Biomedical subjects

N Allocati

Publications and source records attributed to N Allocati.

At least 37 records · Page 2Linked to original sources

PubMed1993

Characterization of glutathione transferase from Xanthomonas campestris.

A single form of glutathione transferase (Xc-GST-4.5) having an isoelectric point at pH 4.5 was resolved from Xanthomonas campestris cytosol by affinity chromatography and isoelectric focusing. HPLC,N-terminal amino acid sequence, and SDS-PAGE analyses indicate that Xc-GST-4.5 is composed of two identical subunits, each with a molecular mass of 22 kDa. As indicated by its substrate specificity, immunological reactivity, and CD spectra, as well as by its N-terminal amino acid sequence, Xc-GST-4.5 appears to be distinct from the other bacterial glutathione transferases, Pm-GST-6.0 and Sm-GST-7.3, previously purified from the cytosolic fraction of Proteus mirabilis and Serratia marcescens. Xc-GST-4.5 also appears to be distinct from the GST so far purified from other sources.

Amino Acid Sequence↗

PubMed1993

Multiphasic denaturation of glutathione transferase B1-1 by guanidinium chloride. Role of the dimeric structure on the flexibility of the active site.

The unfolding and refolding mechanisms of dimeric glutathione transferase GSTB1-1 from Proteus mirabilis, using guanidinium chloride as a denaturant, have been investigated. The protein transitions were monitored by enzyme activity, intrinsic fluorescence, far ultraviolet circular dichroism and gel-filtration chromatography. The non coincidence of denaturation curves at equilibrium indicates that the unfolding of GSTB1-1 is a multistep process, i. e. inactivation of the structured dimer, dissociation into partially structured monomers followed by complete unfolding. In the 50% inactivated enzyme the Km for glutathione increases threefold, while the kcat appears almost the same, indicating that the initial phase of the denaturation involves the binding site of glutathione. The rapid recovery of the folded dimer precedes the complete enzyme reactivation. This indicates that the reconstitution of the native structure of GSTB1-1 is the result of folding and association of compact monomers followed by subtle rearrangements of assembled monomers that build up the active site.

Binding Sites↗

PubMed1993

The amino acid sequence of glutathione transferase from Proteus mirabilis, a prototype of a new class of enzymes.

The complete amino acid sequence of glutathione transferase from Proteus mirabilis was determined. The sequence was reconstructed by analysis of peptides obtained after cleavage by trypsin, Glu-C and Asp-N endoproteinases. The enzyme subunit is composed of 203 amino acid residues corresponding to a molecular mass of 22856 Da. Comparison of this sequence with other known primary structures of the corresponding enzyme from different sources shows a low level of identity (17-26%) with only seven conserved residues in all the sequences considered. This novel glutathione transferase could represent the prototype of a new class, possibly including other bacterial enzymes.

Amino Acid Sequence↗

PubMed1992

New plate medium for growth and detection of urease activity of Helicobacter pylori.

A new medium for detection of urease activity and isolation of Helicobacter pylori is proposed. This medium, containing Columbia Agar Base, was supplemented with IsoVitaleX, hemin, urea, and phenol red (nonselective medium [NSM]). Both bacterial growth and color change were evaluated and compared with growth in the same medium supplemented with cefsulodin, vancomycin, polymyxin B sulfate, and amphotericin B (selective medium [SM]). Twenty-five recent clinical isolates and antral biopsy specimens from 33 patients who underwent endoscopy were examined. The isolates showed a rapid color change and good growth at 5 days of incubation with NSM and SM. H. pylori-positive biopsies revealed a color change within 36 h, and bacterial growth was better appreciated in NSM, but with more contaminating flora than in SM.

Culture Media↗

PubMed1991

Enhanced clearing of Helicobacter pylori after omeprazole plus roxithromycin treatment.

The in vitro antibacterial activity of omeprazole against eight strains of Helicobacter pylori was evaluated. Minimum inhibitory concentration (MIC) values were 32 micrograms/ml and 64 micrograms/ml (MIC50 and MIC90 respectively). We performed a randomized single blind study comparing the efficacy of omeprazole alone (for 4 weeks) or combined with roxithromycin (for 2 weeks) in the treatment of duodenal ulcer and chronic active gastritis associated with H. pylori infection, H. pylori was eradicated in 75% of patients treated with omeprazole alone whereas the patients treated with the combination of these drugs were completely free from H. pylori at the end of the therapy.

