Analysis of photosystem II electron transfer with natural PsbA-variants by redox polymer/protein biophotoelectrochemistry

Photosynthetica - Redox polymer/protein biophotoelectrochemistry was used to analyse forward electron transfer of isolated PSII complexes with natural PsbA-variants. PsbA1- or PsbA3-PSII was...     

The association of insulin resistance with serum osteoprotegerin in obese adolescents

Some data indicate a potential relationship between insulin resistance level and the concentration of osteoprotegerin (OPG) in the body. There have been few studies concerning OPG level and its relationship with insulin resistance and body composition in young people. The aim of this study was to assess serum osteoprotegerin concentration in obese adolescents, and to evaluate its potential association with insulin resistance. Seventy-eight obese adolescents and 20 healthy volunteers aged 12–18 years were recruited in the study. Selected anthropometrical measurements and blood biochemical analyses were performed. Insulin resistance in the participants was evaluated according to the homeostasis model assessment-insulin resistance (HOMA-IR) protocol. Level of OPG was assessed in serum. Obese subjects had significantly higher HOMA-IR indices and OPG levels in serum than the control group. A significant positive correlation between OPG and insulin resistance was found. It was observed that high concentrations of osteoprotegerin are associated with insulin resistance in obese adolescents.     

Synthesis of cyclooctapeptides: constraints analogues of the peptidic neurotoxin, ω‐agatoxine IVB—an experimental point of view

ω‐AGA IVB is an important lead structure when considering the design of effectors of glutamate release inducting P/Q‐type calcium channels. The best route to achieve the analogues possessing the three‐dimensional arrangement corresponding to the native binding loop was the introduction of constraint by ring formation via side chain to side chain lactamization for suitably protected Lys and Glu residues. Since tryptophane residue located at position 14 of this neuropeptide has been suggested as essential for binding, analogues in which this amino acid was replaced by aza‐tryptophane and alanine were synthesized. The synthesis was carried out on various acid‐labile resins (BARLOS chlorotrityl, Rink amide, PEG‐based or Wang resins), by Fmoc strategy. In this paper, we describe optimization of the peptide cyclization with various protecting groups, and on resin or in solution cyclization experimental parameters. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.     

ADP-Ribosylation of Brain Neuronal Proteins Is Altered by In Vitro and In Vivo Exposure to Inorganic Mercury

Abstract: ADP-ribosylation is an essential process in the metabolism of brain neuronal proteins, including the regulation of assembly and disassembly of biological polymers. Here, we examine the effect of HgCl₂ exposure on the ADP-ribosylation of tubulin and actin, both cytoskeletal proteins also found in neurons, and B-50/43-kDa growth-associated protein (B-50/GAP-43), a neuronal tissue-specific phosphoprotein. In rats we demonstrate, with both in vitro and in vivo experiments, that HgCl₂ markedly inhibits the ADP-ribosylation of tubulin and actin. This is direct quantitative evidence that HgCl₂, a toxic xenobiotic, alters specific neurochemical reactions involved in maintaining brain neuron structure.     

Structural and biochemical studies of a plant formamidopyrimidine-DNA glycosylase reveal why eukaryotic Fpg glycosylases do not excise 8-oxoguanine

Formamidopyrimidine-DNA glycosylase (Fpg; MutM) is a DNA repair enzyme widely distributed in bacteria. Fpg recognizes and excises oxidatively modified purines, 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxoguanine (8-oxoG), with similar excision kinetics. It exhibits some lesser activity toward 8-oxoadenine. Fpg enzymes are also present in some plant and fungal species. The eukaryotic Fpg homologs exhibit little or no activity on DNA containing 8-oxoG, but they recognize and process its oxidation products, guanidinohydantoin (Gh) and spiroiminohydantoin (Sp). To date, several structures of bacterial Fpg enzymes unliganded or in complex with DNA containing a damaged base have been published but there is no structure of a eukaryotic Fpg. Here we describe the first crystal structure of a plant Fpg, Arabidopsis thaliana (AthFpg), unliganded and bound to DNA containing an abasic site analog, tetrahydrofuran (THF). Although AthFpg shares a common architecture with other Fpg glycosylases, it harbors a zincless finger, previously described in a subset of Nei enzymes, such as human NEIL1 and Mimivirus Nei1. Importantly the "αF-β9/10 loop" capping 8-oxoG in the active site of bacterial Fpg is very short in AthFpg. Deletion of a segment encompassing residues 213-229 in Escherichia coli Fpg (EcoFpg) and corresponding to the "αF-β9/10 loop" does not affect the recognition and removal of oxidatively damaged DNA base lesions, with the exception of 8-oxoG. Although the exact role of the loop remains to be further explored, it is now clear that this protein segment is specific to the processing of 8-oxoG.     

