Timely DNA vaccine combined with systemic IL-12 prevents parotid carcinomas before a dominant-negative p53 makes their growth independent of HER-2/neu expression

Double transgenic mice overexpressing the transforming rat HER-2/neu oncogene and the mutated p53, with both dominant-negative and a gain-of-function properties, display early aggressive and metastasizing parotid tumors. Multiple acinar and ductal hyperplasia foci overexpressing the HER-2/neu gene product are evident at wk 5 and progress to poorly differentiated carcinoma by wk 7. Mice die before wk 18 with invasive carcinomas and multiple metastases that no longer express HER-2/neu. A combination of repeated electroporations of plasmids coding for the extracellular and transmembrane domains of the rat HER-2/neu receptor with systemic IL-12 administrations started when the parotids that present diffuse hyperplasia protected all female and 50% of the male mice until the close of the experiment at wk 40. This combined treatment began when multifocal in situ carcinomas that were already present cured 33% of the females and 25% of the males. The most prominent immunologic features associated with the antitumor protection were the production of high titers of anti-HER-2/neu Abs and the nonappearance of cell-mediated cytotoxic reactivity. In conclusion, anti-HER-2/neu vaccination combined with systemic IL-12 control parotid carcinomas as far as p53 mutation makes their growth independent of HER-2/neu expression.     

A new modified thrombin binding aptamer containing a 5'-5' inversion of polarity site

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5′–5′ inversion of polarity site, namely d(^3′GGT^5′-^5′TGGTGTGGTTGG³′), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(^3′GGT^5′-^5′TGGTGTGGTTGG^3′) is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4°C higher than the corresponding unmodified TBA. d(^3′GGT^5′-^5′TGGTGTGGTTGG^3′) continues to display an anticoagulant activity, even if decreased with respect to the TBA.     

Accurate energies of hydrogen bonded nucleic acid base pairs and triplets in tRNA tertiary interactions

Tertiary interactions are crucial in maintaining the tRNA structure and functionality. We used a combined sequence analysis and quantum mechanics approach to calculate accurate energies of the most frequent tRNA tertiary base pairing interactions. Our analysis indicates that six out of the nine classical tertiary interactions are held in place mainly by H-bonds between the bases. In the remaining three cases other effects have to be considered. Tertiary base pairing interaction energies range from -8 to -38 kcal/mol in yeast tRNA(Phe) and are estimated to contribute roughly 25% of the overall tRNA base pairing interaction energy. Six analyzed posttranslational chemical modifications were shown to have minor effect on the geometry of the tertiary interactions. Modifications that introduce a positive charge strongly stabilize the corresponding tertiary interactions. Non-additive effects contribute to the stability of base triplets.     

Anxiolytic-like effect of the selective neuropeptide Y Y2 receptor antagonist BIIE0246 in the elevated plus-maze

The involvement of Neuropeptide Y (NPY) in the pathophysiology of mood disorders has been suggested by clinical and preclinical evidence. NPY Y1 and Y2 receptors have been proposed to mediate the NPY modulation of stress responses and anxiety related behaviors. To further investigate the role of Y2 receptors in anxiety we studied the effect of BIIE0246, a selective Y2 receptor antagonist, in the elevated plus-maze test. Rats treated with 1.0 nmol BIIE0246 showed an increase in the time spent on the open arm of the maze. In addition, to study the effects of the Y2 antagonism on NPY protein level, NPY-like immunoreactivity was measured in different brain regions following treatment with BIIE0246, but no statistically significant effects were observed. These results suggest that BIIE0246 has an anxiolytic-like profile in the elevated plus-maze.     

Determinants of conformational dimerization of Mad2 and its inhibition by p31comet

The spindle assembly checkpoint (SAC) monitors chromosome attachment to spindle microtubules. SAC proteins operate at kinetochores, scaffolds mediating chromosome-microtubule attachment. The ubiquitous SAC constituents Mad1 and Mad2 are recruited to kinetochores in prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate anaphase onset. Its function is counteracted by p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2 (Open) to C-Mad2 (Closed). A Mad1-bound C-Mad2 template, to which O-Mad2 binds prior to being converted into Cdc20-bound C-Mad2, assists this process. A molecular understanding of this prion-like property of Mad2 is missing. We characterized the molecular determinants of the O-Mad2:C-Mad2 conformational dimer and derived a rationalization of the binding interface in terms of symmetric and asymmetric components. Mutation of individual interface residues abrogates the SAC in Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that O-Mad2 undergoes a major conformational rearrangement upon binding C-Mad2, suggesting that dimerization facilitates the structural conversion of O-Mad2 required to bind Cdc20. We also show that the negative effects of p31comet on the SAC are based on its competition with O-Mad2 for C-Mad2 binding.     

