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Biomedical subjects

E Cundari

Publications and source records attributed to E Cundari.

At least 37 records · Page 2Linked to original sources
PubMed1995

Low environmental radiation background impairs biological defence of the yeast Saccharomyces cerevisiae to chemical radiomimetic agents.

Background radiation is likely to constitute one of the factors involved in biological evolution since radiations are able to affect biological processes. Therefore, it is possible to hypothesize that organisms are adapted to environmental background radiation and that this adaptation could increase their ability to respond to the harmful effects of ionizing radiations. In fact, adaptive responses to alkylating agents and to low doses of ionizing radiation have been found in many organisms. In order to test for effects of adaptation, cell susceptibility to treatments with high doses of radiomimetic chemical agents has been studied by growing them in a reduced environmental radiation background. The experiment has been performed by culturing yeast cells (Saccharomyces cerevisiae D7) in parallel in a standard background environment and in the underground Gran Sasso National Laboratory, with reduced environmental background radiation. After a conditioning period, yeast cells were exposed to recombinogenic doses of methyl methanesulfonate. The yeast cells grown in the Gran Sasso Laboratory showed a higher frequency of radiomimetic induced recombination as compared to those grown in the standard environment. This suggests that environmental radiation may act as a conditioning agent.

Background Radiation↗
PubMed1994

Analysis of susceptibility of mature human T lymphocytes to dexamethasone-induced apoptosis.

We present evidence that dexamethasone (Dex), a synthetic glucocorticosteroid, causes apoptosis in mature human T cells, similarly to what has been reported for murine T lymphocytes. Human T cell clones and short-term activated T lymphocytes treated with Dex show the characteristic pattern of apoptotic cells, such as hypodiploid nuclei, chromatin condensation and DNA fragmentation into oligonucleosomal fragments. However, Dex susceptibility of T cells to apoptosis is cell cycle-dependent. The progression in the proliferative cell cycle (G1 versus S) rescues Dex-treated T cells from apoptosis. Moreover, occupancy of the T cell receptor reverses Dex-induced apoptotic phenomena. These observations suggest that glucocorticoids contribute to the regulation of the proliferative or the suicidal response of antigen-activated human T cells.

Animals↗
PubMed1994

Different regions of the N-terminal domains of HLA-DR1 influence recognition of individual peptide-DR1 complexes.

The contributions of individual amino acids in the polymorphic beta chain and the conserved alpha chain of HLA-DR1 to influenza HA-specific DR1-restricted and anti-DR1 allospecific T-cell recognition were analyzed. The genes encoding HLA-DR1 were subjected to site-directed mutagenesis in order to introduce single amino acid substitutions at 12 positions in the beta 1 domain and 11 positions in the alpha 1 domain. The beta 1-domain substitutions were all at polymorphic positions and introduced residues that are found in DR4 alleles. The amino acids introduced into the DR alpha 1 domain were based on the sequences of other human and mouse class II alpha chains. The responses of 12 DR1-restricted T-cell clones specific for two peptides of HA and seven anti-DR1 allospecific clones were studied. Substitutions at positions that point up from and into the peptide-binding site in the third variable region of the beta 1-domain alpha-helix caused substantial reduction in the responses of all of the clones. Substitutions at multiple positions in the beta 1-domain floor and in the alpha 1 domain influenced the anti-DR1 responses of the alloreactive and of the HA100-115-specific T-cell clones. In contrast, very few changes outside of the beta 1 domain third variable region affected the responses of the HA306-324-specific DR1-restricted T-cell clones. These results suggest that a surprisingly limited region of the HLA-DR1 molecule is critically involved in T-cell recognition of HA306-324 by DR1-restricted T cells. However, the susceptibility of the HA100-115-specific and the anti-DR1 allospecific T-cell clones to substitutions at multiple positions in both N-terminal domains shows that the response to DR1-HA306-324 is unusual and may reflect the promiscuity with which this peptide binds to HLA-DR molecules.

Amino Acid Sequence↗
PubMed1994

[Changes in the radiation-induced apoptotic response in homozygotes and heterozygotes for the ataxia-telangiectasia gene].

Ataxia-telangiectasia is a progressive recessive disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation hypersensitivity and increased predisposition to cancer. Impaired induction of the tumor suppressor protein p53 after gamma-irradiation was recently reported. All together these characteristics may be compatible with an inability to correctly regulate the apoptotic pathway of cell death in this syndrome. We show here that lymphocyte cultures from AT patients are characterized by a 3 times more elevated spontaneous level of apoptotic cells compared to normal ones. In spite of this, 24 h after exposure to gamma-irradiation (5 to 10 Gy), AT lymphocytes show a dramatically reduced capacity to undergo apoptosis compared to normal cells. We obtained similar results on EBV-transformed lymphoblasts. Interestingly, lymphoblasts from obligate heterozygous for the AT mutation(s) show the same features as AT lymphoblasts, i.e. an elevated frequency of spontaneous and a reduced level of radio-induced apoptotic figures in comparison to normal cultured cells. In conclusion, we show here, for the first time, that mutation(s) in AT gene(s) results in an impaired ability to correctly regulate the apoptotic pathway of cell death.

