[Vaccines on mucosal surfaces].
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Adjuvants, Immunologic↗
Biomedical subjects
M Lebrun
Publications and source records attributed to M Lebrun.
Prospects for a human Toxoplasma vaccine.
Human toxoplasmosis is usually benign, but may occasionally lead to severe or lethal damages when combined with immunosuppressive states or when transmitted to the fetus during pregnancy. Only a vaccine could prevent these harmful effects. The oral route is the natural portal of entry of T. gondii. A protective immune response at the mucosal level is required to kill the parasite as soon as it penetrates the intestinal barrier thus preventing toxoplasma from invading the host and settling into tissues. The probable major roles played by both CD8 T cells and antibodies, specially IgA, suggest that the best strategy would be to stimulate both the cellular and humoral arms of the mucosal immune system. Mucosal dendritic cells have been shown to induce good protection against oral toxoplasma challenge. Our hypothesis is that an acceptable and effective human vaccine would have to carry the optimized synthetic vaccine (subunit, DNA or replicon) plus an appropriate adjuvant and to target the mucosal dendritic cells by means of an inert delivery system such as polymer microparticles, which can be endocytosed by M cells of the gut or nasal-associated lymphoid tissues.
Animals↗
Nickel resistance and chromatin condensation in Saccharomyces cerevisiae expressing a maize high mobility group I/Y protein.
Expression of a maize cDNA encoding a high mobility group (HMG) I/Y protein enables growth of transformed yeast on a medium containing toxic nickel concentrations. No difference in the nickel content was measured between yeast cells expressing either the empty vector or the ZmHMG I/Y2 cDNA. The ZmHMG I/Y2 protein contains four AT hook motifs known to be involved in binding to the minor groove of AT-rich DNA regions. HMG I/Y proteins may act as architectural elements modifying chromatin structure. Indeed, a ZmHMG I/Y2-green fluorescent protein fusion protein was observed in yeast nuclei. Nickel toxicity has been suggested to occur through an epigenetic mechanism related to chromatin condensation and DNA methylation, leading to the silencing of neighboring genes. Therefore, the ZmHMG I/Y2 protein could prevent nickel toxicity by interfering with chromatin structure. Yeast cell growth in the presence of nickel and yeast cells expressing the ZmHMG I/Y2 cDNA increased telomeric URA3 gene silencing. Furthermore, ZmHMG I/Y2 restored a wild-type level of nickel sensitivity to the yeast (Delta)rpd3 mutant. Therefore, nickel resistance of yeast cells expressing the ZmHMG I/Y2 cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA.
Amino Acid Sequence↗
Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii.
Host-cell invasion by apicomplexan parasites is extremely rapid and relies on a sequence of events that are tightly controlled in time and space. In most Apicomplexa, the gliding motility and host-cell invasion are tightly coupled to the release of microneme proteins at the apical tip of the parasites and their redistribution toward the posterior pole. This movement is dependent on an intact parasite actomyosin system. Micronemes are involved in the trafficking and storage of ligands (MICs) for host-cell receptors that are not only structurally related but also functionally conserved among the Apicomplexa. In Toxoplasma gondii, the repertoire of membrane-spanning microneme proteins includes adhesins such as TgMIC2 and escorters such as TgMIC6. The latter forms a complex with the soluble adhesins, TgMIC1 and TgMIC4 and assures their proper sorting to the mironemes. Escorters are also anticipated to bridge host-cell receptors to the parasite membrane during invasion. Most TgMICs are proteolytically cleaved either during their transport along the secretory pathway and/or after exocytosis. The biological significance of these processing events is largely unknown. One of these processing events targets a conserved motif close to the membrane-spanning domain causing the release of the processed form of the micronemes from the parasite surface. The cleavages occurring after release might contribute to the disassembly of the complexes and thus to fission between the parasitophorous vacuole and the host plasma membrane at the end of the invasion process. Gliding motility and host-cell penetration involve the redistribution of the micronemes toward the posterior pole of the parasites. This capping process involves actin polymerisation, myosin adenosine triphosphatase activation and the establishment of a connection between the MICs-receptor complexes and the actomyosin system of the parasite. The most carboxy-terminal end of the MICs cytoplasmic tails is implicated in this process, but the precise nature of the connection with the actomyosin system remains to be elucidated.
