Cancer metabolism and the dynamics of metastasis

Cancer growth dynamics, commonly simulated with a Gompertzian model, is analyzed in the framework of a more recent and realistic model. In particular, we consider the setting of a tumor embedded in a host organ and investigate their interaction. We assume that, at least in some cases, tumor metastasis may be triggered by an ‘energetic crisis’, when the tumor exceeds the ‘carrying capacity’ of the host organ. As a consequence, dissemination of clusters of cancer cells is set in motion, with a statistical probability given by a Poisson distribution. The model, although still at a preclinical level, is fully quantitative and is applied, as an example, to the case of prostate cancer. The results confirm that, at least for the more aggressive cancers, metastasis starts very early during tumorigenesis and a quantitative link is found between the tumor's doubling time, its ‘aggressiveness’ and the metastatic potential.     

Evidence for aerobic ATP synthesis in isolated myelin vesicles

Even though brain represents only 2–3% of the body weight, it consumes 20% of total body oxygen, and 25% of total body glucose. This sounds surprising, in that mitochondrial density in brain is low, while mitochondria are thought to be the sole site of aerobic energy supply. These data would suggest that structures other than mitochondria are involved in aerobic ATP production. Considering that a sustained aerobic metabolism needs a great surface extension and that the oxygen solubility is higher in neutral lipids, we have focused our attention on myelin sheath, the multilayered membrane produced by oligodendrocytes, hypothesizing it to be an ATP production site. Myelin has long been supposed to augment the speed of conduction, however, there is growing evidence that it exerts an as yet unexplained neuro-trophic role. In this work, by biochemical assays, Western Blot analysis, confocal laser microscopy, we present evidence that isolated myelin vesicles (IMV) are able to consume O₂ and produce ATP through the operation of a proton gradient across their membranes. Living optic nerve sections were exposed to MitoTracker, a classical mitochondrial dye, by a technique that we have developed and it was found that structures closely resembling nerve axons were stained. By immunohystochemistry we show that ATP synthase and myelin basic protein colocalize on both IMV and optic nerves. The complex of data suggests that myelin sheath may be the site of oxygen absorption and aerobic metabolism for the axons.     

The D4Z4 Macrosatellite Repeat Acts as a CTCF and A-Type Lamins-Dependent Insulator in Facio-Scapulo-Humeral Dystrophy

Both genetic and epigenetic alterations contribute to Facio-Scapulo-Humeral Dystrophy (FSHD), which is linked to the shortening of the array of D4Z4 repeats at the 4q35 locus. The consequence of this rearrangement remains enigmatic, but deletion of this 3.3-kb macrosatellite element might affect the expression of the FSHD-associated gene(s) through position effect mechanisms. We investigated this hypothesis by creating a large collection of constructs carrying 1 to >11 D4Z4 repeats integrated into the human genome, either at random sites or proximal to a telomere, mimicking thereby the organization of the 4q35 locus. We show that D4Z4 acts as an insulator that interferes with enhancer–promoter communication and protects transgenes from position effect. This last property depends on both CTCF and A-type Lamins. We further demonstrate that both anti-silencing activity of D4Z4 and CTCF binding are lost upon multimerization of the repeat in cells from FSHD patients compared to control myoblasts from healthy individuals, suggesting that FSHD corresponds to a gain-of-function of CTCF at the residual D4Z4 repeats. We propose that contraction of the D4Z4 array contributes to FSHD physio-pathology by acting as a CTCF-dependent insulator in patients.     

Replication Timing of Human Telomeres Is Chromosome Arm–Specific,Influenced by Subtelomeric Structures and Connected to Nuclear Localization

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of β-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4–associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm–specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.     

Distribution and correlation of three oenological traits inSaccharomyces cerevisiae

The improvement of yeast starters for wine making, with classical genetic techniques, relays on the effective independence of the main oenological traits in order to combine them optimally. The analysis of three characters (ethanol production, volatile acidity and fermenting rate) in 787Saccharomyces cerevisiae strains, isolated from several parts of the world and substrates, shows moderate correlation between the volatile acidity and the other two traits, whereas ethanol production and fermenting rate appear more correlated. The minimum number of strains necessary to start selection or genetic improvement programs is discussed. The correlation between the oenological traits and the source of isolation showed that the best strains can be obtained from wineries and wine, whereas the isolates from grapes and must are less performing on the average.     

