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.

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.

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.

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.

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.

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 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.

[Percutaneous treatment under echographic guidance of toxic thyroid nodules. Technique for its performance and preliminary results].

Six patients affected by toxic thyroid nodules (Plummer disease) were treated by percutaneous ethanol injection (PEI). Treatment was performed injecting under ultrasound guidance 2-4 mL of 95% sterile ethyl alcohol through a spinal needle (22 gauge, 75 mm length). Treatment was performed once-twice weekly and repeated as an outpatient procedure 4-6 times.

[Fine needle aspiration biopsy (FNAG) guided with tomodensitometry of pulmonary and mediastinal masses. Personal experience].

CT-guided fine-needle aspiration biopsy (FNAB) was performed on the patients with pulmonary or mediastinal masses to obtain material for cytologic/histologic diagnosis. Diagnostic accuracy and safety of the technique were evaluated in 75 patients affected with thoracic lesions still undiagnosed after thorough radiological and endoscopic investigations. The cytologic and/or microhistologic samples allowed a correct diagnosis to be made in 61 cases (81%), with no false positives and 7 false negatives (9%).

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.

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.

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.

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.

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.

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.

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.

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.

Circulating immune complexes in serum and in cerebrospinal fluid of patients with multiple sclerosis. Characterization and correlation with the clinical course.

We studied circulating immune complexes (IC) in the serum and cerebrospinal fluid (CSF) of patients with clinically defined multiple sclerosis (MS), in order to establish a correlation with the clinical course of the disease and to investigate the molecular composition of the IC isolated from patients in active phase of the disease. Serum IC levels were found to be significantly increased in patients from the progressive and active relapsing-remittent subgroups with both the CIC-conglutinin and C1q-binding methods. High levels of IC in CSF were detected only in the subgroup consisting of the relapsing-remittent patients in disease exacerbation when IC were determined by the C1q-binding test.

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 2. Hydrophobic photolabelling and cell intoxication.

The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 1. Liposome aggregation and fusion.

The interaction of diphtheria toxin and its cross-reacting mutants crm 45,228 and 1001 with small unilamellar vesicles has been followed by a turbidity assay, electron microscopy, fluorescence energy transfer and membrane permeability. All toxins at pH lower than 6 induce the aggregation and fusion of liposomes containing negatively charged phospholipids; crm 45 and crm 1001 are less potent than diphtheria toxin. Isolated diphtheria toxin fragment B is very effective while isolated fragment A is ineffective.

Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids.

The interaction of diphtheria toxin and its enzymatically deficient mutants crm 176 and crm 197 with liposomes has been studied by turbidity measurement and hydrophobic photolabelling with photoactivatable phosphatidylcholines. Diphtheria toxin and crm 176 at neutral pH bind to the surface of lipid bilayers while crm 197 also appears to interact with the fatty acid chains of phospholipids. All proteins undergo a change in conformation over the same range of acidic pH and become able to insert in the lipid bilayer.

Presence of cytochrome b-245 in NADPH oxidase preparations from human neutrophils.

The composition of NADPH oxidase purified by Red Sepharose chromatography of extracts from human neutrophil membranes was investigated. In contrast to that was recently reported by others, the enzyme isolated according to this procedure contained a high concentration of cytochrome b-245 and little FAD. The results reinforce the belief that cytochrome b-245 is a major component of the NADPH oxidase and plays a fundamental role in the formation of O2-by neutrophils.

Respiratory response of phagocytes: terminal NADPH oxidase and the mechanisms of its activation.

The chemical composition, properties and activation mechanism of the O2(-)-forming NADPH oxidase of phagocytes were investigated, using partially purified enzyme preparations. Highly active NADPH oxidase was extracted as an aggregate of high Mr from the membranes of neutrophils and macrophages. The enzyme complex contained phospholipids and cytochrome b-245, very little FAD and almost no quinones or NAD(P)H-dye reductase activity.