The effects of human sera conditioned by high-intensity exercise sessions and training on the tumorigenic potential of cancer cells.

PURPOSE: There is growing evidence of an association between physical activity and a reduced risk of cancer and cancer recurrence. The aim of this study was to assess the effects of exercise-conditioned human serum (HS) effects on the proliferative and tumorigenic potential of triple-negative breast cancer (TNBC) and prostate cancer (PC) cells. Moreover, modulated mechanisms and several physiological factors that can predict exercise effects were investigated. METHODS: Thirty healthy sedentary subjects were recruited for the study. The subjects performed two high-intensity endurance cycling (HIEC) sessions before and after a nine-week period of high-intensity interval training (HIIT). Cell tumorigenic capacity affected by HS collected before (t0), immediately after (t1), 4 h (t2), and 24 h (t3) after the HIEC sessions was evaluated by in vitro three-dimensional colony formation. The modulation of molecular pathways was analyzed by western blotting and qPCR in TNBC and PC cells, and in TNBC xenografts in exercised mice. RESULTS: All of the HIEC-conditioned HS (t1, t2, and t3) markedly impacted the proliferative and the microtumor-forming capacity of both TNBC and PC cell lines, while the HS collected from the subjects at rest did not. Modulation of the Hippo and Wnt/ß-catenin pathways by HIEC-conditioned HS before and after the period of HIIT was shown. Multiple linear regression analysis showed relationships between the effects of HIEC-conditioned HS in PC cells, lactate threshold and VO2max. CONCLUSIONS: These results highlight the potential of HIEC bouts in tumor progression control and the importance of optimizing an approach to identify physiological predictors of the effects of acute exercise in tertiary cancer prevention.

Evolution of lymphocytes. Immunoglobulin T of the teleost sea bass (Dicentrarchus labrax): Quantitation of gene expressing and immunoreactive cells.

Immunoglobulin T (IgT) is one of the key effector molecules of jawed vertebrate's adaptive immune system, and in this work we describe the quantitative distribution of IgT-expressing and IgT-producing cells in tissues of the European seabass Dicentrarchus labrax by using mRNA riboprobes and a specific anti-IgT antibody. A polyclonal antiserum (pAb) was prepared by immunizing rabbits with three synthetic peptides deduced from the full length IgT cDNA sequence and located in a surface-exposed CH3 domain of IgT constant region. The obtained antiserum, named RAIgT1, was able to recognize by ELISA immunization antigens and IgT from intestinal mucus and serum. In western blots of head kidney leukocytes lysates the antiserum recognized a 180 kDa polypeptide in non-reducing, and a 75 kDa peptide in reducing conditions. Interestingly, the RAIgT1 pAb crossreacted intensely in western blots with rainbow trout IgT purified from mucus and serum. Antisense mRNA IgT oligonucleotide sequences were employed in in situ hybridization to detect IgT-expressing cells in sections from lymphoid tissues, and positive cells were observed in head kidney, spleen, intestine and gills. By employing RAIgT1 in quantitative immunohistochemistry, the highest number of IgT-producing cells was observed in the gills (9.5 ± 0.7%), followed by intestine (8.4 ± 1.2%), head kidney (6.2 ± 1.4%), and spleen (4.1 ± 0.7%). Interestingly, the number of IgT-B cells showed a regionalization in the intestine, increasing from the proximal to the terminal part. By immunofluorescence and flow cytometry of live leukocytes, the percentages of RAIgT1 stained cells were 34 ± 11% in the intestine, 22 ± 5% in head kidney, 16 ± 7% in spleen, and 9 ± 5% in gills. At the fluorescence microscope, live cells from these tissues showed a typical membrane-associated positivity and a lymphocytic morphology, and no IgT/IgM double positive cells were detected. Immunoreactive cells have been purified from head kidney using magnetic beads, and IgT-enriched cells showed by RT-PCR an enhanced expression of the IgT gene, whereas IgT-depleted cells had an highest expression of IgM and TRß genes. These data describe for the first time a quantitative panel of IgT-expressing and IgT-immunoreactive cells in tissues of a teleost fish species.

Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures.

Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A(2A) and D(2) receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D(2)R-CFP or A(2A)R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D(2)R-CFP and A(2A)R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A(2A)R positive MVs were treated with the adenosine A(2A) receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A(2A)Rs were functionally competent in target cells. These findings demonstrate that A(2A) receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

Sulfate metabolism in Tuber borchii: characterization of a putative sulfate transporter and the homocysteine synthase genes.

