Long-term engraftment and expansion of tumor-derived memory T cells following the implantation of non-disrupted pieces of human lung tumor into NOD-scid IL2Rgamma(null) mice

Non-disrupted pieces of primary human lung tumor implanted into NOD-scid IL2Rgamma(null) mice consistently result in successful xenografts in which tissue architecture, including tumor-associated leukocytes, stromal fibroblasts, and tumor cells are preserved for prolonged periods with limited host-vs-graft interference. Human CD45(+) tumor-associated leukocytes within the xenograft are predominantly CD3(+) T cells with fewer CD138(+) plasma cells. The effector memory T cells that had been shown to be quiescent in human lung tumor microenvironments can be activated in situ as determined by the production of human IFN-gamma in response to exogenous IL-12. Plasma cells remain functional as evidenced by production of human Ig. Significant levels of human IFN-gamma and Ig were detected in sera from xenograft-bearing mice for up to 9 wk postengraftment. Tumor-associated T cells were found to migrate from the microenvironment of the xenograft to the lung, liver, and primarily the spleen. At 8 wk postengraftment, a significant portion of cells isolated from the mouse spleens were found to be human CD45(+) cells. The majority of CD45(+) cells were CD3(+) and expressed a phenotype consistent with an effector memory T cell, consisting of CD4(+) or CD8(+) T cells that were CD45RO(+), CD44(+), CD62L(-), and CD25(-). Following adoptive transfer into non-tumor bearing NOD-scid IL2Rgamma(null) mice, these human T cells were found to expand in the spleen, produce IFN-gamma, and maintain an effector memory phenotype. We conclude that the NOD-scid IL2Rgamma(null) tumor xenograft model provides an opportunity to study tumor and tumor-stromal cell interactions in situ for prolonged periods.     

Tyrosine modification enhances metal-ion binding

Tyrosine sulfation is a common modification of many proteins, and the ability to phosphorylate tyrosine residues is an intrinsic property of many growth-factor receptors. In the present study, we have utilized the peptide hormone CCK(8) (cholecystokinin), which occurs naturally in both sulfated and unsulfated forms, as a model to investigate the effect of tyrosine modification on metal-ion binding. The changes in absorbance and fluorescence emission on Fe(3+) binding indicated that tyrosine sulfation or phosphorylation increased the stoichiometry from 1 to 2, without greatly affecting the affinity (0.6-2.8 microM at pH 6.5). Measurement of Ca(2+) binding with a Ca(2+)-selective electrode revealed that phosphorylated CCK(8) bound two Ca(2+) ions. CCK(8) and sulfated CCK(8) each bound only one Ca(2+) ion with lower affinity. Binding of Ca(2+), Zn(2+) or Bi(3+) to phosphorylated CCK(8) did not cause any change in absorbance, but substantially increased the change in absorbance on subsequent addition of Fe(3+). The results of the present study demonstrate that tyrosine modification may increase the affinity of metal-ion binding to peptides, and imply that metal ions may directly regulate many signalling pathways.     

Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry

The GIVA phospholipase A(2) (PLA(2)) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA(2) activity is Ca(2+)-dependent in that calcium binding promotes protein docking to the phospholipid membrane. In addition, the catalytic domain has a lid that covers the active site, presumably regulating GIVA PLA(2) activity. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D) exchange coupled with liquid chromatography-mass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA(2). We also analyzed the changes in H/D exchange of the intact GIVA PLA(2) upon Ca(2+) binding. The DXMS results showed a fast H/D-exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the "cleft" region between the C2 and catalytic domains. The slow exchanging region of the C2 domain is in tight proximity to the catalytic domain. The effects of Ca(2+) binding on GIVA PLA(2) are localized in the C2 domain and suggest that binding of two distinct Ca(2+) ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step in GIVA PLA(2) activation.     

Prolonged root hypoxia induces ammonium accumulation and decreases the nutritional quality of tomato fruits

Here we examined the effects of root hypoxia (1-2% oxygen) on the physiology of the plant and on the biochemical composition of fruits in tomato (Solanum lycopersicum cv. Micro-Tom) plants submitted to gradual root hypoxia at first flower anthesis. Root hypoxia enhanced nitrate absorption with a concomitant release of nitrite and ammonium into the medium, a reduction of leaf photosynthetic activity and chlorophyll content, and an acceleration of fruit maturation, but did not affect final fruit size. Quantitative metabolic profiling of mature pericarp extracts by (1)H NMR showed that levels of major metabolites including sugars, organic acids and amino acids were not modified. However, ammonium concentration increased dramatically in fruit flesh, and ascorbate and lycopene concentrations decreased. Our data indicate that the unfavorable effects of root hypoxia on fruit quality cannot be explained by two of the well-known effects of root hypoxia on the plant, namely a decrease in photosynthesis or an excess in ethylene production, but may instead result from disturbances in the supply of either growth regulators or ammonium, by the roots.     

Expression of a modified Mannheimia haemolytica GS60 outer membrane lipoprotein in transgenic alfalfa for the development of an edible vaccine against bovine pneumonic pasteurellosis

The GS60 antigen is one of the protective antigens of Mannheimia haemolytica A1. GS60 contains conserved domains belonging to the LppC family of bacterial outer membrane lipoproteins. A high antibody titer to GS60 has been shown to be significantly correlated with resistance to pneumonic pasteurellosis. Calves vaccinated with a commercial vaccine (Presponse) and demonstrating protection against M. haemolytica A1 produced antibodies directed against GS60. Alfalfa was chosen as the platform for an edible vaccine. Agrobacterium tumefaciens was used to mediate the transformation of alfalfa with sequences encoding a slightly shortened derivative of the GS60 antigen (GS60(54)). Stable transgenic alfalfa lines were recovered and production of GS60(54) was examined by Western immunoblot analysis. The antigen is stable in dried transgenic plant material stored at ambient temperature for more than a year. The plant-produced GS60(54) protein was shown to be immunogenic when injected into rabbits. Feeding of the dried transgenic alfalfa expressing the GS60(54) to rabbits is capable of inducing seroconversion, suggesting that GS60(54) could be an effective oral antigen for stimulating mucosal immune responses.