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Biomedical subjects

Dominique Michaud

Publications and source records attributed to Dominique Michaud.

18 recordsLinked to original sources
PubMed2006

Polymorphisms in GSTT1, GSTM1, NAT1 and NAT2 genes and bladder cancer risk in men and women.

BACKGROUND: Cigarette smoking is an established risk factor for bladder cancer. Epidemiological and biological data suggest that genetic polymorphisms in activating and detoxifying enzymes may play a role in determining an individual's susceptibility to bladder cancer in particular when in combination with specific environmental exposures such as cigarette smoking. N-acetyltransferase (NAT) enzymes, NAT1 and NAT2, are involved in the activation and detoxification of tobacco smoke constituents. Polymorphisms in these genes alter the ability of these enzymes to metabolize carcinogens, as certain allelic combinations result in a slow or rapid acetylation phenotype. Glutathione S-transferases (GSTs) also detoxify tobacco smoke constituents, and polymorphisms within the GSTM1 and GSTT1 genes can result in a complete lack of enzyme activity. METHODS: We assessed the association between common polymorphisms identified in the GSTM1, GSTT1, NAT1, and NAT2 genes and the risk of bladder cancer in two nested case-control studies within the Nurses' Health Study (n = 78 female cases, 234 female controls) and the Health Professionals' Follow-up Study (n = 139 male cases, 293 male controls). We also evaluated whether cigarette smoking modified the associations of the genotypes and bladder cancer risk in men and women. RESULTS: Overall, we observed no statistically significant associations between the polymorphisms and bladder cancer risk among men and women, although given our sample size, we had limited power to detect small to moderate effects. There was however the suggestion of an increased risk among female ever smokers with the NAT2 slow genotype and an increased risk in male never smokers with the GSTM1 null genotype. CONCLUSION: In summary, these prospective results are consistent with previous literature supporting associations between bladder cancer and the NAT2 slow acetylation and the GSTM1 null genotypes.

Adult↗
PubMed2006

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites.

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.

Amino Acids↗
PubMed2006

Protein phosphorylation and membrane association of sucrose synthase in developing tomato fruit.

Calcium-dependent protein kinase (CDPK) activities were detected both in the soluble and the membrane fraction of various tomato (Lycopersicon esculentum Mill.) organs, using a synthetic peptide mimicking the serine 11 phosphorylation site of a tomato sucrose synthase (SS, EC 2.4.1.13) isoform as substrate. The levels of membrane and soluble Ser-CDPK activities were differentially regulated during fruit development. The membrane Ser-CDPK activity was maximal in young fruit but decreased as the fruit developed, suggesting a specific role during fruit growth. Using an in gel assay with purified tomato SS as substrate, we showed that partially purified soluble and membrane Ser-CDPK preparations both contained a SS-kinase polypeptide of 55 kDa. The membrane and soluble Ser-CDPK activities were largely inactivated in the absence of calcium or when MgCl(2) was replaced by MnCl(2). Both soluble and membrane Ser-CDPK activities were very sensitive to staurosporine. Using Fe(III)-immobilized metal chromatography to determine the apparent phosphorylation status of the enzyme in vivo, we showed that soluble SS was largely dephosphorylated in fruits fed EGTA or staurosporine, compared to fruits fed water or sucrose. Moreover, the level of SS increased by about two-fold in the membrane fraction of fruits fed the Ser-CDPK inhibitors, compared to the control. The level of SS protein in the membrane and soluble fractions of tomato fruit was developmentally regulated, the membrane form being specifically detected in actively growing fruits. Together, our results suggest that a mechanism involving protein phosphorylation/dephosphorylation and/or calcium would in part control the association of SS isoforms with membranes in developing tomato fruit.

Calcium↗
PubMed2006

An in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants.

Proteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a 'mouse trap' against the host proteases during extraction. After confirming the importance of trypsin- and chymotrypsin-like activities in crude protein extracts of potato (Solanum tuberosum L.) leaves, transgenic lines of potato expressing either tomato cathepsin D inhibitor (CDI) or bovine aprotinin, both active against trypsin and chymotrypsin, were generated by Agrobacterium tumefaciens-mediated genetic transformation. Leaf crude protein extracts from CDI-expressing lines, showing decreased levels of cathepsin D-like and ribulose 1,5-bisphosphate carboxylase/oxygenase hydrolysing activities in vitro, conducted decreased turnover rates of the selection marker protein neomycin phosphotransferase II (NPTII) relative to the turnover rates measured for transgenic lines expressing only the marker protein. A similar stabilizing effect on NPTII was observed in leaf protein extracts from plant lines coexpressing bovine aprotinin, confirming the ability of ectopically expressed broad-spectrum serine proteinase inhibitors to reproduce the protein-stabilizing effect of low-molecular-weight proteinase inhibitors generally added to protein extraction media.

Agrobacterium tumefaciens↗
PubMed2005

Aphid clonal resistance to a parasitoid fails under heat stress.

Parasitoid virulence and host resistance are complex interactions depending on metabolic rate and cellular activity, which in aphids additionally implicate heritable secondary symbionts among the Enterobacteriaceae. As performance of the parasitoid, the aphid host and its symbionts may differentially respond to temperature, the success or failure of aphid parasitism is difficult to predict when temperature varies. We tested the hypothesis that resistance of the pea aphid Acyrthosiphon pisum to the parasitoid Aphidius ervi, which is linked to aphid secondary symbionts, may depend on temperature in several resistant and non-resistant aphid clonal lineages of different geographic origin and of known bacterial symbiosis, using experiments in controlled environments. Complete immunity to A. ervi at 20 degrees C in three different aphid clones whose symbiosis is characterized by the possession of Hamiltonella defensa reversed to high susceptibility at 25 degrees C and especially 30 degrees C, suggesting that the aphid's immune responses to the establishment and early development of the parasitoid is strongly reduced at moderately high temperatures. There was no evidence that a pea aphid control genotype that was susceptible to A. ervi at 20 degrees C could become more resistant as temperature increases, as has been suggested for insect fungal pathogens. By contrast, our results suggest that aphid clonal resistance to A. ervi and related parasitoids is characteristic of cool temperature conditions, similar to various other fitness attributes of aphids. Based on evidence that H. defensa symbionts characterized all three A. ervi resistant pea aphid clones studied, but was absent in control aphids that remained susceptible at all temperatures, we suggest that secondary symbiosis plays a key role in the heat sensitivity of aphid clonal resistance. Our study may also indicate that aphid natural control of variably susceptible host populations by aphid parasitoids is more likely at moderate to high temperatures.

Animals↗
PubMed2005

Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming.

Plants have become, over the last ten years, a suitable alternative to microbial and animal cell factories for the production of clinically-useful, therapeutic proteins. Besides the well known advantage of low-cost and large-scale production of safe and biologically active mammalian proteins, plants also are able to perform most post-translational maturations required for biological activity and suitable pharmacokinetics of recombinant therapeutic proteins. In this short review we focus on glycosylation and proteolytic processing of plant-made pharmaceuticals during their transport through the plant cell's secretory pathway. We also address the practical implications of these important processes on the effectiveness of plant molecular pharming systems.

Animals↗
PubMed2005

A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases.

Protein engineering approaches are currently being devised to improve the inhibitory properties of plant proteinase inhibitors against digestive proteinases of herbivorous insects. Here we engineered a potent hybrid inhibitor of aspartate and cysteine digestive proteinases found in the Colorado potato beetle, Leptinotarsa decemlineata Say. Three cathepsin D inhibitors (CDIs) from stressed potato and tomato were first compared in their potency to inhibit digestive cathepsin D-like activity of the insect. After showing the high inhibitory potency of tomato CDI (M(r) approximately 21 kDa), an approximately 33-kDa hybrid inhibitor was generated by fusing this inhibitor to the N terminus of corn cystatin II (CCII), a potent inhibitor of cysteine proteinases. Inhibitory assays with recombinant forms of CDI, CCII, and CDI-CCII expressed in Escherichia coli showed the CDI-CCII fusion to exhibit a dual inhibitory effect against cystatin-sensitive and cathepsin D-like enzymes of the potato beetle, resulting in detrimental effects against 3rd-instar larvae fed the hybrid inhibitor. The inhibitory potency of CDI and CCII was not altered after their fusion, as suggested by IC(50) values for the interaction of CDI-CCII with target proteinases similar to those measured for each inhibitor. These observations suggest the potential of plant CDIs and cystatins as functional inhibitory modules for the design of effective broad-spectrum, hybrid inhibitors of herbivorous insect cysteine and aspartate digestive proteinases.

