Binding of myotoxin a to sarcoplasmic reticulum Ca(2+)-ATPase: a structural study.

The interaction of myotoxin alpha with intact sarcoplasmic reticulum (SR) components was investigated, and two SR proteins were identified that associated with myotoxin a. One of the proteins has an apparent molecular weight similar to the Ca(2+)-ATPase, the major SR protein responsible for calcium loading. Ca(2+)-ATPase was purified, and its interaction with myotoxin a was studied. Evidence for specific binding of myotoxin a to Ca(2+)-ATPase was established by isolating chemically cross-linked myotoxin a-Ca(2+)-ATPase complexes and further proving their association with anti-myotoxin a antibodies. The binding region of myotoxin a was further delineated by cleaving the protein with cyanogen bromide (CNBr) into two fragments, a larger N-terminal fragment of 28 residues and a smaller C-terminal fragment of 14 residues. Competition experiments with 125I-myotoxin a showed that the C-terminal fragment competed better against 125I-myotoxin a than the N-terminal fragment for SR protein binding. Two overlapping peptides covering the sequence of the N-terminal fragment were synthesized to clarify the interaction of the N-terminal fragment of myotoxin a with SR proteins. A 16-residue peptide corresponding to residues 1-16 competed strongly with 125I-myotoxin a, while a second peptide (residues 13-28) did not.     

Cloning and characterization of the nuclear AC115 gene of Chlamydomonas reinhardtii

The nuclear ac115 mutant of Chlamydomonas reinhardtii is specifically blocked in the synthesis of the chloroplast encoded D2 protein of the photosystem II reaction center at a point after translation initiation. Here, we report the identification of the AC115 gene through complementation rescue of the ac115 mutant strain, using an indexed cosmid library of Chlamydomonas genomic DNA. AC115 is a small, novel, intronless nuclear gene which encodes a protein of 113 amino acids. The amino terminal end of the Ac115 protein is rich in basic amino acids and has features which resemble a chloroplast transit sequence. A hydrophobic stretch of amino acids at the protein's carboxyl terminus is sufficiently large to be a membrane spanning or a protein/protein interaction domain. Various models are discussed to account for the mechanism by which Ac115p works in D2 synthesis. The ac115 mutant allele was sequenced and determined to be an A-to-T transversion at the first position of the fourth codon of the coding sequence. This mutation changes an AAG codon to a TAG nonsense codon and results in a null phenotype.     

Burkholderia pseudomallei interferes with inducible nitric oxide synthase (iNOS) production: a possible mechanism of evading macrophage killing

Burkholderia pseudomallei is a causative agent of melioidosis, a life threatening disease which affects humans and animals in tropical and subtropical areas. This bacterium is known to survive and multiply inside cells such as macrophages. The mechanism of host defense against this bacterium is still unknown. In this study, we demonstrated that B. pseudomallei exhibited unique macrophage activation activity compared with Escherichia coli and Salmonella typhi. The mouse macrophage cell line (RAW 264.7) infected with B. pseudomallei at MOI of 0.1:1, 1:1 and 10:1 did not express a detectable level of inducible nitric oxide synthase (iNOS). Moreover, the B. pseudomallei infected cells released TNF-alpha only when they were infected with high MOI (10:1). Unlike the cells infected with B. pseudomallei, the cells infected with E. coli, and S. typhi expressed iNOS even at MOI of 0.1:1. These infected cells also released a significantly higher level of TNF-alpha at the low MOI ratio. The cells that were preactivated with IFN-gamma prior to being infected with B. pseudomallei exhibited an enhanced production of iNOS and TNF-alpha release. The increased macrophage activation activity in the presence of IFN-gamma also correlated with the restriction of the intracellular bacteria survival. Moreover, IFN-gamma also prevented cell fusion and multinucleated cell formation induced by B. pseudomallei, a phenomenon recently described by our group. Altogether, these results indicate that internalization of B. pseudomallei failed to trigger substantial macrophage activation, a phenomenon which could prolong their survival inside the phagocytic cells and facilitate a direct cell to cell spreading of B. pseudomallei to neighboring cells.     

