Obraz ponowoczesnego świata z perspektywy współczesnej młodzieży

Modern reality is ambivalent, heterogenous, ambiguous and unpredictable. The living conditions it creates aren’t easy for adolescing people forced to realize many developmental tasks. Among theese tasks is forming a mature vision of reality. This article presents the postmodern view of the world from the perspective of young people, based on research conducted on high school graduates. It shows young people as very reflective, critical towards certain aspects of postmodernity but at the same time, due to highly developed adaptational skills, able to find their place in the world and make use of its possibilities

Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA

Surface plasmon resonance imaging of cells and surface-associated fibronectin

<p>Abstract</p> <p>Background</p> <p>A critical challenge in cell biology is quantifying the interactions of cells with their extracellular matrix (ECM) environment and the active remodeling by cells of their ECM. Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. A label-free imaging method would provide an alternative that would eliminate the requirement of transfected cells and modified biological molecules, and if collected nondestructively, would allow long term observation and analysis of live cells.</p> <p>Results</p> <p>Using surface plasmon resonance imaging (SPRI), the deposition of protein by vascular smooth muscle cells (vSMC) cultured on fibronectin was quantified as a function of cell density and distance from the cell periphery. We observed that as much as 120 ng/cm²of protein was deposited by cells in 24 h.</p> <p>Conclusion</p> <p>SPRI is a real-time, low-light-level, label-free imaging technique that allows the simultaneous observation and quantification of protein layers and cellular features. This technique is compatible with live cells such that it is possible to monitor cellular modifications to the extracellular matrix in real-time.</p

Structure-Based Stabilization of HIV-1 gp120 Enhances Humoral Immune Responses to the Induced Co-Receptor Binding Site

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions) into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region

Soluble CD4 and CD4-Mimetic Compounds Inhibit HIV-1 Infection by Induction of a Short-Lived Activated State

Binding to the CD4 receptor induces conformational changes in the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein. These changes allow gp120 to bind the coreceptor, either CCR5 or CXCR4, and prime the gp41 transmembrane envelope glycoprotein to mediate virus–cell membrane fusion and virus entry. Soluble forms of CD4 (sCD4) and small-molecule CD4 mimics (here exemplified by JRC-II-191) also induce these conformational changes in the HIV-1 envelope glycoproteins, but typically inhibit HIV-1 entry into CD4-expressing cells. To investigate the mechanism of inhibition, we monitored at high temporal resolution inhibitor-induced changes in the conformation and functional competence of the HIV-1 envelope glycoproteins that immediately follow engagement of the soluble CD4 mimics. Both sCD4 and JRC-II-191 efficiently activated the envelope glycoproteins to mediate infection of cells lacking CD4, in a manner dependent on coreceptor affinity and density. This activated state, however, was transient and was followed by spontaneous and apparently irreversible changes of conformation and by loss of functional competence. The longevity of the activated intermediate depended on temperature and the particular HIV-1 strain, but was indistinguishable for sCD4 and JRC-II-191; by contrast, the activated intermediate induced by cell-surface CD4 was relatively long-lived. The inactivating effects of these activation-based inhibitors predominantly affected cell-free virus, whereas virus that was prebound to the target cell surface was mainly activated, infecting the cells even at high concentrations of the CD4 analogue. These results demonstrate the ability of soluble CD4 mimics to inactivate HIV-1 by prematurely triggering active but transient intermediate states of the envelope glycoproteins. This novel strategy for inhibition may be generally applicable to high–potential-energy viral entry machines that are normally activated by receptor binding

Protection in Macaques Immunized with HIV-1 Candidate Vaccines Can Be Predicted Using the Kinetics of Their Neutralizing Antibodies

A vaccine is needed to control the spread of human immunodeficiency virus type 1 (HIV-1). An in vitro assay that can predict the protection induced by a vaccine would facilitate the development of such a vaccine. A potential candidate would be an assay to quantify neutralization of HIV-1.We have used sera from rhesus macaques that have been immunized with HIV candidate vaccines and subsequently challenged with simian human immunodeficiency virus (SHIV). We compared neutralization assays with different formats. In experiments with the standardized and validated TZMbl assay, neutralizing antibody titers against homologous SHIV(SF162P4) pseudovirus gave a variable correlation with reductions in plasma viremia levels. The target cells used in the assays are not just passive indicators of virus infection but are actively involved in the neutralization process. When replicating virus was used with GHOST cell assays, events during the absorption phase, as well as the incubation phase, determine the level of neutralization. Sera that are associated with protection have properties that are closest to the traditional concept of neutralization: the concentration of antibody present during the absorption phase has no effect on the inactivation rate. In GHOST assays, events during the absorption phase may inactivate a fixed number, rather than a proportion, of virus so that while complete neutralization can be obtained, it can only be found at low doses particularly with isolates that are relatively resistant to neutralization.Two scenarios have the potential to predict protection by neutralizing antibodies at concentrations that can be induced by vaccination: antibodies that have properties close to the traditional concept of neutralization may protect against a range of challenge doses of neutralization sensitive HIV isolates; a window of opportunity also exists for protection against isolates that are more resistant to neutralization but only at low challenge doses

Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides

Structure of S. aureus HPPK and the Discovery of a New Substrate Site Inhibitor

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, Kd, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC50 of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and 15N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including 15N-1H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway

Contribution of Intrinsic Reactivity of the HIV-1 Envelope Glycoproteins to CD4-Independent Infection and Global Inhibitor Sensitivity

Human immunodeficiency virus (HIV-1) enters cells following sequential activation of the high-potential-energy viral envelope glycoprotein trimer by target cell CD4 and coreceptor. HIV-1 variants differ in their requirements for CD4; viruses that can infect coreceptor-expressing cells that lack CD4 have been generated in the laboratory. These CD4-independent HIV-1 variants are sensitive to neutralization by multiple antibodies that recognize different envelope glycoprotein epitopes. The mechanisms underlying CD4 independence, global sensitivity to neutralization and the association between them are still unclear. By studying HIV-1 variants that differ in requirements for CD4, we investigated the contribution of CD4 binding to virus entry. CD4 engagement exposes the coreceptor-binding site and increases the “intrinsic reactivity” of the envelope glycoproteins; intrinsic reactivity describes the propensity of the envelope glycoproteins to negotiate transitions to lower-energy states upon stimulation. Coreceptor-binding site exposure and increased intrinsic reactivity promote formation/exposure of the HR1 coiled coil on the gp41 transmembrane glycoprotein and allow virus entry upon coreceptor binding. Intrinsic reactivity also dictates the global sensitivity of HIV-1 to perturbations such as exposure to cold and the binding of antibodies and small molecules. Accordingly, CD4 independence of HIV-1 was accompanied by increased susceptibility to inactivation by these factors. We investigated the role of intrinsic reactivity in determining the sensitivity of primary HIV-1 isolates to inhibition. Relative to the more common neutralization-resistant (“Tier 2-like”) viruses, globally sensitive (“Tier 1”) viruses exhibited increased intrinsic reactivity, i.e., were inactivated more efficiently by cold exposure or by a given level of antibody binding to the envelope glycoprotein trimer. Virus sensitivity to neutralization was dictated both by the efficiency of inhibitor/antibody binding to the envelope glycoprotein trimer and by envelope glycoprotein reactivity to the inhibitor/antibody binding event. Quantitative differences in intrinsic reactivity contribute to HIV-1 strain variability in global susceptibility to neutralization and explain the long-observed relationship between increased inhibitor sensitivity and decreased entry requirements for target cell CD4