Amino Acid Sequence↗
PubMed2004

Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato.

We reported earlier the importance of digestive cathepsin D-like activity for initiating dietary protein hydrolysis in Colorado potato beetle, Leptinotarsa decemlineata Say [Brunelle et al. (1999) Arch. Insect Biochem. Physiol. 42:88-98]. We assessed here whether transgenic lines of potato (Solanum tuberosum L.) expressing a cathepsin D inhibitor (CDI) from tomato would show resistance to the beetle, or if the insect would compensate for the loss of cathepsin D activity after ingesting the recombinant inhibitor. Transgenic potato lines expressing tomato CDI were developed by Agrobacterium tumefaciens genetic transformation, and selected based on their relative amount of CDI. After confirming the absence of detectable visible effects of CDI on the plant's phenotype, diet assays with control and transgenic lines were carried out to assess the impact of the inhibitor on growth and development of the insect. Leaf consumption, relative growth rate, molting incidence, and digestive protease activity were monitored at 12-h intervals over 132 h for 3rd-instar larvae provided with transgenic potato foliage. Leaf consumption and relative growth rate were slightly reduced during the first 12 h for larvae fed CDI, but no significant differences were observed thereafter. In contrast, time for molting to the 4th larval stage was significantly longer for larvae fed modified plants, with developmental delays of approximately 10 h (0.5 day) compared to control larvae. Recombinant CDI also had an impact on the insect's digestive physiology, readily inducing overproduction of digestive proteases (rubiscases), followed by a gradual decrease of total and pepstatin-sensitive activity. Overall, these observations show the ability of Colorado potato beetle to compensate for the loss of cathepsin D activity by modulating its digestive protease complement in response to aspartate-type inhibitors in the diet. From a practical viewpoint, these data stress the importance of devising improved strategies for the effective inhibition of insect digestive proteinases in vivo, based on the use of hybrid inhibitors active against different protease classes.

Animal Feed↗
PubMed2004

Colorado potato beetles show differential digestive compensatory responses to host plants expressing distinct sets of defense proteins.

Herbivorous insects fed plants expressing proteinase inhibitors (PIs) compensate for the loss of digestive proteolytic functions by producing novel proteinases. We assessed here whether such compensatory responses represent a general, non-specific adaptation to defense-related proteins in host plant tissues, or if distinct responses occur depending on the stress exerted on the plant. As a model, growth, development, and digestive proteases of the Colorado potato beetle (Leptinotarsa decemlineata Say) were monitored after feeding larvae with plants pre-treated with either methyl jasmonate or arachidonic acid, two compounds inducing different sets of defense genes in potato. In brief, larvae fed plants treated with jasmonate or arachidonate were negatively affected compared to larvae fed non-treated plants, suggesting the potency of both molecules to induce partial resistance to potato beetles in potato. On the other hand, larvae fed treated plants partially compensated for the presence of defense-related proteins by adapting their digestive proteolytic system, both quantitatively and qualitatively. These compensatory processes varied depending on the treatment, the larvae fed arachidonate-treated plants showing the most dramatic response. Compensation to jasmonate and arachidonate was also influenced by a cysteine PI from rice expressed in the plant, pointing out the possible indirect effects of recombinant defense proteins on naturally-occurring plant-insect interactions. These observations, while showing the potential of jasmonate and arachidonate as inducers of partial resistance to the potato beetle in potato, also suggest that digestive compensation in herbivorous insects is determined, at least in part, by defense-related compounds found in the plant in response to different stress stimuli or as a result of ectopic expression in transgenic plants.

Acetates↗
PubMed2004

Inhibitory effects of cystatins on proteolytic activities of the Plum pox potyvirus cysteine proteinases.

