Production of active recombinant eIF5A: reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.     

Electrodeposition of porous Co layers and their conversion to electrocatalysts for methanol oxidation by spontaneous deposition of Pd

Electrocatalysts for methanol oxidation were prepared through a two stage deposition process: porous cobalt layers were deposited, by cathodic reduction of Co²⁺ ions, and then modified by spontaneous deposition of Pd. A basic sulphate solution and a mildly acid chloride solution were compared as media for the electrodeposition of Co. Deposits with the highest surface roughness were obtained in the chloride solution, at large current densities. Pd was deposited onto the Co porous layers by immersing them in acid deaerated PdCl₂ solutions, at open circuit. The Pd loading and the Pd surface area were estimated by UV–visible spectroscopy and by cyclic voltammetry, respectively. The Pd-modified Co electrodes were tested as anodes for methanol oxidation and compared to the similarly prepared Pd-modified Ni electrodes. The former exhibited better stability of performance and higher methanol oxidation peak currents per unit Pd mass, ca. 200 A g⁻¹.     

Peptidyl Vinyl Ketone Irreversible Inhibitors of Rhodesain: Modifications of the P2 Fragment

In this paper, we report the design, synthesis and biological investigation of a series of peptidyl vinyl ketones obtained by modifying the P2 fragment of previously reported highly potent inhibitors of rhodesain, the main cysteine protease of Trypanosoma brucei rhodesiense. Investigation of the structure–activity relationship led us to identify new rhodesain inhibitors endowed with an improved selectivity profile (a selectivity index of up to 22 000 towards the target enzyme), and/or an improved antitrypanosomal activity in the sub-micromolar range.     

Synthesis and Biological Evaluation of CTP Synthetase Inhibitors as Potential Agents for the Treatment of African Trypanosomiasis

Acivicin analogues with an increased affinity for CTP synthetase (CTPS) were designed as potential new trypanocidal agents. The inhibitory activity against CTPS can be improved by increasing molecular complexity, by inserting groups able to establish additional interactions with the binding pocket of the enzyme. This strategy has been pursued with the synthesis of α‐amino‐substituted analogues of Acivicin and N1‐substituted pyrazoline derivatives. In general, there is direct correlation between the enzymatic activity and the in vitro anti‐trypanosomal efficacy of the derivatives studied here. However, this cannot be taken as a general rule, as other important factors may play a role, notably the ability of uptake/diffusion of the molecules into the trypanosomes.     

Isolation and Characterization of Microsatellite Markers for Passiflora contracta

Passiflora contracta Vitta (Passifloraceae) is an endemic species of the Atlantic Rainforest, one of the most species-rich ecoregions in the world, although extremely endangered. We have developed an enriched microsatellite library in order to fine-scale studies of the genetic structure of P. contracta. Twelve pairs of microsatellite primers were designed, and seven loci were successfully amplified and characterized by genotyping two wild populations of P. contracta. All seven loci were polymorphic, with an average number of alleles found being 4.8 and 5 per population. The cross-species transferability was tested using sister species Passiflora ovalis Vell. Ex Roemer. The development of these markers will contribute to the studies of population genetics in P. contracta as well as future studies concerning diversity patterns in the Atlantic Rainforest, and may also help to establish strategies for the conservation of this species.     

Impedance of passive oxide films with graded thickness: Influence of the electrode and cell geometry

The impedance of cylindrical electrodes and of rectangular sheet electrodes coated by passive films with either a constant or a graded thickness has been calculated using a finite element method for solving Laplace's equation. The comparison of different electrode/cell geometries characterized by homogenous (on cylindrical electrodes) or inhomogeneous distributions of primary current (on rectangular sheet electrodes) and by homogeneous or graded thickness has allowed to clarify the origin of frequency dispersion phenomena. Calculated curves were compared with experimental impedance plots obtained with Nb and Ta sheet electrodes and with Nb cylindrical electrodes. The results of the finite element calculations for rectangular sheet electrodes are in better agreement with the experiments than those of a previous analytical calculation developed under a simplifying assumption (i.e. identical local electrolyte resistance at each point of the electrode surface) which was equivalent to neglecting current distribution induced by electrode and cell geometry.     

