Studies on metrizamide-protein interactions

1. The apparent density of catalase after isopycnic centrifugation in metrizamide gradients is dependent on the metrizamide concentration into which the enzyme is dissolved at the beginning of the centrifugation. 2. This different behaviour of the enzyme in metrizamide gradients is due to the formation of a metrizamide-protein complex which is more dense than the uncomplexed catalase. 3. A bimodal distribution of the catalase, with additional heavy bands, was only observed in metrizamide gradients in light water, where rather high metrizamide concentrations are needed even for a banding of the uncomplexed enzyme. 4. The half-life of the metrizamide-protein complex is less than 5 min. This was shown by spectroscopical measurements and band sedimentation analysis in an analytical ultracentrifuge.     

Studies on human plasma antithrombin isolated on heparin gel

1. A study has been made of peptide excretion in twenty cases of Wilson's disease, ligand-exchange column chromatography being used to separate peptides from free amino acids. Previous reports of excess of peptide output in the disease were confirmed and the excess was shown to be highly significant statistically. 2. A considerable fraction of the excess of peptide output was shown to consist of hydroxyproline-containing peptides derived from collagen degradation. 3. The method of rank correlation showed that the difference both in free amino acid and peptide-bound amino acid output in cases of Wilson's disease and in control subjects was mainly quantitative; the pattern of amino acid excretion was qualitatively similar in both groups. 4. Evidence is presented that the increase copper output in the urine in the disease is not secondary to peptiduria.     

Effect of heparin on the interaction between platelets with collagen

An effect of a standard heparin preparation on the interaction between blood platelets and collagen has been investigated. The experiments have shown, that the addition of heparin in the concentration of 0.3; 0.6 or 0.9 IU/mL did not change the interaction between blood platelets and collagen. Such interaction increased, when heparin concentration was 1,2 IU/mL, and remained unchanged despite the further increase in heparin concentration. The authors suggest that such a course of this interaction results from the stimulating action of heparin added in adequate concentration on protein release from platelet alpha granulations--which bound GP II b/III a complex with collagen.     

Structural aspects of heparin responsible for interactions with von Willebrand factor

Unfractionated heparin (UFH) binds von Willebrand factor (vWF) and inhibits the vWF-platelet GP Ib interaction. For vWF, a heparin-binding domain has been identified, but for heparin, the structures that confer such activity are unknown. To investigate this, UFH was depolymerized by methods that yield structurally distinct fragments. The glycosaminoglycans (GAGs) produced were separated into five groups of homogeneous molecular weight (MW). Anti-Xa activity, vWF binding affinity, and vWF-dependent platelet agglutination were measured. Periodate oxidation but not heparinase digestion destroyed anti-Xa activity. At all MWs, periodate conferred greater vWF binding affinity and greater ability to inhibit platelet agglutination than heparinase. As an example, at MW 6100, the binding IC50 was 100+/-19 micromol/L for a periodate-derived GAG and 527+/-70 micromol/L for a heparinase-derived GAG. At the same MW, the agglutination IC50 was 17+/-5 micromol/L for periodate and 135+/-18 micromol/L for heparinase. This suggests that the disaccharide GlcNS[6S]-IdoA2S, destroyed by heparinase but not periodate, is crucial to heparin-vWF interactions. An MW dependency was also noted, with a minimum dodecasaccharide required for activity inhibition. To further investigate the heparin/vWF interaction, affinity fractionation of heparins was performed with an immobilized peptide derived from a heparin-binding domain of vWF. Disaccharide analysis of high-affinity heparins revealed an increased ratio of IdoA2S-GlcN[S/Ac]6S to IdoA2S-GlcN[S/Ac]. Affinity fractionation of oligosaccharides (MW 3500) diminished the relative content of all disaccharides except IdoA2S-GlcNS6S, which was increased. These data suggest that the disaccharide structures IdoA2S-GlcNS6S and GlcNS6S-IdoA2S are crucial to heparin/vWF interactions. Understanding the structural aspects that confer such activity may be useful in designing heparin-based antithrombotic drugs.     

Curvature-mediated interactions between membrane proteins

Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions.     

Studies on Gonococcus infection. X. Pili and leukocyte association factor as mediators of interactions between gonococci and eukaryotic cells in vitro

Two independent gonococcal surface components, pili and leukocyte association factor, appear to mediate in vitro interactions of Neisseria gonorrhoeae with tissue culture cells and human peripheral blood leukocytes, respectively.     

