Biomedical subjects

G Grandi

Publications and source records attributed to G Grandi.

At least 37 records · Page 2Linked to original sources

PubMed1999

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure.

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.

Amino Acid Sequence↗

PubMed1998

Characterization of the syringomycin synthetase gene cluster. A link between prokaryotic and eukaryotic peptide synthetases.

With this work we have completed the characterization of the syringomycin synthetase gene cluster. In particular, by sequencing additional 28.5 kilobase pairs we show that the nine modules involved in the binding of the nine amino acids of syringomycin are localized on SyrB and SyrE, with SyrE carrying eight modules. The recombinant SyrB and the first and second modules of SyrE (SyrE1 and SyrE2) have been expressed in Escherichia coli and purified. The biochemical data indicate that SyrB binds threonine, the putative precursor of the last amino acid of syringomycin, whereas SyrE1 and SyrE2 bind serine, the first and the second amino acids of syringomycin, respectively. On the basis of the sequence analysis and the biochemical data presented here, it appears that syringomycin synthetase is unique among peptide synthetases in that its genetic organization does not respect the "colinearity rule" according to which the order of the amino acid binding modules along the chromosome parallels the order of the amino acids on the peptide. This feature, together with the absence of a single transcription unit and the absence of epimerase-like domains make syringomycin synthetase more related to the eukaryotic peptide synthetases than to the bacterial counterparts.

Amino Acid Sequence↗

PubMed1998

Binding of hepatitis C virus to CD81.

Chronic hepatitis C virus (HCV) infection occurs in about 3 percent of the world's population and is a major cause of liver disease. HCV infection is also associated with cryoglobulinemia, a B lymphocyte proliferative disorder. Virus tropism is controversial, and the mechanisms of cell entry remain unknown. The HCV envelope protein E2 binds human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Binding of E2 was mapped to the major extracellular loop of CD81. Recombinant molecules containing this loop bound HCV and antibodies that neutralize HCV infection in vivo inhibited virus binding to CD81 in vitro.

Amino Acid Sequence↗

PubMed1998

Coordinate transcription and physical linkage of domains in surfactin synthetase are not essential for proper assembly and activity of the multienzyme complex.

Bacterial peptide synthetases have two common features that appear to be strictly conserved. 1) The enzyme subunits are co-regulated at both transcriptional and translational level. 2) The organization of the different enzymatic domains constituting the enzyme fulfills the "colinearity rule" according to which the order of the domains along the chromosome parallels their functional hierarchy. Considering the high degree of conservation of these features, one would expect that mutations such as transcription uncoupling and domain dissociations, deletions, duplications, and reshuffling would result in profound effects on the quality and quantity of synthesized peptides. To start testing this hypothesis, we designed two mutants. In one mutant, the operon structure of surfactin synthetase was destroyed, thus altering the concerted expression of the enzyme subunits. In the other mutant, the thioesterase domain naturally fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted by the mutant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical to it, indicating that specific domain-domain interactions rather than coordinated transcription and translation play the major role in determining the correct assembly and activity of peptide synthetases.

Bacillus subtilis↗

PubMed1998

Multi-plasmid DNA vaccination avoids antigenic competition and enhances immunogenicity of a poorly immunogenic plasmid.

DNA immunization is a very promising approach to the formulation of multivalent vaccines. However, little information is currently available on the immunogenicity of multi-plasmid formulations. To address this issue, we immunized mice with a combination of four plasmids encoding malarial antigens and we compared antibody responses with those obtained with single-plasmid injections. We found that when four plasmids encoding Plasmodium falciparum circumsporozoite protein, thrombospondin-related anonymous protein, major merozoite surface protein (MSP)1 and Pfs25 are co-injected into mice, Ab responses against each antigen are elicited at levels at least as high as the level obtained with single-plasmid injection. The quality of antibody production, as determined by isotype analysis, was similar when single- and multi-plasmid administrations were compared, indicating the priming of the same cytokine profile for CD4+ T helper cells. The sera from mice immunized with the four-plasmid formulation specifically recognized sporozoites, blood stage schizonts and gametes, indicating that DNA immunization induced antibody responses relevant to the native conformation. Finally and of particular interest, in the case of MSP1, the antibody response appears to be strongly potentiated by the presence of additional plasmids, indicating an adjuvant effect of DNA.

