Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin's mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.

Transcriptomic and genetic studies identify IL-33 as a candidate gene for Alzheimer's disease.

The only recognized genetic determinant of the common forms of Alzheimer's disease (AD) is the epsilon 4 allele of the apolipoprotein E gene (APOE). To identify new candidate genes, we recently performed transcriptomic analysis of 2741 genes in chromosomal regions of interest using brain tissue of AD cases and controls. From 82 differentially expressed genes, 1156 polymorphisms were genotyped in two independent discovery subsamples (n=945). Seventeen genes exhibited at least one polymorphism associated with AD risk, and following correction for multiple testing, we retained the interleukin (IL)-33 gene. We first confirmed that the IL-33 expression was decreased in the brain of AD cases compared with that of controls. Further genetic analysis led us to select three polymorphisms within this gene, which we analyzed in three independent case-control studies. These polymorphisms and a resulting protective haplotype were systematically associated with AD risk in non-APOE epsilon 4 carriers. Using a large prospective study, these associations were also detected when analyzing the prevalent and incident AD cases together or the incident AD cases alone. These polymorphisms were also associated with less cerebral amyloid angiopathy (CAA) in the brain of non-APOE epsilon 4 AD cases. Immunohistochemistry experiments finally indicated that the IL-33 expression was consistently restricted to vascular capillaries in the brain. Moreover, IL-33 overexpression in cellular models led to a specific decrease in secretion of the A beta(40) peptides, the main CAA component. In conclusion, our data suggest that genetic variants in IL-33 gene may be associated with a decrease in AD risk potentially in modulating CAA formation.

Evidence for induction of the ornithine transcarbamylase expression in Alzheimer's disease.

To more rapidly identify candidate genes located within chromosomal regions of interest defined by genome scan studies in Alzheimer's disease (AD), we have developed a customized microarray containing all the ORFs (n=2741) located within nine of these regions. Levels of gene expression were assessed in total RNA from brain tissue of 12 controls and 12 AD patients. Of all genes showing differential expression, we focused on the ornithine transcarbamylase (OTC) gene on Xp21.1., a key enzyme of the urea cycle which we found to be expressed in AD brains but not in controls, as confirmed by RT-PCR. We also detected mRNA expression of all the other urea cycle enzymes in AD brains. Immunochemistry experiments revealed that the OTC expression was strictly restricted to vascular endothelial cells in brain. Furthermore, OTC activity was 880% increased in the CSF of probable AD cases compared with controls. We analysed the association of the OTC -389 G/A and -241 A/G promoter polymorphisms with the risk of developing AD. We observed that rare haplotypes may be associated with the risk of AD through a possible modulation of the methylation of the OTC promoter. In conclusion, our results suggest the involvement of a new pathway in AD brains involving the urea cycle.

The Dijon Physical Activity Score: reproducibility and correlations with physical fitness in patients with coronary artery disease.

OBJECTIVES: To study the clinimetric properties of the Dijon Physical Activity Score (PAS) in patients with coronary artery disease (CAD). PATIENTS: Two populations of patients with CAD: one group of stabilized patients from the RICO county-wide monitoring program and one group in the initial phase of a cardiovascular rehabilitation program (CVR group). METHODS: The patients carried out a maximal effort test on a cycle ergometer, plus two walking tests (a six-minute walk test and a 200 m fast walk test). They completed the Dijon PAS questionnaire on two occasions at an interval of 10 days. The reproducibility of the score and the latter's correlations with physical parameters were analyzed. RESULTS: Sixty-seven subjects were included and 52 answered the questionnaire both times. The average time spent answering the questionnaire was 173+/-37 seconds and reproducibility was satisfactory in the RICO group only. In this group, there were significant correlations between total score and maximal power during the effort test (r=0.41; P<0.05) and between the "sports/leisure activities" sub-score and maximal power (r=0.57; P<0.01). No correlations were found in the CVR group. CONCLUSION: The Dijon PAS is a simple, generic, reproducible and reliable score for measuring physical activity in patients with stable coronary artery disease but, because of the conjunction of confounding factors, it is not suitable for subjects who experienced a recent acute cardiac event. It could thus be used in epidemiological studies to determine the impact of a sedentary lifestyle and the efficacy of methods intended to counter sedentariness and to help design personalized secondary prevention programs.

