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Biomedical subjects

C Montecucco

Publications and source records attributed to C Montecucco.

At least 181 records · Page 10Linked to original sources
PubMed1992

The role of the intercellular matrix in dermal calcinosis of the CRST syndrome. An electron-microscopic study.

We performed an electron-microscopic study on calcium deposits in two patients with calcifying scleroderma (CRST syndrome). Calcium deposits were detected both intracellularly in the mitochondria of phagocytic cells and extracellularly. Needle-like crystals measuring up to 4,500 A in length and approximately 60 A in width were present in both the Von Kossa-positive regions and the apparently normal dermal areas around the calcification sites. Although the fibrillar matrix's ultrastructure was normal, hollow oxytalan fibrils were detected. Slightly electron-opaque, star-shaped material was observed among the fibrillar component of the matrix (matrix granules), which is the ultrastructural expression of some types of proteoglycans containing keratan sulphate and chondroitin sulphate. These granules cannot be detected in normal dermis. The extrafibrillary calcium deposits on these mucopolysaccharide structures may represent an early event in the complex pathogenesis of calcification in the CRST syndrome.

Actin Cytoskeleton↗
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↗
PubMed1992

Antiphospholipid antibody associated thrombosis in juvenile chronic arthritis.

A child with systemic onset juvenile chronic arthritis (JCA) who developed a bilateral femuropopliteal vein thrombosis after plaster immobilisation following a tibial fracture is described. When the thrombosis was diagnosed, antiphospholipid antibodies detected either as lupus anticoagulant and anticardiolipin antibodies were found. This suggests that short term prophylatic antithrombotic treatment should be considered in antiphospholipid antibody positive JCA patients who require immobilisation after fractures of demineralised bones.

Antibodies, Antiphospholipid↗
PubMed1992

Sex hormones and bone metabolism in postmenopausal rheumatoid arthritis treated with two different glucocorticoids.

To investigate the effect of low doses of 2 different glucocorticoids on bone mass, sex hormone status and bone metabolic indices, a study was undertaken in 16 postmenopausal women with rheumatoid arthritis (RA) receiving < 15 mg/day of deflazacort and in 16 patients with RA matched for age, years postmenopause and disease duration, receiving < 10 mg/day of prednisone. Sixteen healthy postmenopausal women and 16 nonsteroid treated patients with RA were also studied as control groups. Vertebral bone density (vBMD) was lower (mean +/- SD: 0.65 +/- 0.07 vs 0.73 +/- 0.09 g/cm2; p < 0.02) in prednisone treated patients than in deflazacort treated patients, whose vBMD values were similar to those of nonsteroid treated RA. No significant difference was found as for radial bone mineral content. Circulating levels of estradiol, dehydroepiandrosterone sulfate, androstenedione and progesterone were low in all patient groups with RA when compared with healthy controls. The prednisone treated patients showed significantly lower values of all sex hormones with respect to deflazacort treated patients. Osteocalcin values were also lower (3.0 +/- 1.4 vs 3.9 +/- 1.6 ng/ml; p < 0.05) in prednisone treated patients with respect to deflazacort treated group. Glucocorticoid treated patients showed a direct correlation (r2 = 0.39) between vBMD and plasma estradiol levels, while no correlation was found with osteocalcin values. In conclusion, our postmenopausal patients with RA treated with low dose prednisone had reduced levels of sex hormones and osteocalcin and reduced vertebral bone mass. Comparable doses of deflazacort showed only a mild inhibitory effect on sex hormones and osteocalcin, and did not show any detectable effect on bone mass.

Adult↗
PubMed1991

Tetanus toxin receptor. Specific cross-linking of tetanus toxin to a protein of NGF-differentiated PC 12 cells.

A subclone of rat pheochromocytoma cells expresses high affinity receptors for tetanus toxin on differentiation with NGF [Walton, K.M., Sandberg, K., Rogers, T.B. and Schnaar, R.L. (1988) J. Biol. Chem. 263, 2055-2063]. In the presence of protein cross-linking agents, [125I]tetanus toxin, bound to these cells at 0 degree C, forms a cross-linked product with apparent molecular weight of 120 kDa. The formation of [125I]tetanus toxin conjugate involves the heavy chain of the toxin, is prevented by cold toxin and it is largely reduced by pretreating cells with proteases. The cross-linked product is formed only upon incubation of the toxin with NGF-differentiated cells. These results suggest that a protein with apparent molecular weight of 20 kDa is involved in the neurospecific binding of tetanus toxin.

Animals↗
PubMed1991

On the role of polysialoglycosphingolipids as tetanus toxin receptors. A study with lipid monolayers.

