Combined effects of vasoactive intestinal peptide and dopamine on adenylate cyclase in prolactin-secreting cells

VIP stimulates adenylate cyclase activity of male and female rat anterior pituitaries and human prolactinomas, while dopamine inhibits the enzyme activity of female rat pituitaries and prolactinomas. A dopamine inhibited cyclase can be detected also in male rats provided the enzyme activity is increased by VIP. The analysis of the dose-response curves for one agent (VIP or dopamine) in the absence or in the presence of the other indicates that the two agents exhibit a different pattern of interaction in the different systems. In fact, in female rat pituitaries and in human prolactinomas, the curves for dopamine±VIP and for VIP±dopamine were parallel, indicating that the two agents exherted their effects independently from one another. On the contrary, in male rat pituitaries, the curves were definitively non parallel, that is, the inhibitory effect of dopamine was greatly amplified by VIP. In no case was the apparent affinity (EC₅₀) of one agent modified by the presence of the other. It is concluded that two different modes of interaction between stimulatory and inhibitory neurohormones might exist at the level of adenylate cyclase from anterior pituitary cells.     

Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation

To attain its native conformation, the cytoskeletal protein tubulin needs the concourse of several molecular chaperones, among others the cytosolic chaperonin CCT. It has been previously described that denatured tubulin interacts with CCT in a quasi-folded conformation using several loops located throughout its sequence. These loops are also involved in microtubule formation and are absent in its prokaryote homologue FtsZ, which in vitro folds by itself and does not interact with CCT. Several FtsZ/tubulin chimeric proteins were generated by inserting consecutively one, two or three of the CCT-binding domains of tubulin into the corresponding sequence of FtsZ from Methanococccus jannaschii. The insertion of any of the CCT-binding loops generates in the FtsZ/tubulin chimeras the ability to interact with CCT. The accumulation of CCT-binding loops induces in the FtsZ/tubulin chimeras unfolding and refolding properties that are more similar to tubulin than to its prokaryote counterpart. Finally, the insertion of some of these loops generates in the FtsZ/tubulin chimeras more complex polymeric structures than those found for FtsZ. These results reinforce the notion that CCT has coevolved with tubulin to deal with the folding problems encountered by the eukaryotic protein with the appearance of the new sequences involved in microtubule formation.     

THI1, a protein involved in the biosynthesis of thiamin in Arabidopsis thaliana: Structural analysis of THI1(A140V) mutant

In eukaryotes, there are still steps of the vitamin B₁ biosynthetic pathway not completely understood. In Arabidopsis thaliana, THI1 protein has been associated with the synthesis of the thiazole ring, a finding supported by the identification of a thiamine pyrophosphate (TPP)-like compound in its structure. Here, we investigated THI1 and its mutant THI1(A140V), responsible for the thiamin auxotrophy in a A. thaliana mutant line, aiming to clarify the impact of this mutation in the stability and activity of THI1. Recently, the THI1 orthologue (THI4) was revealed to be responsible for the donation of the sulfur atom from a cysteine residue to the thiazole ring in the thiamine intermediate. In this context, we carried out a cysteine quantification in THI1 and THI1(A140V) using electron spin resonance (ESR). These data showed that THI1(A140V) contains more sulfur-containing cysteines than THI1, indicating that the function as a sulfur donor is conserved, but the rate of donation reaction is somehow affected. Also, the bound compounds were isolated from both proteins and are present in different amounts in each protein. Unfolding studies presented differences in melting temperatures and also in the concentration of guanidine at which half of the protein unfolds, thus showing that THI1(A140V) has its conformational stability affected by the mutation. Hence, despite keeping its function in the early steps during the synthesis of TPP precursor, our studies have shown a decrease in the THI1(A140V) stability, which might be slowing down the biological activity of the mutant, and thus contributing to thiamin auxotrophy.     

