Primary Interaction Site Research Articles

Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 1 Signaling Requires G Protein-coupled Receptor Kinase 2 Binding to the Second Intracellular Loop

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.

Oxidative stress and ozone: perception, signalling and response

ABSTRACTThe primary site of ozone interaction with plant cells is the extracellular matrix where ozone challenges the antioxidant protection of the cells. Accordingly, ozone sensitivity generally correlates with the ascorbate status of the apoplast, which is an important signal initiation point. In addition, ozone sensing takes place by covalent modification of redox‐sensitive components of the plasma membrane, for example ion channels like the plasma membrane Ca2+‐channels. Subsequent intracellular signal transduction is an intriguing network of hormone, Ca2+ and MAPK signalling pathways, significantly overlapping with oxidative burst‐induced pathogen signalling. Comparison of recent transcriptome analysis revealed that in addition to genes generally induced by all kinds of oxidative stress, for example, transcripts for PR‐proteins and most antioxidant enzymes, approximately one‐third of the responsive transcripts are ozone specific, indicating jasmonic acid, salicylic acid and ethylene‐independent redox signalling triggered by extracellular redox sensing.

Evaluation of the 17-kDa Prenyl-binding Protein as a Regulatory Protein for Phototransduction in Retinal Photoreceptors

The mammalian rod photoreceptor phosphodiesterase (PDE6) holoenzyme is isolated in both a membrane-associated and a soluble form. Membrane binding is a consequence of prenylation of PDE6 catalytic subunits, whereas soluble PDE6 is purified with a 17-kDa prenyl-binding protein (PDEdelta) tightly bound. This protein, here termed PrBP/delta, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/delta in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/delta in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/delta was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/delta. In contrast to bovine ROS, all of the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/delta can solubilize PDE6 and prevent its activation by transducin. PrBP/delta also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis of the PrBP/delta content of purified ROS reveals insufficient amounts of PrBP/delta (<0.1 PrBP/delta per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/delta in frog and bovine retina shows greatest PrBP/delta immunolabeling outside the photoreceptor cell layer. Within photoreceptors, only the inner segments of frog double cones are strongly labeled, whereas bovine photoreceptors reveal more PrBP/delta labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/delta as a PDE6 subunit and implicate PrBP/delta in the transport and membrane targeting of prenylated proteins (including PDE6) from their site of synthesis in the inner segment to their final destination in the outer segment of rods and cones.

Plasmin(ogen)-binding α-Enolase from Streptococcus pneumoniae: Crystal Structure and Evaluation of Plasmin(ogen)-binding Sites

Alpha-enolases are ubiquitous cytoplasmic, glycolytic enzymes. In pathogenic bacteria, alpha-enolase doubles as a surface-displayed plasmin(ogen)-binder supporting virulence. The plasmin(ogen)-binding site was initially traced to the two C-terminal lysine residues. More recently, an internal nine-amino acid motif comprising residues 248 to 256 was identified with this function. We report the crystal structure of alpha-enolase from Streptococcus pneumoniae at 2.0A resolution, the first structure both of a plasminogen-binding and of an octameric alpha-enolase. While the dimer is structurally similar to other alpha-enolases, the octamer places the C-terminal lysine residues in an inaccessible, inter-dimer groove restricting the C-terminal lysine residues to a role in folding and oligomerization. The nine residue plasminogen-binding motif, by contrast, is exposed on the octamer surface revealing this as the primary site of interaction between alpha-enolase and plasminogen.

Role of Protein Phosphatase Type 1 in Contractile Functions: Myosin Phosphatase

Role of Protein Phosphatase Type 1 in Contractile Functions: Myosin Phosphatase

Surface-enhanced Raman spectroscopy study of the interaction of the antitumoral drug emodin with human serum albumin.

Surface-enhanced Raman spectroscopy was employed in this work to study the interaction between the antitumoral drug emodin and human serum albumin (HSA), as well as the influence of fatty acids in this interaction. We demonstrated that the drug/protein interaction can take place through two different binding sites which are probably localized in the IIA and IIIA hydrophobic pockets of HSA and which correspond to Sudlow's I and II binding sites, respectively. The primary interaction site of this drug seems to be site II in the defatted albumin. Fatty acids seem to displace the drug from site II to site I in nondefatted HSA, due to the high affinity of fatty acids for site II. The drug interacts with the protein through its dianionic form in defatted HSA (when placed in the site II) and through its neutral form in the site I of nondefatted albumins.

