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G Satta

Publications and source records attributed to G Satta.

At least 37 records · Page 2Linked to original sources
PubMed1995

Engineering human monoclonal antibody fragments: a recombinant enzyme-linked Fab.

A new plasmid vector, pCRP, allowing the expression of human recombinant monoclonal antibody Fab fragments fused with a bacterial acid phosphatase has been constructed. pCRP can accept heavy- and light-chain cDNAs cloned from combinatorial antibody libraries displayed on filamentous phages with the pCombIII system and is able to direct expression to soluble Fabs in which the carboxy-terminus of the heavy chain is fused to the amino-terminus of the mature PhoN nonspecific acid phosphatase of Providencia stuartii. Using the pCRP vector, we expressed two different human recombinant Fabs cloned from combinatorial libraries (one anti-tetenus toxoid and the other anti-HIV-1 gp120) fused with the acid phosphatase. In both cases chimeric antibodies were obtained which retained the antigen-binding ability and the enzymatic activity. Similar Fab-enzyme fusions can be successfully used, even unpurified, in enzyme immunoassays.

Acid Phosphatase↗
PubMed1995

Cloning and characterization of human recombinant antibody Fab fragments specific for types 1 and 2 herpes simplex virus.

Twenty-six bacterial clones producing human recombinant Fab fragments specific for Herpes Simplex virus (HSV) antigens were obtained from an IgG1k human antibody combinatorial library displayed on filamentous phage, following panning against an HSV lysate. All the Fabs reacted against the HSV lysate in enzyme-linked immunosorbent assay and were able to recognize both type 1 and type 2 HSV in an indirect immunofluorescence assay (IFA). DNA sequencing of the heavy chain variable regions showed that these Fabs were different from those already described. One of these Fabs (Fab19) was purified and subjected to further characterization. Purified Fab19 was able to specifically recognize several different HSV-1 and HSV-2 strains (including 2 reference strains and 12 clinical isolates) in IFA. It was also able to neutralize the infectivity of both HSV-1 and HSV-2 strains, although the neutralizing activity was somewhat lower against HSV-2. In fact, 100% neutralization of infectivity was observed at a Fab concentration of 2 micrograms/100 TCD50 for the majority of HSV-1 strains, while a concentration of 8 micrograms/100 TCD50 was needed for 100% neutralization of all the HSV-2 strains tested. Owing to the above properties, Fab19 appears to be useful for diagnostic purposes and might also prove useful for in vivo immunoprophylaxis and therapy of HSV infections.

Animals↗
PubMed1995

A new plasmid cloning vector for direct detection of recombinant clones, based on inactivation of a bacterial acid phosphatase-encoding gene.

A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii. Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E. coli strains carrying pPhoR. Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium. The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation. Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E. coli host.

Acid Phosphatase↗
PubMed1994

fimA-folD genes linkage in Salmonella identifies a putative junctional site of chromosomal rearrangement in the enterobacterial genome.

Analysis of the Salmonella chromosomal region located upstream of the fimA gene (coding for the major type 1 fimbrial subunit) showed a close linkage of this gene to the folD gene (coding for the enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase), indicating that the fim gene cluster of Salmonella, unlike that of Escherichia coli, has no regulatory genes located upstream of fimA and apparently terminates with this gene. The respective locations of the fim and folD genes in the E. coli and Salmonella genetic maps suggests that the fimA-folD intergenic region of Salmonella encompasses a junctional site of a genetic rearrangement that probably originated from the different chromosomal location of the fim genes in these species.

Amino Acid Sequence↗
PubMed1994

Identification of an Escherichia coli periplasmic acid phosphatase containing of a 27 kDa-polypeptide component.

An acid phosphatase containing a 27-kDa polypeptide component has been identified in Escherichia coli by means of a zymogram technique. The enzyme is secreted in the periplasmic space and is able to hydrolyze several organic phosphate esters, but not diesters, showing preferential activity on p-nitrophenyl phosphate and other phenolic phosphate esters. Production of the enzyme apparently occurs only in cells growing on carbon sources other than glucose.

Acid Phosphatase↗
PubMed1994

Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequence information.

The performance of oligonucleotide primers containing deoxyinosine (dl) at all ambiguous positions for polymerase chain reaction, based on ambiguous sequence information derived either from compilations of consensus nucleotide sequences or from amino acid sequences, has been evaluated in two model systems represented respectively by amplification of conserved genomic regions from different types of human papillomavirus and by amplification of a region of the human lysozyme cDNA on the basis of the protein amino acid sequence. In both instances the dl-containing primers obtained the expected amplification products. When using short primers or primers with very high dl contents, however, peculiar reaction conditions had to be adopted to obtain successful amplification and, in the latter case, performance remained suboptimal. Comparison of results with those obtained using corresponding degenerate primers showed that the use of dl-containing primers can be advantageous in terms of both specificity and yield of the amplification product. Sequence analysis of amplification products showed that dG residues are always found at positions corresponding to the dl residues of the primers.

