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## Molecular basis for a link between complement and the vascular complications of diabetes

Acosta, Juan; Hettinga, Judith; Flückiger, Rudolf; Krumrei, Nicole; Goldfine, Allison; Angarita, Luis; Halperin, Jose
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
16.69%
Activated terminal complement proteins C5b to C9 form the membrane attack complex (MAC) pore. Insertion of the MAC into endothelial cell membranes causes the release of growth factors that stimulate tissue growth and proliferation. The complement regulatory membrane protein CD59 restricts MAC formation. Because increased cell proliferation characterizes the major chronic vascular complications of human diabetes and because increased glucose levels in diabetes cause protein glycation and impairment of protein function, we investigated whether glycation could inhibit CD59. Glycation-inactivation of CD59 would cause increased MAC deposition and MAC-stimulated cell proliferation. Here, we report that (i) human CD59 is glycated in vivo, (ii) glycated human CD59 loses its MAC-inhibitory function, and (iii) inactivation of CD59 increases MAC-induced growth factor release from endothelial cells. We demonstrate by site-directed mutagenesis that residues K41 and H44 form a preferential glycation motif in human CD59. The presence of this glycation motif in human CD59, but not in CD59 of other species, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.

## Antibodies to CD9, a Tetraspan Transmembrane Protein, Inhibit Canine Distemper Virus-Induced Cell-Cell Fusion but Not Virus-Cell Fusion

Schmid, Erik; Zurbriggen, Andreas; Gassen, Uta; Rima, Bert; ter Meulen, Volker; Schneider-Schaulies, Jürgen
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
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Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected.

## CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus.

Löffler, S; Lottspeich, F; Lanza, F; Azorsa, D O; ter Meulen, V; Schneider-Schaulies, J
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
17.65%
Canine distemper virus (CDV), a lymphotropic and neurotropic negative-stranded RNA virus of the Morbillivirus genus, causes a life-threatening disease in several carnivores, including domestic dogs. To identify the cellular receptor(s) involved in the uptake of CDV by susceptible cells, we isolated a monoclonal antibody (MAb K41) which binds to the cell surface and inhibits the CDV infection of several cell lines from various species. Pretreatment of cells with MAb K41 reduces the number of infectious centers and the size of the syncytia. Using affinity chromatography with MAb K41, we purified from HeLa and Vero cell extracts a 26-kDa protein which contained the amino acid sequence TKDEPQRETLK of human CD9, a member of the tetraspan transmembrane or transmembrane 4 superfamily of cell surface proteins. Transfection of NIH 3T3 or MDBK cells with a CD9 expression plasmid rendered these cells permissive for viral infection and raised virus production by a factor of 10 to 100. The mechanism involved is still unclear, since we were unable to detect direct binding of CDV to CD9 by using immunoprecipitation and a virus overlay protein binding assay. These findings indicate that human CD9 and its homologs in other species are necessary factors for the uptake of CDV by target cells...

## A Recombinant Influenza A Virus Expressing an RNA-Binding-Defective NS1 Protein Induces High Levels of Beta Interferon and Is Attenuated in Mice

Donelan, Nicola R.; Basler, Christopher F.; García-Sastre, Adolfo
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
16.69%
Previously we found that the amino-terminal region of the NS1 protein of influenza A virus plays a key role in preventing the induction of beta interferon (IFN-β) in virus-infected cells. This region is characterized by its ability to bind to different RNA species, including double-stranded RNA (dsRNA), a known potent inducer of IFNs. In order to investigate whether the NS1 RNA-binding activity is required for its IFN antagonist properties, we have generated a recombinant influenza A virus which expresses a mutant NS1 protein defective in dsRNA binding. For this purpose, we substituted alanines for two basic amino acids within NS1 (R38 and K41) that were previously found to be required for RNA binding. Cells infected with the resulting recombinant virus showed increased IFN-β production, demonstrating that these two amino acids play a critical role in the inhibition of IFN production by the NS1 protein during viral infection. In addition, this virus grew to lower titers than wild-type virus in MDCK cells, and it was attenuated in mice. Interestingly, passaging in MDCK cells resulted in the selection of a mutant virus containing a third mutation at amino acid residue 42 of the NS1 protein (S42G). This mutation did not result in a gain in dsRNA-binding activity by the NS1 protein...

## A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies.

Groves, P.; Linse, S.; Thulin, E.; Forsén, S.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
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Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.

