[GA2LEN (Global Allergy and Asthma European Network)]. / Le réseau d'excellence de l'Union Européenne GA2LEN (Global Allergy and Asthma European Network).

Allergic diseases represent a major health problem in Europe. They are increasing in prevalence, severity and costs. GA2LEN (Global Allergy and Asthma European Network), an FP6 Network of Excellence, was created in 2005 as a vehicle to ensure excellence in research bringing together research and clinical institutions to combat fragmentation in the European research area and to tackle Allergy in its globality. GA2LEN benefited greatly from the voluntary efforts of researchers who are strongly committed to this model of pan-European collaboration. The network was organized in order to increase networking for scientific projects in allergy and asthma around Europe and to make GA2LEN the world leader in the field. Besides these activities, research has been jointly made and the first papers are being published. GA2LEN achievements in general can be grouped as those for a durable infrastructure built up during the project phase those which are project-related work based on these novel infrastructures, and the development and implementations of guidelines. The major achievements of GA2LEN are reported in this paper.

GA2LEN (Global Allergy and Asthma European Network) addresses the allergy and asthma 'epidemic'.

Allergic diseases represent a major health problem in Europe. They are increasing in prevalence, severity and costs. The Global Allergy and Asthma European Network (GA(2)LEN), a Sixth EU Framework Program for Research and Technological Development (FP6) Network of Excellence, was created in 2005 as a vehicle to ensure excellence in research bringing together research and clinical institutions to combat fragmentation in the European research area and to tackle allergy in its globality. The Global Allergy and Asthma European Network has benefited greatly from the voluntary efforts of researchers who are strongly committed to this model of pan-European collaboration. The network was organized in order to increase networking for scientific projects in allergy and asthma around Europe and to make GA(2)LEN the world leader in the field. Besides these activities, research has also been carried out and the first papers are being published. Achievements of the Global Allergy and Asthma European Network can be grouped as follows: (i) those for a durable infrastructure built up during the project phase, (ii) those which are project-related and based on these novel infrastructures, and (iii) the development and implementation of guidelines. The major achievements of GA(2)LEN are reported in this paper.

The iodocyanopindolol and SM-11044 binding protein belongs to the TM9SF multispanning membrane protein superfamily.

SM-11044 is the only beta-adrenergic agonist that inhibits guinea pig eosinophil chemotaxis and induces relaxation of depolarized rat colon tonus. We have previously reported the purification of a 34 kDa photoaffinity-labeled SM-11044 binding protein (SMBP) from rat colon that may mediate the biological effects of the ligand and that differs from all known monoamine receptors (Sugasawa et al., J. Biol. Chem. 272 (1997) 21244). The present report describes partial amino acid sequence of rat SMBP and molecular cloning of corresponding human SMBP (hSMBP) cDNA. This cDNA encodes a 588 amino acid residue polypeptide comprising a signal peptide, a long hydrophilic amino-terminal region, and a highly hydrophobic C-terminal portion organized into nine putative transmembrane domains. The sequence and structure of hSMBP shows homology to members of a new transmembrane protein 9 superfamily (TM9SF). Comparison of hSMBP with related protein sequences from yeast, plant and human revealed two subgroups within TM9SF. The members of these groups differ in length and have characteristic amino acid sequence motifs in their amino-terminal portion. Northern blot analysis revealed two major SMBP mRNAs, at 3.4 and 3.8 kb, that were present in all the human tissues examined. Western blot experiments detected SMBP as a 70 kDa protein that may be further cleaved into an active 34 kDa N-terminal polypeptide. Stable Chinese Hamster Ovary cell transfectants expressing hSMBP cDNA displayed specific binding of [(125)I]iodocyanopindolol that was displaced by SM-11044 in a dose-dependent manner. Thus, SMBP is the first member of TM9SF with functional ligand binding properties, suggesting that some of these integral membrane proteins may function as channels, small molecule transporters or receptors.

The protein-protein interaction map of Helicobacter pylori.

With the availability of complete DNA sequences for many prokaryotic and eukaryotic genomes, and soon for the human genome itself, it is important to develop reliable proteome-wide approaches for a better understanding of protein function. As elementary constituents of cellular protein complexes and pathways, protein-protein interactions are key determinants of protein function. Here we have built a large-scale protein-protein interaction map of the human gastric pathogen Helicobacter pylori. We have used a high-throughput strategy of the yeast two-hybrid assay to screen 261 H. pylori proteins against a highly complex library of genome-encoded polypeptides. Over 1,200 interactions were identified between H. pylori proteins, connecting 46.6% of the proteome. The determination of a reliability score for every single protein-protein interaction and the identification of the actual interacting domains permitted the assignment of unannotated proteins to biological pathways.