Drug Combinations↗

PubMed1991

Purification of a GSH-affinity binding protein from Bacteroides fragilis devoid of glutathione transferase activity.

An affinity binding protein from the cytosolic fraction of Bacteroides fragilis was purified by using epoxy activated-Sepharose 6B resin immobilized with GSH or with hexyl-GSH. This protein showed a subunit molecular mass (22 kDa) similar to that of glutathione transferase purified from Proteus mirabilis (22.5 kDa). However, the affinity binding protein of Bacteroides fragilis, unlike the GSH-affinity binding protein of Proteus mirabilis, was devoid of the capacity to conjugate GSH to the most commonly used glutathione transferase substrates. The GSH-affinity binding protein of Bacteroides fragilis was also antigenically different from the GSH-affinity bound protein of Proteus mirabilis. It was concluded that the anaerobic microorganism is not able to express glutathione transferase even though it contains a GSH-affinity binding protein with a structural characteristic reminiscent of aerobic glutathione transferase.

Anaerobiosis↗

PubMed1991

Purification and characterization of a novel glutathione transferase from Serratia marcescens.

Four forms of glutathione transferase were resolved from the cytosol of Serratia marcescens CIP 6755 by GSH-affinity chromatography followed by isoelectric focusing. The major isoenzyme, named Sm-GST-7.3, is composed of two subunits each with a molecular mass of 22 kDa and has an isoelectric point at pH 7.3. Sm-GST-7.3, appears to be distinct from Pm-GST-6.0, previously characterized from Proteus mirabilis AF 2924 as indicated by its substrate specificity, immunological reactivity, subunit molecular mass as well as by its N-terminal amino acid sequence. None of the antisera raised against a number of human, rat and mouse GSTs cross-reacted with Sm-GST-7.3 indicating major structural differences between them and bacterial GST. This is further supported by the fact that the N-terminal sequence of Sm-GST-7.3 also differs significantly from the known sequences of mammalian GSTs of alpha, mu and pi classes. In addition, comparison with the known N-terminal amino acid sequences of helminth, plant and insect GSTs demonstrate that the latter enzymes are distantly related (less than 25% identity) to the Sm-GST-7.3. Immunoblotting experiments performed with antisera raised against Sm-GST-7.3 indicate that a GST immunologically identical to Sm-GST-7.3 is present in a number of other bacterial strains. All together the results obtained suggest that Sm-GST-7.3 is distinct from any known GST, including microbial and mammalian GSTs.

Amino Acid Sequence↗

PubMed1991

Enterosistem 18-R: description and comparative evaluation with conventional methods for identification of members of the family Enterobacteriaceae.

The efficiency and accuracy of Enterosistem 18-R (Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy) were compared with those of conventional biochemical methods to identify 360 members (38 species) of the family Enterobacteriaceae. Overall, 329 strains (91.3%) were correctly identified (percentage of identification, greater than or equal to 90.0), with 37 (11.2%) requiring additional tests for complete identification. For 11 isolates (3.1%), Enterosistem 18-R gave only genus identifications, and for 14 (3.9%), the strains did not correspond to any key in the codebook and could not be identified by the manufacturer's computer service. Only six isolates (1.7%) were misidentified. The new system accurately identified common and several newly described isolates of the family Enterobacteriaceae, such as Enterobacter gergoviae, Providencia rustigianii, Serratia odorifera, and Serratia rubidaea. The system is highly reproducible, simple to perform, easy to handle, and inexpensive. With adjustments in supplementary code numbers for some strains, Enterosistem 18-R is a suitable alternative for identification of members of the Enterobacteriaceae in clinical laboratories.

Bacteriological Techniques↗

PubMed1989

N-terminal region of Proteus mirabilis glutathione transferase is not homologous to mammalian and plant glutathione transferases.

