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ABSTRACT: The present work aimed to investigate whether exposure to static magnetic field (SMF) and extremely low frequency magnetic field (ELF-MF) can induce biomolecular changes on Tuber borchii hyphal growth. Tuber borchii mycelium was exposed for 1 h for 3 consecutive days to a SMF of 300 mT or an ELF-MF of 0.1 mT 50 Hz. Gene expression and biochemical analyses were performed. In mycelia exposed to ELF-MF, some genes involved in hyphal growth, investigated using quantitative real-time polymerase chain reaction, were upregulated, and the activity of many glycolytic enzymes was increased. On the contrary, no differences were observed in gene expression after exposure to SMF treatment, and only the activities of glucose 6-phosphate dehydrogenase and hexokinase increased. The data herein presented suggest that the electromagnetic field can act as an environmental factor in promoting hyphal growth and can be used for applicative purposes, such as the set up of new in vitro cultivation techniques.
Canadian Journal of Microbiology 10/2012; · 1.36 Impact Factor
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ABSTRACT: The present work aimed to investigate whether exposure to static magnetic field (SMF) and extremely low frequency magnetic field (ELF-MF) can induce biomolecular changes on Tuber borchii hyphal growth. Tuber borchii mycelium was exposed for 1 h for 3 consecutive days to a SMF of 300 mT or an ELF-MF of 0.1 mT 50 Hz. Gene expression and biochemical analyses were performed. In mycelia exposed to ELF-MF, some genes involved in hyphal growth, investigated using quantitative real-time polymerase chain reaction, were upregulated, and the activity of many glycolytic enzymes was increased. On the contrary, no differences were observed in gene expression after exposure to SMF treatment, and only the activities of glucose 6-phosphate dehydrogenase and hexokinase increased. The data herein presented suggest that the electromagnetic field can act as an environmental factor in promoting hyphal growth and can be used for applicative purposes, such as the set up of new in vitro cultivation techniques.
Canadian Journal of Microbiology 09/2012; 58(10):1174-82. · 1.36 Impact Factor
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ABSTRACT: An aqueous extract of V. vinifera L. tendrils was evaluated for its ability to enrich the antioxidant capacity of cultured cells. The long-time antioxidant capability of the extract was measured by in vitro chemical methods, and its influence on reduced glutathione levels and plasma membrane oxido reductase activity was determined in cultured human keratinocytes (NCTC 2544). Keratinocytes are cells normally exposed to oxidative stress, and for this reason adequately equipped with antioxidant defences. However, it has long been suggested that exogenous antioxidants may play an important role in minimizing the adverse effects of oxidative stress on skin.We demonstrated that V. vinifera tendril aqueous extract was able to increase, in a time- and dose-dependent manner, the reduced glutathione concentration and activity of trans plasma membrane oxido reductase as an indirect evaluation of the intracellular redox status of the cells demonstrating a relevant antioxidant activity of this phytocomplex.
Natural product communications 09/2011; 6(9):1315-9. · 1.24 Impact Factor
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Lucia Potenza,
Cinzia Calcabrini,
Roberta De Bellis,
Michele Guescini,
Umberto Mancini,
Luigi Cucchiarini,
Gennaro Nappo,
Rossana Alloni,
Roberto Coppola,
Laura Dugo,
Marina Dacha
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ABSTRACT: Anastomotic dehiscence is one of the most severe complications of colorectal surgery. Gaining insight into the molecular mechanisms responsible for the development of anastomotic dehiscence following colorectal surgery is important for the reduction of postoperative complications.
Based on the close relationship between surgical stress and oxidative stress, the present study aimed to determine whether a correlation exists between increased levels of reactive oxygen species and colorectal anastomotic dehiscence.
Patients who underwent surgical resection for colorectal cancer were divided into three groups: patients with anastomotic dehiscence (group 1); patients without dehiscence who underwent neoadjuvant radiochemotherapy (group 2); and patients without anastomotic dehiscence who did not undergo neoadjuvant radiochemotherapy (group 3). Quantitative polymerase chain reaction and real-time polymerase chain reaction assays were performed to measure nuclear DNA and mitochondrial DNA (mtDNA) content, and possible oxidative damage to nonmalignant colon and rectal tissues adjacent to the anastomoses.
mtDNA content was reduced in the colon tissue of patients in groups 1 and 2. Rectal mtDNA was found to be more damaged than colonic mtDNAs in all groups. The 4977 bp common deletion was observed in the mtDNA of tissues from both the colon and rectum of all patients.
