The first enantioselective synthesis of the amavadin ligand and its complexation to vanadium

The ligand of the naturally occurring vanadium compound amavadin found in Amanita muscaria, (2S, 2'S)-N-hydroxyimino-2,2'-dipropionic acid (1), was synthesized stereoselectively in two steps with 43% overall yield. After complexation of this ligand to vanadyl acetate, amavadin was isolated in quantitative yield. Due to the chirality at vanadium amavadin consists of a mixture of delta and lambda diastereoisomers. Directly after its synthesis, the delta to lambda ratio of amavadin is 2.27 and it decreases to 0.80 after equilibrium has been reached. During this epimerization the optical rotation for V[(2S,2'S)-N-hydroxyimino-(2,2')-dipropionate]2 (=amavadin) changes from [alpha](D)25 = +36 degrees to +114.0 degrees (c = 0.5, H2O). For V[(2R,2'R)-N-hydroxyimino-(2,2')-dipropionate] the optical rotation changes from [alpha](D)25 = -36 degrees to -113.2 degrees (c = 0.5, H2O).     

Large complex formation of the inhibitor of caspase-activated DNase

Inhibitor of caspase-activated DNase (ICAD) is required for correctly folding of CAD and inhibits nuclease activity of CAD in non-apoptotic cells. From proteomic analysis of the ICAD binding proteins, we revealed that over-expressed flag-ICAD bound other ICAD molecules}. Purified recombinant ICAD protein showed three bands, 66 KDa, 132 KDa and 450 KDa, by native-PAGE. ICAD fused with glutathione-S-transferase (GST) was immunoprecipitated with anti-flag antibody from Jurkat cell lysates cotransfected with ICAD fused with either GST or flag expression vectors. When purified recombinant ICAD protein was separated by gel chromatography, the molecular weight of ICAD was detected at 440 and 45 K. ICAD in extracts of wild type Jurkat cells also existed at 440 and 45 K as measured by gel chromatography; so that fractions of CAD coincided with fractions of 440 K of ICAD. These results indicate that ICAD and/or CAD appeared to form large complexes in Jurkat cells.     

Nuclear transfer of perchloric acid-soluble protein by endoplasmic reticulum stressors

Perchloric acid-soluble protein (PSP) is highly conserved during evolution from bacteria to mammals. Although PSP has been recognized as an inhibitor of translation and proliferation in vitro, its precise biological role has not yet been elucidated. Since we previously found similar distributions for PSP and the endoplasmic reticulum (ER) and Golgi complex, the intracellular distribution of PSP was analyzed in more detail. Immunofluorescence studies indicated that PSP co-localized with the ER and Golgi complex, since the distribution pattern of PSP was well matched to both of these organelles. An immunoelectron microscopic study revealed PSP was located not only in the cytosol but also on the surface of the outer ER membrane. Since PSP was present on the ER, we speculated that it may be associated with ER function. Therefore, we analyzed whether or not the ER stress response, which is one of the ER functions, affected PSP expression. The results showed that various ER stressors (thapsigargin, A23187, tunicamycin, brefeldin A, and cisplatin) provoked a dramatic change in the localization of PSP from outside of the nucleus to inside the nucleus within 3 h. Moreover, the ER stressors induced PSP expression. These results suggest that PSP is involved in the cellular response to ER stressors, and that the change in localization of PSP from the ER to the nucleus may be associated with ER stress responses.     

Nucleotide-binding oligomerization domain-2 modulates specific TLR pathways for the induction of cytokine release

The recognition of peptidoglycan by cells of the innate immune system has been controversial; both TLR2 and nucleotide-binding oligomerization domain-2 (NOD2) have been implicated in this process. In the present study we demonstrate that although NOD2 is required for recognition of peptidoglycan, this leads to strong synergistic effects on TLR2-mediated production of both pro- and anti-inflammatory cytokines. Defective IL-10 production in patients with Crohn's disease bearing loss of function mutations of NOD2 may lead to overwhelming inflammation due to a subsequent Th1 bias. In addition to the potentiation of TLR2 effects, NOD2 is a modulator of signals transmitted through TLR4 and TLR3, but not through TLR5, TLR9, or TLR7. Thus, interaction between NOD2 and specific TLR pathways may represent an important modulatory mechanism of innate immune responses.     

A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa

A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F(2) population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.     

RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.     

Analysis of autoreactive CD4 T cells in experimental autoimmune encephalomyelitis after primary and secondary challenge using MHC class II tetramers

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is primarily mediated by CD4 T cells specific for Ags in the CNS. Using MHC class II tetramers, we assessed expansion and phenotypic differentiation of polyclonal self-reactive CD4 T cells during EAE after primary and secondary challenge with the specific Ag. After EAE induction in SJL mice with proteolipid protein 139-151, CNS-specific T cells up-regulated activation markers and expanded in the draining lymph nodes and in the spleen. Less than 20% of total autoreactive T cells entered the CNS simultaneously with Th cells of other specificities. Almost all tetramer-positive cells in the CNS were activated and phenotypically distinct from the large peripheral pool. When EAE was induced in Ag-experienced mice, disease symptoms developed earlier and persisted longer; autoreactive T cells were more rapidly activated and invaded the CNS earlier. In striking contrast to specific CTLs that respond after secondary viral challenge, the absolute numbers of autoreactive CD4 T cells were not increased, indicating that the accelerated autoreactivity in Ag-experienced mice is not related to higher frequencies of autoreactive CD4 T cells.     

The movement protein of cowpea mosaic virus binds GTP and single-stranded nucleic acid in vitro

The movement protein (MP) of Cowpea mosaic virus forms tubules in plasmodesmata to enable the transport of mature virions. Here it is shown that the MP is capable of specifically binding riboguanosine triphosphate and that mutational analysis suggests that GTP binding plays a role in the targeted transport of the MP. Furthermore, the MP is capable of binding both single-stranded RNA and single-stranded DNA in a non-sequence-specific manner, and the GTP- and RNA-binding sites do not overlap.     

Generation of peptide-MHC class I complexes through UV-mediated ligand exchange

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.