Effects of acrolein on the quadruplex forming d(TTAGGG)4 telomeric repeat sequence

HPLC and ESI-MS analysis have been used to investigate the effect of acrolein exposure on d(TITAGGG)4 human telomeric repeat. Preliminary results disclosed a novel relationship between the structure assumed by oligodeoxynucleotides (ODNs) and the capability of their nucleobase residues to react with acrolein.     

Unusual monomolecular DNA quadruplex structures using bunch-oligonucleotides

The chemical synthesis of several G-rich bunch-oligonucleotides and the structural characterization of the corresponding monomolecular G-quadruplexes (I-IV) have been reported. The synthetic method allow the achievement of monomolecular DNA quadruplex structures having unusual and predeterminable oligodeoxyribonucleotide (ODN) strand orientation.     

Synthesis and characterization of monomolecular DNA G-quadruplexes formed by tetra-end-linked oligonucleotides

Guanine-rich DNA sequences are widely dispersed in the eukaryotic genome and are abundant in regions with relevant biological significance. They can form quadruplex structures stabilized by guanine quartets. These structures differ for number and strand polarity, loop composition, and conformation. We report here the syntheses and the structural studies of a set of interconnected d(TG(4)T) fragments which are tethered, with different orientations, to a tetra-end-linker in an attempt to force the formation of specific four-stranded DNA quadruplex structures. Two synthetic strategies have been used to obtain oligodeoxyribonucleotide (ODN) strands linked with their 3'- or 5'-ends to each of the four arms of the linker. The first approach allowed the synthesis of tetra-end-linked ODN (TEL-ODN) containing the four ODN strands with a parallel orientation, while the latter synthetic pathway led to the synthesis of TEL-ODNs each containing antiparallel ODN pairs. The influence of the linker at 3'- or 5'-ODN, on the quadruplex typology and stability, in the presence of sodium or potassium ions, has been investigated by circular dichroism (CD), CD thermal denaturation, (1)H NMR experiments at variable temperature, and molecular modeling. All synthesized TEL-ODNs formed parallel G-quadruplex structures. Particularly, the TEL-ODN containing all parallel ODN tracts formed very stable parallel G-quadruplex complexes, whereas the TEL-ODNs containing antiparallel ODN pairs led to relatively less stable parallel G-quadruplexes. The molecular modeling data suggested that the above antiparallel TEL-ODNs can adopt parallel G-quadruplex structures thanks to a considerable folding of the tetra-end-linker around the whole quadruplex scaffold.     

An apical actin-rich domain drives the establishment of cell polarity during cell adhesion

One of the most important questions in cell biology concerns how cells reorganize after sensing polarity cues. In the present study, we describe the formation of an actin-rich domain on the apical surface of human primary endothelial cells adhering to the substrate and investigate its role in cell polarity. We used confocal immunofluorescence procedures to follow the redistribution of proteins required for endothelial cell polarity during spreading initiation. Activated Moesin, vascular endothelial cadherin and partitioning defective 3 were found to be localized in the apical domain, whereas podocalyxin and caveolin-1 were distributed along the microtubule cytoskeleton axis, oriented from the centrosome to the cortical actin-rich domain. Moreover, activated signaling molecules were localized in the core of the apical domain in tight association with filamentous actin. During cell attachment, loss of the apical domain by Moesin silencing or drug disruption of the actin cytoskeleton caused irregular cell spreading and mislocalization of polarity markers. In conclusion, our results suggest that the apical domain that forms during the spreading process is a structural organizer of cell polarity by regulating trafficking and activation of signaling proteins.     

G-quadruplex-forming oligonucleotide conjugated to magnetic nanoparticles: synthesis, characterization, and enzymatic stability assays

In the present work, we report the conjugation of superparamagnetic nanoparticles to a fluorescently labeled oligodeoxyribonucleotide (ODN) able to fold into stable unimolecular guanine quadruple helix under proper ion conditions by means of its thrombin-binding aptamer (TBA) sequence. The novel modified ODN, which contained a fluorescent dU(Py) unit at 3'-end and a 12-amino-dodecyl spacer (C(12)-NH(2)) at 5' terminus, was characterized by ESI-MS and optical spectroscopy (UV, CD, fluorescence), and analyzed by RP-HPLC chromatography and electrophoresis. From CD and fluorescence experiments, we verified that dU(Py) and C(12)-NH(2) incorporation does not interfere with the conformational stability of the G-quadruplex. Subsequently, the conjugation of the pyrene-labeled ODN with the magnetite particles was performed, and the ODN-conjugated nanoparticles were studied through optical spectroscopy (UV, CD, fluorescence) and by enzymatic and chemical assays. We found that the nanoparticles enhanced the stability of the TBA ODN to enzymatic degradation. Finally, we evaluated the amount of the TBA-conjugated nanoparticles immobilized on a magnetic separator in view of the potential use of the nanosystem for the magnetic capture of thrombin from complex mixtures.     

