Components of sterol biosynthesis assembled on the oxygen-avid hemoglobin of Ascaris.

The parasitic nematode Ascaris infests a billion people worldwide. Much of its proliferative success is due to prodigious egg production, up to 10(6) sterol-replete eggs per day. Sterol synthesis requires molecular oxygen for squalene epoxidation, yet oxygen is scarce in the intestinal folds the worms inhabit. Ascaris has an oxygen-avid hemoglobin in the perienteric fluid that bathes its reproductive organs. Purified hemoglobin contained tightly bound squalene and functioned as an NADPH-dependent, ferrihemoprotein reductase. All components of the squalene epoxidation reaction--squalene, oxygen, NADPH, and NADPH-dependent reductase--are assembled on the hemoglobin. This molecule may thus function in sterol biosynthesis.

Compensatory ahpC gene expression in isoniazid-resistant Mycobacterium tuberculosis.

Mutations that eliminate KatG catalase-peroxidase activity prevent activation of isoniazid and are a major mechanism of resistance to this principal drug for the treatment of Mycobacterium tuberculosis infections. However, the loss of KatG activity in clinical isolates seemed paradoxical because KatG is considered an important factor for the survival of the organism. Expression of either KatG or the recently identified alkyl hydroperoxidase AhpC was sufficient to protect bacilli against the toxic effects of organic peroxides. To survive during infection, isoniazid-resistant KatG mutants have apparently compensated for the loss of KatG catalase-peroxidase activity by a second mutation, resulting in hyperexpression of AhpC.

Transcription of the procyclic acidic repetitive protein genes of Trypanosoma brucei.

The procyclic acidic repetitive protein (parp) genes of Trypanosoma brucei encode a small family of abundant surface proteins whose expression is restricted to the procyclic form of the parasite. They are found at two unlinked loci, parpA and parpB; transcription of both loci is developmentally regulated. The region of homology upstream of the A and B parp genes is only 640 base pairs long and may contain sequences responsible for transcriptional initiation and regulation. Transcription upstream of this putative promoter region is not developmentally regulated and is much less active than that of the parp genes; the polymerase responsible is inhibited by alpha-amanitin, whereas that transcribing the parp genes is not. Transcription of the parp genes is strongly stimulated by low levels of UV irradiation. The putative parp promoter, when placed upstream of the chloramphenicol acetyltransferase gene, is sufficient to cause production of chloramphenicol acetyltransferase in a T. brucei DNA transformation assay. Taken together, these results suggest that a promoter for an alpha-amanitin-resistant RNA polymerase lies less than 600 nucleotides upstream of the parp genes.

Novel gene structure and evolutionary context of Caenorhabditis elegans globin.

Animal and plant globin-encoding genes (Glo) contain two introns in strictly conserved positions. Plant Glo genes possess an additional, centrally located intron. We have determined the cDNA sequence and gene structure of a putative Glo gene from the free-living nematode, Caenorhabditis elegans. The gene encodes a one-domain globin with a single intron, corresponding to the central intron of plant Glo genes. The two introns common to virtually all animal and plant Glo genes are missing. Comparison with the related organisms Trichostrongylus colubriformis, Ascaris suum and Pseudoterranova decipiens, provides evidence of gene duplication, intron loss, and functional divergence within the Glo genes of the nematode phylum. It is now apparent that differential intron loss during evolution has generated Glo genes with a panoply of exon/intron permutations.

The psychometric and cardiac effects of pseudoephedrine in the hyperbaric environment.

STUDY OBJECTIVES: To examine the psychometric and cardiac effects of pseudoephedrine at 1 and 3 atmospheres (atm) of pressure (0 and 66 feet of sea water, respectively), and to make recommendations about the agent's safety in the diving environment. DESIGN: Double-blind, placebo-controlled, crossover study. SETTING: Monoplace hyperbaric chamber of a university hospital. SUBJECTS: Thirty active divers (mean age 38 yrs). INTERVENTION: A bank of seven tests was used to assess cognitive function during four different simulated dive combinations: placebo-1 atm, placebo3 atm, pseudoephedrine-1 atm, and pseudoephedrine-3 atm. MEASUREMENTS AND MAIN RESULTS: Heart rate and cardiac rhythm were recorded during all dives. Repeated-measures analysis of variance was used to analyze the effects of pseudoephedrine, depth, and drug-depth interaction. No significant, independent effects of pseudoephedrine were seen on any of the seven psychometric test scores (p>0.05), although the drug tended to increase anxiety scores (p=0.092). Depth resulted in a significant increase in anxiety scores (p=0.021) and a significant decrease in verbal fluency test scores (p=0.041); it had no significant effects on the other five psychometric tests (p>0.05). Pseudoephedrine caused a significant increase (p=0.036) in mean heart rate, and depth caused a significant decrease (p=0.013). Neither pseudoephedrine nor depth affected cardiac rhythm. CONCLUSION: Pseudoephedrine does not cause significant alterations in psychometric performance at 3 atm of pressure that might increase the risk of diving. Depth causes significant adverse effects on anxiety levels and semantic memory at 3 atm. Pseudoephedrine and depth have significant but opposite effects on heart rate; although, these effects are unlikely to be clinically significant during diving. It is unlikely that pseudoephedrine adds significant risk to the diver.

