DNA damage response, checkpoint activation and dysfunctional telomeres: face to face between mammalian cells and Drosophila.

Eukaryotic cells evolved telomeres, specialized nucleoproteic complexes, to protect and replicate chromosome ends. In most organisms, telomeres consist of short, repetitive G-rich sequences added to chromosome ends by a reverse transcriptase with an internal RNA template, called telomerase. Specific DNA-binding protein complexes associate with telomeric sequences allowing cells to distinguish chromosome ends from sites of DNA damage. When telomeres become dysfunctional, either through excessive shortening or due to defects in the proteins that form their structure, they trigger p53/pRb pathways that limits proliferative lifespan and eventually leads to chromosome instability. Drosophila lacks telomerase, telomeres are assembled in a sequence-independent fashion and their length is maintained by transposition of three specialized retroelements. Nevertheless, fly telomeres are maintained by a number of proteins involved in telomere metabolism as in other eukaryotic systems and that are required to prevent checkpoint activation and end-to-end fusion. Uncapped Drosophila telomeres induce a DNA damage response just as dysfunctional human telomeres. Most interestingly, uncapped Drosophila telomeres also activate the spindle assembly checkpoint (SAC) by recruiting the SAC kinase BubR1. Here we review parallelisms and variations between mammalian and Drosophila cells in the crosstalks between telomeres and cell cycle regulation.

Organization and maintenance of Drosophila telomeres: the roles of terminin and non-terminin proteins.

Drosophila telomeres are elongated by occasional transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the sequence of the DNA termini. Drosophila telomeres are capped by terminin, a complex formed by the HOAP, Moi, Ver and HipHop proteins that localize exclusively at telomeres and protect them from fusion events. Other proteins required to prevent end-to-end fusion include HP 1 Eff/UbcD 1, ATM, the components of the Mrel 1-Rad50-Nbs (MRN) complex, and the Woc transcription factor. The terminin proteins are encoded by fast-evolving genes and are not evolutionarily conserved outside the Drosophila species. In contrast, the non-terminin telomere capping proteins are not fast-evolving, do not localize only at telomeres and are conserved from yeasts to mammals. We propose that following telomerase loss, Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent manner, and that non-terminin proteins did not evolve as rapidly as terminin because of the functional constraints imposed by their involvement in diverse cellular processes. This hypothesis suggests that the Drosophila non-terminin proteins might correspond to ancestral telomere-associated proteins with homologues in other organisms including humans.

Low dose rate γ-irradiation protects fruit fly chromosomes from double strand breaks and telomere fusions by reducing the esi-RNA biogenesis factor Loquacious.

It is still continuously debated whether the low-dose/dose-rate (LDR) of ionizing radiation represents a hazard for humans. Model organisms, such as fruit flies, are considered valuable systems to reveal insights into this issue. We found that, in wild-type Drosophila melanogaster larval neuroblasts, the frequency of Chromosome Breaks (CBs), induced by acute γ-irradiation, is considerably reduced when flies are previously exposed to a protracted dose of 0.4 Gy delivered at a dose rate of 2.5 mGy/h. This indicates that this exposure, which is associated with an increased expression of DNA damage response proteins, induces a radioadaptive response (RAR) that protects Drosophila from extensive DNA damage. Interestingly, the same exposure reduces the frequency of telomere fusions (TFs) from Drosophila telomere capping mutants suggesting that the LDR can generally promote a protective response on chromatin sites that are recognized as DNA breaks. Deep RNA sequencing revealed that RAR is associated with a reduced expression of Loquacious D (Loqs-RD) gene that encodes a well-conserved dsRNA binding protein required for esiRNAs biogenesis. Remarkably, loss of Loqs mimics the LDR-mediated chromosome protection as it decreases the IR-induced CBs and TFs frequency. Thus, our molecular characterization of RAR identifies Loqs as a key factor in the cellular response to LDR and in the epigenetic routes involved in radioresistance.

Telomere capping and cellular checkpoints: clues from fruit flies.