Mechanical and dynamic mechanical analysis of hybrid composites molded by resin transfer molding

This work aims to evaluate the performance of glass/sisal hybrid composites focusing on mechanical (flexural and impact) and dynamic mechanical analyses (DMTA). Hybrid composites with different fiber loadings and different volume ratios between glass and sisal were studied. The effect of the fiber length has also been investigated. The densities of the composites were compared with the theoretical values, showing agreement with the rule of mixtures. The results obtained in the flexural and impact analysis revealed that, in general, the properties were always higher for higher overall reinforcement content. By DMTA, an increase in the storage and loss modulus was found, as well as a shift to higher values for higher glass loading and overall fiber volume. It was also noticed an increase in the efficiency of the filler and the calculated activation energy for the relaxation process in the glass transition region. The fiber length did not significantly change the results observed in all analyses carried out in this work. The calculated adhesion factor increased for higher glass loadings, meaning the equation may not be applied for the system studied and there are other factors, besides adhesion influencing energy dissipation of the composites. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Modulation on C‐ and N‐Terminal Moieties of a Series of Potent and Selective Linear Tachykinin NK2 Receptor Antagonists

Herein we describe the synthesis of a series of new potent tachykinin NK₂ receptor antagonists by the modulation of the C‐ and N‐terminal moieties of ibodutant (MEN 15596, 1 ). The N‐terminal benzo[b]thiophene ring was replaced by different substituted naphthalenes and benzofurans, while further modifications were evaluated at the C‐terminal tetrahydropyran moiety. Most compounds demonstrated a high affinity for the human NK₂ receptor and high in vitro antagonist potency, indicating that a wide range of substituents at both termini can be incorporated in the molecule without detrimental effects on the interactions with the NK₂ receptor. Selected compounds were tested in vivo confirming their activity as NK₂ antagonists. In particular, after both iv and id administration to guinea pig, compound 61 b was able to antagonize NK₂‐induced colonic contractions with a potency and duration‐of‐action fully comparable to the reference compound 1 (MEN 15596, ibodutant).     

A backoff strategy for model‐based experiment design under parametric uncertainty

Model‐based experiment design techniques are an effective tool for the rapid development and assessment of dynamic deterministic models, yielding the most informative process data to be used for the estimation of the process model parameters. A particular advantage of the model‐based approach is that it permits the definition of a set of constraints on the experiment design variables and on the predicted responses. However, uncertainty in the model parameters can lead the constrained design procedure to predict experiments that turn out to be, in practice, suboptimal, thus decreasing the effectiveness of the experiment design session. Additionally, in the presence of parametric mismatch, the feasibility constraints may well turn out to be violated when that optimally designed experiment is performed, leading in the best case to less informative data sets or, in the worst case, to an infeasible or unsafe experiment. In this article, a general methodology is proposed to formulate and solve the experiment design problem by explicitly taking into account the presence of parametric uncertainty, so as to ensure both feasibility and optimality of the planned experiment. A prediction of the system responses for the given parameter distribution is used to evaluate and update suitable backoffs from the nominal constraints, which are used in the design session to keep the system within a feasible region with specified probability. This approach is particularly useful when designing optimal experiments starting from limited preliminary knowledge of the parameter set, with great improvement in terms of design efficiency and flexibility of the overall iterative model development scheme. The effectiveness of the proposed methodology is demonstrated and discussed by simulation through two illustrative case studies concerning the parameter identification of physiological models related to diabetes and cancer care. © 2009 American Institute of Chemical Engineers AIChE J, 2010     

Aramid pulp treated with imidazolium ionic liquids as a filler in rigid polyurethane bio-foams

This article reports an aramid pulp (AP) treated with two ionic liquids (IL), namely 1-n-butyl-3-methylimidazolium chloride (C₄.Cl) and 1-carboxymethyl-3-methylimidazolium chloride (HO₂C), and its use as a filler in reinforced rigid polyurethane foams (RPUF). The RPUF were incorporated with the treated AP at three weight fractions (c.a. 0.1, 0.5, and 1.0 wt%) and were produced by the free rising method. The results showed that the studied IL promoted a better interaction between the AP and the RPUF system, which increased the overall reactivity, imparting a higher cell anisotropy. This also yielded a positive effect in mechanical properties and thermal stability of the RPUF. Compared to the neat RPUF, outstanding increases of approximately 50 and 20% were achieved in compressive modulus and strength, respectively. In all, the use of IL promoted increased compatibility between matrix and reinforcement, especially that HO₂C IL.