Beta2-microglobulin isoforms display an heterogeneous affinity for type I collagen

It has been claimed that beta2-microglobulin (beta2-m) interacts with type I and type II collagen, and this property has been linked to the tissue specificity of the beta2-m amyloid deposits that target the osteo-articular system. The binding parameters of the interaction between collagen and beta2-m were determined by band shift electrophoresis and surface plasma resonance by using bovine collagen of type I and type II and various isoforms of beta2-m. Wild-type beta2-m binds collagen type I with a Kd of 4.1 x 10(-4) M and type II with 2.3 x 10(-3) M. By the BIAcore system we monitored the binding properties of the conformers of the slow phase of folding of beta2-m. The folding intermediates during the slow phase of folding do not display any significant difference with respect to the binding properties of the fully folded molecule. The affinity of beta2-m truncated at the third N-terminal residue does not differ from that reported for the wild-type protein. Increased affinity for collagen type I is found in the case of N-terminal truncated species lacking of six residues. The Kd of this species is 3.4 x 10 (-5) M at pH 7.4 and its affinity increases to 4.9 x 10(-6) M at pH 6.4. Fluctuations of the affinity caused by beta2-m truncation and pH change can cause modifications of protein concentration in the solvent that surrounds the collagen, and could contribute to generate locally a critical protein concentration able to prime the protein aggregation.     

Expression and characterization of soluble amino-terminal domain of NR2B subunit of N-methyl-D-aspartate receptor

N-methyl-D-aspartate (NMDA) receptors are involved in mediating excitatory synaptic transmissions in the brain and have been implicated in numerous neurologic disorders. The proximal amino-terminal domains (ATDs) of NMDA receptors constitute many modulatory binding sites that may serve as potential drug targets. There are few biochemical and structural data on the ATDs of NMDA receptors, as it is difficult to produce the functional proteins. Here an optimized method was established to reconstitute the insoluble recombinant ATD of NMDA receptor NR2B subunit (ATD2B) through productive refolding of 6xHis-ATD2B protein from inclusion bodies. Circular dichroism and dynamic light scattering characterizations revealed that the solubilized and refolded 6xHis-ATD2B adopted well-defined secondary structures and monodispersity.More significantly, the soluble 6xHis-ATD2B specifically bound ifenprodil to saturation. Ifenprodil bound to 6xHis-ATD2B with a dissociation constant (KD) of 127.5+/-45 nM, which was within the range of the IC50 determined electrophysiologically. This is the first report on a functional recombinant ATD2B with a characterized KD.     

Differential expression of inflammatory chemokines by Th1- and Th2-cell promoting dendritic cells: a role for different mature dendritic cell populations in attracting appropriate effector cells to peripheral sites of inflammation

Protective immunity to pathogens depends on efficient immune responses adapted to the type of pathogen and the infected tissue. Dendritic cells (DC) play a pivotal role in directing the effector T cell response to either a protective T helper type 1 (Th1) or type 2 (Th2) phenotype. Human monocyte-derived DC can be differentiated into Th1-, Th2- or Th1/Th2-promoting DC in vitro upon activation with microbial compounds or cytokines. Host defence is highly dependent on mobile leucocytes and cell trafficking is largely mediated by the interactions of chemokines with their specific receptors expressed on the surface of leucocytes. The production of chemokines by mature effector DC remains elusive. Here we assess the differential production of both inflammatory and homeostatic chemokines by monocyte-derived mature Th1/Th2-, Th1- or Th2-promoting DC and its regulation in response to CD40 ligation, thereby mimicking local engagement with activated T cells. We show that mature Th1- and Th1/Th2-, but not Th2-promoting DC, selectively express elevated levels of the inflammatory chemokines CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta and CCL5/RANTES, as well as the homeostatic chemokine CCL19/MIP-3beta. CCL21/6Ckine is preferentially expressed by Th2-promoting DC. Production of the Th1-attracting chemokines, CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC, is restricted to Th1-promoting DC. In contrast, expression of Th2-associated chemokines does not strictly correlate with the Th2-promoting DC phenotype, except for CCL22/MDC, which is preferentially expressed by Th2-promoting DC. Because inflammatory chemokines and Th1-associated chemokines are constitutively expressed by mature Th1-promoting DC and CCL22/MDC is constitutively expressed by mature Th2-promoting DC, we propose a novel role for mature DC present in inflamed peripheral tissues in orchestrating the immune response by recruiting appropriate leucocyte populations to the site of pathogen entry.     

