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E Papini

Publications and source records attributed to E Papini.

At least 55 records · Page 3Linked to original sources
PubMed1996

Bacterial protein toxins and cell vesicle trafficking.

A group of bacterial protein toxins interfere with vesicular trafficking inside cells. Clostridial neurotoxins affect mainly the highly regulated fusion of neurotransmitter- and hormone-containing vesicles with the plasma membrane. They cleave the three SNARE proteins: VAMP, SNAP-25 and syntaxin, and this selective proteolysis results in a blockade of exocytosis. The Helicobacter pylori cytotoxin is implicated in the pathogenesis of gastroduodenal ulcers. It causes a progressive and extensive vacuolation of cells followed by necrosis, after a cytotoxin-induced alteration of membrane trafficking by late endosomes. Vacuoles originate from this compartment in a rab7-dependent process and swell because they are acidic and accumulate membrane-permeant amines.

Bacterial Toxins↗
PubMed1996

The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors.

The lethal factor of Bacillus anthracis is central to the pathogenesis of anthrax. Its mechanism of action is still unknown. Recently, on the basis of sequence similarities, we suggested that lethal factor might act similarly to leukotriene A4 hydrolase (LTA4), a bifunctional enzyme also endowed with a metallopeptidase activity. Here we show that some inhibitors of the LTA4 hydrolase and metallopeptidase activities of LTA4 hydrolase also affect the cytotoxicity of the anthrax lethal factor on macrophage cell lines, without interfering with the ability of the lethal factor to enter cells. These results support the proposal that anthrax lethal factor might display in the cytosol of intoxicated cells a peptidase activity similar to that of LTA4 hydrolase.

Animals↗
PubMed1996

Morphological changes in a hyperfunctioning thyroid adenoma after percutaneous ethanol injection: histological, enzymatic and sub-microscopical alterations.

Ultrasound-guided ethanol injection (PEI) is an effective treatment of autonomously functioning thyroid nodules (AFTN) but little is known about the tissue lesions induced by alcohol. We report morphological changes of an AFTN which was removed after successful PEI treatment. At histological evaluation, the adenoma showed multiple areas of coagulative necrosis with eosinophilic ghost follicles, hemorrhages, small vessel thrombosis and a wedge-shaped hemorrhagic infarction. The peripheral adenomatous tissue and the surrounding thyroid parenchyma were free of regressive changes and/or lympho-monocytic inflammatory reactions. Enzyme histochemistry confirmed the hyperfunctioning character of the lesion and showed reduction of succinic dehydrogenase and cytochrome-c-oxydase activity in the viable tissue surrounding the central necrotic areas. By electron microscopy, the hyperfunctioning follicular epithelium showed submicroscopical alterations of the adenomatous cells surrounding the areas of coagulative necrosis. These results suggest that control of hyperthyroidism, after PEI, is due to: a) directly induced, irreversible damage (coagulative necrosis and vascular thrombosis with hemorrhagic infarction) in the central areas of the lesion; b) potentially reversible damage (reduction of intracellular enzyme activity and ultrastructural changes) in the peripheral areas.

Acid Phosphatase↗
PubMed1996

The vacuolar ATPase proton pump is present on intracellular vacuoles induced by Helicobacter pylori.

Cytotoxic strains of Helicobacter pylori cause an intense vacuolar degeneration of cells, due to the enlargement of late endosomes in the presence of membrane permeant weak bases. Bafilomycins, specific inhibitors of the vacuolar-type (V-) ATPase proton pump, prevent vacuole formation. The presence of the V-ATPase on vacuolar membranes was demonstrated by immunofluorescence with a monoclonal antibody (MAb) specific for the human 116-kDa regulatory subunit. The V-ATPase co-localised with the late endosomal marker rab7 on vacuolar membranes. In contrast, the early recycling endosomal compartment was not altered by the VacA cytotoxin, although it was endowed with the V-ATPase. Endocytosis of a MAb against the 116-kDa regulatory subunit of V-ATPase blocked endosomal acidification in HeLa cells and prevented VacA action. These results indicate that selective swelling of late endosomes, due to accumulation of osmotically active weak bases driven by the V-ATPase, is essential for vacuole formation.