Genomic cloning and species-specific properties of the recombinant canine beta3-adrenoceptor.

A molecular clone encoding a beta3-adrenoceptor was isolated from a canine genomic library. The cloned receptor exhibited a pharmacological profile similar to that of other species: in particular, high efficiency of the two selective beta3-adrenoceptor agonists, CL 316,243 (disodium(R,R)-5[2[[2-(chlorophenyl)-2hydroxyethyl]-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate) and ICI 201651 ((R)4-(2-hydroxy-3-phenoxypropylaminoethoxy)-N-(2-methoxyethyl)phe noxy acetic acid) and a low affinity for the radioligand (-)-[3-(125)I]-iodocyanopindolol. Interestingly, CGP 12177A ((+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one), which is described as a partial agonist for the human receptor, was a full agonist for the canine receptor. After expression and stimulation of the canine beta3-adrenoceptor in stably transfected Chinese hamster ovary cells there was a very low accumulation of cAMP, suggesting weak coupling to Gs-protein and adenylyl cyclase. However, the response was much better in human embryonal kidney cells transfected with the canine beta3-adrenoceptor gene. The cloning of the canine beta3-adrenoceptor and the insights gained from its pharmacological characterization may allow the development of selective compounds for use in the treatment of obese dogs.

Molecular cloning and pharmacological characterization of the bovine beta 3-adrenergic receptor.

A full-length clone encoding a beta-adrenergic receptor was isolated from a bovine brown adipose tissue cDNA library. By comparative sequence analysis, and pharmacological characterization of a Chinese hamster ovary cell line expressing the full-length cDNA, it was shown that the product of the cloned gene is the bovine equivalent of the atypical beta 3-adrenergic receptor previously described in human, mouse, and rat [Strosberg, A. D. (1993) Prot. Sci. 2, 1198-1209]. The cloned receptor exhibits a pharmacological profile very similar to those from other species. In particular, the receptor has high affinity for BRL 37344 [(RR,SS)-(+/-)-4-(2'-[2-hydroxy-2-(3- chlorophenyl)ethylamino]propyl)phenoxyacetate sodium salt sesquihydrate], and low affinity for the iodinated ligand(-)-[3-125I]-iodocyanopindolol. The bovine beta 3-adrenergic receptor has high affinity for beta 1-adrenergic receptor and beta 2-adrenergic receptor antagonists including ICI 201651 [(R)-4-(2-hydroxy-3-phenoxypropylaminoethoxy)-N-(2- methoxyethyl)phenoxy acetic acid], carazolol, and CGP 12177A [(+/-)-4-(3-t-butylamino-2- hydroxypropoxy)benzimidazol-2-one]. In contrast to the murine beta 3-adrenergic receptor, both bupranolol and (-)-propranolol were partial agonists of the bovine receptor. The isolation of the bovine beta 3-adrenergic receptor, and information obtained from detailed pharmacological profiling may allow for the development of selective compounds for producing beef cattle with a low-body-mass index, and also aid the ongoing search for more selective agonists for the human receptor.

Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA.

The induction of anti-influenza cytotoxic T lymphocytes (CTL) in vivo by immunizing mice with liposomes containing messenger RNA (mRNA) encoding the influenza virus nucleoprotein (NP) is described. NP mRNA, obtained by in vitro transcription, was encapsulated into simple cholesterol/phosphatidylcholine/phosphatidylserine liposomes by the detergent removal technique. The dependence of the route of mRNA-liposomes delivery on CTL induction was studied. The CTL induced were identical to those obtained in vivo with infectious virus in terms of specificity, lysing both peptide-sensitized and virus-infected targets. Furthermore, with the same mRNA-liposome preparation, virus-specific CTL responses could be also elicited in mice of three different haplotypes each of them known to present a distinct NP peptide in an MHC-restricted fashion. The relevance of these results in the context of vaccine development is discussed.

Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences.

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.

Molecular characterization of a 23-kilodalton major antigen secreted by Toxoplasma gondii.