Ablation of capsaicin-sensitive afferent nerves affects insulin response during an intravenous glucose tolerance test

We investigated the role of sensory nerves in glucose tolerance in conscious Wistar rats neonatally treated with neurotoxin capsaicin or vehicle. Intravenous glucose tolerance tests (IVGTT, 150, 300 and 450 mg in 30 min) were performed to measure glucose tolerance, and glucose, insulin and glucagon levels were measured. Higher glucose concentration resulted in a greater insulin response in both capsaicin- and vehicle-treated rats. However, glucose-stimulated insulin secretion was attenuated in capsaicin-treated animals, even though glucose levels did not differ. Glucagon levels did not differ between both groups. These results show that capsaicin-sensitive nerves are involved in glucose-stimulated insulin secretion, but are not directly involved in the regulation of blood glucose levels. Moreover, they suggest that capsaicin-sensitive nerves could be involved in the regulation of insulin sensitivity. We hypothesize that sensory afferents could play a role in the aetiology of pathologies where glucohomeostatic mechanisms are disturbed, as is in type 2 diabetes mellitus.     

Lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type 1 transmission by blocking the DC-SIGN--gp120 interaction

One of the cell types first encountered by human immunodeficiency virus type 1 (HIV-1) following sexual transmission are dendritic cells (DC). DC capture HIV-1 through C-type lectin receptors, of which the best studied example is DC-SIGN, which mediates HIV-1 internalization. DC can keep the virus infectious for several days and are able to transmit HIV-1 to CD4(+) T cells. We tested proteins from milk and serum for their ability to block DC-mediated HIV-1 transmission, of which bovine lactoferrin (bLF) is the most potent inhibitor. bLF binds strongly to DC-SIGN, thus preventing virus capture and subsequent transmission. Interestingly, bLF is a much more efficient inhibitor of transmission than human lactoferrin. Since bLF is nontoxic and easy to purify in large quantities, it is an interesting candidate microbicide against HIV-1. Another advantage of bLF is its ability to block HIV-1 replication in T cells. DC-mediated capture of a bLF-resistant HIV-1 variant that was selected during long-term culturing in T cells could still be blocked by bLF. This underscores the usefulness of bLF as a microbicide drug to prevent HIV-1 transmission.     

DRAGON, a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)beta superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFbeta ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFbeta signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFbeta ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling.     

Modulation of DNA synthesis in Saccharomyces cerevisiae nuclear extract by DNA polymerases and the origin recognition complex

We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol alpha, the catalytic subunit of pol delta, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of polepsilon. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, alpha and delta, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.     

EhCP112 is an Entamoeba histolytica secreted cysteine protease that may be involved in the parasite-virulence

EhCP112 is an Entamoeba histolytica protease that together with the EhADH112 protein forms the EhCPADH complex involved in trophozoite virulence. Here, we produced the recombinant EhCP112 and studied its relationships with extracellular matrix components and with target cells. A DNA fragment containing the pro-peptide and the mature enzyme was expressed in bacteria as an active enzyme (rEhCP112), whereas the full gene containing the signal peptide, the pro-peptide and the mature enzyme expressed a non-active protein. The fragment only with the mature enzyme was not expressed. rEhCP112 purified by affinity columns digested azocasein and had a strong autoproteolytic activity. Four hours after purification the protein appeared degraded. Anti-tag antibodies, monoclonal antibodies against the EhCP112 and sera from human patients with amoebiasis recognized rEhCP112. rEhCP112 digested gelatin, collagen type I, fibronectin and haemoglobin; it destroyed MDCK cell monolayers and bound to red blood cells. The native EhCP112 was poorly expressed in a virulence-deficient mutant, and in the wild-type clone it was located in secreted vesicles, forming the EhCPADH complex. Altogether these results show that EhCP112 is a molecule able to disrupt cell monolayers and digest proteins of the extracellular matrix and haemoglobin, and it is secreted by the trophozoites.