High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein

Listeriolysin O (LLO) is a cholesterol-binding sulfhydryl-activated hemolysin encoded by Listeria monocytogenes hlyA gene. After analyzing the nucleotide coding sequence of this gene from the ATCC 9525 L. monocytogenes strain, we cloned it in a pET vector for expression in Escherichia coli. Thanks to the optimization of the induction protocol, we achieved a high-level LLO synthesis (about 10% of total cell proteins) in hemolytically active form. The expressed hemolysin was then purified to homogeneity, as revealed by SDS-PAGE and Western blot analysis, by a hydroxyapatite adsorption chromatography, followed by an SP Sepharose ion-exchange chromatography. The recombinant protein showed the same properties determined for LLO purified from L. monocytogenes cultures and the characteristics of the sulfhydryl-activated toxins such as inactivation by oxidation and by reaction with cholesterol. By a combination of the pET expression system and the simple purification method, we obtained a significant amount of toxin (4.5 mg/litre cell culture) in a hemolytically active form (1.25 x 10(6)HU/mg protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures, which is a difficult task, mainly owing to the low levels of toxin released in the culture media. The recombinant hemolysin, purified in sufficient quantities, could be very useful for structural studies and for diagnostic and pharmaceutical applications.     

Helicobacter pylori Infection and Gastric Autoimmune Diseases: Is There a Link?

Background. Helicobacter pylori is thought to be involved in atrophic body gastritis. We explored the prevalence of H. pylori infection in asymptomatic subjects with gastric parietal cell antibodies, as well as in patients with pernicious anemia, to evaluate a possible role of H. pylori gastric infection in gastric autoimmunity. Patients and Methods. We studied 79 consecutive asymptomatic subjects with parietal cell antibodies, 24 patients with pernicious anemia, and 66 parietal cell antibody‐negative controls. All patients underwent gastric biopsies for histology and detection of H. pylori. Red blood cell count and volume, serum levels of gastrin, pepsinogen I, iron, folic acid, vitamin B₁₂, and circulating antibodies to H. pylori and to intrinsic factor were also determined. Results. We found an atrophic body gastritis in 14 of the 79 asymptomatic subjects with parietal cell antibodies (18%) and in 2 of the 66 controls (3%) (p = .01). Mean levels of gastrin were increased (p < .0001), while those of pepsinogen were reduced (p < .001) compared with controls. H. pylori was identified at the gastric level and/or circulating anti‐H. pylori antibodies were detected in 46 parietal cell antibody‐positive subjects (58%) compared with 26 controls (39%) (p = .03). In patients with pernicious anemia we found an atrophic body gastritis in 18 of 24 cases (75%) (p < .001 vs. controls). Mean levels of gastrin were markedly increased (p < .0001) and those of pepsinogen I decreased (p < .0001) relative to controls. Only five of these patients (21%) had evidence of H. pylori infection compared with 46 of the parietal cell antibody‐positive subjects (58%) (p = .003) and 26 of the controls (39%). Considering all patients with gastric autoimmunity (i.e. with parietal cell antibodies and/or with pernicious anemia), H. pylori was found in 44 of 72 of those without atrophy (61%) but in 6 of 31 with gastric body atrophy (19%) (p < .001), indicating that H. pylori infection is greatly reduced when gastric acid secretion decreases. Conclusions. The frequent detection of H. pylori infection in subjects with early gastric autoimmunity, indicated by the presence of parietal cell antibodies, suggests that H. pylori could have a crucial role in the induction and/or the maintenance of autoimmunity at the gastric level.     

Fibrinogen conformation and platelet reactivity in relation to material-blood interaction: effect of stress hormones

The performance of many biomaterials in hemocompatibility tests is altered when blood is drawn from stressed subjects. A salient physiological response during stress is one in which hormones are released into plasma by the hypothalamo-pituitary-adrenal axis. We investigated the influence of basal and stress levels of epinephrine and beta-endorphin on the conformation of fibrinogen (Fbg), both in saline solution (under physiological conditions) and after its adsorption to polyethylene (PE), by FT-IR spectroscopy. Moreover, as Fbg is one of the major mediators of platelet adhesion, the behavior of platelets in contact with PE was also evaluated as a function of the two different hormone concentrations. Epinephrine was found to affect Fbg conformation and to increase platelet adhesion to PE at stress level. Basal and stress levels of beta-endorphin did not significantly affect the Fbg conformation and only induced adhesion of isolated platelets to the PE surface. A direct relationship was therefore found between Fbg conformation and platelet behavior. The response of platelets was affected by the stress status of donors through the influence of epinephrine on Fbg conformation.     

Three highly oxygenated caryophyllene sesquiterpenes from Pestalotiopsis sp., a fungus isolated from bark of Pinus taeda

A Pestalotiopis sp. was isolated from the trunk bark of Pinus taeda. The fungus was cultivated in liquid medium and produced three highly oxygenated caryophyllene sequiterpene derivatives, named pestalotiopsolide A, taedolidol and 6-epitaedolidol, respectively. The sesquiterpenes were isolated by silica gel based chromatographic procedures and their structures were elucidated by NMR spectroscopic data.     

The effect of Cyclosporine A chronic administration on the antioxidant pattern of rat liver mitochondria: structural and functional consequences

Cyclosporine A (CsA) plays a pivotal role in controlling Ca2+ movement in the cell modulating also the mitochondrial permeability transition pore. We investigated if chronic administration of CsA may have some effects on the lipophilic and hydrophilic antioxidant pattern of rat liver mitochondria and on their morphological structure. It seems that CsA administration does not statistically affect the redox status of the antioxidants investigated and their amounts (vitamin E, CoQ9, CoQ10, glutathione, uric acid and ascorbic acid) despite the variety of effects that this treatment produces at physiological and morphological levels. However, some kind of derangement could occur in the liver biochemical machinery since CsA treatment induces a markedly increased variability in antioxidant contents.     

Programmed cell death in 2',3'-dideoxycytidine-resistant human monoblastoid U937 cells

2,3-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O₂ and nitric oxide.Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.     

Molecular structure and evolution of DNA sequences located at the alpha satellite boundary of chromosome 20

We have isolated and characterised one PAC clone (dJ233C1) containing a linkage between alphoid and non-alphoid DNA. The non-alphoid DNA was found to map at the pericentromeric region of chromosome 20, both on p and q sides, and to contain homologies with one contig (ctg176, Sanger Centre), also located in the same chromosome region. At variance with the chromosome specificity shown by the majority of non-alphoid DNA, a subset of alphoid repeats derived from the PAC yielded FISH hybridisation signals located at the centromeric region of several human chromosomes, belonging to three different suprachromosomal families. The evolutionary conservation of this boundary region was investigated by comparative FISH experiments on chromosomes from great apes. The non-alphoid DNA was found to have undergone events of expansion and transposition to different pericentromeric regions of great apes chromosomes. Alphoid sequences revealed a very wide distribution of FISH signals in the great apes. The pattern was substantially discordant with the data available in the literature, which is essentially derived from the central alphoid subset. These results add further support to the emerging opinion that the pericentromeric regions are high plastics, and that the alpha satellite junctions do not share the evolutionary history with the main subsets.