Generation and characterisation of functional recombinant antibody fragments against RNA replicase NIb from plum pox virus

A monoclonal antibody (mAb 2A) able to react against the RNA replicase NIb from plum pox virus (PPV) was obtained and used for generating a specific scFv fragment. The VH and VL coding sequences were cloned and expressed as a fusion scFv protein to alkaline phosphatase. This fusion protein was able to recognise viral NIb in both Western and tissue-print ELISA blots. The affinity and specificity of scFv2A for NIb was similar to that of the parental mAb and the region YLEAFY from PPV-NIb was identified by PEPSCAN assay as the putative epitope. Isolated VH domains from scFv2A were also expressed as fusion to alkaline phosphatase. However, their ability to react against NIb was greatly altered. scFv2A fragments were transiently expressed in the cytosol of Nicotiana benthamiana and although they accumulated to low levels, inhibition-ELISA results indicated that they retained antigen-binding activity.     

Leads for development of new naphthalenesulfonate derivatives with enhanced antiangiogenic activity: crystal structure of acidic fibroblast growth factor in complex with 5-amino-2-naphthalene sulfonate

Inhibition of angiogenesis-promoting factors such as fibroblast growth factors is considered to be a potential procedure for inhibiting solid tumor growth. Although several peptide-based inhibitors are currently under study, the development of antiangiogenic compounds of small molecular size is a pharmacological goal of considerable interest. We have already shown that certain naphthalene sulfonates constitute minimal functional substitutes of the antiangiogenic compounds of the suramin and suradista family. Using those data as a lead, we have carried out a rational search for new angiogenesis inhibitors that could provide new pharmacological insights for the development of antiangiogenic treatments. The results of the study strongly underline the relevance of the stereochemistry for an efficient inhibition of acidic fibroblast growth factor mitogenic activity by the naphthalene sulfonate family and allow us to formulate rules to aid in searching for new inhibitors and pharmaceutical developments. To provide further leads for such developments and acquire a detailed insight into the basis of the inhibitory activity of the naphthalene sulfonate derivatives, we solved the three-dimensional structure of acidic fibroblast growth factor complexed to 5-amino-2-naphthalenesulfonate, the most pharmacologically promising of the identified inhibitors. The structure shows that binding of this compound would hamper the interaction of acidic fibroblast growth factor with the different components of the cell membrane mitogenesis-triggering complex.     

A novel membrane-associated glycovariant of BEHAB/brevican is up-regulated during rat brain development and in a rat model of invasive glioma

BEHAB (brain-enriched hyaluronan-binding protein)/brevican is the most abundant chondroitin sulfate proteoglycan in the extracellular matrix of the adult rat brain. BEHAB/brevican expression is up-regulated coincident with glial cell proliferation and/or motility, including during early central nervous system development and in invasive glioma. An understanding of the molecular interactions that mediate BEHAB/brevican function is still in its infancy because of the existence of several BEHAB/brevican isoforms, each of which may mediate different functions. Here, we describe a novel BEHAB/brevican isoform, B/b130, and demonstrate that it is neither the glycosylphosphatidylinositol-linked splice variant of BEHAB/brevican nor a cleavage product of the full-length protein (B/b150). B/b130 is an underglycosylated isoform of BEHAB/brevican, lacking glycosaminoglycan chains as well as most of the sugars that invest B/b150. B/b130 localizes exclusively to the particulate fraction of rat brain and associates with the cell membrane by a previously undescribed calcium-independent mechanism. In addition, B/b130 is the major isoform of BEHAB/brevican that is up-regulated in a rat model of invasive glioma and may therefore contribute to the invasive ability of glioma cells. Further understanding of BEHAB/brevican isoforms will advance our knowledge of the function of this ECM component and may help identify new potential therapeutic targets for primary brain tumors.     

Release of arachidonic acid by stimulation of opsonic receptors in human monocytes: the FcgammaR and the complement receptor 3 pathways

