Functional specific roles of H-ras and N-ras. A proteomic approach using knockout cell lines

Ras small GTPases function as transducers of extracellular signals regulating cell survival, growth and differentiation. There are three major ras isoforms: H-, N- and K-Ras. To improve the understanding of H- and N-Ras protein signalling networks, we compared total proteome changes in mouse embryonic fibroblasts knock out for H-ras and/or N-ras, using proteomics tools combining 2DE, semi-quantitative image analysis, in-gel trypsin digestion and mass spectrometry. There are four up-regulated proteins due to the loss of expression of H-Ras (including cyclin-dependent kinase inhibitor 2A) and eight down-regulated (including stress-70 protein, dihydropyrimidinase-related-protein 3, heat shock cognate 71 kDa protein, tropomyosin beta chain, Rho GDP-dissociation inhibitor 1) and six up-regulated proteins (e.g. leukocyte elastase inhibitor A, L-lactate dehydrogenase B chain, c-Myc-responsive protein Rcl, interleukin-1 receptor antagonist protein) due to the loss of expression of both N- and H-Ras. Most of these proteins are related to Ras signalling in one way or another. Changes in expression of some of these proteins were further confirmed by Western blot. This proteomic comparative analysis from loss of function of H- and N-Ras knockout fibroblasts yields interpretable data to elucidate the differential protein expression, and contributes to evaluate the possibilities for physiological and therapeutic targets.     

Synthesis and characterization of novel second-order NLO-chromophores bearing pyrrole as an electron donor group

Two series of novel push–pull 1-(4-(thiophen-2-yl)phenyl)-1H-pyrroles 3–5 were designed to explore the consequence of using different electron accepting moieties linked to the thiophene at the arylthiophene bridge or to the pyrrole heterocycle, which plays the role of donor group. Compound 2 showed a different reactivity behavior in the presence of the Vilsmeier reagent or with tetracyanoethylene (TCNE) giving compounds 4a and 4b functionalized, respectively, at the 2 or on the 3-position of the pyrrole heterocycle. Their optical (linear and first hyperpolarizability), electrochemical, and thermal properties have been examined. Hyper-Rayleigh scattering (HRS) in dioxane solutions using a fundamental wavelength of 1064 nm was employed to evaluate their second-order nonlinear optical properties. Of these systems, thiobarbituric acid derivative 5b functionalized in the thiophene ring exhibits the largest first hyperpolarizability (β=2480×10^?30 esu, T convention) compared to the corresponding compound 4c substituted on the pyrrole heterocycle (β=290×10^?30 esu, T convention). Good to excellent thermal stabilities were also obtained for push–pull compounds 4 and 5 (270–288 °C). This multidisciplinary study shows that modulation of the optical and electronic properties can be achieved by introduction of the acceptor groups in the thiophene of the arylthiophene bridge. The measured molecular first hyperpolarizabilities and the observed electrochemical behavior are quite sensitive to the position of acceptor group on the heterocyclic system (on the thiophene or on the pyrrole ring) as well as the strength of the acceptor moieties. Moreover, the combination of their good nonlinearity and high thermal stability make them good candidates for second-order nonlinear optical applications.     

Synthesis and evaluation of fluorimetric and colorimetric chemosensors for anions based on (oligo)thienyl-thiosemicarbazones

A family of heterocyclic thiosemicarbazone dyes (3a–d) containing thienyl groups has been synthesized, characterized, and their chromo-fluorogenic response in acetonitrile in the presence of selected anions was studied. Acetonitrile solutions of 3a–d show absorption bands in the 338–425 nm range, which are modulated by the groups attached to the thiosemicarbazone moiety. The fluoride, chloride, bromide, iodide, dihydrogen phosphate, hydrogen sulfate, nitrate, acetate, and cyanide anions were used in the recognition studies. Only sensing features were observed for fluoride, cyanide, acetate, and dihydrogen phosphate anions. Two different chromogenic responses were found, (i) a small shift of the absorption band due to coordination of the anions with the thiourea protons and (ii) the appearance of a new red-shifted band due to deprotonation of the receptor. For the latter process changes in the color solutions from pale-yellow to orange-red were observed. Fluorescence studies showed a different emission behavior according to the number of thienyl rings in the π-conjugated bridges. Stability constants for the two processes (complex formation+deprotonation) for receptors 3a–d in the presence of fluoride and acetate anions were determined from spectrophotometric titrations using the HypSpec program. The interaction of 3d with fluoride was studied through ¹H NMR titrations. Semiempirical calculations to evaluate the hydrogen-donating ability of the receptors were also performed.     

Proline imidazolidinones and enamines in Hajos-Wiechert and Wieland-Miescher ketone synthesis

Readily available aromatic prolinamides obtained from the acid chloride of proline hydrochloride and anilines induce large enantiomeric excesses in intramolecular aldol condensations. Imidazolidinones derived from the reaction of the catalyst and enamines have been found as intermediates in these reactions.     

