Biomedical subjects

M Drolet

Publications and source records attributed to M Drolet.

At least 19 recordsLinked to original sources

PubMed2003

Consequence of Hoechst 33342-mediated Leishmania DNA topoisomerase-I inhibition on parasite replication.

This study reports that inhibition of Leishmania Topo-I with the minor groove-binding ligands (MGBLs) Hoechst 33342 (Ho342) blocks parasite growth in culture by mechanisms involving DNA breakage. While Ho342 inhibited the replication of several species of Leishmania in a dose- and time-dependent manner, Ho258 was not effective. Cytofluorometric analysis suggested that superior effectiveness of Ho342 over Ho258 was attributed to Leishmania parasites being more permeable toward Ho342. This observation was supported by the ability of both Ho342 and Ho258 to block the relaxation of supercoiled pBR322 DNA by Leishmania Topo-I. The Ho342 specificity toward L. donovani Topo-I was reinforced by the observation that increased Topo-I gene expression and Topo-I activity in Leishmania was paralleled by augmented resistance for this compound. Furthermore, the capacity of NaCl treatment to reverse MGBL-mediated DNA break suggests that Ho342 targetted Topo-I. Moreover, we observed that Ho342-inducible arrest of Leishmania growth was accompanied by G1 arrest and induction of cell death that closely resembles apoptosis. Taken together, our results suggest that MGBL compounds show promise as Topo-I inhibitors against Leishmania infection.

Animals↗

PubMed2001

Sexual maturation and fertility of male Nigerian Dwarf goat (Capra hircus) clones produced by somatic cell nuclear transfer.

Three, genetically identical, Nigerian Dwarf bucks produced by somatic cell nuclear transfer (NT) of fetal fibroblasts were monitored for sexual maturation and fertility. Starting at four months of age, these male clones were trained to serve an artificial vagina (AV). Average age of the NT-derived bucks at first semen collection was 20 weeks, which was not different from that of other young bucks of this breed (average age at first collection = 20 weeks). Average sperm production at 5 months of age for the NT-derived bucks was 5.0 x 10(8) spermatozoa, which was comparable to that of dwarf bucks of similar age (3.4 x 10(8) spermatozoa). At seven months of age, semen collected from two NT-derived bucks was used to artificially inseminate six females (three does per buck). Five does were confirmed pregnant by ultrasound at day 42. Nine healthy kids, four males and five females, were born in March and April 2000. Viable spermatozoa were collected from one of the F1 males at 28 weeks of age. These results demonstrated that NT-derived bucks and one of their male offspring developed sexually within the normal timeframe for their breed and that the clones were fertile.

Animals↗

PubMed2001

Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR.

Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 x 10(2) CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 x 10(3) CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.

Animals↗

PubMed2000

RNase H overproduction corrects a defect at the level of transcription elongation during rRNA synthesis in the absence of DNA topoisomerase I in Escherichia coli.

It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation.

DNA Topoisomerases, Type I↗

PubMed2000

Feasibility of an eight-week dance-based exercise program and its effects on locomotor ability of persons with functional class III rheumatoid arthritis.

OBJECTIVES: The main objectives of this experimental case series were to evaluate the feasibility of a modified dance-based exercise program with low ground impacts in persons with rheumatoid arthritis (RA) functional class III and to describe its effects on locomotor ability. The relationship between 3 locomotor tests and their responsiveness also were addressed. METHODS: Ten female subjects participated in an 8-week exercise program. Locomotor ability was measured before and after the program using the 50-foot test of walking time, the 6-minute test of walking distance, and the locomotion biomechanical analysis. RESULTS: All subjects showed a high compliance (92.5% presence at sessions) over the 8 weeks of exercise without any aggravation in disease status. They were able to train efficiently at moderate intensity up to 25 minutes. Significant improvements were found in locomotor ability, with a higher responsiveness measured by the locomotion biomechanical analysis, followed by the 6-minute gait test and the 50-foot gait test. Inconsistent relationships between tests suggested that different locomotor abilities are required during tests. CONCLUSION: These results support the feasibility of a modified dance-based exercise program for persons with severe RA. With high levels of responsiveness, the detailed biomechanical analysis and the 6-minute gait test are recommended for the assessment of locomotor ability.

Aged↗

PubMed2000

Effects of global regulatory proteins and environmental conditions on fimbrial gene expression of F165(1) and F165(2) produced by Escherichia coli causing septicaemia in pigs.

Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and possess at least two types of fimbriae. F165(1) fimbriae belong to the P fimbrial family and F165(2) fimbriae belong to the S fimbrial family. Regulatory regions of foo (F165(1)) and fot (F165(2)) fimbrial gene clusters from wild-type strain 4787 were sequenced and characterised. Expression of F165(1) and F165(2) fimbrial genes was analysed by using lacZ and/or luxAB as reporter genes under the control of the native fimbrial promoters. Differential expression of fimbrial genes was observed. Global regulatory mechanisms such as catabolite repression, leucine-responsive regulatory protein (Lrp), methylation and DNA supercoiling were demonstrated to influence foo and fot expression. foo and fot expression was optimal at 37 degrees C and under aerobic conditions. Expression of foo was higher on minimal medium, whereas fot expression was higher on complex Luria-Bertani medium. This could reflect an in vivo differential expression.

Amino Acid Sequence↗

PubMed2000

Isolation of the topB gene encoding DNA topoisomerase III as a multicopy suppressor of topA null mutations in Escherichia coli.

One major function of DNA topoisomerase I in Escherichia coli is to repress R-loop formation during transcription elongation, which may otherwise inhibit cell growth. We have previously shown that the growth problems of topA mutants can be corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop. The goal of the present study was to identify other potential regulators of R-loop formation. To this end, we have screened for multicopy suppressors of topA null mutations. As expected using this procedure, we cloned the rnhA gene encoding RNase H. In addition, we also identified the topB gene encoding DNA topoisomerase III as an efficient suppressor of topA null mutations and, hence, of R-loop formation. We show that DNA topoisomerase III is able to relax transcription-induced negative supercoiling both in vitro and in vivo. An R-loop is also shown to be a hot-spot for relaxation by DNA topoisomerase III, and we found that R-loop-dependent hypernegative supercoiling can be prevented by the activity of this topoisomerase in vivo. It is also shown that the topB gene can act synergistically with the rnhA gene to correct the growth defect of topA null mutants efficiently. This synergistic effect can be explained by the fact that some R-loops must not be degraded in order for the RNA to be available for protein synthesis. Topoisomerase III can presumably repress the formation of such R-loops or cause their destabilization to prevent RNA degradation. This is supported by the fact that overproduction of this topoisomerase corrects the negative effect of overexpressing RNase H activity on the growth of topA null mutants at low temperatures. Moreover, the fact that DNA topoisomerase III does not relax global supercoiling supports our previous conclusion that R-loop formation, and therefore the essential function of DNA topoisomerase I, involves local, rather than global, supercoiling.

Base Sequence↗

PubMed1999

R-loop-dependent hypernegative supercoiling in Escherichia coli topA mutants preferentially occurs at low temperatures and correlates with growth inhibition.

We have recently presented evidence that inhibitory R-loops form during transcription in topA null mutants when the nascent RNA anneals with the template DNA strand behind the moving RNA polymerase. This was supported by the results of in vitro transcription assays and by in vivo studies in which R-loop formation was shown to be inhibited by coupled transcription-translation. The results presented here support this model and further demonstrate the link between R-loop formation and growth inhibition of topA null mutants. First, we show that RNase H activity is essential in the absence of DNA topoisomerase I. This was observed even if the growth of the topA null mutant is compensated for by naturally selected mutations, that also reduce global supercoiling below the wild-type level. Second, we show that R-loop-dependent hypernegative supercoiling increases as the temperature decreases and correlates with growth inhibition of topA null mutants. In fact, RNase H overproduction is shown to be detrimental to cell growth at 21 degrees C. Presumably, several mRNAs are being sequestered in R-loops and their degradation by RNase H significantly impedes protein synthesis. We propose that a reduced transcription velocity at low temperatures favors the annealing of the nascent RNA with the template strand behind the moving RNA polymerase, in agreement with the results of previous studies. Finally, based on the currently available data on R-loop formation, we present a model that explains the sensitivity of topA null mutants to various environmental changes that are often accompanied by transient inhibition of translation.

Cell Division↗

PubMed1999

Characterization of a Leishmania donovani gene encoding a protein that closely resembles a type IB topoisomerase.