In most organisms, telomeres consist of repetitive G-rich sequences that are elongated by a specific reverse transcriptase, telomerase. A large number of proteins are recruited by these terminal repeats, forming specialized structures that regulate telomerase activity and protect telomeres from degradation and recombination. Drosophila lacks telomerase and telomere length is maintained by transposition of three specialized retrotransposons. In addition, unlike yeast and mammals, Drosophila telomeres are epigenetically determined, sequence-independent structures. However, several proteins required for Drosophila telomere behavior are evolutionarily conserved. These include the Mre11-Rad50-Nbs (MRN) complex and the Ataxia Telangiectasia Mutated (ATM) kinase, which are required to prevent telomeric fusions. In addition, recent studies have provided evidence that Drosophila uncapped telomeres elicit a DNA damage response (DDR) just as dysfunctional yeast and human telomeres. Uncapped Drosophila telomeres also activate the spindle assembly checkpoint (SAC) by recruiting the SAC kinase BubR1. Telomere-induced DDR and SAC both require the wild type function of the MRN complex. In addition, while DDR is mediated by ATR kinase, SAC activation requires both the ATM and ATR activities. These results indicate that the DNA repair systems play multiple roles at Drosophila telomeres, highlighting the importance of this model organism for investigations on the relationships between DNA repair and telomere maintenance.

Effect of natural early menopause on bone mineral density.

OBJECTIVES: Early menopause (EM) is included among the risk factors for osteoporosis. Several studies have shown that women with early menopause have lower bone mineral density (BMD) than those with normal expected age of menopause. The aim of our cross-sectional study was to investigate the effects of time of menopause on vertebral bone mass in healthy postmenopausal women and to evaluate if early menopause is a risk factor for lower vertebral BMD. METHOD: We studied 782 who had never received drugs acting on bone mass. The study population was divided into three groups: women with early, normal (NM), and late (LM) menopause. Our study population was further categorized in 5-year age segments between 45 and >75. RESULTS: The three groups examined did not differ for age, age at menarche, body mass index (BMI), and vertebral BMD, while there were significant differences in age at menopause and years since menopause. Our study showed that women with EM presented significantly lower vertebral BMD than NM and LM in 50-54 age segments. Beyond 55 years, EM, NM, and LM women had no differences in lumbar BMD values. CONCLUSIONS: In conclusion, controversial data demonstrated that the absolute amount of bone loss is greater after early menopause than after normal or late menopause, even if a slight effect of early menopause on bone mass cannot be excluded.

In vitro inhibitory activity of probiotic spore-forming bacilli against genotoxins.

AIMS: To investigate the ability of bacilli of various species (Bacillus clausii, Bacillus subtilis, Bacillus lentus, Bacillus pumilus. Bacillus megaterium, Bacillus firmus, Bacillus sp.) and origins (probiotic and collection strains) to counteract the activity of some representative DNA-reactive agents. METHODS AND RESULTS: The inhibitory effect of 21 bacilli strains, previously characterized by tDNA-PCR, on four genotoxins, was examined in vitro using the short-term assay SOS-Chromotest. All strains had a high inhibitory activity against 4-nitroquinoline-1-oxide and N-methyl-N'-nitro-nitrosoguanidine (direct agents), whereas the inhibitory activity was high or moderate against 2-amino-3,4-dimethylimidazo[4,5-f]quinoline and aflatoxin B1 (indirect agents). Antigenotoxicity was observed in vegetative cells, but not heat-treated cells or spore suspensions. The spectroscopic properties of compounds were modified after cell co-incubation and all the strains maintained high viability after exposure to the genotoxins. CONCLUSIONS: No relevant differences in antigenotoxicity were evidenced among strains of the examined species or between probiotic and collection strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Although derived from an in vitro model, the results suggest that Bacillus-based probiotics could be useful for reducing the gastrointestinal risk originating from genotoxic agents.

[Occupational accidents among immigrant workers in the Fabriano areas]. / II fenomeno infortunistico nei lavoratori extracomunitari della zona territoriale n. 6 Fabriano.