A selective radiobrominated cocaine analogue for imaging of dopamine uptake sites: pharmacological evaluation and PET experiments

(E)-N-(3-bromoprop-2-enyl)-2beta-carbomethoxy-3beta-4'-tolyl -nortropane or PE2Br, an analogue of cocaine was labelled with the positron emitter 76Br (T1/2=16 h) for pharmacological evaluation in the rat and PET investigation in the monkey. [76Br]PE2Br was obtained by electrophilic substitution from the tributylstannyl precursor with radiochemical yield of 80%. In vivo biodistribution studies of [76Br]PE2Br (20 MBq/nmol) in rats showed a high uptake in the striatum (2.2% ID/g tissue at 15 min p.i.). The striatum to cerebellum radioactivity ratio was 6 at 1 hour p.i. Striatal uptake of [76Br]PE2Br was almost completely prevented by pretreatment with GBR 12909, but citalopram and maprotiline had no effect, confirming the selectivity of the radioligand for the dopamine transporter. PET imaging of the biodistribution of [76Br]PE2Br in the baboon demonstrated rapid and high uptake in the brain (5% ID at 3 min p.i.). The striatal radioactivity concentration reached a plateau at 20 min p.i. (7% ID/100 mL). The uptake in the cortex and cerebellum was very low. A significantly higher uptake in the thalamus was observed. At 1h p.i., the striatum to cerebellum ratio and thalamus to cerebellum ratio were 8 and 1.9 respectively. In competition experiments the radioactivity in the striatum and the thalamus was displaced by 5 mg/kgof cocaine and 5 mg/kg of GBR 12909, but citalopram and maprotiline had no effect. These results showed that [76Br]PE2Br is in vivo a potent and selective radioligand suitable for PET imagingof the dopamine transporter.     

Structure and orientation of pardaxin determined by NMR experiments in model membranes

Pardaxins are a class of ichthyotoxic peptides isolated from fish mucous glands. Pardaxins physically interact with cell membranes by forming pores or voltage-gated ion channels that disrupt cellular functions. Here we report the high-resolution structure of synthetic pardaxin Pa4 in sodium dodecylphosphocholine micelles, as determined by (1)H solution NMR spectroscopy. The peptide adopts a bend-helix-bend-helix motif with an angle between the two structure helices of 122 +/- 9 degrees , making this structure substantially different from the one previously determined in organic solvents. In addition, paramagnetic solution NMR experiments on Pa4 in micelles reveal that except for the C terminus, the peptide is not solvent-exposed. These results are complemented by solid-state NMR experiments on Pa4 in lipid bilayers. In particular, (13)C-(15)N rotational echo double-resonance experiments in multilamellar vesicles support the helical conformation of the C-terminal segment, whereas (2)H NMR experiments show that the peptide induces considerable disorder in both the head-groups and the hydrophobic core of the bilayers. These solid-state NMR studies indicate that the C-terminal helix has a transmembrane orientation in DMPC bilayers, whereas in POPC bilayers, this domain is heterogeneously oriented on the lipid surface and undergoes slow motion on the NMR time scale. These new data help explain how the non-covalent interactions of Pa4 with lipid membranes induce a stable secondary structure and provide an atomic view of the membrane insertion process of Pa4.     

Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins

It was determined that flavan-3-ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan-3-ols and procyanidins was competitive with the two substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 microM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface.     

Effect of beta-amyloid on endothelial cells: lack of direct toxicity,enhancement of MTT-induced cell death and intracellular accumulation

Several cerebrovascular alterations have been described in Alzheimer's disease (AD) including an accumulation of beta-amyloid (betaA) on the vascular walls in the brain. To investigate the potential toxic activity of betaA on endothelial cells (EC), two endothelial murine cell lines derived from heart and brain were exposed to betaA1-42 and the biologically active fragment betaA25-35 in the range from 5nM to 50 microM. In a low concentration range (50 nM to 2.5 microM) both peptides significantly reduced the 3-(4,5-dimethylthiazol-2y1)-2-5-diphenyltetrazolium bromide (MTT) signal in the endothelial cell lines exposed for 24h. However, microscopic examination, lactate dehydrogenase (LDH) release determination and Neutral Red assay did not confirm any toxic effect associated with inhibition of MTT formazan reduction. The effect on MTT was not susceptible to anti-oxidant treatment and did not increase the sensitivity to oxidative stress. However, when the EC were exposed to betaA and MTT for 1h, cell viability, determined by LDH release, was strongly reduced, while in normal conditions MTT-induced cell death only after 2h. An inhibitor of lysosomal ATPase activity, bafilomycin A1, completely antagonized this effect. The morphological examination showed that the functional activation by betaA in EC enhanced the production of MTT formazan crystals. To verify the accumulation of betaA in the lysosomal compartment we analyzed the subcellular distribution of betaA1-42 at different exposure times of EC to the peptide. The peptide was found in several organelles and was absent in the cytoplasmic compartment; co-treatment with bafilomycin A1 did not reduce the intracellular presence of betaA1-42. In our condition, the exposure of EC to betaA induced an intracellular accumulation of the peptide and a vasoactive effect that did not appear associated with direct toxic activity.