Interaction of Ly-49D+ NK cells with H-2Dd target cells leads to Dap-12 phosphorylation and IFN-gamma secretion

Murine Ly-49D augments NK cell function upon recognition of target cells expressing H-2Dd. Ly-49D activation is mediated by the immunoreceptor tyrosine-based activation motif-containing signaling moiety Dap-12. In this report we demonstrate that Ly-49D receptor ligation can lead to the rapid and potent secretion of IFN-gamma. Cytokine secretion can be induced from Ly-49D+ NK cells after receptor ligation with Ab or after interaction with target cells expressing their H-2Dd ligand. Consistent with the dominant inhibitory function of Ly-49G, NK cells coexpressing Ly-49D and Ly-49G show a profound reduction in IFN-gamma secretion after interaction with targets expressing their common ligand, H-2Dd. Importantly, we are able to demonstrate for the first time that effector/target cell interactions using Ly-49D+ NK cells and H-2Dd targets result in the rapid phosphorylation of Dap-12. However, Dap-12 is not phosphorylated when Ly-49D+ NK cells coexpress the inhibitory receptor, Ly-49G. These studies are novel in describing Ly-49 activation vs inhibition, where two Ly-49 receptors recognize the same class I ligand, with the dominant inhibitory receptor down-regulating phosphorylation of Dap-12, cytokine secretion, and cytotoxicity in NK cells.     

Expression of a synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize

We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R₁ kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R₁ kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R₃ seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R₃ seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens.     

Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle

Oscillations in the rate of photosynthesis of sunflower (Helianthus annuus L.) leaves were induced by subjecting leaves, whose photosynthetic apparatus had been activated, to a sudden transition from darkness or low light to high-intensity illumination, or by transfering them in the light from air to an atmosphere containing saturating CO₂. It was found that at the first maximum, light-and CO₂-saturated photosynthesis can be much faster than steady-state photosynthesis. Both Q_A in the reaction center of PS II and P₇₀₀ in the reaction center of PS I of the chloroplast electron-transport chain were more oxidized during the maxima of photosynthesis than during the minima. Maxima of P₇₀₀ oxidation slightly preceded maxima in photosynthesis. During a transition from low to high irradiance, the assimilatory force F_A, which was calculated from ratios of dihydroxyacetone phosphate to phosphoglycerate under the assumption that the reactions catalyzed by NADP-dependent glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase are close to equilibrium, oscillated in parallel with photosynthesis. However, only one of its components, the calculated phosphorylation potential (ATP)/(ADP)(P_i), paralleled photosynthesis, whereas calculated NADPH/NADP ratios exhibited antiparallel behaviour. When photosynthetic oscillations were initiated by a transition from low to high CO₂, the assimilatory force F_A declined, was very low at the first minimum of photosynthesis and increased as photosynthesis rose to its second maximum. The observations indicate that the minima in photosynthesis are caused by lack of ATP. This leads to overreduction of the electron-transport chain which is indicated by the reduction of P₇₀₀. During photosynthetic oscillations the chloroplast thylakoid system is unable to adjust the supply of ATP and NADPH rapidly to demand at the stoichiometric relationship required by the carbonreduction cycle.Abbreviations PGA 3-phosphoglycerate - DHAP dihydroxyacetone phosphate - P₇₀₀ electron-donor pigment in the reaction enter of PS I - Q_A quinone acceptor in the reaction center of PS II This work received support from the Estonian Academy of Sciences, the Bavarian Ministry of Science and Art and the Sonderforschungsbereich 251 of the University of Würzburg. We are grateful for criticism by D.A. Walker, Robert Hill Institute, University of Sheffield, U.K. and by Mark Stitt, Institute of Botany, University of Heidelberg, FRG.     

Cupric-amyloid beta peptide complex stimulates oxidation of ascorbate and generation of hydroxyl radical

A growing body of evidence supports an important role for oxidative stress in the pathogenesis of Alzheimer's disease. Recently, a number of papers have shown a synergistic neurotoxicity of amyloid beta peptide and cupric ions. We hypothesized that complexes of cupric ions with neurotoxic amyloid beta peptides (Abeta) can stimulate copper-mediated free radical formation. We found that neurotoxic Abeta (1-42), Abeta (1-40), and Abeta (25-35) stimulated copper-mediated oxidation of ascorbate, whereas nontoxic Abeta (40-1) did not. Formation of ascorbate free radical was significantly increased by Abeta (1-42) in the presence of ceruloplasmin. Once cupric ion is reduced to cuprous ion, it can be oxidized by oxygen to generate superoxide radical or it can react with hydrogen peroxide to form hydroxyl radical. Hydrogen peroxide greatly increased the oxidation of cyclic hydroxylamines and ascorbate by cupric-amyloid beta peptide complexes, implying redox cycling of copper ions. Using the spin-trapping technique, we have shown that toxic amyloid beta peptides led to a 4-fold increase in copper-mediated hydroxyl radical formation. We conclude that toxic Abeta peptides do indeed stimulate copper-mediated oxidation of ascorbate and generation of hydroxyl radicals. Therefore, cupric-amyloid beta peptide-stimulated free radical generation may be involved in the pathogenesis of Alzheimer's disease.     

Determining the role of specific signaling molecules during lymphocyte development in vivo: instant transgenesis

A common method of determining the role of specific signaling molecules during lymphocyte development is to generate a transgenic mouse. This procedure, while informative, is time consuming, expensive and ultimately does not guarantee a defined answer. Here we present a protocol in which the in vivo effects of a gene of interest on both B and T lymphocyte development may be determined simultaneously in a relatively short time period. This is achieved by introducing a defined gene, such as a wild-type or mutated signaling molecule, into a lymphoid progenitor population by retroviral infection. The retrovirus generates a bicistronic message encoding the gene of interest and GFP, thus enabling identification of retrovirally transduced cells in subsequent lymphocyte lineages. The cells are then introduced into mice deficient for recombinase activating gene 1 (Rag-/- mice), thus allowing the development of donor-derived B and T lymphocytes in vivo. Using this technique, results can be obtained within 3-8 weeks.     

Exploring routes to stabilize a cationic pyridoxamine in an artificial transaminase: site-directed mutagenesis versus synthetic cofactors

Two artificial transaminases were assembled by linking a pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between alpha-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The k(cat) values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group.     

Multidomain Human Peroxidasin 1 Is a Highly Glycosylated and Stable Homotrimeric High Spin Ferric Peroxidase

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of −233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (k_cat/K_M^app) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking.