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S Forti

Publications and source records attributed to S Forti.

25 records · Page 2Linked to original sources
PubMed1994

Quantitative growth fraction evaluation with MIB1 and Ki67 antibodies in breast carcinomas.

Ki67 and MIB1 monoclonal antibodies are directed against different epitopes of the same proliferation-related antigen. Whereas Ki67 works only on frozen sections, MIB1 may be used also on fixed sections. The authors immunostained a series of 40 breast carcinomas with MIB1 and Ki67 antibodies on serial frozen sections and on fixed material. The Ki67 labeling index (LI) was 12.9 +/- 8.9 and 12 (mean +/- SD and median, respectively). MIB1 LI was 21.2 +/- 11.9 and 19.5 on frozen sections and 24 +/- 15.2 and 21.5 on fixed sections (mean +/- SD and median, respectively). Ki67 LI and MIB1 LI on frozen and fixed sections were strictly correlated (P < .001). The results are in keeping with the reported coincidental nuclear staining pattern of Ki67 and MIB1, but the mean and median values of MIB1 LI are almost twice the values of Ki67 LI. The cut-off values to define high and low proliferative activity with the two antibodies are therefore different. The differences in immunolabeling may be due to better survival of the MIB1 epitope in freezing and acetone fixation or to differing accessibilities of the MIB1 and Ki67 epitopes during the cell cycle due to molecular conformational modifications. The MIB1 monoclonal antibody is a reasonable substitute for the Ki67 monoclonal antibody. The advantages of MIB1 immunostaining on paraffin sections include the feasibility of retrospective studies and of obtaining clear morphologic specimens that are optimal for use with computer-assisted image analysis systems. Our image-processing system allows automatic nuclear counting, detects positive nuclei and measures their staining intensity.

Adenocarcinoma↗
PubMed1991

Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.

ADP Ribose Transferases↗
PubMed1989

Staphylococcal alpha-toxin increases the permeability of lipid vesicles by cholesterol- and pH-dependent assembly of oligomeric channels.

alpha-Toxin, a lethal hemolytic toxin secreted by Staphylococcus aureus, forms ionic channels of large size in lipid membranes. To investigate the mechanism of channel assembly we have studied the kinetics of pore formation on small unilamellar vesicles. We have used two assays of vesicle permeabilization: one is the release of a fluorescent molecule trapped in their inner compartment; the other is the dissipation of an imposed potential. Both methods indicate that the kinetics are complex consisting of an initial delay followed by a non-linear relaxation. The dependence of the pore formation rate and the extent of permeabilization on the toxin/vesicle ratio indicates that aggregation of 4-10 preinserted toxin monomers underlies channel assembly. The pH dependence of permeabilization suggests that protonation of an acidic group of the toxin is a prerequisite to channel formation. Inclusion of cholesterol in the target vesicles potentiates alpha-toxin effects, in a dose-dependent way, possibly by facilitating its protonation. The location of the proton-binding site on the two adjacent aspartic acid residues in positions 127 and 128 of the toxin monomer is proposed.

Binding Sites↗
PubMed1989

Interaction of tetanus toxin with lipid vesicles. Effects of pH, surface charge, and transmembrane potential on the kinetics of channel formation.

We investigated the interaction of tetanus toxin with small unilamellar vesicles composed of different phospholipids as a function of pH, toxin concentration, temperature, and ionic strength of the solution. Tetanus toxin increased the permeability of the vesicles to fluorescent markers of molecular weight up to 700. The time course of the permeabilization was described as the sum of two exponential components of which the faster accounts for more than 70% of the total effect. Both time constants decreased when the pH of the solution was lowered and when vesicles contained negative lipids. These results can be explained in terms of a phenomenological model based on reaction rate theory. The model assumes that tetanus toxin, after equilibrating with the local pH existing at the surface of the vesicles, inserts into the lipid bilayer forming an ionic channel through which solutes can diffuse. Trigger event for the insertion of the toxin is the protonation, and consequent neutralization of one charged group which makes the molecule more hydrophobic. The intrinsic pK of this group was found to be 3.4 +/- 0.2, suggesting that it may be a carboxyl group. Since the toxin equilibrates with the local pH, the enhancing effect of acidic phospholipids is merely explained by the creation of a negative surface potential which increases the local proton concentration. This was confirmed by the inhibitory effect of high Na+ concentration which reduced the surface charge by screening and specific binding. We found still small differences between the lipids tested and the following order of sensitivity to the action of the toxin: phosphatidylinositol greater than phosphatidylserine greater than phosphatidylcholine approximately cholesterol. The activation energy for the two time constants was found to be 19.8 and 14.8 kcal/mol, fast and slow component, respectively, i.e., slightly larger than that for pure diffusion through the bilayer. The permeabilization induced by tetanus toxin is a voltage-dependent process because vesicles bearing an inner negative potential were depolarized very quickly whereas those bearing an inner positive voltage were barely depolarized at all.

Cholesterol↗
PubMed1989

Kinetics of phototransduction in retinal rods of the newt Triturus cristatus.

1. The kinetics of photoresponses to flashes and steps of light of rods, from the retina of the newt Triturus cristatus, were analysed by recording the membrane current with a suction electrode. 2. In dark-adapted conditions the relation between the normalized amplitude of the photoresponse at a fixed time 1 s after the onset of light and the light intensity could be fitted by an exponential or a polynomial relation. In the presence of a steady bright light the same relation could be fitted by a Michaelis-Menten relation. 3. The kinetics of photoresponses to light stimuli was reconstructed using a model in which: (i) three molecules of guanosine 3'.5'-cyclic monophosphate (cyclic GMP) open a light-sensitive channel; (ii) light activates the enzyme phosphodiesterase, which hydrolyses cyclic GMP, thus closing light-sensitive channels: (iii) Ca2+ ions permeate through light-sensitive channels: and (iv) intracellular Ca2+ inhibits, in a co-operative way, the enzyme cyclase, which synthesizes cyclic GMP. 4. The model reproduces the shortening of the time to peak of brief flash photoresponses from about 1080 ms to about 690 ms with brighter lights. The model also explains the shortening of the time to peak to 350 ms observed in the presence of a steady light and the lack of a further acceleration with brighter flashes of lights. 5. The presence in the model of an intracellular calcium buffer accounts for the partial reactivation of the photocurrent following a step of light, lasting several seconds. The time course of this reactivation is not accelerated by a steady bright light both experimentally and in the model. 6. After the extinction to a long step of light the photocurrent showed a rapid partial reactivation, which was followed by a slow component of the photoresponse which extinguished with a rate constant of about 0.05 s-1. The model explains the origin of this slow component by assuming that the inactivation of excited rhodopsin is partially reversible. 7. The model is also able to explain the particular changes of kinetics when different amounts of exogenous calcium buffers are incorporated into rods (Torre, Matthews & Lamb, 1986).

Animals↗