Examination of the kinetics of herpes simplex virus glycoprotein D binding to the herpesvirus entry mediator, using surface plasmon resonance

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (KD) of 3.2 x 10(-6) M and gD2(306t) had a KD of 1.5 x 10(-6) M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Delta290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (KD = 3.3 x 10(-8) M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Delta290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.     

The regulation of mouse liver ornithine decarboxylase by metabolites

The enzyme ornithine decarboxylase (L-Ornithine carboxy-lyase, EC 4.1.1.17), has been partially purified from the livers of mice subjected to partial hepatectomy (6-8 h previously). Mouse liver ornithine decarboxylase requires pyridoxal phosphate, and dithiothreitol for maximal activity. The enzyme has a pH optimum of 7.3, it is inhibited in the presence of 0.3 M phosphate, glycine, Tricine and Tris. It shows no dependence on metal ions and is inhibited by high salt concentrations, particularly ammonium salts. The kinetics of the enzyme have been studied with putrescine (and analogs), spermidine and spermine, in the presence of both high and low levels of pyridoxal phosphate. High concentrations of pyridoxal phosphate inhibit the enzyme. The enzyme is also inhibited by low concentrations of putrescine (1 mM). As the concentration of putrescine increased to 10 mM, non-competitive inhibition was observed, this could be reversed by addition of higher levels of pyridoxal phosphate. Spermidine and spermine inhibit (noncompetitively) only at high concentrations (10 mM). Ornithine inhibits at high concentrations (2 mM). Spectral studies have shown that the observed kinetics of competitive inhibition at low concentrations of polyamine changing to noncompetitive inhibition at high polyamine concentrations are due to competition between enzyme and substrate (or inhibitor) for free (non-enzyme bound) pyridoxal phosphate. Noncompetitive inhibition arises through the formation of transient Schiff base complexes between amines and free pyridoxal phosphate. It also appears that the binding of substrate to the active site takes place through Schiff base formation with enzyme bound pyridoxal phosphate.     

Linguistic and location effects in compliance with pesticide warning labels for amateur and professional users

Three studies explored amateur and professional users' compliance with pesticide warning labels. Professionals were classified as people working in a profession in which the use of pesticides is a necessary part of their job. Amateurs used pesticides only in their leisure time. The first study showed that the wording used affected perception of the appropriateness of hazard statements, one of the most effective variations being the use of the personal pronoun (statements beginning "You should..."). The location of warning information was also found to affect actual compliance: Compliance increased when warning information was presented in the directions for use section. A supplemental directive increased compliance only for professional users. In a final study, "best-case" and "worst-case" linguistic variations were combined with best-case and worst-case locations for safety information. Instruction statements using the personal pronoun and presented in the directions for use section resulted in the highest levels of compliance. The differences in compliance between amateur and professional users are interpreted within the framework of Rasmussen's (1986) distinction among skill-, rule-, and knowledge-based behavior. Actual or potential applications of this research include the design of warning labels and safety information.     

Decoding two-dimensional complex multicomponent separations by autocovariance function

A new method for decoding two-dimensional (2D) multicomponent separations based on the use of the 2D Autocovariance function (2D-ACVF) has been developed. Theoretical models of single component (SC) spot distributions in 2D separations, both random and structured, are developed as the basis for a nonlinear estimation of both sample and separation system parameters from experimental 2D separations. The number of SCs, the average spot size, the spot capacity, and the saturation factor can be evaluated in the case of random SC spot patterns. The procedure was validated by extensive numerical simulation under conditions close to those usually found in GC x GC or 2D-polyacrylamid gel electrophoresis of proteins. The worse precision degree was no greater than 10% in the case of maximum spot density. This imprecision was fully accounted for, and it seems acceptable owing to the intrinsic statistical character of the estimation method. Structured multicomponent 2D separations, where SCs are linked by linear relationships, give rise to specific structured patterns in 2D-ACVF plots from which the parameters (phase and frequency) of the structured SC sequences can be evaluated: the study of 2D-ACVF makes it possible to decode multicomponent 2D separation, that is, to determine the number, relative abundance, and structural similarities of the single components. Pertinent expressions of the theoretical 2D-ACVF were derived for simple cases, and a procedure for decoding cases of structured 2D separations was developed and applied. It was shown that 2D separations containing both random and structured patterns of SC spots give rise to 2D-EACVF, which is the superimposition of the two component parts. This feature allows one, in principle, to decode the two components. The relevance of these results for Giddings sample dimensionality and separation dimensionality and their effective experimental evaluation is discussed.     

Fortran computer program for inextensional bending of a doubly curved shell triangular element

A Fortran computer program is described which calculates physical quantities for a class of shell triangular elements undergoing inextensional bending. These elements are in quadratic parametric representation and may have positive, zero or negative Gaussian curvature. The exact inextensional bending solutions for the rectangular displacement components are cubic in the surface co-ordinates and the curvature changes are relatively slowly varying.     

Vacuum metallurgy 1967—Part 2

The following is Part 2 of a 2-part review article of the International Conference on Vacuum Metallurgy. Part 1 appeared in the October issue, pages 35 to 39, and covered the first 3 topics of the conference: I—Plenary Session; II—Vacuum Equipment and Technology and Vacuum Metallurgy Operations; and III—Electron Beam Processing.     

A formal framework for connective stability of highly decentralized cooperative negotiations

Multiagent cooperative negotiation is a promising technique for modeling and controlling complex systems. Effective and flexible cooperative negotiations are especially useful for open complex systems characterized by high decentralization (which implies a low amount of exchanged information) and by dynamic connection and disconnection of agents. Applications include ad hoc network management, vehicle formation, and physiological model combination. To obtain an effective control action, the stability of the negotiation, namely the guarantee that an agreement will be eventually reached, is of paramount importance. However, the techniques usually employed for assessing the stability of a negotiation can be hardly applied in open scenarios. In this paper, whose nature is mainly theoretical, we make a first attempt towards engineering stable cooperative negotiations proposing a framework for their analysis and design. Specifically, we present a formal protocol for cooperative negotiations between a number of agents and we propose a criterion for negotiation stability based on the concept of connective stability. This is a form of stability that accounts for the effects of structural changes on the composition of a system and that appears very suitable for multiagent cooperative negotiations. To show its possible uses, we apply our framework for connective stability to some negotiations taken from literature.     

Children's differentiation between beliefs about matters of fact and matters of opinion.

Two experiments investigated children's implicit and explicit differentiation between beliefs about matters of fact and matters of opinion. In Experiment 1, 8- to 9-year-olds' (n = 88) explicit understanding of the subjectivity of opinions was found to be limited, but their conformity to others' judgments on a matter of opinion was considerably lower than their conformity to others' views regarding an ambiguous fact. In Experiment 2, children aged 6, 8, or 10 years (n = 81) were asked to make judgments either about ambiguous matters of fact or about matters of opinion and then heard an opposing judgment from an expert. All age groups conformed to the opposing judgments on factual matters more than they did to the experts' views on matters of opinion. However, only the oldest children explicitly recognized that opinions are subjective and cannot be "wrong." Implications of these results for models of children's reasoning about epistemic states are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)