Underwhelming the immune response: effect of slow virus growth on CD8+-T-lymphocyte responses

The speed of virus replication has typically been seen as an advantage for a virus in overcoming the ability of the immune system to control its population growth. Under some circumstances, the converse may also be true: more slowly replicating viruses may evoke weaker cellular immune responses and therefore enhance their likelihood of persistence. Using the model of lymphocytic choriomeningitis virus (LCMV) infection in mice, we provide evidence that slowly replicating strains induce weaker cytotoxic-T-lymphocyte (CTL) responses than a more rapidly replicating strain. Conceptually, we show a "bell-shaped" relationship between the LCMV growth rate and the peak CTL response. Quantitative analysis of human hepatitis C virus infections suggests that a reduction in virus growth rate between patients during the incubation period is associated with a spectrum of disease outcomes, from fulminant hepatitis at the highest rate of viral replication through acute resolving to chronic persistence at the lowest rate. A mathematical model for virus-CTL population dynamics (analogous to predator [CTL]-prey [virus] interactions) is applied in the clinical data-driven analysis of acute hepatitis B virus infection. The speed of viral replication, through its stimulus of host CTL responses, represents an important factor influencing the pathogenesis and duration of virus persistence within the human host. Viruses with lower growth rates may persist in the host because they "sneak through" immune surveillance.     

Detection of aptamer-protein interactions using QCM and electrochemical indicator methods

We report novel method of detection thrombin-aptamer interaction based on measurement the charge consumption from the electrode covered by DNA aptamers to an electrochemical indicator methylene blue (MB), that is bounded to a thrombin. The binding of thrombin to an aptamers has been detected also by QCM method in flow measuring cell. We showed that using MB it is possible to detect thrombin with high sensitivity and selectivity.     

Hierarchy of simulation models in predicting molecular recognition mechanisms from the binding energy landscapes: structural analysis of the peptide complexes with SH2 domains.

Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature-dependent binding free energy landscapes. Once some native peptide-SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel-like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide-SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C-terminal to the pTyr group. The topological features of the peptide-protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino-terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy-terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide-SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region.     

The photoproduction of superoxide radicals and the superoxide dismutase activity of Photosystem II. The possible involvement of cytochrome b559

In the present study the light induced formation of superoxide and intrinsic superoxide dismutase (SOD) activity in PS II membrane fragments and D1/D2/Cytb559-complexes from spinach have been analyzed by the use of ferricytochrome c (cyt c(III)) reduction and xanthine/xanthine oxidase as assay systems. The following results were obtained: 1.) Photoreduction of Cyt c (III) by PS II membrane fragments is induced by addition of sodium azide, tetracyane ethylene (TCNE) or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP) and after removal of the extrinsic polypeptides by a 1M CaCl₂-treatment. This activity which is absent in control samples becomes completely inhibited by the addition of exogenous SOD. 2.) The TCNE induced cyt c(III) photoreduction by PS II membrane fragments was found to be characterized by a half maximal concentration of c_1/2=10 M TCNE. Simultaneously, TCNE inhibits the oxygen evolution rate of PS II membrane fragments with c_1/2 3 M. 3.) The photoproduction of O₂ ^– is coupled with H⁺-uptake. This effect is diminished by the addition of the O₂ ^–-trap cyt c(III). 4.) D1/D2/Cytb559-complexes and PS II membrane fragments deprived of the extrinsic proteins and manganese exhibit no SOD-activity but are capable of producing O₂ ^– in the light if a PS II electron donor is added.Based on these results the site(s) of light induced superoxide formation in PS II is (are) inferred to be located at the acceptor side. A part of the PS II donor side and Cyt b559 in its HP-form are proposed to provide an intrinsic superoxide dismutase (SOD) activity.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - ANT-2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - BCP bromocresol purple - cyt cytochrome - Cyt c cytochrome c - DCIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DEDTC Diethyldithiocarbamate - DMBQ 2,5-dimethyl-p-benzoquinone - DPC 1,5-diphenylcarbazide - FCCP carbonylcyanide-p-trifluoro/methoxy-phenylhydrazone - HP high potential - LP low potential - MES 2-(N-morpholino)ethanesulfonic acid - NADP nicotinamide adenine dinucleotide phosphate - SOD superoxide dismutase - TCNE tetracyane ethylene - TEMED N,N,N,N-tetramethylethylenediamine     

Desiccation tolerant lichens facilitate in vivo H/D isotope effect measurements in oxygenic photosynthesis

We have used the desiccation-tolerant lichen Flavoparmelia caperata, containing the green algal photobiont Trebouxia gelatinosa, to examine H/D isotope effects in Photosystem II in vivo. Artifact-free H/D isotope effects on both PSII primary charge separation and water oxidation yields were determined as a function of flash rate from chlorophyll-a variable fluorescence yields. Intact lichens could be reversibly dehydrated/re-hydrated with H₂O/D₂O repeatedly without loss of O₂ evolution, unlike all isolated PSII preparations. Above a threshold flash rate, PSII charge separation decreases sharply in both D₂O and H₂O, reflecting loss of excitation migration and capture by PSII. Changes in H/D coordinates further slow charge separation in D₂O (?23% at 120?Hz), attributed to reoxidation of the primary acceptor Q_A^?. At intermediate flash rates (5–50?Hz) D₂O decreases water oxidation efficiency (O₂ evolution) by ?2–5%. No significant isotopic difference is observed at slow flash rates (<5?Hz) where charge recombination dominates. Slower D₂O diffusion, changes in hydrogen bonding networks, and shifts in the pK_a's of ionizable residues may all contribute to these systematic variations of H/D isotope effects. Lichens' reversible desiccation tolerance allows highly reproducible H/D exchange kinetics in PSII reactions to be studied in vivo for the first time.     

Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating residues. This study has outlined mechanisms by which inhibitor binding could modulate resilience and efficiency of allosteric interactions in the kinase structures, while preserving structural topology required for catalytic activity and regulation.     

New analysis of the Pleistocene carnivores from Petralona Cave (Macedonia, Greece) based on the Collection of the Thessaloniki Aristotle University

New taxonomic study of the “old collection” of Carnivora from Petralona Cave, associated to the well-known hominid skull, housed in the Geology School of the Thessaloniki Aristotle University since 1960, revealed 11 species (Canis arnensis, Lycaon lycaonoides, Vulpes praeglacialis, Ursus deningeri, U. spelaeus, U. arctos, Pliocrocuta perrieri, Pachycrocuta brevirostris, Crocuta crocuta, Panthera leo spelaea, and Felis silvestris), which are described in detail. The species composition is typical of the eastern part of the European Mediterranean and may be divided into three biostratigraphic assemblages: early Middle Pleistocene, late Middle Pleistocene and Late Pleistocene.     

Estimation of Uptake of Humic Substances from Different Sources by Escherichia coli Cells under Optimum and Salt Stress Conditions by Use of Tritium-Labeled Humic Materials