The homocysteine synthase (tbhos) and putative sulfate transporter (tbsul1) genes have been characterized in order to understand the sulfate metabolism and regulation in the ectomycorrhizal fungus Tuber borchii. The analyses of tbsul1 and tbhos nucleotide and deduced amino acid sequences led to the identification of the typical domains shown in homologous proteins. Sulfate starvation condition upregulates both genes. The real-time PCR assay of tbsul1 revealed that gene expression was about threefold higher in mycelia grown under sulfate starvation for 2 days than in the mycelial control and in the same starvation condition, the sulfate uptake increased. Real-time PCR and enzymatic assays showed regulation of tbhos when sulfur sources were lacking, suggesting that a transcriptional regulation of this gene rather than a post-transcriptional one occurred. Furthermore, the tbsul1 and tbhos expression patterns were evaluated during the truffle life cycle, revealing an over-expression in the mature ascomata for both genes. In the ectomycorrhizal tissue, only tbhos was upregulated suggesting its substantial role in T. borchii cysteine synthesis. The regulation of tbsul1 and tbhos occurs primarily at the transcriptional level both during vegetative and fruiting phases and these genes could be directly involved in VOCs production.

Molecular characterisation of the small GTPase CDC42 in the ectomycorrhizal fungus Tuber borchii Vittad.

The small GTPase CDC42 is ubiquitously expressed in eukaryotes, where it participates in the regulation of the cytoskeleton and a wide range of cellular processes, including cytokinesis, gene expression, cell cycle progression, apoptosis, and tumorigenesis. As very little is known on the molecular level about mycorrhizal morphogenesis and development and these events depend on a tightly regulated reorganisation of the cytoskeleton network in filamentous fungi, we focused on the molecular characterisation of the cdc42 gene in Tuber borchii Vittad., an ascomycetous hypogeous fungus forming ectomycorrhizae. The entire gene was isolated from a T. borchii cDNA library and Southern blot analyses showed that only one copy of cdc42 is present in the T. borchii genome. The predicted amino acid sequence is very similar to those of other known small GTPases and the similar domain structures suggest a similar function. Real-time PCR analyses revealed an increased expression of Tbcdc42 during the phase preparative to the instauration of symbiosis, in particular after stimulation with root exudate extracts. Immunolocalisation experiments revealed an accumulation of CDC42 in the apical tips of the growing hyphae. When a constitutively active Tbcdc42 mutant was expressed in Saccharomyces cerevisiae, morphological changes typical of pseudohyphal growth were observed. Our results suggest a fundamental role of CDC42 in cell polarity development in T. borchii.

Characterization and biological activity of the main flavonoids from Swiss Chard (Beta vulgaris subspecies cycla).

The molecular components of a phenolic fraction (P2), obtained from liquid chromatography of a Swiss Chard (Beta vulgaris subsp. cycla) extract, were identified using HPLC-ESI-MS/MS. The primary P2 components were: vitexin-2''O-rhamnoside, its demethylated form 2''-xylosylvitexin, isorhamnetin 3-gentiobioside, and rutin. P2 "in toto" and the single components were characterized for antioxidant capacity, antimitotic activity on MCF-7 human breast cancer cells and for toxicity to human lymphocytes and macrophages. P2 inhibited MCF-7 cell proliferation (IC(50) value = 9 microg/ml) without inducing apoptosis, showed no toxicity to human lymphocytes and slight toxicity to macrophages. Vitexin-2''O-rhamnoside strongly inhibited DNA synthesis in MCF-7 cells, whereas 2''-xylosylvitexin and isorhamnetin 3-gentiobioside were activators; combinations of activators and inhibitors maintained the over-all inhibitory effect.

Determination of microbial volatile organic compounds from Staphylococcus pasteuri against Tuber borchii using solid-phase microextraction and gas chromatography/ion trap mass spectrometry.

The mycelium of Tuber borchii Vittad., a commercial truffle species, is used as a model system for in vitro ectomycorrhizal synthesis, infected seedling production and biotechnological applications. Our fungal cultures were accidentally contaminated with a Staphylococcus pasteuri strain, showing a strong antifungal activity against T. borchii mycelium. In order to identify the antifungal volatile agents produced by S. pasteuri, solid-phase microextraction (SPME) with gas chromatography and mass spectrometry (GC/MS) was used. Using this method 65 microbial volatile organic compounds (MVOCs), synthesized by this bacterium in either single or in fungal-bacterial dual culture, were identified. SPME combined with GC/MS may be a useful method for the determination of MVOCs involved in the antifungal activity. These results showed that bacteria with unusual biological activities could be a major problem during large-scale production of inoculum for truffle-infected seedling.

A putative mitochondrial fission gene from the ectomycorrhizal ascomycete Tuber borchii Vittad.: cloning, characterisation and phylogeny.

Mitochondrial binary division is a complex process occurring in multiple steps, mediated by several proteins. In Saccharomyces cerevisiae, a mitochondrial membrane protein, Fis1p, is required for the proper assembly of the mitochondrial division apparatus. In this study, we report the cloning, characterisation and phylogenetic analysis of Tbfis1, a gene from the ectomycorrhizal ascomycetous truffle Tuber borchii, encoding for an orthologue of S. cerevisiae Fis1p. The Tbfis1 coding region consists of a 468-nucleotide open reading frame interrupted by four introns, which encodes for a polypeptide of 155 amino acids, having a predicted transmembrane domain structure typical of the Fis1p Family. Southern blot analysis revealed that Tbfis1 is a single-copy gene in the T. borchii genome. Tbfis1 is highly expressed during the first stages of T. borchii fruit body ripening, while its expression decreases during T. borchii mycelium ageing. Also, Virtual Northern blot analysis revealed Tbfis1 expression in the symbiotic phase of the fungus life cycle. Phylogenetic analysis allowed the identification of Tbfis1 orthologues in filamentous fungi, yeasts, plants, worms, flies and mammals, indicating that the function of the protein coded by this gene has been conserved during evolution.

Solid-phase microextraction gas chromatography/ion trap mass spectrometry and multistage mass spectrometry experiments in the characterization of germacrene D.

Germacrene D is a vegetable pheromone utilized in interactions among organisms belonging to different species. For the first time, using solid-phase microextraction/gas chromatography/ion trap mass spectrometry, the presence of this compound was detected in an in vitro mycorrhizal synthesis system where the mycelium of the ectomycorrhizal fungus Tuber borchii Vittad. interacts with the plant Tilia Americana L. From this symbiosis, a new structure, called ectomycorrhiza, is formed where the two symbionts exchange nutrients and metabolites. It seems that only after this interaction can the mycelium develop the fruitbody, commonly known as truffle. The results obtained allowed us to ascertain that germacrene D was synthesized by the plant exclusively in the presence of T. borchii. The originality of these data prompted us to hypothesize that this compound could be involved in the first step of ectomycorrhiza formation, as it is able to stimulate specific fungi receptors. In fact, plants release hundreds of secondary metabolites that are important in their interactions with other organisms.

Cloning, expression, and characterization of the hxk-1 Gene from the white truffle Tuber borchii vittad.: A first step toward understanding sugar metabolism.

Recent biochemical investigations of Tuber borchii Vittad. mycelium have demonstrated the presence of three distinct forms of hexokinase (HK(M1), HK(M2), and HKM3). In the investigation described here, a gene coding for hexokinase (hxk-1) from T. borchii was isolated and characterized. The hxk-1 gene is characterized by an ORF of 1494 nucleotides and codes for a polypeptide of 497 aa. The gene was overexpressed in Escherichia coli, and the recombinant protein was kinetically characterized. The K(cat) value for fructose is in agreement with the data reported for the hexokinase of Yarrowia lipolytica, the Km for ATP is not dependent on the sugar used, and the enzyme is not inhibited by trehalose 6-phosphate or glucose 6-phosphate. The biochemical characteristics confirm that this enzyme is a hexokinase, as suggested by the Pileup results, and it corresponds to the HKM1 isoform. This work represents the first characterization of the key enzyme of the glycolytic pathway and the related gene in a Tuber species.

One-step purification of a fully active hexahistidine-tagged human hexokinase type I overexpressed in Escherichia coli.

The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa. Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate. In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions. In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg. The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation.

Possible involvement of Pseudomonas fluorescens and Bacillaceae in structural modifications of Tuber borchii fruit bodies.

Previous studies on Tuber borchii fruit bodies in early maturation stages suggested a role of bacteria in sporocarp structural modifications. In order to verify this hypothesis, in the present study we investigated by means of microbial and ultrastructural approaches, the bacterial population of T. borchii sporocarps from intermediate maturation phases to advanced decomposition stages, paying particular attention to chitinolytic and cellulolytic bacteria and to their relationships with ascii and ascospores. We found that Pseudomonas fluorescens and spore-forming Bacillaceae, both able to degrade cellulose and chitin, are present inside the sporocarps in all maturation stages investigated. Moreover, rod-shaped bacteria seem able to erode ascus walls and colonize the interior of ascii containing mature spores. These results suggest a possible role of these bacteria in the process of ascus opening. Moreover, the presence of P. fluorescens and Bacillaceae on isolated mature spores after decontamination suggests an intimate association between these bacteria and the ascospores.

Immunolocalization of hydrophobin HYDPt-1 from the ectomycorrhizal basidiomycete Pisolithus tinctorius during colonization of Eucalyptus globulus roots.

• The immunolocalization of one of the hydrophobins of Pisolithustinctorius (HYDPt-1) is reported. Hydrophobin proteins play key roles in adhesion and aggregation of fungal hyphae, and it is already known that formation of ectomycorrhizas on eucalypt roots enhances the accumulation of hydrophobin mRNAs in the mycelium of Pisolithus tinctorius. • Purification of SDS-insoluble proteins from the mycelium of P. tinctorius showed the presence of a 13 kDa polypeptide with properties of class I hydrophobin. • Polyconal antibodies were raised against a recombinant HYDPt-1 polypeptide, and these were used for immunofluorescence-coupled transmission electron microscopy. • HYDPt-1 is a cell wall protein located at the surface of the hyphae with no preferential accumulation in the fungal cells of the different tissues of the ectomycorrhiza (i.e. extraradical hyphae, mantle or Hartig net).

Dehydroascorbic acid irreversibly inhibits hexokinase activity.

The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pKa and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues. Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.

In vitro and in vivo effects of heavy metals on mussel digestive gland hexokinase activity: the role of glutathione.

Hexokinase (E.C. 2.7.1.1), the enzyme responsible for glucose phosphorylation to G-6P, is inactivated by SH reagents and oxyradicals, and its inhibition has been involved in heavy metal toxicity in mammalian systems. In this work, the possibility that hexokinase activity could be affected by both heavy metal binding and oxidative stress conditions also in mussel tissues (Mytilus galloprovincialis Lam.) was investigated. The results obtained in vitro demonstrate that heavy metals inhibited digestive gland hexokinase (with Cd2+ > Cu2+ > Hg2+ > Zn2+ > Pb2+) and suggest a role for GSH in the protection against the heavy metal effects. Hexokinase activity was also reduced by addition of iron/ascorbate, indicating a susceptibility of the enzyme to metal-mediated oxyradical production. The effects of Cu2+ treatment (3 days, 40 micrograms l-1 per animal) on hexokinase activity and on the GSH/GSSG status were then evaluated in mussels exposed to a cycle of air exposure/reimmersion. In Cu-exposed mussels, a significant decrease in hexokinase activity and a parallel reduction in tissue GSH levels were observed, suggesting that the two effects of metal treatment could be related; however, hexokinase activity progressively recovered during air exposure and reimmersion, whereas the level of GSH showed a further decrease during air exposure followed by recovery after reimmersion. The in vitro results therefore indicate that mussel digestive gland hexokinase is susceptible to inactivation by heavy metal binding and suggest a role for GSH in the protection against the effects of heavy metals. The effects of copper were confirmed by the results obtained in vivo. The possible relationship between hexokinase activity and the level of GSH in the digestive gland of control and Cu-exposed mussels during air exposure and reimmersion are discussed, taking into account the balance between pro-oxidant and antioxidant processes at different stages of exposure.

Substrates of hexokinase, glucose-6-phosphate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase prevent the inhibitory response induced by ascorbic acid/iron and dehydroascorbic acid in rabbit erythrocytes.

