T-DNA tagging in the model legume Medicago truncatula allows efficient gene discovery

The annual legume Medicago truncatula has been proposed as a model plant to study various aspects of legume biology including rhizobial and mycorrhizal symbiosis because it is well suited for the genetic analysis of these processes . To facilitate the characterization of M. truncatula genes participating in various developmental processes we have initiated an insertion mutagenesis program in this plant using three different T-DNAs as tags. To investigate which type of vector is the most suitable for mutagenesis we compared the behavior of these T-DNAs. One T-DNA vector was a derivative of pBin19 and plant selection was based on kanamycin resistance. The two other vectors carried T-DNA conferring Basta resistance in the transgenic plants. For each T-DNA type, we determined the copy number in the transgenic lines, the structure of the T-DNA loci and the sequences of the integration sites. The T-DNA derived from pBin19 generated complex T-DNA insertion patterns. The two others generally gave single copy T-DNA inserts that could result in gene fusions for the pGKB5 T-DNA. Analysis of the T-DNA borders revealed that several M. truncatula genes were tagged in these transgenic lines and in vivo gus fusions were also obtained. These results demonstrate that T-DNA tagging can efficiently be used in M. truncatula for gene discovery.     

Subunits asymmetry in the ternary complex of lamb liver 6-phosphogluconate dehydrogenase detected by a NADP analogue.

Incubation of lamb liver 6-phosphogluconate dehydrogenase, a dimeric enzyme with periodate-oxidized NADP causes the inactivation of the enzyme due to the covalent binding of 2 mol of inhibitor/mol of dimer. In the presence of substrate, the inactivation is faster and is almost complete after the labelling of only one subunit. These results not only confirm the hypothesis of a 'half-of-the-sites' mechanism of action of the enzyme, but also suggest that the formation of the ternary complex (enzyme-substrate-coenzyme) in one subunit causes a conformational change that makes the other subunit unable to bind the coenzyme (and even the adenylic part of it) and, thus, this subunit becomes inactive. It appears that while one subunit catalyses the oxidation of 6-phosphogluconate the other is inactive in this reaction.     

SslE Elicits Functional Antibodies That Impair In Vitro Mucinase Activity and In Vivo Colonization by Both Intestinal and Extraintestinal Escherichia coli Strains

SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslE_IHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslE_IHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.     

A simplified procedure for the isolation of a highly active crystalline glucose-6-phosphate dehydrogenase from Candida utilis

A simple procedure for the isolation of crystalline glucose-6-phosphate dehydrogenase from dried Candida is described. The procedure allows the simultaneous purification of phosphogluconate dehydrogenase and ribose phosphate isomerase. The glucose phosphate dehydrogenase is electrophoretically homogeneous; its specific activity is about twice that of the best preparations reported previously. Isoelectric focusing over a distance of 1 m reveals 2 peaks of glucose-6-phosphate dehydrogenase activity.     

Seroprevalence of HBV and HCV markers among intravenous drug abusers and subjects affected by chronic active hepatitis

In order to investigate the seroprevalence of HBV and HCV in neapolitan area, we studied blood specimens of 180 IVDAs, 115 CAH patients and 72 healthy subjects using hepatitis B core (anti-HBs) and hepatitis C antibody (anti-HCV). High frequency of anti-HBs (80.9%) and strong association with an unexpected seroprevalence of anti-HCV (67%) was found in CAH patients. Our study does not explain these results, but suggest either a possible inference between both viruses or that serum from CAH subjects contains a component that gives false positivity.     

Landscape fragmentation and habitat suitability in endangered Italian hare (<Emphasis Type="Italic">Lepus corsicanus</Emphasis>) and European hare (<Emphasis Type="Italic">Lepus europaeus</Emphasis>) populations

During the last years, the population of Italian hare decreased significantly in central and south Italy. This is imputable to harvest, poaching, habitat fragmentation, and the probable competition with congeneric European hare introduced in the last decades by man for hunt. The goal of our work is to define the ecological characteristics of the two aforementioned species in order to understand how landscape facilitates or impedes movement. Spatially explicit models are used to identify a species ecological niche and to build a landscape model of suitability. To validate ecological modeling of landscape, we performed a population genetic analysis. Results suggest that the Italian hare shows an ecological requirement close to average of available resources in the considered landscape. The genetic structure of this autochthonous species validates the habitat suitability model and highlights the differences with European hare. This work analyzes for the first time the ecological relationship between those two sympatric species.     

Specific DNA binding and regulation of its own expression by the AidB protein in Escherichia coli

Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. While the biological roles of the Ada, AlkA, and AlkB proteins have been defined, despite many efforts, the molecular functions of AidB remain largely unknown. In this study, we focused on the biological role of the AidB protein, and we demonstrated that AidB shows preferential binding to a DNA region that includes the upstream element of its own promoter, PaidB. The physiological significance of this specific interaction was investigated by in vivo gene expression assays, demonstrating that AidB can repress its own synthesis during normal cell growth. We also showed that the domain architecture of AidB is related to the different functions of the protein: the N-terminal region, comprising the first 439 amino acids (AidB "I-III"), possesses FAD-dependent dehydrogenase activity, while its C-terminal domain, corresponding to residues 440 to 541 (AidB "IV"), displays DNA binding activity and can negatively regulate the expression of its own gene in vivo. Our results define a novel role in gene regulation for the AidB protein and underline its multifunctional nature.