Looking at structure, stability, and evolution of proteins through the principal eigenvector of contact matrices and hydrophobicity profiles

We review and further develop an analytical model that describes how thermodynamic constraints on the stability of the native state influence protein evolution in a site-specific manner. To this end, we represent both protein sequences and protein structures as vectors: structures are represented by the principal eigenvector (PE) of the protein contact matrix, a quantity that resembles closely the effective connectivity of each site; sequences are represented through the "interactivity" of each amino acid type, using novel parameters that are correlated with hydropathy scales. These interactivity parameters are more strongly correlated than the other hydropathy scales that we examine with: (1) the change upon mutations of the unfolding free energy of proteins with two-states thermodynamics; (2) genomic properties as the genome-size and the genome-wide GC content; (3) the main eigenvectors of the substitution matrices. The evolutionary average of the interactivity vector correlates very strongly with the PE of a protein structure. Using this result, we derive an analytic expression for site-specific distributions of amino acids across protein families in the form of Boltzmann distributions whose "inverse temperature" is a function of the PE component. We show that our predictions are in agreement with site-specific amino acid distributions obtained from the Protein Data Bank, and we determine the mutational model that best fits the observed site-specific amino acid distributions. Interestingly, the optimal model almost minimizes the rate at which deleterious mutations are eliminated by natural selection.     

Production of volatile organic compounds (VOCs) by yeasts isolated from the ascocarps of black (<Emphasis Type="Italic">Tuber melanosporum</Emphasis> Vitt.) and white (<Emphasis Type="Italic">Tuber magnatum</Emphasis> Pico) truffles

Twenty-nine yeast strains were isolated from the ascocarps of black and white truffles (Tuber melanosporum Vitt. and Tuber magnatum Pico, respectively), and identified using a polyphasic approach. According to the conventional taxonomic methods, MSP-PCR fingerprinting and sequencing of the D1/D2 domain of 26S rDNA, the strains were identified as Candida saitoana, Debaryomyces hansenii, Cryptococcus sp., Rhodotorula mucilaginosa, and Trichosporon moniliiforme. All isolates assimilated l-methionine as a sole nitrogen source and produced the volatile organic compounds (VOCs), 2-methyl butanol, 3-methyl butanol, methanethiol, S-methyl thioacetate, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, dihydro-2-methyl-3(2H)-thiophenone and 3-(methylthio)-1-propanol (MTP). ANOVA analysis of data showed significant (P<0.01) differences in VOCs produced by different yeasts, with MTP as the major component (produced at concentrations ranging from 19.8 to 225.6 mg/l). In addition, since some molecules produced by the isolates of this study are also characteristic of truffle complex aroma, it is possible to hypothesize a complementary role of yeasts associated with this ecosystem in contributing to final Tuber spp. aroma through the independent synthesis of yeast-specific volatile constituents.     

Biodiversity in model ecosystems, II: species assembly and food web structure

This is the second of two papers dedicated to the relationship between population models of competition and biodiversity. Here, we consider species assembly models where the population dynamics is kept far from fixed points through the continuous introduction of new species, and generalize to such models the coexistence condition derived for systems at the fixed point. The ecological overlap between species and shared preys, that we define here, provides a quantitative measure of the effective interspecies competition and of the trophic network topology. We obtain distributions of the overlap from simulations of a new model based both on immigration and speciation, and show that they are in good agreement with those measured for three large natural food webs. As discussed in the first paper, rapid environmental fluctuations, interacting with the condition for coexistence of competing species, limit the maximal biodiversity that a trophic level can host. This horizontal limitation to biodiversity is here combined with either dissipation of energy or growth of fluctuations, which in our model limit the length of food webs in the vertical direction. These ingredients yield an effective model of food webs that produce a biodiversity profile with a maximum at an intermediate trophic level, in agreement with field studies.     