The primary goal of this paper is to demonstrate potential strengths of the use of tritium-labeled humic substances (HS) to quantify their interaction with living cells under various conditions. A novel approach was taken to study the interaction between a model microorganism and the labeled humic material. The bacterium Escherichia coli was used as a model microorganism. Salt stress was used to study interactions of HS with living cells under nonoptimum conditions. Six tritium-labeled samples of HS originating from coal, peat, and soil were examined. To quantify their interaction with E. coli cells, bioconcentration factors (BCF) were calculated and the amount of HS that penetrated into the cell interior was determined, and the liquid scintillation counting technique was used as well. The BCF values under optimum conditions varied from 0.9 to 13.1 liters kg⁻¹ of cell biomass, whereas under salt stress conditions the range of corresponding values increased substantially and accounted for 0.2 to 130 liters kg⁻¹. The measured amounts of HS that penetrated into the cells were 23 to 167 mg and 25 to 465 mg HS per kg of cell biomass under optimum and salt stress conditions, respectively. This finding indicated increased penetration of HS into E. coli cells under salt stress.Humic substances (HS) are natural organic compounds comprising 50 to 90% of the organic matter of peat, coal, and sapropel (i.e., sludge that accumulates at the bottom of lakes), as well as of the nonliving organic matter of soil and water ecosystems (9, 34, 53). Being the products of stochastic synthesis, HS are characterized as polydispersed substances having elemental compositions that are nonstoichiometric and structures which are irregular and heterogeneous. Thus, it is not possible to assign an exact structure to HS. Instead, they are operationally defined using a model structure predicated on available compositional, structural, functional, and behavioral data: a model structure containing all the same basic structural units and types of reactive functional groups (46). HS have been demonstrated to contain a large amount of residues resembling the original building blocks (aromatic subunits, amino acids, carbohydrates, etc.) (51) as well as polyphenolic components with nonhydrolyzable C-C and ether bonds (51, 16). Since humic matter is a complex mixture of organic substances, HS yield extremely high polydispersity values (i.e., the ratio of weight-average molecular weight to the number-average molecular weight [M_w/M_n]), which vary within the range of 1.64 to 4.40 (38). These extremely high polydispersity values mean that even though they yield relatively high values of molecular weight, HS contain a low-molecular-weight fraction.HS are known to play important roles in protecting microorganisms and higher plants from climatic and technogenic stresses, such as pollution, draught, UV irradiation, and pathogen and viral infections (2, 22, 31). However, mechanisms underlying protective functions of these natural systems are still poorly understood. The primary reason for that is a lack of experimental tools for tracking uptake and distribution of natural organic mixtures in living cells and tissues, which makes it extremely difficult to link structure and functions in systems of such high complexity. Besides, predicting HS behavior in biological systems is extremely arduous, as HS are complex mixtures with a number of concurrent properties, such as polyanionic and polyelectrolyte character, hydrophilic and hydrophobic moieties, different functional groups, etc.The most straightforward hypothesis is that the biological activity of HS depends on both hydrophobic and hydrophilic characteristics of structural components (56). This hypothesis implies that the biological effects of HS are connected to membrane activity (12, 44, 56). Sorption of HS onto cells is the best documented phenomenon; numerous studies include phytoplankton (7, 15, 36, 57), isolated fish gill cells (7), bacteria (13, 14, 27, 57), fungi (60), and plants (12, 31, 44). This suggests that the sorption of HS onto biological membranes is a general process, but very few quantitative estimates are available (13, 36). Moreover, the penetration of HS into the living cells is still questionable, and to the best of our knowledge, only one study has reported a direct estimate of HS uptake by microorganisms (10).The main complication that arises in the study of the interactions of HS with living cells is the lack of a reliable analytical technique for determination of HS in the presence of biomolecules (e.g., proteins, lipids, and saccharides). To overcome the problem, radioactive labeling of HS is being used widely for this purpose. However, the reported studies deal predominantly with synthetic rather than with native humic materials (49, 50, 58). This is because of the approaches used for the radioactive labeling of the humics used in those studies, which involve either composting of a labeled precursor (e.g., ¹⁴C-glucose) with a soil sample (17) or the synthesis of model polymeric compounds. The polymeric compounds either are synthesized by enzyme-mediated oxidative polymerization of phenolic compounds, which is initiated by adding H₂O₂ in the presence of horseradish peroxidase (19), or proceed spontaneously in the presence of oxygen or other oxidants at an alkaline pH (48). When phenolics are polymerized with nonaromatic precursors (e.g., proteins, peptides, amino acids, carbohydrates, and amino sugars), the resulting humic-like materials are very similar to natural HS (19). These methods can be used for producing both ¹⁴C- and ¹⁵N-labeled humic-like substances. Their substantial disadvantage is that the resulting materials are similar but not identical to natural HS. Given high structural heterogeneity and irregularity inherent within HS, the availability of a broad set of labeled humic materials identical to their natural counterparts is a prerequisite for disclosing the mechanism of their interactions with living organisms on the cellular and organismal levels.The goal of this work was to study the behavior of humics in a simple microbial system using humic substances from various natural sources. For this purpose, six natural humic materials with different molecular features and properties were isolated from various natural sources and then labeled with tritium using a technique developed in previous studies (3, 4). A strain of the well-studied bacterium Escherichia coli was used as a model bacterial culture. The molecular weight of humics is quite high and is generally considered to be the leading factor restricting their uptake by living cells. In view of our study, prokaryotic bacteria seemed to be the most appropriate model, as recent evidence suggests that size differences among eukaryotic homologues of integral membrane proteins are consistently larger than their bacterial counterparts (8).To demonstrate the potential strengths of using tritium-labeled HS for biological study, uptake of HS by bacterial cells under stress conditions was also studied. One way in which bacteria respond to environmental change is to regulate cell membrane permeability. Thus, another goal of this work was to monitor changes in HS-bacterium interactions under salt stress conditions. For quantitative indicators, bioconcentration factors, maximum adsorption, and the amount of HS that penetrated into the cell interior were used.     