Lipid monolayers of different compositions were used to study the interaction of tetanus toxin with membrane lipids and to evaluate the role of polysialoglycosphingolipids as membrane receptors. At neutral pH, the toxin binds to dioleoylglycerophosphocholine monolayers and inserts into the phospholipid layer. This effect is potentiated by acidic phospholipids without an apparent preference for a single class of phospholipids. Polysialoglycosphingolipids further increase the fixation and penetration of tetanus toxin in lipid monolayers, but no specific requirement for a particular ganglioside was identified. The ganglioside effect is abolished in the presence of other nervous tissue lipids: cerebrosides and glycosphingolipid sulfates are partially responsible for this effect. The penetration of tetanus toxin in the lipid monolayer is pH dependent. It increases with lowering pH, it is facilitated by acidic phospholipids and by glycosphingolipid sulfates and it is mediated both by hydrophobic and electrostatic interactions as deduced from an analysis of the effect of ionic strength. Fragment B of tetanus toxin the low-pH-driven lipid interaction of the toxin. On the basis of the present findings, the possible role of polysialoglycosphingolipids in the neurospecific binding of tetanus toxin is discussed.

Gangliosides↗
PubMed1991

Lipid interaction of diphtheria toxin and mutants. A study with phospholipid and protein monolayers.

To study the structural change of diphtheria toxin (DT) induced by low pH and its influence on the interaction with membrane lipids, protein and lipid monolayers were formed and characterized. DT at neutral and acidic pH forms stable monolayers, whose surface-pressure-increase curves allow an estimation of the apparent molecular area of 29.5 nm2/molecule at pH 7.4 (corresponding to a radius of 3.06 nm) and 34.5 nm2/molecule at pH 5.0 (corresponding to a radius of 3.32 nm). DT at pH 7.4 does not insert into phospholipid monolayers, while at pH 5.0 it penetrates into the lipid layer with a portion of apparent molecular area of 21.0 nm2/molecule (corresponding to a radius of 2.6 nm). The low-pH driven lipid interaction of the toxin is favoured by the presence of acidic phospholipids, without an apparent requirement for a particular class of negative lipids. The DT mutants crm 45 and crm 197 are capable of hydrophobic interaction already at neutral pH and cause an increase of surface pressure with a further increase upon acidification.

Diphtheria Toxin↗
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

Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding.

Five ADP-ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site-directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three-dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.

ADP Ribose Transferases↗
PubMed1991

Prevalence of anticardiolipin antibodies in juvenile chronic arthritis.

The prevalence of anticardiolipin antibodies was evaluated in 70 children with juvenile chronic arthritis (JCA), in 25 adult patients with rheumatoid arthritis, in 42 healthy children and in 40 adult controls. Thirty seven (53%) patients with JCA were positive for IgG or IgM anticardiolipin antibodies, or both, and 30 (43%) for IgG anticardiolipin antibodies. In contrast, only seven (28%) adult patients with rheumatoid arthritis presented anticardiolipin antibodies, which were of IgG class in four (16%) cases. IgG anticardiolipin antibodies were negative in all control subjects while IgM anticardiolipin antibodies were detected in two (5%) children and in four (10%) adult controls. No correlations were found in patients with JCA between the presence or titres of anticardiolipin antibodies and various clinical or laboratory variables. No patient with anticardiolipin antibodies showed any feature of the anticardiolipin syndrome.

Adolescent↗
PubMed1990

Antibodies from patients with rheumatoid arthritis and systemic lupus erythematosus recognize different epitopes of a single heterogeneous nuclear RNP core protein. Possible role of cross-reacting antikeratin antibodies.

Antibodies to recombinant heterogeneous nuclear RNP core protein A1 were detected in sera from 27 of 58 patients with rheumatoid arthritis (RA) and from 7 of 31 patients with systemic lupus erythematosus, by immunoblotting and enzyme-linked immunosorbent assay. Protein A1 consists of 2 distinct domains: The N-terminal sequence is identical to a single-stranded DNA binding protein termed UP1, and the C-terminal domain shows a partial homology with keratin. All 7 A1-positive systemic lupus erythematosus sera reacted with UP1, whereas 9 of the 27 A1-positive RA sera did not. In RA, anti-A1 activity was significantly associated with antikeratin antibodies (AKA); these antibodies were present in 23 of 27 A1-positive sera and 10 of 31 A1-negative sera (P less than 0.01). Immunoabsorption with recombinant protein A1 resulted in a significant reduction of AKA titers in 6 of 10 RA sera tested, suggesting that AKA from RA patients may cross-react with the C-terminal portion of the heterogeneous nuclear RNP protein A1.

Adolescent↗
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↗