The psychometric and cardiac effects of dimenhydrinate in the hyperbaric environment.

STUDY OBJECTIVES: To examine the psychometric and cardiac effects of dimenhydrinate at 1 and 3 atmospheres (atm) of pressure (0 and 66 feet of sea water, respectively), and to make recommendations about the drug's safety in the diving environment. DESIGN: Double-blind, placebo-controlled, crossover study SETTING: Monoplace hyperbaric chamber of a university hospital. SUBJECTS: Thirty active divers (mean age 38 yrs). INTERVENTIONS: A bank of seven tests was used to assess cognitive function during four different dive combinations: placebo-1 atm, placebo-3 atm, dimenhydrinate-1 atm, and dimenhydrinate-3 atm. MEASUREMENTS AND MAIN RESULTS: Heart rate and cardiac rhythm were recorded during all dives. Repeated-measures multivariate analysis of variance was used to analyze the effects of dimenhydrinate, depth, and drug-depth interaction. Dimenhydrinate resulted in a significant decrease in scores of mental flexibility (trail-making, part B, p<0.05) but had no effect on scores in the six other psychometric tests (p>0.05). It had no effect on mean heart rate (p>0.05), although frequent unifocal ventricular ectopic beats occurred in two subjects after ingestion of the drug. Depth resulted in a significant decrease in verbal memory test scores (p=0.001) and mean heart rate (p<0.001). CONCLUSION: Dimenhydrinate adversely affects mental flexibility. This effect, when added to the adverse effect of depth on memory, may contribute to the dangers of diving.

AhpC, oxidative stress and drug resistance in Mycobacterium tuberculosis.

The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin-dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front-line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid-resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild-type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid-resistant, AhpC-overexpressing strains were also deficient in activity of the mycobacterial catalase-peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG-deficient, isoniazid-resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.

Mutually dependent secretion of proteins required for mycobacterial virulence.

The ESX-1 locus is a region critical for full virulence in Mycobacterium tuberculosis, which encodes two secreted proteins as well as other genes involved in their secretion. The mechanism of secretion of the two proteins, ESAT-6 and CFP-10, and their function remain unknown. Using proteomic methods to search for additional proteins secreted by the ESX-1 locus, we discovered that a protein encoded by a chromosomally unlinked gene, espA, is also secreted by strains that contain the ESX-1 locus but not by strains with ESX-1 deletions. Mutations in individual ESX-1 genes, including those that encode ESAT-6 and CFP-10, were found to block EspA secretion. Surprisingly, mutants that lack espA reciprocally failed to secrete ESAT-6 and CFP-10 and were as attenuated as ESX-1 mutants in virulence assays. The results indicate that secretion of these proteins, which are each critical for virulence of pathogenic mycobacteria, is mutually dependent. The results further suggest that discerning the nature of the interaction and the structure of macromolecular complexes will provide insights into both an alternative mechanism of protein secretion and mycobacterial virulence.

How do retinoids work?

The main physiological function of natural retinoids, i.e. the dietary micronutrient retinol and its metabolite retinoic acid, is in proper differentiation and maintenance of differentiated state of many if not all epithelia. Also, it appears that the metabolism of nonepithelial cells can be affected by these compounds. The effects are diverse. Although the exact molecular mechanism causing these effects remain to be elucidated, recent research indicates that both retinol and retinoic acid affect genomic expression by activating and simultaneously repressing specific genes. Such action results in the appearance and disappearance of more than 40 specific proteins. The effect on the genomic expression is time-dependent. Some of the genes are influenced very soon in a matter of a few hours. Effects on other genes require considerable time. Whether natural retinoids interact with the genome directly remains to be shown. How such interactions could occur is discussed.

Anatomy of the parp gene promoter of Trypanosoma brucei.

While growing in the tsetse fly, Trypanosoma brucei expresses a major surface glycoprotein, the procyclic acidic repetitive protein (PARP). The parp genes are transcribed by an alpha-amanitin-resistant RNA polymerase. We have determined the sequence requirements for parp promoter activity. Studies of RNA produced from input DNA in transiently transfected trypanosomes indicate that the RNA is correctly processed by trans-splicing and polyadenylation. Deletion analyses show that 330 bp are sufficient for full promoter and splicing activity and that the promoter structure is complex, involving at least three elements whose mutual spacing is important. Mutagenesis pin-pointed two sequences vital for promoter activity; neither bears any resemblance to known prokaryotic or eukaryotic promoter elements.

Regulation of Saccharomyces cerevisiae flavohemoglobin gene expression.

