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E Palla

Publications and source records attributed to E Palla.

12 recordsLinked to original sources
PubMed1999

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus.

An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Deltaasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain chi4072, while in strain chi3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus.

Bacterial Toxins↗
PubMed1998

Levels of expression and immunogenicity of attenuated Salmonella enterica serovar typhimurium strains expressing Escherichia coli mutant heat-labile enterotoxin.

The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium delta cya delta crp delta asd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated delta cya delta crp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence.

Adjuvants, Immunologic↗
PubMed1996

Synthetic alleles at position 121 define a functional domain of human interleukin-1 beta.

The non-conservative substitution of the tyrosine residue at position 121 of human interleukin-1 beta (IL-1 beta) generates protein mutants showing strong reduction of the capacity to induce (a) prostaglandin E2 (PGE2) release from fibroblasts and smooth muscle cells, (b) murine T-cells proliferation and (c) activation of interleukin-6 (IL-6) gene expression. It is generally accepted that these functions are mediated by the type-I interleukin-1 receptor (IL-1RI). However, the mutant proteins maintain the binding affinity to the types-I and II IL-1 receptors, which is the same as the control IL-1 beta, suggesting that this amino acid substitution does not alter the structure of the molecule, except locally. Thus we have identified a new functional site of IL-1 beta different from the known receptor binding region, responsible for fundamental IL-1 beta functions. Moreover, we show that the same mutants maintain at least two hypothalamic functions, that is, the in vitro short-term PGE2 release from rat hypothalamus and the induction of fever in rabbits. This result suggests that there is yet another site of the molecule responsible for the hypothalamic functions, implying that multiple active sites on the IL-1 beta molecule, possibly binding to more than one receptor chain, trigger different signals.

Alleles↗
PubMed1993

Loop substitution as a tool to identify active sites of interleukin-1 beta.

By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor.

Amino Acid Sequence↗
PubMed1992

Processing of interleukin-1 in cells of monocytic lineage is differentiation-dependent.

The interleukin-1 (IL-1) alpha and beta precursor proteins are processed and released from several cell types in the absence of a canonical signal peptide. To gain some insight into the mechanisms that allow the production of IL-1 alpha and beta, we have investigated by immunoprecipitation the synthesis, their release and processing in a promyeloblastic cell line of tumoral origin, U937, and in peripheral blood monocytes. We show that U937 monocytic cells, on induction with a tumor-promoting agent, synthesize and release into the culture medium proIL-1 beta but do not process it. Similarly, peripheral blood monocytes left in adherence for 24 h or longer, prior to addition of lipopolysaccharide, synthesize and release proIL-1 alpha and beta without detectable processing of either cytokine. Processing and release of IL-1 alpha and beta by peripheral blood monocytes can be observed when monocytes are left to adhere for periods less than 15 h before lipopolysaccharide addition. IL-1 alpha and beta show similar kinetics of release from the cells, suggesting the existence of a common mechanism regulating their secretion. Since peripheral blood monocytes left in adherence in the presence of lipopolysaccharide differentiate into macrophages, we conclude that release and processing of IL-1 can occur independently and that processing depends on the stage of differentiation of monocytes, i.e. only the monocytes at an early stage of differentiation produce 17-kDa IL-1 alpha and beta.

Cell Adhesion↗
PubMed1990

Characterization of the human lipocortin-2-encoding multigene family: its structure suggests the existence of a short amino acid unit undergoing duplication.

Human genomic clones of the gene encoding lipocortin (LIP) 2 (p36) and of three pseudogenes have been isolated and characterized. The LIP2 gene is at least 40 kb long and consists of 13 exons. The three pseudogenes present typical features of retroposons and, together with the gene, probably represent the entire LIP2 multigene family. Chromosomal assignment of the four loci is proposed. The hypothesis that an ancestral unit coding for 15 to 20 amino acids may have been involved in the evolution of the gene is discussed.

