Search PubMed⌕ Search

Biomedical subjects

G Bottiroli

Publications and source records attributed to G Bottiroli.

At least 73 records · Page 4Linked to original sources
PubMed1984

The time-dependent behaviour of hematoporphyrin-derivative in saline: a study of spectral modifications.

Hematoporphyrin-Derivative (HpD), a widely-used tumor-specific photosensitizer, is a complex mixture of porphyrins whose composition has yet to be clarified. This paper reports on the behaviour of HpD in saline. From a spectroscopic point of view, the fresh solution is characterized by two main absorption peaks, attributable to monomeric and dimeric forms. With aging, a new porphyrin species (NPS) appears. To define the NPS, absorption, excitation and emission spectra were measured in different conditions and time-resolved fluorescence measurements were also performed. This species exhibits an absorption/excitation peak at 405 nm, an emission peak at 575 nm and a fluorescence decay time of approximately 3.5 ns. Its formation is strongly influenced by many environmental factors: in particular, gases diluted in the solution, temperature, pH and concentration. The presence of Oxygen and a pH value outside the 6-8 range may be considered inhibiting factors. The NPS seems to be quite important in the understanding of HpD tumor-specificity, since the presence of an emission band similar to the NPS one seems to be favoured in tumor cells as compared with normal cells.

Hematoporphyrin Derivative↗
PubMed1984

Time-resolved fluorescence spectroscopy of hematoporphyrin-derivative in human lymphocytes.

This paper reports on time-resolved microfluorimetric measurements on hematoporphyrin-derivative (HpD)-treated lymphocytes. HpD is at present widely used as a tumor-locating and photosensitizing drug. It is therefore of great importance to study the extent to which the HpD uptake process depends on cell functional and structural properties. Time-resolved fluorescence measurements in single cells are very useful in this respect, since they give information on the content of fluorescent molecules through fluorescence peak-intensity, and, indirectly, on the binding properties through the fluorescence decay times. In particular, we studied the dependence of HpD fluorescence on the cellular functional state. To this end, we performed in-cell fluorescence measurements on human lymphocytes, both in quiescent conditions and in the pre-replicative phase, after stimulation with phytohemagglutinin (PHA). We found a higher HpD content in stimulated lymphocytes. Moreover, we found a spectral band around 575 nm, corresponding to a particular porphyrin species, in which the differences between normal and stimulated lymphocytes are more striking. The porphyrin species emitting in this band seems to play a role in the specific interaction of HpD with tumors, since a similar emission band has also been found in tumor cells containing HpD.

Hematoporphyrin Derivative↗
PubMed1984

Spectroscopic studies of hematoporphyrin-derivative in culture medium.

This work reports on studies of hematoporphyrin-derivative (HpD) behaviour in culture medium. Absorption, excitation and emission spectra, together with time-resolved fluorescence measurements, were performed. In previous works, similar studies had been carried out on HpD in saline and in lymphocytes: a new porphyrin species (NPS) and the environmental conditions for its formation in saline were studied. A fluorescent emission similar to that presented by the NPS is reported to be more likely in tumor rather than in normal HpD-treated cells, it was also found in greater amounts in lymphocytes in the pre-replicative phase, as compared with quiescent ones. The higher NPS content in stimulated rather than in quiescent lymphocytes may be due either to a differential uptake, as compared with other HpD components, or to a differential formation rate in cells, because of different microenvironmental conditions. To distinguish between these two main assumptions, the formation of NPS in culture medium was studied. The process was very slow: no NPS appeared within the first 40 h. The incubation time of lymphocytes in culture medium added with HpD in the experiments performed was only 1 h and therefore a differential formation rate of NPS may explain the higher content found in stimulated lymphocytes.

Culture Media↗
PubMed1984

An automatic pulsed laser microfluorometer with high spatial and temporal resolution.

The paper describes an automatic pulsed laser microfluorometer with high spatial and temporal resolution, developed in our laboratories. The instrument consists of: (i) a nitrogen-laser-pumped dye-laser for the excitation of the fluorescence, (ii) a microscope with additional optics to focus the excitation beam on the sample and to collect the fluorescence, (iii) filters or monochromators to select the output wavelength, (iv) a fast photomultiplier tube to detect the signal, and (v) a dual time-scale microprocessor-controlled signal averager for the acquisition and processing of the signal. Examples are given that show the potential of the time-resolved fluorescence microscopy in studying, quantitatively and qualitatively, the properties of fluorescent molecules.

Animals↗
PubMed1983

Enhanced membrane fluorescence of CDC-labelled paramecium subsequent to removal of surface components.