High susceptibility of human dendritic cells to avian influenza H5N1 virus infection and protection by IFN-alpha and TLR ligands

There is worldwide concern that the avian influenza H5N1 virus, with a mortality rate of >50%, might cause the next influenza pandemic. Unlike most other influenza infections, H5N1 infection causes a systemic disease. The underlying mechanisms for this effect are still unclear. In this study, we investigate the interplay between avian influenza H5N1 and human dendritic cells (DC). We showed that H5N1 virus can infect and replicate in monocyte-derived and blood myeloid DC, leading to cell death. These results suggest that H5N1 escapes viral-specific immunity, and could disseminate via DC. In contrast, blood pDC were resistant to infection and produced high amounts of IFN-alpha. Addition of this cytokine to monocyte-derived DC or pretreatment with TLR ligands protected against infection and the cytopathic effects of H5N1 virus.     

Immunisation with recombinant PfEMP1 domains elicits functional rosette-inhibiting and phagocytosis-inducing antibodies to Plasmodium falciparum

Background

Rosetting is a Plasmodium falciparum virulence factor implicated in the pathogenesis of life-threatening malaria. Rosetting occurs when parasite–derived P. falciparum Erythrocyte Membrane Protein One (PfEMP1) on the surface of infected erythrocytes binds to human receptors on uninfected erythrocytes. PfEMP1 is a possible target for a vaccine to induce antibodies to inhibit rosetting and prevent severe malaria.

Methodology/Findings

We examined the vaccine potential of the six extracellular domains of a rosette-mediating PfEMP1 variant (ITvar9/R29var1 from the R29 parasite strain) by immunizing rabbits with recombinant proteins expressed in E. coli. Antibodies raised to each domain were tested for surface fluorescence with live infected erythrocytes, rosette inhibition and phagocytosis-induction. Antibodies to all PfEMP1 domains recognized the surface of live infected erythrocytes down to low concentrations (0.02–1.56 µg/ml of total IgG). Antibodies to all PfEMP1 domains except for the second Duffy-Binding-Like region inhibited rosetting (50% inhibitory concentration 0.04–4 µg/ml) and were able to opsonize and induce phagocytosis of infected erythrocytes at low concentrations (1.56–6.25 µg/ml). Antibodies to the N-terminal region (NTS-DBL1α) were the most effective in all assays. All antibodies were specific for the R29 parasite strain, and showed no functional activity against five other rosetting strains.

Conclusions/Significance

These results are encouraging for vaccine development as they show that potent antibodies can be generated to recombinant PfEMP1 domains that will inhibit rosetting and induce phagocytosis of infected erythrocytes. However, further work is needed on rosetting mechanisms and cross-reactivity in field isolates to define a set of PfEMP1 variants that could induce functional antibodies against a broad range of P. falciparum rosetting parasites.     

Chondroitin sulfate A-adhering Plasmodium falciparum-infected erythrocytes express functionally important antibody epitopes shared by multiple variants

