The inverse relationship between protein dynamics and thermal stability

Protein powders that are dehydrated or mixed with a glassy compound are known to have improved thermal stability. We present elastic and quasielastic neutron scattering measurements of the global dynamics of lysozyme and ribonuclease A powders. In the absence of solvation water, both protein powders exhibit largely harmonic motions on the timescale of the measurements. Upon partial hydration, quasielastic scattering indicative of relaxational processes appears at sufficiently high temperature. When the scattering spectrum are analyzed with the Kohlrausch-Williams-Watts formalism, the exponent beta decreases with increasing temperature, suggesting that multiple relaxation modes are emerging. When lysozyme was mixed with glycerol, its beta values were higher than the hydrated sample at comparable temperatures, reflecting the viscosity and stabilizing effects of glycerol.     

Influenza virus ns1 protein induces apoptosis in cultured cells

The importance of influenza viruses as worldwide pathogens in humans, domestic animals, and poultry is well recognized. Discerning how influenza viruses interact with the host at a cellular level is crucial for a better understanding of viral pathogenesis. Influenza viruses induce apoptosis through mechanisms involving the interplay of cellular and viral factors that may depend on the cell type. However, it is unclear which viral genes induce apoptosis. In these studies, we show that the expression of the nonstructural (NS) gene of influenza A virus is sufficient to induce apoptosis in MDCK and HeLa cells. Further studies showed that the multimerization domain of the NS1 protein but not the effector domain is required for apoptosis. However, this mutation is not sufficient to inhibit apoptosis using whole virus.     

Ebola virus VP40-induced particle formation and association with the lipid bilayer

Viral protein 40 (VP40) of Ebola virus appears equivalent to matrix proteins of other viruses, yet little is known about its role in the viral life cycle. To elucidate the functions of VP40, we investigated its ability to induce the formation of membrane-bound particles when it was expressed apart from other viral proteins. We found that VP40 is indeed able to induce particle formation when it is expressed in mammalian cells, and this process appeared to rely on a conserved N-terminal PPXY motif, as mutation or loss of this motif resulted in markedly reduced particle formation. These findings demonstrate that VP40 alone possesses the information necessary to induce particle formation, and this process most likely requires cellular WW domain-containing proteins that interact with the PPXY motif of VP40. The ability of VP40 to bind cellular membranes was also studied. Flotation gradient analysis indicated that VP40 binds to membranes in a hydrophobic manner, as NaCl at 1 M did not release the protein from the lipid bilayer. Triton X-114 phase-partitioning analysis suggested that VP40 possesses only minor features of an integral membrane protein. We confirmed previous findings that truncation of the 50 C-terminal amino acids of VP40 results in decreased association with cellular membranes and demonstrated that this deletion disrupts hydrophobic interactions of VP40 with the lipid bilayer, as well as abolishing particle formation. Truncation of the 150 C-terminal amino acids or 100 N-terminal amino acids of VP40 enhanced the protein's hydrophobic association with cellular membranes. These data suggest that VP40 binds the lipid bilayer in an efficient yet structurally complex fashion.     

Silicon and heavy metal tolerance of higher plants

The heavy metal tolerant Cardaminopsis halleri, grown on Zn and Cu polluted soil, showed electron dense metal containing precipitates (Zn, Cu, Sn, Fe, Al) on the leaf surface, in the intercellular spaces (Zn, Cu, Sn), the cell walls and the cell wall thickenings of the xylem vessels (Zn, traces of Cu and Fe). Large amounts of Zn were measured in the vacuoles, the main storage compartment for this metal in Cardarminopsis. The cytoplasm and nuclei contained small precipitates, including mainly Zn and Si. As shown by ESI Zn was co-localized with Si in these structures. The EEL-spectra of the cytoplasmic precipitates corresponded with the spectra of Zn-silicate. Besides Zn-silicate, electron translucent structures in the cytoplasm were identified as SiO2 by their EEL spectra. It was concluded that in the cytoplasm of Cardaminopsis Zn is transiently accumulated as silicate, being slowly degraded to SiO2. Zn is translocated into the vacuole and accumulated in an unknown form. A second Si and Zn-uptake mechanism was found, excluding a membrane and cytoplasm passage. Pinocytotic vesicles, formed by the plasmamembrane and the tonoplast, enable a direct translocation of Si and Zn from extracellular compartments into the vacuole. The formation of Zn-silicate is part of the heavy metal tolerance mechanism and may be responsible for the amelioration of the Zn toxicity in Cardaminopsis.     