Patients in groups 1 and 2 were more similar to one another than to group 3, probably due to higher levels of reactive oxygen species in the mitochondria; the greater damage found in the rectum suggests that dehiscence originates primarily from the rectal area.
The present study of mtDNA analyses of normal human colon and rectal tissues from patients with colorectal cancer is among the first of its kind.
Canadian journal of gastroenterology = Journal canadien de gastroenterologie 08/2011; 25(8):433-9. · 1.21 Impact Factor
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ABSTRACT: In a previous investigation we reported that exposure to a moderate (300 mT) static magnetic field (SMF) causes transient DNA damage and promotes mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs). To better understand the response of HUVECs to the 300 mT SMF, a high-quality subtracted cDNA library representative of genes induced in cells after 4 h of static magnetic exposure was constructed. The global gene expression profile showed that several genes were induced after the SMF exposure. The characterized clones are involved in cell metabolism, energy, cell growth/division, transcription, protein synthesis, destination and storage, membrane injury, DNA damage/repair, and oxidative stress response. Quantitative real-time polymerase chain reaction (qRT-PCR) experiments were performed at 4 and 24 h on four selected genes. Their expression profiles suggest that HUVEC's response to SMF exposure is transient. Furthermore, compared to control cells, an up-regulation of several genes involved in cell growth and division was observed. This up-regulation is likely to be the cause of the slight, but significant, increase in cell proliferation at 12 h post-treatment. These results provide additional support to the notion that SMFs may be harmless to human health, and could support the rationale for their possible use in medical treatments.
Bioelectromagnetics 07/2011; 33(1):65-74. · 1.84 Impact Factor
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ABSTRACT: Surgical resection at any location in the body leads to stress response with cellular and subcellular change, leading to tissue damage. The intestine is extremely sensitive to surgical stress with consequent postoperative complications. It has been suggested that the increase of reactive oxygen species as subcellular changes plays an important role in this process. This article focuses on the effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer. Mitochondrial DNA copy number, mitochondrial common deletion and nuclear and mitochondrial 8-oxo-2'-deoxyguanosine content were measured. Both the colon and rectal tissue were significantly damaged either at the nuclear or mitochondrial level. In particular, mitochondrial DNA was more damaged in rectum than in colon. The present investigation found an association between surgical stress and nuclear and mitochondrial DNA damage, suggesting that surgery may generate an increase in free radicals, which trigger a cascade of molecular changes, including alterations in DNA.
Journal of Biosciences 06/2011; 36(2):243-51. · 1.65 Impact Factor
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ABSTRACT: This study describes the effects of a static magnetic field (SMF) on cell growth and DNA integrity of human umbilical vein endothelial cells (HUVECs). Fast halo assay was used to investigate nuclear damage; quantitative polymerase chain reaction (QPCR), standard PCR, and real-time PCR were used to evaluate mitochondrial DNA integrity, content, and gene expression. HUVECs were continually exposed to a 300 mT SMF for 4, 24, 48, and 72 h. Compared to control samples (unexposed cultures) the SMF-exposed cells did not show a statistically significant change in their viability. Conversely, the static field was shown to be significant after 4 h of exposure, inducing damage on both the nuclear and mitochondrial levels, reducing mitochondrial content and increasing reactive oxygen species. Twenty-four hours of exposure increased mitochondrial DNA content as well as expression of one of the main genes related to mitochondrial biogenesis. No significant differences between exposed and sham cultures were found after 48 and 72 h of exposure. The results suggest that a 300 mT SMF does not cause permanent DNA damage in HUVECs and stimulates a transient mitochondrial biogenesis.