Acquired protection from Plasmodium falciparum placental malaria, a major cause of maternal, fetal, and infant morbidity, is mediated by IgG specific for the P. falciparum erythrocyte membrane protein 1 variant VAR2CSA. This protein enables adhesion of P. falciparum-infected erythrocytes to chondroitin sulfate A in the intervillous space. Although interclonal variation of the var2csa gene is lower than that among var genes in general, VAR2CSA-specific Abs appear to target mainly polymorphic epitopes. This has raised doubts about the feasibility of VAR2CSA-based vaccines. We used eight human monoclonal IgG Abs from affinity-matured memory B cells of P. falciparum-exposed women to study interclonal variation and functional importance of Ab epitopes among placental and peripheral parasites from East and West Africa. Most placental P. falciparum isolates were labeled by several mAbs, whereas peripheral isolates from children were essentially nonreactive. The mAb reactivity of peripheral isolates from pregnant women indicated that some were placental, whereas others had alternative sequestration foci. Most of the mAbs were comparable in their reactivity with bound infected erythrocytes (IEs) and recombinant VAR2CSA and interfered with IE and/or VAR2CSA binding to chondroitin sulfate A. Pair-wise mAb combinations were more inhibitory than single mAbs, and all of the mAbs together was the most efficient combination. Each mAb could opsonize IEs for phagocytosis, and a combination of the eight mAbs caused phagocytosis similar to that of plasma IgG-opsonized IEs. We conclude that functionally important Ab epitopes are shared by the majority of polymorphic VAR2CSA variants, which supports the feasibility of VAR2CSA-based vaccines against placental malaria.     

Plasma and synovial fluid inflammatory cytokine profiles in primary knee osteoarthritis

Objective: The objective of this study is to compare inflammatory cytokine levels in primary knee osteoarthritis (OA) patients and healthy controls.

Methods: A total of 32 knee OA patients and 14 healthy controls were enrolled. A multiplex immunoassay was utilized for 10 cytokines in plasma and synovial fluid.

Results: Plasma IL-2, IL-4, and IL-6 concentrations were significantly greater in knee OA patients than controls. Moreover, both plasma IL-4 and IL-6 were positively correlated with the radiographic severity of knee OA.

Conclusions: Plasma IL-4 and IL-6 may serve as biomarkers reflecting the severity of OA.     

Age-Related Decrease of the Phosphorus Content in the Ligamentum Capitis Femoris of Monkeys

To elucidate compositional changes of the ligament with aging, the authors investigated age-related changes of elements in the ligamentum capitis femoris (LCF) of monkeys with a wide range of ages by direct chemical analysis. Used rhesus and Japanese monkeys consisted of 9 males and 22 females, ranging in age from newborn to 31 years (average age?=?10.4?±?10.9 years). After incineration with nitric acid and perchloric acid, element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that the P content decreased significantly in the LCFs of monkeys with aging, but other six element contents, Ca, S, Mg, Zn, Fe, and Na, did not change significantly with aging. Assuming that the P content indicated the active cell density and the S content indicated the protein amount, an age-related change of the mass ratio of P/S was examined in the LCFs. The mass ratio of P/S decreased significantly in the LCFs in childhood. Regarding the relationships among elements, significant direct correlations were found among the Ca, P, S, and Mg contents in the LCFs. It was suggested that the active cell density of the connective tissue cells might decrease significantly in the LCF in childhood.     

Structural Comparison of Two CSPG-Binding DBL Domains from the VAR2CSA Protein Important in Malaria during Pregnancy

Severe malaria during pregnancy is associated with accumulation of parasite-infected erythrocytes in the placenta due to interactions between VAR2CSA protein, expressed on the surface of infected-erythrocytes, and placental chondroitin sulfate proteoglycans (CSPG). VAR2CSA contains multiple CSPG-binding domains, including DBL3X and DBL6?. Previous structural studies of DBL3X suggested CSPG to bind to a positively charged patch and sulfate-binding site on the concave surface of the domain. Here we present the structure of the DBL6? domain from VAR2CSA. This domain displays the same overall architecture and secondary structure as that of DBL3X but differs in loop structures, disulfide bond positions and surface charge distribution. In particular, despite binding to CSPG, DBL6? lacks the key features of the CSPG-binding site of DBL3X. Instead DBL6? binds to CSPG through a positively charged surface on the distal side of subdomain 2 that is exposed in intact VAR2CSA on the erythrocyte surface. Finally, unlike intact VAR2CSA, both DBL3X and DBL6? bind to various carbohydrates, with greatest affinity for ligands with high sulfation and negative charge. These studies provide further insight into the structure of DBL domains and suggest a model for the role of individual domains in CSPG binding by VAR2CSA in placental malaria.