Spike-Frequency Adaptation in the Inferior Colliculus

We investigated spike-frequency adaptation of neurons sensitive to interaural phase disparities (IPDs) in the inferior colliculus (IC) of urethane-anesthetized guinea pigs using a stimulus paradigm designed to exclude the influence of adaptation below the level of binaural integration. The IPD-step stimulus consists of a binaural 3,000-ms tone, in which the first 1,000 ms is held at a neuron's least favorable ("worst") IPD, adapting out monaural components, before being stepped rapidly to a neuron's most favorable ("best") IPD for 300 ms. After some variable interval (1-1,000 ms), IPD is again stepped to the best IPD for 300 ms, before being returned to a neuron's worst IPD for the remainder of the stimulus. Exponential decay functions fitted to the response to best-IPD steps revealed an average adaptation time constant of 52.9 +/- 26.4 ms. Recovery from adaptation to best IPD steps showed an average time constant of 225.5 +/- 210.2 ms. Recovery time constants were not correlated with adaptation time constants. During the recovery period, adaptation to a 2nd best-IPD step followed similar kinetics to adaptation during the 1st best-IPD step. The mean adaptation time constant at stimulus onset (at worst IPD) was 34.8 +/- 19.7 ms, similar to the 38.4 +/- 22.1 ms recorded to contralateral stimulation alone. Individual time constants after stimulus onset were correlated with each other but not with time constants during the best-IPD step. We conclude that such binaurally derived measures of adaptation reflect processes that occur above the level of exclusively monaural pathways, and subsequent to the site of primary binaural interaction.

Structure of complement receptor (CR) 2 and CR2-C3d complexes.

Using X-ray crystallography, we have determined the structure of the first two short consensus repeats (SCRs) of human complement receptor (CR) 2 in complex with C3d. These studies revealed: (i) a primary site of interaction for C3d within SCR2 of CR2, (ii) a hydrophobic patch holding SCR1 to SCR2 in a rigid V-shape, (iii) a dimer formed by interactions between SCR1 of each molecule, (iv) several non-linear sequences on C3d that interact with CR2 and (v) mutations of C3d amino acids within the co-crystal interface that resulted in decreased binding. In addition, a polymorphism that results in decreased C3d binding and introduces a new glycosylation site predicted to disrupt the dimer interface was found in the New Zealand White autoimmune mouse strain. Although the co-crystal complex results are in agreement with a subset of prior studies, our additional findings, which demonstrate an extended SCR1-SCR2 structure in solution and differences in the kinetics of ligand-receptor interactions with longer forms of CR2, have suggested a more complex receptor-ligand interaction. To characterize this interaction further, several approaches directed at the determination of solution phase interactions as well as the analysis of the three-dimensional structure of CR2 alone and key CR2 mutants will be necessary.

Jun dimerization protein 2 functions as a progesterone receptor N-terminal domain coactivator.

The progesterone receptor (PR) contains two transcription activation function (AF) domains, constitutive AF-1 in the N terminus and AF-2 in the C terminus. AF-2 activity is mediated by a hormone-dependent interaction with a family of steroid receptor coactivators (SRCs). SRC-1 can also stimulate AF-1 activity through a secondary domain that interacts simultaneously with the primary AF-2 interaction site. Other protein interactions and mechanisms that mediate AF-1 activity are not well defined. By interaction cloning, we identified an AP-1 family member, Jun dimerization protein 2 (JDP-2), as a novel PR-interacting protein. JDP-2 was first defined as a c-Jun interacting protein that functions as an AP-1 repressor. PR and JDP-2 interact directly in vitro through the DNA binding domain (DBD) of PR and the basic leucine zipper (bZIP) region of JDP-2. The two proteins also physically associate in mammalian cells, as detected by coimmunoprecipitation, and are recruited in vivo to a progesterone-inducible target gene promoter, as detected by a chromatin immunoprecipitation (ChIP) assay. In cell transfection assays, JDP-2 substantially increased hormone-dependent PR-mediated transactivation and worked primarily by stimulating AF-1 activity. JDP-2 is a substantially stronger coactivator of AF-1 than SRC-1 and stimulates AF-1 independent of SRC-1 pathways. The PR DBD is necessary but not sufficient for JDP-2 stimulation of PR activity; the DBD and AF-1 are required together. JDP-2 lacks an intrinsic activation domain and makes direct protein interactions with other coactivators, including CBP and p300 CBP-associated factor (pCAF), but not with SRCs. These results indicate that JDP-2 stimulates AF-1 activity by the novel mechanism of docking to the DBD and recruiting or stabilizing N-terminal PR interactions with other general coactivators. JDP-2 has preferential activity on PR among the nuclear receptors tested and is expressed in progesterone target cells and tissues, suggesting that it has a physiological role in PR function.