Adenosine Triphosphatases↗
PubMed1996

Treatment of solitary autonomous thyroid nodules by percutaneous ethanol injection: results of an Italian multicenter study. The Multicenter Study Group.

Percutaneous intranodular ethanol injection (PEI) has been proposed for the therapy of autonomously functioning thyroid nodules. In 1992, an Italian multicenter study was undertaken to confirm the usefulness and the feasibility of this procedure. The study included 429 patients: 242 (56.4%) were affected by a toxic adenoma (TA) and 187 (43.5%) by pretoxic adenoma (PTA). Free thyroid hormone levels (FT4, FT3) and thyroid stimulating hormone (TSH) were measured before and 3, 6, 12 months after the end of treatment; thyroid ultrasound and thyroid scintiscan were performed in the majority of patients before and after treatment. Patients underwent 2-12 sessions of ethanol injection under sonographic guidance (median 4). The total amount of ethanol administered per patient (1.5 mL/mL nodular volume) was 2-50 mL (mean +/- SD, 17 +/- 9 mL), and the amount per each injection was 1-8 mL (3.2 +/- 1.3 mL). The treatment was judged successful when both TSH and free thyroid hormone serum levels returned within the normal range and recovery of tracer uptake in extranodular tissue was observed at scintiscan, at any time during the follow-up period. The treatment was considered unsuccessful when no change was observed at scintiscan and/or serum TSH levels remained less than 0.4 mU/L. A successful treatment was achieved in 66.5% of patients with TA and in 83.4% of patients with PTA, when assessed after a 12-month follow-up. In all cases a reduction of the nodular size was observed. Almost all positive results were obtained in nodules whose initial volume was less than 15 mL; large nodules responded less favorably. The treatment was generally well tolerated, only transient side-effects, mainly local pain at the time of injection, were observed. Once normalization of scintigraphic image and of FT4, FT3 and TSH serum concentrations was achieved, no recurrence of hyperthyroidism nor development of hypothyroidism were observed for the length of the study. In conclusion, percutaneous ethanol injection for treatment of autonomously functioning thyroid nodules is effective and safe. Better results are obtained in patients with PTA than in patients with TA, particularly when the initial volume of the nodule is less than or equal to 15 mL. PEI may be considered as an alternative to surgery and to radioiodine for treatment of autonomously functioning thyroid nodules.

Adenoma↗
PubMed1995

Lipid interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pylori cytotoxin.

Helicobacter pylori cytotoxin vacA (95 kDa) causes a vacuolar degeneration of epithelial cells. There is evidence that this protein toxin acts inside cells, and hence has to cross a cell membrane. This cytotoxin is frequently obtained as two fragments of 58 kDa (p58) and 37 kDa (p37) and it is available only in minute amounts. Here, its membrane interaction was studied with the two fragments, produced in Escherichia coli. Light scattering and energy transfer experiments show that p37 and p58 cause aggregation and fusion of small unilamellar lipid vesicles; only a reversible aggregation is induced at neutral pH, whereas at acid pH fusion also takes place. p58, but not p37, causes potassium efflux from liposomes and this occurs only at acid pH. Hydrophobic photolabelling with photoactivatable phosphatidylcholines inserted into liposomes shows that both fragments are labelled at neutral pH. The amount of labelling of the two fragments is much higher at acid pH, consistent with a further penetration into the hydrophobic core of the lipid bilayer. Tryptophan fluorescence measurements indicate that the two fragments undergo a pH-driven conformational change. These data are consistent with cytotoxin entry in the cell cytosol via an intracellular acidic compartment.

Bacterial Proteins↗
PubMed1995

Low pH activates the vacuolating toxin of Helicobacter pylori, which becomes acid and pepsin resistant.

