Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

The role of zinc in the binding of killer cell Ig-like receptors to class I MHC proteins

The binding of killer cell Ig-like Receptors (KIR) to their Class I MHC ligands was shown previously to be characterized by extremely rapid association and dissociation rate constants. During experiments to investigate the biochemistry of receptor–ligand binding in more detail, the kinetic parameters of the interaction were observed to alter dramatically in the presence of Zn2+ but not other divalent cations. The basis of this phenomenon is Zn2+-induced multimerization of the KIR molecules as demonstrated by BIAcore, analytical ultracentrifugation, and chemical cross-linking experiments. Zn2+-dependent multimerization of KIR may be critical for formation of the clusters of KIR and HLA-C molecules, the “natural killer (NK) cell immune synapse,” observed at the site of contact between the NK cell and target cell.

Recent improvements in prediction of protein structure by global optimization of a potential energy function

Recent improvements of a hierarchical ab initio or de novo approach for predicting both α and β structures of proteins are described. The united-residue energy function used in this procedure includes multibody interactions from a cumulant expansion of the free energy of polypeptide chains, with their relative weights determined by Z-score optimization. The critical initial stage of the hierarchical procedure involves a search of conformational space by the conformational space annealing (CSA) method, followed by optimization of an all-atom model. The procedure was assessed in a recent blind test of protein structure prediction (CASP4). The resulting lowest-energy structures of the target proteins (ranging in size from 70 to 244 residues) agreed with the experimental structures in many respects. The entire experimental structure of a cyclic α-helical protein of 70 residues was predicted to within 4.3 Å α-carbon (Cα) rms deviation (rmsd) whereas, for other α-helical proteins, fragments of roughly 60 residues were predicted to within 6.0 Å Cα rmsd. Whereas β structures can now be predicted with the new procedure, the success rate for α/β- and β-proteins is lower than that for α-proteins at present. For the β portions of α/β structures, the Cα rmsd's are less than 6.0 Å for contiguous fragments of 30–40 residues; for one target, three fragments (of length 10, 23, and 28 residues, respectively) formed a compact part of the tertiary structure with a Cα rmsd less than 6.0 Å. Overall, these results constitute an important step toward the ab initio prediction of protein structure solely from the amino acid sequence.

Methylation of the Protein Phosphatase 2A Catalytic Subunit Is Essential for Association of Bα Regulatory Subunit But Not SG2NA, Striatin, or Polyomavirus Middle Tumor Antigen

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Bα subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Bα subunit binding without greatly affecting methylation, indicating that Bα subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Bα subunit but not the amount that could be coimmunoprecipitated with Aα subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Bα subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Bα subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.

Substrate-specific inhibition of RecQ helicase

The RecQ helicases constitute a small but highly conserved helicase family. Proteins in this family are of particular interest because they are critical to maintenance of genomic stability in prokaryotes and eukaryotes. Eukaryotic RecQ helicase family members have been shown to unwind not only DNA duplexes but also DNAs with alternative structures, including structures stabilized by G quartets (G4 DNAs). We report that Escherichia coli RecQ can also unwind G4 DNAs, and that unwinding requires ATP and divalent cation. RecQ helicase is comparably active on duplex and G4 DNA substrates, as measured by direct comparison of protein activity and by competition assays. The porphyrin derivative, N-methyl mesoporphyrin IX (NMM), is a highly specific inhibitor of RecQ unwinding activity on G4 DNA but not duplex DNA: the inhibition constant (Ki) for NMM inhibition of G4 DNA unwinding is 1.7 µM, approximately two orders of magnitude below the Ki for inhibition of duplex DNA unwinding (>100 µM). NMM may therefore prove to be a valuable compound for substrate-specific inhibition of other RecQ family helicases in vitro and in vivo.

Tbx19, a tissue-selective regulator of POMC gene expression

Pituitary cell types arise in a temporally and spatially specific fashion, in response to combinatorial actions of transcription factors induced by transient signaling gradients. The critical transcriptional determinants of the two pituitary cell types that express the pro-opiomelanocortin (POMC) gene, the anterior lobe corticotropes, producing adrenocorticotropin, and the intermediate lobe melanotropes, producing melanocyte-stimulating hormone (MSHα), have remained unknown. Here, we report that a member of the T-box gene family, Tbx19, which is expressed only in the rostral ventral diencephalon and pituitary gland, commencing on e11.5, marks pituitary cells that will subsequently express the POMC gene and is capable of altering progression of ventral cell types and inducing adrenocorticotropin in rostral tip cells. It is suggested that Tbx19, depending on the presence of synergizing transcription factors, can activate POMC gene expression and repress the α glycoprotein subunit and thyroid-stimulating hormone β promoters.

