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Biomedical subjects

E Papini

Publications and source records attributed to E Papini.

At least 91 records · Page 5Linked to original sources
PubMed1988

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. No significant increase in serum or in CSF were found using the mRF-I test. The preliminary results of a qualitative investigation on serum IC in MS indicated that they are heterogeneous in nature, their size is mainly of the intermediate type, and they contain IgG, IgM, complement components and beta 2-microglobulins, the latter presenting an observation both new and interesting for studies on serum IC in MS patients.

Adolescent↗
PubMed1987

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. Liposome fusion induced by the toxins at low pH occurs without release of the internal content implying that fusion does not involve vesicle breakage and resealing. The pH dependence of the membrane interaction of diphtheria toxin monitored by turbidity is in close agreement with that monitored by fluorescence energy transfer. It shows that diphtheria toxin can alter the lipid bilayer structure in the pH interval 5-6. This pH range occurs in endosomes and suggests that histidyl and carboxyl residues are likely to be involved in the conformational change of diphtheria toxin triggered by acidic pH.

Diphtheria Toxin↗
PubMed1987

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 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half-maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate-controlling step in the process of membrane translocation of diphtheria toxin. crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid-treated cells, suggesting that the 12-kDa COOH-terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin. It is suggested that crm 1001 is non-toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.

Animals↗
PubMed1987

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. The tighter lipid interaction of crm 197 may account for its higher cell association constant. The possibility is discussed that the binding of diphtheria toxin to cells is mediated by both a protein receptor and an interaction with the head group of phospholipids.

Diphtheria Toxin↗
PubMed1986

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.

Cell Membrane↗
PubMed1986

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. The purification of a polypeptide with a relative molecular mass of 31 500 strictly paralleled the purification of NADPH oxidase, suggesting that this polypeptide is a component of the enzyme. This protein was identified as cytochrome b -245 after dissociation of the proteolipid complex and purification of the cytochrome moiety. The 31 500 Mr protein was phosphorylated in enzyme preparations from activated but not from resting cells. The results indicate that: cytochrome b-245 is a major component of NADPH oxidase; the involvement of NAD(P)H dye reductases in the O2(-)-forming activity is questionable; the cytochrome b-245: FAD ratio in the enzyme complex is much higher than that indicated in crude preparations; the Mr of pig neutrophil cytochrome b-245 is 31 500; the activation of the O-2-forming system involves a process of phosphorylation of cytochrome b-245.

Animals↗
PubMed1985

Partial purification of the superoxide-generating system of macrophages. Possible association of the NADPH oxidase activity with a low-potential (-247 mV) cytochrome b.

NADPH oxidase activity was solubilized by detergent treatment of subcellular particles obtained from guinea-pig peritoneal macrophages stimulated with phorbol myristate acetate. Gel filtration of the material containing the NADPH oxidase activity gave two peaks of proteins, one of which eluted with the void and the other with the included volume of an AcA 22 column. The material eluted in the void volume contained more than 50% of the NADPH oxidase activity and less than 10% of the NAD(P)H cytochrome c reductase activity. A b-type cytochrome with peaks of absorption at 558, 528 and 426 nm was also enriched in the fraction which contained the NADPH oxidase activity. The distribution of flavoproteins as revealed by the measurement of FAD was different from that of NADPH oxidase and cytochrome b, and followed the elution profile of NADH cytochrome c reductase. Studies in subcellular particles showed that the b cytochromes of mitochondria and endoplasmic reticulum reduced by selective biochemical means accounted for only a minor part of the total b-type cytochromes and that the new cytochrome b previously described in neutrophils is the major chromophore also in macrophages. Oxidation-reduction midpoint potential of the partially purified cytochrome b was shown to be -247 mV. Association of cytochrome b with the NADPH oxidase activity and its very low Em7.0 makes it a suitable candidate to be part of the superoxide-generating system also in macrophages.

Animals↗
PubMed1985

Protein kinase C phosphorylates a component of NADPH oxidase of neutrophils.

A protein of 31.5 kDa belonging to the NADPH oxidase of neutrophils was phosphorylated following stimulation of the cells with phorbol myristate acetate. The same protein was phosphorylated in vitro in the presence of cytosol and of Ca2+ and phosphatidylserine. The phosphorylation in vitro of the 31.5 kDa protein was increased by phorbol myristate acetate and was inhibited by trifluoperazine. The data are compatible with an involvement of protein kinase C in the activation of NADPH oxidase.

Animals↗
PubMed1985

Independence with respect to Ca2+ changes of the neutrophil respiratory and secretory response to exogenous phospholipase C and possible involvement of diacylglycerol and protein kinase C.

Exogenous phospholipase C induces in human neutrophils the activation of a respiratory burst, measured as O2 consumption and O-2 production, and of secretion of specific granules, measured as release of vitamin B-12 binding protein. The secretory response is minimal and follows the onset of the respiratory response. Studies carried out using cells prelabeled with [3H]glycerol and 32P on the molecular mechanism of the stimulations demonstrate that the effects are dependent on the formation of diacylglycerol by hydrolysis of different classes of glycerophospholipids. They are, however, independent of the activation of a 'phosphoinositide turnover' as occurs in cells stimulated with fMet-Leu-Phe. Furthermore, the respiratory and secretory responses to exogenous phospholipase C are not associated with modifications of cytosolic Ca2+ concentration, measured with the Quin-2 method, and of the release of bound Ca2+, measured with the membrane probe, chlorotetracycline. Apart from a quantitative difference, mostly regarding the ratio of the intensity of the respiratory and secretory responses, the effects caused by exogenous phospholipase C are qualitatively similar to those induced by phorbol myristate acetate and are probably linked to an involvement of protein kinase C, activated by diacylglycerol liberated in the plasma membrane.