Apoptosis↗
PubMed1993

Chromatin bridges between sister chromatids induced in late G2 mitosis in CHO cells by trimethylpsoralen + UVA.

A hypothesis is proposed that sister chromatid pairing is due, at least in part, to the pairing between DNA strands belonging to each of the two sister chromatids (conservative pairing). To test this hypothesis interstrand DNA cross-links were induced in late G2-mitosis in CHO cells in order to bind covalently paired DNA strands eventually coming from both sister chromatids and detect the consequent chromatin bridges between sister chromatids (SCCBs). Therefore cells were treated with trimethylpsoralen (TMP) + UVA (365 or 405 nm). Chromatin bridges in ana-telophase were induced by an UVA irradiation at 365 nm, which gives rise to both monoadducts and cross-links, but not by a 405-nm irradiation, which gives rise only to monoadducts. An analysis of colchicine-induced c-anaphases demonstrated that such chromatin bridges were really SCCBs and that terminal regions of chromosomes were particularly involved. The evolution of SCCBs was studied to rule out that they were masked isochromatid exchanges. So TMP + UVA-treated cells were induced to polyploidize with colchicine and labeled with 5-bromodeoxyuridine. Cells treated with TMP + UVA in G2-mitosis appeared as M1 tetraploid c-metaphases; in such cell populations there was not an increase in isodicentric chromosomes, which are derived from isochromatid exchanges. The present data, as a whole, support the hypothesis that a "conservative pairing" between DNA strands of sister chromatids can be present in mitosis.

Animals↗
PubMed1993

The generation of nitric oxide participates in gamma IFN-induced MHC class II antigen expression by cultured astrocytoma cells.

The effects of interferon gamma (gamma IFN) on the MHCII antigen expression by human cultured astrocytoma cells were investigated. The co-incubation of gamma IFN with T67 astrocytoma cells produced a dose-dependent increase of MHCII antigen expression as evaluated by flow cytometric (FACS) analysis and confocal laser microscopy analysis. The number of MHCII molecules expressed by gamma IFN-pretreated astrocytoma cells was reduced by co-incubation with N omega-nitro-L-arginine methyl ester (L-NAME), a selective inhibitor of the nitric oxide (NO)-synthesizing enzyme. In addition, methylene blue, which inhibits the biological activity of NO acting at the guanylate cyclase level, strongly antagonized the MHCII antigen expression on astrocytoma cells induced by gamma IFN. Furthermore, gamma IFN increased the activity of the inducible isoform of NO-synthase as well as the concentration of nitrite, one of the breakdown products of NO and the antiplatelet activity of astrocytoma cells. In conclusion, the present data show that gamma IFN increases the synthesis and release of NO by cultured astrocytoma cells and this could co-participate in the MHCII antigen expression by this cell type. Therefore, the generation of NO by cultured astrocytoma cells may represent an important step in the development of the immunocompetent activity of astrocytes.

Amino Acid Oxidoreductases↗
PubMed1991

Repair of 8-methoxypsoralen photoinduced cross-links in yeast. Analysis by alkaline step-elution and electron microscopy.

The repair of interstrand cross-links induced by 8-methoxypsoralen plus UVA (365 nm) radiation DNA was analyzed in diploid strains of the yeast Saccharomyces cerevisiae. The strains employed were the wild-type D7 and derivatives homozygous for the rad18-1 or the rad3-12 mutation. Alkaline step-elution and electron microscopy were performed to follow the process of induction and removal of photoinduced cross-links. In accordance with previous reports, the D7 rad3-12 strain failed to remove the induced lesions and could not incise cross-links. The strain D7 rad18-1 was nearly as efficient in the removal of 8-MOP photoadducts after 2 h of post-treatment incubation as the D7 RAD+ wild-type strain. However, as demonstrated by alkaline step-elution and electron microscopic analysis, the first incision step at DNA cross-links was three times more effective in D7 rad18-1 than in D7 RAD+. This is consistent with the hypothesis that the RAD18 gene product is involved in the filling of gaps resulting from persistent non-informational DNA lesions generated by the endonucleolytic processing of DNA cross-links. Absence of this gene product may lead to extensive strand breakage and decreased recognition of such lesions by structural repair systems.