Amino Acid Sequence↗
PubMed1987

Human interleukin-1 beta gene.

We report the nucleotide sequence of the human chromosomal gene which encodes the interleukin-1 beta protein (IL-1 beta). The gene spans a region of 7.5 kb and the coding part is divided into seven exons. Comparison with the homologous mouse gene reveals that the structural organization is conserved through evolution. In addition to this, human and murine IL-1 beta genes show extensive sequence homology within the intervening sequences.

Amino Acid Sequence↗
PubMed1986

The murine interleukin 1 beta gene: structure and evolution.

We have isolated from a genomic library a murine recombinant clone containing the gene coding for interleukin-1 beta m-RNA. A 7000 b.p. DNA fragment has been sequenced. Sequences homologous with human IL-1 beta cDNA have been found distributed within 7 exons. The translation of these sequences allows the prediction of a protein 269 aminoacids long. Hybridization of P388D1 RNA from cells stimulated with phorbol myristic acetate with a genomic DNA probe shows the existence of a 1.6 Kb murine IL-1 beta mRNA which is absent in the unstimulated cells. The comparative analysis between the murine IL-1 beta and the human IL-1 alpha genes shows extreme conservation of the aminoacids at the exon junctions. This observation together with the similarity in number and size of the exons suggests that these genes have diverged from a common ancestor.

Amino Acid Sequence↗
PubMed1986

New plasmid expression vectors for Bacillus subtilis.

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.

Bacillus subtilis↗
PubMed1982

Sequence analysis of heteropolymeric DNA synthesized in vitro by the enzyme terminal deoxynucleotidyl transferase and cloned in Escherichia coli.

We have synthesized in vitro single strands of DNA in reaction mixtures containing the terminal deoxynucleotidyl transferase (Bollum's enzyme), an oligo-dG as primer, the four common deoxynucleoside triphosphates and both Mg and Co ions. The resulting heteropolymers have been converted into double strands, tailed with oligo-dC sequences, annealed with an oligo-dG tailed plasmid vector and cloned in E. coli. Six recombinant plasmids have been isolated and characterized. Two of them have been sequenced. The heteropolymeric chains produced by the terminal transferase, ranging in size between 200 and 400 nucleotides, are richer in purines than in pyrimidines, except in the last portions. Open reading frames for 7-20 amino acids with repeated, in phase translational stop codons are present in these sequences and in their complements.

Base Sequence↗
PubMed1980

DNA cloning in Bacillus subtilis. III. Efficiency of random-segment cloning and insertional inactivation vectors.

Random segments of Bacillus amyloliquefaciens and yeast Saccharomyces cerevisiae DNA were used to determine two parameters pertinent to cloning in Bacillus subtilis, the yield of hybrids and the mean size of cloned segments. 10(3) to 10(4) hybrids/micrograms of DNA segments were obtained. Hybrids represented 11--18% of transformants. Mean m. wt. of cloned DNA segments was about 1 x 10(6), substantially lower than 3 x 10(6) found for donor DNAs after digestion with restriction endonucleases. We have cloned a B. amyloliquefaciens DNA segment which complemented a deficiency in B. subtilis hisH and E. coli hisC genes, which encode imidazolylacetolphosphate aminotransferase. The cloning efficiency for this gene was 10 transformed hosts/micrograms of donor DNA. Several B. subtilis insertional-inactivation cloning vectors were examined. One, pHV41, allows inactivation of the kanamycin-resistance (KmR) gene by insertion into its unique Bg/II site. In two other vectors, pHV11 and pHV23, insertion in their unique Kpn site inactivates the tetracycline-resistance (TcR) gene. pHV23 replicates both in E. coli and B. subtilis, and carries unique sites for seven restriction endonucleases (BamHI, EcoRI, HpaI, KpnI, PstI, SalI, XbaI). This makes it one of the most versatile B. subtilis cloning vectors yet described.

Bacillus↗