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Biomedical subjects

C E Barry

Publications and source records attributed to C E Barry.

50 records · Page 3Linked to original sources
PubMed1996

Drug sensitivity and environmental adaptation of mycobacterial cell wall components.

The intrinsic resistance of many mycobacterial species to chemotherapy is largely attributable to their impermeable cell wall. The composition of the cell wall of a particular species appears to be influenced by the environmental niche that the species occupies. The complex regulatory and biosynthetic pathways involved in cell wall biosynthesis and construction offer useful chemotherapeutic targets against mycobacteria.

Adaptation, Physiological↗
PubMed1996

Biochemical and genetic data suggest that InhA is not the primary target for activated isoniazid in Mycobacterium tuberculosis.

An examination of the pattern of lipid biosynthetic responses to isoniazid (INH) treatment of Mycobacterium tuberculosis and Mycobacterium smegmatis suggests that the mode of action of activated INH differs between these 2 organisms. Transformation of M. smegmatis with inhA on a plasmid construct conferred high-level resistance to INH, while the same construct failed to confer resistance upon M. tuberculosis. The inhA region from 2 clinical isolates whose resistance has been attributed to changes in the upstream promoter region has been cloned and was not sufficient to impart INH resistance to the level of the parent strain on sensitive M. tuberculosis. These putative mutant promoter elements appear to elevate expression levels of gene fusion reporter constructs, suggesting some noncausal connection between the observed mutations and the lipid metabolism of drug-resistant organisms. These results suggest that InhA is not the major target for activated INH in M. tuberculosis.

Antitubercular Agents↗
PubMed1996

Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog.

The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.

Antigens, Bacterial↗
PubMed1995

The biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis. Identification and functional analysis of CMAS-2.

The major mycolic acid produced by Mycobacterium tuberculosis contains two cis-cyclopropanes in the meromycolate chain. The gene whose product cyclopropanates the proximal double bond was cloned by homology to a putative cyclopropane synthase identified from the Mycobacterium leprae genome sequencing project. This gene, named cma2, was sequenced and found to be 52% identical to cma1 (which cyclopropanates the distal double bond) and 73% identical to the gene from M. leprae. Both cma genes were found to be restricted in distribution to pathogenic species of mycobacteria. Expression of cma2 in Mycobacterium smegmatis resulted in the cyclopropanation of the proximal double bond in the alpha 1 series of mycolic acids. Coexpression of both cyclopropane synthases resulted in cyclopropanation of both centers, producing a molecule structurally similar to the M. tuberculosis alpha-dicyclopropyl mycolates. Differential scanning calorimetry of purified cell walls and mycolic acids demonstrated that cyclopropanation of the proximal position raised the observed transition temperature by 3 degrees C. These results suggest that cyclopropanation contributes to the structural integrity of the cell wall complex.

Amino Acid Sequence↗
PubMed1995

Disparate responses to oxidative stress in saprophytic and pathogenic mycobacteria.

To persist in macrophages and in granulomatous caseous lesions, pathogenic mycobacteria must be equipped to withstand the action of toxic oxygen metabolites. In Gram-negative bacteria, the OxyR protein is a critical component of the oxidative stress response. OxyR is both a sensor of reactive oxygen species and a transcriptional activator, inducing expression of detoxifying enzymes such as catalase/hydroperoxidase and alkyl hydroperoxidase. We have characterized the responses of various mycobacteria to hydrogen peroxide both phenotypically and at the levels of gene and protein expression. Only the saprophytic Mycobacterium smegmatis induced a protective oxidative stress response analogous to the OxyR response of Gram-negative bacteria. Under similar conditions, the pathogenic mycobacteria exhibited a limited, nonprotective response, which in the case of Mycobacterium tuberculosis was restricted to induction of a single protein, KatG. We have also isolated DNA sequences homologous to oxyR and ahpC from M. tuberculosis and Mycobacterium avium. While the M. avium oxyR appears intact, the oxyR homologue of M. tuberculosis contains numerous deletions and frameshifts and is probably nonfunctional. Apparently the response of pathogenic mycobacteria to oxidative stress differs significantly from the inducible OxyR response of other bacteria.

Amino Acid Sequence↗
PubMed1995

Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis.

Mycolic acids represent a major constituent of the mycobacterial cell wall complex, which provides the first line of defense against potentially lethal environmental conditions. Slow-growing pathogenic mycobacteria such as Mycobacterium tuberculosis modify their mycolic acids by cyclopropanation, whereas fast-growing saprophytic species such as Mycobacterium smegmatis do not, suggesting that this modification may be associated with an increase in oxidative stress experienced by the slow-growing species. We have demonstrated the transformation of the distal cis double bond in the major mycolic acid of M. smegmatis to a cis-cyclopropane ring upon introduction of cosmid DNA from M. tuberculosis. This activity was localized to a single gene (cma1) encoding a protein that was 34% identical to the cyclopropane fatty acid synthase from Escherichia coli. Adjacent regions of the DNA sequence encode open reading frames that display homology to other fatty acid biosynthetic enzymes, indicating that some of the genes required for mycolic acid biosynthesis may be clustered in this region. M. smegmatis overexpressing the cma1 gene product significantly resist killing by hydrogen peroxide, suggesting that this modification may be an important adaptation of slow-growing mycobacteria to oxidative stress.

Amino Acid Sequence↗
PubMed1994

Where do all the placentas go?

Re-examining the disposal of placentas as pathological waste is suggested by the author. Placentas can be reprocessed safely for the production of blood derivatives necessary for human treatment by following accepted infection control guidelines in collection, transportation and reprocessing in approved medical laboratories. Money can be saved by removing placentas from the pathological waste stream. Historically, infection control reasons have often been cited as the rationale for preventing changes in the perinatal area. It may be time for infection control teams, administrators and obstetrical staff to review the ethical, legal and infection control issues related to disposal of placentas.