Cross-Linking Reagents↗
PubMed1991

Non-specific incision of DNA due to the presence of 8-methoxypsoralen photoinduced interstrand cross-links in Saccharomyces cerevisiae.

The repair of DNA interstrand cross-links (CL) induced by 8-methoxypsoralen (8-MOP) plus UVA irradiation was analyzed by the alkaline step elution technique. A double-exposure protocol was used with 8-MOP, starting with exposure to monochromatic 405-nm radiation inducing only DNA monoadducts (MA), followed, after washing out of unbound 8-MOP molecules, by a second exposure to 365-nm radiation inducing varying relative amounts of CL at a constant level of total photoadducts. In the range of doses used for the second exposure, repair of CL took place; however, in the presence of increased relative amounts of CL induced non-specific incision of DNA occurred. This endonucleolytic cleavage appears to be related to the increased mutagenic and recombinogenic effects observed at increased levels of CL.

Cross-Linking Reagents↗
PubMed1991

Metabolism of 5-methoxypsoralen by Saccharomyces cerevisiae.

Incubation of methoxypsoralen (5-MOP) in the presence of diploid yeast cells (Saccharomyces cerevisiae) before UV-A exposure leads to an incubation-time dependent decrease of photoinduced genotoxic effects. The reduction in photoinduced genotoxicity is stronger in cells grown in the presence of 20% glucose and containing high levels of cytochrome P-450 than in cells grown in the presence of 0.5% glucose and containing undetectable levels of cytochrome P-450. Inhibition of P-450 activity by specific inhibitors, such as tetrahydrofuran and metyrapone, strongly affects the observed decrease in 5-MOP genotoxicity, indicating the involvement of P-450 in 5-MOP metabolism. As demonstrated by spectrophotometric and chromatographic (HPLC) analysis during incubation of 5-MOP with P-450 containing yeast cells, 5-MOP gradually disappears from the cell supernatant of the incubation mixture. The reduction in the chromatographic peak corresponding to 5-MOP is accompanied by the appearance of a new peak that probably corresponds to a metabolite. As shown by the use of P-450 specific inhibitors, the metabolite appears to be due to P-450 mediated 5-MOP metabolisation. Its UV absorption spectrum suggests an alteration of the pyrone moiety of the 5-MOP molecule.

5-Methoxypsoralen↗
PubMed1990

Genetic effects and repair of DNA photo-adducts induced by 8-methoxypsoralen and homopsoralen (pyranocoumarin) in diploid yeast.

The relationship between DNA mono- and di-adducts and genetic effects induced by the pyranocoumarin 8,8-desmethylxanthyletine (homopsoralen) HP and 365 nm radiation (UVA) was investigated in the diploid yeast strain D7 (Saccharomyces cerevisiae) taking 8-methoxypsoralen (8-MOP) as a reference compound. The number of DNA cross-links (CLs) induced was determined using alkaline step elution analysis. The induction and removal of total photo-adducts was followed using radioactively labelled compounds. HP showed the same photobinding capacity as 8-MOP. As a function of UVA dose, it was less effective than 8-MOP for the induction of CLs and genetic effects. However, as a function of CLs induced, HP was shown to be more effective for the induction of lethal effects and mitotic recombination than 8-MOP but equally effective for the induction of mutations. The results suggest that, although CLs are recognized as genetically effective lesions, at a given number of CLs, HP induced mono-adducts efficiently contribute to the induction of lethal effects and mitotic recombination but less to the induction of mutations. Using a re-irradiation protocol, HP was brought to yield the same relative amounts of CLs at the same number of total adducts as single UVA exposures with 8-MOP. In these conditions, mutation induction and the kinetics for the removal of photo-adducts were the same for both agents indicating that not only the removal of adducts but also mutation induction are highly dependent on the relative level of CLs induced.

DNA Repair↗
PubMed1988

Some photochemical and photobiological properties of 8,8-desmethylxanthyletine (homopsoralen), a potential photochemotherapeutic agent.

In this paper the photochemical and photobiological properties of 8,8-desmethyl-xanthyletine (homopsoralen) have been studied. This drug forms a molecular complex with DNA undergoing intercalation between two base pairs of the macromolecule. By subsequent irradiation (365 nm) the compound photobinds covalently to the macromolecule showing an initial DNA-photobinding rate higher than that of 8-methoxypsoralen. In the photoreaction with DNA the drug effectively forms inter-strand cross-linkages. Its ability to generate singlet oxygen when irradiated with ultraviolet light is markedly higher than that of 8-MOP. Homopsoralen when irradiated in water solution undergoes effective photolysis. The drug shows a lower antiproliferative activity on diploid yeast (D 7) than 8-MOP and an almost parallel mutagenic activity. Also the gene convertogenic activity, determined on the same yeast strain, is lower than that of 8-MOP. Homopsoralen does not show any skin phototoxicity on guinea-pig skin. Taking into account its DNA-photobinding capacity, its antiproliferative activity, its reduced genotoxicity and lack of skin-phototoxicity homopsoralen may deserve further studies for evaluating its photochemotherapeutic activity.

