Biomedical subjects

E Papini

Publications and source records attributed to E Papini.

At least 73 records · Page 4Linked to original sources

PubMed1993

Cell vacuolization induced by Helicobacter pylori: inhibition by bafilomycins A1, B1, C1 and D.

All available bafilomycins (A1, B1, C1 and D) inhibit and revert macroscopic vacuolization induced by Helicobacter pylori cell-free extracts. Bafilomycin A1 displays the highest activity, followed by bafilomycin B1, C1 and D. The different potency of bafilomycins correlates with their ability to inhibit the vacuolar-type ATPase (V-ATPase) and to dissipate the membrane pH gradient of intracellular acidic organelles. These results suggest that bafilomycins should be considered as possible therapeutic agents in the treatment of gastritis.

Adenosine Triphosphatases↗

PubMed1993

Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer.

Helicobacter pylori has been associated with gastritis, peptic ulcer, and gastric adenocarcinoma. We report the nucleotide sequence and expression of an immunodominant antigen of H. pylori and the immune response to the antigen during disease. The antigen, named CagA (cytotoxin-associated gene A), is a hydrophilic, surface-exposed protein of 128 kDa produced by most clinical isolates. The size of the cagA gene and its protein varies in different strains by a mechanism that involves duplication of regions within the gene. Clinical isolates that do not produce the antigen do not have the gene and are unable to produce an active vacuolating cytotoxin. An ELISA to detect the immune response against a recombinant fragment of this protein detects 75.3% of patients with gastroduodenal diseases and 100% of patients with duodenal ulcer (P < 0.0005), suggesting that only bacteria harboring this protein are associated with disease.

Amino Acid Sequence↗

PubMed1993

Cell penetration of diphtheria toxin. Reduction of the interchain disulfide bridge is the rate-limiting step of translocation in the cytosol.

The pathway of cell penetration of diphtheria toxin (DT) was studied in Vero cells by following the kinetics of uptake, reduction, degradation, and sub-cellular distribution of 125I-DT in the absence or presence of bafilomycin A1 (baf-A1), a powerful inhibitor of the endosomal H(+)-ATPase. After a lag phase of 4 min, DT, bound to Vero cells, reached an acidic intracellular compartment, where about one-third of it underwent a transition to a state competent for subsequent reduction and membrane translocation. After further 4 min, this DT fraction was reduced in a baf-A1-insensitive reaction and DT-A, the intracellularly active protomer of DT, was immediately released in the cytosol. The present data indicate that cell-mediated reduction of the toxin is the rate-determining step of the DT cell intoxication process. Subcellular fractionation showed that DT underwent the low pH-driven conformational change in an early endosome, distinct from the subsequent endosomal compartment where reduction took place. DT-B remained endosome-bound and was proteolyzed at low pH as well as the portion of DT which was not reduced after the exposure to low pH in early endosomes.

Animals↗

PubMed1993

The sensitivity of cystic fibrosis cells to diphtheria toxin.

Cystic fibrosis (CF) cells have been reported to have a defective acidification and it has been suggested that they may be less sensitive than normal cells to diphtheria toxin (DT). A comparative analysis of DT toxicity in CF and normal cells, both in terms of kinetics of cell intoxication and of cell survival, shows very little difference, which makes it unlikely that diphtheria epidemics contribute to accounting for the large diffusion of the CF trait. DT penetrates cells from endosomal compartments different from those defective in CF cells.

Cell Division↗

PubMed1993

A prospective randomized trial of levothyroxine suppressive therapy for solitary thyroid nodules.

OBJECTIVE: We examined the effects of L-thyroxine therapy versus placebo over a 12-month period on volume of solitary thyroid nodules. DESIGN: Prospective randomized clinical trial; placebo and control groups followed for one year in three centres. PATIENTS: One hundred and one euthyroid patients with single palpable colloid thyroid nodules. MEASUREMENTS: Serum thyroid hormones, TSH, thyroglobulin, anti-thyroglobulin and anti-thyroid peroxidase antibodies measured at the first visit and after 2, 6 and 12 months; nodule volume and contralateral thyroid lobe thickness measured by ultrasound in double blind fashion; nodule diameter measured by palpation every 6 months. RESULTS: In the treatment group the nodule volume measured by ultrasound decreased progressively but not significantly; in the placebo group no changes were demonstrated. The number of nodules which decreased in size was however significantly larger in the treatment group. None of the initial parameters was predictive for the response. In the treatment group the nodule size assessed by palpation showed a significant decrease after 12 months when compared to baseline. CONCLUSIONS: In non-endemic areas TSH suppression induces nodule volume reduction measured by ultrasound which, although not significant, seems to be progressive and occurring only in a subgroup of patients. Significant palpatory nodule reduction is probably related to decreased thickness of the surrounding thyroid tissue still responsive to TSH.

