Search PubMed⌕ Search

Biomedical subjects

G Bottiroli

Publications and source records attributed to G Bottiroli.

At least 37 records · Page 2Linked to original sources
PubMed1994

DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ.

The influence of DNA topology on stainability with the externally binding fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investigated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the relaxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-linear form of pBR322, compared with the supercoiled-circular molecule. With MI, the stainability of HeLa nuclei did not depend on the DNA topology, whereas the stainability of the plasmid was similar to that of HO. In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, fluorescence resonance energy transfer (FRET) studies were performed in nuclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I digestion, the relative FRET efficiency between donor (HO) and acceptor molecules (PI or MI) was reduced significantly only when MI was the acceptor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainability with base-specific fluorochromes may be affected by the topology of chromatin regions.

Binding Sites↗
PubMed1994

Fluorochromes with long-lived fluorescence as potential labels for pulsed laser immunocytofluorometry: photophysical characterization of pyrene derivatives.

An apparatus for laser-induced time-resolved fluorescence measurements, in conjunction with pyrene derivatives endowed with long-lived excited singlet states, was employed for immunocytofluorometric measurements. N-(1-pyrene)maleimide, 1-pyrenesulfonyl chloride and 1-pyreneisothiocyanate were conjugated with immunoglobulin, antimouse-IgG, and the fluorescence decays of both free and conjugated forms were investigated. Bi-exponential decays were obtained in all cases with time constants of the short-lived component in the range 3-4.7 ns and the long-lived one in the range 20-55 ns. Only the spectral distribution of the two components is essentially affected upon conjugation. The persistence of the long-lived component, well above the lifetime of autofluorescence, and of the antibody specificity, as shown by immunodiffusion tests, upon conjugation indicates that this technique could be advantageously adopted in immunocytofluorometry.

Animals↗
PubMed1994

Fluorescence excimer formation imaging: a new technique to investigate association to cells and membrane behavior of glycolipids.

A new fluorescence ratio imaging technique aiming to monitor the lateral distribution of pyrene-labeled lipids in the membranes through visualization of the excimer/monomer (E/M) intensity ratio, has been set up, studying the association of a fluorescent derivative of GM1 ganglioside (pyreneGM1) to rat cerebellar granule cells in culture. The imaging results show that the mean E/M ratio value, under experimental conditions leading to the association of pyreneGM1 with the plasma membrane, is significantly higher in neuritic processes than in cell bodies and, moreover, locally distributed in patches. Fluorescence antisotropy imaging of the fluorescent probe TMA-DPH (trimethylaminodiphenylhexatriene) shows the presence of domains having different fluidity and that the average fluidity is higher in cell bodies than in neuritic processes. Additional experiments using TMA-DPH as a marker of fluid-phase pinocytosis, suggest that the rate of endocytosis is comparable in the two regions of the cell. Taken together, on the one hand these data indicate that the fluorescent ganglioside is present in a more clustered state at the level of neuritic processes than in cell bodies and locally distributed in patches of different size and enrichment. On the other hand, they point out the potentiality of the excimer formation imaging technique to study membrane behavior and dynamics of pyrene-labeled lipids, with a particular insight into their aggregative properties.

Animals↗
PubMed1993

Characterisation of biotinylated liposomes for in vivo targeting applications.

Liposomes containing monosialoganglioside (GM1) or polyethylene glycol (PEG) lipid derivatives have prolonged circulation in the blood. This favours liposome extravasation to tumour sites. In this report it is shown that inclusion of GM1, PEG550-DPPE or PEG2000-DPPE in liposomes containing biotin-DPPE significantly diminished the ability of vesicles to bind to streptavidin in vitro. Steric inhibition due to the bulky head group of these lipids was least for biotin-DPPE liposomes containing GM1. Biodistribution studies in C26 tumour-bearing mice showed that GM1-liposomes containing small amounts of biotin-DPPE have long circulation life-times in the blood. Using fluorescent microscopic techniques, liposomes containing both GM1 and biotin-DPPE were detected within extra-vascular spaces in tumours. In addition it was shown that biotin-DPPE in GM1-liposomes bound streptavidin in situ. These results suggest that GM1-liposomes containing biotin-DPPE have potential use as diagnostic or therapeutic reagents in pre-targeting applications dependent on the high-affinity interaction of biotin with streptavidin.

Animals↗
PubMed1993

Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry.

DNA sensitivity to digestion with nuclease S1 was investigated in cells irradiated with gamma rays, or treated with the antitumor drug adriamycin (Adr). The nuclease-resistant DNA fraction was determined by propidium iodide staining. Treated cells were found to be more sensitive to nuclease digestion than the undamaged controls. Gamma ray-induced strand breaks were detectable at doses up to 10 Gy; an increase in the reaction temperature, from 37 degrees to 63 degrees C, was necessary in order to detect higher levels of damage. Nuclease S1 sensitivity in Adr-treated cells showed a single-peak, concentration-dependent relationship, in agreement with the known self-inhibitory effect exerted by high drug doses. Determination of DNA digestion could be performed in combination with other cellular parameters (e.g., protein content). Detection of drug-resistant cells in a heterogeneous population of small-cell lung carcinoma was achieved on the basis of the different sensitivity of the cells to enzymatic digestion. These results indicate that nuclease S1 may be a useful probe for studying in single cells DNA alterations induced by drugs or radiation.