Actomyosin↗
[Identification and molecular characterization of a Toxoplasma gondii microneme].
Protozoan of the phylum Apicomplexa are of high medical and veterinary importance, causing diseases such as malaria, toxoplasmosis and cryptosporidiosis. Invasive stages of apicomplexans possess organelles named micronemes, which are involved in the invasion process. We have recently characterized a protein in micronemes of Toxoplasma gondii, TgMIC3, which possess adhesive properties to host cell surface. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that TgMIC3 is exocytosed and re-localised on the surface of the parasite during invasion. By being able to bind both the putative host cells and the parasites, TgMIC3 could be involved in invasion by acting as a bridge between the parasite and the host cell. Gene sequence analysis of TgMIC3 has revealed 5 partially overlapping EGF-like domains and a lectin binding-like domain, which can be involved in protein-protein or protein-carbohydrate interactions respectively. TgMIC3 is a homodimer synthetized with a N-terminal propeptide that is cleaved during trafficking to the organelle, presumably in the trans-Golgi network. The processing involves a serine protease and is required for correct binding function of TgMIC3. The exact role of this propeptide remains unexplained. It may be involved in the targetting of the protein to the micronemes by masking the region involved in interaction with membranes to avoid binding of the protein in the trafficking pathway.
Adhesins, Bacterial↗
The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite.
Assay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein-protein or protein-carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii.
Adhesins, Bacterial↗
Hyperhomocysteinemia in hemodialysis patients: effects of 12-month supplementation with hydrosoluble vitamins.
BACKGROUND: High-efficiency hemodialysis may induce a deficiency in hydrosoluble vitamins. Supplementation with B-complex vitamins has been shown to lower serum homocysteine concentrations in several groups, but relatively few studies have concerned hemodialysis patients. Our objectives were to determine the status in B-complex vitamins in a large cohort of unsupplemented hemodialysis patients and to assess the effects of supplementation with hydrosoluble vitamins on serum homocysteine over one year. METHODS: Serum total homocysteine (tHcy), vitamin B12, folate, pyridoxal-5'-phosphate (P-5'-P; the active moiety of vitamin B6), as well as red blood cell folate concentrations, were measured in 168 chronic dialysis patients on three times weekly high-efficiency hemodialysis and not supplemented with hydrosoluble vitamins. Their methylenetetrahydrofolate reductase C677T (MTHFR) genotypes were also determined (homozygotes TT, heterozygotes CT, without mutation CC). All involved patients were then supplemented with hydrosoluble vitamins (once daily by mouth, DiaVite; R&D Laboratories, Minneapolis, MN, USA), and half of them were randomized to receive in addition 10 mg intravenously of folic acid posthemodialysis (30 mg intravenously per week). Serum tHcy was monitored after 6 and 12 months of supplementation in the 140 and 128 patients available for follow-up. RESULTS: At baseline, serum and red blood cell folate concentrations were within normal limits in all patients except for two with borderline serum folate (mean values of 21 +/- 8 and 1195 +/- 454 nmol/L), whereas serum vitamin B12 and P-5'-P were below normal in 11 and 65 patients, respectively (mean values of 327 +/- 215 pmol/L and 19 +/- 16 nmol/L for the 168 patients). Initial tHcy levels were increased in all patients (mean 33.3 +/- 16.6 for a normal below 11.8 +/- 1.5 micromol/L); tHcy significantly decreased to 23.5 +/- 7.6 micromol/L after six months (P < 0.0001 vs. baseline) and to 21.7 +/- 6.1 micromol/L after 12 months (P < 0.0001 vs. baseline) for the entire