Functional in vivo gene transfer into the myofibers of adult skeletal muscle

The postmitotic nature and longevity of skeletal muscle fibers permit stable expression of any transfected gene. Direct in vivo injection of plasmid DNA, in both adult and regenerating muscles, is a safe, inexpensive, and easy approach. Here we present an optimized electroporation protocol based on the use of spatula electrodes to transfer cDNA in vivo into the adult myofibers of an anatomically defined muscle, which could be functionally characterized. In our hands, about 80% of adult myofibers were transfected in vivo by different plasmids for GFP fusion proteins or for beta-galactosidase. The luciferase activity increased several orders of magnitude when compared to standard DNA delivery. In an anatomical defined muscle, the wide gene transfer was comparable to or better than that of retrovirus delivery, that recently has been shown to be prone to severe side-effects in human clinical studies. Furthermore, with our method the tissue damage was greatly decreased. Thus, the present work describes in vivo functional electrotransfer of genes in adult skeletal muscle fibers by a protocol that is of great potential for gene therapy, as well as for basic research.     

Crystal structures of engrailed homeodomain mutants: implications for stability and dynamics

We report the crystal structures and biophysical characterization of two stabilized mutants of the Drosophila Engrailed homeodomain that have been engineered to minimize electrostatic repulsion. Four independent copies of each mutant occupy the crystal lattice, and comparison of these structures illustrates variation that can be partly ascribed to networks of correlated conformational adjustments. Central to one network is leucine 26 (Leu26), which occupies alternatively two side chain rotameric conformations (-gauche and trans) and different positions within the hydrophobic core. Similar sets of conformational substates are observed in other Engrailed structures and in another homeodomain. The pattern of structural adjustments can account for NMR relaxation data and sequence co-variation networks in the wider homeodomain family. It may also explain the dysfunction associated with a P26L mutation in the human ARX homeodomain protein. Finally, we observe a novel dipolar interaction between a conserved tryptophan and a water molecule positioned along the normal to the indole ring. This interaction may explain the distinctive fluorescent properties of the homeodomain family.     

The denatured state of Engrailed Homeodomain under denaturing and native conditions

Protein folding starts from the elusive form of the denatured state that is present under conditions that favour the native state. We have studied the denatured state of Engrailed Homeodomain (En-HD) under mildly and strongly denaturing conditions at the level of individual residues by NMR and more globally by conventional spectroscopy and solution X-ray scattering. We have compared these states with a destabilized mutant, L16A, which is predominantly denatured under conditions where the wild-type is native. This engineered denatured state, which could be directly studied under native conditions, was in genuine equilibrium with the native state, which could be observably populated by changing the conditions or introducing a stabilizing mutation. The denatured state had extensive native secondary structure and was significantly compact and globular. But, the side-chains and backbone were highly mobile. Non-cooperative melting of the residual structure on the denatured state of En-HD was observed, both at the residue and the molecular level, with increasingly denaturing conditions. The absence of a co-operative transition could result from the denatured state ensemble progressing through a series of intermediates or from a more general slide (second-order transition) from the compact form under native conditions to the more extended at highly denaturing conditions. In either case, the starting point for folding under native conditions is highly structured and already poised to adopt the native structure.     

Regulation of constitutive TCR internalization by the zeta-chain

The ability of a T cell to be activated is critically regulated by the number of TCRs expressed on the plasma membrane. Cell surface TCR expression is influenced by dynamic processes such as synthesis and transport of newly assembled receptors, endocytosis of surface TCR, and recycling to the plasma membrane of internalized receptors. In this study, the internalization of fluorescently labeled anti-TCR Abs was used to analyze constitutive endocytosis of TCRs on T cells, and to investigate the role of the zeta-chain in this process. We found that cell surface TCRs lacking zeta were endocytosed more rapidly than completely assembled receptors, and that reexpression of full-length zeta led to a dose-dependent decrease in the rate of TCR internalization. Rapid TCR internalization was also observed with CD4(+)CD8(+) thymocytes from zeta-deficient mice, whereas TCR internalization on thymocytes from CD3-delta deficient animals was slow, similar to that of wild-type thymocytes. This identifies a specific role for zeta in the regulation of constitutive receptor internalization. Furthermore, chimeric zeta molecules containing non-native intracellular amino acid sequences also led to high levels of TCR expression and reduced TCR cycling. These effects were dependent solely on the length of the intracellular tail, ruling out a role for intracellular zeta-specific interactions with other molecules as a mechanism for regulating TCR internalization. Rather, these findings strongly support a model in which the zeta-chain stabilizes TCR residency on the cell surface, and functions to maintain cell surface receptor expression by sterically blocking internalization sequences in other TCR components.     

