Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3-4, 2003

To develop approaches to prophylaxis/protection, mitigation and treatment of radiation injuries, appropriate models are needed that integrate the complex events that occur in the radiation-exposed organism. While the spectrum of agents in clinical use or preclinical development is limited, new research findings promise improvements in survival after whole-body irradiation and reductions in the risk of adverse effects of radiotherapy. Approaches include agents that act on the initial radiochemical events, agents that prevent or reduce progression of radiation damage, and agents that facilitate recovery from radiation injuries. While the mechanisms of action for most of the agents with known efficacy are yet to be fully determined, many seem to be operating at the tissue, organ or whole animal level as well as the cellular level. Thus research on prophylaxis/protection, mitigation and treatment of radiation injuries will require studies in whole animal models. Discovery, development and delivery of effective radiation modulators will also require collaboration among researchers in diverse fields such as radiation biology, inflammation, physiology, toxicology, immunology, tissue injury, drug development and radiation oncology. Additional investment in training more scientists in radiation biology and in the research portfolio addressing radiological and nuclear terrorism would benefit the general population in case of a radiological terrorism event or a large-scale accidental event as well as benefit patients treated with radiation.     

Development of species diagnostic SNP markers for quality control genotyping in four rice (<Emphasis Type="Italic">Oryza</Emphasis> L.) species

Species misclassification (misidentification) and handling errors have been frequently reported in various plant species conserved at diverse gene banks, which could restrict use of germplasm for correct purpose. The objectives of the present study were to (i) determine the extent of genotyping error (reproducibility) on DArTseq-based single-nucleotide polymorphisms (SNPs); (ii) determine the proportion of misclassified accessions across 3134 samples representing three African rice species complex (Oryza glaberrima, O. barthii, and O. longistaminata) and an Asian rice (O. sativa), which are conserved at the AfricaRice gene bank; and (iii) develop species- and sub-species (ecotype)-specific diagnostic SNP markers for rapid and low-cost quality control (QC) analysis. Genotyping error estimated from 15 accessions, each replicated from 2 to 16 times, varied from 0.2 to 3.1%, with an overall average of 0.8%. Using a total of 3134 accessions genotyped with 31,739 SNPs, the proportion of misclassified samples was 3.1% (97 of the 3134 accessions). Excluding the 97 misclassified accessions, we identified a total of 332 diagnostic SNPs that clearly discriminated the three indigenous African species complex from Asian rice (156 SNPs), O. longistaminata accessions from both O. barthii and O. glaberrima (131 SNPs), and O. sativa spp. indica from O. sativa spp. japonica (45 SNPs). Using chromosomal position, minor allele frequency, and polymorphic information content as selection criteria, we recommended a subset of 24 to 36 of the 332 diagnostic SNPs for routine QC genotyping, which would be highly useful in determining the genetic identity of each species and correct human errors during routine gene bank operations.     

Characterization of toad liver glutathione transferase

The major form of glutathione transferase from the toad liver previously designed as Bufo bufo liver GST-7.6 (A. Aceto, B. Dragani, T. Bucciarelli, P. Sacchetta, F. Martini, S. Angelucci, F. Amicarelli, M. Miranda and C. Di Ilio, Biochem. J. 289 (1993) 417-422) has been characterized. According to its partial amino acid sequence, the toad enzyme may be included in the pi class GST and named bbGST P2-2. However, bbGST P2-2 appears to be immunologically, structurally and kinetically distinct from any other members of pi family, including bbGST P1-1, suggesting that it may constitute a subset of pi class GST. The data support the hypothesis that the transition from aquatic to terrestrial life causes a switch of the GST amphibian pattern promoting the expression of a GST form (bbGST P2-2) able to counteract, with higher efficiency, the toxic effects of reactive metabolites of oxidative metabolism and those of hydrophobic xenobiotics.     

Characterization and expression analysis of genes encoding alpha and beta carbonic anhydrases in Arabidopsis

Carbonic anhydrases (CAs) are Zn-containing metalloenzymes that catalyse the reversible hydration of CO(2). We investigated the alphaCA and betaCA families in Arabidopsis, which contain eight alphaCA (At alphaCA1-8) and six betaCA genes (At betaCA1-6). Analyses of expressed sequence tags (ESTs) from The Arabidopsis Information Resource (TAIR) database indicate that all the betaCA encoding sequences, but only three of the At alphaCA, are expressed. Using semi-quantitative PCR experiments, functional CA genes were more strongly expressed in green tissue, but strong expression was also found in roots for betaCA3, betaCA6 and alphaCA2. Two alphaCA genes were shown to respond to the CO(2) environment, while the others were unresponsive. Using the green fluorescent reporter protein gene fused with cDNA sequences coding for betaCAs, we provided evidence that betaCAs were targeted to specific subcellular compartments: betaCA1 and betaCA5 were targeted to the chloroplast, betaCA2 and betaCA3 to the cytosol, betaCA4 to the plasma membrane and betaCA6 to the mitochondria. The targeting and the pattern of gene expression suggest that CA isoforms play specific roles in subcellular compartments, tissues and organs. The data indicate that other CA isoforms than the well-characterized betaCA1 may contribute to the CO(2) transfer in the cell to the catalytic site of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco).     