Impact of iron toxicity on oxidative metabolism in young Eugenia uniflora L. plants

In excess, iron can induce the production and accumulation of reactive oxygen species (ROS), causing oxidative stress. The objective of this work was to evaluate the impact of toxic concentrations of iron (Fe) on the antioxidative metabolism of young Eugenia uniflora plants. Forty-five-day-old plants grown in Hoagland nutrient solution, pH 5.0, were treated with three Fe concentrations, in the form of FeEDTA, during three periods of time. At the end of the treatment, the plants were harvested and relative growth rate, iron content, lipid peroxidation and enzymes and metabolites of the antioxidative metabolism were determined. Iron-treated plants showed higher iron contents, reduced relative growth rates and iron toxicity symptoms in both leaves and roots. There was an increase in lipid peroxidation with increasing Fe, only in the leaves. The enzymatic activities of superoxide dismutase (SOD) and glutathione reductase (GR) increased with increasing Fe concentration and treatment exposure time. The activities of catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APX) also increased with increasing Fe concentration but decreased with increasing treatment exposure time. Glutathione peroxidase activity (GPX) decreased with increasing Fe concentration and exposure time. The ascorbate (AA) and reduced glutathione (GSH) contents and the AA/DHA and GSH/GSSG ratios, in general, increased with increasing Fe concentration and treatment exposure time. The results indicate that under toxic levels of Fe, young E. uniflora plants suffer increased oxidative stress, which is ameliorated through changes in the activities of antioxidative enzymes and in the contents of the antioxidants AA and GSH.     

Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in Indica rice (cv. CO39)

Based on blast pathogen population dynamics and lineage exclusion assays, we found that the major blast resistance genes Pi-1 and Piz-5 confer resistance against most Magnaporthe grisea lineages. Near-isogenic rice lines C101LAC and C101A51 carrying these two major genes for blast resistance in the background of a most blast-susceptible genotype were used for developing the pyramids. The closely linked RFLP marker RZ536 and NBS-LRR r10 marker for Pi-1 and a PCR-based SAP marker RG64 for Piz-5 were used to identify the genes in the parents and in marker-assisted breeding of the pyramided populations. To achieve multiple resistance against blast and blight in this cultivar, these blast-resistant pyramids were transformed with the cloned bacterial blight resistance gene Xa21 known to confer resistance to all races of Xanthomonas oryzae pv. oryzae (Xoo). Bioassays with six independent transformants showed that transgenic CO39 plants were resistant to both pathogens, M. grisea and Xoo. We report here the stacking of three major genes (Pi-1 + Piz-5 + Xa21) into rice using two different approaches of molecular breeding: marker-assisted selection (MAS) and genetic transformation.     

Prediction of protein-protein interactions using distant conservation of sequence patterns and structure relationships

MOTIVATION: Given that association and dissociation of protein molecules is crucial in most biological processes several in silico methods have been recently developed to predict protein-protein interactions. Structural evidence has shown that usually interacting pairs of close homologs (interologs) physically interact in the same way. Moreover, conservation of an interaction depends on the conservation of the interface between interacting partners. In this article we make use of both, structural similarities among domains of known interacting proteins found in the Database of Interacting Proteins (DIP) and conservation of pairs of sequence patches involved in protein-protein interfaces to predict putative protein interaction pairs. RESULTS: We have obtained a large amount of putative protein-protein interaction (approximately 130,000). The list is independent from other techniques both experimental and theoretical. We separated the list of predictions into three sets according to their relationship with known interacting proteins found in DIP. For each set, only a small fraction of the predicted protein pairs could be independently validated by cross checking with the Human Protein Reference Database (HPRD). The fraction of validated protein pairs was always larger than that expected by using random protein pairs. Furthermore, a correlation map of interacting protein pairs was calculated with respect to molecular function, as defined in the Gene Ontology database. It shows good consistency of the predicted interactions with data in the HPRD database. The intersection between the lists of interactions of other methods and ours produces a network of potentially high-confidence interactions.     

Collective Electronic Excitations in Thin Ag Films on Ni(111)

The collective electronic excitations in thin Ag films deposited onto the Ni(111) surface were studied by high-resolution electron energy loss spectroscopy. A broad loss peak at 7.7 eV was assigned to the Ag multipole plasmon, in excellent agreement with calculations based on s-d polarization model. Ag multipole plasmon was excited only at grazing incidence. Furthermore, a strong dependence on the impinging energy exists. Multipole plasmon could be measured only for a very strict range of primary electron beam energies and it was excited by electrons scattered at a reflection plane located just underneath the jellium edge. Such mode was found to be dramatically more sensible to the state of the surface with respect to ordinary surface plasmon. Moreover, we report experimental evidence of interference effects in surface plasmon excitation.