Chromosomal map of the model legume Lotus japonicus

Lotus japonicus is a model plant for the legume family. To facilitate map-based cloning approaches and genome analysis, we performed an extensive characterization of the chromosome complement of the species. A detailed karyotype of L. japonicus Gifu was built and plasmid and BAC clones, corresponding to genetically mapped markers (see the accompanying article by Sandal et al. 2002, this issue), were used for FISH to correlate genetic and chromosomal maps. Hybridization of DNA clones from 32 different genomic regions enabled the assignment of linkage groups to chromosomes, the comparison between genetic and physical distances throughout the genome, and the partial characterization of different repetitive sequences, including telomeric and centromeric repeats. Additional analysis of L. filicaulis and its F(1) hybrid with L. japonicus demonstrated the occurrence of inversions between these closely related species, suggesting that these chromosome rearrangements are early events in speciation of this group.     

Mechanisms underlying the neuronal calcium sensor-1-evoked enhancement of exocytosis in PC12 cells

Neuronal calcium sensor-1 (NCS-1) or the originally identified homologue frequenin belongs to a superfamily of EF-hand calcium binding proteins. Although NCS-1 is thought to enhance synaptic efficacy or exocytosis mainly by activating ion channel function, the detailed molecular basis for the enhancement is still a matter of debate. Here, mechanisms underlying the NCS-1-evoked enhancement of exocytosis were investigated using PC12 cells overexpressing NCS-1. NCS-1 was found to have a broad distribution in the cells being partially distributed in the cytosol and associated to vesicles and tubular-like structures. Biochemical and immunohistochemical studies indicated that NCS-1 partially colocalized with the light synaptic vesicle marker synaptophysin. When stimulated with UTP or bradykinin, agonists to phospholipase C-linked receptors, NCS-1 enhanced the agonist-mediated elementary and global Ca2+ signaling and increased the levels of downstream signals of phosphatidylinositol 4-kinase. NCS-1 enhanced the UTP-evoked exocytosis but not the depolarization-evoked Ca2+ responses or exocytosis, suggesting that the enhancement by NCS-1 should involve phospholipase C-linked receptor-mediated signals rather than the Ca2+ channels or exocytotic machinery per se. Taken together, NCS-1 enhances phosphoinositide turnover, resulting in enhancement of Ca2+ signaling and exocytosis. This is a novel regulatory mechanism of exocytosis that might involve the activation of phosphatidylinositol 4-kinase.     

A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals

An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees C) in a 30-60% sucrose gradient. Using a combination of SDS-PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS-PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes.     

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.     

Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.     

Chemotactic responsiveness toward ligands for CXCR3 and CXCR4 is regulated on plasma blasts during the time course of a memory immune response

Plasma blasts formed during memory immune responses emigrate from the spleen to migrate into the bone marrow and into chronically inflamed tissues where they differentiate into long-lived plasma cells. In this study, we analyze the chemokine responsiveness of plasma blasts formed after secondary immunization with OVA. Starting from day 4 and within approximately 48 h, OVA-specific plasma blasts emigrate from spleen and appear in the bone marrow. Although these migratory cells have lost their responsiveness to many B cell attracting chemokines, e.g., CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant), they migrate toward CXCL12 (stromal cell-derived factor 1 alpha), and toward the inflammatory chemokines CXCL9 (monokine induced by IFN-gamma), CXCL10 (IFN-gamma-inducible protein 10), and CXCL11 (IFN-inducible T cell alpha chemoattractant). However, the responsiveness of plasma blasts to these chemokines is restricted to a few days after their emigration from the spleen, indicating a role for these molecules and their cognate receptors, i.e., CXCR3 and CXCR4, in the regulation of plasma blast migration into the bone marrow and/or inflamed tissues.     

A single nucleotide polymorphism in the promoter region of the human gene for osteoprotegerin is related to vascular morphology and function

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor receptor family, and has previously been shown to regulate bone mass by inhibiting osteoclast differentiation and activation. Recent evidence indicates that OPG also plays a role in the vascular system, since ablation of the OPG gene in mice results in calcification of the aorta and renal arteries, and association has been found between serum levels of OPG and cardiovascular mortality. This study presents a novel single nucleotide polymorphism, a T/C transition located 129 bp upstream the TATA-box of the human OPG gene, detected by sequence analysis. The OPG genotype was determined by restriction fragment length polymorphism in a cohort consisting of 59 healthy subjects. The intima-media thickness (IMT) in the common carotid artery and maximal post-ischemic forearm blood flow (FBF) were investigated. Subjects with the CC genotype showed a significantly increased IMT (p<0.05) and a concommitantly reduced maximal FBF (p<0.01) as compared to those with the T allele. Thus, our results show that the polymorphism in the promoter region of OPG is associated with both vascular morphology and function in apparently healthy subjects.