In order to clone the gene encoding a type I DNA topoisomerase from Leishmania donovani, a PCR-amplified DNA fragment obtained with degenerate oligodeoxyribonucleotides was used to screen a genomic library from this parasite. An open reading frame of 1905 bases encoding a putative protein of 635 amino acid residues was isolated. A substantial part of the protein shares a significant degree of homology with the sequence of other known members of the IB topoisomerase family, in a highly conserved region of these enzymes termed the core domain. However, homology is completely lost after this conserved central core. Moreover, no conventional active tyrosine site could be identified. In fact, the protein expressed in Escherichia coli did not show any relaxation activity in vitro and was unable to complement a mutant deficient in topoisomerase I activity. The results of Southern blot experiments strongly suggested that the cloned gene was not a pseudogene. Northern analysis revealed that the gene was transcribed in its full length and also excluded the possibility that some form of splicing is necessary to produce a mature messenger. Furthermore, our results indicate that the gene is preferentially expressed in actively growing L.donovani promastigotes and that it is also expressed in other kinetoplastid parasites.

Amino Acid Sequence↗

PubMed1999

Relaxation of transcription-induced negative supercoiling is an essential function of Escherichia coli DNA topoisomerase I.

It has been suggested that the essential function of DNA topoisomerase I in Escherichia coli is to prevent chromosomal DNA from reaching an unacceptably high level of global negative supercoiling. However, other in vivo studies have shown that DNA topoisomerase I is very effective in removing local negative supercoiling generated during transcription elongation. To determine whether topoisomerase I is essential for controlling global or local DNA supercoiling, we have prepared a set of topA null mutant strains in combination with different plasmid DNAs. Although we found a correlation between the severity of the growth defect with both transcription-induced and global supercoiling, near to complete growth inhibition correlated only with transcription-induced supercoiling. This result strongly suggests that the major function of DNA topoisomerase I is to relax local negative supercoiling generated during transcription elongation.

Antiporters↗

PubMed1999

Escherichia coli DNA topoisomerase I inhibits R-loop formation by relaxing transcription-induced negative supercoiling.

It has recently been shown that RNase H overproduction can partially compensate for the growth defect due to the absence of DNA topoisomerase I in Escherichia coli (Drolet, M., Phoenix, P., Menzel, R., Massé, E., Liu, L. F., and Crouch, R. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3526-3530). This result has suggested a model in which inhibitory R-loops occur during transcription in topA mutants. Results presented in this report further support this notion and demonstrate that transcription-induced supercoiling is involved in R-loop formation. First, we show that stable R-loop formation during in vitro transcription with E. coli RNA polymerase only occurs in the presence of DNA gyrase. Second, extensive R-loop formation in vivo, revealed by the production of RNase H-sensitive hypernegatively supercoiled plasmid DNAs, is observed under conditions where topA mutants fail to grow. Furthermore, we have demonstrated that the coupling of transcription and translation in bacteria is an efficient way of preventing R-loop formation.

Antiporters↗

PubMed1999

Muscle strength changes as measured by dynamometry following functional rehabilitation in individuals with spinal cord injury.

OBJECTIVE: To objectively quantify muscle strength changes over the course of functional rehabilitation and up to 15 months postdischarge in individuals with spinal cord injury (SCI). METHOD: Hand-held dynamometry was used to evaluate the strength of six muscle groups in 31 individuals after acute SCI (tetraplegia, n = 13, paraplegia, n = 18). Assessment was performed by a single research therapist at admittance and discharge from functional rehabilitation and 3 months and 15 months after discharge. RESULTS: There were significant increases of mean strength values for all muscle groups between admittance and discharge in individuals with parapalegia (median value between 13% and 22%) and tetraplegia (median value between 33% and 83%). Three months after discharge, only individuals with tetraplegia continued to significantly improve their mean strength for four muscle groups (elbow flexors-extensors and shoulder flexors-extensors). One year later, elbow flexors were significantly improved in both paraplegic and tetraplegic persons, while shoulder extensors showed significant gains only in individuals with paraplegia. Study results showed a large variability in the individual profiles of upper limb strength recovery, particularly in tetraplegic individuals. Although some individuals showed strength gains exceeding 200%, some strength decreases were noted. CONCLUSION: Recovery of muscle strength in individuals with tetraplegia over individuals with parapalegia continues for a longer period since it depends initially on recovery of muscle innervation. This study quantified strength improvements during rehabilitation and clearly showed that these gains can be maintained or even increased when the person returns to community living.

