Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom

Summary Two-dimensional ¹H NMR techniques were used to determine the spatial structure of ectatomin, a toxin from the venom of the ant Ectatomma tuberculatum. Nearly complete proton resonance assignments for two chains of ectatomin (37 and 34 amino acid residues, respectively) were obtained using 2D TOCSY, DQF-COSY and NOESY experiments. The cross-peak volumes in NOESY spectra were used to define the local structure of the protein and generate accurate proton-proton distance constraints employing the MARDIGRAS program. Disulfide bonds were located by analyzing the global fold of ectatomin, calculated with the distance geometry program DIANA. These data, combined with data on the rate of exchange of amide protons with deuterium, were used to obtain a final set of 20 structures by DIANA. These structures were refined by unrestrained energy minimization using the CHARMm program. The resulting rms deviations over 20 structures (excluding the mobile N- and C-termini of each chain) are 0.75 ? for backbone heavy atoms, and 1.25 ? for all heavy atoms. The conformations of the two chains are similar. Each chain consists of two α-helices and a hinge region of four residues; this forms a hairpin structure which is stabilized by disulfide bridges. The hinge regions of the two chains are connected together by a third disulfide bridge. Thus, ectatomin forms a four-α-helical bundle structure.     

Fusion Peptide Interaction with Lipid Bilayer: Modeling with Monte Carlo Simulation and Continuum Electrostatics Calculation

Abstract

Conformation of 20-residue peptide E5, an analog of the fusion peptide of influenza virus hemagglutinin, was explored by Monte-Carlo technique starting with the fully buried in the membrane ideal α-helix. The lipid bilayer (of 30 Å width) together with surrounding water were modeled by the atomic solvation parameters. During the simulation, residues 2–18 of the peptide retained α-helical conformation, and the peptide was found to be partially immersed into the bilayer. In the resulting low-energy conformers, the N-terminus was buried inside the membrane, its position with respect to the bilayer surface (Z_NT) being varied from 2.5 to 7.5 Å, and the orientation of the helical axis relative to the membrane plane (Θ) – from 10 to 35°. The low-energy conformers (below -200kcal/mol) were clustered in the space (Z_NT, Θ) into 4 groups. To select low-energy states of the peptide compatible with NMR data, we calculated pKa values of E5 ionizable groups and compared them with the experimental values. It was shown that the best correlation coefficient (0.87) and rmsd (0.68 in pH units) were obtained for the group of states which is characterized by Θ = 15–19° and Z_NT = 3.5–4.5Å.     

Modeling of Peptides in Implicit Membrane-Mimetic Media

Abstract

Lipid bilayer plays a crucial role in folding of membrane peptides and their stabilization in the membrane-bound state. Correct treatment of the media effects is thus essential for realistic simulations of peptides in bilayers. Previously (Volynsky et al., 1999), we proposed an efficient solvation model which mimics heterogeneous membrane-water system. The model is based on combined employment of atomic solvation parameters for water and hydrocarbon, which approximate hydrated headgroups and acyl chains of lipids, respectively. In this study, the model is employed in non-restrained Monte Carlo simulations of several peptides: totally apolar 20-residue poly-L-Leu, hydrophobic peptide with polar edges, and strongly amphiphilic pep-tide. The principal goals are: to explore energy landscape of these peptides in membrane; to characterize the structures of low-energy states and their orientations with respect to the bilayer. Simulations were performed starting from different structures (unordered or helical) and orientations. It was found that the membrane environment significantly promotes an α-helical conformation for all the peptides, while their energetically favourable orientations are quite different. Thus, poly-Leu was immobilized inside the membrane, the hydrophobic peptide with polar termini adapted transbilayer orientation, whereas the amphiphilic peptide stayed on the lipid-water interface in peripherial orientation. Energy barriers between different states were characterized. The computational results were compared with the experimental structural data.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates

?. P. Marin?ek, N. Nolde, I. Kardum‐Skelin, R. Nizzoli, B. Önal, T. Rezanko, E. Tani, K. T. Ostovi?, P. Vielh, F. Schmitt and G. Kocjan
Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates Objectives: To collect data on the variability of immunocytochemical (ICC) procedures used to detect oestrogen/progesterone receptors (ER/PR) on cytological material; to test the reproducibility of results; and to identify the crucial points in the ICC procedures that affect the result. Methods: Ten laboratories from eight countries participated in a two‐part study. In the first part, one of the participants (the coordinator) prepared and distributed cytospins from a fine needle aspirate of a primary breast carcinoma. Laboratories performed ICC staining for ER/PR according to their own methods on the test slides and in‐house positive controls. Slides were returned to the coordinator together with information on the preparation of positive control slides and the ICC methodology used. In the second part, obligatory methods of fixation and antigen retrieval were specified. Evaluation of results included grading the number of positive cells, staining intensity, background staining, cytoplasmic staining, sample condition and cellularity. Participants evaluated their own results, which were subsequently evaluated by the coordinator. Results: There was great variability in the preparation of slides for in‐house controls and ICC methodology. The outcome of ICC staining of in‐house control slides was excellent in two laboratories, adequate in three, sub‐optimal in four and inadequate in one. Only six obtained a positive reaction on the test slides and not all were of a high quality. Results of the second run were greatly improved in terms of cellularity of in‐house positive control slides, and scores for the percentage of stained cells and staining intensity of control and test slides. Cytospins and monolayer (ThinPrep^®) preparations were superior to direct smears; methods of fixation and antigen retrieval were the key points in the staining process. Conclusions: Our experience points to the need for guidelines for hormonal receptor determination and external quality control on cytological material, in order for cytological methods to be used in routine clinical practice with a suitable degree of confidence.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3'UTR-mediated repression of the let-7 microRNA target gene, hbl-1