## Identification of Glucose-Derived Cross-Linking Sites in Ribonuclease A

Dai, Zhenyu; Wang, Benlian; Sun, Gang; Fan, Xingjun; Anderson, Vernon E.; Monnier, Vincent M.
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
17.32%

## ISG15 conjugation system targets the viral NS1 protein in influenza A virus–infected cells

Zhao, Chen; Hsiang, Tien-Ying; Kuo, Rei-Lin; Krug, Robert M.
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
17.65%
ISG15 is an IFN-α/β–induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-α, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-β. We also show that the NS1A protein directly and specifically binds to Herc5...

## Prevalence and serodiversity of the pandemic clone among the clinical strains of Vibrio parahaemolyticus isolated in southern Thailand.

Laohaprertthisan, V.; Chowdhury, A.; Kongmuang, U.; Kalnauwakul, S.; Ishibashi, M.; Matsumoto, C.; Nishibuchi, M.
Fonte: Cambridge University Press Publicador: Cambridge University Press
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
17.32%
We collected diarrhoea specimens in two hospitals in southern Thailand in 1999 to examine whether infection by the Vibrio parahaemolyticus pandemic clone is prevalent. V. parahaemolyticus was isolated from 317 specimens. Seventy-six per cent of the isolated strains had the pandemic clone-specific characteristics (tdh+, trh-, and an unique toxRS sequence detectable by GS-PCR) and an associated characteristic (the ORF8 sequence of f237 phage). These strains belonged to the three pandemic servovars with the O3:K6 strains being dominant and three other serovars (O1:K25, O1:K41 and O4:K12). We also found O1:K25 and O1:K41 strains with the pandemic clone-specific characteristics among the strains isolated from the international travellers who left Thailand and three other Asian countries between 1998 and 1999, verifying pandemic potential of these strains. The results demonstrate prevalence of infection by the pandemic clone in southern Thailand and suggest emergence of various serovariants in this area and their implication in international spread.

## CD9 clustering and formation of microvilli zippers between contacting cells regulates virus-induced cell fusion

Singethan, Katrin; Müller, Nora; Schubert, Sabine; Lüttge, Doreen; Krementsov, Dimitry N; Khurana, Sandhya R; Krohne, Georg; Schneider-Schaulies, Sibylle; Thali, Markus; Schneider-Schaulies, Jürgen
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
17.32%
Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific mAb, inhibits the release of human immunodeficiency virus (HIV-1), and canine distemper virus (CDV)-, but not measles virus (MV)-induced cell-cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop (LEL) of CD9, induces rapid relocation and clustering of CD9 in net-like structures at cell-cell contact areas. High resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins β1-integrin and EWI-F were co-clustered with CD9 at cell-cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell-cell fusion by controlling the access of the fusion machinery to cell contact areas.

## Ribonuclease A: Revealing Structure–Function Relationships with Semisynthesis

Messmore, June M.; Fuchs, Dana N.; Raines, Ronald T.
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
17.65%
Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P–O5′ bonds in RNA. Structural analyses had suggested that the active-site lysine residue (K41) may interact preferentially with the transition state for covalent bond cleavage, thus facilitating catalysis. Here, site-directed mutagenesis and semisynthesis were combined to probe the role of K41 in the catalysis of RNA cleavage. Recombinant DNA techniques were used to replace K41 with an arginine residue (K41R) and with a cysteine residue (K41C), which had the only sulfhydryl group in the native protein. The value of kcat/Km for cleavage of poly(C) by K41C RNase was 105-fold lower than that by the wild-type enzyme. The sulfhydryl group of K41C RNase A was alkylated with 5 different haloalkylamines. The value of kcat/Km for the resulting semisynthetic enzymes and K41R RNase A were correlated inversely with the values of pKa for the side chain of residue 41. Further, no significant catalytic advantage was gained by side chains that could donate a second hydrogen bond. These results indicate that residue 41 donates a single hydrogen bond to the rate-limiting transition state during catalysis.

## Exact Resummations in the Theory of Hydrodynamic Turbulence: I The Ball of Locality and Normal Scaling

L'vov, Victor; Procaccia, Itamar
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
17.32%
This paper is the first in a series of three papers that aim at understanding the scaling behaviour of hydrodynamic turbulence. We present in this paper a perturbative theory for the structure functions and the response functions of the hydrodynamic velocity field in real space and time. Starting from the Navier-Stokes equations (at high Reynolds number Re) we show that the standard perturbative expansions that suffer from infra-red divergences can be exactly resummed using the Belinicher-L'vov transformation. After this exact (partial) resummation it is proven that the resulting perturbation theory is free of divergences, both in large and in small spatial separations. The hydrodynamic response and the correlations have contributions that arise from mediated interactions which take place at some space- time coordinates. It is shown that the main contribution arises when these coordinates lie within a shell of a "ball of locality" that is defined and discussed. We argue that the real space-time formalism developed here offers a clear and intuitive understanding of every diagram in the theory, and of every element in the diagrams. One major consequence of this theory is that none of the familiar perturbative mechanisms may ruin the classical Kolmogorov (K41) scaling solution for the structure functions. Accordingly...