The protein toxin VacA, produced by cytotoxic strains of Helicobacter pylori, causes a vacuolar degeneration of cells, which eventually die. VacA is strongly activated by a short exposure to acidic solutions in the pH 1.5-5.5 range, followed by neutralization. Activated VacA has different CD and fluorescence spectra and a limited proteolysis fragmentation pattern from VacA kept at neutral pH. Moreover, activated VacA is resistant to pH 1.5 and to pepsin. The relevance of these findings to pathogenesis of H. pylori-induced gastrointestinal ulcers is discussed.

Bacterial Proteins↗
PubMed1995

Insertion of diphtheria toxin in lipid bilayers studied by spin label ESR.

The pH dependence of the insertion of diphtheria toxin into bilayers of dioleoylphosphatidylglycerol (DOPG) has been studied by using ESR spectroscopy of spin-labeled phosphatidylglycerol with the reporter group at either the 5-position or the 14-position of the sn-2 chain (5-PGSL and 14-PGSL, respectively). At neutral pH, addition of diphtheria toxin has little effect on the ESR spectra of either spin label in large unilamellar vesicles of DOPG. At acidic pH, the outer hyperfine splitting of the 5-PGSL label is increased, and a second component corresponding to lipids whose chain motion is selectively restricted appears in the spectra of the 14-PGSL label, in the presence of diphtheria toxin. The motionally restricted component of 14-PGSL has a large outer hyperfine splitting (2Amax approximately 61 G) and corresponds to spin-labeled lipids the chains of which are in direct contact with the membrane-penetrant part of the inserted toxin. This restricted component is present, although to a lesser extent, in vesicles containing 90% of the zwitterionic lipid dioleoylphosphatidylcholine and displays a limited selectivity for negatively charged relative to zwitterionic spin-labeled phospholipids. The fraction of lipids which are motionally restricted by the toxin increases with decreasing pH, titrating in DOPG vesicles with an apparent pKa of approximately 6.1. The outer hyperfine splitting of the 5-PGSL label titrates with an apparent pKa of approximately 5.5, suggesting that this might be preferentially sensitive to a later stage in the insertion of the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Diphtheria Toxin↗
PubMed1995

Vesicle-associated membrane protein (VAMP)/synaptobrevin-2 is associated with dense core secretory granules in PC12 neuroendocrine cells.

The presence and intracellular distribution of vesicle-associated membrane protein-1 (VAMP-1) and VAMP-2 were investigated in the PC12 neuroendocrine cell line using isotype-specific polyclonal antibodies. VAMP-2 was detected in the total membrane fraction, while VAMP-1 was undetectable. Subcellular fractionation demonstrates that a substantial amount of the VAMP-2 (24-36%) is associated with dense core, catecholamine-containing granules (DCGs). This was confirmed by immunofluorescence microscopy. The L chain of tetanus neurotoxin, known to inhibit granule mediated secretion in permeabilized PC12 cells, as well as botulinum neurotoxins F and G, effectively cleaved DCG-associated VAMP-2. These data demonstrate that VAMP-2 is present on the secretory granules of PC12 cells.

Animals↗
PubMed1995

Percutaneous ethanol injection (PEI): what is its role in the treatment of benign thyroid nodules?

Ultrasound-guided percutaneous ethanol injection (PEI) was first proposed by Livraghi in 1990 as a possible therapy for autonomously functioning thyroid nodules (AFTN). The procedure is performed on out-patients; is rapid; there is no need of anesthesia, nor of bed rest or patient observation after treatment. Under direct sonographic control a limited amount of 95% sterile ethanol (1-5 ml) is slowly injected into the nodule. In predominantly cystic nodules complete fluid removal is preliminarily performed, and thereafter ethanol is injected on the basis of the aspirated fluid volume without removing the needle. PEI-induced thyroid damage is characterized by coagulative necrosis and haemorrhagic infarction due to vascular thrombosis, and is well defined from the surrounding thyroid parenchyma. Several papers have confirmed the effectiveness of PEI. Normalization of serum TSH and thyroid hormones, marked decrease of nodule volume, and effacement of the previously hyperfunctioning area at thyroid scintiscan are reported in 64-85% of toxic AFTN. Nontoxic AFTN are successfully treated in 80-100% of the cases. PEI significantly superior to aspiration alone in inducing volume reduction of benign cystic thyroid nodules, and the recurrence rate is only 2.5-5%. Complications requiring hospitalization were not reported, but the importance of experienced operators must be stressed to avoid transient dysphonia due to recurrent laryngeal nerve damage. Prior to PEI malignancy must be ruled out by fine needle aspiration in all patients. Radioiodine and surgery remain the treatments of choice of large toxic thyroid nodules, but PEI can be effectively used in selected cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol↗
PubMed1995

Helicobacter pylori cytotoxin: importance of native conformation for induction of neutralizing antibodies.

