Differential recognition and scavenging of native and truncated macrophage-derived chemokine (macrophage-derived chemokine/CC chemokine ligand 22) by the D6 decoy receptor

The promiscuous D6 receptor binds several inflammatory CC chemokines and has been recently proposed to act as a chemokine-scavenging decoy receptor. The present study was designed to better characterize the spectrum of CC chemokines scavenged by D6, focusing in particular on CCR4 ligands and analyzing the influence of NH(2)-terminal processing on recognition by this promiscuous receptor. Using D6 transfectants, it was found that D6 efficiently bound and scavenged most inflammatory CC chemokines (CCR1 through CCR5 agonists). Homeostatic CC chemokines (CCR6 and CCR7 agonists) were not recognized by D6. The CCR4 agonists CC chemokine ligand 17 (CCL17) and CCL22 bound to D6 with high affinity. CCL17 and CCL22 have no agonistic activity for D6 (chemotaxis and calcium fluxes), but were rapidly scavenged, resulting in reduced chemotactic activity on CCR4 transfectants. CD26 mediates NH(2) terminus processing of CCL22, leading to the production of CCL22 (3-69) and CCL22 (5-69) that do not interact with CCR4. These NH(2)-terminal truncated forms of CCL22 were not recognized by D6. The results presented in this study show that D6 recognizes and scavenges a wide spectrum of inflammatory CC chemokines, including the CCR4 agonists CCL22 and CCL17. However, this promiscuous receptor is not engaged by CD26-processed, inactive, CCL22 variants. By recognizing intact CCL22, but not its truncated variants, D6 expressed on lymphatic endothelial cells may regulate the traffic of CCR4-expressing cells, such as dendritic cells.     

Analysis of integration sites of Jaagsiekte sheep retrovirus in ovine pulmonary adenocarcinoma

Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.     

The origin of the tRNA molecule: implications for the origin of protein synthesis

A model for tRNA molecule origin is discussed. The model postulates that this molecule originated simply by direct duplication (and subsequent evolution) of a gene coding for an RNA hairpin structure, which can thus be hypothesized as the evolutionary precursor of the tRNA molecule. The main properties are defined for these hairpin structures and it is suggested that these structures might have housed, near their 3' end, anticodons that were transferred to the loop of the tRNA anticodon during duplication of the hairpin structures. Moreover, the main characteristics are given for the evolutionary intermediary formed by direct duplication of the hairpin structure, i.e. the double hairpin. The evolutionary stages envisaged by this model for tRNA origin seem to naturally imply some evolutionary transitions through which the origin of protein synthesis passed. Finally, some strong historical evidence is provided to corroborate the model.     

Human papillomavirus type 16 E6 promotes retinoblastoma protein phosphorylation and cell cycle progression

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G(1)/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16(INK4a), CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16(INK4a). The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21(WAF1/CIP1) were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21(WAF1/CIP1) gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21(WAF1/CIP1) levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G(1) arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16(INK4a)) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.     

Hydrogen abstraction ability of different aromatic nitroxides

Indolinonic aromatic nitroxides have been shown to efficiently inhibit free radical mediated oxidation reactions in biological systems. Since all antioxidants also possess pro-oxidant activity, possibly through a hydrogen abstraction process from suitable substrates, the relative hydrogen abstraction abilities of these compounds were evaluated. Different hydrogen donors were reacted with an indolinic and two indolinonic nitroxides and the rates of hydrogen abstraction were determined using UV-Vis spectroscopy. From the data obtained, a structure-activity relationship was found. In addition, the hydrogen abstraction ability of these compounds was found to be much greater than that of the aliphatic nitroxide TEMPO, despite existing reports indicating that these two classes of compounds show similar antioxidant activities in biological systems.     

Isolation of functional RNA from small amounts of different grape and apple tissues

An efficient, simple, and small-scale procedure for isolating functional ribonucleic acid (RNA) was successfully applied to many different tissues of grape and apple. These woody plants are rich in polyphenolic compounds and polysaccharides that could impair the RNA extraction. The method chosen is based on the use of hot borate buffer at alkaline pH supplemented with several adjuvants and followed by selective precipitations. Starting with only 0.4 g of fresh tissue and working with small tubes (2 mL), we were able to obtain good yields of high-quality RNA suitable for further applications. The procedure can be proposed for many applications, and it is particularly highly recommended when isolating RNA from a large number of samples.     

Morphological responses to simulated wind in the genus Brassica (Brassicaceae): allopolyploids and their parental species

Thigmomorphogenesis refers to the widespread ability of sessile organisms to modify their morphology in response to a variety of mechanical stimulations, from direct contact with the stem by insects or other plants to flexure caused by wind, water, or snow. In this paper we investigated the differences in the reaction norms to wind exposure of seven species of the Brassicaceae that constitute a well-studied complex of known phylogenetic relationships. The goals included the characterization of differences between allopolyploids and their parental species and the comparison of wild and fast-cycling accessions within each species. We found statistically significant variation for plasticity among species or accessions for several characters, but the majority of the phenotypic variance was accounted for by overall (across-environment) differences among species and accessions and not by variation in plasticity. Allopolyploids displayed an array of behaviors when compared to their parents, from co-dominance to complete dominance to exceeding both parental means. Furthermore, fast-cycling plants showed distinct features from their wild relatives, suggesting that wild populations should be included with artificially selected lines in ecological studies. We proposed further steps to gain a more comprehensive understanding of thigmomorphogenetic responses, by integrating current research on the molecular bases of thigmomorphogenesis with insights into the ecology and evolution of plants exposed to wind.     

Domain 2 of the urokinase receptor contains an integrin-interacting epitope with intrinsic signaling activity: generation of a new integrin inhibitor

We investigated the interaction between the urokinase receptor (uPAR) and the integrin alphavbeta3. Vitronectin (VN) induces cell migration by binding to alphavbeta3, but expression of the uPAR boosts its efficacy. Thus, uPAR may regulate VN-induced cell migration by interacting laterally with alphavbeta3. In contrast, cells expressing a uPAR mutant lacking domain 2 do not migrate in response to VN. This effect is overcome by D2A, a synthetic peptide derived from the sequence of domain 2. In addition, D2A has chemotactic activity that requires alphavbeta3 and activates alphavbeta3-dependent signaling pathways such as the Janus kinase/Stat pathway. Moreover, D2A disrupts uPAR-alphavbeta3 and uPAR-alpha5beta1 co-immunoprecipitation, indicating that it can bind both of these integrins. We also identify the chemotactically active epitope harbored by peptide D2A. Mutating two glutamic acids into two alanines generates peptide D2A-Ala, which lacks chemotactic activity but inhibits VN-, FN-, and collagen-dependent cell migration. In fact, the GEEG peptide has potent chemotactic activity, and the GAAG sequence has inhibitory capacities. In summary, we have identified an integrin-interacting sequence located in domain 2 of uPAR, which is also a new chemotactic epitope that can activate alphavbeta3-dependent signaling pathways and stimulate cell migration. This sequence thus plays a pivotal role in the regulation of uPAR-integrin interactions. Moreover, we describe a novel, very potent inhibitor of integrin-dependent cell migration.