group, but was normalized in only four patients at 12 months. After six months, the mean reduction in tHcy was slightly but significantly greater for patients receiving intravenous folic acid (12.2 +/- 18.5 micromol/L) compared with patients not receiving it (8.3 +/- 9.8 micromol/L, P < 0.05). However, at 12 months, no difference between both subgroups persisted. When considering the different genotypes, tHcy at baseline tended to be higher for TT than CT and CC (39.8 +/- 30.9 vs. 31.4 +/- 10.5 vs. 31.6 +/- 11.8 micromol/L) and decreased to respective values of 21.1 +/- 6.9 versus 21.4 +/- 6.1 versus 22.2 +/- 5.9 micromol/L at 12 months. The impact of the addition of folic acid to DiaVite appeared particularly significant in TT patients at six months. CONCLUSIONS: (1) Hyperhomocysteinemia was present in 100% of our hemodialysis patients. (2) Nearly 40% of our unsupplemented hemodialysis patients were deficient in vitamin B6. (3) Supplementation with DiaVite(R) has resulted in significant tHcy reductions for all three genotypes. (4) The impact of the proposed supplementation protocol was found after six months and was maintained, but did not increase further after 12 months of the same regimen. (5) The addition of intravenous folic acid has been associated with a more pronounced decrease in tHcy in TT patients.
Aged↗
The transmembrane protein bacterioopsin affects the polarity of the hydrophobic core of the host lipid bilayer.
Influence of the transmembrane protein bacterioopsin (the retinal-free form of bacteriorhodopsin) on the polarity of egg-phosphatidylcholine bilayers was studied by means of a steady-state and time-resolved fluorescence approach exploiting the solvatochromic properties of the 2-anthroyl fluorophore. Introduced in phosphatidylcholine molecules in the form of 8-(2-anthroyl)octanoic acid, this fluorophore probed the hydrocarbon core of the lipid bilayer. As previously shown (E. Pérochon et al., Biochemistry 31 (1992) 7672-7682), water molecules were detected in this region of the terminal part of the lipid acyl chains. Their number was considerably reduced upon addition of bacterioopsin to the lipids. This was assessed by a blue shift in the fluorescence emission spectra of the probe and a marked decrease in the fractional population of fluorophores interacting with water, to the benefit of those experiencing a hydrophobic environment. In agreement with current theories, this decrease in the hydration of the bilayer may be linked to an increase in the acyl chain order and a decrease in the lateral diffusion coefficient of lipids near the protein. The data obtained at high protein concentration accounts for a protein/lipid interface which is much less hydrated than the hydrophobic core of a protein-free lipid bilayer.
Bacteriorhodopsins↗
Plant responses to metal toxicity.
Metal toxicity for living organisms involves oxidative and/or genotoxic mechanisms. Plant protection against metal toxicity occurs, at least in part, through control of root metal uptake and of long distance metal transport. Inside cells, proteins such as ferritins and metallothioneins, and glutathion-derived peptides named phytochelatins, participate in excess metal storage and detoxification. Low molecular weight organic molecules, mainly organic acids and amino acids and their derivatives, also play an important role in plant metal homeostasis. When these systems are overloaded, oxidative stress defense mechanisms are activated. Molecular and cellular knowledge of these processes will be necessary to improve plant metal resistance. Occurrence of naturally tolerant plants which hyperaccumulate metals provides helpful tools for this research.
Metals↗
Haemodialysis for severe hyperammonaemic coma complicating urinary diversions.
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Aged↗
Expression cloning in Fe2+ transport defective yeast of a novel maize MYC transcription factor.
