Serendipitous discovery and X-ray structure of a human phosphate binding apolipoprotein

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is copurified with the enzyme paraoxonase. Its X-ray structure is similar to the prokaryotic phosphate solute binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The systematic absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation between genes belonging to evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the only known transporter capable of binding phosphate ions in human plasma and may become a new predictor of or a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.     

LPS response and endotoxin tolerance in Flt-3L-induced bone marrow-derived dendritic cells

Fms-like tyrosine kinase-3 ligand (Flt-3L) stimulates the differentiation of bone marrow cells into dendritic cells (DCs) and was used as an adjuvant therapy in the experimental model of burn wound sepsis. In this study, we describe the phenotypical characteristics of an Flt-3L-dependent DC culture (FLDC) system following LPS stimulation, which induces an inflammatory response, and after a second LPS stimulation, which induces tolerance. Priming of FLDCs with LPS via TLR4 has been shown to induce the activation of all three mitogen-activated protein kinase (MAPK) families and enhance NF-κB complex translocation into the nucleus. Stimulated FLDCs express all maturation markers and exhibit an increase in IL-12p40 production and to a lesser extent, IL-10 production. In contrast, LPS stimulation of tolerized FLDCs was not associated with TLR4 up-regulation and led to MAPK inhibition. The decrease in p38 and JNK activation was correlated with an impairment of IL-12p40 production. Endotoxin tolerance in FLDCs was associated with enhanced ERK1/2 activation, an increase in MKP-1 phosphatase expression, a decrease in NF-κB translocation to the nucleus and an increase in IL-10 production. Overall, DCs generated from bone marrow with Flt-3 ligand have similar characteristics to DC subtypes found in the steady state in vivo, which can acquire endotoxin tolerance in some circumstances.     

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood ³³. To help improve this understanding, proton magnetic resonance spectroscopy (¹H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner ⁴¹. In fact, a recent study demonstrated that ¹H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration ³⁴. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with ¹H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices ^27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.     

Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.     

Involvement of tyrosine phosphatase CD45 in apoptosis

CD45 is a transmembrane molecule with phosphatase activity expressed in all nucleated haematopoietic cells and plays a major role in immune cells. It is a protein tyrosine phosphatase that is essential for antigen-receptor-mediated signal transduction by regulating Src family members that initiate TCR signaling. CD45 is being attributed a new emerging role as an apoptosis regulator. Cross-linking of the extracellular portion of the CD45 by monoclonal antibodies and by galectin-1, can induce apoptosis in T and B cells. Interestingly, this phosphatase has also been involved in nuclear apoptosis induced by mitochondrial perturbing agents. Furthermore, it is involved in apoptosis induced by HIV-1. CD45 defect is implicated in various diseases such as severe-combined immunodeficiency disease (SCID), acquired immunodeficiency syndrome (AIDS), lymphoma and multiple myelomas. The understanding of the mechanisms by which CD45 regulates apoptosis would be very useful in disease treatment.     

Generation of anti-idiotypic and anti-anti-idiotypic monoclonal antibodies in the same fusion. Support of Jerne's Network Theory

Upon immunization of mice with a mAb (290A-167) directed against an epitope of Lol p I (the major allergenic determinant of Lolium perenne), both anti-idiotypic (aId) mAb (Ab2) and anti-aId mAb (Ab3) were produced. The Ab2 displayed the following internal image properties of Lol p I: it can be affinity-purified on an immobilized Id column; its binding to the anti-Lol p I mAb (290A-167) is inhibited by Lol p I; it inhibits in a dose-response fashion the binding of the specific Id to Ag. It is recognized by anti-Lol p I antisera from different species such as mouse, human, and goat. The Ab3 which binds to Lol p I was also produced from the same fusion. This binding was inhibited significantly by aId mAb (Ab2), anti-Lol p I mAb (290A-167) and Lol p I. These data indicate that the two mAb with specificity for Lol p I (290A-167 and Ab3) share similar reactivity to the Ag and that aId mAb is the internal image of the epitope recognized by the Id. We showed also that the capacity of rabbit aId Ab directed against the 290A-167 Id to inhibit the binding of Ab1 and Ab3 to Ag was almost abolished by passage over a Ab3-coated Sepharose column. This would suggest that not only are the two mAb with reactivity to Lol p I (Ab1 and Ab3) directed against identical epitopes, but that they in fact shared identical idiotopes as well. The production of identical mAb upon immunization with either the Ag or the aId mAb supports that the conceptual framework proposed by Jerne finds its biologic application in the course of an immune response.     

Activation of replicon origins as a possible target for cytokinins in shoot meristems of Sinapis

Whilst the cytokinins are important promoters of plant cell division in vitro and in vivo, their mode of action remains unknown. Here we report the results of a study showing that a single application of a low dose of a cytokinin to the shoot apical meristem of Sinapis alba L. activates new replicon origins in chromosomal DNA, resulting in the halving of replicon size, and synchronizes the activation of replicon origins. These effects cause a 3.5-fold shortening of the duration of chromosomal DNA replication (S phase of the cell cycle). We hypothesize that one of the proteins involved in the initiation of DNA replication is a target for cytokinins.Abbreviations BA N⁶-benzyladenine - F fork rate - R size ofmost replicons - Rs time taken for replicon to replicate its allotedDNA - TdR [³H]thymidine - Ts duration of S phase C. Houssa is grateful to I.R.S.I.A. for the award of a research fellowship. This research was supported by the Belgian Government (Concerted Research Actions and FRFC).