The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation: USE-DEPENDENT BLOCK BY BENZOTHIAZEPINES

The role of inactivated channel conformation and use dependence for diltiazem, a specific benzothiazepine calcium channel inhibitor, was studied in chimeric constructs and point mutants created in the IVS5 transmembrane segment of the L-type cardiac calcium channel. All mutations, chimeric or point mutations, were restricted to IVS5, while the YAI-containing segment in IVS6, i.e. the primary interaction site with benzothiazepines, remained intact. Slowed inactivation rate and incomplete steady state inactivation, a behavior of some mutants, were accompanied by a reduced or by a complete loss of use-dependent block by diltiazem. Single channel properties of mutants that lost use dependence toward diltiazem were characterized by drastically elongated mean open times and distinctly slower time constants of open time distribution. Mutation of individual residues of the IVMLF segment in IVS5 did not mimic the complete loss of use dependence as observed for the replacement of the whole stretch. These results establish evidence that amino acids that govern inactivation and the drug-binding site and other amino acids that are located distal from the putative drug-binding site contribute significantly to the function of the benzothiazepine receptor region. The data are consistent with a complex "pocket" conformation that is responsive to a specific class of L-type calcium channel inhibitors. The data allow for a concept that multiple sites within regions of the alpha(1) subunit contribute to auto-regulation of the L-type Ca(2+) channel.

Partial identification of the mab (CD)Amp1 antigen at the epithelial-mesenchymal interface in the developing kidney.

The nature of the primary functional events of nephron induction is still unknown, making it impossible to completely understand the mechanism of tissue interaction between collecting duct ampulla and the surrounding nephrogenic mesenchyme. Soluble morphogenic substances are known to be exchanged in the process and it is assumed that nephron induction requires close contact between both tissues involved. Contrasting with that assumption our previous investigation revealed a thick fibrous meshwork separating nephron inducer and mesenchyme. Our present investigation focused on the molecular characterization of the mab (CD)Amp1 antigen, which is found only in this meshwork. The protein was shown immunohistochemically to be located exclusively at the embryonic collecting duct ampulla and could be clearly distinguished from other extracellular matrix proteins such as collagen type IV, laminin, reticulin, and fibronectin. Two-dimensional electrophoresis of the soluble form of P(CD)Amp1 showed a molecular weight of 87,000 and an isoelectric point of 4.3-4.4. Results from N-terminal sequencing indicated a partial sequence homology of P(CD)Amp1 to collagen type IV alpha 2-chain precursor but additionally yielded unknown sequences. Thus P(CD)Amp1 is a novel, collagen-related protein, restricted to the fibrous meshwork at the mesenchymal-epithelial interphase, which is the site of primary epithelial-mesenchymal interaction.

The Carboxyl Terminus of Protein Kinase C Provides a Switch to Regulate Its Interaction with the Phosphoinositide-dependent Kinase, PDK-1