Female↗
PubMed1993

Diversity in the Chlamydia trachomatis histone homologue Hc2.

Chlamydia trachomatis elementary bodies contain two developmentally expressed histone H1 homologues. An 18-kDa histone homologue, Hc1, is conserved among C. trachomatis serovars and C. psittaci. The other histone homologue, Hc2 (encoded by hctB), varies in size between C. trachomatis serovars but is present in reduced amounts or absent from C. psittaci. The variation in Hc2 size among C. trachomatis serovars was found to be due to internal deletions from a region of the hctB gene encoding lysine- and alanine-rich pentameric repeats.

Amino Acid Sequence↗
PubMed1993

Hc1-mediated effects on DNA structure: a potential regulator of chlamydial development.

Chlamydiae are obligate intracellular bacteria which undergo a unique developmental cycle, alternating between non-replicative elementary bodies (EBs) and replicative reticulate bodies (RBs). The transition from RB to EB is characterized by condensation of the chromosome into a dense nucleoid structure. The chlamydial histone homologue Hc1 is sufficient to induce formation of a similar structure in Escherichia coli. High-level Hc1 expression in E. coli is self-limiting and down-regulates transcription, translation, and replication at concentrations similar to those observed in chlamydial elementary bodies. Expression of Hc1 at sub-structural levels may have specific regulatory functions through its interaction with chromosomal DNA. In E. coli this is reflected in a dramatic shift in the pattern of gene expression. The differential expression of the outer membrane porin proteins OmpC and OmpF and analysis of lacZ fusions with promoter regions sensitive to supercoiling suggests that low-level Hc1 expression results in a net relaxation of chromosomal DNA. Topological analysis of plasmid DNA from both E. coli and Chlamydia trachomatis supports a decrease in superhelicity preceding nucleoid formation. In vitro analysis of purified Hc1-DNA interactions supports preferential binding based upon DNA conformation. These results suggest a dual role in which Hc1-mediated changes in gene expression may precede metabolic inactivity.

Amino Acid Transport Systems↗
PubMed1993

Molecular cloning and expression of hctB encoding a strain-variant chlamydial histone-like protein with DNA-binding activity.

Two DNA-binding proteins with similarity to eukaryotic histone H1 have been described in Chlamydia trachomatis. In addition to the 18-kDa histone H1 homolog Hc1, elementary bodies of C. trachomatis possess an antigenically related histone H1 homolog, which we have termed Hc2, that varies in apparent molecular mass among strains. We report the molecular cloning, expression, and nucleotide sequence of the hctB gene encoding Hc2 and present evidence for in vivo DNA-binding activity of the expressed product. Expression of Hc2 in Escherichia coli induces a compaction of bacterial chromatin that is distinct from that observed upon Hc1 expression. Moreover, isolated nucleoids from Hc2-expressing E. coli exhibit markedly reduced sensitivity to DNase I. These properties of Hc2 are consistent with a postulated role in establishing the nucleoid structure of elementary bodies.

Amino Acid Sequence↗
PubMed1992

Nucleoid condensation in Escherichia coli that express a chlamydial histone homolog.

Chlamydial cell types are adapted for either extracellular survival or intracellular growth. In the transcriptionally inert elementary bodies, the chromosome is densely compacted; in metabolically active reticulate bodies, the chromatin is loosely organized. Condensation of the chlamydial nucleoid occurs concomitant with expression of proteins homologous to eukaryotic histone H1. When the Chlamydia trachomatis 18-kilodalton histone homolog Hc1 is expressed in Escherichia coli, a condensed nucleoid structure similar to that of chlamydiae is observed with both light and electron microscopy. These results support a role for Hc1 in condensation of the chlamydial nucleoid.

Blotting, Western↗
PubMed1989

Phenoxazinone synthase: mechanism for the formation of the phenoxazinone chromophore of actinomycin.

Phenoxazinone synthase is a copper-containing oxidase that catalyzes the coupling of 2-aminophenols to form the 2-aminophenoxazinone chromophore. This reaction constitutes the final step in the biosynthesis of the potent antineoplastic agent actinomycin. The mechanism of this complex 6-electron oxidation was determined by using a variety of substituted 2-aminophenols, designed to block the reaction at intermediate stages. Thus, with 3,5-di-tert-butyl-2-aminophenol as substrate, the reaction was blocked at the o-quinone imine 17; with 5-tert-butyl-2-aminophenol (19) as substrate, the reaction was blocked at the p-quinone imine 20; and with 5-methyl-2-aminophenol (21) as substrate, the reaction was blocked at the dihydro-2-aminophenoxazinone 22. These findings suggested a mechanism in which 2-aminophenoxazinone formation proceeded via a quinone imine intermediate 4 that was trapped by a second molecule of 2-aminophenol. Oxidation of the adduct 5 to the p-quinone imine 6 was followed by a second conjugate addition and a final 2-electron oxidation to give the product, 2-aminophenoxazinone. The role of the enzyme in the catalysis of each of these steps was examined. It was found that the second conjugate addition generated a racemic center at C4a, suggesting that this reaction did not occur at the active site. A deuterium isotope effect on the cleavage of the C4-H bond of 2-aminophenol suggested that partial dissociation of an intermediate from the enzyme occurred after the first conjugate addition. It is proposed that 2-aminophenoxazinone synthesis proceeds via a sequence of three consecutive 2-electron aminophenol oxidations and that the aminophenol moiety is regenerated during the reaction sequence by facile tautomerization reactions. Thus, what initially appears to be an impressively complex mechanism may, in fact, be ingeniously simple.

Aminophenols↗