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A Aprile

Publications and source records attributed to A Aprile.

At least 19 recordsLinked to original sources
PubMed2005

Mapping regulatory genes as candidates for cold and drought stress tolerance in barley.

Cereal crop yield is greatly affected in many growing areas by abiotic stresses, mainly low temperature and drought. In order to find candidates for the tolerance genes for these stresses, 13 genes encoding for transcription factors and upstream regulators were screened by amplification and SSCP on six parental genotypes of three barley mapping populations ('Nure' x 'Tremois', 'Proctor' x 'Nudinka', and 'Steptoe' x 'Morex'), and mapped as newly developed STS, SNP, and SSCP markers. A new consensus function map was then drawn using the three maps above, including 16 regulatory candidate genes (CGs). The positions of barley cold and drought tolerance quantitative trait loci (QTLs) presently described in the literature were added to the consensus map to find positional candidates from among the mapped genes. A cluster of six HvCBF genes co-mapped with the Fr-H2 cold tolerance QTL, while no QTLs for the same trait were positioned on chromosome 7H, where two putative barley regulators of CBF expression, ICE1 and FRY1, found by homology search, were mapped in this work. These observations suggest that CBF gene(s) themselves, rather than their two regulators, are at present the best candidates for cold tolerance. Four out of 12 drought tolerance QTLs of the consensus map are associated with regulatory CGs, on chromosomes 2H, 5H, and 7H, and two QTLs with effector genes, on chromosomes 5H and 6H. The results obtained could be used to guide MAS applications, allowing introduction into an ideal genotype of favourable alleles of tolerance QTLs.

Chromosome Mapping↗
PubMed2001

p53 mutants can often transactivate promoters containing a p21 but not Bax or PIG3 responsive elements.

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fisher's exact test), more rarely mutated (P<0.006, Fisher's exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fisher's exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fisher's exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.

Amino Acid Sequence↗
PubMed2000

p53 mutations experimentally induced by 8-methoxypsoralen plus UVA (PUVA) differ from those found in human skin cancers in PUVA-treated patients.

8-Methoxypsoralen (8-MOP) plus UVA irradiation (PUVA therapy) has been used for the treatment of psoriasis. PUVA therapy has been associated with an increased risk of developing skin squamous cell carcinoma (SCC). In order to determine the PUVA-induced p53 mutation spectrum, a yeast expression vector harbouring a human wild-type p53 cDNA was incubated with 8-MOP, and UVA irradiated in vitro. PUVA-damaged and undamaged DNA was transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. An 8-MOP concentration-dependent decrease in survival and increase in mutant frequency were observed. At a fixed 8-MOP concentration, survival decreased and mutant frequency increased as UVA irradiation increased. Eleven mutant clones contained 11 mutations: 10 were single base pair substitutions, the remaining one being a complex mutation. All eight T:A-targeted mutations were at 5'-TpA sites, hallmark mutations of PUVA mutagenesis. Through a rigorous statistical test, the PUVA-induced p53 mutation spectrum appears to differ significantly (P < 0.0002) from that observed in SCC in PUVA-treated patients. The present work demonstrates that a specific PUVA-induced mutational fingerprint could be obtained and recognized on human p53 cDNA. This result may suggest that PUVA therapy can be a risk factor for the development of SCC in psoriasis patients through a mechanism not involving the induction of p53 mutations.

DNA Mutational Analysis↗
PubMed1999

5-methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation.

Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (lambda(max): 254 nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P = 0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solarlight and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a "spontaneous" origin.

5-Methylcytosine↗
PubMed1999

Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent.

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT --> TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10(-6), Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.

Adenine↗
PubMed1999

N-(2-chloroethyl)-N-nitrosourea tethered to lexitropsin induces minor groove lesions at the p53 cDNA that are more cytotoxic than mutagenic.

