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.

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.

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.

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.

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)

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.

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.

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli.

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.

Cloning and nucleotide sequence of the phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032.

As a first step in determining the importance of the anaplerotic function of phosphoenolpyruvate carboxylase (PEPC) in amino acid biosynthesis, the ppc gene coding for PEPC of Corynebacterium glutamicum ATCC13032 has been cloned by complementation of an Escherichia coli ppc mutant strain. PEPC activity encoded by the cloned gene is not affected by acetyl-CoA under conditions where the E. coli enzyme is strongly activated, whereas acetyl-CoA is able to relieve inhibition by L-aspartate used singly or in combination with alpha-ketoglutarate. Amplification of the ppc gene in a C. glutamicum lysine-excreting strain resulted in increased PEPC-specific activity and lysine productivity. The nucleotide sequence of a DNA fragment of 4885 bp encompassing the ppc gene has been determined. At the amino acid level, PEPC from C. glutamicum presents overall a high degree of similarity with corresponding enzymes from three different organisms. The location of some strictly conserved regions may have important implications for PEPC activity and allostery.

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.

Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin.

Using 8.8 kb of genetic information from Bacillus sphaericus, it was possible to confer to Escherichia coli bio- strains, including delta bioA-D, bioC-, bioH-, the ability to convert exogenous pimelate into biotin. The bio genes were borne on two recombinant plasmids with inserts of 4.3 kb and 4.5 kb, which had been isolated from a genomic bank of HindIII-digested B. sphaericus DNA, by phenotypic complementation of various E. coli bio mutants. The B. sphaericus bioD and bioA genes were unambiguously identified within the 4.3-kb insert and shown to be closely linked to bioY (coding for a protein with a presently unknown function) and to bioB [Ohsawa et al., Gene 80 (1989) 39-48]. These genes are clustered in the order bioDAYB. The 4.5-kb fragment contains genetic information for three different proteins, the products of bioX, bioW and bioF. Complementation studies using an E. coli bioF mutant and a B. subtilis bio112TG3 strain, revealed that the third ORF of this cluster encodes 7-keto-8-aminopelargonic acid synthetase. A combination of bioW and bioF allows an efficient complementation of E. coli bioC and bioH mutants, provided that pimelate is added to the biotin-depleted growth medium. No function could be identified for the product of bioX. The gene order of this cluster is bioXWF. By sequence analysis, the two cloned DNA fragments were shown to bear overlapping open reading frames and secondary structures at their 3' ends, typical of transcription terminators.(ABSTRACT TRUNCATED AT 250 WORDS)

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).

Development of an efficient spheroplast transformation procedure for S. thermophilus: the use of transfection to define a regeneration medium.

The conditions for the efficient polyethylene glycol (PEG)-induced spheroplast transformation of three strains of Streptococcus thermophilus have been established. This required the careful optimization of various experimental parameters, the most important being the choice of the lytic enzyme (lysozyme versus mutanolysin), the extent of cell wall digestion and the conditions for the PEG shock which were found to be strain-specific. The transfection assay we had previously developed for S. thermophilus represented a key step and powerful tool in our transformation studies. It allowed individual and stepwise adjustment of the above mentioned factors, but was also compulsory for the establishment of an effective regeneration medium for the strains we examined. Among various potential osmotic protectors tested, raffinose in combination with CaCl2 and MgCl2 was most efficient and routinely supported regeneration with up to 10% efficiency, after PEG treatment. With the spheroplast transformation procedure described in this paper, shuttle vectors and recombinant plasmids could be introduced into three industrial yogurt starters, with maximal efficiencies of 7.5 x 10(4) transformants/micrograms of liposome encapsulated, covalently closed circular DNA. A striking, yet unexplained, reduction in transformation rates was observed when erythromycin rather than chloramphenicol was used as the selecting agent.

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.

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.

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.

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.

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.

The regulation of urea amidolyase of Saccharomyces cerevisiae: mating type influence on a constitutivity mutation acting in cis.

Constitutivity for the synthesis of the urea amidolyase bienzymatic complex is obtained by durOh mutations located in the regulatory genetic region adjacent to the dur1, dur2 gene cluster. The durOh mutations act only in cis and are a new case of cis effect strongly cancelled in alpha/a diploid, similar to cargA+Oh mutation shown previously to lead to arginase constitutivity. Illegitimate diploids do not show such a cancellation of constitutivity. The constitutivity produced by durOh mutation comprises the process of induction and the release of the glutamine effect. It does not impair the NH+4 effect.