Direct effect of a gonadotropin-releasing hormone agonist on the growth of canine mammary tumour cells

Gonadotropin-releasing hormone (GnRH) agonist exert "in vivo" an inhibitory action on the growth of hormone-dependent canine mammary tumours (Lombardi et al. [1999] J. Vet. Pharmacol Ther. 22(1):56-61). The present experiments have been performed "in vitro" in order to investigate the mechanisms involved in this direct antiproliferative action of GnRH agonists. In particular, the aim was to study whether these compounds might exert their antiproliferative effect by interfering with the stimulatory action of epidermal growth factor (EGF). To this purpose, the effects of GnRH agonist, Goserelin (GnRH-A), on the mitogenic action of EGF, on EGF-activated intracellular signaling mechanisms (intracellular calcium and nitric oxide production) as well as on ATP induced cell proliferation and signalling, and on the binding of EGF receptors have been evaluated in primary culture of canine mammary tumour cells. The results of these "in vitro" studies show that GnRH-A counteracts the mitogenic action of EGF and ATP, decreases the EGF/ATP-induced calcium signalling and reduces EGF binding, probably by means of NO-induced [Ca2+]i downregulation. These data suggest that GnRH agonists may inhibit the proliferation of the tumour cells by interfering with the stimulatory action of EGF.     

Sinorhizobium fredii isolates can be specifically identified by a 260 bp fragment from the nolXWBTUV locus

A pair of primers homologous to the nolXWBTUV locus generated a 260 bp fragment by PCR only in the presence of Sinorhizobium fredii template DNA of different quality. This resulted in a fast and accurate method for the identification of S. fredii either from pure DNA, whole bacterial cells or nodule extracts. By means of two PCR fragments, one specific for S. fredii (260-bp) and the other specific for Bradyrhizobium japonicum (RSalpha), we found that S. fredii strain SMH12 and B. japonicum E109 were equally efficient at developing nodules on soybean plants grown under controlled environmental conditions.     

Prevalence of Toxocara infection in schoolchildren from the Butantã region,São Paulo,Brazil

Visceral larva migrans syndrome by Toxocara affects mainly children between 2 and 5 years of age, it is generally asymptomatic, and the seroprevalence varies from 3 to 86% in different countries. A total of 399 schoolchildren from 14 public schools of the Butant? region, S?o Paulo city, Brazil, were evaluated by Toxocara serology (enzyme-linked immunosorbent assay). Epidemiological data to the Toxocara infection obtained from a protocol were submitted to multiple logistic regression analysis for a risk profile definition. Blood was collected on filter paper by finger puncture, with all samples tested in duplicate. Considering titers > or = 1/160 as positive, the seroprevalence obtained was 38.8%. Among infected children, the mean age was 9.4 years, with a similar distribution between genders. A significant association was observed with the presence of onychophagia, residence with a dirty backyard, living in a slum, previous wheezing episodes, school attended, and family income (p < 0.05). All data, except "living in a slum", were considered to be determinant of a risk profile for the acquisition of Toxocara infection. A monthly income > or = 5 minimum salaries represented a protective factor, although of low relevance. Toxocara eggs were found in at least one of the soil samples obtained from five schools, with high prevalence of Toxocara infections, indicating the frequent soil contamination by this agent.