Adolescent↗

PubMed1993

Bafilomycin A1 inhibits Helicobacter pylori-induced vacuolization of HeLa cells.

Bafilomycin A1, a specific inhibitor of the vacuolar-type H(+)-ATPase, responsible for acidification of intracellular compartments, prevents the vacuolization of Hela cells induced by H. pylori, with an inhibitory concentration giving 50% of maximal (ID50) of 4 nM. Bafilomycin A1 is also very efficient in restoring vacuolated cells to a normal appearance. The vacuolating activity of Helicobacter pylori is not inhibited by a series of specific inhibitors of vacuolar H(+)-ATPases. These findings indicate that a transmembrane pH gradient is needed for the formation and growth of vacuoles caused by the bacterium and that this pH gradient is due to the activity of a vacuolar ATPase proton pump of HeLa cells.

Anti-Bacterial Agents↗

PubMed1993

Percutaneous ultrasound-guided ethanol injection: a new treatment of toxic autonomously functioning thyroid nodules?

Twenty autonomously functioning thyroid nodules were treated with ultrasound-guided percutaneous ethanol injection (PEI) and followed for 12 months. PEI was performed on symptomatic and biochemically proven thyrotoxic patients by injecting 2.0-4.0 mL sterile ethanol and was repeated 3-8 times, depending on nodule size. Serum thyroglobulin increased immediately after PEI, peaked at 6-10 h, and declined thereafter. Free T4 progressed slowly during the first 24 h. Free T3 showed a delayed and not significant increase. By the end of the treatment (4 weeks), PEI had produced clinical improvement and hormonal normalization. Three months later, serum TSH was detectable and responsive to TRH in 17 of the 20 patients. All nodules had a significant shrinkage at ultrasound evaluation. The previous hot thyroid areas manifested hypofunction at technetium-99 m-pertechnetate scintiscan, and the surrounding parenchyma returned to normal function in all but the 3 cases with still suppressed TSH. The histological features of a nodule that had been operated upon and the fine needle aspiration biopsy patterns obtained after PEI showed coagulative necrosis, with scanty follicles, some fibroblasts, but no lymphocytic infiltration. The procedure was generally well tolerated, but in 1 case it induced temporary dysphonia, and in another patient, it caused a transient exacerbation of thyrotoxicosis.

Adult↗

PubMed1993

[Long-term results of echographically guided percutaneous ethanol injection in the treatment of the autonomous thyroid nodule].

Ultrasound-guided percutaneous ethanol injection (PEI) was performed as a therapeutic procedure on twenty-four patients affected by toxic autonomously functioning thyroid nodules (AFTN). After treatment patients were followed up for a mean period of 12 months. PEI induced persistent and complete (clinical and hormonal) disease control in 19/23 cases (82.6%) that completed the procedure, normalization of serum FT4 and FT3 associated with a still suppressed TSH in 2/23 cases (8.6%) and failed to control hyperthyroidism in 2/23 cases (8.6%). After PEI all AFTN became smaller at clinical and US examination with a 60% mean volume decrease. Nodule shrinkage was related to cytological and histological findings of well circumscribed coagulative necrosis, granulomatous inflammation and progressive fibrosis. PEI induced two cases of temporary complications: 1 case of acute worsening of thyrotoxicosis and 1 case of self-resolving vocal cord paresis. No increase of serum autoantibodies (TgAb, TPOAb, TRAb) was detected during the follow-up period.

Adult↗

PubMed1992

Ion channel and membrane translocation of diphtheria toxin.

Diphtheria toxin is the best studied member of a family of bacterial protein toxins which act inside cells. To reach their cytoplasmic targets, these toxins, which include tetanus and botulinum neurotoxins and anthrax toxin, have to cross the hydrophobic membrane barrier. All of them have been shown to form ion channels across planar lipid bilayer and, in the case of diphtheria toxin, also in the plasma membrane of cells. A relation between the ion channel and the process of membrane translocation has been suggested and two different models have been put forward to account for these phenomena. The two models are discussed on the basis of the available experimental evidence and in terms of the focal points of difference, amenable to further experimental investigations.

Animals↗

PubMed1991

Tyrosine 65 is photolabeled by 8-azidoadenine and 8-azidoadenosine at the NAD binding site of diphtheria toxin.