Relevance and limitations of public databases for microarray design: a critical approach to gene predictions.

In conjunction with the completion of the human genome sequence, microarray technology offers a complementary strategy to traditional methodologies used to search for genetic determinants involved in multifactorial diseases such as Alzheimer's disease. In order to gain benefits from this strategy, we have designed home-made microarrays to compare the expression of all ORFs located within loci of interest defined by genome scanning in Alzheimer family studies. Two approaches were selected using either probes amplified by PCR from a cDNA bank or specific oligonucleotides. Here, we report the challenging task of validating, prioritising and selecting the best ORFs derived from the genome sequence. The initial inventory from the NCBI website allowed us to select 5849 ORF's within nine loci. Half of them resulted from prediction models using the GenomeScan software. However, our data have shown that predicted ORFs may not be representative of exonic sequences, or even real genes. These observations have led us to exclude these ORFs from our study, decreasing their number from 5849 to 2748. Microarrays may be only 'snapshots' of our current knowledge of the human genome.

Differential modulation of Bordetella pertussis virulence genes as evidenced by DNA microarray analysis.

The production of most factors involved in Bordetella pertussis virulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg+ phase virulence-activated genes (vags) are expressed, and virulence-repressed genes (vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO(4) or nicotinic acid. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO(4) or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

Astaxanthin accumulation in Haematococcus requires a cytochrome P450 hydroxylase and an active synthesis of fatty acids.

Astaxanthin accumulation by green microalgae is a natural phenomenon known as red snows and blood rains. The fact that astaxanthin synthesis requires oxygen, NADPH and Fe(2+) led Cunningham and Gantt [Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 557-583] to propose that a cytochrome P450-dependent enzyme might be involved in the transformation of beta-carotene to astaxanthin. In Haematococcus only esterified astaxanthin molecules accumulate, but it is not determined whether a fatty acid synthesis should occur simultaneously to allow pigment accumulation. The aim of this contribution was to answer these two questions using specific inhibitors of beta-carotene (norflurazon) and fatty acid (cerulenin) synthesis, and of cytochrome P450 enzyme activity (ellipticine).

Improved liquid chromatographic method for the analysis of photosynthetic pigments of higher plants.

The paper presents an improved reversed-phase LC method for the separation of the pigments from green leaves. A good separation of carotenoids and of their cis- and trans-isomers was achieved, especially for the separation of trans-lutein, zeaxanthin, cis-lutein, which are usually not well separated. No perfect separation of alpha-carotene, beta-carotene and pheophytin a was possible, but conditions for a perfect coelution of pheophytin a with either beta-carotene or alpha-carotene were established. Simultaneous equations allowing the determination of pheophytin a and alpha-carotene or pheophytin a and beta-carotene are also given.

Cloning of Schizosaccharomyces pombe bio2 by heterologous complementation of a Saccharomyces cerevisiae mutant.

A Saccharomyces cerevisiae mutant affected in the last step of the biotin biosynthesis pathway was isolated by using a transposon mutagenesis method. The gene BIO2, encoding a biotin synthase, is shown to be interrupted in this mutant. Heterologous complementation experiment allowed the cloning and the characterization of a novel bio gene: bio2, encoding biotin synthase from Schizosaccharomyces pombe.

Characterization of the biotin biosynthesis pathway in Saccharomyces cerevisiae and evidence for a cluster containing BIO5, a novel gene involved in vitamer uptake.

An engineered mutant of Saccharomyces cerevisiae affected in biotin biosynthesis has been isolated. This mutant allowed the characterization of a bio cluster (BIO3-4-5). We demonstrate that BIO3 (YNR058w) and BIO4 (YNR057c) encode, respectively, a 7, 8-diaminopelargonic acid aminotransferase and a dethiobiotin synthase, involved in the biotin biosynthesis pathway. A novel gene, BIO5 (YNR056c), is present immediately downstream from BIO4. This gene encodes Bio5p, a protein with 11 putative transmembrane regions. Uptake experiments performed with labeled 7-keto 8-aminopelargonic acid indicate that Bio5p is responsible for transport into the cell of 7-keto 8-aminopelargonic acid.

On the presence and role of a molecule of chlorophyll a in the cytochrome b6 f complex.