We have attempted to express the Helicobacter pylori vacuolating cytotoxin in Escherichia coli. Although the 95-kDa VacA polypeptide was expressed abundantly, it completely lacked any biological activity. In addition, this material failed to induce neutralizing antibodies after immunization of rabbits. In contrast, highly purified high-molecular-mass cytotoxin from the supernatant of H. pylori cultures was active in a HeLa cell assay and effectively induced a neutralizing response in rabbits. Neutralizing sera were shown to contain a high proportion of antibodies which recognized conformational epitopes found only on the native toxin. The data indicate that toxin-neutralizing epitopes are conformational and that potential vaccines based on the cytotoxin may benefit from the use of the intact molecule.

Animals↗
PubMed1994

Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments.

Pathogenic strains of Helicobacter pylori cause progressive vacuolation and death of epithelial cells. To identify the nature of vacuoles, the distribution of markers of various membrane traffic compartments was studied. Vacuoles derive from the endocytic pathway since they include the fluid-phase marker Lucifer yellow. Early endosome markers such as rab5, transferrin, and transferrin receptor, as well as the lysosomal hydrolase cathepsin D, are excluded from these structures. In contrast, the vacuolar membrane is specifically stained by affinity-purified antibodies against rab7, a small GTPase, localized to late endosomal compartments. The labeling of rab7 on vacuolar membranes increases as vacuolation progresses, without a concomitant increase of cellular rab7. Cell vacuolation is inhibited by the microtubule-depolymerizing agents nocodazole and colchicine. Taken together, these findings indicate that the vacuoles specifically originate from late endosomal compartments.

Adenine↗
PubMed1994

Bacterial protein toxins penetrate cells via a four-step mechanism.

Bacteria produce several protein toxins that act inside cells. These toxins bind with high affinity to glycolipid or glycoprotein receptors present on the cell surface. Binding is followed by endocytosis and intracellular trafficking inside vesicles. Different toxins enter different intracellular routes, but have the common remarkable property of being able to translocate their catalytic subunit across a membrane into the cytosol. Here, a toxin modifies a specific target with ensuing cell alterations, necessary for the survival and diffusion strategies of the toxin producing bacterium.

Bacterial Toxins↗
PubMed1994

Active-site mutations of the diphtheria toxin catalytic domain: role of histidine-21 in nicotinamide adenine dinucleotide binding and ADP-ribosylation of elongation factor 2.

Diphtheria toxin (DT) has been studied as a model for understanding active-site structure and function in the ADP-ribosyltransferases. Earlier evidence suggested that histidine-21 of DT is important for the ADP-ribosylation of eukaryotic elongation factor 2 (EF-2). We have generated substitutions of this residue by cassette mutagenesis of a synthetic gene encoding the catalytic A fragment (DTA) of DT, and have characterized purified mutant forms of this domain. Changing histidine-21 to alanine, aspartic acid, leucine, glutamine, or arginine diminished ADP-ribosylation activity by 70-fold or greater. In contrast, asparagine proved to be a functionally conservative substitution, which reduced ADP-ribosylation activity by < 3-fold. The asparagine mutant was approximately 50-fold-attenuated in NAD glycohydrolase activity, however. Dissociation constants (Kd) for NAD binding, determined by quenching of the intrinsic protein fluorescence, were 15 microM for wild-type DTA, 160 microM for the asparagine mutant, and greater than 500 microM NAD for the alanine, leucine, glutamine, and arginine mutants. These and previous results support a model of the ADP-ribosylation of EF-2 in which histidine-21 serves primarily a hydrogen-bonding function. We propose that the pi-imidazole nitrogen of His-21 hydrogen-bonds to the nicotinamide carboxamide, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism, and then stabilizing the transition-state intermediate of this reaction.