Many different N-chloroethyl-N-nitrosourea (CENU) derivatives have been synthesized in an attempt to minimize carcinogenic activity while favoring antineoplastic activity. CENU derivatives linked to the dipeptide lexitropsin (lex) showed significant changes in groove- and sequence-selective DNA alkylation inducing thermolabile N3-alkyladenines (N3-Alkyl-As) at lex equilibrium binding sites. CENU-lex sequence specificity for DNA alkylation was determined using 32P-end-labeled restriction fragments of the p53 cDNA. The adducted sites were converted into single-strand breaks by sequential heating at neutral pH and exposure to piperidine. To establish the mutagenic and lethal properties of CENU-lex-specific lesions, a yeast expression vector harboring a human wild-type p53 cDNA was treated in vitro with CENU-lex and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutants were isolated from independent ade- transformants. The results revealed that: (a) CENU-lex preferentially induces N3-Alkyl-A at specific lex equilibrium binding sites, the formations of which are strongly inhibited by distamycin; (b) reactivity toward Gs is still present, albeit to a lesser extent when compared to N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea and to CENU; (c) 91% of the 49 CENU-lex p53 mutations (45 of 49) were bp substitutions, 29 of which were GC-->AT transitions, mainly at 5' purine G sites; (d) all AT-targeted mutations but one were AT-->TA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GC-->AT transitions were observed, being a real (n = 3, P < 0.0002) CENU-lex mutation hot spot; and (f) a shift in DNA alkylation sites between lesion spectra induced by CENU-lex and N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea was associated with an increased lethality and a decreased mutagenicity, whereas no dramatic change in mutational specificity was observed. Hence, it is tempting to conclude that, in this experimental system, N3-Alkyl-A is more lethal than mutagenic, whereas O6-alkylguanine is a common premutational lesion formed at non-lex binding sites. These results suggest that CENU derivatives with virtually absolute specificity for A residues would make targeting of lethal, nonmutagenic lesions at A+T-rich regions possible, and this may represent a new strategy for the development of new chemotherapeutic agents with a higher therapeutic index.

Alkylation↗
PubMed1998

Ultraviolet-light induced p53 mutational spectrum in yeast is indistinguishable from p53 mutations in human skin cancer.

Ultraviolet (UV) light has been associated with the development of human non-melanoma skin cancers (NMSC). Such cancers often exhibit mutations in the p53 tumour suppressor gene. In order to determine the UV-induced p53 mutation spectrum, a yeast expression vector that harbours a human wild-type p53 cDNA was UV-irradiated in vitro and transfected into a yeast strain that contained the ADE2 gene regulated by a p53-responsive promoter. Forty-five mutant clones contained 51 mutations. Seven mutations were tandem base pair substitutions, four of which being CC-->TT, hallmark mutations of UV mutagenesis. Eighty percent (41/51) of the mutations were single or non-tandem base pair substitutions, the majority of which (27/41) were C-->T transitions. Ninety-five percent of such mutations occurred at dipyrimidine sites. Through a rigorous statistical test, the UV-induced p53 mutation spectrum appears to differ significantly (P < 0.008) from the one induced by the antineoplastic drug chloroethyl-cyclohexyl-nitrosourea, and to be indistinguishable from the one observed in NMSC (P = 0.4). These results demonstrate that the assay allows the determination of carcinogen-specific p53 mutation fingerprints and represents a new tool for molecular epidemiology.

DNA, Complementary↗
PubMed1997

Simple identification of dominant p53 mutants by a yeast functional assay.

Analysis of families with germline p53 mutations shows that the mutant p53 allele behaves as a dominant oncogene at the genetic level, although it behaves as a recessive oncogene at the cellular level, since tumours invariably show mutation or loss of both wild-type alleles. At the biochemical level it is possible that some clinically important mutant p53 proteins may be carcinogenic through a dominant mechanism. We show that p53 mutants can be readily classified according to their dominant potential using a simple yeast functional assay. Wild-type p53 is constitutively expressed from a TRP1 vector, p53 mutants are expressed from an otherwise identical LEU2 vector and net transcriptional activity is scored using an ADE2-based reporter. Twenty seven p53 mutants were tested: 19 were recessive, i.e. gave white colonies, and eight showed dominant activity, i.e. gave pink/red colonies. This simple assay should facilitate studies on p53 dominance.

DNA, Complementary↗
PubMed1996

p53 is frequently mutated in Barrett's metaplasia of the intestinal type.