Bioelectromagnetics 12/2010; 31(8):630-9. · 1.84 Impact Factor
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ABSTRACT: During the life cycle of mycorrhizal fungi, morphological, genetic and metabolic modifications are induced in the fungus and its symbiotic partner. These changes are influenced by environmental factors: light, gravity, oxygen, temperature, soil type, nutrients, root exudates and the presence of particular bacterial and perhaps fungal and viral populations in the mycorrhizosphere. To determine whether different carbohydrates lead to cell-signalling events and morphofunctional changes in cultured Tuber borchii mycelia, the expression level of genes involved in morphological modifications was investigated using a macroarray technique and real-time RT-PCR. The morphological study showed an increased growth of Tuber mycelia in glucose, while the hyphae were thinner and less branched in sucrose and maltose. This was accompanied by an upregulation of the genes involved in the general cell metabolism, detoxification processes, hyphal growth and cytoskeleton organization. Since glucose is also present in root exudates, the increased expression of these genes might support the hypothesis that glucose can act as a signal for the fungus to indicate the presence of the plant, and to trigger the complex symbiotic process. These mechanisms can lead to morphological modifications, including increased branching of the root which is necessary for the fungus to establish the symbiosis.
Journal of Molecular Microbiology and Biotechnology 03/2010; 18(2):120-8. · 1.95 Impact Factor
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ABSTRACT: Keratinocytes are cells strongly exposed to oxidative stress, but normally good equipped for antioxidant responses. However, it has long been suggested that exogenous antioxidants could play a useful role in minimizing the adverse skin responses associated with such oxidant species. In this work it was paid attention to the extract of Rhodiola rosea L. roots by using the phytocomplex as a whole because of the important activity of its composition and mutual distribution of its components. We have measured the protection afforded by the extract to reduced glutathione levels, glyceraldehyde-3-phosphate dehydrogenase activity, and thiobarbituric acid reactive substances levels in cultured human keratinocytes (NCTC 2544) exposed to different oxidative insults: Fe(II)/ascorbate, Fe(II)/H(2)O(2), and tert-butyl-hydroperoxide. We also have investigated the influence of the R. rosea extract on the production of intracellular reactive oxygen species and on the activity of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase). Furthermore, we have demonstrated that R. rosea extract was able to increase in a time- and dose-dependent manner the activity of the trans plasma membrane oxido reductase activity as an indirect evaluation of the intracellular redox status and this effect was already evident with small concentration of the extract and in a long time. As a result, NCTC 2544 are able to better counteract to several oxidative insults if incubated with R. rosea extract demonstrating a very good antioxidant activity of this phytocomplex.
Archives for Dermatological Research 09/2009; 302(3):191-200. · 2.28 Impact Factor
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ABSTRACT: Quercetin is a well-investigated antioxidant known to protect cells against oxidative nuclear DNA damage. There is no knowledge regarding its effect on oxidative mitochondrial DNA damage. In this study we investigated the effect of quercetin on oxidatively-injured DNA. Cell-free and cell studies were performed. Cell-free analyses carried out on plasmidic DNA showed that quercetin protects from all oxidative challenges used. Cellular studies were carried out on NCTC 2544 cells which were insulted with hydrogen peroxide and UVC radiations. Nuclear and mitochondrial DNAs were analysed by measuring DNA damage with a quantitative polymerase chain reaction. Quercetin supplementation showed significant genoprotective activity on mitochondrial DNA when hydroperoxide was used. The evidence of the protection afforded by quercetin suggests that this flavonoid may play an important role on mitochondrial genome stability.
BioFactors 08/2009; 33(1):33 - 48. · 4.93 Impact Factor
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ABSTRACT: Quercetin is a well-investigated antioxidant known to protect cells against oxidative nuclear DNA damage. There is no knowledge regarding its effect on oxidative mitochondrial DNA damage. In this study we investigated the effect of quercetin on oxidatively-injured DNA. Cell-free and cell studies were performed. Cell-free analyses carried out on plasmidic DNA showed that quercetin protects from all oxidative challenges used. Cellular studies were carried out on NCTC 2544 cells which were insulted with hydrogen peroxide and UVC radiations. Nuclear and mitochondrial DNAs were analysed by measuring DNA damage with a quantitative polymerase chain reaction. Quercetin supplementation showed significant genoprotective activity on mitochondrial DNA when hydroperoxide was used. The evidence of the protection afforded by quercetin suggests that this flavonoid may play an important role on mitochondrial genome stability.
BioFactors 02/2008; 33(1):33-48. · 4.93 Impact Factor
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ABSTRACT: Creatine is a naturally occurring compound obtained in humans from endogenous production and consumption through the diet. It is used as an ergogenic aid to improve exercise performance and increase fat-free mass. Lately, creatine's positive therapeutic benefits in various oxidative stress-associated diseases have been reported in literature and, more recently, creatine has also been shown to exert direct antioxidant effects. Oxidatively-challenged DNA was analysed to show possible protective effects of creatine. Acellular and cellular studies were carried out. Acellular assays, performed using molecular approaches, showed that creatine protects circular and linear DNA from oxidative attacks. Nuclear and mitochondrial DNAs from oxidatively-injured human umbilical vein endothelial cells were analyzed. Creatine supplementation showed significant genoprotective activity on mitochondrial DNA. This evidence suggests that creatine may play an important role in mitochondrial genome stability in that it could normalize mitochondrial mutagenesis and its functional consequences. Thus, creatine supplementation could be used to prevent or ameliorate diseases related to mitochondrial DNA mutations, and possibly to delay aging.
Biochimica et Biophysica Acta 02/2008; 1780(1):16-26. · 4.66 Impact Factor
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Michele Guescini,
Cristina Fatone,
Laura Stocchi,
Chiara Guidi, Lucia Potenza,
Massimiliano Ditroilo,
Anna Ranchelli,
Chiara Di Loreto,
Davide Sisti,
Pierpaolo De Feo,
Vilberto Stocchi
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ABSTRACT: In this study we developed a new methodology for obtaining human skeletal muscle samples to evaluate gene expression. This approach is based on a fine needle aspiration technique, which allows us to extract a small tissue sample in a significantly less invasive manner than with classic biopsy.
Multiplex tandem RT-PCR was used to determine the mRNA levels of genes involved in ATP production and mitochondrial biogenesis in muscle tissue. Samples of vastus lateralis muscle were obtained from 21 healthy subjects with different fitness levels. The principal findings in our study show a strong correlation between PGC-1alpha and COX5B (p<0.001) and between PGC-1alpha and MT-CO2 (p=0.017) expression. Furthermore, a significant positive correlation between mtDNA content and the percentage of MHCI present in the aspired samples were found (p=0.028). These data are in agreement with current knowledge on skeletal muscle physiology and show the reliability of the proposed method.
This painless methodology can be used to investigate, in vivo, human muscle RNA and DNA adaptations in response to either physiological and/or pharmacological stimuli. This method has major clinical relevance, such as its application in clarifying the mechanisms underling metabolic and systemic disorders.
Nutrition, metabolism, and cardiovascular diseases: NMCD 07/2007; 17(5):383-93. · 3.52 Impact Factor
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ABSTRACT: Ectomycorrhizae are mutualistic associations of several species of fungi with higher plants. Their formation involves alterations in the morphology and cell structure of the plant root and fungal mycelium. These modifications are correlated with mRNA and protein synthesis in the two symbionts. To gain more information about structural and functional traits involved in ectomycorrhizal formation, two "in vitro" ectomycorrhizal systems, set up by the inoculation of Tilia platyphyllos Scop. roots with either Tuber brumale Vittad. or T. borchii Vittad. mycelia, were investigated. Different parameters such as, fungal volume ratio, fungal biomass, plant and fungal transcript levels, specific enzymes activity and protein patterns were evaluated. In T. platyphyllos-T. brumale ectomycorrhizal tissue all the molecular and morphometrical approaches revealed a higher fungal biomass, volume and transcript as well as higher fungal protein levels respect to the host plant, suggesting that the fungal genes and proteins are up regulated after the establishment of symbiosis. These results are completely divergent from that obtained in T. platyphyllos Scop.-T. borchii Vittad. ectomycorrhizal system, leading us to hypothesise a different role of the fungal partner in the mycorrhization process according to the species it belongs to.