Highly purified preparations of cytochrome b6 f complex from the unicellar freshwater alga Chlamydomonas reinhardtii contain about 1 molecule of chlorophyll a/cytochrome f. Several lines of evidence indicate that the chlorophyll is an authentic component of the complex rather than a contaminant. In particular, (i) the stoichiometry is constant; (ii) the chlorophyll is associated with the complex at a specific binding site, as evidenced by resonance Raman spectroscopy; (iii) it does not originate from free chlorophyll released from thylakoid membranes upon solubilization; and (iv) its rate of exchange with free, radioactive chlorophyll a is extremely slow (weeks). Some of the putative functional roles for a chlorophyll in the b6f complex are experimentally ruled out, and its possible evolutionary origin is briefly discussed.

The ATP binding cassette transporters Pdr5 and Snq2 of Saccharomyces cerevisiae can mediate transport of steroids in vivo.

Multiple or pleiotropic drug resistance in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 and Snq2 ATP binding cassette multidrug transporters. Expression of Pdr5 and Snq2 is regulated by the two transcription factors Pdr1 and Pdr3, as multidrug-resistant pdr1 and pdr3 gain-of-function mutants overexpress both drug efflux pumps. One such pdr1 mutant allele was previously cloned in a genetic screen by its ability to suppress the squelching toxicity mediated by an estradiol-inducible chimeric VP16-human estrogen receptor (VEO) expressed in yeast (Gilbert, D. M. , Heery, D. M., Losson, R., Chambon, P., and Lemoine, Y. (1993) Mol. Cell. Biol. 13, 462-472). In this study, we demonstrate that relief of estradiol toxicity in yeast cells expressing VEO requires functional PDR5 and SNQ2 genes, since a Deltapdr5 Deltasnq2 double deletion leads to an increased estradiol toxicity. Furthermore, using URA3 as an estradiol-inducible reporter gene, we show that Pdr5 and Snq2, when overexpressed from high-copy plasmids, can reduce the intracellular concentration of estradiol. In contrast, a Deltapdr5 Deltasnq2 double deletion mutant accumulates almost 30-fold more intracellular estradiol than the isogenic wild type. Indirect immunofluorescence showed that a pdr1-3 mutant massively overexpresses Pdr5 at the plasma membrane, suggesting that estradiol efflux from the cells occurs across the plasma membrane. Our data demonstrate that Pdr5 and Snq2 can transport steroid substrates in vivo and suggest that steroids and/or related membrane lipids could represent physiological substrates for certain yeast ABC transporters, which are otherwise involved in the development of pleiotropic drug resistance.

Estradiol-inducible squelching and cell growth arrest by a chimeric VP16-estrogen receptor expressed in Saccharomyces cerevisiae: suppression by an allele of PDR1.

We have constructed and characterized a flexible system for analyzing the phenomenon of squelching and estrogen receptor function in the yeast Saccharomyces cerevisiae. The A/B region of the human estrogen receptor was replaced with the transcriptional activating domain of VP16 and expressed in yeast cells from high-copy-number plasmids. Addition of hormone resulted in an immediate inhibition of expression (squelching) of a chromosomally integrated GAL1:lacZ reporter gene and the eventual arrest of cell growth (toxicity). In order to determine whether a relationship exists between toxicity and squelching, mutations were made in this chimeric receptor (VEO) and their effects on transcriptional activation, squelching, and toxicity were compared. A direct correlation was found between mutations in VEO that reduced VP16 transactivation ability in yeast cells and those that reduced both squelching and toxicity. Surprisingly, mutations in the DNA binding domain (DBD) of VEO dramatically reduced squelching and completely relieved toxicity, suggesting a role for the DBD in squelching and strengthening the correlation between squelching and toxicity. To demonstrate the utility of this system for carrying out genetic selection, a plasmid-based yeast genomic bank was screened for genes that can relieve the toxicity of VEO by means of an elevated copy number, resulting in the repeated cloning of an allele of the PDR1 (pleiotropic drug resistance) gene. We present evidence that mutations in PDR1 can modulate the intracellular availability of estradiol by the same mechanism that leads to multiple drug resistance in yeast cells. Taken together, our results provide evidence that cell growth arrest occurs when squelching exceeds a certain threshold and that strong squelching requires both a DBD and a transcriptional activating domain. Furthermore, we show that growth arrest can provide a useful phenotype for carrying out the genetic analysis of both squelching and estrogen receptor function in yeast cells.

Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate.

The pimeloyl-CoA synthase from Bacillus sphaericus has been purified to homogeneity from an overproducing strain of Escherichia coli. The purification yielded milligram quantities of the synthase with a specific activity of 1 unit/mg of protein. Analysis of the products showed that this enzyme catalysed the transformation of pimelate into pimeloyl-CoA with concomitant hydrolysis of ATP to AMP. Using a continuous spectrophotometric assay, we have examined the catalytic properties of the pure enzyme. The pH profile under Vmax. conditions showed a maximum around 8.5. Apparent Km values for pimelate, CoASH, ATP.Mg2- and Mg2+ were respectively 145 microM, 33 microM, 170 microM and 2.3 mM. The enzyme was inhibited by Mg2+ above 10 mM. This acid-CoA ligase exhibited a very sharp substrate specificity, e.g. neither GTP nor pimelate analogues (di- or mono-carboxylic acids) were processed. The bivalent metal ion requirement was also investigated: Mn2+ (73%) and Co2+ (32%) but not Ca2+ could replace Mg2+. The enzyme was inhibited by metal chelators such as 1,10-phenanthroline and EDTA. The synthase was a homodimer with a 28,000-M(r) subunit. N-Terminal sequencing definitely proved that this enzyme was encoded by the bioW gene. A careful study of pimelate uptake by B. sphaericus, E. coli and Pseudomonas dentrificans showed that this metabolite crossed the membrane of these microorganisms by passive diffusion, ruling out the involvement of the bioX gene product as pimelate carrier.

Functional analysis of the human estrogen receptor using a phenotypic transactivation assay in yeast.

We have constructed yeast strains in which the expression of the Saccharomyces cerevisiae URA3 gene is induced by the human estrogen receptor (hER). Promoter sequences required for both basal and activated transcription of URA3 were replaced with one or three estrogen-response elements (EREs) positioned upstream of the native TATA box. These constructs were each integrated at the TRP1 locus of a yeast strain in which the natural URA3 gene had been deleted, and the integrants were transformed with low- or high-copy-number shuttle plasmids expressing wild-type or truncated derivatives of hER. Transformants were assayed for growth on uracil-deficient medium plus or minus estradiol (E2), for resistance to 5-fluoroorotic acid (5-FOA) and for activity of OMPdecase (orotidine-5'-monophosphate decarboxylase), the product of the URA3 gene. We show that the growth and 5-FOA-resistance (5-FOAR) phenotypes of these strains are strictly dependent upon the function of the receptor derivatives. Induction of URA3, measured by OMPdecase activity, was observed over a 20- to 2500-fold range depending on the receptor derivative, its expression level and the number of EREs in the responsive promoter. Both one- and three-ERE reporter strains expressing the full-length receptor are completely E2-dependent for growth, and display a 5-FOAR phenotype in the absence of the hormone. We demonstrate that the individual hER transactivation functions, TAF1 and TAF2, are both functional in yeast, and that the hormone-dependent TAF2 is the more potent activator on our reporters. We show that hER displays strong homosynergism in yeast, and discuss the contributions of the two TAFs in hER synergism.(ABSTRACT TRUNCATED AT 250 WORDS)

Promoter specificity of the two transcriptional activation functions of the human oestrogen receptor in yeast.

We have previously demonstrated that the human oestrogen receptor (hER) contains two transcriptional activation functions located in the N-terminal region (TAF-1) and in the hormone binding domain (TAF-2), which can act both independently and synergistically in a promoter- and cell-specific manner in animal cells. We have also demonstrated that hER can activate transcription from chimaeric oestrogen-responsive GAL1 promoters in yeast, and shown that transcriptional activation was due to TAF-1, whereas TAF-2 showed little, if any, transcriptional activity on these promoters. By using a more complex promoter derived from the URA3 gene, we now show that TAF-2 is also active in yeast, and that the activities of TAF-1 and TAF-2 are promoter-context-specific in yeast. We also confirm that the agonistic activity of 4-hydroxytamoxifen (OHT) can be ascribed to the activity of TAF-1.