Adenosine Diphosphate Ribose↗
PubMed1994

Effects of herpes simplex virus type 1 infection on the plasma membrane and related functions of HeLa S3 cells.

In this study we evaluated modifications of various structural and functional properties of the plasma membrane of HeLa S3 cells following infection by the lytic virus herpes simplex virus type 1 (HSV-1). Na+/K(+)-ATPase activity considerably decreased during the first few hours post-infection (p.i.), whereas Na+ and K+ concentrations were not significantly affected until a much later period. By 8 h p.i., a partial membrane depolarization in infected cells had occurred, as indicated by a small change in the transmembrane potential. HSV infection induced a time-dependent lipid peroxidation of HeLa cell plasma membranes temporally correlated with the progressive reduction in Na+/K(+)-ATPase activity. Moreover, a significant decrease of membrane fluidity appeared at a late phase of the viral replicative cycle probably representing cumulative membrane damage. These results demonstrate that HSV-1 infection induced the production of free radicals in non-phagocytic cells. Since lipid peroxidation begins at an early stage of the virus replicative cycle, it may be directly related to viral cytopathicity.

Cell Membrane↗
PubMed1994

Ultrasound guided percutaneous ethanol injection in the treatment of cystic thyroid nodules.

OBJECTIVE: The management of cystic lesions in the thyroid remains controversial. We examined the efficacy and safety of ultrasound guided percutaneous ethanol injection for the treatment of benign cystic thyroid nodules in euthyroid patients. STUDY DESIGN: (A) Preliminary randomized trial: 20 patients with predominantly cystic thyroid nodules were randomized in two groups and followed up for 1 month. Group 1 underwent fine needle aspiration of the fluid component of the nodules; Group 2 underwent percutaneous ethanol injection at the end of fine needle aspiration of the cyst fluid. (B) Prospective study: 32 patients with the same clinical characteristics (Group 3) were treated by fine needle aspiration and percutaneous ethanol injection, and followed up for 12 months. METHODS: Groups 1 and 2: clinical evaluation, thyroid ultrasonography, thyroid scintiscan and serum thyroid hormone profile were performed before treatment and one month later. Group 3: clinical evaluation, thyroid ultrasonography and serum thyroid hormone profile were performed in basal conditions and 1, 3, 6, 9, 12 months after treatment. Thyroid scintiscan was performed before treatment and 3-6 months later. RESULTS: (A) Randomized study: one month after treatment, nodule volume decrease in Group 2 was significantly higher than in Group 1 (Group 2: median 14.75 range 6-29.9 ml; Group 1: median 3.65 range 0.2-18.5 ml; P < 0.01). Nodule volume reduction was greater than 50% versus baseline in 8 (80%) patients in Group 2 and in 3 (30%) patients in Group 1 (P < 0.01). (B) Prospective study (Group 3): a significant nodule volume reduction (P < 0.01 vs pretreatment) was observed 1 and 12 months after percutaneous ethanol injection (pretreatment: median 14.5, range 1.5-65.8 ml; 1 month: median 3.5, range 0.4-38.9 ml; 12 months: median 2.5, range 0.4-34.5 ml). Nodule volume reduction greater than 50% was recorded in 24 (80%) patients. Groups 1-3: recurrence of cyst fluid was demonstrated by ultrasonography in 8 patients of Group 1, in 3 patients of Group 2 and in 1 patient of Group 3. No relevant adverse effects or significant biochemical changes were observed. CONCLUSIONS: Ultrasound guided percutaneous ethanol injection of cystic thyroid nodules is a safe, low-cost and effective therapeutic procedure in patients with benign thyroid cystic nodules. Indeed, nodule volume reduction was significantly greater and more frequent in patients treated by percutaneous ethanol injection than in those patients who underwent fluid aspiration alone. These results were confirmed in a long-term prospective trial and only one fluid recurrence was observed during 12 months of follow-up.

Adolescent↗