Systemic versus cartilage-specific expression of a type II collagen-specific T-cell epitope determines the level of tolerance and susceptibility to arthritis.

Immunization of mice with rat type II collagen (CII), a cartilage-specific protein, leads to development of collagen-induced arthritis (CIA), a model for rheumatoid arthritis. To define the interaction between the immune system and cartilage, we produced two sets of transgenic mice. In the first we point mutated the mouse CII gene to express an earlier defined T-cell epitope, CII-(256-270), present in rat CII. In the second we mutated the mouse type I collagen gene to express the same T-cell epitope. The mice with mutated type I collagen showed no T-cell reactivity to rat CII and were resistant to CIA. Thus, the CII-(256-270) epitope is immunodominant and critical for development of CIA. In contrast, the mice with mutated CII had an intact B-cell response and had T cells which could produce gamma interferon, but not proliferate, in response to CII. They developed CIA, albeit with a reduced incidence. Thus, we conclude that T cells recognize CII derived from endogenous cartilage and are partially tolerized but may still be capable of mediating CIA.

In vitro integration of human immunodeficiency virus type 1 cDNA into targets containing protein-induced bends.

Integration of human immunodeficiency virus type 1 cDNA into a target DNA can be strongly influenced by the conformation of the target. For example, integration in vitro is sometimes favored in target DNAs containing sequence-directed bends or DNA distortions caused by bound proteins. We have analyzed the effect of DNA bending by studying integration into two well-characterized protein-DNA complexes: Escherichia coli integration host factor (IHF) protein bound to a phage IHF site, and the DNA binding domain of human lymphoid enhancer factor (LEF) bound to a LEF site. Both of these proteins have previously been reported to bend DNA by approximately 140 degrees. Binding of IHF greatly increases the efficiency of in vitro integration at hotspots within the IHF site. We analyzed a series of mutants in which the IHF site was modified at the most prominent hotspot. We found that each variant still displayed enhanced integration upon IHF binding. Evidently the local sequence is not critical for formation of an IHF hotspot. LEF binding did not create preferred sites for integration. The different effects of IHF and LEF binding can be rationalized in terms of the different proposed conformations of the two protein-DNA complexes.

Expression cloning of a human polysialyltransferase that forms the polysialylated neural cell adhesion molecule present in embryonic brain.

Polysialic acid is a developmentally regulated posttranslational modification of the neural cell adhesion molecule (N-CAM). It has been suggested that this large anionic carbohydrate modulates the adhesive property of N-CAM, but the precise function of polysialic acid is not known. Here we describe the isolation and functional expression of a cDNA encoding a human polysialyltransferase. For this expression cloning, COS-1 cells were cotransfected with a human fetal brain cDNA library and a cDNA encoding human N-CAM. Transfected COS-1 cells were stained with a monoclonal antibody specific for polysialic acid and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of polysialic acid on the cell surface. The deduced amino acid sequence indicates that the polysialyltransferase shares a common sequence motif with other sialyltransferases cloned so far. The polysialyltransferase is, however, distinct by having two clusters of basic amino acids. The amount of the polysialyltransferase transcripts correlates well with the formation of polysialic acid in various human tissues, and is abundant in the fetal brain but not in the adult brain. Moreover, HeLa cells stably expressing polysialic acid and N-CAM promoted neurite outgrowth and sprouting. These results indicate that the cloned polysialyltransferase forms polysialylated, embryonic N-CAM, which is critical for plasticity of neural cells.

Sustainability in the Upper Colorado River Basin: A Struggle Between Water Resource Use and Remnants of the Natural Ecosystem

This study evaluated whether development of the Colorado River system has exceeded sustainability by comparing the trends in water use in the Colorado River. Two sustainable areas were identified in the upper basin and one in the lower-- the mainstream Colorado River, Green and Yampa rivers, and the Little Colorado River. These areas are also high priority recovery areas for four endangered fishes and protected by critical habitat provisions of the ESA. Unfortunately, the endangered fishes are declining because of habitat destruction and non-native species. If increasing water demand causes the fishes to go extinct the few sustainable areas will be lost. It will take careful management of the endangered fishes and water users to ensure these areas are maintained.

