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R Ramponi

Publications and source records attributed to R Ramponi.

At least 19 recordsLinked to original sources
PubMed1994

Propidium iodide and the thiol-specific reagent DACM as a dye pair for fluorescence resonance energy transfer analysis: an application to mouse sperm chromatin.

The dyes N-(-7-dimethyl-amino-4-methyl-coumarinyl) maleimide and propidium iodide, specific for the thiol group and DNA, respectively, were considered as a donor-acceptor couple suitable for investigating "in situ" the relative spatial distribution of DNA and protamines in mouse spermatozoa chromatin. The two dyes are characterized by favourable spectral properties, so that a simplified analytical procedure, based on the measurement of both donor and acceptor emission in double-stained samples, can be applied to evaluate the relative efficiency of the energy transfer process and its topological distribution. The results obtained indicate that during the maturation process: 1) the basic arrangement of protamine-DNA complex does not undergo structure changes, and 2) the oxidation of sulfhydryl to disulfide groups, resulting in chromatin stabilization, first involves the protamine thiols spatially closer to DNA. Fluorescence energy transfer imaging suggests that chromatin stabilization starts in the midportion of the sperm head, then spreads towards the periphery.

Animals↗
PubMed1992

Distribution of di-sulfonated aluminum phthalocyanine and Photofrin II in living cells: a comparative fluorometric study.

Microspectrofluorometric and fluorescence imaging techniques have been employed to study the internalization and intracellular distribution of both Photofrin II, an experimental drug used in photodynamic therapy, and di-sulfonated aluminum phthalocyanine, a very promising photosensitizer. The results obtained by microscopic techniques in living cells have been compared with those obtained in solution on cell extracts. Experimental results indicated that the complexity of the drug-cell interaction can be explained according to the chemico-physical nature of the drugs. In particular, the presence of both monomeric and aggregated fractions, which are supposed to be internalized through different mechanisms, accounts for the intracellular distributions observed for both drugs, depending on the treatment conditions. Equilibria among the drug fractions take place within the cells, resulting in the persistence of the intracellular fluorescence. On the whole, the behavior of the two drugs appears very similar, except for some aspects related to the intracellular distribution, which can be explained in terms of different degree of lipophilicity of the drugs.

Cells, Cultured↗
PubMed1991

Time-gated fluorescence spectroscopy of porphyrin derivatives and aluminium phthalocyanine incorporated in vivo in a murine ascitic tumour model.

The effect of systemic administration on drug uptake at cellular level was evaluated using time-gated fluorescence spectroscopy performed on a murine ascitic tumour model. Mice bearing L1210 leukaemia were injected intraperitoneally or intravenously with 25 mg per kg body weight hematoporphyrin derivative (HpD), 12.5 mg per kg body weight photofrin II (PII), 25 or 5 mg per kg body weight disulphonated aluminium phthalocyanine (AlS2Pc). Every 2 h and for up to 22 or 30 h, mice were sacrificed, leukaemic cells extracted from the peritoneum, washed, and resuspended in buffer for fluorescence measurements. HpD and PII emission spectra were almost identical 12 h after intraperitoneal injection with main peaks at 630 nm and no appreciable changes afterwards. In the first 12 h, the PII fluorescence spectrum was constant, while in the case of HpD a shoulder at 615 nm was detectable. Similar fluorescence behaviour was observed after intravenous administration of porphyrin derivatives. These results seem to confirm that the tumour localizing fraction is the part actually retained by the cells. The AlS2Pc spectrum peaked at 685 nm and did not change in any of our experiments. AlS2Pc is incorporated more rapidly with respect to porphyrins, as was clearly observed in the case of intravenous administration, where the AlS2Pc fluorescence was readily detectable after 2 h, whereas the PII emission became apparent only after 4-6 h.

Aluminum↗
PubMed1991

Age-related changes in the fluorescence of melanin and lipofuscin granules of the retinal pigment epithelium: a time-resolved fluorescence spectroscopy study.

The photophysical properties of purified populations of melanin and lipofuscin granules from human retinal pigment epithelium, and their changes with donor age, have been investigated using high-sensitivity time-resolved fluorescence spectroscopy techniques with picosecond gating capabilities. The overall fluorescence intensity of both melanin and lipofuscin granules clearly increased with increasing donor age, the increase being most marked for melanin. In all granule populations the fluorescence decays were multiexponential with subnanosecond and nanosecond decay components. The resultant time-integrated and time-gated spectra also exhibited marked age-variations for each type of granule. Young melanin showed spectral patterns similar to those of bovine melanin, while a yellow-orange fluorescence band appeared in melanin samples from older age groups. Lipofuscin granules exhibited a blue, a yellow and an orange band whose relative amounts were age-related. The results demonstrate the potential of time-resolved techniques for discriminating fluorophores in vitro and in situ, and have confirmed results previously obtained using extraction techniques. Furthermore, the ability of this technique to identify and quantify individual fluorophores within granules may provide an important insight into the origin and development of lipofuscin within the retinal pigment epithelium and ultimately into the mechanisms of age-related retinal diseases.