Plant Physiology and Biochemistry 08/2005; 43(7):709-16. · 2.84 Impact Factor
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ABSTRACT: Escherichia coli cultures exposed to a 300mT static magnetic field (SMF) were studied in order to analyse possible induced changes in cellular growth and gene expression. Biomass was evaluated by visible-light spectrometry and gene expression analyses were carried out by use of RNA arbitrarily primed PCR. The bacterial strain XL-1Blue, cultivated in traditional and modified Luria-Bertani medium, was exposed to SMF generated by permanent neodymium magnetic disks. The results show alterations induced by SMF in terms of increased cell proliferation and changes in gene expression compared with control groups. Three cDNAs were found to be expressed only in the exposed cells, whereas one cDNA was more expressed in the controls. One clone, expressed only in the exposed cells, corresponds to a putative transposase. This is of particular interest in that it suggests that exposure to a magnetic field may stimulate transposition activity.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 08/2004; 561(1-2):53-62. · 2.85 Impact Factor
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ABSTRACT: The effects of magnetic fields produced by permanent magnets on different DNA sources were investigated in vivo and in vitro. Escherichia coli DNA, plasmid, and amplification products of different lengths were used as the magnetic field target. The in vivo assays did not reveal any DNA alterations following exposure, demonstrating the presence of cell dependent mechanisms, such as the repair system and the buffering action of the heat shock proteins DNA K/J (Hsp 70/40). The in vitro assays displayed interactions between the magnetic field and DNA, revealing principally that magnetic field exposure induces DNA alterations in terms of point mutations. We speculate that the magnetic field can perturb DNA stability interacting with DNA directly or potentiating the activity of oxidant radicals. This genotoxic effect of the magnetic field, however, is minimized in living organisms due to the presence of protective cellular responses.
Bioelectromagnetics 08/2004; 25(5):352-5. · 1.84 Impact Factor
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ABSTRACT: In order to analyse gene expression during fruit body development of the ectomychorrizal fungus Tuber borchii Vittad., a modified differential display procedure was set up. The procedure used is easier and faster than the traditional one and generates reproducible cDNA banding patterns that can be resolved on a standard ethidium bromide-agarose gel. From 16 cDNA fingerprints, 25 amplicons with apparent differential expression were identified and cloned without a previous reamplification. Fifteen clones showed significant similarity to known proteins that are involved in dikaryosis and fruiting, cell division, transport across membranes, mitochondrial division, intermediary metabolism, biosynthesis of isoprenoid compounds and putative RNA/DNA binding. Northern blot analyses confirmed that seven cDNAs were indeed differentially expressed during fruit body development. The characterisation of these cDNAs represents a starting point in understanding the molecular mechanisms of cellular differentiation leading to the development of the T. borchii fruit body.
Current Genetics 01/2003; 42(3):161-8. · 2.56 Impact Factor
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ABSTRACT: Molecular identification techniques were applied in order to analyse food products containing fragments of some Tuber species. Samples of fungal DNA were processed by analyses of the internal transcribed spacer (ITS) region. The polymerase chain reaction (PCR) using truffle species-specific primers, multiplex PCR, restriction fragment length polymorphism (RFLP) analysis, sequencing of the ITS region and specific oligonucleotide probe hybridisation were used. The results obtained demonstrate the applicability of these molecular strategies to the identification of truffles, even when their morphological characteristics are difficult to interpret owing to the drastic treatments utilised in food preparation or the use of unripe fruit bodies (lacking spores). Furthermore, testing was also possible starting from very small amounts of sample and degraded DNA. The methods described have important applications in both the production and sale of such food products, in order to avoid fraud and reveal the possible presence of other fungal species.© 2002 Society of Chemical Industry
Journal of the Science of Food and Agriculture 08/2002; 82(12):1391 - 1397. · 1.44 Impact Factor
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ABSTRACT: The sequence and characterisation of the entire nuclear rDNA intergenic spacer (IGS) for the genus Tuber are presented. Sequence analyses showed that the organisation of the Tuber borchii rDNA IGS is typical of rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. Direct repeats, symmetry elements, tandem repeats and possible areas of recombination were found. The putative ends of the 25S and 17S rDNA were identified. The presence of 5S rDNA in the IGS region was excluded.
Journal of biomolecular structure & dynamics 03/2002; 19(4):701-8. · 4.99 Impact Factor
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ABSTRACT: PCR amplification of the complete intergenic spacer region (IGS) of the Tuber borchii nuclear ribosomal repeat was obtained using universal primers CNL 12 and NS1rev. In order to improve amplification yield a specific primer, T1, was selected from a partial sequence of the IGS product. IGS diversity was characterized both at the intraindividual and intraspecific level. The results obtained at the intraindividual level showed 10% varying repeats on ten screened colonies, while at the intraspecific level the IGS polymorphism was evident as difference in length amplification between mycelial strains and fruit bodies: 3.5 kb and 2 kb respectively.
Microbiological Research 02/2002; 157(1):69-74. · 2.31 Impact Factor