The function of protein kinase C family members depends on two tightly coupled phosphorylation mechanisms: phosphorylation of the activation loop by the phosphoinositide-dependent kinase, PDK-1, followed by autophosphorylation at two positions in the COOH terminus, the turn motif, and the hydrophobic motif. Here we address the molecular mechanisms underlying the regulation of protein kinase C betaII by PDK-1. Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C, by a direct mechanism. The exposed COOH terminus of protein kinase C provides the primary interaction site for PDK-1, with co-expression of constructs of the carboxyl terminus effectively disrupting the interaction in vivo. Disruption of this interaction promotes the autophosphorylation of protein kinase C, suggesting that the binding of PDK-1 to the carboxyl terminus protects it from autophosphorylation. Studies with constructs of the COOH terminus reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH terminus is over an order of magnitude greater than that for unphosphorylated COOH terminus, contrasting with the finding that PDK-1 does not bind phosphorylated protein kinase C effectively. However, effective binding of the phosphorylated species can be induced by the activated conformation of protein kinase C. This suggests that the carboxyl terminus becomes masked following autophosphorylation, a process that can be reversed by the conformational changes accompanying activation. Our data suggest a model in which PDK-1 provides two points of regulation of protein kinase C: 1) phosphorylation of the activation loop, which is regulated by the intrinsic activity of PDK-1, and 2) phosphorylation of the carboxyl terminus, which is regulated by the release of PDK-1 to allow autophosphorylation.

Interface configuration and metal adhesion in Au-polycarbonate bilayer structure: Influence of 27Al+ ion mixing

Structural modification at the Au-polycarbonate (PC) interface upon 100 keV Al+27 ion implantation at a dose and beam current density 5×1016 ions cm−2 and 30–50 nA cm−2, respectively, was studied through x-ray photoelectron spectroscopy (XPS) with a Au thickness of 28.2 nm. XPS depth profiling with 3 keV Ar+ ion sputtering at 1 μA revealed the interface to be sharp covering a few monolayers. A substantial Au atomic concentration of ∼5% in the bulk PC indicated the Ar+ ion assisted diffusion of the metal into the bulk. Existence of weak Au→C charge transfer interactions with Au as the electron injector distributing a net charge density at the C=O bond as the primary interaction site was deduced from the appearance of the 282.4 eV C1s feature [H. A. Mizes, K. G. Loh, R. J. D. Miller, S. K. Ahuja, and E. F. Grabowski, Appl. Phys. Lett. 59, 2901 (1991)]. Al+ ion implantation induced interfacial mixing of the substrate C with the Au film in the bilayer target was observed with the evolution of a broad interface of ∼50 nm thickness, accompanied by the formation of Au–Al, Al–O, and Al–O–C bonds along with dominant graphitization of the polymer. Free carbon transport into and through the Au film to the surface resulted in a diffused interface with an abnormally low oxygen concentration throughout. Au–Al bonding was identified in the Au-rich region, and shifted to Al–O bonding in the C-rich region of the polymer, with the Al atomic concentration reaching a maximum of 1.8%. After ion-beam mixing and surface modification by ion bombardment, force curve measurements performed through atomic force microscopy showed a drastic reduction in the interface adhesion values.

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc

NADPH‐dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero‐length crosslinks mediated by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X‐ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin −P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin−P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc Mass spectrometry and Edman degradation of complexes of the steroidogenic hydroxylase system

NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin -P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin-P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding

Novel glycopeptide analogs are known that have activity on vancomycin resistant enterococci despite the fact that the primary site for drug interaction, d-ala- d-ala, is replaced with d-ala- d-lactate. The mechanism of action of these compounds may involve dimerization and/or membrane binding, thus enhancing interaction with d-ala- d-lactate, or a direct interaction with the transglycosylase enzymes involved in peptidoglycan polymerization. We evaluated the ability of vancomycin (V), desleucyl-vancomycin (desleucyl-V), chlorobiphenyl-vancomycin (CBP-V), and chlorobiphenyl-desleucyl-vancomycin (CBP-desleucyl-V) to inhibit (a) peptidoglycan synthesis in vitro using UDP-muramyl-pentapeptide and UDP-muramyl-tetrapeptide substrates and (b) growth and peptidoglycan synthesis in vancomycin resistant enterococci. Compared to V or CBP-V, CBP-desleucyl-V retained equivalent potency in these assays, whereas desleucyl-V was inactive. In addition, CBP-desleucyl-V caused accumulation of N-acetylglucosamine-β-1,4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II). These data show that CBP-desleucyl-V inhibits peptidoglycan synthesis at the transglycosylation stage in the absence of binding to dipeptide.

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding.