Cytofluorimetric analysis of cycloheptaamylose-dansyl chloride (CDC) labelled Paramecium indicates that after mild trypsin removal of surface components the localization of CDC on the outer surface of living cells was not modified by the treatment. After such treatment the intensity of fluorescence emission was found about 3-fold higher in treated single cell than in the untreated one. These findings indicate that CDC labelling can be used to follow alteration occurred on the membrane of the living cell prior to labelling.

Animals↗
PubMed1983

Uptake kinetics and intracellular distribution of anthracyclines studied by laser cytofluorometry.

The detection of low amounts of anthracyclines in single cells was attained with a microscope-photometer by employing an argon laser as a fluorescence excitation source. The time course of drug uptake was followed by incubating the cells under the microscope and measuring the increase of fluorescence intensity from the beginning of the drug penetration. An intracellular distribution map of the drug fluorescence was obtained by scanning measurements, through which the more specific sites of binding were visualized. This means of detection is compared with two of the most commonly employed biochemical techniques.

Antibiotics, Antineoplastic↗
PubMed1983

Fluorescence histochemical patterns of Purkinje cell layer in rat cerebellum after long-term phenytoin administration.

The effects of long-term Phenytoin (DPH) administration were studied on the cerebellum Purkinje layer of Sprague-Dawley rats. Histochemical qualitative studies were performed in order to investigate fluorescence patterns linked to degenerative pigment storage and to changes in biogenic amines. Specific tests for Falck's method and the microspectrofluorometric analysis of fluorescent bodies showed: a) an increase in storage lipopigment which, generally referred to in the literature as lipofuscin, partially exhibit a more complex structure due to the simultaneous presence of lipofuscin and ceroids in the same granular body; b) a change in neurotransmitter patterns. This change must be typically related to an altered metabolism of biogenic amines and/or to degenerative phenomena causing the accumulation of biogenic amines in nerve terminals. The morphological and histochemical pictures support the idea of a generalized Purkinje cell damage.

Animals↗
PubMed1982

Time-resolved fluorescence microscopy of hematoporphyrin-derivative in cells.

This work presents measurements of time-resolved fluorescence microscopy of hematoporphyrin-derivative (HpD) in single cells of mice tissue (both tumor and normal cells), in HeLa Cells, and in solution. The measurements were performed using a pulsed-laser microfluorometer with high spatial and temporal resolution. In agreement with the results obtained with other techniques, it has been found that the tumor cells examined present an HpD uptake about five times higher than that of the normal cells of the corresponding tissue and that, within a cell, HpD become localized mainly in the cytoplasm. It has also been found that the fluorescence decay time is different in cells as compared with solution, and that the presence of HpD stabilizes cell auto-fluorescence. These results are discussed.

Animals↗
PubMed1982

Membrane processes in myotonic dystrophy during in vitro aging of erythrocytes.

The present communication is devoted to investigating the possibility that in myotonic dystrophy (MyD) a decreased ATP utilization by the membrane may produce modifications in glycoprotein structure and/or in the supramolecular arrangement of some membrane proteins. The study was carried out a) by determining the membrane sialic acid content and the cellular ATP concentration in 10 cases of MyD, b) by evaluating the structural modifications of membrane glycoproteins occurring during in vitro incubation, c) by testing the presence on the membrane of high molecular weight aggregates of proteins, and d) by evaluating the distribution of hydrophobic and hydrophilic domains of the membrane by using 1-anilino-8-naphthalene sulfonate (1,8 ANS) as a fluorescent probe. Our evidence suggests that the ATP concentration and membrane sialic acid content are within normal values. Only in two cases did structural modifications of membrane glycoproteins occur while the supramolecular assembly of membrane proteins could be considered normal. The fluorescent probe behaviour after in vitro aging was indicative of a decreased polarity of its environment.

Adolescent↗
PubMed1981

Cycloheptaamylose-dansyl chloride complex as a fluorescent label of surface membranes in living ciliates.

Labelling of surface membrane of living ciliates: Paramecium aurelia and Tetrahymena pyriformis with fluorescent compound--cycloheptaamylose-dansyl chloride complex (CDC) has been achieved. Fluorescence micrographs of the dried samples showed specific localization of CDC on the cell membrane without any intracellular penetration. On the contrary the ciliates which have been dead during labelling revealed a non-specific fluorescence of their whole bodies. Microspectrofluorimetric analysis of labelled Paramecium cells was performed with Leitz microspectrograph. Spectrum of fluorescence emission measured over the cell membrane level had maximum at 450 nm. Strikingly, the emission maximum of the cells dead at the moment of labelling was shifted 10 nm to a longer wavelength. The rate of photofading measured in this case was almost 3-fold higher than for the ciliates labelled as living ones. Fluorescence excitation spectra did not show any difference in the peak position. Thus CDC staining appears to be an useful method of supravital labelling of cell surface enabling also to distinguish--on the basis of spectral characteristics--the ciliates being alive from those dead at the moment of fluorochrome binding.