Animals↗

PubMed1997

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Bacillus subtilis↗

PubMed1997

Engineering of peptide synthetases. Key role of the thioesterase-like domain for efficient production of recombinant peptides.

Peptide synthetases are large enzymatic complexes that catalyze the synthesis of biologically active peptides in microorganisms and fungi and typically have an unusual structure and sequence. Peptide synthetases have recently been engineered to modify the substrate specificity to produce peptides of a new sequence. In this study we show that surfactin synthetase can also be modified by moving the carboxyl-terminal intrinsic thioesterase region to the end of the internal amino acid binding domains, thus generating strains that produce new truncated peptides of the predicted sequence. Omission of the thioesterase domain results in nonproducing strains, thus showing the essential role of this region and the possibility of obtaining peptides of different lengths by genetic engineering. Secretion of the peptides depends on the presence of a functional sfp gene.

Amino Acid Sequence↗

PubMed1997

Inhibitory activity of IL-1 receptor antagonist depends on the balance between binding capacity for IL-1 receptor type 1 and IL-1 receptor type II.

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.

Animals↗

PubMed1997

Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol.

Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles that originate from massive swelling of membranous compartments of late stages of the endocytic pathway. To determine if the toxin is active from the cell cytosol, cells were either microinjected with toxin or transfected with plasmids encoding VacA. Both procedures cause formation of intracellular vacuoles. Cytosolic localization of the toxin was assessed by indirect immunofluorescence with specific antibodies and by expression of an active green fluorescence protein (GFP)-VacA chimera. Vacuoles induced by internally produced VacA are morphologically and functionally identical to those induced by externally added toxin. It is concluded that VacA is a toxin acting intracellularly by altering a cytosol-exposed target, possibly involved in the control of membrane trafficking.

Bacterial Proteins↗

PubMed1997

Model building of a thermolysin-like protease by mutagenesis.

The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub). An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus thermoproteolyticus (thermolysin) and Bacillus cereus as templates. The largest portion of NP-sub could be modelled satisfactorily, using standard techniques, but several surface-located regions could only be modelled with a high degree of uncertainty. In order to make the model more reliable in these regions a 'model building by mutagenesis' approach was adopted. Mutations were designed such that their effect on thermal stability could indicate how their local environment should be modelled. This approach provided insight in the local structure of several regions in NP-sub that were hard to model on the basis of homology with the two known structures alone.

Amino Acid Sequence↗

PubMed1997

Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker.

Research on Helicobacter pylori has been hindered by the lack of useful genetic tools. Using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows introduction of unmarked mutations in H. pylori. A kan-sacB cassette, consisting of the sacB gene expressed from the H. pylori flagellin promoter and the kanamycin resistance module, was introduced by homologous recombination into a target H. pylori gene. The resultant strains were sucrose sensitive and kanamycin resistant. Following transformation with a mutated allele, growth in sucrose-containing medium allowed the selection of strains that had lost the kan-sacB module and had integrated the unmarked allele. We have used this cassette to perform a site-directed modification of two histidine residues encoded by the vacA gene in a two-step procedure. This system should prove useful in the site-directed mutagenesis of H. pylori genes.

Alleles↗

PubMed1997

Latency periods in asbestos-related mesothelioma of the pleura.