A complementation approach of the yeast fet3fet4 mutant strain, defective in both low- and high-affinity iron transport, was initiated as an attempt to characterize the Fe(III)-mugineic acid (MA) transporter from grasses. A maize cDNA encoding a novel MYC transcription factor, named 7E, was cloned by screening an iron-deficient maize root cDNA expression library on a minimum media containing Fe(III)-deoxyMA as a unique iron source. 7E expression restored growth specifically to the fet3 fet4 mutant strain. It did not affect growth rate of a trk1trk2 potassium transport defective yeast strain or parental W303 strain growth rate. No 55Fe uptake increase was observed in 7E transformed fet3 fet4 yeast during short-term kinetics. However, the iron accumulation in these cells was 1.3-fold higher than in untransformed cells after a 24-h period. The 7E protein contained 694 amino acids and had a predicted molecular mass of 74.2kDa. It had 44% identity with the RAP-1 protein, a 67.9-kDa MYC-like protein from Arabidopsis thaliana which binds the G-box sequence via a basic region helix-loop-helix (bHLH), without requiring heterodimerization with MYB proteins. Phylogenic comparisons revealed that the maize 7E protein was related to the Arabidopsis thaliana RAP-1 protein and to the Phaseolus vulgaris PG1. This similarity was particularly evident for the bHLH domain, which was 95% identical between maize 7E and Arabidopsis thaliana RAP-1. 7E, RAP-1 and PG-1 proteins revealed a plant MYC-like sub-family that was more related to the maize repressor-like IN1 than to maize R proteins. 7E mRNA was detected in both roots and leaves by the Northern analysis. The amount of 7E mRNA increased, in response to iron starvation, by 20 and 40% in roots and leaves, respectively. The relationship between iron metabolism and myc expression in animal cells is discussed.
Amino Acid Sequence↗
Performance of the new plasma-compatible Advantage blood glucose test strips.
OBJECTIVE: To establish a plasma calibration curve for the Advantage Blood Glucose System that would permit direct comparison with results provided by clinical laboratories. DESIGN AND METHODS: The initial study involved the comparison between the Advantage whole blood referenced results and the laboratory plasma results. The second study validated the plasma-compatibility of the first new lot of Advantage test strips with this new calibration curve. RESULTS: Plasma and whole blood glucose data, obtained at Hôpital Maisonneuve-Rosemont in Montréal, demonstrated the typical differences between these two sample types. Mathematical correction of the Advantage results eliminated this difference. This new equation was then encoded into the code keys of a new lot of Advantage test strips and a second study was conducted to verify the comparability to laboratory plasma glucose. CONCLUSION: The results for the Advantage Blood Glucose System may now be compared directly with the laboratory plasma or serum glucose results.
Blood Chemical Analysis↗
Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD.
Listeria monocytogenes is a bacterial pathogen that is able to invade nonphagocytic cells. Two surface proteins, internalin, the inlA gene product, and InlB, play important roles in the entry into cultured mammalian cells. These proteins also have extensive sequence similarities. Previously, Southern hybridization predicted the existence of an internalin multigene family. Recently, InlC, a secreted protein of 30 kDa homologous to InlA and InlB, was identified. In this work, we identified and characterized four new members of the internalin multigene family, inlC2, inlD, inlE, and inlF which encode proteins of 548, 567, 499, and 821 amino acids respectively. inlC2, inlD, and inlE are contiguous on the chromosome of L. monocytogenes EGD, whereas inlF is located in a different chromosomal region. These four inl gene products display the principal features of internalin, namely, a signal sequence, two regions of repeats (or LRR and B repeats), and a putative cell wall anchor sequence containing the sorting motif LPXTG. The four inl genes were maximally expressed albeit at a low level during early exponential growth in bacterial medium at 37 degrees C. The role of these inl genes in L. monocytogenes invasion was assessed by constructing isogenic chromosomal deletion mutants and testing them for entry into various nonphagocytic cells. Unexpectedly, the inlC2, inlD, inlE, and inlF null mutants were not affected for entry into any of the cell lines tested, raising the possibility that these genes are needed for an aspect of pathogenicity other than invasion. The identity of such an aspect remains to be determined.
Amino Acid Sequence↗
Molecular and genetic determinants involved in invasion of mammalian cells by Listeria monocytogenes.
