Immunity in the absence of CD28 and CD137 (4-1BB) molecules

We report the generation and immune regulation of mice that are deficient in CD28 and 4-1BB (CD137) genes. These mice were viable, fertile and did not display any overt abnormalities and had a normal T cell phenotype in thymus and spleen. Proliferative responses to anti-CD3 and ConA were enhanced in 4-1BB-/- but not in either CD28-/- or double mutant mice, while levels of interleukin-2 were decreased in all mutant mice. Although the 4-1BB-/- mice displayed increased basal levels of most immunoglobulin isotypes tested, the plateau levels of immunoglobulin G2a, immunoglobulin G2b and immunoglobulin A were particularly high compared to wild type controls. The immunoglobulin class switch to T-dependent antigen was normal in 4-1BB-/- mice but was greatly affected in both CD28-/- and 4-1BB-/- CD28-/- mice. Vesicular stomatitis virus-specific cytotoxic T lymphocyte responses and plaque reduction neutralizing ability was differentially reduced in all mutant mice. Contact sensitivity to allergens showed marginal but not significant change in ear thickness in 4-1BB-/- mice, but an ability to mount contact hypersensitivity to the same antigens was greatly curtailed in CD28-/- and double mutant mice.     

Stilbenes from the roots of Pleuropterus ciliinervis and their antioxidant activities

Two stilbene glycosides, pieceid-2"-O-gallate and pieceid-2"-O-coumarate, were isolated from the MeOH extract of the roots of Pleuropterus ciliinervis Nakai (Polygonaceae), together with two known compounds, resveratrol and pieceid. Their structures were determined spectroscopically, particularly by 2D NMR spectroscopic analysis. The antioxidant activities of stilbenes isolated were determined in vitro against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, superoxide radicals and by determining their lipid peroxidation inhibitory activities. Among the compounds isolated, pieceid-2"-O-gallate had the most potent inhibitory scavenging effect on DPPH, superoxide radicals and upon lipid peroxidation inhibition with IC50 values of 16.5, 23.9 and 5.1 microM, respectively.     

Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight(-1). This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.     

Syntenic relationships between Medicago truncatula and Arabidopsis reveal extensive divergence of genome organization

Arabidopsis and Medicago truncatula represent sister clades within the dicot subclass Rosidae. We used genetic map-based and bacterial artificial chromosome sequence-based approaches to estimate the level of synteny between the genomes of these model plant species. Mapping of 82 tentative orthologous gene pairs reveals a lack of extended macrosynteny between the two genomes, although marker collinearity is frequently observed over small genetic intervals. Divergence estimates based on non-synonymous nucleotide substitutions suggest that a majority of the genes under analysis have experienced duplication in Arabidopsis subsequent to divergence of the two genomes, potentially confounding synteny analysis. Moreover, in cases of localized synteny, genetically linked loci in M. truncatula often share multiple points of synteny with Arabidopsis; this latter observation is consistent with the large number of segmental duplications that compose the Arabidopsis genome. More detailed analysis, based on complete sequencing and annotation of three M. truncatula bacterial artificial chromosome contigs suggests that the two genomes are related by networks of microsynteny that are often highly degenerate. In some cases, the erosion of microsynteny could be ascribed to the selective gene loss from duplicated loci, whereas in other cases, it is due to the absence of close homologs of M. truncatula genes in Arabidopsis.     

Identification of Arabidopsis rat mutants

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.     

A single nucleotide polymorphism in the<Emphasis Type="Italic"> Emp3</Emphasis> gene defines the H4 minor histocompatibility antigen

Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4^b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4^b presented on K^b class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to CT nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4^a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4^b while the anti-B6 response directed against H4^a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.     

Antigenicity of the region encoded by exon8 of the human serine protease, HtrA2/Omi, is associated with its protein solubility

HtrA2/Omi, a mitochondrial serine protease, is pivotal in regulating apoptotic cell death. To determine the location of antigenic determinants in HtrA2/Omi, we expressed a series of the N-terminally truncated HtrA2/Omi as GST fusion proteins in E. coli. We assessed protein solubility and antigenic reactivity of various N-terminally truncated HtrA2/Omi proteins by binding to glutathione beads and immunoblot analyses, respectively. We identified that the region encoded by exon8 of HtrA2/Omi was expressed as a highly soluble form and contains an antigenic determinant specifically recognized by a polyclonal serum against HtrA2/Omi. Our data provide evidence that protein solubility of the specific region in target proteins may contribute to the antigenicity.     

Extracts of Phellinus linteus grown on germinated brown rice suppress liver damage induced by carbon tetrachloride in rats

Extracts of Phellinus linteus (EPB), grown on germinated brown rice, protected rats from liver injury induced by carbon tetrachloride (CCl4). Peroxidation products in the liver were decreased to 10% by EPB. Catalase and superoxide dismutase activities were significantly decreased to 55% and 39% by CCl4 administration, but EPB blocked this effect, resulting in enzyme activities at control levels. Expression of cytochromeP450 2E1 (CYP2E1) protein was significantly decreased to 88% in CCl4-treated rats but remained at control levels when EPB was also administered. EPB did not affect the altered fatty acid composition induced by CCl4. The hepatoprotective effect of EPB may be mediated by EPB's prevention of CCl4-induced CYP2E1 degradation.