Latency periods (time intervals elapsing between first exposure to asbestos and death) were examined in 421 cases of malignant pleural mesothelioma, diagnosed in the Trieste-Monfalcone area, Italy. Occupational data were collected from the patients or from their relatives by personal or telephone interviews. Routine lung sections were examined for asbestos bodies in 370 cases. Latency periods, calculated in 312 cases, ranged from 14 to 72 years (mean 48.7, median 51). Latency periods differed significantly from one occupational group to another. Mean latency periods were 29.6 among insulators, 35.4 among dock workers, 43.7 in a heterogeneous group defined as various, 46.4 in non-shipbuilding industry workers, 49.4 in shipyard workers, 51.7 among women with a history of domestic exposure to asbestos, and 56.2 in people employed in maritime trades. The ANOVA test indicated a correlation between latency periods and occupational groups. Latency periods in people with asbestos bodies visible in routine lung sections did not differ from those seen in cases with no evidence of asbestos bodies. These data suggest that intensity of exposure is a relevant, but not the only, factor in determining the duration of latency periods.

Adult↗

PubMed1996

Topological mapping of the cysteine residues of N-carbamyl-D-amino-acid amidohydrolase and their role in enzymatic activity.

The N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, the enzyme used for the industrial production Of D-amino acids, was cloned, sequenced, and expressed in Escherichia coli. The protein, a dimer constituted by two identical subunits of 34,000 Da with five cysteines each, was susceptible to aggregation under oxidizing conditions and highly sensitive to hydrogen peroxide. To investigate the role of the cysteines in enzyme stability and activity, mutant proteins were constructed by site-directed mutagenesis in which the five residues were substituted by either Ala or Ser. Only the mutant carrying the Cys172 substitution was catalytically inactive, and the other mutants maintained the same specific activity as the wild type enzyme. The crucial role of Cys172 in enzymatic activity was also confirmed by chemical derivatization of the protein with iodoacetate. Furthermore, chemical derivatizations using both acrylamide and Ellman's reagent revealed that (i) none of the five cysteines is engaged in disulfide bridges, (ii) Cys172 is easily accessible to the solvent, (iii) Cys193 and Cys250 appear to be buried in the protein core, and (iv) Cys243 and Cys279 seem to be located within or in proximity of external loops and are derivatized under mild denaturing conditions. These data are discussed in light of the possible mechanisms of enzyme inactivation and catalytic reaction.

Alanine↗

PubMed1996

Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis.

Using genetically engineered mutants of the neutral protease from Bacillus stearothermophilus (BsteNP), it had been shown that the surface-exposed structural motif constituted by Phe63 embedded in a four amino acid hydrophobic pocket is critical for the thermal stability of the thermophilic neutral proteases from Bacilli. To measure the stabilizing contribution of each hydrophobic interaction taking place between Phe63 and the hydrophobic pocket, we grafted this structural motif in the neutral protease from the mesophile Bacillus subtilis (BsubNP). This was accomplished by first creating the Thr63-->Phe mutant of BsubNP and then generating a series of mutants in which the four amino acids which in thermolysin surround Phe63 and form the hydrophobic pocket were added one after the other. By analysing the thermal stability of each mutant it was found that the 2 degrees C destabilizing effect of the Thr63-->Phe substitution was completely suppressed by the addition of the four amino acid hydrophobic pocket, each replacement providing a stabilizing contribution of approximately 0.8-1 degrees C. These results are discussed in the light of the peculiar mechanism of thermal inactivation of proteolytic enzymes.

Bacillus subtilis↗

PubMed1996

The [Ni-Fe] hydrogenase from the thermophilic bacterium Acetomicrobium flavidum.

Biochemical analysis of the soluble hydrogenase from the thermophilic organism Acetomicrobium flavidum revealed that the enzyme is an alpha 2 beta 2 tetramer, with the alpha and beta subunits having a molecular mass of 50 kDa and 25 kDa, respectively. The most important biochemical properties of the enzyme are a specific activity of 77 mumol min-1 (mg protein)-1, a Km for methylviologen of 0.2 mM, a pH optimum of 7.5 and a T50 of about 70 degrees C. In addition, the enzyme is remarkably stable to oxygen inactivation, retaining full activity after 24 h exposure to air. By using oligodeoxynucleotides designed on the basis of the N-terminal sequences of the two subunits, the corresponding genes have been isolated and sequenced. When compared to the other hydrogenases so far characterized, the A. flavidum hydrogenase appears to be a typical [Ni-Fe] enzyme. The hydrogenase was expressed in Escherichia coli at high levels in a soluble form but it was not active. The analysis of the recombinant large subunit showed that it was not post-translationally processed at its C-terminus.