Exposure of intact rabbit erythrocytes or erythrocyte lysates to ascorbic acid/FeCl3 in a glucose-free saline promoted a rapid decline in reduced glutathione and this response was paralleled by inactivation of hexokinase. Under the same conditions, the activity of the enzymes glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase did not show appreciablevariations in intact cells, but was severely inhibited in the cell-free system. Similar results were obtained by replacing ascorbic acid/FeCl3 with dehydroascorbic acid. In addition, both treatments effectively inhibited the activity of purified hexokinase as well as those of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Further studies using the cell-free system indicated that the inhibition of enzyme activities elicited by either of the two treatments was effectively counteracted by the specific substrates of these enzymes. The fact that the hexokinase substrate glucose freely permeates the plasma membrane, unlike the substrates of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphatedehydrogenase, explains the selective inhibition of hexokinase observed in intact cells. The above results also indicate that dehydroascorbic acid is an inhibitor of these enzymes and strongly suggest that it is at least in part responsible for the effects mediated by the cocktail ascorbic acid/FeCl3.

Hexokinase inactivation induced by ascorbic acid/Fe(II) in rabbit erythrocytes is independent of glutathione-reductive processes and appears to be mediated by dehydroascorbic acid.

Recent studies performed in our laboratory demonstrated that rabbit red blood cell hexokinase was remarkably inhibited by the cocktail ascorbic acid/Fe(II) (Stocchi et al., 1994, Arch. Biochem. Biophys. 311, 160-167) and that the formation of dehydroascorbic acid was a key event in this process (Fiorani et al., 1996, Arch. Biochem. Biophys, 334, 357-361). The present study was undertaken to determine the final hexokinase-inactivating species using cell-free extract as a model. Our results demonstrate superimposable kinetics of hexokinase decay promoted by either ascorbic acid/Fe(II) or dehydroascorbic acid in erythrocyte lysates in which the reduced glutathione (GSH) levels were variously manipulated. In particular, neither removal nor addition of this tripeptide was able to significantly alter the rate or extent of hexokinase inhibition. Thus, GSH-reductive processes are dispensable events in the process of hexokinase inhibition promoted by ascorbic acid/Fe(II) in red blood cells. As a consequence, dehydroascorbic acid appears to be the species which directly inhibits hexokinase. This inference is further supported by the observation that addition of dehydroascorbic acid to the purified enzyme leads to a remarkable inhibition in its activity.

Protein oxidation of a hair sample kept in Alaskan ice for 800-1000 years.

Ancient finds of organic matter are not only of the highest value for palaeochemists and palaeobiologists but can be used to determine basic chemical reactions, such as protein oxidation, over long time periods. We studied oxidation of human hair protein about one thousand years old of an Alaskan child buried in ice, ten hair samples of copts of comparable age buried in graves of hot dry sand and compared the results to ten recent hair samples. Protein oxidation parameters o-tyrosine and cysteic acid of the Alaskan child were comparable to recent samples whereas they were higher in the coptic specimen. N-epsilon-carboxymethyllysine, a parameter for glycoxidation, however, was as high in coptic specimen. We conclude that ice in contrast to soil prevented protein oxidation but failed to inhibit glycoxidation, a reaction initiated by autooxidation of glucose. This study therefore has implications for the interpretation of oxidation and glycoxidation as well as preservation mechanisms of proteins.

In vitro effects of 50 Hz magnetic fields on oxidatively damaged rabbit red blood cells.

The aim of this study was to investigate the effects of 50 Hz magnetic fields (0.2-0.5 mT) on rabbit red blood cells (RBCs) that were exposed simultaneously to the action of an oxygen radical-generating system, Fe(II)/ascorbate. Previous data obtained in our laboratory showed at the exposure of rabbit erythrocytes or reticulocytes to Fe(II)/ascorbate hexokinase inactivation, whereas the other glycolytic enzymes do not show any decay. We also observed depletion of reduced glutathione (GSH) content with a concomitant intracellular and extracellular increase in oxidized glutathione (GSSG) and a decrease in energy charge. In this work we investigated whether 50 Hz magnetic fields could influence the intracellular impairments that occur when erythrocytes or reticulocytes are exposed to this oxidant system, namely, inactivation of hexokinase activity, GSH depletion, a change in energy charge, and hemoglobin oxidation. The results obtained indicate the a 0.5 mT magnetic field had no effect on intact RBCs, whereas it increased the damage with Fe(II)/ascorbate to a 0.5 mT magnetic field induced a significant further decay in hexokinase activity (about 20%) as well as a twofold increase in methemoglobin production compared with RBCs that were exposed to the oxidant system alone. Although further studies will be needed to determine the physiological implications of these data, the results reported in this study demonstrate that the effects of the magnetic fields investigated are able to potentiate the cellular damage induced in vitro by oxidizing agents.