The strategy chosen for cloning potential vaccine antigens of Toxoplasma gondii was based on the hypothesis that the definitive protection observed in natural infection is due to the presence of encysted bradyzoite forms in host tissues throughout life. The antigens released by the bradyzoites would maintain an immune response against the invading tachyzoites. This led us to identify in tachyzoite in vitro translation products a polypeptide of 24 kDa that is an excreted-secreted antigen (ESA) and is cross-reactive with bradyzoites. In addition, the detection of anti-P24 IgG antibodies is correlated with the chronic infection in man. The gene encoding P24 has been isolated, sequenced, and expressed in Escherichia coli and eukaryotic cells. The recombinant proteins were immunogenic in mice, producing anti-native P23 antibodies. Immunocytochemical analysis located the native antigen in the dense granules of both tachyzoite and bradyzoite forms and showed that it is secreted within host-cell-modified phagosome. Moreover 45Ca2+ labeling as well as regional homologies indicate that this protein has Ca2+-binding properties, suggesting its physiological importance in host-cell invasion. P23 is of diagnostic interest as a marker of chronic toxoplasmosis and is proposed as a vaccine component.

Structure and evolution of bacterial adenylate cyclase: comparison between Escherichia coli and Erwinia chrysanthemi.

The cya genes, coding for adenylate cyclase, from Escherichia coli and Erwinia chrysanthemi B374 are compared after determination of a 3632 bp long nucleotide sequence of the hemC-cya region of E. chrysanthemi, encompassing the whole cya gene. In spite of a large divergence between the two organisms, especially visible in non coding regions, the amino acid sequence of the proteins are very similar, except at the very distal carboxyl end. Codon usage is different in the two organisms, and E. chrysanthemi tends to restrict translation to codons ending in G or C. Conservation of the translation initiation start region (including the poor ribosome binding site GGCG, and the TTG start codon), suggests that a specific protein synthesis process controls adenylate cyclase expression. Finally a palindromic unit, of primary sequence differing from the E. coli counterpart, borders the gene in E. chrysanthemi.

A TY1 element is inserted in the CYR1 control region of Saccharomyces cerevisiae strain AB320.

Southern blotting using a 5'-proximal probe of the Saccharomyces cerevisiae CYR1 gene has revealed heterogeneity in laboratory strains. It is demonstrated that strain AB320 contains a Ty1 element inserted in the promoter region of CYR1. The Ty1 orientation suggests that transcription of CYR1 is initiated downstream from the insertion region.

Nucleotide sequence of a tRNA(leu)CAG gene from Rhizobium meliloti.

The nucleotide sequence of a tRNA(leu)CAG gene from Rhizobium meliloti has been determined. Its organization includes a short transcript with a promoter-like structure and two Rho-independent terminators about 10 bp downstream from the cloverleaf structure. The putative promoter shows good homology with the -45 and -35 region of the consensus (sigma 70) promoter sequence of Escherichia coli, but less with the -10 region. A discriminator-like sequence is present between the start of the transcript and the -10 region. The gene shows extensive homology to tRNA(leu)CAG genes from Gram-positive and Gram-negative bacteria. However, significant differences are encountered in the variable loop region. This is the first report of a tRNA sequence from R. meliloti.

Yeast adenylate cyclase catalytic domain is carboxy terminal.

Subcloning of DNA fragments from the gene coding for yeast adenylate cyclase has permitted, after complementation studies in S. cerevisiae cdc35 mutants as well as E. coli cya mutants, to identify the sequence coding for the catalytic domain of the protein. No homology is found between the yeast cyclase catalytic domain and the homologous domain found in E. coli adenylate cyclase. Analysis by Northern blotting of yeast polyA mRNA has shown the existence of multiple transcriptional products of the gene. A putative origin of a major transcript (3.5 kb) would allow synthesis of a ca. 100,000 dalton protein exhibiting cyclase activity in its carboxy terminal domain, and having 7 repeats of 17 amino acids at its amino terminal end. Several note-worthy features, including the possibility of transcriptional control by the general control of amino acids biosynthesis, are present at this putative origin. Data are presented suggesting that a much longer gene product might also be synthesized from the CDC35 gene. Neither the gene organization nor the amino acid sequence of the protein does display any homology with the adenylate cyclase gene and protein of Escherichia coli. This suggests a case of evolutionary convergence for the synthesis of cAMP in prokaryotes and eukaryotes.