Inhibition of cPLA2 and sPLA2 activities in primary cultures of rat cortical neurons by Centella asiatica water extract

Leaf extract of Centella asiatica has been used as an alternative medicine for memory improvement in the Indian Ayurvedic system of medicine for a long time. Although several studies have revealed its effect in ameliorating the cognitive impairment in rat models of Alzheimer's disease, the molecular mechanism of C. asiatica on neuroprotection still remains unexplained. In this study, we investigated the effects of C. asiatica water extract on activity of subtypes of phospholipase A2 (PLA2) in primary cultures of rat cortical neurons and quantified by HPLC a possible molecule responsible for the activity. The cPLA2 and sPLA2 activities were inhibited in vitro by asiaticoside present in the water extract of C. asiatica. This extract may be a candidate for the treatment of neurodegenerative processes because of its pharmacological activity in the brain and its low toxicity, as attested by its long popular use as a natural product.     

An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector

Background  

This study compared the transduction efficiencies of an adeno-associated viral (AAV) vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP), with a lentiviral (LV) vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed), to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 × 10⁸ or 1 × 10⁹ genomic copies of AAV1-GFP and 1 × 10⁵ transducing units of LV-dsRed.     

Backlund transformations and exact solutions for a nonlinear elliptic equation modelling isothermal magnetostatic atmosphere

The equations of magnetostatic equilibria for a plasma in agravitational field are investigated analytically. For equilibriawith an ignorable spatial coordinate, the equations reduce toa single nonlinear elliptic equation for the magnetic potentialu known as the Grad-Shafranov equation. By specifying the arbitraryfunctions in this equation, a Liouville equation is obtained.Bäcklund transformations are described and applied to obtainexact solutions for the Liouville equation modelling an isothermalmagnetostatic atmosphere, in which the current density J isproportional to the exponential of the magnetic potential andmoveover falls off exponentially with distance vertical to thebase with an e-folding distance equal to the gravitational scaleheight.     

Ferrimagnetic resonance of manganese ferrites with iron excess

Ferrimagnetic resonance linewidths of polycrystalline and single crystal manganese ferrites, Mn_xFe_3-xO₄ with x<1.0, were measured at 8.9 GHz in the range 77–320 K. Conductivity measurements were carried out from 100 Hz to 40 MHz. The behavior of the ferrimagnetic resonance linewidth and of the resonance field was different from that obtained for manganese ferrites with x=1. These differences are attributed to the presence of a resonance mode in addition to the uniform resonance mode. The variation of the skin depth as a function of the temperature and the high conductivity of the samples is responsible for the appearance of this extra mode. This assumption is corroborated by the analysis of the asymmetry of the absorption curve. Received: 25 May 2000 / Accepted: 9 October 2000 / Published online: 26 April 2001     

Bioinspired systems for metal-ion sensing: new emissive peptide probes based on benzo[d]oxazole derivatives and their gold and silica nanoparticles

Seven new bioinspired chemosensors (2-4 and 7-10) based on fluorescent peptides were synthesized and characterized by elemental analysis, (1)H and (13)C NMR, melting point, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and IR and UV-vis absorption and emission spectroscopy. The interaction with transition- and post-transition-metal ions (Cu(2+), Ni(2+), Ag(+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Fe(3+)) has been explored by absorption and fluorescence emission spectroscopy and MALDI-TOF-MS. The reported fluorescent peptide systems, introducing biological molecules in the skeleton of the probes, enhance their sensitivity and confer them strong potential for applications in biological fields. Gold and silica nanoparticles functionalized with these peptides were also obtained. All nanoparticles were characterized by dynamic light scattering, transmission electron microscopy, and UV-vis absorption and fluorescence spectroscopy. Stable gold nanoparticles (diameter 2-10 nm) bearing ligands 1 and 4 were obtained by common reductive synthesis. Commercial silica nanoparticles were decorated at their surface using compounds 8-10, linked through a silane spacer. The same chemosensors were also taken into aqueous solutions through their dispersion in the outer layer of silica core/poly(ethylene glycol) shell nanoparticles. In both cases, these complex nanoarchitectures behaved as new sensitive materials for Ag(+) and Hg(2+) in water. The possibility of using these species in this solvent is particularly valuable because the impact on human health of heavy- and transition-metal-ion pollution is very severe, and all analytical and diagnostics investigations involve a water environment.     

Chagas Disease: Perspectives on the Past and Present and Challenges in Drug Discovery

Chagas disease still has no effective treatment option for all of its phases despite being discovered more than 100 years ago. The development of commercial drugs has been stagnating since the 1960s, a fact that sheds light on the question of how drug discovery research has progressed and taken advantage of technological advances. Could it be that technological advances have not yet been sufficient to resolve this issue or is there a lack of protocol, validation and standardization of the data generated by different research teams? This work presents an overview of commercial drugs and those that have been evaluated in studies and clinical trials so far. A brief review is made of recent target-based and phenotypic studies based on the search for molecules with anti-Trypanosoma cruzi action. It also discusses how proteochemometric (PCM) modeling and microcrystal electron diffraction (MicroED) can help in the case of the lack of a 3D protein structure; more specifically, Trypanosoma cruzi carbonic anhydrase.