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Amino Acid Sequence↗
Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells.
Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram-positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C-terminal end. By immunofluorescence and immunogold labelling, we show that in wild-type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C-terminal region of internalin is necessary for cell-surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a 'domain-swapping' strategy-replacement of the cell wall anchor of IniA by the membrane anchor of ActA- we show that the reduced ability to adhere and enter cells of strains expressing IniA-ActA correlates with a lower amount of surface-exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.
Amino Acid Sequence↗
Antibodies to the leucine-rich repeat region of internalin block entry of Listeria monocytogenes into cells expressing E-cadherin.
Internalin, a surface protein essential for entry of Listeria monocytogenes EGD into epithelial cells, was used as an antigen to raise nine monoclonal antibodies. These monoclonal antibodies recognized seven distinct epitopes which were located in three different regions of the protein. Three of them inhibited internalin-mediated entry and recognized the amino-terminal leucine-rich repeat region of the protein, suggesting that this region is essential for entry.
Animals↗
Structure and differential expression of two maize ferritin genes in response to iron and abscisic acid.
In plants, synthesis of the iron-storage protein ferritin in response to iron is not regulated at the translational level; this is in contrast to ferritin synthesis in animals. Part of the response is mediated through a transduction pathway which involves the plant hormone abscisic acid. In this work, we report the cloning and sequencing of two maize ferritin genes (ZmFer1 and ZmFer2) coding for members of the two ferritin mRNA subclasses, FM1 and FM2, respectively. Although plant and animal ferritins are closely related proteins, a major difference is observed between the organisation of the genes. Both maize ferritin genes are organised as eight exons and seven introns, the positions of which are identical within the two genes, while animal ferritin genes are interrupted by three introns, at positions different from those found in maize genes. Sequence divergence between the 3' untranslated regions of these genes has allowed the use of specific probes to study the accumulation of FM1 and FM2 transcripts in response to various environmental cues. Such probes have shown that FM1 and FM2 transcripts accumulate with differential kinetics in response to iron; FM1 mRNA accumulate earlier than FM2 mRNA and only FM2 transcripts accumulate in response to exogenous abscisic acid or water stress. Mapping of the transcriptional initiation region of these two genes defined their 5' upstream regions and allowed a sequence comparison of their promoters, which appeared highly divergent. This raises the possibility that the differential accumulation of FM1 and FM2 mRNAs in response to iron, abscisic acid and drought could be due to differential transcription of ZmFer1 and ZmFer2.
Abscisic Acid↗
Subcellular distribution of serine acetyltransferase from Pisum sativum and characterization of an Arabidopsis thaliana putative cytosolic isoform.
The intracellular compartmentation of serine acetyltransferase, a key enzyme in the L-cysteine biosynthesis pathway, has been investigated in pea (Pisum sativum) leaves, by isolation of organelles and fractionation of protoplasts. Enzyme activity was mainly located in mitochondria (approximately 76% of total cellular activity). Significant activity was also identified in both the cytosol (14% of total activity) and chloroplasts (10% of total activity). Three enzyme forms were separated by anion-exchange chromatography, and each form was found to be specific for a given intracellular compartment. To obtain cDNA encoding the isoforms, functional complementation experiments were performed using an Arabidopsis thaliana expression library and an Escherichia coli mutant devoid of serine acetyltransferase activity. This strategy allowed isolation of three distinct cDNAs encoding serine acetyltransferase isoforms, as confirmed by enzyme activity measurements, genomic hybridizations, and nucleotide sequencing. The cDNA and related gene for one of the three isoforms have been characterized. The predicted amino acid sequence shows that it encodes a polypeptide of M(r) 34,330 exhibiting 41% amino acid identity with the E. coli serine acetyltransferase. Since none of the general features of transit peptides could be observed in the N-terminal region of this isoform, we assume that it is a cytosolic form.
Acetyltransferases↗