Proteomic Profiling Unravels Insights into the Molecular Background Underlying Increased <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>-Tolerance of <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity. Requests concerning seeds from the Medicago truncatula lines F83.005-5 and F83.005-9 should be addressed to Jean-Marie Prospéri, INRA-SGAP Laboratory, Laboratoire de Ressources Génétiques et d’Amélioration des Luzernes méditerranéennes, Mauguio, France, jean-marie.prosperi@ensam.inra.fr.     

Ablation of beta1 integrin in mammary epithelium reveals a key role for integrin in glandular morphogenesis and differentiation

Integrin-mediated adhesion regulates the development and function of a range of tissues; however, little is known about its role in glandular epithelium. To assess the contribution of beta1 integrin, we conditionally deleted its gene in luminal epithelia during different stages of mouse mammary gland development and in cultured primary mammary epithelia. Loss of beta1 integrin in vivo resulted in impaired alveologenesis and lactation. Cultured beta1 integrin-null cells displayed abnormal focal adhesion function and signal transduction and could not form or maintain polarized acini. In vivo, epithelial cells became detached from the extracellular matrix but remained associated with each other and did not undergo overt apoptosis. beta1 integrin-null mammary epithelial cells did not differentiate in response to prolactin stimulation because of defective Stat5 activation. In mice where beta1 integrin was deleted after the initiation of differentiation, fewer defects in alveolar morphology occurred, yet major deficiencies were also observed in milk protein and milk fat production and Stat5 activation, indicating a permissive role for beta1 integrins in prolactin signaling. This study demonstrates that beta1 integrin is critical for the alveolar morphogenesis of a glandular epithelium and for maintenance of its differentiated function. Moreover, it provides genetic evidence for the cooperation between integrin and cytokine signaling pathways.     

The evolutionarily related beta-barrel polypeptide transporters from Pisum sativum and Nostoc PCC7120 contain two distinct functional domains

Several beta-barrel-type channels are involved in the translocation or assembly of outer membrane proteins of bacteria or endosymbiotically derived organelles. Here we analyzed the functional units of the beta-barrel polypeptide transporter Toc75 (translocon in outer envelope of chloroplasts) of the outer envelope of chloroplasts and of a protein, alr2269, from Nostoc PCC7120 with homology to Toc75, both proteins having a similar domain organization. We demonstrated that the N-terminal region functions as a recognition and complex assembly unit, whereas the C terminus forms the beta-barrel-type pore. The pore region is, in turn, modulated by the N terminus of the proteins. The protein from Nostoc PCC7120, which shares a common ancestor with Toc75, is able to recognize precursor proteins destined for chloroplasts. In contrast, the recognition of peripheral translocon subunits by Toc75 is a novel feature acquired through evolution.     

In vivo reconstitution of the FhuA transport protein of Escherichia coli K-12

The FhuA protein in the outer membrane of Escherichia coli actively transports ferrichrome and the antibiotics albomycin and rifamycin CGP 4832 and serves as a receptor for the phages T1, T5, and phi80 and for colicin M and microcin J25. The crystal structure reveals a beta-barrel with a globular domain, the cork, which closes the channel formed by the barrel. Genetic deletion of the cork resulted in a beta-barrel that displays no FhuA activity. A functional FhuA was obtained by cosynthesis of separately encoded cork and the beta-barrel domain, each endowed with a signal sequence, which showed that complementation occurs after secretion of the fragments across the cytoplasmic membrane. Inactive complete mutant FhuA and an FhuA fragment containing 357 N-proximal amino acid residues complemented the separately synthesized wild-type beta-barrel to form an active FhuA. Previous claims that the beta-barrel is functional as transporter and receptor resulted from complementation by inactive complete FhuA and the 357-residue fragment. No complementation was observed between the wild-type cork and complete but inactive FhuA carrying cork mutations that excluded the exchange of cork domains. The data indicate that active FhuA is reconstituted extracytoplasmically by insertion of separately synthesized cork or cork from complete FhuA into the beta-barrel, and they suggest that in wild-type FhuA the beta-barrel is formed prior to the insertion of the cork.     

