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.     

Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon

The majority of all proteins of a living cell is active in complexes rather than in an isolated way. These protein-protein interactions are of high relevance for many biological functions. In addition to many well established protein complexes an increasing number of protein-protein interactions, which form rather transient complexes has recently been discovered. The formation of such complexes seems to be a common feature especially for metabolic pathways. In the Gram-positive model organism Bacillus subtilis, we identified a protein complex of three citric acid cycle enzymes. This complex consists of the citrate synthase, the isocitrate dehydrogenase, and the malate dehydrogenase. Moreover, fumarase and aconitase interact with malate dehydrogenase and with each other. These five enzymes catalyze sequential reaction of the TCA cycle. Thus, this interaction might be important for a direct transfer of intermediates of the TCA cycle and thus for elevated metabolic fluxes via substrate channeling. In addition, we discovered a link between the TCA cycle and gluconeogenesis through a flexible interaction of two proteins: the association between the malate dehydrogenase and phosphoenolpyruvate carboxykinase is directly controlled by the metabolic flux. The phosphoenolpyruvate carboxykinase links the TCA cycle with gluconeogenesis and is essential for B. subtilis growing on gluconeogenic carbon sources. Only under gluconeogenic growth conditions an interaction of these two proteins is detectable and disappears under glycolytic growth conditions.     

BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol

How non-enveloped viruses overcome host cell membranes is poorly understood. Here, we show that after endocytosis and transport to the endoplasmic reticulum (ER), but before crossing the ER membrane to the cytosol, incoming simian virus 40 particles are structurally remodelled leading to exposure of the amino-terminal sequence of the minor viral protein VP2. These hydrophobic sequences anchor the virus to membranes. A negatively charged residue, Glu 17, in the α-helical, membrane-embedded peptide is essential for infection, most likely by introducing an 'irregularity' recognized by the ER-associated degradation (ERAD) system for membrane proteins. Using a siRNA-mediated screen, the lumenal chaperone BiP and the ER-membrane protein BAP31 (both involved in ERAD) were identified as being essential for infection. They co-localized with the virus in discrete foci and promoted its ER-to-cytosol dislocation. Virus-like particles devoid of VP2 failed to cross the membrane. The results demonstrated that ERAD-factors assist virus transport across the ER membrane.     

Letter to the editor: SeqXML and OrthoXML: standards for sequence and orthology information

There is a great need for standards in the orthology field. Users must contend with different ortholog data representations from each provider, and the providers themselves must independently gather and parse the input sequence data. These burdensome and redundant procedures make data comparison and integration difficult. We have designed two XML-based formats, SeqXML and OrthoXML, to solve these problems. SeqXML is a lightweight format for sequence records-the input for orthology prediction. It stores the same sequence and metadata as typical FASTA format records, but overcomes common problems such as unstructured metadata in the header and erroneous sequence content. XML provides validation to prevent data integrity problems that are frequent in FASTA files. The range of applications for SeqXML is broad and not limited to ortholog prediction. We provide read/write functions for BioJava, BioPerl, and Biopython. OrthoXML was designed to represent ortholog assignments from any source in a consistent and structured way, yet cater to specific needs such as scoring schemes or meta-information. A unified format is particularly valuable for ortholog consumers that want to integrate data from numerous resources, e.g. for gene annotation projects. Reference proteomes for 61 organisms are already available in SeqXML, and 10 orthology databases have signed on to OrthoXML. Adoption by the entire field would substantially facilitate exchange and quality control of sequence and orthology information.