The role of the opsonic receptors FcgammaR and CR3 on the release of arachidonic acid (AA) by human monocytes was studied using IgG-ovalbumin (OVA) equivalence immune complexes (IC), anti-OVA IgG bound to OVA-coupled latex beads, and C3bi-bound IC. Release of AA was produced by IC and latex-OVA beads bound to IgG, whereas binding of C3bi to IC inhibited the ability of IC to release AA. In contrast, coating of zymosan particles with C3bi enhanced AA release as compared with that produced by non-coated particles. Masking of C3bi on C3bi-bound IC by incubation with anti-C3 IgG resulted in the recovery of their ability to release AA, thereby suggesting that binding of C3b by IC reduces their flogogenic effects, whereas opsonization of microbial walls by complement may enhance their proinflammatory potential. The binding/uptake of opsonized zymosan particles was inhibited by anti-CR3 Ab and C3bi-bound IC, but not by beta-glucan, mannan, and anti-Toll-like receptor 2 Ab. These findings show that cooperative engagement of CR3 on both the lectin-like site involved in beta-glucan binding and the I-domain involved in C3bi binding, as it can be observed in the innate immune response, produces AA release, whereas the unique interaction of C3bi-bound IC with the I-domain of CR3, as it may occur in the adaptive immune response, diverts the IC lattice from a productive interaction with FcgammaR linked to AA release.     

WW domain HECT E3s target Cbl RING finger E3s for proteasomal degradation

Cbl proteins have RING finger-dependent ubiquitin ligase (E3) activity that is essential for down-regulation of tyrosine kinases. Here we establish that two WW domain HECT E3s, Nedd4 and Itch, bind Cbl proteins and target them for proteasomal degradation. This is dependent on the E3 activity of the HECT E3s but not on that of Cbl. Consistent with these observations, in cells expressing the epidermal growth factor receptor, Nedd4 reverses Cbl-b effects on receptor down-regulation, ubiquitylation, and proximal events in signaling. Cbl-b also targets active Src for degradation in cells, and Nedd4 similarly reverses Cbl-mediated Src degradation. These findings establish that RING finger E3s can be substrates, not only for autoubiquitylation but also for ubiquitylation by HECT E3s and suggest an additional level of regulation for Cbl substrates including protein-tyrosine kinases.     

Regulation of trophoblast beta1-integrin expression by contact with endothelial cells

Background  

In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade uterine matrix, and enter the uterine vasculature. Invasive trophoblasts show increased expression of β1 integrin. Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. To test this, we used an in vitro system consisting of early gestation macaque trophoblasts co-cultured on top of uterine microvascular endothelial cells.     

Genetic diversity of bradyrhizobial populations from diverse geographic origins that nodulate Lupinus spp. and Ornithopus spp

The genetic diversity of 45 bradyrhizobial isolates that nodulate several Lupinus and Ornithopus species in different geographic locations was investigated by 16S rDNA PCR-RFLP and sequence analysis, 16S-23S rDNA intergenic spacer (IGS) PCR-RFLP analysis, and ERIC-PCR genomic fingerprinting. Reference strains of Bradyrhizobium japonicum, B. liaoningense and B. elkanii and some Canarian isolates from endemic woody legumes in the tribe Genisteae were also included. The 16S rDNA-RFLP analysis resolved 9 genotypes of lupin isolates, a group of fourteen isolates presented restriction-genotypes identical or very similar to B. japonicum, while another two main groups of isolates (69%) presented genotypes that clearly separated them from the reference species of soybean. 16S rDNA sequencing of representative strains largely agreed with restriction analysis, except for a group of six isolates, and showed that all the lupin isolates are relatives of B. japonicum, but different lineages were observed. The 16S-23S IGS-RFLP analysis showed a high resolution level, resolving 19 distinct genotypes among 30 strains analysed, and so demonstrating the heterogeneity of the 16S-RFLP groups. ERIC-PCR fingerprint analysis showed an enormous genetic diversity producing a different pattern for each but two of the isolates. Phylogeny of nodC gene was independent from the 16S rRNA phylogeny, and showed a tight relationship in the symbiotic region of the lupin isolates with isolates from Canarian genistoid woody legumes, and in concordance, cross-nodulation was found. We conclude that Lupinus is a promiscuous host legume that is nodulated by rhizobia with very different chromosomal genotypes, which could even belong to several species of Bradyrhizobium. No correlation among genomic background, original host plant and geographic location was found, so, different chromosomal genotypes could be detected at a single site and in a same plant species, on the contrary, an identical genotype was detected in very different geographical locations and plants.     

A survey of temperature and pH effect on colonial growth of Botryodiplodia theobromae RC1

Study of fungal colonial growth is a basic method to examine their behaviour in different cultivation conditions. The influence of temperature and initial pH on growth radial velocity and growth density of Botryodiplodia theobromae RC1, was studied in order to show the growth characteristics of this fungus. Both temperature and culture medium influenced growth density, but radial velocity of growth was only affected by temperatures above 40 degrees C. In addition, initial pH of culture media did not affect either parameter.