Amino Acid Sequence↗

PubMed1995

Mapping of receptor binding sites on IL-1 beta by reconstruction of IL-1ra-like domains.

Upon structure comparison between IL-1 beta and its antagonist IL-1ra, single or multiple residues along the IL-1 beta sequence were replaced with the corresponding amino acids present in the IL-1ra protein, in the attempt to identify sites important for receptor binding and for biologic activity on the two molecules. Ten of fifteen mutant proteins had activity comparable to that of wild-type IL-1 beta in three different biologic assays and in receptor binding, indicating that the introduced changes did not influence the functional structure of the protein. Conversely, three mutants (SMIL-9: 127/263 R/T-->W/Y; SMIL-10: 125/127/263/265 T/R/T/Q-->R/W/Y/E; SMIL-15:222/227 I/E-->S/S) showed an increased binding capacity for IL-1RI, not paralleled by increased agonist activity, indicating that the introduced IL-1ra residues could be involved in the nonagonist IL-1RI binding site. On the other hand, two mutants showed diminished binding capacity with concomitant decrease in biologic activity. Both mutants (SMIL-1, five substitutions in the loop 202-214; and SMIL-3, total replacement of the loop 164-173 with the IL-1ra stretch 52-55) included substitutions of residues allegedly important for agonist binding to IL-1RI. Mutant SMIL-3 showed the most profound reduction in binding capacity for IL-1RI (CDw121a) and a more than 1,000-fold reduced biologic activity both in vitro and in vivo, but it retained full capacity of binding to IL-1RII (CDw121b) and acted as a selective antagonist of IL-1RII. From these results the following conclusions can be drawn. IL-1 beta binds to IL-1RI and to IL-1RII through different sites, and the loop 164-173 appears as one of the areas involved in the selective interaction with IL-1RI. Agonist (IL-1 beta) and nonagonist (IL-1ra) binding to IL-1RI occur through distinct sites, with loops 164-173 and 202-214 of IL-1 beta identified as two of the sites selectively involved in agonist binding to the activating receptor.

Animals↗

PubMed1995

Characterization of the locus encoding the [Ni-Fe] sulfhydrogenase from the archaeon Pyrococcus furiosus: evidence for a relationship to bacterial sulfite reductases.

The hydBGDA genes, which encode the four subunits beta, gamma, delta and alpha of the [Ni-Fe] hydrogenase from the archaeon Pyrococcus furiosus, have been isolated and sequenced using a PCR/IPCR-based strategy. From the sequence analysis it appears that the four structural genes are tightly linked and organized in a single transcription unit. The hydD and hydA gene products are related to the small and the large subunits of several archaeal and eubacterial [Ni-Fe] hydrogenases with an overall degree of sequence relatedness ranging from 35% to 50% (identity + similarity). In particular, the amino acid sequence motifs involved in the accommodation of nickel and iron-sulfur clusters are conserved. In addition, the database search revealed that the hydB and hydG gene products are homologous to the asrA- and asrB-encoded subunits of the sulfite reductase enzyme from Salmonella typhimurium. This is particularly interesting in view of the recent finding that the P. furiosus hydrogenase appears to be a bifunctional enzyme endowed with both proton- and sulfur-reducing activities.

Amino Acid Sequence↗

PubMed1995

A putative new peptide synthase operon in Bacillus subtilis: partial characterization.

A large operon-type structure has been located between the gltA and citB loci on the Bacillus subtilis chromosome. On the basis of the analysis of the 25 kb sequenced so far, it potentially encodes at least three large proteins which contain structural motifs associated with the subunits of all characterized peptide synthases. The amino acid recognition specificity of this new peptide synthase is discussed in the light of sequence homology with other synthases.

Amino Acid Sequence↗