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.

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.

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.

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.

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.

[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.

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.

Implications of alpha-amylase production and beta-glucuronidase expression in Escherichia coli strains.

Two Escherichia coli strains in which alpha-amylase production differed were used to study in depth some characteristics related to beta-glucuronidase induction by starch. The beta-glucuronidase background activity in Luria broth medium was comparable for the two isolates, but only amylase positive S1 was able to grow on starch molecules supplied as the sole carbon source. In this case growth resulted at higher beta-glucuronidase levels (p < 0.01) with respect to basal activity and the induced expression was maximal (6.1-fold) when cultures reached the stationary phase. Growth in the presence of a protein synthesis inhibitor (chloramphenicol) was associated with a marked reduction of activity. The beta-glucuronidase activity of amylase negative M94 remained unchanged during starvation on starch medium, but an induced response was observed with methylumbelliferyl-glucuronide. These results further support the hypothesis that starch metabolism is involved in the complex beta-glucuronidase regulation of E. coli strains. This is relevant not only for basic research but also to investigating gut microbial enzymology.

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.

UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior.

The end-to-end association of chromosomes through their telomeres has been observed in normal cells of certain organisms, as well as in senescent and tumor cells. The molecular mechanisms underlying this phenomenon are currently unknown. We show here that five independent mutant alleles in the Drosophila UbcD1 gene cause frequent telomere-telomere attachments during both mitosis and male meiosis that are not seen in wild type. These telomeric associations involve all the telomeres of the D. melanogaster chromosome complement, albeit with different frequencies. The pattern of telomeric associations observed in UbcD1 mutants suggests strongly that the interphase chromosomes of wild-type larval brain cells maintain a Rab1 orientation within the nucleus, with the telomeres and centromeres segregated to opposite sides of the nucleus. The UbcD1 gene encodes a class I ubiquitin-conjugating (E2) enzyme. This indicates that ubiquitin-mediated proteolysis is normally needed to ensure proper telomere behavior during Drosophila cell division. We therefore suggest that at least one of the targets of UbcD1 ubiquitination is a telomere-associated polypeptide that may help maintain proper chromosomal orientation during interphase.

Chromatin and microtubule organization during premeiotic, meiotic and early postmeiotic stages of Drosophila melanogaster spermatogenesis.

Larval and pupal testes of Drosophila melanogaster were fixed with a methanol/acetone fixation procedure that results in good preservation of cell morphology; fixed cells viewed by phase-contrast optics exhibit most of the structural details that can be seen in live material. Fixed testis preparations were treated with anti-tubulin antibodies and Hoechst 33258 to selectively stain microtubules and DNA. The combined analysis of cell morphology, chromatin and microtubule organization allowed a fine cytological dissection of gonial cell multiplication, spermatocyte development, meiosis and the early stages of spermatid differentiation. We placed special emphasis on the spermatocyte growth phase and the meiotic divisions, providing a description of these processes that is much more detailed than those previously reported. In addition, by means of bromo-deoxyuridine incorporation experiments, we were able to demonstrate that premeiotic DNA synthesis occurs very early during spermatocyte growth.

Comparison of fluorogenic and conventional membrane filter media for enumerating coliform bacteria.

4-Methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide were added to MacConkey broth and their diagnostic powers for total coliforms (TC) and Escherichia coli, respectively, were tested by membrane filtration at primary isolation. Examining water samples from different sources proved the usefulness of fluorogenic rather than reference media both as regards recovery efficiency and rapidity (possible within 12 h) of analyses. The recoveries obtained by fluorogenic and conventional tests for both TC and E. coli were correlated. Values were comparable in surface water samples, while a higher sensitivity of fluorogenic media was observed in samples of shallow contaminated ground water. Results seem to indicate that the use of fluorogenic membrane filtration analysis for colimetric indicators could be favourably considered especially for sanitary surveying of drinking water.

Carbohydrate enriched diets and bacterial glycosidases in rat faeces.

The expression of some faecal hydrolytic enzymes in rats fed for 4 months on sucrose or starch enriched diets was compared with a standard diet. The assay reliability was confirmed for animal and experimental variability. The beta-D-glucuronidase and beta-D-glucosidase activities were always higher in rats fed on starch than on other diets. N-acetyl-beta-D-glucosaminidase and N-acetyl-beta-D-galactosaminidase showed decreased activity when passing from a standard to a sucrose, and from a sucrose to a starch diet. There was little modification in the levels of faecal alpha-D-glucosidase, sulphatase and protease with the various experimental diets.

beta-N-acetylhexosaminidases from Serratia marcescens.

Two forms of beta-N-acetylhexosaminidase from Serratia marcescens with an optimum pH of 5.0 and 6.5, respectively, to 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside were separated by DEAE-cellulose chromatography and Sephacryl S-200 chromatography. On the basis of their molecular weights, thermal stability, substrate specificity and isoelectric points, the form with an acidic pH optimum resembled hexosaminidase B, whereas the form with a neutral pH optimum resembled hexosaminidase C. Lectin binding studies showed that the acidic form does not bind to concanavalin-A-Sepharose, Tetragonolobus purpurea-agarose, wheat germ-agglutinin-Sepharose or Ricinus communis-agglutinin-agarose, whereas the neutral form binds to the last two lectin columns.

Fluorogenic detection of atypical coliforms from water samples.

We examined the effectiveness of fluorogen in detecting bacterial enzymes in atypical or injured coliform strains in environmental water samples. 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide, substrates for beta-galactosidase and beta-glucuronidase respectively, were used as markers for total and faecal coliform bacteria and it was confirmed that fluorogenic assays have a greater sensitivity than reference methods. It was also observed that adding MU-conjugates (50 micrograms/ml) to low selective media for membrane filtration, besides shortening test times, reduces false negative results when detecting sanitary microbial indicators of water pollution.

[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.