BACKGROUND: The growing contribution of immigrant workers to the national economy particularly affects the trend of accidents at work. OBJECTIVES: The aim of this study was to describe the trend of work accidents in the Local Health Area No. 6 - Fabriano (Marche Region), during the period 2000-2003; to define the frequency for each job sector, age, gender, type of work, severity, month, day and week and time of day; to calculate the incidence rate for each year taken into consideration. METHODS: The sources of information used were: 1) The "New Informative Flows" database set up by Italian National Institute of Insurance for Occupational Injury (INAIL), Italian Superior Institute for Work Prevention and Safety (ISPESL) and Regional Governments, and the "EPIWORK software", for the total number of occupational accidents among immigrant workers. 2) The local Jobs and Training Centre of Fabriano. We used three different correction indexes to evaluate the number of hired workers so as to estimate the rate of accident incidence among immigrant workers. RESULTS: The total number of occupational accidents reached its peak in 2001 as a result of the rise in the number of employed people. After this date, the trend started to reverse and in 2002 an increase in the number of employed people--although smaller compared to the previous year--was accompanied by a reduction in the overall number of accidents, a reduction that became even more evident in 2003. Occupational accidents among immigrant workers gradually rose and peaked in 2002. The sectors with high rates of accidents were the mechanical engineering and metallurgic sectors and the construction industry. Accidents occurred mainly among young people (18 to 34years old). As for gender, there was a marked prevalence of men (83.3%) over women (16.7%). Most accidents had a prognosis of 8 to 30 days. The number ofoccupational accidents with a prognosis of 8 to 30 days fell progressively for workers in general but gradually rose for immigrant workers with a peak in 2001. The overall number of occupational accidents that caused permanent invalidity fell by 52.3% for the workforce in general, and by 25% among immigrant workers. CONCLUSIONS: This study shows that immigrant workers employed in the Fabriano area had a higher risk of accidents at work.

Screening of potential lactobacilli antigenotoxicity by microbial and mammalian cell-based tests.

Antigenotoxicity is considered an important property for probiotic lactobacilli. The ability of non probiotic lactobacilli from dairy products and starters to inhibit two reference genotoxins: 4-nitroquinoline-1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine was evaluated. The study was carried out using short-term assays with different targets, such as procaryotic cells (SOS-Chromotest for genotoxicity in Escherichia coli and Ames test for mutagenicity in Salmonella typhimurium) and eucaryotic cells (Comet assay for genotoxicity in Caco-2 enterocytes). A high proportion of strains inhibiting 4-nitroquinoline-1-oxide activity was found in Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus plantarum. Inhibition of N-methyl-N'-nitro-N-nitrosoguanidine activity occurred in only one L. acidophilus strain. All the strains with antigenotoxic properties also demonstrated antimutagenic activity and produced modifications in genotoxin spectroscopic profiles. Strain viability during and after genotoxin exposure was confirmed. Concordance of the results obtained with microbial and mammalian cell-based tests is underlined.

Evaluation of the toxic effect of Cd2+ and Cd(CN)2-4 ions on the growth of mixed microbial population of activated sludges.

The effect has been studied of Cd2+ and Cd(CN)2-4 ions on the growth and enzymatic activity of mixed microflora from activated sludges. Both ions tested significantly inhibited the growth of microorganisms, estimated by means of the optical density, and the inhibitory effect of Cd2+ was significantly greater than that of Cd(CN)2-4 at the same mass and molar concentrations. There was also inhibition of the maximum uptake rate of glucose, but in this case there were no statistically significant differences between the inhibitory effect of the corresponding mass and molar concentrations of Cd2+ and Cd(CN)2-4.

[Development of antibiotic resistance in purified sewage effluents subjected to chlorination]. / Evoluzione dell'antibiotico-resistenza in effluenti fognali depurati sottoposti a clorazione.

Antibiotic-resistance is widely spread phenomenon in the environment because of uncontrolled discharge of urban and animal wastewaters. Sewage treatment can significantly reduce the number of both sensitive and resistant bacteria. A reduction of about 1.5 logarithmic units in faecal coliforms was observed during biological treatment (3, 7), but a simultaneous increase in the percentage of resistant strains occurred because of not well understood selection phenomena. The above reported bacterial reduction is not always sufficient to meet the quality standards of Italian legislation required to discharge the treated effluents into surface waters, and so, chlorination become a compulsory additional treatment whose impact on both sensitive and resistant microflora must be evaluated. The results obtained in the present research have demonstrated that chlorine concentrations in the range of 0.5-2 ppm are able to reduce significantly the faecal coliforms concentrations and, in particular, treatment with 1 ppm of chlorine for 1 hour reduces the concentration of the above reported bacteria to the extent of 2 logarithmic units, so that their final concentration are of the about 10(2)/100 ml. The surviving chlorine tolerant bacteria seem to be antibiotic resistant in higher percentage than the chlorine sensitive ones and so, as a consequence, a significant increase in the antibiotic resistance and multiresistance was observed in the chlorinated effluents. In this context it is interesting to underline the larger variety of resistance patterns observed in the chlorine-resistant bacteria in comparison with the uniformity in the resistance patterns observed in isolated from unchlorinated effluents. The selected chlorine-tolerant strains seem to be less able to transfer their resistances under laboratory conditions, not because of curing effect of chlorine on the plasmids but, probably, because of the damage to cellular cell envelopes.