Reevaluating the CD8 T-Cell Response to Herpes Simplex Virus Type 1: Involvement of CD8 T Cells Reactive to Subdominant Epitopes

In C57BL/6 (B6) mice, most herpes simplex virus (HSV)-specific CD8 T cells recognize a strongly immunodominant epitope on glycoprotein B (gB₄₉₈) and can inhibit HSV type 1 (HSV-1) reactivation from latency in trigeminal ganglia (TG). However, half of the CD8 T cells retained in latently infected TG of B6 mice are not gB₄₉₈ specific and have been largely ignored. The following observations from our current study indicate that these gB₄₉₈-nonspecific CD8 T cells are HSV specific and may contribute to the control of HSV-1 latency. First, following corneal infection, OVA₂₅₇-specific OT-1 CD8 T cells do not infiltrate the infected TG unless mice are simultaneously immunized with OVA₂₅₇ peptide, and then they are not retained. Second, 30% of CD8 T cells in acutely infected TG that produce gamma interferon in response to HSV-1 stimulation directly ex vivo are gB₄₉₈ nonspecific, and these cells maintain an activation phenotype during viral latency. Finally, gB₄₉₈-nonspecific CD8 T cells are expanded in ex vivo cultures of latently infected TG and inhibit HSV-1 reactivation from latency in the absence of gB₄₉₈-specific CD8 T cells. We conclude that many of the CD8 T cells that infiltrate and are retained in infected TG are HSV specific and potentially contribute to maintenance of HSV-1 latency. Identification of the viral proteins recognized by these cells will contribute to a better understanding of the dynamics of HSV-1 latency.The generation and maintenance of a CD8 T-cell response represent an important line of defense against many viral pathogens. Such responses are typically initiated when host antigen-presenting cells at the site of infection capture and process viral proteins and transport them to local draining lymph nodes (DLN). There the antigen-presenting cells either directly present viral antigens to naïve CD8 T cells or pass them to a distinct LN-resident dendritic cell (DC) subset for antigen presentation in the context of major histocompatibility complex class I (1). Antigen-specific CD8 T cells then undergo robust division and differentiation into effector populations armed to infiltrate infected tissue and eliminate the invading pathogen. The magnitude of the CD8 T-cell response against different viral epitopes is typically aligned within a defined hierarchy. Those epitopes recognized by the largest portion of the pathogen-specific CD8 T-cell population are referred to as immunodominant, while those inciting lesser responses are referred to as subdominant (17). Manipulation of this hierarchal system by the elimination of an immunodominant epitope often results in the expansion of a normally silent or “cryptic” determinant (2, 17, 21).Although the HSV-1 genome contains at least 84 open reading frames (13), it is estimated that 70 to 95% of the acute CD8 T-cell response in lymphoid organs of B6 mice is directed against the single immunodominant gB₄₉₈ epitope (11, 21, 24, 26, 27). The remaining HSV-specific CD8 T cells are thought to be directed against a subdominant epitope on the viral ribonucleotide reductase (RR1₈₂₂) (16). These conclusions are derived from studies characterizing the specificity of CD8 T cells at the peak of the effector response in lymphoid tissue. Interestingly, a recombinant HSV-1 lacking the immunodominant gB₄₉₈ epitope induced an HSV-specific CD8 T-cell response of normal magnitude, while the RR1₈₂₂ epitope remained subdominant (21), suggesting the emergence of previously unrecognized or cryptic epitopes.Following HSV-1 corneal infection of B6 mice, virus is transmitted to the trigeminal ganglia (TG), where it replicates briefly (up to 6 days postinfection [dpi]) and then establishes a latent infection. CD8 effector T cells accumulate to peak levels in the TG by 8 dpi and then undergo contraction, and then a memory population of constant size is maintained for the life of the animal. While 50% of both the effector and memory CD8 T-cell populations are specific for the immunodominant gB₄₉₈ epitope (11, 18), the remaining TG-resident CD8 T cells are specific for neither the dominant gB₄₉₈ nor the subdominant RR1₈₂₂ epitope. Although the phenotype and function of the gB₄₉₈-specific CD8 T cells in sensory ganglia and their role in maintaining HSV-1 latency have been well characterized (3, 5, 9, 11, 12, 14, 18, 19, 22, 24, 25, 27), the properties of the gB₄₉₈-nonspecific TG-resident CD8 T-cell population and their role in maintaining viral latency remain unexplored. Here we demonstrate that many of the gB₄₉₈-nonspecific CD8 T cells in latently infected TG proliferate and some produce gamma interferon (IFN-γ) when stimulated with HSV-1 antigens directly ex vivo. These cells also persistently exhibit an activation phenotype within latently infected TG, are expanded in ex vivo cultures of latently infected TG, and can block HSV-1 reactivation in TG neurons in the absence of gB₄₉₈-specific CD8 T cells.     

Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation

An 11.7-A-resolution cryo-EM map of the yeast 80S.eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin-ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S.eEF2.sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.     

Strain fidelity of chronic wasting disease upon murine adaptation

Chronic wasting disease (CWD), a prion disease of deer and elk, is highly prevalent in some regions of North America. The establishment of mouse-adapted CWD prions has proven difficult due to the strong species barrier between mice and deer. Here we report the efficient transmission of CWD to transgenic mice overexpressing murine PrP. All mice developed disease 500 +/- 62 days after intracerebral CWD challenge. The incubation period decreased to 228 +/- 103 days on secondary passage and to 162 +/- 6 days on tertiary passage. Mice developed very large, radially structured cerebral amyloid plaques similar to those of CWD-infected deer and elk. PrP(Sc) was detected in spleen, indicating that murine CWD was lymphotropic. PrP(Sc) glycoform profiles maintained a predominantly diglycosylated PrP pattern, as seen with CWD in deer and elk, across all passages. Therefore, all pathological, biochemical, and histological strain characteristics of CWD appear to persist upon repetitive serial passage through mice. These findings indicate that the salient strain-specific properties of CWD are encoded by agent-intrinsic components rather than by host factors.     

Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.