Cell Separation↗
PubMed1993

High-frequency perfusion fluorometry (HFPF): preliminary results on a new technique for assessing bronchial mucosa blood perfusion.

A new method for estimating airway mucosa blood perfusion is proposed. It is based on the use of a high-sensitivity perfusion fluorometer, which allows us to detect the first intravascular passage of injected fluorochromes. Total measuring time can be sensibly reduced with respect to standard perfusion fluorometry, allowing us to make assessments during fiberoptic bronchoscopy. In vitro studies were performed to set up the optical arrangement of the instrument. The optimal measuring distance (2.2 mm) between the probe tip and the fluorescent mucosa was established using a fluorescent film surface to simulate fluorescent mucosa. The depth of tissue layers involved in the fluorescence emission was assessed by positioning tissue sections of different thickness between the probe tip and the fluorescent film. Tissue layers at depths not exceeding 1200 microns were found to contribute to the fluorescence signal recorded by the instrument. Mucosa blood perfusion was evaluated in rat trachea, as a model, under three different experimental conditions of vascularization: (a) basal condition (BC), (b) partial devascularization (PD), (c) full devascularization (FD). Sodium fluorescein (diffusible into the interstice) and fluorescein isothiocyanate-dextran (not diffusible into the interstice) were used as fluorescent tracers. Under the BC both substances allowed us to detect the first passage of fluorochrome through the mucosa capillary network with the same accuracy. In PD the first pass peak disappeared; total emitted fluorescence decreased as well. In FD a flat tracing was recorded. Consistent results were obtained with both substances.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Stable hybrid myotubes: a new model for studying re-expression of enzymatic activities in vitro.

Heterokaryons represent a stable and reproducible model system for the study of biochemical and molecular aspects responsible for muscle gene activation. Previous experiments have used this fusion system to demonstrate human gene activation in hybrids formed between human and non-human cells. The aim of this research was to apply this experimental model to the correction of a cytoplasmic activity, namely glucose-6-phosphate dehydrogenase (G6PD), in vitro, in hybrid myotubes formed between G6PD-negative and positive myoblasts. Different identification methods were used (Hoechst stain and Fluorescent Latex Microspheres, FLMs) to identify hybrid myotubes formed. We demonstrated the restoration of G6PD activity in all hybrid myotubes formed; we then tried to elucidate the mechanisms underlying the restoration of this specific activity and apply the results obtained to the understanding of more complex mechanisms involved in muscle gene activation.

Animals↗
PubMed1993

Cytoplasmic restoration and persistence of glucose-6-phosphate dehydrogenase activity in stable hybrid myotubes.

In previous experiments, we obtained an in vitro restoration of a cytosolic enzyme glucose-6-phosphate dehydrogenase (G6PD) in hybrid myotubes formed between G6PD-deficient and human or murine myoblasts. In the present series of experiments, the degree of restoration was observed and the persistence of the restored activity was evaluated in the hybrids formed. Quantitative measurements of enzymatic activity in single human-human or human-mouse myotubes, identified by using either fluorescent latex microspheres or Hoechst stain 33258, were made by using a computer-controlled Leitz photometer. Histospectrophotometry and statistical analysis showed a 50% restoration of enzymatic activity in human-human hybrids compared to deficient and normal myotubes. The restored activity was uniformly distributed throughout the cytoplasm and persisted in long-term cultures. No nuclear domain was observed for G6PD. Knowledge of the degree of restoration, of the extent of distribution of the products of "competent" nuclei and the demonstration that the correction is not a transitory event in vitro, supports the potential usefulness of myoblast transfer therapy for this type of myopathy.

Animals↗
PubMed1992

Light-induced fluorescence spectroscopy of adenomas, adenocarcinomas and non-neoplastic mucosa in human colon. I. In vitro measurements.

In an attempt to evaluate whether induced fluorescence could be exploited to discriminate neoplastic from non-neoplastic tissue, fluorescence spectroscopy was performed at 450-800 nm on 83 biopsy specimens of colonic mucosa. Measurements showed that fluorescence spectra of adenoma, adenocarcinoma and non-neoplastic mucosa manifest dissimilar patterns. Nine variables, whose photophysical and/or biological bases need further investigation, were derived from the spectra. Discriminant functions between the groups of lesions were determined by using a stepwise discriminant analysis. The diagnostic test had a sensitivity of 80.6% and 88.2%, and a specificity of 90.5% and 95.2% in discriminating neoplastic from non-neoplastic mucosa and adenoma from non-neoplastic mucosa respectively. These results suggest that fluorescence spectroscopy has the potential to improve endoscopic diagnosis of premalignant and malignant lesions of colonic mucosa.

Adenocarcinoma↗
PubMed1992

Comparative studies on the effects of doxorubicin and differentiation inducing agents on B16 melanoma cells.