Increased Serum Hepcidin Levels in Subjects with the Metabolic Syndrome: A Population Study

The recent discovery of hepcidin, the key iron regulatory hormone, has changed our view of iron metabolism, which in turn is long known to be linked with insulin resistant states, including type 2 diabetes mellitus and the Metabolic Syndrome (MetS). Serum ferritin levels are often elevated in MetS (Dysmetabolic hyperferritinemia - DHF), and are sometimes associated with a true mild-to-moderate hepatic iron overload (dysmetabolic iron overload syndrome - DIOS). However, the pathophysiological link between iron and MetS remains unclear. This study was aimed to investigate, for the first time, the relationship between MetS and hepcidin at population level. We measured serum hepcidin levels by Mass Spectrometry in 1,391 subjects from the Val Borbera population, and evaluated their relationship with classical MetS features. Hepcidin levels increased significantly and linearly with increasing number of MetS features, paralleling the trend of serum ferritin. In multivariate models adjusted for relevant variables including age, C-Reactive Protein, and the HFE C282Y mutation, ferritin was the only significant independent predictor of hepcidin in males, while in females MetS was also independently associated with hepcidin. Overall, these data indicate that the fundamental iron regulatory feedback is preserved in MetS, i.e. that hepcidin tends to progressively increase in response to the increase of iron stores. Due to recently discovered pleiotropic effects of hepcidin, this may worsen insulin resistance and contribute to the cardiovascular complications of MetS.     

Vascular endothelial growth factor receptor-3 activity is modulated by its association with caveolin-1 on endothelial membrane

Vascular endothelial growth factor receptor-3 (VEGFR-3) is constitutively expressed in lymphatic vessels and transiently in endothelial cells of blood vessels during angiogenesis. Here we report that VEGFR-3 localizes in the caveolae membrane of endothelial cells and co-immunoprecipitates with caveolin-1. Caveolin-1 silencing or its depletion from the cell membrane by cholesterol increases VEGFR-3 autophosphorylation, suggesting that caveolin acts as a negative regulator of VEGFR-3 activity. Receptor activation induces caveolin-1 phosphorylation on tyrosine residues including tyrosine 14. Cell treatment with Src or Abl inhibitors PP2 or STI571, prior to receptor stimulation, affects caveolin-1 phosphorylation without affecting receptor autophosphorylation, suggesting that both Src and Abl are involved in VEGFR-3-dependent caveolin-1 phosphorylation. Caveolin-1 phosphorylation in Src/Fyn/Yes knockout cells demonstrated that Abl phosphorylates caveolin-1 independently from Src family members. These results suggest a functional interaction between VEGFR-3 and caveolin-1 to modulate endothelial cell activation during angiogenesis.     

Oxygen free radical scavenger properties of dehydroepiandrosterone

The microsomes from dehydroepiandrosterone (DHEA)-supplemented animals are good hydroxyl radical scavengers, as demonstrated through electron spin resonance and deoxyribose degradation. The ability of DHEA-supplemented microsomes to react with superoxide radical was also demonstrated through the inhibition of nitro-blue tetrazolium reduction determined by superoxide radicals produced in a hypoxanthine–xanthine oxidase system. DHEA-enriched microsomes, obtained from acutely DHEA-treated rats, become resistant to iron-dependent lipid peroxidation triggered by H₂O₂/FeSO₄ and ascorbate/FeSO₄. The direct addition of DHEA to microsomes from untreated rats failed to prevent iron-dependent lipid peroxidation, even if the microsomes were preincubated with DHEA for up to 15 min, indicating that in vivo transformation is required before antioxidant action can be exerted. © 1998 John Wiley & Sons, Ltd.