Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer.     

Thiosemicarbazones derived from 1-indanones as new anti-Trypanosoma cruzi agents

In the present work, we synthesized a series of thiosemicarbazones derived from 1-indanones with good anti-Trypanosoma cruzi activity. Most of them displayed remarkable trypanosomicidal activity. All the compounds showed nonspecific cytotoxicity on human erythrocytes. The ability of the new compounds to inhibit cruzipain, the major cysteine protease of T. cruzi, was also explored. Thiosemicarbazones 12 and 24 inhibited this enzyme at the dose assayed. This interaction was also studied in terms of molecular docking.     

Pasteurella multocida involved in respiratory disease of wild chimpanzees

Pasteurella multocida can cause a variety of diseases in various species of mammals and birds throughout the world but nothing is known about its importance for wild great apes. In this study we isolated P. multocida from wild living, habituated chimpanzees from Taï National Park, Côte d''Ivoire. Isolates originated from two chimpanzees that died during a respiratory disease outbreak in 2004 as well as from one individual that developed chronic air-sacculitis following this outbreak. Four isolates were subjected to a full phenotypic and molecular characterisation. Two different clones were identified using pulsed field gel electrophoresis. Multi Locus Sequence Typing (MLST) enabled the identification of previous unknown alleles and two new sequence types, ST68 and ST69, were assigned. Phylogenetic analysis of the superoxide dismutase (sodA) gene and concatenated sequences from seven MLST-housekeeping genes showed close clustering within known P. multocida isolated from various hosts and geographic locations. Due to the clinical relevance of the strains described here, these results make an important contribution to our knowledge of pathogens involved in lethal disease outbreaks among endangered great apes.     

Influence of inflammation-related changes on conformational characteristics of HLA-B27 subtypes as detected by IR spectroscopy

Inflammatory processes are accompanied by the post-translational modification of certain arginine residues to yield citrulline, and a pH decrease in the affected tissue, which might influence the protonation of histidine residues within proteins. We employed isotope-edited IR spectroscopy to investigate whether conformational features of two human major histocompatibility antigen class I subtypes, HLA-B*2705 and HLA-B*2709, are affected by these changes. Both differ only in residue 116 (Asp vs. His) within the peptide-binding grooves, but are differentially associated with inflammatory rheumatic disorders. Our analyses of the two HLA-B27 subtypes in complex with a modified self-peptide containing a citrulline RRKWURWHL (U = citrulline) revealed that the heavy chain is more flexible in the HLA-B*2705 subtype than in the HLA-B*2709 subtype. Together with our previous studies of HLA-B27 subtypes complexed with the unmodified self-peptide RRKWRRWHL, these findings support the existence of subtype-specific conformational features of the heavy chains under physiological conditions, which are undetectable by X-ray crystallography and exist irrespective of the sequence of the bound peptide and its binding mode. They might thus influence antigenic properties of the respective HLA-B27 subtype. Furthermore, a decrease in the pH from 7.5 to 5.6 during the analyses had an influence only on HLA-B*2709 complexed with the unmodified self-peptide, where His116 is not contacted by any peptide side chain. This permits us to conclude that histidines, and in particular His116, influence the stability of MHC:peptide complexes. The conditions prevailing in inflammatory environments in vivo might thus also exert an impact on selected conformational features of HLA-B27:peptide complexes.     

The novel immunosuppressive protein kinase C inhibitor sotrastaurin has no pro-viral effects on the replication cycle of hepatitis B or C virus

The pan-protein kinase C (PKC) inhibitor sotrastaurin (AEB071) is a novel immunosuppressant currently in phase II trials for immunosuppression after solid organ transplantation. Besides T-cell activation, PKC affects numerous cellular processes that are potentially important for the replication of hepatitis B virus (HBV) and hepatitis C virus (HCV), major blood-borne pathogens prevalent in solid organ transplant recipients. This study uses state of the art virological assays to assess the direct, non-immune mediated effects of sotrastaurin on HBV and HCV. Most importantly, sotrastaurin had no pro-viral effect on either HBV or HCV. In the presence of high concentrations of sotrastaurin, well above those used clinically and close to levels where cytotoxic effects become detectable, there was a reduction of HCV and HBV replication. This reduction is very likely due to cytotoxic and/or anti-proliferative effects rather than direct anti-viral activity of the drug. Replication cycle stages other than genome replication such as viral cell entry and spread of HCV infection directly between adjacent cells was clearly unaffected by sotrastaurin. These data support the evaluation of sotrastaurin in HBV and/or HCV infected transplant recipients.     

Progression of pathogenic events in cynomolgus macaques infected with variola virus

Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections - an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions.     

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer''s patches ^1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes ⁴.Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.     

Benzotriazoles and indazoles are scaffolds with biological activity against Entamoeba histolytica

Parasitic infections caused by Entamoeba histolytica are still major threats against public health, especially in developing countries. Although current therapies exist, the problems associated with parasite resistance and negative side effects make it imperative to search for new therapeutic agents. A systematic scaffold analysis reported herein of a public database containing 474 antiamoebic compounds reveals that benzimidazole is the most active scaffold reported thus far. To gain insights into the antiamoebic activity of novel compounds, the authors report herein the biological activity of 12 compounds, including benzotriazole and indazole derivatives, scaffolds not previously tested against E. histolytica. Compounds with the benzotriazole and indazole scaffolds showed low micromolar activity (IC(50) = 0.304 and 0.339 μM) and are more active than metronidazole, which is the drug of choice used for the treatment of amebiosis. The novel compounds have similar properties to approved drugs. Compounds with novel scaffolds represent promising starting points of an optimization program against E. histolytica.