Methods for molecular dynamics simulations of protein folding/unfolding in solution

All atom molecular dynamics simulations have become a standard method for mapping equilibrium protein dynamics and non-equilibrium events like folding and unfolding. Here, we present detailed methods for performing such simulations. Generic protocols for minimization, solvation, simulation, and analysis derived from previous studies are also presented. As a measure of validation, our water model is compared with experiment. An example of current applications of these methods, simulations of the ultrafast folding protein Engrailed Homeodomain are presented including the experimental evidence used to verify their results. Ultrafast folders are an invaluable tool for studying protein behavior as folding and unfolding events measured by experiment occur on timescales accessible with the high-resolution molecular dynamics methods we describe. Finally, to demonstrate the prospect of these methods for folding proteins, a temperature quench simulation of a thermal unfolding intermediate of the Engrailed Homeodomain is described.     

HIV-1-induced migration of monocyte-derived dendritic cells is associated with differential activation of MAPK pathways

From the site of transmission at mucosal surfaces, HIV is thought to be transported by DCs to lymphoid tissues. To initiate migration, HIV needs to activate DCs. This activation, reflected by intra- and extracellular changes in cell phenotype, is investigated in the present study. In two-thirds of the donors, R5- and X4-tropic HIV-1 strains induced partial up-regulation of DC activation markers such as CD83 and CD86. In addition, CCR7 expression was increased. HIV-1 initiated a transient phosphorylation of p44/p42 ERK1/2 in iDCs, whereas p38 MAPK was activated in both iDCs and mDCs. Up-regulation of CD83 and CD86 on DCs was blocked when cells were incubated with specific p38 MAPK inhibitors before HIV-1-addition. CCR7 expression induced by HIV-1 was sufficient to initiate migration of DCs in the presence of secondary lymphoid tissue chemokine (CCL21) and MIP-3beta (CCL19). Preincubation of DCs with a p38 MAPK inhibitor blocked CCR7-dependent DC migration. Migrating DCs were able to induce infection of autologous unstimulated PBLs in the Transwell system. These data indicate that HIV-1 triggers a cell-specific signaling machinery, thereby manipulating DCs to migrate along a chemokine gradient, which results in productive infection of nonstimulated CD4(+) cells.     

Paradoxical roles of different nitric oxide synthase isoforms in colonic injury

Nitric oxide (NO) is a free radical that is largely produced by three isoforms of NO synthase (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). NO regulates numerous processes in the gastrointestinal tract; however, the overall role that NO plays in intestinal inflammation is unclear. NO is upregulated in both ulcerative colitis and Crohn's disease as well as in animal models of colitis. There have been conflicting reports on whether NO protects or exacerbates injury in colitis or is simply a marker of inflammation. To determine whether the site, timing, and level of NO production modulate the effect on the inflammatory responses, the dextran sodium sulfate model of colitis was assessed in murine lines rendered deficient in iNOS, nNOS, eNOS, or e/nNOS by targeted gene disruption. The loss of nNOS resulted in more severe disease and increased mortality, whereas the loss of eNOS or iNOS was protective. Furthermore, concomitant loss of eNOS reversed the susceptibility found in nNOS-/- mice. Deficiencies in specific NOS isoforms led to distinctive alterations of inflammatory responses, including changes in leukocyte recruitment and alterations in colonic lymphocyte populations. The present studies indicate that NO produced by individual NOS isoforms plays different roles in modulating an inflammatory process.     

kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila

Drosophila myoblast fusion proceeds in two steps. The first one gives rise to small syncytia, the muscle precursor cells, which then recruit further fusion competent myoblasts to reach the final muscle size. We have identified Kette as an essential component for myoblast fusion. In kette mutants, founder cells and fusion-competent myoblasts are determined correctly and overcome the very first fusion. But then, at the precursor cell stage, fusion is interrupted. At the ultrastructural level, fusion is characterised by cell-cell recognition, alignment, formation of prefusion complexes, electron dense plaques and membrane breakdown. In kette mutants, electron dense plaques of aberrant length accumulate and fusion is interrupted owing to a complete failure of membrane breakdown. Furthermore, we show that kette interacts genetically with blown fuse (blow) which is known to be required to proceed from prefusion complexes to the formation of the electron dense plaques. Interestingly, a surplus of Kette can replace Blow function during myogenesis. We propose a model in which Dumbfounded/Sticks and stones-dependent cell adhesion is mediated over Rolling Pebbles, Myoblast city, Crk, Blown fuse and Kette, and thus induces membrane fusion.     

Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function

Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.     

Leaf axil sampling of midwest U.S. maize for mycotoxigenic Fusarium fungi using PCR analysis

PCR analysis was used to detect Fusarium species generically, as well as the mycotoxin-producing species F.␣subglutinans, F. proliferatum, and F. verticillioides in leaf axil and other maize tissues during ear fill in a multiyear study in central Illinois. The frequency of Fusarium detected varied from site to site and year to year. Fusarium was generically detected more frequently in leaf axil material than in leaf/husk lesions. In two growing seasons, the leaf axil samples were also tested for the presence of the mycotoxin producing species F. proliferatum, F. subglutinans, and F. verticillioides. Overall, F. proliferatum and F. verticillioides were detected less often than F. subglutinans. Fusarium was generically and specifically detected most commonly where visible fungal growth was present in leaf axil material. Disclaimer: The mention of firm names or trade products in this article does not imply that they are endorsed or recommended by the United States Department of Agriculture over other firms or similar products not mentioned.     

Sequence-matched probes produce increased cross-platform consistency and more reproducible biological results in microarray-based gene expression measurements

Cancer derived microarray data sets are routinely produced by various platforms that are either commercially available or manufactured by academic groups. The fundamental difference in their probe selection strategies holds the promise that identical observations produced by more than one platform prove to be more robust when validated by biology. However, cross-platform comparison requires matching corresponding probe sets. We are introducing here sequence-based matching of probes instead of gene identifier-based matching. We analyzed breast cancer cell line derived RNA aliquots using Agilent cDNA and Affymetrix oligonucleotide microarray platforms to assess the advantage of this method. We show, that at different levels of the analysis, including gene expression ratios and difference calls, cross-platform consistency is significantly improved by sequence- based matching. We also present evidence that sequence-based probe matching produces more consistent results when comparing similar biological data sets obtained by different microarray platforms. This strategy allowed a more efficient transfer of classification of breast cancer samples between data sets produced by cDNA microarray and Affymetrix gene-chip platforms.