Effect of anti-NGF on ovarian expression of alpha1- and beta2-adrenoceptors, TrkA, p75NTR, and tyrosine hydroxylase in rats with steroid-induced polycystic ovaries

Estradiol valerate (EV)-induced polycystic ovaries (PCO) in rats are associated with higher ovarian release and content of norepinephrine, decreased beta2-adrenoceptors (ARs), and dysregulated expression of alpha1-AR subtypes, all preceded by an increase in the production of ovarian NGF. The aim of this study was to further elucidate the role of NGF in the ovaries by blocking the action of NGF during development of EV-induced PCO in rats. Control and EV-injected rats were treated with intraperitoneal injections of IgG (control and PCO groups) or with anti-NGF antibodies (anti-NGF and PCO anti-NGF groups) every third day for 5 wk starting from the day of PCO induction. Rat weight, estrous cyclicity, ovarian morphology, ovarian mRNA, and protein expression of alpha1-AR subtypes, beta2-AR, the NGF receptor tyrosine kinase A (TrkA), p75 neurotrophin receptor (p75NTR), and tyrosine hydroxylase (TH) were analyzed. Ovaries in both PCO and PCO anti-NGF groups decreased in size as well as in number and size of corpora lutea. mRNA expression of alpha1a-AR and TrkA in the ovaries was lower, whereas expression of alpha1b- and alpha1d-AR and TH was higher, in the PCO group than in controls. Protein quantities of alpha1-ARs, TrkA, p75NTR, and TH were higher in the PCO group compared with controls, whereas the protein content of beta2-AR was lower. Anti-NGF treatment in the PCO group restored all changes in mRNA and protein content, except that of alpha1b-AR and TrkA mRNAs, to control levels. The results indicate that the NGF/NGF receptor system plays a role in the pathogenesis of EV-induced PCO in rats.     

Functionalized carbon nanotubes as emerging nanovectors for the delivery of therapeutics

Functionalized carbon nanotubes (f-CNT) are emerging as a new family of nanovectors for the delivery of different types of therapeutic molecules. The application of CNT in the field of carrier-mediated delivery has become possible after the recent discovery of their capacity to penetrate into the cells. CNT can be loaded with active molecules by forming stable covalent bonds or supramolecular assemblies based on noncovalent interactions. Once the cargos are carried into various cells, tissues and organs they are able to express their biological function. In this review, we will describe the potential of f-CNT to deliver different types of therapeutic molecules.     

Benthic communities on a sandy Ligurian beach (NW Mediterranean)

The different components of the benthic community of a sandy microtidal beach (Arenzano) in Liguria (NW Mediterranean) were investigated during late spring (May) 2002 and 2003. Sampling was carried out in two transects, chosen in order to represent the characteristics of the entire beach and their eventual spatial variations. Each transect included two stations: one placed in the swash zone (SW) and one in the surf zone (SF). Although no significant differences were found in the sediment texture over the 2 years (t-tests p > 0.1 for all the granulometric fractions), notwithstanding an increase in the mean grain size (from 0.8 to 1.1 mm) between the sampling periods, 2002 was characterised by a higher quantity of organic matter (on average 14.4 vs. 3.6 gC m⁻² for the sum of proteins, carbohydrates and lipids) and higher bacterial biomass (on average 1.9 vs. 0.9 gC m⁻²). The metazoan assemblages (meiofauna and macrofauna) were also richer (density = 2.9 × 10⁵ vs. 1.0 × 10⁵ ind. m⁻², biomass = 0.09 vs. 0.03 gC m⁻² for meiofauna; density = 1988 vs. 739 ind. m⁻², biomass = 0.14 vs. 0.03 gC m⁻² for macrofauna) in 2002. A significant quantitative reduction (t-test for proteins, carbohydrates and lipids, at least p = 0.004) in the food supply in 2003 affected the abundance of the metazoans, as confirmed by a multivariate analysis that clearly differentiated the 2 years, and seemed to inhibit their relationships within the benthic food web. The bacterial biomass was always dominant, even under the least favourable trophic conditions, due to the ability of bacteria to adapt to a very harsh environment. Our results suggest that the food supply played an important role in the benthic community structures of the beach during late spring, bacteria being the key organisms within the benthic system. The communities seemed to be bottom-up controlled, while predation seemed to be irrelevant.