Activities of Daily Living↗

PubMed1999

Age-related evolution of the contribution of the right hemisphere to language: absence of evidence.

The age difference observed between Wernicke's and Broca's aphasics has been understood by some authors as an indication of a progressive diminution of the contribution of the right hemisphere to language throughout the life span. To test this hypothesis, 24 right-hemisphere-damaged (RHD) and 24 normal control adults were submitted to six tasks looking at different aspects of language abilities. Results showed that RHDs performed less well than normal subjects on 3 of these tasks, but that this difference was not linked with age (younger than 55 versus older than 65 years). Consequently, these results do not support models of functional brain organization suggesting a decreasing contribution of the right hemisphere to language abilities with age.

Acoustic Stimulation↗

PubMed1997

DNA topoisomerases regulate R-loop formation during transcription of the rrnB operon in Escherichia coli.

Recent in vivo and in vitro studies have suggested an important role for DNA topoisomerases in regulating R-loop formation during transcription in Escherichia coli. In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity. We found that a multicopy plasmid (pBR322) carrying an heavily transcribed portion of the rrnB operon cannot be transformed in topA mutants unless RNase H is overproduced. Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular level of RNase H activity. When DNA gyrase is sufficiently active, hypernegatively supercoiled plasmid DNA is produced if the same DNA fragment is transcribed in a topA mutant. The formation of such topoisomers most likely reflect the presence of extensive R-loops since it is sensitive to the intracellular level of RNase H activity. Finally, the formation of R-looped plasmid DNAs in an in vitro transcription system using phage RNA polymerases is also detected when the 567-base pair HindIII fragment is transcribed on a negatively supercoiled DNA template.

DNA Topoisomerases, Type I↗

PubMed1997

Roles of DNA topoisomerases in the regulation of R-loop formation in vitro.

We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation. Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level. In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process. By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase. When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation. This result suggests that more stable R-loops are being produced in this orientation. Our results also suggest that DNA gyrase can participate in the process of R-loop elongation. The strong transcription-induced relaxing activity of E. coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping. In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.

DNA Topoisomerases, Type I↗

PubMed1997

Incompatibility of Escherichia coli rho mutants with plasmids is mediated by plasmid-specific transcription.

The Escherichia coli rho-15 mutant (deficient in transcription termination) is known to be incompatible with pBR322 and other plasmids (J. S. Fassler, G. F. Arnold, and I. Tessman, Mol. Gen. Genet. 204:424-429, 1986). We show that failure of pBR322 to transform rho-15 is mediated by transcription from the tet promoter and readthrough from the tet gene into the rom region. Using an isopropyl-beta-D-thiogalactopyranoside-inducible promoter to replace the tet promoter, we have demonstrated that plasmid-specific transcription inhibits growth of the rho-15 host, possibly due to the expression of the Rom protein. The involvement of Rom protein in pBR322-rho-15 incompatibility is further indicated by the following two experiments. (i) Functional inactivation of the rom gene in pBR322 enabled plasmids to transform E. coli rho-15. (ii) Specific overexpression of the rom gene abolished plasmid transformation into E. coli rho-15. An rpoB8(Ts) mutant RNA polymerase which compensated for the termination defect in E. coli rho-15 also restored plasmid-host compatibility, suggesting that Rom-mediated plasmid-host incompatibility is linked to a defect in transcription termination.

Bacterial Proteins↗

PubMed1995

Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I.

Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces of genetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression of RNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.

Cold Temperature↗

PubMed1994

Hypernegative supercoiling of the DNA template during transcription elongation in vitro.

Supercoiled plasmid DNAs with negative superhelicity several times higher than normal have been isolated from Escherichia coli topA mutants. The formation of these hypernegatively supercoiled plasmid DNAs is apparently induced by transcription. We show that hypernegatively supercoiled plasmid DNAs isolated from topA mutants contain R-loop(s). To study the mechanism of formation of hypernegatively supercoiled plasmid DNA, we have been able to reproduce hypernegatively supercoiled DNA in vitro using purified RNA polymerase and DNA gyrase. The formation of hypernegatively supercoiled plasmid DNA template in vitro is shown to require transcription elongation and is tightly linked to R-loop formation. We propose that one of the roles of topoisomerase I is to suppress R-loop formation during transcription elongation.

DNA Topoisomerases, Type I↗