A new signal peptide useful for secretion of heterologous proteins from yeast and its application for synthesis of hirudin.

The BGL2 gene from Saccharomyces cerevisiae encodes a beta-glucanase which is localized to the yeast cell wall. The ability of a 23-amino acid (aa) signal peptide derived from the BGL2 gene to direct a heterologous protein to the secretory pathway of yeast has been compared to that of the MF alpha 1-encoded signal peptide in a series of gene fusions. As a model protein, the leech anticoagulant, recombinant hirudin variant 2-Lys47 (HIR) has been studied. From a multicopy plasmid chimaeric proteins were produced which carry the BGL2 signal peptide (or the artificial BGL2 pre-Val7 variant) (i) in front of the MF alpha 1 pro sequence (or modified versions of MF alpha 1 pro), i.e., a prepro signal, or (ii) joined directly to the heterologous protein. Accumulation of active HIR in yeast culture supernatants was observed when the BGL2 (or the BGL2 pre-Val7) signal peptide were used in combination with either of three versions of the MF alpha 1 pro peptide: the authentic MF alpha 1 pro, a partially deleted MF alpha 1 pro-delta 22-61, or a pro bearing an aa change (MF alpha 1 pro-Gly22). In each case the BGL2 signal peptide (or its variant) has proven equally productive to the corresponding MF alpha 1 peptide. Four times more active HIR was detected in the culture supernatant when either signal peptide was fused directly to the recombinant protein, as compared to a prepro protein version. Correct signal peptide cleavage was obtained when HIR was produced as a BGL2 pre-Val7::fusion protein.

Isolation of Bacillus sphaericus biotin synthesis control mutants: evidence for transcriptional regulation of bio genes.

The genes involved in biotin synthesis have recently been isolated from Bacillus sphaericus [Gloeckler et al., Gene 87 (1990) 63-70]. Sequence analysis revealed that they are organized into two gene clusters, designated bioXWF and bioDAYB. The 5'-noncoding region of the bioD locus fused to the xylE reporter gene was inserted into the Gram-positive pUB110 replicon and the resulting plasmid was introduced into B. sphaericus IFO3525. Transformants expressed the xylE gene only if the biotin concentration in the growth medium remained below 50 ng/ml. After mutagenesis, colonies were screened for their ability to express the chromogenic marker in the presence of an excess of biotin. Most of these mutants escaped biotin repression of xylE gene expression. Classical genetic analysis showed they formed two main categories: chromosomal mutations, pleiotropically acting in trans on both bioXWF and bioDAYB 5'-noncoding regions, in which a 15-bp region common to both promoters represented a hot-spot for the second class of plasmid-associated mutations. These results, completed by the identification of transcription start points for the bioD and bioX genes, strongly suggest that this 15-bp sequence overlaps the site of a biotin-mediated negative regulation circuit controlling the transcription of the bio genes.

Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus.

A promoter probe vector, pTG244, was constructed with the aim of isolating transcription initiation signals from Streptococcus thermophilus (Streptococcus salivarius subsp. thermophilus). pTG244 is based on the Escherichia coli-streptococcus shuttle vector pTG222, into which the promoterless chloramphenicol acetyltransferase gene of Bacillus pumilus (cat-86) was cloned. Random Sau3A fragments from the S. thermophilus A054 chromosomal DNA were cloned upstream of the cat-86 gene by using E. coli as the host. The pool of recombinant plasmids were introduced into S. thermophilus and Lactococcus lactis subsp. lactis in order to search for promoter activity in these hosts. For S. thermophilus, it was necessary to first select erythromycin-resistant transformants and then to screen for chloramphenicol resistance among these. Direct selection of chloramphenicol-resistant clones was, however, possible in L. lactis subsp. lactis. Six fragments exhibiting promoter activity were characterized in S. thermophilus by measuring the levels of cat-86 transcription and/or chloramphenicol acetyltransferase specific activity. Three of the promoter-carrying fragments were sequenced. The 5' ends of their corresponding mRNAs were determined by S1 mapping and shown to correspond to a purine residue in all cases. Upstream from these potential transcription start points, sequences homologous to the E. coli sigma 70 and the Bacillus subtilis vegetative sigma 43 (or sigma A) consensus promoters were identified.