The N-terminal amino acid sequence of glutathione transferase, Pm-GST-6.0, purified from Proteus mirabilis [(1988) Biochem. J. 255, 971-975] up to residue 38 and a comparative peptide fingerprint are reported. No obvious homology with the sequences of alpha, pi and mu classes of mammalian glutathione transferases as well as with those of plant glutathione transferases has been noted. These results suggest that the classification so far adopted for glutathione transferases cannot be extended to the bacterial enzyme.

Amino Acid Sequence↗

PubMed1989

In vitro susceptibility to quinolones and other antimicrobial agents of Staphylococcus species isolated from immunocompromised patients.

A total of 237 Staphylococcus strains were isolated from different kinds of body tissues and fluids from immunocompromised patients admitted to the Hematology Department of Pescara Hospital (Italy). These strains, collected from November 1987 to September 1988, were studied for their susceptibility to methicillin and other drugs commonly used in therapy, and the minimum inhibitory and minimum bactericidal concentrations of five quinolones were determined. The killing curve of ciprofloxacin compared with nalidixic acid was determined. The results show a considerable activity of fluoroquinolones against all strains studied.

4-Quinolones↗

PubMed1989

Glutathione transferase in bacteria: subunit composition and antigenic characterization.

The presence of glutathione transferase (GST; EC 2.5.1.18) in Escherichia coli ATCC 25922, E. coli ATCC 25422, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Klebsiella oxytoca CIP 666, K. oxytoca AF 101, Enterobacter cloacae CIP 6085, Serratia marcescens CIP 6755, and Proteus mirabilis AF 2924 was investigated. Using 1-chloro-2,4-dinitrobenzene as substrate, GST activity was found in the glutathione-(GSH-)affinity-purified fraction of all strains tested. SDS-PAGE analysis of GSH-affinity-purified enzyme indicated that the GSTs of all these bacteria are dimers of two identical subunits of Mr about 22,500. Rabbit antiserum directed against the major isoenzyme present in Proteus mirabilis AF 2924, Pm-GST-6.0, was used to investigate the antigenic properties of bacterial GSTs. Western blot analysis indicated that a GST antigenically identical to Pm-GST-6.0 is present in Enterobacter cloacae CIP 6085, Escherichia coli ATCC 25422 and Proteus vulgaris ATCC 8427, but absent in Escherichia coli ATCC 25922, Klebsiella oxytoca CIP 666, K. oxytoca AF 101 and Serratia marcescens CIP 6755. The presence of Pm-GST-6.0, but not mammalian GST, increased the MIC values of amikacin, ampicillin, cefotaxime, cephalothin and nalidixic acid for E. coli ATCC 25922. It is suggested that bacterial GST may represent a defense against the effects of antibiotics.

Anti-Bacterial Agents↗

PubMed1989

In vitro activity of netilmicin alone and in combination with azlocillin, mezlocillin and imipenem against 149 coagulase-negative staphylococci.

Coagulase-negative staphylococci (CNS) have long been regarded as innocuous skin commensals with little pathogenic potential but they have recently become, under appropriate conditions, an important cause of infections. In fact, infections caused by CNS are an increasing problem especially, but not exclusively, in immuno-compromised patients. A total of 149 strains of CNS were identified from 47 patients admitted to the Haematology Department of Pescara Hospital from October 1986 to November 1987. The strains, isolated from different parts of the body and characterized by their methicillin susceptibility, were classified by API-Staph in 11 different groups. MICs and MBCs of netilmicin alone and combined with azlocillin, mezlocillin and imipenem were studied. For all combinations FIC and FBC indices were determined. The killing kinetics of the drugs mentioned above were also determined. Except for a few microorganisms (less than 5%), the associations showed a synergic or additive effect.

Azlocillin↗

PubMed1988

Purification and characterization of three forms of glutathione transferase from Proteus mirabilis.