## Quasi-Gaussian Statistics of Hydrodynamic Turbulence in 3/4+\epsilon dimensions

L'vov, Victor S.; Pomyalov, Anna; Procaccia, Itamar
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
17.32%
The statistics of 2-dimensional turbulence exhibit a riddle: the scaling exponents in the regime of inverse energy cascade agree with the K41 theory of turbulence far from equilibrium, but the probability distribution functions are close to Gaussian like in equilibrium. The skewness $\C S \equiv S_3(R)/S^{3/2}_2(R)$ was measured as $\C S_{\text{exp}}\approx 0.03$. This contradiction is lifted by understanding that 2-dimensional turbulence is not far from a situation with equi-partition of enstrophy, which exist as true thermodynamic equilibrium with K41 exponents in space dimension of $d=4/3$. We evaluate theoretically the skewness $\C S(d)$ in dimensions ${4/3}\le d\le 2$, show that $\C S(d)=0$ at $d=4/3$, and that it remains as small as $\C S_{\text{exp}}$ in 2-dimensions.; Comment: PRL, submitted, REVTeX 4, 4 pages

## Chaotic Cascades with Kolmogorov 1941 Scaling

Biferale, L.; Blank, M.; Frisch, U.
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
17.32%
We define a (chaotic) deterministic variant of random multiplicative cascade models of turbulence. It preserves the hierarchical tree structure, thanks to the addition of infinitesimal noise. The zero-noise limit can be handled by Perron-Frobenius theory, just as the zero-diffusivity limit for the fast dynamo problem. Random multiplicative models do not possess Kolmogorov 1941 (K41) scaling because of a large-deviations effect. Our numerical studies indicate that deterministic multiplicative models can be chaotic and still have exact K41 scaling. A mechanism is suggested for avoiding large deviations, which is present in maps with a neutrally unstable fixed point.; Comment: 14 pages, plain LaTex, 6 figures available upon request as hard copy (no local report #)

## Scaling Laws and Intermittency in Highly Compressible Turbulence

Kritsuk, Alexei G.; Padoan, Paolo; Wagner, Rick; Norman, Michael L.
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
17.32%
We use large-scale three-dimensional simulations of supersonic Euler turbulence to study the physics of a highly compressible cascade. Our numerical experiments describe non-magnetized driven turbulent flows with an isothermal equation of state and an rms Mach number of 6. We find that the inertial range velocity scaling deviates strongly from the incompressible Kolmogorov laws. We propose an extension of Kolmogorov's K41 phenomenology that takes into account compressibility by mixing the velocity and density statistics and preserves the K41 scaling of the density-weighted velocity v=rho^{1/3}u. We show that low-order statistics of 'v' are invariant with respect to changes in the Mach number. For instance, at Mach 6 the slope of the power spectrum of 'v' is -1.69 and the third-order structure function of 'v' scales linearly with separation. We directly measure the mass dimension of the "fractal" density distribution in the inertial subrange, D_m=2.4, which is similar to the observed fractal dimension of molecular clouds and agrees well with the cascade phenomenology.; Comment: 7 pages, 3 figures; in press, AIP Conference Proceedings: "Turbulence and Nonlinear Processes in Astrophysical Plasmas", Waikiki Beach, Hawaii, March 21...

## Fluid Particle Accelerations in Fully Developed Turbulence

La Porta, A.; Voth, Greg A.; Crawford, Alice M.; Alexander, Jim; Bodenschatz, Eberhard
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
17.32%
The motion of fluid particles as they are pushed along erratic trajectories by fluctuating pressure gradients is fundamental to transport and mixing in turbulence. It is essential in cloud formation and atmospheric transport, processes in stirred chemical reactors and combustion systems, and in the industrial production of nanoparticles. The perspective of particle trajectories has been used successfully to describe mixing and transport in turbulence, but issues of fundamental importance remain unresolved. One such issue is the Heisenberg-Yaglom prediction of fluid particle accelerations, based on the 1941 scaling theory of Kolmogorov (K41). Here we report acceleration measurements using a detector adapted from high-energy physics to track particles in a laboratory water flow at Reynolds numbers up to 63,000. We find that universal K41 scaling of the acceleration variance is attained at high Reynolds numbers. Our data show strong intermittency---particles are observed with accelerations of up to 1,500 times the acceleration of gravity (40 times the root mean square value). Finally, we find that accelerations manifest the anisotropy of the large scale flow at all Reynolds numbers studied.; Comment: 7 pages, 4 figures

