p63/p73 in the control of cell cycle and cell death.

The p53 family apparently derives from a common ancient ancestor that dates back over a billion years, whose function was protecting the germ line from DNA damage. p63 and p73 would maintain this function through evolution while acquiring novel roles in controlling proliferation and differentiation of various tissues. p53 on the other hand would appear in early vertebrates to protect somatic cells from DNA damage with similar mechanism used by its siblings to protect germ line cells. For the predominant role played by p53 mutations in cancer this was the first family member to be identified and soon became one of the most studied genes. Its siblings were identified almost 20 years later and interestingly enough their ancestral function as guardians of the germ-line was one of the last to be identified. In this review we shortly summarize the current knowledge on the structure and function of p63 and p73.

A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications.

BACKGROUND: Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. RESULTS: Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. CONCLUSIONS: The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.

Bat-man disease transmission: zoonotic pathogens from wildlife reservoirs to human populations.

Bats are natural reservoir hosts and sources of infection of several microorganisms, many of which cause severe human diseases. Because of contact between bats and other animals, including humans, the possibility exists for additional interspecies transmissions and resulting disease outbreaks. The purpose of this article is to supply an overview on the main pathogens isolated from bats that have the potential to cause disease in humans.

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.

Die for the community: an overview of programmed cell death in bacteria.

Programmed cell death is a process known to have a crucial role in many aspects of eukaryotes physiology and is clearly essential to their life. As a consequence, the underlying molecular mechanisms have been extensively studied in eukaryotes and we now know that different signalling pathways leading to functionally and morphologically different forms of death exist in these organisms. Similarly, mono-cellular organism can activate signalling pathways leading to death of a number of cells within a colony. The reason why a single-cell organism would activate a program leading to its death is apparently counterintuitive and probably for this reason cell death in prokaryotes has received a lot less attention in the past years. However, as summarized in this review there are many reasons leading to prokaryotic cell death, for the benefit of the colony. Indeed, single-celled organism can greatly benefit from multicellular organization. Within this forms of organization, regulation of death becomes an important issue, contributing to important processes such as: stress response, development, genetic transformation, and biofilm formation.

Analysis by limited proteolysis of domain organization and GSH-site arrangement of bacterial glutathione transferase B1-1.

Limited proteolysis method has been used to study the structure-function relationship of bacterial glutathione transferase (GSTB1-1). In absence of three-dimensional structural data of prokaryote GST, the results represent the first information concerning the G-site and domains organization of GSTB1-1. The tryptic cleavages occur mainly at the peptide bonds Lys35-Lys36 and Phe43-Leu44, generating two major molecular species of 20-kDa, 3-kDa and traces of 10-kDa. 1-chloro-2,4-dinitrobenzene favoured the proteolysis of the 20-kDa fragment markedly enhancing the production of the 10-kDa peptide by cleaving the chemical bonds Lys87-Ala88 and Arg91-Tyr92. The tryptic cleavage sites of GSTB1-1 was found to be located close to those previously found for the mammalian GSTP1-1 isozyme. It was concluded that despite their low sequence homology (18%), GSTB1-1 and GSTP1-1 displayed similar structural features in their G-site regions and probably a common organization in structural domains.

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.

Immunogold localization of glutathione transferase B1-1 in Proteus mirabilis.

By using the immunolabelling technique, the cellular localization of glutathione transferase in Proteus mirabilis was investigated. Evidence was obtained indicating a significant higher content of glutathione transferase in the periplasmic than cytoplasmic space. This result further support the idea that bacterial glutathione transferase is involved in xenobiotic detoxication.

Site-directed mutagenesis of the Proteus mirabilis glutathione transferase B1-1 G-site.

In order to investigate the roles of near N-terminus Tyr, Cys, and Ser residues in the activity of bacterial glutathione transferase (GSTB1-1) site-directed mutagenesis was used to replace the following residues: Tyr-4, Tyr-5, Ser-9, Cys-10, Ser-11, and Ser-13. The results presented here show that, unlike all other alpha, mu, pi, theta and sigma classes of glutathione transferases so far investigated, GSTB1-1 does not utilise any Tyr, Ser or Cys residue to activate glutathione. These results also suggest that the bacterial glutathione transferases may require classification into their own class.

Functional analysis of the evolutionarily conserved proline 53 residue in Proteus mirabilis glutathione transferase B1-1.

The role of the evolutionarily conserved residue Pro-53 in Proteus mirabilis glutathione transferase B1-1 has been examined by replacing it with a serine residue using site-directed mutagenesis. The effect of the replacement on the activity, thermal stability and antibiotic binding capacity of the enzyme was examined. The results presented support the view that Pro-53 participates in the maintenance of the proper conformation of the enzyme fold rather than playing a direct role in the catalytic reaction. Furthermore, this residue appears to be an important determinant of the antibiotic binding to the enzyme. Experiments with wild type and mutated enzymes provide evidence that glutathione transferases may play an important role in antibiotic resistance exhibited by bacteria.

Failure to detect Helicobacter pylori in nasal mucus in H pylori positive dyspeptic patients.

