Partition of protein solvation into group contributions from molecular dynamics simulations

Linear response theory coupled to molecular dynamics simulations with an explicit solvent representation is used to derive fractional contributions of amino acid residues to the solvation of proteins. The new fractional methods developed here are compared with standard approaches based on empirical 1D and 3D statistical potentials, as well as with estimates obtained from the analysis of classical molecular interaction potentials. The new fractional methods, which have a clear physical basis and explicitly account for the effects due to protein structure and flexibility, provide an accurate picture of the contribution to solvation of different regions of the protein.     

Hybrid xerogel films as novel coatings for antifouling and fouling release

Hybrid sol-gel-derived xerogel films prepared from 45/55 (mol ratio) n-propyltrimethoxysilane (C3-TMOS)/tetramethylorthosilane (TMOS), 2/98 (mol ratio) bis[3-(trimethoxysilyl)propyl]-ethylenediamine (enTMOS)/tetraethylorthosilane (TEOS), 50/50 (mol ratio) n-octyltriethoxysilane (C8-TEOS)/TMOS, and 50/50 (mol ratio) 3,3,3-trifluoropropyltrimethoxysilane (TFP-TMOS)/TMOS were found to inhibit settlement of zoospores of the marine fouling alga Ulva (syn. Enteromorpha) relative to settlement on acid-washed glass and give greater release of settled zoospores relative to glass upon exposure to pressure from a water jet. The more hydrophobic 50/50 C8-TEOS/TMOS xerogel films had the lowest critical surface tension by comprehensive contact angle analysis and gave significantly greater release of 8-day Ulva sporeling biomass after exposure to turbulent flow generated by a flow channel than the other xerogel surfaces or glass. The 50/50 C8-TEOS/TMOS xerogel was also a fouling release surface for juveniles of the tropical barnacle Balanus amphitrite. X-ray photon electron data indicated that the alkylsilyl residues of the C3-TMOS-, C8-TEOS-, and TFP-TMOS-containing xerogels were located on the surface of the xerogel films (in a vacuum), which contributes to the film hydrophobicity. Similarly, the amine-containing silyl residues of the enTMOS/TEOS films were located at the surface of the xerogel films, which contributes to the more hydrophilic character and increased critical surface tension of these films.     

Contributing factors and variability of energy expenditure in non-obese, obese, and post-obese adolescents

Energy expenditure (EE) is a major determinant of energy balance and body composition. The objectives of this paper were to review the contributing factors of the main components of daily EE (DEE) and the inter-individual variability in these components in non-obese (NOb), obese (Ob), and post-obese (POb) adolescents. Body composition especially fat-free mass (FFM), is the major determinant of the basal metabolic rate which contributes 50-70% of DEE, whereas fat mass (FM) is a significant factor only in obese subjects. Physical activity is the second main variation factor of DEE, whereas growth, the thermic effect of food, and thermoregulation are generally of marginal importance. The energy costs and EE associated with various sedentary and physical activities were assessed in NOb, Ob and POb subjects both in standardised and in free-living conditions. The interindividual variability of DEE is high, even after adjustment for body composition, mainly because of great differences in time devoted to the various physical activities. DEE and EE associated with sleep and sedentary activities are significantly higher in Ob than in NOb, but not after adjustment for FFM. On the contrary, EE associated with physical activities is not significantly different between Ob and NOb adolescents, but 61% lower in Ob subjects after adjustment for body composition. Multidisciplinary weight-reduction programmes including moderate energy restriction and physical training result in great FM loss, maintenance of FFM, improvement of physical capacities, but reductions in organ and tissue metabolic rate and in EE associated with the various sedentary and physical activities, which may favour body weight regain in the less active POb subjects.     

Millisecond protein folding studied by NMR spectroscopy

Proteins are involved in virtually every biological process and in order to function, it is necessary for these polypeptide chains to fold into the unique, native conformation. This folding process can take place rapidly. NMR line shape analyses and transverse relaxation measurements allow protein folding studies on a microsecond-to-millisecond time scale. Together with an overview of current achievements within this field, we present millisecond protein folding studies by NMR of the cold shock protein CspB from Bacillus subtilis.     

Identification, cloning and expression of a Cry1Ab cadherin receptor from European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae)

Transgenic corn expressing the Cry1Ab toxin from Bacillus thuringiensis is highly toxic to European corn borer, Ostrinia nubilalis, larvae. A putative Cry1Ab receptor (OnBt-R(1)) molecule was cloned and sequenced from a cDNA library prepared from midgut tissue of O. nubilalis larvae. The 5.6 Kb gene is homologous with a number of cadherin genes identified as Cry1 binding proteins in other lepidopterans. Brush border membrane vesicles were prepared using dissected midguts from late instars. A 220-kDa protein was identified as a cadherin-like molecule, which bound to Cry1Ab toxin and cross-reacted with an anti-cadherin serum developed from recombinant expression of a partial O. nubilalis cadherin peptide. Two additional proteins of smaller size cross-reacted with the anti-cadherin serum indicating that Cry1Ab binds to multiple receptors or to different forms of the same protein. Spodoptera frugiperda (SF9) cells transfected with the OnBt-R(1) gene were shown to express the receptor molecule which caused functional susceptibility to Cry1Ab at concentrations as low as 0.1 microg/ml. These results in combination suggest strongly that a cadherin-like protein acts as receptor and is involved with Cry1Ab toxicity in O. nubilalis.     