The Saccharomyces cerevisiae hemoglobin is a flavoprotein of unknown function. It shares extensive sequence homology with the globin of Candida as well as those of several bacterial species. We have studied its gene regulation in order to better understand its purpose in the cell. Transcriptional analyses indicate that, in sharp contrast to the bacterial globins of Vitreoscilla and Alcaligenes eutrophus, the S. cerevisiae globin message is induced during logarithmic growth and under oxygen-replete conditions. Transcription of the S. cerevisiae hemoglobin gene is positively regulated by the transcription factors heme-activated protein (HAP) 1 and HAP2/3/4, which respond to intracellular heme levels. Anaerobically, there is a low level, HAP-independent induction of hemoglobin mRNA. Unlike other systems influenced by the HAP2/3/4 transcription factor complex, no activation of hemoglobin expression by growth in non-fermentable carbon sources is observed. Flavohemoglobin gene disruption does not alter cell viability or growth in a variety of oxygen conditions and carbon sources. Physical and genetic mapping of the S. cerevisiae flavohemoglobin gene places it on chromosome seven near the formyltetrahydrofolate synthase (ADE3) locus. These data indicate that, despite the high degree of homology, the S. cerevisiae globin may have a function distinct from those proposed for bacterial globins.

Rat cellular retinol-binding protein: cDNA sequence and rapid retinol-dependent accumulation of mRNA.

Cellular retinol-binding protein (CRBP) may be an important mediator of vitamin A action. We report here the identification of a cDNA clone corresponding to the rat CRBP gene. The cDNA is 695 nucleotides long, with an open reading frame corresponding to a protein of 134 amino acids. The deduced amino acid sequence is identical with that of rat CRBP. The nucleotide sequence shows 90.5% similarity with the human CRBP cDNA sequence. Genomic DNA analysis indicates that CRBP is present in one, or at most two, copies within the rat genome. Analysis of mRNA reveals a single species in every tissue tested and suggests that the isolated cDNA is full-length. Finally, when retinol-deficient rats are fed retinyl acetate for 4 hr, about 4-fold accumulation of CRBP-specific mRNA is observed in the lungs. This rapid effect suggests that the micronutrient retinol may directly influence the expression of its specific intracellular binding protein.

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.

Ascaris hemoglobin gene: plant-like structure reflects the ancestral globin gene.

Animal globin genes have two introns at strictly conserved positions, while plant globin genes have both of these as well as an additional, central intron. It has been proposed that a common ancestor gene had three introns, one of which was subsequently lost from animal but not plant globin genes. We have elucidated the cDNA sequence and gene structure of a hemoglobin from the parasitic nematode Ascaris suum and found a plant-like central intron, providing strong evidence for a three-intron ancestor of modern globin genes.

The vitamin D receptor interacts with general transcription factor IIB.

The vitamin D receptor (VDR) heterodimerizes with retinoid X receptors (RXR) on many vitamin D-responsive promoter elements, suggesting that this complex is the active factor in vitamin D-mediated transcription. However, the mechanism of transcriptional regulation following VDR-RXR binding to DNA is not well characterized. Using a yeast two-hybrid protein interaction assay, we demonstrate that VDR forms specific protein: protein contacts with the basal transcription factor TFIIB. Deletion analysis indicated that the carboxyl-terminal ligand binding domain of VDR interacted with a 43-residue amino-terminal domain in TFIIB. The interaction with TFIIB showed selectivity for the ligand binding domain of VDR as similar regions of RXR alpha or of retinoic acid receptor alpha did not couple with TFIIB. Binding assays with purified proteins showed a direct interaction between VDR and TFIIB in vitro. These data suggest a mechanism for VDR-dependent transcription in which protein contacts between VDR and TFIIB may impart regulatory information to the transcription preinitiation complex.

Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin.

Unlike many pathogens that are overtly toxic to their hosts, the primary virulence determinant of Mycobacterium tuberculosis appears to be its ability to persist for years or decades within humans in a clinically latent state. Since early in the 20th century latency has been linked to hypoxic conditions within the host, but the response of M. tuberculosis to a hypoxic signal remains poorly characterized. The M. tuberculosis alpha-crystallin (acr) gene is powerfully and rapidly induced at reduced oxygen tensions, providing us with a means to identify regulators of the hypoxic response. Using a whole genome microarray, we identified >100 genes whose expression is rapidly altered by defined hypoxic conditions. Numerous genes involved in biosynthesis and aerobic metabolism are repressed, whereas a high proportion of the induced genes have no known function. Among the induced genes is an apparent operon that includes the putative two-component response regulator pair Rv3133c/Rv3132c. When we interrupted expression of this operon by targeted disruption of the upstream gene Rv3134c, the hypoxic regulation of acr was eliminated. These results suggest a possible role for Rv3132c/3133c/3134c in mycobacterial latency.

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.

Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase.

Intraerythrocytic malaria parasites rapidly degrade virtually all of the host cell hemoglobin. We have cloned the gene for an aspartic hemoglobinase that initiates the hemoglobin degradation pathway in Plasmodium falciparum. It encodes a protein with 35% homology to human renin and cathepsin D, but has an unusually long pro-piece that includes a putative membrane spanning anchor. Immunolocalization studies place the enzyme in the digestive vacuole and throughout the hemoglobin ingestion pathway, suggesting an unusual protein targeting route. A peptidomimetic inhibitor selectively blocks the aspartic hemoglobinase, prevents hemoglobin degradation and kills the organism. We conclude that Plasmodium hemoglobin catabolism is a prime target for antimalarial chemotherapy and have identified a lead compound towards this goal.

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.