Alpha-anomeric configuration of GT oligodeoxynucleotide leads to loss of the specific aptameric and cytotoxic properties retained by the beta-anomeric analog

The development of antisense, antigene, or aptameric oligonucleotides to modulate in vivo cellular functions depends on using stable biologic molecules. Previous investigations showed that GT oligonucleotides could exert a specific, dose-dependent cytotoxic effect on human cancer cell lines. This is tightly related to the ability of these oligomers to specifically bind nuclear proteins, giving a complex of apparent molecular weight of 45 kDa. We demonstrated that with respect to the cytotoxic GT-beta-oligomer, alpha-anomeric GT analog did not alter the growth of the T lymphoblastic CCRF-CEM cell line, although the cells took it up efficiently. In agreement with this, GT-alpha-oligomer did not form the cytotoxicity-related 45-kDa complex with nuclear proteins. These findings likely could be related to the ability of GT-alpha to structure under nondenaturing conditions because of the high number of T in the sequence.     

Increase in therapeutic index of doxorubicin and vinblastine by aptameric oligonucleotide in human T lymphoblastic drug-sensitive and multidrug-resistant cells

Aptameric GT oligomers are a new class of potential anticancer molecules that inhibit the growth of human cancer cell lines by binding to specific nuclear proteins. We demonstrated that an aptameric GT oligonucleotide increased the therapeutic index of doxorubicin and vinblastine in T lymphoblastic drug-sensitive and multidrug-resistant (MDR) cells. The doxorubicin ID50 decreased 6.5-fold by coadministration of 1 microM GT to CCRF-CEM cells and by 24-fold by coadministration of 0.75 microM GT to CEM-VLB300 cells. In CEM-VLB300 cells, the vinblastine ID50 decreased 11-fold by coadministration of 0.5 microM GT. Control CT sequence did not potentiate the drugs in either CCRF-CEM or CEM-VLB300 cells. The ability of GT to bind to specific nuclear proteins in cancer cells related to the increase in the therapeutic index of doxorubicin and vinblastine. No cooperation was detected by the administration of GT oligomer together with doxorubicin to rat differentiated thyroid FRTL-5 cells and to normal human lymphocytes. These cells did not show binding of GT to the specific nuclear proteins, and they were not sensitive to the cytotoxic action of the GT sequence. Drug potentiation by GT not involving normal human lymphocytes might be exploited to develop a more selective treatment of drug-sensitive and MDR tumors.     

Effect of cations on purine.purine.pyrimidine triple helix formation in mixed-valence salt solutions.

The effect of various monovalent, divalent and oligovalent cations on the reaction of triplex formation by GT and AG motif triplex-forming oligonucleotides, designed to bind to biologically relevant polypurine-polypyrimidine sequences occurring in the promoters of the murine Ki-ras and human bcr genes, has been investigated by means of electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments. We found that in the presence of 10 mm MgCl2 the triple helices were progressively destabilized by adding increasing amounts of NaCl, from 20 to 140 mm, to the solution. We also observed that, while the total monovalent-ion concentration was constant at 100 mm, the exchange of sodium with potassium, but not lithium, results in a further destabilization of the triple helices, due to self-association equilibria involving the G-rich triplex-forming oligonucleotides. Potassium was found to destabilize triplex DNA even when the triple helices are preformed in the absence of K+. However, footprinting experiments also showed that the inhibitory effect of K+ on triplex DNA is partially compensated for by millimolar amounts of divalent transition metal ions such as Mn2+ and Ni2+, which upon coordinating to N7 of guanine are expected to enhance hydrogen-bond formation between the target and the third strand, and to reduce the assembly in quadruple structures of G-rich triplex-forming oligonucleotides. Triplex enhancement in the presence of potassium was also observed, but to a lesser extent, when spermine was added to the reaction mixture. Here, the ion effect on triplex DNA is rationalized in terms of competition among the different valence cations to bind to triplex DNA, and differential cation stabilization of unusual quadruplex structures formed by the triplex-forming oligonucleotides.     

Modulation of human neutrophil function by C-reactive protein

Investigation of the effect of C-reactive protein (CRP), an acute-phase reactant, on the functional capacities of human neutrophils was carried out as the basis for elucidating the biological function of C-reactive protein. An initial stimulation at low concentrations, followed by inhibition of superoxide production, and secretion of vitamin-B12-binding protein in the presence of two stimulants, phorbol myristate acetate and concanavalin A, and of neutrophil chemotaxis with increasing concentration of CRP was observed. Correlation between modulation of cell function, at least at relatively high CRP concentrations (greater than 50 micrograms/ml) and an increase in the intracellular level of cAMP is suggested. CRP was also found to enhance neutrophil phagocytosis of particles not containing phosphorylcholine, the native CRP ligand. The proposed role of CRP in neutrophil function is one of regulation and as a negative feedback for potential cytotoxic neutrophil functions.     

Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers.

GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain.     

Anxiety stress and nociceptive responses in mice

Nociception in laboratory animals appears to be influenced by physical or emotional stressors. Nevertheless, the reported data are not univocal. Discrepancies seem to be caused by some kind of stress model and/or by the timing of stressor application. The aim of the present work is to study the influence of chronic application of a well-controlled and defined anxiety stress paradigm (rotational stress) on the behavioral formalin pain responses in mice maintained in a low-stress environment. The results indicate that emotional chronic stress increases specific pain responses in the late inflammatory phase and, correspondingly, decreases self-grooming. Locomotor activity appears influenced by pain presence only. The hormonal and neural mechanisms that could be involved in the observed nonspecific and specific nociceptive responses to stress are discussed.     

Territorial male gobies respond aggressively to sneakers but do not adjust their sperm expenditure

We investigated whether mating behavior (sperm expenditure,courtship rate, and nest guarding) varied according to differentlevels of sperm competition in territorial males of two gobyspecies, the grass and the black gobies. We measured sperm expenditure(sperm released after 30 min from the beginning of the spawning),male courtship rate, and nest-guarding behavior in territorialmales of both species during simulated spawnings, in which wevaried the number of attending sneakers. Our results showedthat, in both species, territorial males adjusted their effortin nest guarding to the presence of rival sneakers by increasingthe time spent patrolling the territory and attacking the sneakers.In contrast, sperm expenditure and male courtship rate werenot influenced by the number of attending sneakers. These resultsare in agreement with those reported for other fish with alternativemating tactics and help to interpret previous inconsistenciesbetween theoretical predictions and measured levels of spermreleased at different levels of sperm competition by sneakersof the two gobiids studied here.     

Variability of GnRH secretion in two goby species with socially controlled alternative male mating tactics

Male reproductive phenotypic plasticity related to environmental-social conditions is common among teleost fish. In several species, males adopt different mating tactics depending on their size, monopolizing mates when larger, while parasitizing dominant male spawns when smaller. Males performing alternative mating tactics are often characterized by a strong dimorphism in both primary and secondary reproductive traits. According to studies on sex-changing species and on species where only one male morph is reproductively active, male alternative phenotypes are expected to vary also in gonadotropin-releasing hormone (GnRH) neurons in forebrain preoptic area (POA). Here, we compared the intra- and inter-sexual variations in number and size of GnRH neurons, along with gonads and male accessory structure investment, in two goby species, the grass goby, Zosterisessor ophiocephalus, and the black goby, Gobius niger, characterized by male alternative mating phenotypes. In both species, older and larger males defend nests, court and perform parental care, while younger and smaller ones try to sneak territorial male spawning. We found that grass goby and black goby have different patterns of GnRH expression. Grass goby presents a clear intra-sexual dimorphism in GnRH expression, related to the occurrence of alternative mating tactics, while in the black goby, only inter-sexual differences are observed. The inter- and intra-specific variability in the GnRH neurons in these two goby species is discussed in light of the differences in migratory behavior, nest type, and mating system.     

Interaction of G-rich GT oligonucleotides with nuclear-associated eEF1A is correlated with their antiproliferative effect in haematopoietic human cancer cell lines

G-rich GT oligonucleotides with a different content of G clusters have been evaluated for their ability to exert cytotoxicity and to bind to nuclear-associated proteins in T-lymphoblast CCRF-CEM cells. Only the oligomers that did not form G-based structures or had a poor structure, under physiological conditions, were able to exert significant cellular growth inhibition effect. The cytotoxicity of these oligomers was related to their binding to the nuclear-associated eEF1A protein, but not to the recognition of nucleolin or other proteins. In particular, GT oligomers adopting a conformation compatible with G-quadruplex, did not exert cytotoxicity and did not bind to eEF1A. The overall results suggest that the ability of oligomers to adopt a G-quadruplex-type secondary structure in a physiological buffer containing 150 mM NaCl is not a prerequisite for antiproliferative effect in haematopoietic cancer cells. The cytotoxicity of G-rich GT oligomers was shown to be tightly related to their binding affinity for eEF1A protein.