Search PubMed⌕ Search

Biomedical subjects

G Bottiroli

Publications and source records attributed to G Bottiroli.

At least 55 records · Page 3Linked to original sources
PubMed1990

Equilibrium among hematoporphyrin derivative components--II. Effect of esterase activity.

Photofrin II is the hematoporphyrin-derivative fraction enriched in covalently-linked oligomers, characterized by a high degree of folding. Interaction with hydrophobic structures, such as biomolecules and cell structures, results in a modification of the equilibria among the different species, as a consequence of an unfolding effect exerted towards the electrostatic aggregates. The effect of esterase activity was evaluated, taking into account the nature suggested for the covalent linkage of the oligomers (ether and/or ester). The study was performed in Photofrin II aqueous solution by means of absorption and fluorescence spectral analysis. The results showed that the esterase is active only towards the unfold oligomers: that is, in Photofrin II solution supplemented with albumin. In these conditions, spectral analysis revealed the presence of a monomerization process, which is clearly evident during the first four hours of incubation. The monomerization effect induced by the enzyme was also proven by both equilibrium-dialysis measurements and zinc ion complexation. Zinc ion complexes with high affinity for monomeric species, giving rise to a very distinct emission band at 580 nm. The amount of ester linkage shown in the oligomers through enzyme hydrolysis appeared to be less than might have been expected, owing to the inhibiting effect of the monomer produced on the enzyme. The results are a step toward clarifying the intracellular and intratissue turnover of the drug observed after administration.

Esterases↗
PubMed1989

Flow cytometric evaluation of DNA stainability with propidium iodide after histone H1 extraction.

A flow cytometric evaluation of the effect of the histone H1 extraction on DNA stainability with propidium iodide was performed on isolated HeLa nuclei. Selective removal of the lysine-rich protein was attained by using two established techniques involving treatment with 0.7 M NaCl or low pH. DNA stainability was monitored at different dye/DNA-P ratios, varying from low to high saturating concentrations. Depletion of the histone H1from nuclei results in the transition from low to high affinity of a portion of binding sites, as shown by 1) the increase in fluorescence intensity after staining with the dye at low saturating concentrations and 2) the higher value of the fluorescence intensity ratio (FI5/FI50) exhibited by H1-depleted nuclei stained with a low (5 micrograms/ml) vs. a high (50 micrograms/ml) concentration, as compared with control samples.

Cell Nucleus↗
PubMed1989

DNA double staining for a fluorescence energy transfer study of chromatin in liver cells.

Methodological aspects related to the application of techniques based on fluorescence energy transfer in the study of chromatin structure, were first examined. Fluorochromes specific for DNA with different interaction mechanisms were employed, both in single and double stainings. The following dye pairs were considered as donor/acceptor couples: Hoechst 33342 or DAPI/Mithramycin A or Chromomycin A3, Hoechst 33342 or DAPI/Propidium Iodide, and Mithramycin A or Chromomycin A3/Propidium Iodide. Spectrofluorometric analysis showed that the spectral distribution of the dye pair Ho/PI is more suitable for the evaluation of energy transfer efficiency. This dye pair was used in the study of the chromatin microstructure in rat hepatocytes isolated from livers at two different growth stages. In particular, diploid mono- and binucleated cells from young and adult rats were considered. The results indicated the existence of a more homogeneous situation in young than in adult rats. In the latter case, the statistical analysis indicates the presence of two groups of energy transfer values. The different efficiency values in energy transfer can be considered a consequence of chromatin structure rearrangement and are tentatively interpreted according to the functional role of the diploid cells in the two stages of liver growth.

Animals↗
PubMed1988

Limitations of the quantitative cytochemical assay of catechol oxidase in melanoma cells.