Stomach biopsies and samples of nasal mucus were cultured in patients with dyspeptic symptoms who underwent endoscopy to evaluate the possible route of transmission of Helicobacter pylori (H pylori). 42 patients were examined. For each patient two biopsies from the stomach corpus and antrum were taken and, before endoscopy, one nasal swab was obtained. Biopsy samples were tested for urease test, microbiological culture, and histological examination. The nasal swab was processed for microbiological examination. H pylori was not found in the nasal mucus of any of the patients, including the 36 who had H pylori in gastric biopsies.

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.

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.

Effect of anaerobic environment on the glutathione transferase isoenzymatic pattern in Proteus mirabilis.

When Proteus mirabilis was cultured anaerobically in the presence of nitrate as terminal electron acceptor, a dramatic reduction of glutathione transferase production occurred. The analysis of the glutathione affinity purified materials in terms of substrate specificity, SDS-PAGE pattern, IEF pattern and immunoblotting revealed that a significantly different glutathione transferase pattern also occurred: two new glutathione transferase forms with an isoelectric point at pH 4.8 and 5.0 appeared. Their N-terminal amino acid sequence analysis as well as the ability to bind to a glutathione affinity column indicate that major differences between anaerobic and aerobic glutathione transferase forms are mainly located in the C-terminal region of the primary structure. In contrast, no significant changes occurred in the production of glutathione transferase isoenzymes when P. mirabilis was grown anaerobically in the absence of a terminal electron acceptor. These results support the idea that bacterial glutathione transferase expression is not strictly related to the absence of oxygen stress.

Inhibition of Helicobacter pylori by garlic extract (Allium sativum).

The antibacterial effect of aqueous garlic extract (AGE) was investigated against Helicobacter pylori. Sixteen clinical isolates and three reference strains of H. pylori were studied. Two different varieties of garlic were used. The concentration of AGE required to inhibit the bacterial growth was between 2-5 mg ml-1. The concentration, for both AGE types, to inhibit 90% (MIC90) of isolates was 5 mg ml-1. The minimum bactericidal concentration (MBC) was usually equal to, or two-fold higher than, minimum inhibitory concentration (MIC). Heat treatment of extracts reduced the inhibitory or bactericidal activity against H. pylori; the boiled garlic extract showed a loss of efficacy from two- to four-fold the values of MIC and the MBC obtained with fresh AGE. The antibacterial activity of garlic was also studied after combination with a proton pump-inhibitor (omeprazole) in a ratio of 250:1. A synergistic effect was found in 47% of strains studied; an antagonistic effect was not observed.

Helicobacter pylori isolated from stomach corpus and antrum: comparison of DNA patterns.

The genomic DNA of Helicobacter pylori was studied in strains isolated from two different sites of the stomach: the corpus and the antrum. 70 strains of H. pylori were found in 36 patients; 34 out of the 36 patients harboured the strain in both districts analysed. Restriction endonuclease analysis with Hae III and Hind III was used to compare the DNA patterns of strains isolated from the anatomical sites studied. Two pairs of DNA samples were not digested by these enzymes. 27 of the 32 pairs of the digested DNA appeared similar to each other. The analysis of chromosomal DNA in the remaining five pairs showed different electrophoretic patterns. These results indicate that the gastric mucosa can be colonized, at the same time, by strains of H. pylori with different genomic patterns, and this aspect can be important for epidemiological studies.

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.

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.

Microbiological evidence of Helicobacter pylori from dental plaque in dyspeptic patients.

To evaluate the possible route of transmission of Helicobacter pylori, stomach biopsies and dental plaques were cultured from patients with dyspeptic symptoms who underwent endoscopy. A total of 31 patients were examined. Twenty patients out of thirty one (64%) were H. pylori positive in gastric biopsy. Among the microorganisms isolated in dental plaque only one sample (corresponding to a patients with duodenal ulcer H. pylori positive) showed colonies morphologically and biochemically compatible with H. pylori. Proteic patterns of whole cells and restriction endonuclease analysis with Hind III and Hae III endonucleases of DNA extracted from the strain subcultured from a stomach biopsy and from dental plaque of the same patient indicated that both sites were infected with the same strain of H. pylori.

Study of the in vitro activity of norfloxacin and other drugs on amoxicillin-resistant uropathogenic isolates.

The in vitro activity of a quinolinecarboxylic acid compound, norfloxacin, was compared with those of amikacin, carbenicillin, cefazolin, cefoxitin, gentamicin, nalidixic acid, and oxolinic acid against 243 Gram-positive and Gram-negative amoxicillin-resistant (MIC 800 greater than or equal to micrograms/ml) uropathogenic isolates. Norfloxacin showed remarkable activity against the majority of the bacteria tested. Ninety percent of strains of Staphylococcus aureus, Streptococcus faecalis, Citrobacter freundii and Serratia spp. were inhibited by 3.12 micrograms/ml or less. Among the Gram-negative urinary isolates, only a few Enterobacter, Escherichia coli, Klebsiella, Proteus and Pseudomonas aeruginosa strains required norfloxacin levels between 12.5 and 25 micrograms/ml. The MBC was within one dilution of the MIC for 100% of the Gram-positive strains and 95% of the Gram-negative isolates against norfloxacin and aminoglycosides. Norfloxacin had a markedly higher inhibitory index than all other antimicrobial agents for Gram-positive cocci and Gram-negative bacteria. From its in vitro activity, norfloxacin appears to be a potentially valuable agent for the treatment of serious urinary tract infections.