytotoxin. The dermonecrotic toxin of B. avium is a 155,000-molecular-weight, heat-labile protein which was lethal for mice, guinea pigs, young chickens, and turkey poults and produced dermonecrosis when injected intradermally into guinea pigs, chickens, and turkey poults. High-pressure liquid chromatography of B. avium culture supernatant fluid revealed the presence of a tracheal cytotoxin chemically identical to that produced by B. pertussis. B. avium isolates were negative for B. pertussis-like filamentous hemagglutinin and pertussis toxin when assayed with antibody against B. pertussis filamentous hemagglutinin and pertussis toxin. Furthermore, B. avium failed to induce the clustered CHO cell morphology characteristic of pertussis toxin. Adenylate cyclase assays indicated that B. avium does not produce an extracytoplasmic adenylate cyclase, even after passage through embryonated turkey eggs. Since production of virulence proteins by B. pertussis is regulated by growth in media containing nicotinamide or MgSO4 or by growth at reduced temperatures, we determined the effect of these supplements and growth conditions on production of dermonecrotic toxin by B. avium. Production of dermonecrotic toxin in B. avium was not altered by growth in media containing 100 microM FeSO4 or 500 micrograms of nicotinamide per ml or by growth at 25 or 42 degrees C, but production was significantly decreased by growth in media containing 20 mM MgSO4 and slightly reduced by growth in media containing 500 micrograms of nicotinic acid per ml. These studies revealed that B. avium is similar to B. pertussis in that both species produce a dermonecrotic toxin and a tracheal cytotoxin and production of dermonecrotic toxin is regulated by nicotinamide and MgSO4. The presence of dermonecrotic toxin and tracheal cytotoxin in all Bordetella species indicates that these products may be important virulence factors in bordetellosis.

Adenylate Cyclase Toxin↗

PubMed1988

Structure and functions of the Bordetella tracheal cytotoxin.

Of the various toxins and virulence-related factors produced by Bordetella pertussis, only one has been demonstrated to reproduce the specific respiratory epithelial cytopathology characteristic of the pertussis syndrome. That molecule is tracheal cytotoxin (TCT), which is released by B. pertussis during log phase growth. An HPLC-based method has allowed us to purify TCT from culture supernatants, resulting in a preparation with undetectable levels of endotoxin and which is homogeneous by all analytical criteria, including fast atom bombardment-mass spectrometry (FAB-MS). Exposure to purified TCT specifically damages ciliated epithelial cells, causing ciliostasis and extrusion of these cells. Other species of Bordetella, which generate remarkably similar respiratory tract infections and ciliated cell-specific pathology, produce a chemically identical TCT. Compositional analysis and FAB-MS have unambiguously defined the structure of TCT as N-acetylglucosaminyl-1, 6-anhydro-N-acetylmuramylalanyl-gamma-glutamyl-diaminopimelylalanine+ ++. This particular disaccharide-tetrapeptide composition and arrangement reveals that TCT is apparently formed by cleavage of peptidoglycan. Unlike other gram-negative bacteria, however, B. pertussis seems to be very selective in its release of cell wall fragments: greater than 95% of soluble peptidoglycan in culture supernatants is TCT. The structure of TCT places it in the "muramyl peptide" family, a group of structurally related molecules that are responsible for a diverse array of biological activities. Neisseria gonorrhoeae also releases muramyl peptides (one of which is identical to TCT) that can cause ciliated cell-specific damage like that seen during gonococcal infection of fallopian tube mucosa. In addition, TCT is absolutely identical in structure to FSu, a potent sleep-promoting factor isolated from humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Major fragment of soluble peptidoglycan released from growing Bordetella pertussis is tracheal cytotoxin.

Bordetella pertussis is known to release a factor which promotes the loss of ciliated respiratory epithelium and copurifies with a soluble peptidoglycan (PG) fragment termed tracheal cytotoxin (TCT). The objective of this study was to determine whether pertussis organisms turn over and release PG derivatives in addition to TCT. B. pertussis Tohama (phase III) was grown in liquid Stainer-Scholte medium containing [3H]diaminopimelic acid (DAP) to label PG specifically, washed to remove free label, and suspended in fresh medium without [3H]DAP. Molecular sieve chromatography of supernatants obtained from such cultures revealed a single included peak of 3H, the elution volume of which corresponded roughly to a disaccharide peptide monomer standard (ca. 10(3) daltons). This material (i) contained [3H]DAP in acid-hydrolyzable linkage, (ii) comigrated with 1,6-anhydro-N-acetylmuramic acid-containing disaccharide peptides on paper chromatography, (iii) was resistant to degradation by mild alkali, and (iv) was indistinguishable from authentic TCT by high-voltage paper electrophoresis and two reversed-phase high-performance liquid chromatography systems. Together, the data suggest that B. pertussis releases a markedly homogeneous set of PG fragments, consisting principally of TCT, and that TCT is possibly a nonreducing, anhydromuramic acid-containing fragment or a cyclic PG derivative.

Bacterial Toxins↗

PubMed1987

Genetic characterization and regulation of the nadB locus of Salmonella typhimurium.

The nadB locus encodes the first enzyme of NAD synthesis. It has been reported that this gene and nadA are regulated by a positive regulatory protein encoded in the nadB region. In pursuing this regulatory mechanism, we constructed a fine-structure genetic map of the nadB gene. The region appears to include a single complementation group; no evidence for a positive regulatory element was found. Several mutations causing resistance to the analog 6-aminonicotinamide mapped within the structural gene and probably cause resistance to feedback inhibition. Regulatory mutations for nadB were isolated. These mutants mapped far from nadB near the pnuA gene, which encodes a function required for nicotinamide mononucleotide transport. The regulatory mutations appear to affect a distinct function encoded in the same operon as pnuA.

Alleles↗

PubMed1983

6-Aminonicotinamide-resistant mutants of Salmonella typhimurium.

Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD.

6-Aminonicotinamide↗

PubMedDate not supplied

Computer programs that teach the interpretation of image-based laboratory tests.

OBJECTIVE: To review the effort of the University of Washington (UW) Department of Laboratory Medicine to develop and use personal computer programs to teach the interpretation of image-based clinical laboratory tests to medical technologists and other health care workers. DATA SOURCES: Professional journals and books; Software owned by and licensed by the University of Washington. STUDY SELECTION: Not applicable. DATA EXTRACTION: Not applicable. DATA SYNTHESIS: We have been developing interactive personal computer (PC) programs for teaching image-based laboratory tests to medical technologists and other health care workers. The programs, called "Laboratory Tutors," are useful for teaching microscope-based tests and tests based on electrophoresis. Our programs include ANA-Tutor, which teaches the immunofluorescence assay for anti-nuclear antibodies; Gram Stain-Tutor, which teaches the direct Gram stain; Electrophoresis-Tutor, which teaches the interpretation of agarose gel protein electrophoretic patterns; Urinalysis-Tutor, which teaches the microscopic examination of urine sediment; in addition to other programs. The tutorials are all based on high-quality digital images that were acquired and processed using digital imaging systems. They require minimal computer literacy and have a number of advantages over standard approaches to teaching image-based laboratory tests. The computer tutorials are used in UW's medical technology and medical school curriculum, where they are used as supplements to traditional instruction. CONCLUSION: Laboratory tutors are computer programs that use high resolution digital images to teach the interpretation of image-based laboratory tests. We plan to continue to develop these programs, study their educational effectiveness, and update them periodically.

Clinical Laboratory Information Systems↗