## Conditional Eulerian and Lagrangian velocity increment statistics of fully developed turbulent flow

Homann, Holger; Schulz, Daniel; Grauer, Rainer
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
17.32%
Conditional statistics of homogeneous isotropic turbulent flow is investigated by means of high-Reynolds number direct numerical simulations performed with $2048^3$ collocation points. Eulerian as well as Lagrangian velocity increment statistics under several conditions are analyzed and compared. In agreement with experimental data longitudinal probability density functions $P(\delta^{||}_l u|\epsilon_l)$ conditioned on a scale-averaged energy dissipation rate are close to Gaussian distributions over all scales within the inertial range of scales. Also transverse increments conditioned on either the dissipation rate or the square of the vorticity have quasi-Gaussian probability distribution functions (PDFs). Concerning Lagrangian statistics we found that conditioning on a trajectory averaged energy-dissipation rate $\epsilon_\tau$ significantly reduces the scale dependence of the increment PDFs $P(\delta_\tau u_i|\epsilon_\tau)$. By means of dimensional arguments we propose a novel condition for Lagrangian increments which is shown to reduce even more the flatness of the corresponding PDFs and thus intermittency in the inertial range of scales. The conditioned Lagrangian PDF corresponding to the smallest increment considered is reasonably well described by the K41-prediction of the PDF of acceleration. Conditioned structure functions show approximately K41-scaling with a larger scaling range than the unconditioned ones.; Comment: 7 pages...

## Analytic Calculation of the Anomalous Exponents in Turbulence: Using the Fusion Rules to Flush Out a Small Parameter

L'vov, Victor S.; Procaccia, Itamar
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
17.32%
The main difficulty of statistical theories of fluid turbulence is the lack of an obvious small parameter. In this paper we show that the formerly established fusion rules can be employed to develop a theory in which Kolmogorov's statistics of 1941 acts as the zero order, or background statistics, and the anomalous corrections to the K41 scaling exponents $\zeta_n$ of the $n$th order structure functions can be computed analytically. The crux of the method consists of renormalizing a 4-point interaction amplitude on the basis of the fusion rules. The novelty is that this amplitude includes a small dimensionless parameter, which is shown to be of the order of the anomaly of $\zeta_2$, $\delta_2= \zeta_2-2/3\approx 0.03$. Higher order interaction amplitudes are shown to be even smaller. The corrections to K41 to $O(\delta_2)$ result from standard logarithmically divergent ladder-diagrams in which the 4-point interaction acts as a "rung". The theory allows a calculation of the anomalous exponents $\zeta_n$ in powers of the small parameter $\delta_2$. The $n$-dependence of the scaling exponents $\zeta_n$ stems from pure combinatorics of the ladder diagrams. We calculate the exponents $\zeta_n$ up to $O(\delta_2^3)$. Previously derived bridge relations allow a calculation of the anomalous exponents of correlations of the dissipation field and of dynamical correlations in terms of the same parameter $\delta_2$. The actual evaluation of the small parameter $\delta_2$ from first principles requires additional developments that are outside the scope of this paper.; Comment: PRE submitted...

## Intermittency in the large N-limit of a spherical shell model for turbulence

Pierotti, D.
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
17.32%
A spherical shell model for turbulence, obtained by coupling $N$ replicas of the Gledzer, Okhitani and Yamada shell model, is considered. Conservation of energy and of an helicity-like invariant is imposed in the inviscid limit. In the $N \to \infty$ limit this model is analytically soluble and is remarkably similar to the random coupling model version of shell dynamics. We have studied numerically the convergence of the scaling exponents toward the value predicted by Kolmogorov theory (K41). We have found that the rate of convergence to the K41 solution is linear in 1/N. The restoring of Kolmogorov law has been related to the behaviour of the probability distribution functions of the instantaneous scaling exponent.; Comment: 10 pages, Latex, 3 Postscript figures, to be published on Europhys. Lett

## Remarks on the K41 scaling law in turbulent fluids

Flandoli, F.; Gubinelli, M.; Hairer, M.; Romito, M.
Tipo: Artigo de Revista Científica