Phosphorylation inhibits turnover of the tau protein by the proteasome: influence of RCAN1 and oxidative stress

Hyperphosphorylated tau proteins accumulate in the paired helical filaments of neurofibrillary tangles seen in such tauopathies as Alzheimer's disease. In the present paper we show that tau turnover is dependent on degradation by the proteasome (inhibited by MG132) in HT22 neuronal cells. Recombinant human tau was rapidly degraded by the 20 S proteasome in vitro, but tau phosphorylation by GSK3beta (glycogen synthase kinase 3beta) significantly inhibited proteolysis. Tau phosphorylation was increased in HT22 cells by OA [okadaic acid; which inhibits PP (protein phosphatase) 1 and PP2A] or CsA [cyclosporin A; which inhibits PP2B (calcineurin)], and in PC12 cells by induction of a tet-off dependent RCAN1 transgene (which also inhibits PP2B). Inhibition of PP1/PP2A by OA was the most effective of these treatments, and tau hyperphosphorylation induced by OA almost completely blocked tau degradation in HT22 cells (and in cell lysates to which purified proteasome was added) even though proteasome activity actually increased. Many tauopathies involve both tau hyperphosphorylation and the oxidative stress of chronic inflammation. We tested the effects of both cellular oxidative stress, and direct tau oxidative modification in vitro, on tau proteolysis. In HT22 cells, oxidative stress alone caused no increase in tau phosphorylation, but did subtly change the pattern of tau phosphorylation. Tau was actually less susceptible to direct oxidative modification than most cell proteins, and oxidized tau was degraded no better than untreated tau. The combination of oxidative stress plus OA treatment caused extensive tau phosphorylation and significant inhibition of tau degradation. HT22 cells transfected with tau-CFP (cyan fluorescent protein)/tau-GFP (green fluorescent protein) constructs exhibited significant toxicity following tau hyperphosphorylation and oxidative stress, with loss of fibrillar tau structure throughout the cytoplasm. We suggest that the combination of tau phosphorylation and tau oxidation, which also occurs in tauopathies, may be directly responsible for the accumulation of tau aggregates.     

First evidence of miniature transposable elements in sponges (Porifera)

Transposable elements play a vital role in genome evolution and may have been important for the formation of the early metazoan genome, but only little is known about transposons at this interface between unicellular opisthokonts and Metazoa. Here, we describe the first miniature transposable elements (MITEs, Queen1 and Queen2) in sponges. Queen1 and Queen2 are probably derived from Tc1/mariner-like MITE families and are represented in more than 3,800 and 1,700 copies, respectively, in the Amphimedon queenslandica genome. Queen elements are located in intergenic regions as well as in introns, providing the potential to induce new splicing sites and termination signals in the genes. Further possible impacts of MITEs on the evolution of the metazoan genome are discussed.