Barrett's Esophagus (BE) is a complication of gastroesophageal reflux in which the normal squamous epithelium of the lower esophagus is replaced by metaplastic tissue. The clinical significance of this condition is the associated predisposition to adenocarcinomas (ADCs). Three types of BE have been characterized: the gastric fundic (F) type, the gastric cardial (C) type, and the intestinal (I) type. The latter is the most closely associated with the development of ADCs; the causes of this bias remain unknown. To determine whether p53 and/or K-ras gene alterations (a) are present in preneoplastic lesions and (b) are associated with a specific histotype, we performed PCR-based denaturing gradient gel electrophoresis (DGGE) analysis of exon 1 (codons 12-13) of K-ras gene and of exons 5-8 of the p53 gene in biopsies obtained from 30 patients with BE of the I type (9 patients), combined I type (I + C +/- F; 10 patients) and non-I type (C, F, or C + F; 11 patients). None of the cases under study revealed K-ras mutations, whereas biopsies from 12 patients showed at least one p53 DGGE variant. Four patients showed the exact same variants in leukocytes also (polymorphisms), whereas eight cases revealed specific DGGE variants only in biopsies. The molecular characterization of these variants revealed that four of them showed a single base pair substitution, and four showed multiple mutations. Of 17 somatic mutations, all but 1 were base pair substitutions located mainly in exons 7 and 8. The majority of these mutations were GC targeted (13 of 16; 81%), 54% (7 of 13) of which were transitions occurring at CpG sites. All somatic mutations were found in BE with at least one I component. The association with the histotype was statistically significant (P < 0.03; pure I type versus non-I type; P < 0.04, combined I type versus non-I type; Fisher's exact test). Loss of heterozygosity in the vicinity of the p53 locus was evaluated by PCR using a highly polymorphic variable number of tandem repeats marker on 25 out of 30 cases. Ninety-two % of the cases analyzed were informative, and none of them showed LOH. In conclusion, we showed that p53 mutations are frequently observed in specimens from BE patients of the I-type, whereas no involvement of K-ras (exon 1) mutational activation was observed. In light of the key roles that the p53 protein plays in controlling cell cycle and cell diploidy, this result may suggest why this type of metaplasia is the most closely associated to the development of ADCs.

Adult↗
PubMed1995

Induction of kinetochore-containing micronuclei by exogenous O6-methylguanine requires conversion of the methylated base to a nucleotide.

It has been reported that exogenous alkylated purines, such as O6-methylguanine (O6meG), induce aneuploidy in mammalian cells. It is shown here that the aneugenic effect of O6meG, evidenced by its ability to induce micronuclei in rodent cells, is dependent on its conversion to O6-methyl-guanosine-5'-monophosphate (O6me-5'-GMP) by hypoxanthine-guanine phosphoribosyl transferase (HPRT). This conclusion, in contrast with previous in vitro data showing that O6meG does not seem to be a substrate for HPRT, was based on the following observations: 1) O6meG did not induce micronuclei in HPRT-deficient Chinese hamster cells, but did induce micronuclei in HPRT-proficient cells, and in mouse cells partially or totally deficient in adenine phosphoribosyl transferase; 2) O6meG was not metabolized in HPRT-deficient cells, while in wild-type cells a number of metabolites were detected by high performance liquid chromatography (HPLC) analysis of cold acid extracts, one of them coeluting with O6me-5'-GMP used as a marker; 3) when de novo synthesis of purine nucleotides was inhibited by aminopterin, O6meG sustained the growth of HPRT-proficient, but not of HPRT-deficient, cells; and 4) when HPRT-deficient cells were treated with liposomes charged with O6me-5'-GMP, induction of micronuclei was shown. The finding that methylated guanine exerts its aneugenic action through methylated nucleotide(s) provides an important, though indirect, support to the hypothesis that alkylating agents may induce aneuploidy via nucleotide pool alkylation.

Adenine↗
PubMed1989

Interaction retinol-chromatin: an analysis of DNA from vitamin A-treated V79 Chinese hamster cells.