Kinetic analysis of peptide binding to the TAP transport complex: evidence for structural rearrangements induced by substrate binding

The transporter associated with antigen processing (TAP) plays a key role in the class I major histocompatibility complex (MHC) mediated immune surveillance. It translocates peptides generated by the proteasome complex into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. At the cell surface these MHC complexes are monitored for their antigenic cargo by cytotoxic T-lymphocytes. Peptide binding to TAP is the essential step for peptide selection and for subsequent ATP-dependent translocation into the ER lumen. To examine the pathway of substrate recognition by TAP, we employed peptide epitopes, which were labeled with an environmentally sensitive fluorophore. Upon binding to TAP, a drastic fluorescence quenching of the fluorescent substrate was detected. This allowed us to analyze TAP function in real-time by using a homogeneous assay. Formation of the peptide-TAP complex is composed of a fast association step followed by a slow isomerization of the transport complex. Proton donor groups moving in proximity to the fluorescence label cause fluorescence quenching. Taken together, this peptide-induced structural reorganization may reflect the crosstalk of structural information between the peptide binding site and both nucleotide-binding domains within the TAP complex.     

Polycyclic nitroarenes (nitro-PAHs) as biomarkers of exposure to diesel exhaust

Diesel exhaust contains numerous genotoxic carcinogens. It is essentially unknown to which extent this source contributes to the total load of these chemicals in humans. One possible approach to the problem is to find suitable biomarkers. To this end five polycyclic mononitroarenes (nitro-PAH) were selected and methods developed to determine the sulfinic acid-type hemoglobin adducts they form in vivo. The nitro-PAHs are: 1-nitropyrene, 2-nitrofluorene, 3-nitrofluoranthene, 9-nitrophenanthrene, and 6-nitrochrysene. Hydrolysis of the hemoglobin adducts yields the respective arylamines which were analyzed by gas chromatography/mass spectrometry. The detection limit was 0.01-0.08 pmol/g Hb. Blood samples were analyzed from 29 bus garage workers, occupationally exposed to diesel exhaust, and from 20 urban hospital workers and 14 rural council workers as controls. Hb adducts above the detection limit were found in most blood samples. The most abundant cleavage products were 1-aminopyrene and 2-aminofluorene with levels ranging from 0.01 to 0.68 pmol/g Hb. However, there was no significant difference between the groups for 1-nitropyrene and 2-nitrofluorene supporting the conclusion that both are widespread environmental contaminants resulting in significant background exposures. A significant difference on a group from individuals from urban and rural areas was found only if all five adducts were added, this may indicate an additional exposure from traffic. The new specific nitro-PAH Hb adducts are proposed to be used as biomarkers to trace the sources and to identify above-background exposures.     

Recurrent V1–V2 interaction in early visual boundary processing

A majority of cortical areas are connected via feedforward and feedback fiber projections. In feedforward pathways we mainly observe stages of feature detection and integration. The computational role of the descending pathways at different stages of processing remains mainly unknown. Based on empirical findings we suggest that the top-down feedback pathways subserve a context-dependent gain control mechanism. We propose a new computational model for recurrent contour processing in which normalized activities of orientation selective contrast cells are fed forward to the next processing stage. There, the arrangement of input activation is matched against local patterns of contour shape. The resulting activities are subsequently fed back to the previous stage to locally enhance those initial measurements that are consistent with the top-down generated responses. In all, we suggest a computational theory for recurrent processing in the visual cortex in which the significance of local measurements is evaluated on the basis of a broader visual context that is represented in terms of contour code patterns. The model serves as a framework to link physiological with perceptual data gathered in psychophysical experiments. It handles a variety of perceptual phenomena, such as the local grouping of fragmented shape outline, texture surround and density effects, and the interpolation of illusory contours. Received: 28 October 1998 / Accepted in revised form: 19 March 1999     