Animals↗
PubMed1981

Improvement in microscope photometry by voltage to frequency conversion: analogue measurement and digital processing.

A system for digital measurement of microscope photometric signals, based on Voltage to Frequency Conversion (VFC) and suitable for automatic data processing, has been devised. Advantages and disadvantages are illustrated with special emphasis on the comparison between photocurrent measuring and photon counting techniques. A statistical analysis of the results allows the calibration of VFC net counts versus detected photons. A biological application is reported; this shows the suitability of the system even to low light levels.

Animals↗
PubMed1980

On chromatin availability to hydrolysis of the Feulgen reaction in Purkinje neuron population: microdensitometric and microfluorometric data.

The earlier finding that the chromatin organization of Purkinje neurons is not the same in the whole population is confirmed in this study. This was found to be independent of the type of Feulgen reaction hydrolysis kinetics, analytical method (microdensitometry or microfluorometry) and tissue preparation (sections, isolated cells, isolated nuclei). This heterogeneity, possibly linked to different functional stages, leads to Feulgen-DNA contents which, at optimal hydrolysis times, range from 2c to 4c values. The results obtained are discussed in relation to those in the literature.

Animals↗
PubMed1979

Erythrocyte spectrofluorometric abnormalities in Duchenne patients and carriers. A new approach to carrier detection.

Duchenne muscular dystrophy (DMD) is a disorder whose pathogenesis is attributed, according to the most recent theories, to generalized membrane abnormalities, including red cell membrane. The study of erythrocyte ghosts in patients affected by DMD showed alterations in ion transport and in various enzymatic activities. Employing a spectrofluorometric method using 1-anilino-8-naphthalene sufonate (1.8-ANS) we have tried to characterize the erythrocyte ghosts of DMD patients, definite DMD carriers and normal controls, matched for age and sex. We found an increase of membrane polarity in DMD and definite DMD-carrier erythrocyte ghosts. Using this new method we were able to: - confirm by means of a different technique the previous observations of many authors on erythrocyte ghost alterations in DMD - find a new technique useful in detecting the DMD carriers. It is noteworthy that in such a way the detection rate of definite DMD carriers was higher than with the CPK test.

Adult↗
PubMed1979

Employment of bis-intercalating dyes for the "in situ" study of DNA composition.

Spectrofluorometric characteristics of DNA-RO4 complexes have been studied in order to evaluate the possibility of employment of RO4 (a bis-intercalating dye) for the determination of the DNA base composition. The results have shown the usefulness of this compound for DNA with AT percentage between 30% and 60%. RO4 has been also applied to the study of the chromosome banding mechanism.

Aminoacridines↗
PubMed1978

Low temperature microspectrofluorometry: design of a 'cold chamber'.

Low temperature microspectrofluorometry allows an improvement of spectral resolution and an increase of fluorescence intensity. Suppression of fluorescence fading, or at least a marked reduction, is also obtained. A cooling chamber for microspectrofluorometric measurement is described which allows the cooling, under a microscope, of a biological sample down to liquid nitrogen temperature. Objectives with numerical apertures better than 1.0 and a magnification power up to 100X can be used. Low temperature measurements on a histological sample are presented and discussed.

Bacillus cereus↗
PubMed1977

Cytofluorometric quantification of the activity and reaction kinetics of acid phosphatase.

This paper describes how fluorogenic substrates derived from naphthol AS can be used for the microscopic demonstration and cytofluorometric quantification of the activity and reaction kinetics of acid phosphatase in single living cells. A special study has been made of acid naphthol AS-BI phosphatase. However, the method can be extended to other hydrolytic enzymes. The method is sensitive and accurate because: quantification of very low enzyme activity is possible; the reaction kinetics can be evaluated with a good degree of precision inasmuch as the initial reaction velocity is derived over short times; there is an absence of distributional error; and the errors due to extra-cellular diffusion of the hydrolysed substrate, to photodecomposition, and to autofluorescence can be contained within very narrow limits. The procedures for determining enzyme activity and reaction kinetics, and the instrumental characteristics and devices required for carrying out these measurements, are described. Some possible applications are indicated.

Acid Phosphatase↗