Sabrina Zeppa

Exercise as a new physiological stimulus for brown adipose tissue activity

BACKGROUND AND AIM Brown adipose tissue (BAT) plays a major role in body energy expenditure counteracting obesity and obesity-associated morbidities. BAT activity is sustained by the sympathetic nervous system (SNS). Since a massive activation of the SNS was described during physical activity, we investigated the effect of endurance running training on BAT of young rats to clarify the role of exercise training on the activity and recruitment state of brown cells. METHODS AND RESULTS Male, 10-week-old Sprague Dawley rats were trained on a motor treadmill (approximately 60% of VO2max), 5 days/week, both for 1 and 6 weeks. The effect of endurance training was valuated using morphological and molecular approaches. Running training affected on the morphology, sympathetic tone and vascularization of BAT, independently of the duration of the stimulus. Functionally, the weak increase in the thermogenesis (no difference in UCP-1), the increased expression of PGC-1α and the membrane localization of MCT-1 suggest a new function of BAT. Visceral fat increased the expression of the FOXC2, 48 h after last training session and some clusters of UCP-1 paucilocular and multilocular adipocytes appeared. CONCLUSION Exercise seemed a weakly effective stimulus for BAT thermogenesis, but surprisingly, without the supposed metabolically hypoactive effects. The observed browning of the visceral fat, by a supposed white-to-brown transdifferentiation phenomena suggested that exercise could be a new physiological stimulus to counteract obesity by an adrenergic-regulated brown recruitment of adipocytes.

Identification of differentially expressed cDNA clones in Tilia platyphyllos-Tuber borchii ectomycorrhizae using a differential screening approach.

Abstract. No information is presently available on the molecular mechanisms that control the morphogenesis of the truffle, an ectomycorrhizal ascomycetous fungus of great economic interest not only for forestry and agronomy but also for the organoleptic properties of its hypogeous fruitbodies. A Tilia platyphyllos-Tuber borchii model system was used in order to identify genes induced or up-regulated during symbiosis, since their isolation is a prerequisite for the understanding of the molecular bases of mycorrhizal development and regulation. The strategy applied involved the construction of an ectomycorrhizal cDNA library and random selection of clones, followed by a differential screening procedure to analyse cDNA expression in uninfected roots, ectomycorrhizae and free-living mycelia. The results revealed that many genes – and more plant genes than fungal genes – are expressed at higher levels during the symbiotic phase. Several clones were also investigated in order to understand their biological function. This study represents the first attempt to extend our knowledge of the molecular mechanisms underlying the establishment of ectomycorrhiza in Tuber species.

Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation.

This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α, and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

Identification of putative genes involved in the development of Tuber borchii fruit body by mRNA differential display in agarose gel

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.

The expression profile of the Tuber borchii nitrite reductase suggests its positive contribution to host plant nitrogen nutrition

Ectomycorrhizal symbiosis is a ubiquitous association between plant roots and numerous fungal species. One of the main aspects of the ectomycorrhizal association are the regulation mechanisms of fungal genes involved in nitrogen acquisition. We report on the genomic organisation of the nitrate gene cluster and functional regulation of tbnir1, the nitrite reductase gene of the ectomycorrhizal ascomycete Tuber borchii. The sequence data demonstrate that clustering also occurs in this ectomycorrhizal fungus. Within the TBNIR1 protein sequence, we identified three functional domains at conserved positions: the FAD box, the NADPH box and the two (Fe/S)-siroheme binding site signatures. We demonstrated that tbnir1 presents an expression pattern comparable to that of nitrate transporter. In fact, we found a strong down-regulation in the presence of primary nitrogen sources and a marked tbnir1 mRNA accumulation following transfer to either nitrate or nitrogen limited conditions. The real-time PCR assays of tbnir1 and nitrate transporter revealed that both nitrate transporter and nitrite reductase expression levels are about 15-fold and 10-fold higher in ectomycorrhizal tissues than in control mycelia, respectively. The results reported herein suggest that the symbiotic fungus Tuber borchii contributes to improving the host plant’s ability to make use of nitrate/nitrite in its nitrogen nutrition.

Effects of a 300 mT static magnetic field on human umbilical vein endothelial cells

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.

Competitive PCR for Quantitation of a Cytophaga-Flexibacter-Bacteroides Phylum Bacterium Associated with the Tuber borchii Vittad. Mycelium

ABSTRACT An uncultured bacterium associated with the ectomycorrhizal fungus Tuber borchii Vittad. was identified as a novel member of the Cytophaga-Flexibacter-Bacteroides group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 106 cells per 30-day-old T. borchii mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus.

Gene expression profile in cultured human umbilical vein endothelial cells exposed to a 300 mT static magnetic field.

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 HUVECs 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.

Analysis of gene expression in the vegetative and fructification phases of the white truffle, Tuber borchii Vittad., by mRNA differential display

The mRNA differential display technique was used to compare mRNA populations from fruit body and mycelium of a white truffle species in the attempt to identify and clone differentially expressed genes. The differential expression of five out of 30 amplicons was confirmed. One fragment (Tbm 56) corresponded to a part of the ribosomal genes. Three cDNA fragments (Tbf 12, Tbf 20, Tbf 21) were expressed only in the fructification phase, while the other cDNA (Tbf 55) was expressed strongly in fruit body and also detectable in the mycelium. These clones correspond to part of the single-copy genes in the Tuber borchii Vittad. genome.

Effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer

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.