Adolescent↗
PubMed1990

Age-related changes in the morphology, absorption and fluorescence of melanosomes and lipofuscin granules of the retinal pigment epithelium.

The morphological and spectral characteristics of purified populations of melanosomes and lipofuscin granules from the human retinal pigment epithelium (RPE) were studied with respect to donor age. All melanosome and lipofuscin fractions exhibited the typical ultrastructural appearance associated with these granules. Absorption profiles of both melanin and lipofuscin granules demonstrated an increased optical density of the granules with increasing age. The former was associated with an overall increase of melanin within the granules. Melanosomes were weakly fluorescent; emission in the blue decreased with increasing age while emission in the red increased. The fluorescent intensity of lipofuscin granules increased with age. These results provide support for the concept that melanogenesis is occurring within the human RPE throughout life and that pigment granules within the RPE undergo age-related modifications during life.

Adolescent↗
PubMed1990

Comparative study of the therapeutic effect of photoactivated hematoporphyrin derivative and aluminum disulfonated phthalocyanines on tumor bearing mice.

Although the hematoporphyrin derivative (Hpd) is one of the most studied photosensitisers for photodynamic therapy (PDT), it is far from ideal. Therefore, many laboratories have been investigating a new group of sensitisers, the phthalocyanines. Particularly, in our laboratory we decided to study the aluminum disulfonated phthalocyanines (AlS2PC). They are chemically stable, readily soluble in water and have a strong absorption in the red part of the spectrum at 675 nm. Mice bearing the MS-2 fibrosarcoma treated with 5 mg/kg of AlS2PC survived indefinitely also using a low laser power of 100 mW/cm2 X 10' of exposure time, in contrast to experiments carried out with Hpd where the optical therapeutic laser power was 400 mW/cm2 X 10' and the dose of Hpd was 25 mg/kg. Furthermore, treatment of mice bearing the highly metastatic tumor, B16 melanoma, with 5 mg/kg of AlS2PC and laser light (100 mW/cm2 X 10'), significantly prolonged the survival time in respect to mice treated with 25 mg/kg of Hpd and laser light (400 mW/cm2 X 10').

Animals↗
PubMed1990

Time-gated fluorescence spectroscopy of porphyrin derivatives incorporated into cells.

Time-gated fluorescence spectroscopy was performed on the tumour-localizing fraction (TLF) of haematoporphyrin derivative (HPD) incorporated into cells. Three different cell lines were incubated with 20 and 5 micrograms ml-1 of TLF for various time periods; they were then washed and resuspended in buffer. Fluorescence decay measurements and time-integrated and time-gated spectra were then obtained from the cell suspensions. Similar experiments were repeated using HPD containing 60% of the active material. The experimental results show a modification of the emission spectra for both drugs depending on the incubation time; this modification is more significant for the TLF. In particular, the emission peak observed in aqueous solution at 615 nm is shifted to 630 nm as a consequence of incorporation into cells, and the gated spectra indicate that the fluorescence emission is mainly related to monomers and unfolded polymeric chains. The ratio between the intensities of the two peaks depends on the relative amount of the TLF; the peak at 615 nm is more pronounced for HPD. The results obtained seem to indicate that both the composition of the drug and the metabolic properties of the biological environment strongly influence the uptake process and the fluorescence behaviour of the incorporated sensitizer.

Animals↗
PubMed1989

Time-gated fluorescence spectroscopy of the tumor localizing fraction of HpD in the presence of cationic surfactant.

Time-gated fluorescence spectroscopy was performed on the tumor localizing fraction (TLF) of HpD in buffer at different concentrations of cationic surfactant. This technique obtains emission spectra with programmable delay relative to the excitation pulse. According to the measured fluorescence decay-time constants (approximately 0.7, approximately 3 and approximately 15 ns) three gates were considered, delayed by 0, 5 and 18 ns, respectively, to evaluate the contribution of the emitting molecular species to the spectra. Simultaneous to these measurements, fluorescence decay waveforms and time-integrated spectra were also detected. In buffer and in detergent micelles the fluorescence spectra are given by the superposition of the emission of the different molecular species present in the solution, and no appreciable interaction among the chromophores is observed. On the contrary, in the pre-micellar range of the surfactant, evidence for the existence of an energy transfer mechanism was found. This effect seems to be related to the configurational state of the TLF polymeric chains and depends on the relative TLF/surfactant concentration.

Cetrimonium↗
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↗
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↗