Spectroscopic characterization of arrestin interactions with competitive ligands: study of heparin and phytic acid binding

A combination of intrinsic fluorescence and circular dichroic (CD) spectroscopy has been used to characterize the complexes formed between bovine retinal arrestin and heparin or phytic acid, two ligands that are known to mimic the structural changes in arrestin attending receptor binding. No changes in the CD spectra were observed upon ligand binding, nor did the degree of tryptophan fluorescence quenching change significantly in the complexes. These data argue against any large-scale changes in protein secondary or tertiary structure accompanying ligand binding. The change in tyrosine fluorescence intensity was used to determine the dissociation constants for the heparin and phytic acid complexes of arrestin. The only change observed was a saturable diminution of tyrosine fluorescence signal from the protein. For both ligands, the data suggest two distinct binding interactions with the protein--a high--affinity interaction with Kd between 200 and 300 nM, and a lower affinity interaction with Kd between 2 and 8 microM. Study of collisional quenching of tyrosine fluorescence in free arrestin and the ligand-replete complexes indicates that 10 of the 14 tyrosine residues of the protein are solvent-exposed in the free protein; this value drops to between 5 and 6 solvent-exposed residues in the high-affinity complexes of the two ligands. These data suggest that ligand binding leads to direct occlusion of between 4 and 5 tyrosine residues on the solvent-exposed surface of the protein, but not to any large-scale changes in protein structure. The large activation energy previously reported to be associated with arrestin-receptor interactions may therefore reflect localized movements of the N- and C-termini of arrestin, which are proposed to interact in the free protein through electrostatic interactions. Binding of the anionic ligands heparin, phytic acid, or phosphorylated rhodopsin may compete with the C-terminus of arrestin for these electrostatic interactions, thus allowing the C-terminus to swing out of the binding region.     

Adhesive interactions between medically important yeasts and bacteria

Yeasts are being increasingly identified as important organisms in human infections. Adhesive interactions between yeasts and bacteria may contribute to yeast retention at body sites. Methods for studying adhesive interactions between bacterial strains are well known, and range from simple macroscopic methods to flow chamber systems with complex image analysis capabilities. The adhesive interactions between bacteria and yeasts have been studied employing several of the methods originally developed for studying adhesive interactions between bacteria. However, in many of the methods employed the larger size of the yeasts as compared with bacteria results in strong sedimentation of the yeasts, often invalidating the method adapted. In addition, most methods are semi-quantitative and do not properly control mass transport. Consequently, adhesive interaction mechanisms between yeasts and bacteria identified hitherto, including lectin binding and protein-protein interactions, must be regarded with caution. Extensive physico-chemical characteristics of yeast cell surfaces are not available and a physico-chemical mechanism has not yet been put forth. A new method for quantifying adhesive interactions between yeasts and bacteria is proposed, based on the use of a parallel plate flow chamber, in which the influence of adhering bacteria upon the kinetics of yeast adhesion and aggregation of the adhering yeasts is quantitatively evaluated, under carefully controlled mass transport.     

Perceptual interactions between musical pitch and timbre.

Three experiments examined perceptual interactions between musical pitch and timbre. Exp 1, through the use of the Garner classification tasks, found that pitch and timbre of isolated tones interact. Classification times showed interference from uncorrelated variation in the irrelevant attribute and facilitation from correlated variation; the effects were symmetrical. Exps 2 and 3 examined how musical pitch and timbre function in longer sequences. In recognition memory tasks, a target tone always appeared in a fixed position in the sequences, and listeners were instructed to attend to either its pitch or its timbre. For successive tones, no interactions between timbre and pitch were found. That is, changing the pitches of context tones did not affect timbre recognition, and vice versa. The tendency to perceive pitch in relation to other context pitches was strong and unaffected by whether timbre was constant or varying. In contrast, the relative perception of timbre was weak and was found only when pitch was constant. These results suggest that timbre is perceived more in absolute than in relative terms. Perceptual implications for creating patterns in music with timbre variations are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Inhibitory interactions between appetitive and aversive stimuli.