The Puf family of RNA-binding proteins directs cell fates by regulating gene expression at the level of translation and RNA stability. Here, we report that the Caenorhabditis elegans pumilio homolog, puf-9, controls the differentiation of epidermal stem cells at the larval-to-adult transition. Genetic analysis reveals that loss-of-function mutations in puf-9 enhance the lethality and heterochronic phenotypes caused by mutations in the let-7 microRNA (miRNA), while suppressing the heterochronic phenotypes of lin-41, a let-7 target and homolog of Drosophila Brat. puf-9 interacts with another known temporal regulator hbl-1, the Caenorhabditis elegans ortholog of hunchback. We present evidence demonstrating that puf-9 is required for the 3'UTR-mediated regulation of hbl-1, in both the hypodermis and the ventral nerve cord. Finally, we show that this regulation is dependent on a region of the hbl-1 3'UTR that contains putative Puf family binding sites as well as binding sites for the let-7 miRNA family, suggesting that puf-9 and let-7 may mediate hypodermal seam cell differentiation by regulating common targets.     

A solvent model for simulations of peptides in bilayers. I. Membrane-promoting alpha-helix formation

We describe an efficient solvation model for proteins. In this model atomic solvation parameters imitating the hydrocarbon core of a membrane, water, and weak polar solvent (octanol) were developed. An optimal number of solvation parameters was chosen based on analysis of atomic hydrophobicities and fitting experimental free energies of gas-cyclohexane, gas-water, and octanol-water transfer for amino acids. The solvation energy term incorporated into the ECEPP/2 potential energy function was tested in Monte Carlo simulations of a number of small peptides with known energies of bilayer-water and octanol-water transfer. The calculated properties were shown to agree reasonably well with the experimental data. Furthermore, the solvation model was used to assess membrane-promoting alpha-helix formation. To accomplish this, all-atom models of 20-residue homopolypeptides-poly-Leu, poly-Val, poly-Ile, and poly-Gly in initial random coil conformation-were subjected to nonrestrained Monte Carlo conformational search in vacuo and with the solvation terms mimicking the water and hydrophobic parts of the bilayer. All the peptides demonstrated their largest helix-forming tendencies in a nonpolar environment, where the lowest-energy conformers of poly-Leu, Val, Ile revealed 100, 95, and 80% of alpha-helical content, respectively. Energetic and conformational properties of Gly in all environments were shown to be different from those observed for residues with hydrophobic side chains. Applications of the solvation model to simulations of peptides and proteins in the presence of membrane, along with limitations of the approach, are discussed.     

Factors important for fusogenic activity of peptides: molecular modeling study of analogs of fusion peptide of influenza virus hemagglutinin

Nine analogs of fusion peptide of influenza virus hemagglutinin whose membrane perturbation activity has been thoroughly tested [Murata et al. (1992) Biochemistry 31, 1986-1992; Murata et al. (1993) Biophys. J. 64, 724-734] were characterized by molecular modeling techniques with the aim of delineating any specific structural and/or hydrophobic properties inherent in peptides with fusogenic activity. It was shown that, regardless of characteristics common to all analogs (peripheral disposition at the water-lipid interface, amphiphilic nature, alpha-helical structure, etc.), only fusion active peptides reveal a specific 'tilted oblique-oriented' pattern of hydrophobicity on their surfaces and a certain depth of penetration to the non-polar membrane core. The conclusion was reached that these factors are among the most important for the specific destabilization of a bilayer, which is followed by membrane fusion.     

Monte Carlo simulations of voltage-driven translocation of a signal sequence

CD1d-deficient (CD1d-/-) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a V(alpha)19.1-J(alpha)26 (AV19-AJ33) invariant TCR alpha chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.1+ but not NK1.1- T cell population isolated from CD1d-/- mice. Moreover, more than half (11/21) of the hybrid cell lines established from CD1d-/- NKT cells expressed the V(alpha)19.1-J(alpha)26 invariant TCR alpha chain. The expression of the invariant V(alpha)19.1-J(alpha)26 mRNA was absent in beta2-microglobulin-deficient mice. Collectively, the present findings suggest the presence of a second NKT cell repertoire characterized by an invariant TCR alpha chain (V(alpha)19.1-J(alpha)26) that is selected by an MHC class I-like molecule other than CD1d.