Calcium↗
PubMed1985

Mechanism of production of toxic oxygen radicals by granulocytes and macrophages and their function in the inflammatory process.

The paper deals with 1) the features of the respiratory burst (increase of the respiration with production of O2 metabolites, O2-, H2O2, OH) of the inflammatory cells; 2) the factors responsible for its activation; 3) the methods for its measurement; 4) the molecular events which take place at the level of the plasma membrane following the interaction between the stimuli and the cell surface (the Ca++ changes, the modification of membrane potential, the activation of phospholipid turnover) and the hypothesis of the activation of the protein kinase C; 5) the nature of the NADPH oxidase whose activation is responsible for the respiratory burst and the production of O2 metabolites; 6) the defensive, toxic, proinflammatory and modulatory effects due to the reactivity of the oxygen metabolites.

Animals↗
PubMed1985

Studies on the nature and activation of O2(-)-forming NADPH oxidase of leukocytes. Identification of a phosphorylated component of the active enzyme.

Highly active superoxide (O2-)-forming NADPH oxidase was extracted from plasmamembranes of phorbol-12-myristate-13-acetate-activated pig neutrophils and was partially purified by gel filtration chromatography. Oxidase activity copurified with cytochrome b-245 in an aggregate containing phospholipids and was almost completely separated from FAD and NAD(P)H-cytochrome c reductase. A polypeptide with molecular weight of 31,500 strictly paralleled the purification of NADPH oxidase, suggesting that it is a major component of the enzyme. The enzyme complex was then dissociated by high detergent and salt concentration and cytochrome b-245 was isolated by a further gel filtration chromatography, with a 147 fold purification with respect to the initial preparation. The cytochrome b-245 showed a 31,500 molecular weight by SDS electrophoresis, indicating that it is actually the component previously identified in the partially purified enzyme. The 31,500 protein was phosphorylated in enzyme preparations from activated but not from resting neutrophils, suggesting that phosphorylation of cytochrome b-245 is involved in the activation mechanism of the O2(-) -forming enzyme responsible for the respiratory burst in phagocytes.

Animals↗
PubMed1984

Composition of partially purified NADPH oxidase from pig neutrophils.

The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.

Animals↗
PubMed1984

NADPH oxidase of neutrophils forms superoxide anion but does not reduce cytochrome c and dichlorophenolindophenol.

Superoxide (O-2) production by partially purified NADPH oxidase from guinea pig neutrophils was markedly increased when the cells were activated by exposure to phorbol-myristate acetate. On the contrary, NADPH-dependent cytochrome c and 2,6-dichlorophenolindophenol (DCIP) reductase activities in preparations from resting and activated neutrophils were similar. The apparent Km values for NADH and NADPH of the reductase activities were different from those of the O-2 producing enzyme. The electron acceptors did not inhibit the oxygen consumption by NADPH oxidase in the presence of superoxide dismutase. Even in anaerobiosis the oxidase failed to reduce cytochrome c and DCIP. These results suggest that NAD(P)H-dependent dye reductase activities are not involved in the electron transport system responsible for the O-2 production by neutrophils.

2,6-Dichloroindophenol↗
PubMed1983

IgG rheumatoid factors behaviour in young normal subjects following vaccination.

In order to contribute to explain the finding of Rheumatoid Factors of the IgG class in all normal subjects, even at low levels, a study on their behaviour in a group of young subjects serving in the army, at the time of anti-typhoid and antitetanic vaccination, and in the following two months, was conducted. Parallely some aspecific serological parameters of autoimmunity were determined. The results demonstrate, first of all, a good antibody response to the vaccine in all subjects observed. Immunoglobulins of the IgG, IgA, and IgM class were normal in all samples. Complement factors C3 and C4 were slightly decreased after the second dose of vaccine, suggesting a modest consumption probably due to immune complexes formation. Factor C3A showed a slight increase, may be to be considered as its property of an acute phase reactant. Autoantibodies determined by immunofluorescence were constantly negative. IgM rheumatoid factors became transitory positive in a significant proportion. IgG rheumatoid factors demonstrated a two-phase increase, more evident after the second dose of vaccine. We can conclude, mainly for the latest parameter, that these results reveal an autoimmune serological response, even quantitatively modest, concomitant to the specific sensitization resulting from vaccination.

Adult↗
PubMedDate not supplied

[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. PEI induced clinical improvement and hormonal control by the end of the treatment (one month) and no complications took place. After PEI all hyperfunctioning thyroid nodules became smaller at clinical and ultrasound examination. Three months after PEI hot areas appeared cold at 99mTc and 1311 scintiscan. Serum levels of FT3, FT4, AbT, AbM, TG reached the normal range and serum TSH levels were significantly increased and responsive to TRH stimulation.

Adult↗