RESUMEN
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.
Asunto(s)
Asma , Hipersensibilidad , Cooperación Internacional , Investigación , Alergia e Inmunología , Europa (Continente) , HumanosRESUMEN
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.
Asunto(s)
Asma/epidemiología , Hipersensibilidad/epidemiología , Cooperación Internacional/legislación & jurisprudencia , Desarrollo de Programa , Alérgenos/inmunología , Asma/genética , Asma/inmunología , Ensayos Clínicos como Asunto , Conducta Cooperativa , Exposición a Riesgos Ambientales , Europa (Continente)/epidemiología , Femenino , Humanos , Hipersensibilidad/genética , Hipersensibilidad/inmunología , Masculino , Factores SexualesRESUMEN
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.
Asunto(s)
Proteínas Portadoras/genética , Catecoles/metabolismo , Yodocianopindolol/metabolismo , Proteínas de la Membrana/genética , Serina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Células CHO , Proteínas Portadoras/metabolismo , Clonación Molecular , Colon/química , Cricetinae , ADN Complementario/química , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Humanos , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Serina/análogos & derivados , Distribución TisularRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Helicobacter pylori/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Bases de Datos Factuales , Escherichia coli/genética , Biblioteca de Genes , Humanos , Internet , Datos de Secuencia Molecular , Unión Proteica , Proteoma , Alineación de Secuencia , Programas Informáticos , Ureasa/metabolismoRESUMEN
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.
Asunto(s)
Receptores Adrenérgicos beta/genética , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Células CHO , Bovinos , Células Cultivadas , Clonación Molecular , Cricetinae , ADN , Perros , Proteínas de Unión al GTP/metabolismo , Humanos , Datos de Secuencia Molecular , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 3 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad de la Especie , TransfecciónRESUMEN
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.
Asunto(s)
Receptores Adrenérgicos beta/genética , Tejido Adiposo Pardo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Bovinos , Clonación Molecular , Cricetinae , Datos de Secuencia Molecular , Receptores Adrenérgicos beta/biosíntesis , Receptores Adrenérgicos beta/química , Receptores Adrenérgicos beta 3 , Proteínas Recombinantes/biosíntesisRESUMEN
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.
Asunto(s)
Antígenos Virales/genética , Virus de la Influenza A/inmunología , Nucleoproteínas , ARN Mensajero/administración & dosificación , Linfocitos T Citotóxicos/fisiología , Proteínas del Núcleo Viral/genética , Animales , Antígenos Virales/inmunología , Citotoxicidad Inmunológica , Inmunidad Celular , Liposomas/administración & dosificación , Ratones , Ratones Endogámicos , Proteínas de la Nucleocápside , Vacunas Sintéticas/inmunología , Proteínas del Núcleo Viral/inmunología , Vacunas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Escherichia coli/genética , Operón Lac , Lactobacillus/genética , Streptococcus/genética , beta-Galactosidasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/enzimología , Lactobacillus/enzimología , Datos de Secuencia Molecular , Alineación de Secuencia , Streptococcus/enzimologíaRESUMEN
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.
Asunto(s)
Antígenos de Protozoos/genética , Toxoplasma/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios , Antígenos de Protozoos/análisis , Secuencia de Bases , ADN/genética , Biblioteca de Genes , Genes , Ratones , Datos de Secuencia Molecular , Peso Molecular , Sondas de Oligonucleótidos , Biosíntesis de Proteínas , Toxoplasma/inmunología , Toxoplasma/ultraestructura , Toxoplasmosis/inmunologíaRESUMEN
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.
Asunto(s)
Adenilil Ciclasas , Erwinia/enzimología , Escherichia coli/enzimología , Adenilil Ciclasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Biológica , Clonación Molecular , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Elementos Transponibles de ADN , ADN de Hongos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Secuencia de Bases , Clonación Molecular , Regulación de la Expresión Génica , Hibridación de Ácido Nucleico , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Genes Bacterianos , ARN de Transferencia/genética , Rhizobium/genética , Secuencia de Bases , Clonación Molecular , Regulación de la Expresión Génica , Conformación de Ácido Nucleico , ARN Bacteriano/genéticaRESUMEN
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.