Host T cells resist graft-versus-host disease mediated by donor leukocyte infusions

Delayed lymphocyte infusions (DLIs) are used to treat relapse occurring post bone marrow transplantation (BMT) and to increase the donor chimerism in recipients receiving nonmyeloablative conditioning. As compared with donor lymphocytes given early post-BMT, DLIs are associated with a reduced risk of graft-vs-host disease (GVHD). The mechanism(s) responsible for such resistance have remained incompletely defined. We now have observed that host T cells present 3 wk after lethal total body irradiation, at the time of DLI, contribute to DLI-GVHD resistance. The infusion of donor splenocytes on day 0, a time when host bone marrow (BM)-derived T cells are absent, results in greater expansion than later post-BMT when host and donor BM-derived T cells coexist. Selective depletion of host T cells with anti-Thy1 allelic mAb increased the GVHD risk of DLI, indicating that a Thy1(+) host T cell regulated DLI-GVHD lethality. The conditions by which host T cells are required for optimal DLI resistance were determined. Recipients unable to express CD28 or 4-1BB were as susceptible to DLI-GVHD as anti-Thy1 allelic mAb-treated recipients, indicating that CD28 and 4-1BB are critical to DLI-GVHD resistance. Recipients deficient in both perforin and Fas ligand but not individually were highly susceptible to DLI-GVHD. Recipients that cannot produce IFN-gamma were more susceptible to DLI-GVHD, whereas those deficient in IL-12 or p55 TNFRI were not. Collectively, these data indicate that host T cells, which are capable of generating antidonor CTL effector cells, are responsible for the impaired ability of DLI to induce GVHD. These same mechanisms may limit the efficacy of DLI in cancer therapy under some conditions.     

Demographic features, distribution, and habitat selection of the gray mouse opossum (Tlacuatzin canescens) in Colima, Mexico

In western Mexico, gray mouse opossums Tlacuatzin canescens typically are not only in tropical deciduous and semideciduous woodlands but also in croplands and orchards. We conducted mark-recapture studies in January 2003–2007 and 2010 in coastal, northern, and central Colima, Mexico. Each year, five grids, established in areas of thick vegetation within a mosaic of habitats, had 100 stations (10?×?10), each with two Sherman traps, one on the ground and another elevated 1–2 m. On 24 of 30 grids, 82 individuals were captured 126 times (85.7 % in elevated traps). Sex ratio did not deviate from 1:1; there was no sexual dimorphism in mass (average for males, 28.21 g; average for females, 25.64 g); 46.3 % of animals were adults, 27.3 % of adult females were reproductively active, and 77.5 % of males had scrotal testes. Distance from centroid of trap locations averaged 15.24 m, with mean minimum distance moved between captures being 33.19 m. Densities usually were low (0.67–8.03/ha), with the species widespread in habitats studied. We assessed 14 environmental characteristics for each station using ANOVA, logistic regression, and nonparametric multiplicative regression (NPMR) to characterize habitat selection. T. canescens was more likely found where percent grass was about 30 % and litter over 50 %, with height of canopy less than 10 m and about 40 % closed. NPMR, being able to recognize hump-shaped response curves where intermediate variable values are preferred, identified two variables (percent grass and percent canopy closed) not detected by other techniques as important in characterizing habitat selection of T. canescens.     

Modulation of E-cadherin monomer folding by cooperative binding of calcium ions

Classical cadherins are transmembrane glycoproteins involved in calcium-dependent cell-cell adhesion. Calcium ions are coordinated at the interface between successive modules of the cadherin ectodomain and are thought to regulate the adhesive interactions of cadherins when present at millimolar concentrations. It is widely accepted that calcium plays a critical role in cadherin-mediated cell-cell adhesion, but the nature of cadherin-calcium binding remains a matter of debate. We investigated the parameters of noncovalent cadherin-calcium binding, using the two N-terminal modules of E-cadherin (E/EC12) with a native N-terminal end and nondenaturing electrospray ionization mass spectrometry. By directly visualizing the molecular complexes, we demonstrated that E/EC12 binds three calcium ions, with an average KD of 20 +/- 0.7 microM. These calcium ions bound cooperatively to E/EC12 in its monomeric state, and these properties were not modified by an N-terminal extension consisting of a single methionine residue. This binding induced specific structural changes, as shown by assessments of protease sensitivity, circular dichroism, and mass spectrometry. Furthermore, the D103A mutation (a residue involved in E-cadherin adhesive function) modified calcium binding and led to a loss of cooperativity and the absence of structural changes, despite calcium binding. As the amino acids involved in calcium binding are found within the cadherin consensus motif, our findings may be relevant to other members of the cadherin family.     

Endogenous DNA damage clusters in human skin, 3-D model, and cultured skin cells

Clustered damages-two or more oxidized bases, abasic sites, or strand breaks on opposing DNA strands within a few helical turns-are formed in DNA by ionizing radiation. Clusters are difficult for cells to repair and thus pose significant challenges to genomic integrity. Although endogenous clusters were found in some permanent human cell lines, it was not known if clusters accumulated in human tissues or primary cells. Using high-sensitivity gel electrophoresis, electronic imaging, and number average length analysis, we determined endogenous cluster levels in DNA from human skin, a 3-D skin model, and primary cultured skin cells. DNA from dermis and epidermis of human skin contained extremely low levels of endogenous clusters (a few per gigabase). However, cultured skin fibroblasts and keratinocytes-whether in monolayer cultures or in 3-D model skin cultures-accumulated oxidized pyrimidine, oxidized purine, and abasic clusters. The levels of endogenous clusters were decreased by growing cells in the presence of selenium or by increasing cellular levels of Fpg protein, presumably by increasing processing of clustered damages. These results imply that the levels of endogenous clusters can be affected by the cells' external environment and their ability to deal with DNA damage.