8-Azidoadenine and 8-azidoadenosine, two photoactivatable derivatives of adenine and adenosine, are competitive inhibitors of diphtheria toxin of similar potency with respect to their parent compounds. On irradiation, the two tritium-labeled photoactivatable azidoadenines bind covalently and specifically to an enzymic fragment of diphtheria toxin that is known to bind to NAD. This photolabeling is protected by the enzyme substrate NAD. The radiolabeled protein was fragmented, and the radioactive fragments were sequenced. Tyr-65 is labeled specifically by both photoreagents, and its labeling was reduced strongly when NAD was present during irradiation. Labeling is also reduced strongly by adenine, adenosine, and nicotinamide. These results suggest that Tyr-65 is at the NAD binding site of diphtheria toxin and that the competitive inhibitors adenine, adenosine, and nicotinamide bind to the same portion of the catalytic center of the toxin.

Adenine↗

PubMed1991

Determination of diphtheria toxin neutralizing antibody titers with a cell protein synthesis inhibition assay.

A method which allows an accurate determination of very low titers of antibodies able to neutralize diphtheria toxin has been developed. The assay is based on the incubation of a reference amount of diphtheria toxin with different dilution of the serum and the evaluation of the residual toxicity of diphtheria toxin on Vero cells. The cells are seeded in 96-well plastic plates with the toxin-serum mixtures and the residual toxin activity is measured as the block of cell protein synthesis after incubation with [14C]leucine. Antitoxin titers as low as 0.002 IU can be reproducibly and accurately measured and the method gives a linear response in the range 0.002 to 8.0 IU/ml.

Animals↗

PubMed1991

A randomized trial of ultrasound-guided anterior subcostal liver biopsy versus the conventional Menghini technique.

An ultrasound-guided double pass biopsy technique using a large bore cutting needle via an anterior subcostal route (USAB) is described. The diagnostic adequacy of this biopsy procedure was evaluated in comparison with the traditional Menghini technique in 200 cases of suspected chronic liver disease randomly assigned to the two different procedures. Retrieval rate was better in the USAB group. The sample length was less than 15 mm in four cases in the USAB group and in 19 cases in the Menghini group. In three cases in the USAB group the second pass modified the diagnosis of the first specimen from chronic active hepatitis to active cirrhosis. In order to evaluate the safety and discomfort of the two procedures 340 patients assigned to the different techniques had liver ultrasound scans 6 and 24 h following percutaneous biopsy. USAB was associated with a reduced frequency of complications (one out of 170 patients vs. seven out of 170 patients who had the intercostal Menghini procedure). In our study, USAB seems to offer better sampling and size of individual samples, together with reduced frequency of major and minor complications.

Biopsy↗

PubMed1990

Histidine-21 is involved in diphtheria toxin NAD+ binding.

Histidine-21 is the sole histidine present in the A chain of diphtheria toxin and recent evidence suggests that it is involved in NAD+ binding. Fluorimetric assays of NAD+ binding and diethylpyrocarbonate modification performed at different pH values provide further insights on the role of this residue and indicate that its pKa value is 6.3. Conformational changes of subunit A of diphtheria toxin have been detected by analysis of tryptophan fluorescence in the pH 2.5-4 and pH 9-10.5 ranges. This indicates that histidine-21 is unlikely to be involved in the low pH-driven conformational change of diphtheria toxin.

Binding Sites↗

PubMed1990

An intact interchain disulfide bond is required for the neurotoxicity of tetanus toxin.

Tetanus toxin is composed of a heavy chain (100 kDa) and a light chain (50 kDa) held together by a single interchain disulfide bridge. An additional intrachain disulfide is present in the carboxy-terminal part of the heavy chain. Reduction of the two disulfide bonds in tetanus toxin with both chemical and proteinaceous reducing agents was studied. Dithiothreitol and 2-mercaptoethanol cleaved both the inter- and intrachain disulfide bridges of the toxin, while glutathione and cysteine were ineffective. Specific reduction of the single interchain disulfide link was achieved with the thioredoxin-thioredoxin reductase system, thus indicating that this bond is exposed at the protein surface. Also, dead or permeabilized cells were able to reduce the toxin. Such reduced toxin bound to neuronal membranes as well as the native toxin but was not neurotoxic. These findings open the possibility that reduction by cytoplasmic agents released by dead cells contributes to detoxification of tetanus toxin. Moreover, together with the notion that the light chain is the active form of the toxin in the cytoplasm, these results suggest that the interchain disulfide bond of tetanus toxin plays a role in nerve cell penetration.