Asymmetric effects of online consumer reviews

Consumers tend to seek heuristic information cues to simplify the amount of information involved in tourist decisions. Accordingly, star ratings in online reviews are a critical heuristic element of the perceived evaluation of online consumer information. The objective of this article is to assess the effect of review ratings on usefulness and enjoyment. The empirical application is carried out on a sample of 5,090 reviews of 45 restaurants in London and New York. The results show that people perceive extreme ratings (positive or negative) as more useful and enjoyable than moderate ratings, giving rise to a U-shaped line, with asymmetric effects: the size of the effect of online reviews depends on whether they are positive or negative.

The ‘Lisbonisation’ of the European Parliament: Assessing progress, shortcomings and challenges for democratic accountability in the area of freedom, security and justice. CEPS Paper on Liberty and Security in Europe No. 58, September 2013

This paper examines the performance of the European Parliament in EU AFSJ law and policy-making from the entry into force of the Lisbon Treaty until the end of the first half of 2013. The paper situates the EP in the new post-Lisbon institutional setting, documenting its transition to ‘AFSJ decision-maker’, and its new powers to shape and make policy covering the EU’s internal and external security agenda. While the paper finds that the EP has become an active co-owner of the EU AFSJ post-Lisbon, with the Parliament demonstrating a dynamic adjustment to its new post-Lisbon role and powers, the authors identify a set of new developments and challenges that have arisen in the conduct of democratic accountability by the EP in the AFSJ since 2009, which call for critical reflection ahead of the new parliamentary term 2014-2019 and the post-2014 phase of the EU’s AFSJ.

Perspective maternelle de la consultation anténatale pour travail préterme : une approche relationnelle

Une femme à risque d’un accouchement prématuré vit un enjeu de santé très éprouvant et stressant ; elle sera souvent hospitalisée pour recevoir des traitements médicaux visant à prolonger la grossesse et améliorer le pronostic du bébé. Dans ce contexte, une consultation avec un néonatalogiste est demandée. Plusieurs associations professionnelles médicales ont émis des lignes directrices sur cette consultation, insistant sur le besoin d’informer les parents au sujet des complications potentielles de la prématurité pour leur enfant. Ces recommandations s’inspirent du point de vue médical, et très peu d’études ont examiné la perspective, les attentes et les besoins des parents à risque d’un accouchement prématuré. Ce projet de thèse a pour objectif de proposer un modèle de relation médecin-patient informé de la perspective maternelle de la consultation anténatale, pour développer une approche clinique répondant à leurs besoins. Afin d’examiner cette problématique de façon complète, un travail constant de va-et-vient a été effectué entre la recension de données empiriques et une réflexion normative bioéthique féministe. Un projet de recherche empirique a d’abord permis d’explorer les attentes et le vécu de ces femmes. Les participantes espéraient recevoir plus que de l’information sur les complications de la prématurité. Elles souhaitaient que le néonatologiste soit attentif à leur situation particulière et qu’il développe une relation de confiance avec elles, leur permettant d’explorer leurs futurs rôles de mères et les encourageant à formuler leurs propres questions. Le cadre théorique féministe d’autonomie relationnelle a ensuite permis de proposer une approche de soin qui sache répondre aux besoins identifiés par les patientes, tout en adressant des enjeux de pouvoir intrinsèques à la clinique, qui influencent la santé et l’autonomie de ces femmes. Cette approche insiste sur l’importance de la relation de soin en clinique, contrastant avec un modèle encourageant une vision réductrice de l’autonomie, dans laquelle un simple transfert de données scientifiques serait équivalent au respect de la norme médicolégale du consentement éclairé. Ce modèle relationnel propose des actions concrètes et pratiques, encourageant les cliniciens à entrer en relation avec chaque patiente et à considérer l’influence qu’ils exercent sur l’autonomie relationnelle de leurs patientes.

Private diary of travels, personal services, and public events, during mission and employment with the European armies in the campaigns of 1812, 1813, 1814. From the invasion of Russia to the capture of Paris.

Mode of access: Internet.

The mystical element of religion : as studied in Saint Catherine of Genoa and her friends /

Includes bibliographical references and index.