The cytochemical quantification of catechol oxidase activity in fixed B16 melanoma cells was investigated using dopa as the substrate. Inhibitors showed that peroxidases do not significantly interfere. The kinetics of melanin formation were studied initially in solution with purified catechol oxidase. Two key parameters were identified: lag-time and the rate of melanin formation. The lag-time was taken as the time required by intermediates to reach a critical concentration at which the polymerization process starts and melanin production becomes measurable (at 640 nm). In solution, the lag-time decreases as the enzyme activity increases, particularly when the activity is very low. The rate at which melanin is formed by pure enzyme in solution is independent of dopa concentration when its activity is low but increases linearly with dopa concentration when the activity is comparatively high. In fixed melanoma cells, the lag-time decreases linearly with increases of dopa concentrations up to 20 mM; at concentrations higher than this, the lag decreases more slowly. In contrast, the rate of melanin production is unaffected by changes in dopa concentration. The lag-times of different cells lines incubated at the same substrate concentration decrease as the enzyme activity of the cells increases. The rate of melanin production seems to be affected by factors other than catechol oxidase activity, such as the intracellular organization and distribution of the enzyme.

Catechol Oxidase↗
PubMed1987

Topical administration of tetrasodium-meso-tetraphenyl-porphinesulfonate (TPPS): correlation between drug penetration and depth of necrosis in skin of nude mice following red light irradiation.

The main side effect in photodynamic therapy is photosensitization of the patient's skin following systemic administration of the photosensitizing agent. In the case of superficial lesions, this problem can be avoided by topically applying the drug: in this way a local treatment can be performed. We tested the photosensitizing properties of a 2% solution of TPPS (tetrasodium-tetraphenylporphinesulfonate) in a vehicle containing a penetration enhancer, Azone, on skin of nude mice. An aliquot of 0.1 ml/cm2 of the solution was painted on the skin overlying an s.c. implanted NMU-1 tumor. Subsequently, animals were sacrificed at different times after application. Fluorescence microscopy revealed that TPPS penetration depth was related to time elapsed after application and to painting modalities. Solution penetration was enhanced by wiping with ether immediately before painting. Irradiation at 80 mW/cm2 for 20 min with a dye laser emitting at 640 nm, 4 h after TPPS application, produced necrosis of the upper skin layers, up to 0.2 mm in depth. These findings suggest that topical TPPS administration, followed by laser irradiation, may be a suitable treatment modality for skin lesions involving epithelial layers, even though several aspects of this metodology need further investigation.

Adenocarcinoma↗
PubMed1987

Influence of cis-dichlorodiamineplatinum on glioma cell morphology and cell cycle kinetics in tissue culture.

C6 glioma cells (CCL 107) were cultured for three days and then treated with cis-dichlorodiamineplatinum (cis-DDP) at doses of 0.2-10 micrograms/ml medium. Changes in DNA synthesis and DNA content, as well as morphology of cells and chromatin distribution, were examined from the first post-treatment day onwards. The number of cells labelled with [3H]thymidine, detected autoradiographically, decreased after treatment with 0.2-10 micrograms/ml by approximately one half on post-treatment day 1 and diminished further by the third day after treatment. The labelled cells were entirely absent only after treatment with 10 micrograms/ml, 7 days post-treatment. Mitoses decreased from 1.4-0.6% by post-treatment day 1 and completely disappeared by day 3 (1 microgram/ml). Feulgen cytophotometry and propidium iodide cytofluorimetry revealed accumulation of cells in the S-phase, especially the latter part (0.5 and 1.0 micrograms/ml, post-treatment day 1) and subsequently also in G2 phase (post-treatment day 3). Incomplete cyto- and karyokinesis in some cycling cells was indicated by an increased number of binucleate cells and nuclei of higher ploidy classes. Labelled cells with intermediate DNA values were, on average, labelled less intensively, as was revealed by simultaneous measurements of DNA content and [3H]thymidine incorporation. Some cells displayed reduction in grain density over heterochromatin clumps. This would be in agreement with the late S-phase block of DNA replication. After post-treatment day 3 the density of cells in cultures was substantially lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Flow cytometric evaluation of DNA digestion with S1 nuclease.