Induction of primary virus-cross-reactive human immunodeficiency virus type 1-neutralizing antibodies in small animals by using an alphavirus-derived in vivo expression system

We have studied the induction of neutralizing antibodies by in vivo expression of the human immunodeficiency virus type 1 (HIV-1) envelope by using a Venezuelan equine encephalitis virus (VEE) replicon system with mice and rabbits. The HIV-1 envelope, clone R2, has broad sensitivity to cross-reactive neutralization and was obtained from a donor with broadly cross-reactive, primary virus-neutralizing antibodies (donor of reference serum, HIV-1-neutralizing serum 2 [HNS2]). It was expressed as gp160, as secreted gp140, and as gp160deltaCT with the cytoplasmic tail deleted. gp140 was expressed in vitro at a high level and was predominantly uncleaved oligomer. gp160deltaCT was released by cells in the form of membrane-bound vesicles. gp160deltaCT induced stronger neutralizing responses than the other forms. Use of a helper plasmid for replicon particle packaging, in which the VEE envelope gene comprised a wild-type rather than a host range-adapted sequence, also enhanced immunogenicity. Neutralizing activity fractionated with immunoglobulin G. This activity was cross-reactive among a panel of five nonhomologous primary clade B strains and a Chinese clade C strain and minimally reactive against a Chinese clade E (circulating recombinant form 1) strain. The comparative neutralization of these strains by immune mouse sera was similar to the relative neutralizing effects of HNS2, and responses induced in rabbits were similar to those induced in mice. Together, these results demonstrate that neutralizing antibody responses can be induced in mice within 2 to 3 months that are similar in potency and cross-reactivity to those found in the chronically infected, long-term nonprogressive donor of HNS2. These findings support the expectation that induction of highly cross-reactive HIV-1 primary virus-neutralizing activity by vaccination may be realized.     

Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections

The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.     

Blockade of geranylgeranylation by rosuvastatin upregulates eNOS expression in human venous endothelial cells

Endothelial dysfunction is associated with a reduction in nitric oxide (NO) bioavailability. Positive effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on the improvement of endothelial dysfunction have been shown. We investigated the effects of rosuvastatin and isoprenoid metabolites on endothelial NO synthase (eNOS) mRNA and protein expression in human umbilical venous endothelial cells after exposure to 10(-8)-10(-5) mol/l rosuvastatin for 8 and 12 h. Cell viability was not significantly altered after exposure to the statin for 12h. In a concentration-dependent manner, rosuvastatin upregulated eNOS mRNA and protein expression. The effects on eNOS expression mediated through rosuvastatin could be reversed by treatment with mevalonate indicating inhibition of HMG-CoA reductase as the underlying mechanism. Treatment with geranylgeranylpyrophosphate, but not farnesylpyrophosphate, reversed the increase of eNOS expression induced by rosuvastatin. Rosuvastatin may have beneficial effects on endothelial dysfunction associated with cardiovascular diseases beyond its effects on lowering cholesterol.     

Stochastic tunnels in evolutionary dynamics

We study a situation that arises in the somatic evolution of cancer. Consider a finite population of replicating cells and a sequence of mutations: type 0 can mutate to type 1, which can mutate to type 2. There is no back mutation. We start with a homogeneous population of type 0. Mutants of type 1 emerge and either become extinct or reach fixation. In both cases, they can generate type 2, which also can become extinct or reach fixation. If mutation rates are small compared to the inverse of the population size, then the stochastic dynamics can be described by transitions between homogeneous populations. A "stochastic tunnel" arises, when the population moves from all 0 to all 2 without ever being all 1. We calculate the exact rate of stochastic tunneling for the case when type 1 is as fit as type 0 or less fit. Type 2 has the highest fitness. We discuss implications for the elimination of tumor suppressor genes and the activation of genetic instability. Although our theory is developed for cancer genetics, stochastic tunnels are general phenomena that could arise in many circumstances.