Tolerance to challenges miming gastrointestinal transit by spores and vegetative cells of Bacillus clausii.

AIMS: To study Bacillus clausii from a pharmaceutical product (Enterogermina O/C, N/R, SIN, T) and reference strains (B. clausii and Bacillus subtilis) for eco-physiological aspects regarding the gut environment. METHODS AND RESULTS: Spores and vegetative cells were challenged in vitro miming the injury of gastrointestinal transit: pH variations, exposure to conjugated and free bile salts, microaerophilic and anaerobic growth. No relevant differences were found studying the growth at pH 8 and 10, whereas at pH 7 the yields obtained for O/C and SIN were higher than those obtained for N/R and T strains. The spores were able to germinate and grow in the presence of conjugated bile salts (up to 1%, w/v) or free bile salts (0.2%) and also exhibited tolerance for the combined acid-bile challenge. As evidenced by lag-time, growth rate and cell yield the tolerance of Enterogermina isolates for conjugated salts was comparable with that of B. clausii type strain (DSM 8716(T)), and resulted higher than that observed for B. subtilis (ATCC 6051(T)). All the considered B. clausii strains demonstrated microaerophilic growth, but only some grew anaerobically in a nitrate medium. CONCLUSIONS: The ability of B. clausii spores to germinate after an acid challenge and grow as vegetative cells both in the presence of bile and under limited oxygen availability is consistent with the beneficial health effects evidenced for spore-forming probiotics in recent clinical studies. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental evidence from this study emphasizes some functional properties of B. clausii strains regarding their use as probiotics.

Comparison of the toxicity of some metals and their tetracyanide complexes on the respiration of non acclimated activated sludges.

The toxic effect of the metal ions of cadmium, zinc, nickel and mercury and their tetracyanide salt complexes, on the activated sludge not previously acclimated, has been studied. The evaluation of the effect was carried out using both the Warburg and TTC-method. The results obtained have shown that the toxicity of the cadmium and zinc complexes is higher than that of the corresponding metals, while the toxicity of Ni(CN)4(2-) is lower than that of the corresponding metals. No differences have been found between the effect of mercury and the corresponding tetracyanide complex. From the data obtained it appears that it is not possible to generalize about the biological effect of complexation with the CN- group, but it should be stated that, generally, there are substantial differences between metals and their cyanide complexes as far as toxicity for activated sludge is concerned.

The validity of the TTC-test for dehydrogenase activity of activated sludges in the presence of chemical inhibitors.

Dehydrogenase activity of activated sludges coming from a laboratory plant, has been evaluated by the TTC-test in the presence of some chemical toxics. The results obtained have shown that, for all inhibitors tested, there exists a range of concentrations in which the dose-response relationship is linear. As far as the comparison of the inhibition data from the TTC-test and from the Oxygen-respiration rate is concerned, the results obtained show that the inhibitors tested may be divided into three groups: (i) inhibitors which inhibit the Oxygen-respiration rate more than th Dehydrogenase activity (Zn2+, Cd2+, Ni2+, Hg2+, Zn(CN)4(2-), Cd(CN)4(2-)); (II) inhibitors for which there are no significant differences in the inhibition value obtained by the two methods (CN-, C6H5O-, C6Cl5O-, C6H3ClO-, Hg(CN)4(2-)); (iii) inhibitors which inhibit the dehydrogenase activity more than theoxygen-respiration rate (Cu2+, CrO4(2-), Ni(CN)4(2-)). The inhibition data obtained by other authors evaluating by direct methods (COD reduction, turbidity of the final effluent etc.) the performance of pilot or laboratory plants in the presence of some of the toxics used in the present research, are in sufficient agreement with the dehydrogenase inhibition data obtained in this work.