The differentiation-inducing activity of doxorubicin on B16 melanoma cells grown in vitro was compared with that of other known differentiation inducers, such as theophylline, retinoic acid, and melanocyte-stimulating hormone (MSH). At drug concentrations resulting in cytostatic effects, doxorubicin and theophylline induced morphological changes (dendritic-like structures with a terminal melanin granule) with an enhancement of total melanin content and tyrosinase activity. Retinoic acid did not alter melanin content and cell morphology, although it affected cell growth. MSH enhanced total melanin content and tyrosinase activity, with no significant morphological changes. Flow cytometric analysis showed that MSH led to an accumulation of cells in G1 phase whereas doxorubicin induced an accumulation of cells in G2 + M. Studies on DNA content in doxorubicin-treated cells, selected on the basis of a morphologically differentiated pattern, showed a clustering of these cells in G2 + M, probably due to a cytokinesis block. Thus doxorubicin can induce cell differentiation comparable with other differentiation inducers.

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

Nuclease-induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide.

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strand-specific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enzymes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nuclease-induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios greater than 0.11. Below this value (2 micrograms/ml PI), the fluorescence intensity of digested samples was 15-30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation- but not sequence-specific nucleases induce a relaxation of DNA supercoils.

Coloring Agents↗
PubMed1991

Changes in cell cycle and chromatin distribution in C6 glioma cells treated by dibutyryl cyclic AMP.

C6 glioma cells in culture were treated with 1 mM dibutyryl cyclic AMP (Db-cAMP) for 5, 8, 24 and 72 h. The cells were labelled with [3H]-thymidine before either the end, or the beginning, of the Db-cAMP treatment. The cell cycle passage was monitored by the simultaneous determination of DNA content and DNA synthesis in propidium iodide stained autoradiograms. The data revealed an early (t less than or equal to 3-8 h) and moderate inhibitory effect of Db-cAMP on all phases of the cell cycle except mitosis; some cells (2%) were completely blocked in the S phase. Later (8 less than t less than 24-72 h), the cycling of a substantial part of the population became inhibited in G1 phase. Microdensitometric texture analysis of Feulgen-stained nuclei, performed 24 h after administration of Db-cAMP, showed a higher inhomogeneity of the DNA distribution in cell nuclei, caused by the condensation of a part of the chromatin. This may reflect either changes in genome expression taking part in the process of cAMP induced differentiation or transit of some cells into quiescent G0 or S0 phases.

Animals↗
PubMed1990

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1.

Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.

Cell Nucleus↗
PubMed1990

Differences in the organization and chromosomal allocation of satellite DNA between the European long tailed house mice Mus domesticus and Mus musculus.

We compared the organization of satellite DNA (stDNA) and its chromosomal allocation in Mus domesticus and in Mus musculus. The two stDNAs show similar restriction fragment profiles after digestion (probed with M. domesticus stDNA) with some endonucleases of which restriction sequences are present in the 230-240 bp repetitive unit of the M. domesticus stDNA. In contrast, EcoRI digestion reveals that M. musculus stDNA lacks most of the GAATTC restriction sites, particularly at the level of the half-monomer. The chromosome distribution of stDNA (revealed by an M. domesticus stDNA probe) shows different patterns in the M. domesticus and M. musculus karyotypes, with about 60% of M. domesticus stDNA retained in the M. musculus genome. It is particularly noteworthy that the pericentromeric regions of M. musculus chromosomes 1 and X are totally devoid of M. domesticus stDNA sequences. In both groups, the differences in energy transfer between the stDNA-bound fluorochromes Hoechst 33258 and propidium iodide suggest that AT-rich repeated sequences have a much more clustered array in the M. domesticus stDNA, as if they are organized in tandem repeats longer than those of M. musculus. Considering the data as a whole, it seems likely that the evolutionary paths of the two stDNAs diverged after the generation of the ancestral 230-240 bp stDNA repetitive unit through the amplification, in the M. domesticus genome, of a family repeat which included the EcoRI GAATTC restriction sequence.

Animals↗
PubMed1990

Flow cytometric evaluation of DNA digestion with micrococcal nuclease on isolated HeLa nuclei.

Flow cytometric assessment of DNA digestion with micrococcal nuclease has been performed on isolated HeLa nuclei by determining the relative reduction in stainability with the DNA-specific fluorochrome, propidium iodide. At the nuclease concentrations used, DNA histograms of digested nuclei showed the typical bimodal pattern, when the enzymatic reaction was performed in a medium maintaining chromatin in its native (i.e. condensed) or partially decondensed from. In contrast, when nuclei were digested in a buffer lacking both the mono- and divalent cations K+ and Mg2+, an extensive decrease in fluorescence intensity, with loss of the histogram shape, was observed. In nuclei with native chromatin, DNA stainability decreased as a function of time and enzyme concentration, to reach a lower limit of about 46%, as compared with undigested control samples. Removal of the histone H1 induced a significant increase (approximately by a factor of 2) in the extent of digestion, although only in nuclei with partially decondensed chromatin. These results suggest that the sensitivity of DNA to digestion with micrococcal nuclease can be quantitatively monitored with flow cytometry when appropriate reaction conditions are chosen.

Cell Nucleus↗