Reviews the behavioral evidence that a stimulus of a given affective value will exert a central inhibitory influence on responding maintained by stimuli of the opposite affective value. The effects of aversive stimuli on appetitively motivated behavior and of appetitive stimuli on aversively motivated behavior are considered separately. The evidence for a true inhibitory action is evaluated in terms of 3 behavioral criteria: the summation, retardation, and counterconditioning tests. Special attention is paid to the role of peripheral response interactions in determining the outcome of these tests. Although aversive stimuli meet all 3 criteria as inhibitors of appetitive behavior, the evidence that appetitive stimuli inhibit aversively motivated behavior is far less consistent. The strongest evidence for the inhibitory effect of appetitive stimuli comes from studies attempting to countercondition the reinforcing properties of aversive stimuli. It is concluded that this line of research supports general motivational theories that argue for the functional equivalence of excitors and inhibitors of opposite affective value. (4? p ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Inhibitory interactions between perigeniculate GABAergic neurons

Perigeniculate neurons form an interactive sheet of cells that inhibit one another as well as thalamocortical neurons in the dorsal lateral geniculate nucleus (LGNd). The inhibitory influence of the GABAergic neurons of the perigeniculate nucleus (PGN) onto other PGN neurons was examined with intracellular recordings in vitro. Intracellular recordings from PGN neurons during the generation of spindle waves revealed barrages of EPSPs and IPSPs. The excitation of local regions of the PGN with the local application of glutamate resulted in activation of IPSPs in neighboring PGN neurons. These IPSPs displayed an average reversal potential of -77 mV and were blocked by application of bicuculline methiodide or picrotoxin, indicating that they are mediated by GABAA receptors. In the presence of GABAA receptor blockade, the activation of PGN neurons with glutamate could result in slow IPSPs that were mediated by GABAB receptors in a subset (40%) of cells. Similarly, application of specific agonists muscimol and baclofen demonstrated that PGN neurons possess both functional GABAA and GABAB receptors. Examination of the axon arbors of biocytin-filled PGN neurons often revealed the presence of beaded axon collaterals within the PGN, suggesting that this may be an anatomical substrate for PGN to PGN inhibition. Functionally, activation of inhibition between PGN neurons could result in a shortening or a complete abolition of the low threshold Ca2+ spike or an inhibition of tonic discharge. We suggest that the mutual inhibition between PGN neurons forms a mechanism by which the excitability of these cells is tightly controlled. The activation of a point within the PGN may result in the inhibition of neighboring PGN neurons. This may be reflected in the LGNd as a center of inhibition surrounded by an annulus of disinhibition, thus forming a "center-surround" mechanism for thalamic function.     

Identification of multiple sites of interaction between heparin and the complement system

Many diverse effects of heparin on the complement system have been reported. In only a few cases have the sites or the mechanisms of these effects been identified. In order to understand these results we sought to comprehensively analyze which complement proteins interact with heparin and which do not. Purified components of the classical, alternative and terminal pathways of complement were radiolabeled and their affinity for heparin determined. Affinity chromatography of normal human serum on heparin-agarose allowed a complete analysis of complement proteins and confirmed the results obtained with radiolabeled purified components. Of the 22 complement proteins examined, 13 bound heparin (C1q, C2, C4, C4bp, C1INH, B, D, H, P, C6, C8, C9, and vitronectin) while 9 did not bind heparin (C1r, C1s, C3, Factor I, C5, C7, C3b, Ba and Bb). These observations help explain the many effects heparin has on the complement system and they identify the proteins which need to be examined in order to explain these effects.     

Imidazole binding to horse metmyoglobin: dependence upon pH and ionic strength

A series of substituted chalcone oxides (1,3-diphenyl-2-oxiranyl propanones) and structural analogs was synthesized to investigate the mechanism by which they inhibit soluble epoxide hydrolases (sEH). The inhibitor potency and inhibition kinetics were evaluated using both murine and human recombinant sEH. Inhibition kinetics were well described by the kinetic models of A. R. Main (1982, in Introduction to Biochemical Toxicology, pp. 193-223, Elsevier, New York) supporting the formation of a covalent enzyme-inhibitor intermediate with a half-life inversely proportional to inhibitor potency. Structure-activity relationships describe active-site steric constraints and support a mechanism of inhibition consistent with the electronic stabilization of the covalent enzyme-inhibitor intermediate. The electronic effects induced by altering the ketone functionality and the para-substitution of the phenyl attached to the epoxy C1 (i.e., the alpha-carbon) had the greatest influence on inhibitor potency. The direction of the observed influence was reversed for the inhibitory potency of glycidol (1-phenyl-2-oxiranylpropanol) derivatives. Recent insights into the mechanism of epoxide hydrolase activity are combined with these experimental results to support a proposed mechanism of sEH inhibition by chalcone oxides.     

Studies on the Interaction between Yttrium and Human Erythrocyte Membrane

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