Animals↗

PubMed1990

Hepatocellular carcinoma: magnetic resonance imaging after ultrasonically guided percutaneous alcohol treatment. Preliminary report.

Percutaneous alcohol injection (PAI) is reported as a safe, inexpensive and effective method of treatment of small HCC in severely ill patients. Nevertheless, residual cancer can persist after treatment and multiple bioptic manoeveurs are needed to ascertain the actual completeness of treatment. In two cases of HCC treated by ethanol injections, MRI on T2 weighted sequences showed a characteristic change from the previous hyperintense or isointense signal to a hypointense one. This MRI pattern, quite different from the usual HCC features, was correlated to the histologic findings of complete coagulative necrosis of the tumoural mass. Further observations are needed to assess reproducibility and specificity of this finding and the MRI pattern needs to be evaluated also in unsatisfactory percutaneous alcohol treatment of HCC in order to demonstrate that cases with persistent neoplastic tissue display a different pattern. If our report should be confirmed, MRI might be a not invasive tool in evaluating the effectiveness of PAI in patients at risk for multiple histologic samplings. Furthermore MRI could be very useful in assessing the degree and extent of tissutal changes in response to local therapy also after the tumour and its margin are masked by US guided ethanol injections.

Biopsy, Needle↗

PubMed1989

Staphylococcal alpha-toxin: a study of membrane penetration and pore formation.

Cell lysis by staphylococcal alpha-toxin, a potent virulence factor of most pathogenic strains of Staphylococcus aureus, follows a three-step sequence: binding of toxin to the membrane, leaking of ions caused by membrane injury, and rupturing of the membrane caused by osmotic swelling. The membrane injury step is composed of two separate events, membrane penetration and membrane perturbation. The membrane penetration event involves conversion of the soluble toxin monomer into an amphipathic molecule, which inserts into the lipid bilayer of the membrane. The membrane perturbation event involves association of the toxin monomers, in the plane of the membrane, to form hexameric transmembrane pores. In this study, we demonstrate that, in an asolectin liposome system, controlling the pH of the external buffer permits these two events to be temporally resolved. Using Controlled-Pore Glass bead-purified alpha-toxin, four events are measured as a function of pH: (a) release of potassium from prelabeled asolectin vesicles, (b) conversion of the toxin to a globally hydrophobic molecule, (c) binding of detergent by the toxin, and (d) labeling of the toxin with photoactivable, radiolabeled, hydrophobic probes. Two of these events, potassium release and conversion to a net hydrophobic state, are paired in that, for the event to occur, each requires a pH of 4.6 or less. In contrast, photolabeling with the membrane probes PC I and PC II (where PC represents phosphatidylcholine) is easily detectable at pH values as high as 5.0 and 6.0. These results demonstrate that, as the pH is lowered, two distinct changes in the physical properties of alpha-toxin occur. The first, which occurs under mild acidic conditions, converts the toxin from a water-soluble molecule into an amphipathic molecule. The second, requiring relatively more acidic conditions, converts the amphipathic toxin molecule into a globally hydrophobic molecule. Correlated with these physical changes in the alpha-toxin molecule is the acquisition of two new biological properties. The conversion of alpha-toxin into an amphipathic conformation correlates with the acquisition of the biological property of the reversible penetration into the bilayer of the asolectin liposome membrane, as evidenced by labeling with the photoactivable probes. At lower pH, the conversion of the toxin into a globally hydrophobic molecule correlates with the biological property of causing damage to the cell membrane, as measured by the release of internal potassium ions, presumably by the formation of transmembrane hexamer pores.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacterial Toxins↗

PubMed1989

Histidine 21 is at the NAD+ binding site of diphtheria toxin.

Treatment of fragment A chain of diphtheria toxin (DT-A) with diethylpyrocarbonate modifies His-21, the single histidine residue present in the chain, without alteration of other residues. Parallel to histidine modification, NAD+ binding and the NAD-glycohydrolase and ADP-ribosyltransferase activities of DT-A are lost. Both NAD+ and adenosine are very effective in protecting DT-A from histidine modification and in preserving its biological properties, while adenine is ineffective. Reversal of histidine modification with hydroxylamine restores both NAD+ binding and enzymatic activities of the toxin. The possible role of His-21 in the activity of diphtheria toxin is discussed in relation to the available three-dimensional structure of the related toxin produced by Pseudomonas aeruginosa.

Adenine↗

PubMed1988

On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells.

Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed.

Animals↗