Opposite effect of cAMP signaling in endothelial barriers of different origin

cAMP-mediated signaling mechanisms may destabilize or stabilize the endothelial barrier, depending on the origin of endothelial cells. Here, microvascular coronary [coronary endothelial cells (CEC)] and macrovascular aortic endothelial cell (AEC) monolayers with opposite responses to cAMP were analyzed. Macromolecule permeability, isometric force, activation state of contractile machinery [indicated by phosphorylation of regulatory myosin light chains (MLC), activity of MLC kinase, and MLC phosphatase], and dynamic changes of adhesion complex proteins (translocation of VE-cadherin and paxillin) were determined. cAMP signaling was stimulated by the adenosine receptor agonist 5'-N-(ethylcarboxamido)-adenosine (NECA), the -adrenoceptor agonist isoproterenol (Iso), or by the adenylyl cyclase activator forskolin (FSK). Permeability was increased in CEC and decreased in AEC on stimulation with NECA, Iso, or FSK. The effects could be inhibited by the PKA inhibitor Rp-8-CPT-cAMPS and imitated by the PKA activator Sp-cAMPS. Under cAMP/PKA-dependent stimulation, isometric force and MLC phosphorylation were reduced in monolayers of either cell type, due to an activation of MLC phosphatase. In CEC but not in AEC, FSK induced delocalization of VE-cadherin and paxillin from cellular adhesion complexes as indicated by cell fractionation and immunofluorescence microscopy. In conclusion, decline in contractile activation and isometric force contribute to cAMP/PKA-mediated stabilization of barrier function in AEC. In CEC, this stabilizing effect is overruled by cAMP-induced disintegration of cell adhesion structures. endothelial cell adhesion; endothelial permeability; isometric force; myosin light chain kinase; myosin light chain phosphatase     

MprF-mediated biosynthesis of lysylphosphatidylglycerol, an important determinant in staphylococcal defensin resistance

Frequently bacteria are exposed to membrane-damaging cationic antimicrobial molecules (CAMs) produced by the host's immune system (defensins, cathelicidins) or by competing microorganisms (bacteriocins). Staphylococcus aureus achieves CAM resistance by modifying anionic phosphatidylglycerol with positively charged L-lysine, resulting in repulsion of the peptides. Inactivation of the novel S. aureus gene, mprF, which is found in many bacterial pathogens, has resulted in the loss of lysylphosphatidylglycerol (L-PG), increased inactivation by CAM-containing neutrophils, and attenuated virulence. We demonstrate here that expression of mprF is sufficient to confer L-PG production in Escherichia coli, which indicates that MprF represents the L-PG synthase. L-PG biosynthesis was studied in vitro and found to be dependent on phosphatidylglycerol and lysyl-tRNA, two putative substrate molecules. Further addition of cadaverin, a competitive inhibitor of the lysyl-tRNA synthetases, or of RNase A abolished L-PG biosynthesis, thereby confirming the involvement of lysyl-tRNA. This study forms the basis for further detailed analyses of L-PG biosynthesis and its role in bacterial infections.     

Skin waste, vertex angle, and scar length in excisional biopsies: comparing five excision patterns--fusiform ellipse, fusiform circle, rhomboid, mosque, and S-shaped

The common excision skin pattern is either a fusiform ellipse or another pattern with dissimilar length and width. The purpose of this study was to define the most advantageous skin pattern regarding skin waste, vertex angle, and scar length. Five skin excision patterns used traditionally for closure of round lesions were analyzed: fusiform ellipse, fusiform circle, rhomboid, mosque, and S-shaped. In the analysis, the pattern characteristics were formulated by geometric principles, from which the results were compared. The smallest skin waste was found in rhomboid and mosque patterns, whereas the largest skin waste was found in the fusiform circle and ellipse. The vertex angle was found to decrease monotonously with the excision length-to-width ratio for all patterns except the mosque shape, which is zero per definition. The paradigm stating that a vertex angle of 30 degrees or less is maintained for length-to-width ratios below 4 in the surgical ellipse was found incorrect. It holds only for rhomboid and S-shaped excisions. The scar length was found almost independent of the pattern, with a variance of 3 percent. The authors conclude that the most advantageous surgical skin patterns are the rhomboid and mosque excisions.