Bacterial amylolytic activity enhances beta-glucuronidase expression of amylase-negative Escherichia coli strain in starch-medium.

The possibility of associating starch degradation with bacterial beta-glucuronidase expression was examined. We proved that starving, in starch medium, amylase-negative Escherichia coli (M94) which has constitutive beta-glucuronidase greatly reduces (p < 0.01) its background activity, but the addition of both cell-free supernatants or cells of Bacillus subtilis (B10) producing amylase greatly increases (p < 0.01) the E. coli beta-glucuronidase activity. Increases in activity were maximal when amylase in the medium ranged from 0.3 to 0.8 U ml-1 and pH from 6.8 to 6.3, whereas higher amylase activity interacted with E. coli viability and the effect on beta-glucuronidase was less evident. The impact of B. subtilis amylase on E. coli beta-glucuronidase induction, observed when the organisms were co-cultured, indirectly supports the hypothesis that amylolytic activity of hindgut bacteria may be effective on beta-glucuronidase induction of the climax microflora. This last finding is important in the health field, considering the implication between the deconjugating role of this enzyme and consequent activation of toxic and carcinogenic compounds.

Effect of different starches on Escherichia coli (S1) beta-glucuronidase expression.

Studies with pure cultures growing in laboratory media indicated that beta-glucuronidase expression of Escherichia coli S1 was considerably affected by starch added to the medium as the only carbon source. This result, which may be an aspecific modulation of enzyme expression, was independent of the starch molecular structure and effects were analogous for maize, rice, wheat or potato starches. It was observed that enzyme expression was little affected by the growth rate. The beta-glucuronidase activities of starch-grown bacteria found in the present study agree with those observed in animal and human models performed for in vivo evaluation of effects of dietary starch effects on gut microbial ecosystems.

The tolerance of an environmental strain of Escherichia coli to some heavy metals.

The effect of mercury, cadmium, copper, zinc, chromium and lead on an environmental strain of Escherichia coli was studied. The tolerance growth limits, growth kinetics and viability were determined. It was found that metal-ion concentrations greater than the threshold values (0.005 microM Hg2+, 0.5 microM Cd2+, 5 microM Cu2+, 23 microM Cr3+, 30 microM Zn2+, 45 microM Pb2+) increase the lag-phases but do not modify the growth kinetics (Pb2+ is an exception). During the lag-phase a reduction in cell viability, varying from a maximum of 89% for lead, to a minimum of 13% for cadmium, was observed. The reduction of viability accounts for a cells physiological adaptation to the metals rather than selection of resistant mutants. Loss of the acquired metal resistance following exposure of the bacterial cells to the metals supported this last point.

[Submerged-phase gel electrophoresis in the analysis of plasmids for epidemiologic purposes]. / Gel elettroforesi in fase sommersa nell'analisi plasmidica a fini epidemiologici.

The validity of the submarine agarose gel electrophoresis as routine method for plasmidic epidemiology was considered. Using standard plasmids, the efficiency of a performed protocol conditions (5 V/cm for 3 hours, 0.8% agarose) in screening plasmids of different size was studied. The results provide sharp profile resolution and an accurate estimate of molecular weights in the range from 1.2 to 112 megadaltons.

Resistotype heterogeneity of Escherichia coli strains isolated from humans, animals and sewage waters.

The tests commonly used for bacterial identification, especially in the field of microbial environmental analyses frequently do not provide a sufficient strain differentiation. Considering the importance that accurate characterization of bacterial pollution indicators could have as epidemiological tools, this study used a resistogram subtyping method for Escherichia coli as a tentative method of effecting a good monitoring of the environmental spread of this microorganism. The resistance of 313 E. coli strains of different origin (human, animal, sewage), previously identified by standard biochemical reactions, to 8 chemical compounds (inorganic, organic and dyes) and to 7 antibiotics was tested. The results indicated this method has a higher discriminatory power for chemicals than for drugs. Some typical resistotype patterns for E. coli from various sources are described.