When V79 cells are incubated in the presence of radiolabeled retinol, a small but consistent amount of radioactivity remains associated with nuclear DNA. Chromatographic analysis of enzymatic hydrolysates of DNA shows that no irreversible changes, such as adducts, have taken place on DNA. We present evidence that this radioactive incorporation may occur via a metabolic conversion of retinol, leading mainly to the formation of radiolabeled thymidine, which is then incorporated into newly made DNA. Mutagenic effects by retinol, given at concentrations well above physiological levels, have been also excluded.

Animals↗
PubMed1986

[Psychalgia in rheumatology. Methods of approach and intervention].

The observation of several cases triggers a review of psychological pain in rheumatology. Such situations require a thorough grounding on the part of the rheumatologist who must work with the psychiatrist and clinical psychologist in the treatment of such patients. The importance of teamwork and the need for clinical psychologists and psychiatrists in rheumatology wards is emphasised and the approach and treatment techniques to be adopted for patients suffering from so-called psychological pain are outlined. Essentially, the first (approach) stage involving several phases will be followed by a second stage to be evaluated by the team in an attempt to provide the patient with the most appropriate treatment.

Adolescent↗
PubMedDate not supplied

The use of antileukotrienes in paediatrics.

Allergic diseases include a variety of different illnesses (rhinitis, conjunctivitis, asthma, urticaria, dermatitis) whose physiological and pathological basis is the release of chemical mediators such as histamine, PAF (platelet activating factor), metabolites of arachidonic acid and chemotactic factors from mastocytes, basophils and eosinophils. The numerous drugs used for allergy treatment now include the new pharmacological category of cysteinyl leukotriene antagonists. The cysteinyl leukotrienes (LTC4, LTD4 and LTE4) are chemical mediators of the inflammation involved in the pathogenesis of asthma, the biological effects of which are bronchial constriction and an increase in both mucus secretion and vascular permeability. Recent studies carried out above all on adult patients suggest that antileukotrienes can play an important part not only in the acute phase but also in controlling the chronic development of bronchial asthma. Antileukotrienes have also been successfully used by some authors to control atopic dermatitis and urticaria. Though further controlled testing will be required, these applications broaden the possible range of treatments for allergic disease in all its many aspects.

Adolescent↗
PubMedDate not supplied

[Guidelines for health surveillance of health care workers exposed to biological risks set up by the Italian Society for Occupational Health and Industrial Hygiene: application in health facilities of the Regional Health Administration--District No. 7, Ancona].

BACKGROUND: Evidence Based Medicine and the need to achieve better management of health budgets call for verification and, if necessary, revision of the criteria used in Occupational Health, in order to ensure appropriate measures as regards protection of health at the workplace. In December 2003, the Marche Region initiated a process of reform of the regional health service, which will be completed in two years, and will provide a new regional health organization that will improve the quality and appropriateness of health services available to the population. The reform also involves the Occupational Health Services responsible for prevention activities for 20,000 health care workers employed in regional public health facilities. The need was strongly felt to set up a network that would meet the health needs of health care workers, by adopting a common language among occupational health physicians, by eliminating reported criticism, which is due not only to lack of communication between different structures and profiles, but also to the different methods of approach, evaluation and management of occupational risks. OBJECTIVES AND METHODS: From a historical point of view, the health sector has the biggest as regards prevention of biological risk. Therefore, with a view to harmonizing actions and approach among occupational health physicians in the evaluation of this risk, the publication by the Italian Society for Occupational of Health and Industrial Hygiene of Guidelines for health surveillance of health care workers exposed to biological risks, produced by the working group under the leadership of Prof. Lorenzo Alessio, was considered to offer an interesting opportunity to verify the reproducibility of methods and the quality of results, as applied to health facilities under the Regional Health Administration in Ancona (District No. 7). RESULTS AND CONCLUSION: The Guidelines fully confirmed the need to plan activities, starting from analysis of epidemiological and occupational data, thus assuring good results both in terms of efficacy and efficiency of the health surveillance programme used. This method also assures a high level of appropriateness of medical measures as regards the "safety" target, at the same time avoiding waste and poor management of human and economic resources, which were till now caused by differences in methods used in assessment of biological risk.

Biological Factors↗