The digestion of DNA with the S1 nuclease has been investigated by flow cytometry on two cultured cell lines and on splenic lymphocytes. The nucleolytic activity of the enzyme has been determined by measuring the decrease in fluorescence intensity of DNA stained with Propidium Iodide (PI). The reaction kinetics could be estimated through the shift of the G1 peak, since the shape of the DNA histograms obtained from cell submitted to S1 digestion did not show any particular variation as compared with the histograms of undigested control cells. DNA digestion was found to be more pronounced when cells were previously damaged with agents known to induce strand breaks, such as gamma-rays or Adriamycin. The assessment of DNA sensitivity to S1 nuclease with flow cytometry may thus provide a fast and reliable procedure for evaluating chromatin structural changes, both in physiological and pathological conditions.

Animals↗
PubMed1987

Organization of membrane constituents in human erythrocytes of different age.

The effect on the structural integrity of membrane glycoproteins of different methods for the isolation of human red cell subpopulations has been investigated. Moreover the extent of sialylation of membrane glycoconjugates in red cells of different age has been studied by a quantification of WGA binding sites at the single-cell level by cytofluorometric techniques, using the FITC-labeled lectin; the surface distribution of these sites has also been described by recording fluorescence intensity maps of cell surfaces.

Binding Sites↗
PubMed1986

Flow cytometric analysis of membrane permeability properties influencing intracellular accumulation and efflux of fluorescein.

A flow cytometric investigation has been made on the membrane permeability properties that mediate intracellular turnover of fluorogenic substrates. The accumulation and efflux of fluorescein, consequent to the enzymatic turnover of fluorescein diacetate, were assessed in the presence of metabolic inhibitors and after treatment with membrane-active compounds. The metabolic poisons KCN and rotenone greatly inhibited only the fluorescein efflux, reducing the rate constant to as little as one-tenth in relation to control cells; in the presence of glucose such inhibition was partially removed. Glucose availability also affected fluorescein efflux: an increase of the rate constant was observed in cells treated with 20 mM glucose, and a decrease was measured in cells incubated for 1 hr in glucose-free buffer. Membrane-active compounds Triton X-100 and hydrocortisone reduced fluorescein accumulation. Hydrocortisone strongly blocked also the efflux; the addition of glucose did not restore the rate significantly. The major evidence of these results is that fluorescein efflux is dependent on membrane integrity and on availability of metabolic energy. Fluorescein accumulation is only partially related to permeability properties regulating FDA uptake, due to the influence that treatments exhibit at the same time on FDA hydrolysis and/or fluorescein release.

Biological Transport↗
PubMed1986

Cytochemical evaluation of sperm and lymphocyte DNA content after treatment with 5 N HCl.

In situ as well as extra situm cytochemical methods were used to investigate why the observed Feulgen-DNA value of sperm versus lymphocyte cells is lower than expected. After treatment with 5 N HCl, in situ experiments involving the GCA reaction and the UV cytophotometry showed the loss of DNA in sperm nuclei to be 12% more than that in lymphocyte nuclei. Extra situm study of sperm and lymphocytes treated with 5 N HCl showed the phosphate and DABA contents of sperm to be 35% and 23%, respectively, less than those of lymphocytes. The data suggest that sperm chromatin is much more sensitive than somatic chromatin to HCl depolymerization during the Feulgen reaction, and this can tentatively be attributed to the protein complement of sperm chromatin.

Animals↗
PubMed1986

Effects of hematoporphyrin-derivative on mouse erythroleukemia cells in the absence of light irradiation.

This paper concerns a general study on the effects of hematoporphyrin-derivative (HpD) on mouse erythroleukemia (MEL) cells, in the absence of light irradiation. In particular, HpD intrinsic cytotoxicity was evaluated at different doses and the results correlated with those referring to membrane functional and morphological changes. HpD uptake and release processes were also studied and compared with the above-mentioned results. In order to have an overall picture of HpD-cell interactions, time-resolved fluorescence measurements were performed on both undifferentiated and differentiated MEL cells. The results obtained indicate that, even at HpD doses exhibiting neither any cytotoxicity nor any morphological damage (1-10 micrograms/ml), membrane permeability alterations are observed. Thirty minutes of treatment are sufficient for HpD to develop its toxic effect: indeed, no differences in HpD influence on cell viability can be observed after 30 min, 60 min or 5 days of treatment. HpD cytotoxicity is reduced by high protein content in the incubation culture medium. The presence of both monomeric species and 580 nm emitting species was observed at cellular level. The latter is likelier in undifferentiated MEL cells, which also exhibit higher overall HpD uptake, as compared with differentiated MEL cells.

Animals↗
PubMed1986

Anthracycline-induced inhibition of membrane permeability functions dependent on metabolic energy.

The influence of anthracyclines on membrane permeability functions has been investigated in HeLa cells by monitoring the efflux of fluorescein. Release of the fluorescent dye, dependent on the metabolic energy supply, occurs after the intracellular accumulation and enzymatic hydrolysis of the non-fluorescent substrate fluorescein diacetate (FDA). Flow cytometric evaluation of the efflux kinetics showed that adriamycin (ADR), N-trifluoroacetyladriamycin-14-valerate (AD-32) and daunorubicin (DNR) inhibited the permeability process. The degree of inhibition was dependent, though to different extent, on the intracellular concentration of each drug. An increase in the efflux rate was always observed when the cells were treated with the drugs in the presence of 20 mM glucose. Relationship of these effects with energetic metabolism was supported by the finding that ATP levels were lowered by the drugs and increased by glucose. Evaluation of the cytotoxicity induced by each drug showed that the intracellular amount necessary to inhibit cell survival by 50% was of the same order of magnitude as that which decreases to 50% membrane permeability to fluorescein. These results indicate a correspondence in the concentrations of anthracyclines required for inducing cytotoxicity and for inhibiting membrane permeability functions dependent on the metabolic energy supply.

Adenosine Triphosphate↗
PubMed1985

Internalization of cycloheptaamylose-dansyl chloride complex during labelling of surface membrane in living Paramecium aurelia cells.

Internalization of cycloheptaamylose-dansyl chloride complex during surface labelling of living long-term starved Paramecium aurelia cells has been observed. This process may be inhibited by pretreatment of the ciliates with dichloroisoproterenol. Uptake of cycloheptaamylose-dansyl chloride may be visualized only after UV preirradiation: the appearance of orange-fluorescing vacuoles of diameter 2.3-4.5 micron may then be observed. Microspectrographic analysis performed on the cells and dansyl derivatives indicates that this fluorescence is produced by a photochemical reaction of dansyl chloride - released from CDC complex inside the digestive vacuoles-under the influence of UV irradiation.

Animals↗
PubMed1984

Primary fluorescence of rat muscle after CO2 laser thermal injury.

Observations are reported indicating that CO2 laser radiation induces the appearance of strong primary fluorescence in the tissues surrounding the incidence zone of the beam. The characteristics of this emission were studied by means of cytofluorometric techniques on rat muscles and compared with the fluorescence obtained by a heating procedure or by storage of tissue slices. Laser-induced primary fluorescence was found to be related to the thermal effect of CO2 laser radiation. Moreover, evidence is provided that the processes occurring during heating and those occurring during tissue decomposition most probably produce the same fluorescent products, albeit with different rates. From the results obtained, it is suggested that primary fluorescence can be used as a marker of the damage suffered by the irradiated tissues.

Aging↗
PubMed1984

In situ evaluation of the functional state of chromatin by means of Quinacrine Mustard staining and time-resolved fluorescence microscopy.

A pulsed laser microfluorometer with high spatial and temporal resolution was employed to study the functional state of chromatin, using Quinacrine Mustard as the fluorescent probe. Corresponding segments of polytene chromosomes of embryo suspensor cells of Phaseolus coccineus and parenchymal cells of Helianthus tuberosus, in phases not involving DNA synthesis, were selected as models. The fluorescence decay time turned out to be a discriminating parameter for the chromatin fractions of differing functional engagement. The results were interpreted on the basis of a different accessibility of the DNA to the intercalating agent, as a result of the different structural situation of the chromatin. This fact determines modifications of the energy transfer rates between dye molecules in different quantum efficiency conditions, which results in variations of fluorescence decay times.

Chromatin↗