Amino Acid Sequence↗
PubMed1994

A multicentre study: Staphylococcus and Enterococcus susceptibility to antibiotics.

A multicentre study to evaluate the susceptibility of Gram-positive cocci isolated from clinical samples, was performed by six centres working in different areas of Italy. We examined 4,544 strains of Staphylococcus aureus, 4,381 strains of coagulase-negative staphylococci and 2,478 strains of enterococci. The following antibiotics were tested: penicillin G, ampicillin, amoxicillin, piperacillin, imipenem, oxacillin, ofloxacin, pefloxacin, ciprofloxacin, gentamicin, tobramycin, amikacin, netilmicin, rifampicin, clindamycin, tetracycline, cotrimoxazole, erythromycin, chloramphenicol, vancomycin and teicoplanin. Oxacillin-susceptible staphylococci confirmed their susceptibility to many other antimicrobial agents while oxacillin-resistant strains confirmed their multiple and frequent resistance to antibiotics. Resistance to oxacillin, cotrimoxazole and chloramphenicol was more frequent in coagulase-negative staphylococci than in Staphylococcus aureus. Aminoglycosides, rifampicin and quinolones were more active against coagulase-negative staphylococci than against Staphylococcus aureus. Enterococci were susceptible to penicillins and imipenem, and moderately susceptible to ciprofloxacin. Susceptibility of 70-79% was observed with high levels of aminoglycosides. Excellent results against staphylococci and enterococci were observed with vancomycin and teicoplanin.

Anti-Bacterial Agents↗
PubMed1994

Incidence of lower respiratory tract infections caused by Mycoplasma, Chlamydia and Legionella: an Italian Multicenter Survey.

A collaborative retrospective study based on serologic diagnosis was conducted to assess the etiological role sustained by privileged pathogens in Italy. The results obtained indicate the Mycoplasma, Chlamydia and Legionella are important etiologic agents of lower respiratory tract infections in Italy since they account for about 31% of the cases taken into consideration in this survey. We found a high incidence of M. pneumoniae (12.3%), C. pneumoniae (10.5%) and L. pneumophila (8.3%). These results are in line with similar figures reported in the recent literature. While the data gathered in our survey do not allow us to clarify the nature of the agents involved in the etiology of the majority (70%) of the respiratory infections occurring in Italy, it seems safe to assume that after Streptococcus pneumoniae and Haemophilus influenzae, the privileged pathogens represent the most common cause of lower respiratory tract infections.

Chlamydia Infections↗
PubMed1994

In-vitro evaluation of cefpodoxime.

In-vitro antimicrobial activity of cefpodoxime was evaluated against several microbial species by both conventional tests and additional parameters which take into consideration some of the conditions likely to be encountered in infected tissues. MICs for 414 recent clinical isolates, including staphylococci, streptococci, Haemophilus influenzae, Moraxella catarrhalis, several Enterobacteriaceae, Aeromonas hydrophila and Campylobacter jejuni were determined. MIC values overall were similar to those observed for strains from other geographical areas. Inhibition of growth by cefpodoxime was virtually unaffected by the inoculum size, even using bacterial populations as large as 10(9) cfu of Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, H. influenzae, beta-lactamase-negative M. catarrhalis, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Growth in the presence of human serum as the only source of nutrients did not significantly affect the inhibition exerted by cefpodoxime, even against large bacterial populations of S. aureus, S. pneumoniae, E. coli, and K. pneumoniae. For K. pneumoniae, E. coli, P. mirabilis and beta-lactamase-negative M. catarrhalis it was also found that sub-MIC concentrations of cefpodoxime were still able to inhibit the majority of cells in microbial populations as large as 10(9) cfu. Evaluation of bactericidal activity demonstrated that cefpodoxime concentrations comparable to those achievable in plasma or in the respiratory tract were able to kill rapidly large bacterial populations of S. pneumoniae and S. pyogenes. The bactericidal activity was apparently lower against M. catarrhalis, H. influenzae, S. aureus, E. coli, and K. pneumoniae.

Bacteria↗
PubMed1994

Purification and characterization of type 1 fimbriae of Salmonella typhi.