Three forms of glutathione transferase (GST) with pI values of 6.0, 6.4 and 7.3 were isolated from Proteus mirabilis AF 2924 by glutathione-affinity chromatography followed by isoelectric focusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide-slab-gel electrophoresis, all forms proved to be composed of two subunits of identical (22,500) Mr. GST-6.0 and GST-6.4 together account for about 95% of the total activity, whereas GST-7.3 is present only in trace amounts. Extensive similarities have been found between GST-6.0 and GST-6.4. These include subunit molecular mass, amino acid composition, substrate specificities and immunological characteristics. GST-7.3 also cross-reacted (non-identity) with antisera raised against bacterial GST-6.0. None of the antisera raised against a number of human, rat and mouse GSTs cross-reacted with the bacterial enzymes, indicating major structural differences between them and the mammalian GSTs. This conclusion is further supported by c.d. spectra.

Amino Acids↗

PubMed1988

Plasmid "curing" by some recently synthetized 4-quinolone compounds.

Nalidixic acid and two recently synthetized 4-quinolones eliminated F'lac and R-plasmids from E. coli at concentrations of one half or one quarter of the minimum inhibitory concentration (MIC). Two of the three plasmids tested were cured by all derivatives, although with different frequencies. Pefloxacin was the most effective compound compared with the other quinolones and nalidixic acid the least active.

Escherichia coli↗

PubMed1988

In vitro activity of pefloxacin compared with five other quinolones on nalidixic acid-resistant proteae species.

The in vitro activity of pefloxacin, a new fluoroquinolone, was compared with that of 5 other quinolone compounds (nalidixic and pipemidic acids, norfloxacin, ciprofloxacin, and ofloxacin) against 416 strains of Proteae spp. isolated from urine specimens of hospitalized patients with acute urinary tract infections (UTI). Ciprofloxacin was the most active agent. Norfloxacin, ofloxacin, and pefloxacin were similarly active against Proteus strains (MIC90 = 0.39 microgram/ml). Against Providencia spp. pefloxacin and norfloxacin showed similar activity (MIC90 = 3.12 micrograms/ml). There is minimal discrepancy between minimum inhibitory concentrations and minimum bactericidal concentrations exhibited by the quinolones for all urinary tract pathogens tested. Our in vitro studies indicate that pefloxacin is an active antimicrobial agent and suggest that it will prove useful in the treatment of complicated urinary tract infections due to nalidixic acid-resistant Proteae spp.

Anti-Infective Agents↗

PubMed1988

Effect of pefloxacin on adherence of Proteus-Providencia spp to squamous epithelial cells.

Pefloxacin, a new piperazinyl substituted quinolone derivative, reduced at sub-MICs (Minimal Subinhibitory Concentrations: four times lower than Minimal Inhibitory Concentrations) the in vitro adherence of Proteus-Providencia spp. to squamous epithelial cells obtained from the urinary tract. The decrease of adherence was about 30% and 50% after 1 h and, respectively, 16-18 h of incubation with pefloxacin. This finding suggests that pefloxacin may play a role in determining the in vivo adhesion of Proteus-Providencia spp. on the urinary tract and consequently the expression of bacterial pathogenic potency.

Adult↗

PubMed1987

Comparative in vitro activities of 13 antimicrobial agents against Morganella-Proteus-Providencia group bacteria from urinary tract infections.

We examined 741 urinary tract isolates of the Morganella-Proteus-Providencia group, including the recently defined species Proteus penneri, for susceptibilities to aminoglycosides, semisynthetic penicillins, and cephalosporins. The data emphasize the importance of identification to the species level on the basis of marked species differences in patterns of susceptibility.

Anti-Bacterial Agents↗

PubMed1987

Preliminary studies on the effect of glutathione S-transferase from Providencia stuartii on the antimicrobial activity of different antibiotics.

Glutathione S-transferase has been isolated and purified from a Providencia stuartii CH 114 strain. The effect of the enzyme on the antimicrobial activity of amikacin, cefotaxime, cephalexin, ampicillin, nalidixic acid, and ofloxacin was tested. The efficiency of all antibiotics tested, except nalidixic acid and ofloxacin, is relevantly decreased by the presence of glutathione S-transferase in the medium culture, as proved by the increased value of MIC. The effect of glutathione S-transferase on the drugs is not significantly affected by the addition of exogenous glutathione. The possible mechanism of action of the enzyme is also discussed.

Anti-Bacterial Agents↗