Investigation of CC and CXC chemokine quaternary state mutants

The chemokine family forms two different types of homodimer despite members sharing nearly identical folds. To study the formation of quaternary structure in this family, rational mutagenesis was employed on a representative member of each subfamily (MIP-1beta and IL-8). The variants were studied by analytical ultracentrifugation and NMR, and it was determined that formation of a folded monomer from a natural chemokine dimer is reasonably facile, while conversion between dimer types is not. Monomeric variants of MIP-1beta and IL-8 were randomly mutated and a lambda phage-based selection system was employed in a novel way to screen for dimerization. A total of 6,000,000 random mutants were screened, but no dimers were formed, suggesting again that the chemokine fold is robust and amenable to sequence variation, while the chemokine dimer is much more difficult to attain. This work represents a biophysical analysis of an array of chemokine quaternary state variants.     

Ca2+ ionophores trigger membrane remodeling without a need for store-operated Ca2+ entry

Calcium (Ca2+) ionophores are the most effective agents able to elicit rapid membrane remodeling in vitro. This process exposes aminophospholipids at the surface of platelets and blood cells, thus providing a catalytic surface for coagulation. To explore the underlying mechanism, we examined if cytosolic Ca2+ ([Ca2+]i) increase through store-operated Ca2+ entry (SOCE) was necessary for the potent effect of ionophores. Recent studies have demonstrated that the Ca2+-ATPase inhibitor thapsigargin, although able to elevate [Ca2+]i through SOCE, does not trigger the rapid membrane remodeling. However, it was not known if the additional effect of ionophores to promote the process required SOCE or could it occur independently. We took advantage of two mutant B lymphoblast cell lines, characterized either by defective SOCE or altered membrane remodeling, to simultaneously assess [Ca2+]i increase and membrane remodeling in the presence of ionophores or thapsigargin. Results imply that ionophores trigger membrane remodeling without the requirement for a functional SOCE.     

Epithelial laminin alpha5 is necessary for distal epithelial cell maturation, VEGF production, and alveolization in the developing murine lung

Laminin alpha5 is prominent in the basement membrane of alveolar walls, airways, and pleura in developing and adult lung. Targeted deletion of laminin alpha5 in mice causes developmental defects in multiple organs, but embryonic lethality has precluded examination of the latter stages of lung development. To identify roles for laminin alpha5 in lung development, we have generated an inducible lung epithelial cell-specific Lama5 null (SP-CLama5(fl/-)) mouse through use of the Cre/loxP system, the human surfactant protein C promoter, and the reverse tetracycline transactivator. SP-CLama5(fl/-) embryos exposed to doxycycline from E6.5 died a few hours after birth. Compared to control littermates, SP-CLama5(fl/-) lungs had dilated, enlarged distal airspaces, but basement membrane ultrastructure was preserved. Distal epithelial cell differentiation was perturbed, with a marked reduction of alveolar type II cells and a virtual absence of type I cells. Cell proliferation was reduced and apoptosis was increased. Capillary density was diminished, and this was associated with a decrease in total lung VEGF production. Overall, these findings indicate that epithelial laminin alpha5, independent of its structural function, is necessary for murine lung development, and suggest a role for laminin alpha5 in signaling pathways that promote alveolar epithelial cell differentiation and VEGF expression.     

Electrostatic interactions between the syntaxin membrane anchor and neurotransmitter passing through the fusion pore

Recent experiments have shown that flux through the fusion pore is sensitive to manipulations of the side-chain size of certain residues in the syntaxin (syx) membrane anchor. These residues were proposed to line the wall of the fusion pore of Ca(2+)-triggered exocytosis. Here we continued this line of experimentation to examine possible electrostatic interactions between the pore lining residues and the neurotransmitter norepinephrine (NE). Replacing syx pore-lining residues with aspartate enhanced NE flux above that expected for the size of the aspartate side chain. In contrast, substitution with arginine reduced NE flux below that expected for the size of its side chain. Substituting aspartate and arginine into the nonpore-lining residues did not alter the fusion pore flux. Other amino acids with ionizable side chains had variable effects. These results indicate an electrostatic interaction between the pore-lining residues of syx and NE, and provide additional evidence that the syx membrane anchor is a structural component of the fusion pore.