Type 1 fimbriae have been purified from a Salmonella typhi strain of clinical origin. Purified fimbriae retained their ability to bind to erythrocytes in a mannose-inhibitable fashion and, in doing so, behaved preferentially as a monovalent adhesin. SDS-PAGE analysis of the fimbrial preparation showed the presence of a 20-kDa major polypeptide component (fimbrillin) and of additional larger polypeptides present in smaller amounts. The amino-terminal sequence of fimbrillin was determined and turned out to be very similar but not identical to that of type 1 fimbrillins of other Salmonella serovars. A Western blot analysis of the purified fimbrial preparation using an antiserum raised against native fimbriae suggested that fimbrial proteins did not carry any major sequential epitope and that, in native fimbriae, conformational epitopes, possibly generated between different subunits, might provide for the major immunogenic epitopes. Analysis of different S. typhi clinical isolates using the anti-fimbrial antiserum showed an overall immunological similarity of these structures within this serovar.

Amino Acid Sequence↗
PubMed1994

Overproduction of a low-affinity penicillin-binding protein and high-level ampicillin resistance in Enterococcus faecium.

Five ampicillin-resistant clinical isolates of Enterococcus faecium were analyzed for a correlation between overproduction of the low-affinity penicillin-binding protein (PBP 5) and the level of ampicillin resistance. Comparison was made with one susceptible clinical isolate and its ampicillin-resistant derivative obtained in the laboratory by selection with increasing concentrations of penicillin. Overproduction of the low-affinity PBP relative to the susceptible isolate was noted in moderately resistant strains (MIC, 32 micrograms/ml) but not in highly resistant strains (MIC, 128 micrograms/ml). Polyclonal antibodies specifically reacting with the low-affinity PBP of Enterococcus hirae, Enterococcus faecalis, and Enterococcus faecium (M. Ligozzi, M. Aldegheri, S. C. Predari, and R. Fontana, FEMS Microbiol. Lett. 83:335-340, 1991) were used to determine the amount of this PBP in the E. faecium isolates. In all strains, the antibody preparation reacted with a membrane protein of the same molecular mass as PBP 5. The amount of this protein was very small in the susceptible strain but large in all of the resistant strains. These results suggest that the highly resistant strains also overproduced the low-affinity PBP, which, compared with PBP 5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability.

Ampicillin Resistance↗
PubMed1994

PBP binding and periplasmic concentration as determinants of the antibacterial activities of three new oral cephalosporins in Escherichia coli.

The antibacterial activities of cefetamet, cefixime and cefuroxime were investigated in Escherichia coli with regard to their penetration rates through the outer membrane and their affinities for PBPs in Escherichia coli. The permeability coefficient of cefetamet was measured in E. coli C600 carrying a pUC18 plasmid derivative, in which the gene of a transferable cephamycinase (CMY-2) was cloned, whereas diffusion of cefixime and cefuroxime was measured in E. coli C600 harbouring the OXA-1 beta-lactamase gene. It was found that cefetamet penetrated 5 and 9 times faster than cefixime and cefuroxime, respectively. The correlation between antibacterial activities and PBP affinities was studied in E. coli C600 not producing beta-lactamases. In this strain, cefetamet and cefixime shared the same inhibitory activity (MIC = 1 microgram/ml for both antibiotics) and the same affinity for PBP 3 (ID50 = 0.25 micrograms/ml). Cefuroxime had a lower inhibitory activity (MIC = 4 micrograms/ml) and a lower affinity for PBP 3 (ID50 = 0.5 microgram/ml). Cefixime and cefuroxime had 20 and 10 times higher affinity, respectively, than cefetamet for PBP 1s. It was concluded that the superior PBP affinity of cefixime can counterbalance the better penetration of cefetamet through the outer membrane, whilst differences in either permeability or PBP affinity seem sufficient to explain the lower antibacterial activity of cefuroxime compared to cefixime, which might well be related to the poor stability of the cefuroxime molecule.

Bacterial Proteins↗
PubMed1993

Analysis of the Salmonella fim gene cluster: identification of a new gene (fimI) encoding a fimbrin-like protein and located downstream from the fimA gene.

The fimA gene coding for the major component (fimbrin) of type 1 fimbriae was mapped within the Salmonella typhi fim gene cluster, and its nucleotide sequence determined. The deduced amino acid sequence of S. typhi fimbrin is highly homologous to that of S. typhimurium type 1 fimbrin and showed similarity to that of other enterobacterial type 1 fimbrins. Downstream of fimA, an open reading frame was found, named fimI, able to encode a fimbrin-like protein. The fimI product could represent the counterpart, in type 1 fimbriae, of the PapH protein involved in cell anchoring and length modulation of Escherichia coli Pap pili. This genetic organization was found to be common to other Salmonella serovars, including S. typhimurium and S. choleraesuis.