In an effort to develop new antiviral strategies effective against potyviruses, several cystatins were evaluated for their ability to inhibit the cysteine proteinases of Plum pox potyvirus (PPV) using in vitro proteolytic assays. The following cystatins were purified as GST fusion proteins and shown to be active against papain:oryzacystatins I and II (OCI and OCII), corn cystatin II (CCII), human stefin A (HSA), the domain 8 of tomato multicystatin (TMC-8) and a large 24kDa tomato cystatin (LTCyst). These cystatins did not inhibit the activity of purified recombinant PPV NIa proteinase, a serine-like cysteine proteinases related to the 3C proteinases of picornaviruses and to chymotrypsin. The cystatins were shown to inhibit slightly the activity of the PPV HC-Pro proteinase with CCII being the best inhibitor. However a large excess of the cystatins was required to observe any inhibition. Based on these results and on the documented pleiotropic effects of cystatins on the metabolism of plants, we conclude that they are not the best candidates for antiviral strategies targeted to viral cysteine proteinases. The availability of soluble active recombinant PPV NIa proteinase will be instrumental for the selection of other proteinase inhibitors with increased affinity and specificity for this proteinase.

Cystatins↗
PubMed2003

Molecular interactions between an insect predator and its herbivore prey on transgenic potato expressing a cysteine proteinase inhibitor from rice.

Transgenic plants expressing resistance to herbivorous insects may represent a safe and sustainable pest control alternative if they do not interfere with the natural enemies of target pests. Here we examined interactions between oryzacystatin I (OCI), a proteinase inhibitor from rice genetically engineered into potato (Solanum tuberosum cv. Kennebec, line K52) to increase resistance to insect herbivory, and the insect predator Perillus bioculatus. This stinkbug is a relatively specialized predator of caterpillars and leaf-beetle larvae, and may also include plant sap in its predominantly carnivorous diet. One of its preferred prey is Colorado potato beetle (Leptinotarsa decemlineata), a major target of insect resistance development for potato field crops. Gelatin/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that a major fraction of proteinase (gelatinase) activity in P. bioculatus extracts is OCI-sensitive. Among five gelatinolytic bands detected, the slowest-moving one (proteinase I) was inhibited strongly by purified OCI expressed in Escherichia coli or by OCI-transgenic potato extracts, while three other proteinases were partly sensitive to these treatments. There was also evidence of slight inhibition of proteinase I by untransformed potato foliage, suggesting the presence of a natural inhibitor related to OCI at low level in potato foliage. Interestingly, only about 50% of the maximum potential activity of proteinase I was recovered in extracts of P. bioculatus feeding on L. decemlineata larval prey on a diet of OCI-potato foliage, indicating that the predator was sensitive to OCI in the midgut of its prey. However, P. bioculatus on OCI-prey survived, grew and developed normally, indicating ability to compensate prey-mediated exposure to the OCI inhibitor. Confinement of P. bioculatus to potato foliage provided no evidence that potato plant-derived nutrition is a viable alternative to predation, restriction to potato foliage in fact being inferior to free water for short-term survival of nonfeeding first-instar larvae. These results support the view that OCI, an effective inhibitor of a substantial fraction of digestive enzymatic potential in P. bioculatus, should not interfere with its predation potential when expressed in potato plants fed to its prey at a maximum level of approximately 0.8% of total soluble proteins in mature foliage.

Animals↗
PubMed2003

Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant.

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.

Animals↗
PubMed2003

Intra-abdominal pressure monitoring for critically ill patients.

Increased intra-abdominal pressure (IAP) is associated with a variety of clinical situations and has profound effects, both locally and systemically, that may result in organ dysfunction and failure. If not identified and treated promptly, increased IAP can be fatal. Urinary bladder pressure monitoring is a simple and reliable method of quantifying IAP and is a key factor leading to prompt management and a successful outcome for patients.

Abdomen↗
PubMedDate not supplied

[The impact of a case management approach for total hip and knee arthroplasty patients].

The primary goal of this research was to evaluate the impact of introducing a case management approach for total hip (THA) and knee (TKA) arthroplasty patients, on the number of complications, visits to the emergency clinic and readmissions, as well as on length of stay (LS). The period covered by this retrospective study was fiscal 1999-2000. Subjects were divided into an experimental group, with the case management, and a control group receiving the usual care. The second goal of the research was to compare these groups with non-equivalent groups from fiscal 1996-1997, before case management was introduced. The reference framework used is the case management model in the institution. All in all, 256 files (52%) were reviewed. We found that the LS, for THA and TKA cases in the hospital where the experiment was done, had decreased considerably three years after case management was introduced, and that this reduction was also significant for TKA cases in comparison with the non-case management group at the control group hospital, without compromising certain clinical results.

Academic Medical Centers↗