Animals↗
PubMed1987

Alkaline step elution analysis of gamma-ray induced DNA strand breaks and repair in diploid yeast.

Gamma-ray induction of DNA strand breaks and their repair was analysed in the diploid yeast strain D7 (Saccharomyces cerevisiae) by means of the alkaline step elution technique. A dose-dependent increase of DNA strand breakage was observed in the dose range 25-2000 Gy corresponding to 100 and 0.01 per cent survival. When, after exposure to gamma-irradiation, the cells were incubated for 2 h in liquid growth medium, the elution profiles reached the pattern of unirradiated controls, thus indicating the restoration of cellular DNA due to repair. The alkaline step elution analysis is found to be a useful and reproducible technique for studying the induction of DNA strand breaks and repair in yeast. In comparison with other current methods, such as alkaline sucrose gradients and DNA unwinding, this method appears to be more rapid, versatile and easier to handle.

Cross-Linking Reagents↗
PubMed1986

Inducibility of gene conversion in Saccharomyces cerevisiae treated with MMS.

At high survival levels (85%), point mutation and gene conversion frequencies were determined in strain D7 of Saccharomyces cerevisiae after treatment with methyl methanesulfonate (MMS) either after cells were incubated in complete medium before plating or following a split-dose protocol. It is shown that induction of gene conversion by MMS post-incubation leads to an additional enhancement in frequency. This increase is not observed for point mutation. By fractionation of the MMS dose (1 mM + 1 mM) with incubation in complete medium between the 2 doses the frequency of gene conversion is twice as high as with a single equal total dose (2 mM). This treatment does not modify the frequencies of point mutation. These data support the notion that an inducible recombinogenic function exists in wild-type yeast.

DNA Repair↗
PubMed1986

Specific inhibitors of the monooxygenase system of Saccharomyces cerevisiae modified the mutagenic effect of 4-nitroquinoline 1-oxide and the deethylation activity of the yeast.

A form of cytochrome P-450 is produced in Saccharomyces cerevisiae strain D7 during the logarithmic growth phase in 20% glucose liquid medium. This form was inhibited by metyrapone, tetrahydrofuran and by carbon monoxide, specific inhibitors of cytochrome P-450 in mammals. The inhibition was observed by means of the increase of the genetic activity of 4-nitroquinoline 1-oxide (4-NQO) on logarithmic growth phase cells of D7 strain, adding the inhibitors to the incubation mixture. 4-NQO is a strong direct mutagen on stationary phase cells that is detoxified by the monooxygenase system. The inhibition was measured by determining the decrease of the O-deethylation of 7-ethoxycoumarin in whole cells of yeast depending on different concentrations of inhibitors. A decrease of O-deethylation activity was found in the presence of tetrahydrofuran and metyrapone and this behaviour is typical of the cytochrome P-450 species induced by ethanol, as in mammals. Adding sodium phenobarbital to 0.5% glucose liquid medium, a form inhibited only by metyrapone was obtained. The presence of different inducible forms of cytochrome P-450 is evident.

4-Nitroquinoline-1-oxide↗
PubMed1985

Study of the optimal temperature for the liver microsomal assay with mice S9 fractions.

The effect of temperature on enzymatic activity and stability was studied with respect to the monooxygenase activities of aminopyrine-N-demethylase (APD) and p-nitroanisole O-demethylase (pNAD) under incubation conditions for the liver microsomal assay. The activities of S9 liver fractions of mice induced with sodium phenobarbital and beta-naphthoflavone were determined during a period of preincubation in a range of temperatures from 30 to 44 degrees C. The greatest value of the mean specific activity was found at 40-42 degrees C for both APD and pNAD. The rapid increase of lipid peroxidation after 1 h of incubation at temperatures higher than 42 degrees C can provide an explanation of the enhancement of the rate of inactivation. In order to determine whether biological response is affected by the modifications induced by temperature in the metabolic activating system, tester strain D7 of Saccharomyces cerevisiae was used to assay the genetic activity of the well known premutagenic agent cyclophosphamide by incubating the mixtures both at the traditional temperature of 37 degrees C and at 42 degrees C. We suggest that the use of more favourable conditions for LMA with respect to enzymatic activity, than the traditional ones could improve the reliability and the sensitivity of such tests.

Aminopyrine N-Demethylase↗