Amino Acid Sequence↗
PubMed1993

[Laparoscopic cholecystectomy. Our experience].

This study analysed 90 cases of symptomatic cholelithiasis treated with laparoscopic cholecystectomy. 27% of the cases studied suffered from microlithiasis, 44% from multiple calculosis with calculi measuring 1 to 2 cm in diameter, 25% from single calculi measuring up to 4 cm in diameter, and 4% from a benign proliferative pathology of the cholecystic wall. Calculosis of the main biliary tract was also found in 3 patients and was treated by preoperative endoscopic papillosphincterotomy. In addition to routine tests and ultrasonography, endovenous cholangiography, or retrograde cholangiography (ERCP) in cases of suspected calculosis of the biliary tract, was always performed prior to surgery. Mean operating time was 60 min. Two procedures were converted into laparotomy due to laceration of the cystic artery. In one case laparotomy was performed on day two due to choleperitoneum following a lesion of the cystic duct. Pneumoperitoneum could not be performed in three cases. Mean hospital stay was 48 hours. No other intra- or postoperative complications were reported. These results support the opinion that laparoscopic cholecystectomy is a safe method, with few limitations and represents the treatment of choice in the therapy of symptomatic cholelithiasis.

Adult↗
PubMed1993

In vitro activity of azithromycin against Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis in comparison with erythromycin, roxithromycin and minocycline.

The in vitro activity of azithromycin against 40 strains of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis was investigated in comparison with erythromycin, roxithromycin and minocycline. All C. trachomatis strains were inhibited by azithromycin at a concentration < or = 0.5 microgram/ml. The initial minimum inhibitory concentration (MIC) of the drug for U. urealyticum was 4 microgram/ml, whereas some resistance against the drug was shown by M. hominis. Erythromycin and roxithromycin presented almost comparable activities, whereas minocycline was slightly more active than macrolides against C. trachomatis (MIC < or = 0.25) and more active against M. hominis (initial MIC < or = 1 micrograms/ml). Only 97% of U. urealyticum strains were susceptible to 8 micrograms/ml of minocycline.

Azithromycin↗
PubMed1993

The activity of daptomycin on Enterococcus faecium protoplasts: indirect evidence supporting a novel mode of action on lipoteichoic acid synthesis.

The effect of daptomycin, an acidic lipopeptide antibiotic active against Gram-positives, was studied in Enterococcus faecium protoplasts. This antibiotic killed 99% of the protoplasts within 60 minutes of treatment, while vancomycin was ineffective, thus excluding peptidoglycan synthesis as the only target of the action of daptomycin. As previously seen with whole cells, in protoplasts lipoteichoic acid synthesis was the earliest and most strongly inhibited among types of macro-molecular synthesis. Radioactive daptomycin tightly bound only to the cytoplasmic membrane, in which the enzymes involved in lipoteichoic acid synthesis are located. These conclusions strongly support our previous proposal that daptomycin, though active against peptidoglycan synthesis, primarily inhibits lipoteichoic acid synthesis.

Anti-Bacterial Agents↗
PubMed1993

Micrococci demonstrate a phosphatase activity which is repressed by phosphates and which can be differentiated from that of staphylococci.

The phosphatase activities of 114 micrococcal strains belonging to seven different species and of an additional 150 unspeciated micrococcal strains were evaluated on solid media at various pHs containing or not containing phosphates. In the presence of phosphates, only nine strains (five unspeciated strains, one Micrococcus luteus strain, and three Micrococcus varians strains) yielded a positive reaction on plates at pH 8. In media (at pH 8) deprived of phosphates, in contrast, all but 15 strains demonstrated clear-cut phosphatase activity. Acid phosphatase could not be evaluated on solid media since none of the strains grew satisfactorily on plates at pH 5. The phosphatase activities of seven (one or two for each species, which included phosphatase-negative strains) of the strains whose colonies proved phosphatase negative at pH 8 and of 18 (two or three strains per species) of those with phosphatase-positive colonies were evaluated at pH 5 and 8.5 in toluene-treated cells which had been grown in liquid media at pH 7 containing or not containing phosphates. All strains demonstrated distinct phosphatase activity at both pHs when grown in media not containing phosphates. In contrast, when strains were grown in the presence of such substances, virtually no activity was observed at pH 8.5, and, generally, a much reduced activity was observed at pH 5. The phosphatase activity of micrococci of the various species (three to eight strains per species) was also compared with that of staphylococci of different species (5 to 10 strains per species) by the methyl green-phenolphthalein diphosphate method, the sensitivity of which can be varied by using different enzyme substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