Novel glycopeptide analogs are known that have activity on vancomycin resistant enterococci despite the fact that the primary site for drug interaction, D-ala-D-ala, is replaced with D-ala-D-lactate. The mechanism of action of these compounds may involve dimerization and/or membrane binding, thus enhancing interaction with D-ala-D-lactate, or a direct interaction with the transglycosylase enzymes involved in peptidoglycan polymerization. We evaluated the ability of vancomycin (V), desleucyl-vancomycin (desleucyl-V), chlorobiphenyl-vancomycin (CBP-V), and chlorobiphenyl-desleucyl-vancomycin (CBP-desleucyl-V) to inhibit (a) peptidoglycan synthesis in vitro using UDP-muramyl-pentapeptide and UDP-muramyl-tetrapeptide substrates and (b) growth and peptidoglycan synthesis in vancomycin resistant enterococci. Compared to V or CBP-V, CBP-desleucyl-V retained equivalent potency in these assays, whereas desleucyl-V was inactive. In addition, CBP-desleucyl-V caused accumulation of N-acetylglucosamine-beta-1, 4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II). These data show that CBP-desleucyl-V inhibits peptidoglycan synthesis at the transglycosylation stage in the absence of binding to dipeptide.

Cellular characteristics of temporary partial breakdown of mlo- resistance in barley to powdery mildew

When water-stress is relieved, powdery mildew (Erysiphe graminis f.sp. hordei) infection increases on both Mlo -susceptible and mlo -resistant spring barley cultivars. The breakdown is temporary and is determined by the genetic background rather than the specific resistance allele. The relief of water-stress time-point for maximum mildew infection frequency on mlo -resistant barley is approximately 7 h post-inoculation. The degree of breakdown varies with epidermal cell type; increased infection frequency is greatest on short followed by long and adjacent epidermal cell types, rather than on the stomatal subsidiary cells with which occasional colonies are typically associated. Infection frequency on the short cells of mlo -resistant cv. Atem increases from less than 1% under a non-stressed watering regime to more than 10% after a relief of water-stress at 7 h post-inoculation. Following haustorium formation, colonies develop and reach sporulation within 7 days post-inoculation. In host epidermal cells, the extent of cross-linked protein at interaction sites is reduced under conditions of water-stress or stress-relief. Cross-linked protein is reduced in terms of the frequency of occurrence at primary germ tube interaction sites 7 h post-inoculation and the deposition size at appressorial germ tube interaction sites 24 h post-inoculation. This indicates a delayed or reduced defence response during the recovery period. These data demonstrate a differential cellular response to powdery mildew in barley genotypes prone to resistance expression breakdown following relief of water-stress.

Interaction between GPIbα and FcγIIA Receptor in Human Platelets

Glycoprotein (GP) Ib (α and β) in platelets forms a noncovalent hetero-oligomeric complex with GPIX and GPV and serves as a receptor for von Willebrand factor (vWF), which mediates the initial adhesion of platelets to the subendothelium after vascular damage and also plays a role in thrombin-induced platelet activation. We investigated the interaction between GPIbα and FcγIIA receptor using a yeast two-hybrid system and mutagenesis, and we identified residues R542G543R544 in GPIbα and D298D299D300 in FcγIIA receptor as the primary interaction sites. These results further confirmed the interaction between GPIbα and FcγIIA receptor and support the hypothesis that the signal transduction of GPIb-IX-V that leads to platelet activation may be partially mediated through FcγIIA receptor.

Interface configuration in gold/polycarbonate bilayer structure: an in situ study through Ar + ion depth profiling

The formation, configuration and the interactions at the gold/polycarbonate (Au/PC) interface formed by thermally evaporated Au on PC film, a technologically important polymer, is studied by X-ray photoelectron spectroscopy (XPS) at an Au thickness of 35.4 Å. XPS depth profiling with 3 keV Ar + ion sputtering at 1 μA revealed the interface between the as-deposited 35.4 Å Au film and the PC substrate to be sharp covering a few monolayers. A substantial Au atomic concentration of ∼3% in the bulk PC indicated that Ar + ion assisted diffusion of the metal into the bulk. Existence of weak Au→C charge transfer interactions with Au as the electron injector distributing a net charge density at the CO as the primary interaction site was deduced from the appearance of the 282.6 eV C 1s feature, thus resulting in the formation of Au–CO π back bond. The sputtering experiments revealed the growth-mode of Au on PC to proceed by metal monolayer deposition followed by cluster growth on the already weakly bonded Au onto the CO carbon of the polymeric backbone.