Cytological, genetic, and molecular analysis to characterize compatible and incompatible interactions between Medicago truncatula and Colletotrichum trifolii

In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.     

Thymulin and the neuroendocrine system

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. It consists of a nonapeptide component coupled to the ion zinc, which confers biological activity to this molecule. After its discovery in the early 1970, thymulin was characterized as a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Subsequently, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, an emerging core of information points to thymulin as a hypophysotropic peptide. Here we review the evidence supporting the hypothesis that thymulin is an important player in the hypophyso-thymic axis.     

Central and peripheral changes in catecholamine biosynthesis and turnover in rats after a short period of ozone exposure

We investigated in rat the effects of ozone exposure (0.7 ppm) for 5 h on the catecholamine biosynthesis and turnover in sympathetic efferents and various brain areas. For this purpose, the activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, was assessed in superior cervical ganglia and in two major noradrenergic cell groups, A2 and A6 (locus coeruleus). Tyrosine hydroxylase activity was estimated in vivo by measuring the accumulation of l-dihydroxyphenylalanine after pharmacological blockade of L-aromatic acid decarboxylases by NSD-1015 (100 mg/kg i.p.). The catecholamine turnover rate was measured after inhibition of tyrosine hydroxylase by alpha-methyl-para-tyrosine (AMPT, 250 mg/kg, i.p., 2.5 h) in peripheral sympathetic target organ (heart and lungs) as well as in some brain catecholamine terminal areas (cerebral cortex, hypothalamus and striatum). Ozone caused differential effects according to the structure. Catecholamine biosynthesis was stimulated in superior cervical ganglia (+44%, P < 0.05) and caudal A2 subset (+126%, P < 0.01), whereas catecholamine turnover was increased in heart (+183%, P < 0.01) and cortex (+22%, P < 0.05). On the other hand, catecholamine turnover was inhibited in lungs (-53%, P < 0.05) and striatum (-24%, P < 0.05). A brief exposure to ozone, at a concentration chosen to mimic pollution level encountered in urban areas, can modulate catecholamine biosynthesis and utilization rate in the sympathetic and central neurones.     

Visualization of gene expression of prolactin-receptor (PRL-R) by in situ hybridization in reproductive organs of<Emphasis Type="Italic"> Typhlonectes compressicauda</Emphasis>, a gymnophionan amphibian

The expression of the long form of prolactin receptor (PRL-R) mRNAs was studied in reproductive organs from both male and female Typhlonectes compressicauda, an amphibian, by quantitative in situ hybridization. These PRL-R mRNAs were expressed in all the organs studied. In ovarian tissue, mRNAs coding for the long form of PRL-R were strongly expressed in ovaries with compact and vascularized corpora lutea, i.e., during the middle and the end of pregnancy. During the other periods of cycle, sexual rest, vitellogenesis and beginning of pregnancy, expression of these mRNAs was lower than in other periods. In the testis, mRNAs coding for the long form of PRL-R were expressed in germ cells as well as in Leydig and Sertoli cells. In müllerian glands, important variations of expression in these mRNAs have been observed with a large increase during the breeding period and a decrease during sexual rest. Variations of gene expression of the long form of PRL-R for all tissues studied are correlated to synthesis of PRL in the pituitary in both male and female Typhlonectes compressicauda.     

Transposases are responsible for the target specificity of IS1397 and ISKpn1 for two different types of palindromic units (PUs)

Insertion sequences (IS)1397 and ISKpn1, found in Escherichia coli and Klebsiella pneumoniae, respectively, are IS3 family members that insert specifically into short palindromic repeated sequences (palindromic units or PUs). In this paper, we first show that although PUs are naturally absent from extrachromosomal elements, both ISs are able to transpose from the chromosome or from a plasmid into PUs artificially introduced into target plasmids. We also show that ISKpn1 target specificity is restricted to K.pneumoniae Z¹ PU type, whereas IS1397 target specificity is less stringent since the IS targets the three E.coli Y, Z¹ and Z² PU types indifferently. Experiments of transposition of both ISs driven by both transposases demonstrate that the inverted repeats flanking the ISs are not responsible for this target specificity, which is entirely due to the transposase itself. Implications on ISs evolution are presented.     

Crystal structure of the measles virus phosphoprotein domain responsible for the induced folding of the C-terminal domain of the nucleoprotein

Measles virus is a negative-sense, single-stranded RNA virus belonging to the Mononegavirales order which comprises several human pathogens such as Ebola, Nipah, and Hendra viruses. The phosphoprotein of measles virus is a modular protein consisting of an intrinsically disordered N-terminal domain (Karlin, D., Longhi, S., Receveur, V., and Canard, B. (2002) Virology 296, 251-262) and of a C-terminal moiety (PCT) composed of alternating disordered and globular regions. We report the crystal structure of the extreme C-terminal domain (XD) of measles virus phosphoprotein (aa 459-507) at 1.8 A resolution. We have previously reported that the C-terminal domain of measles virus nucleoprotein, NTAIL, is intrinsically unstructured and undergoes induced folding in the presence of PCT (Longhi, S., Receveur-Brechot, V., Karlin, D., Johansson, K., Darbon, H., Bhella, D., Yeo, R., Finet, S., and Canard, B. (2003) J. Biol. Chem. 278, 18638-18648). Using far-UV circular dichroism, we show that within PCT, XD is the region responsible for the induced folding of NTAIL. The crystal structure of XD consists of three helices, arranged in an anti-parallel triple-helix bundle. The surface of XD formed between helices alpha2 and alpha3 displays a long hydrophobic cleft that might provide a complementary hydrophobic surface to embed and promote folding of the predicted alpha-helix of NTAIL. We present a tentative model of the interaction between XD and NTAIL. These results, beyond presenting the first measles virus protein structure, shed light both on the function of the phosphoprotein at the molecular level and on the process of induced folding.     

Reversible inhibition of the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 by S-nitrosothiols

Human arylamine N-acetyltransferase 1 (NAT1) is a polymorphic phase II xenobiotic-metabolizing enzyme which catalyzes the biotransformation of primary aromatic amines, hydrazine drugs, and carcinogens. Structural and functional studies have shown that the NAT1 and factor XIII transglutaminase catalytic pockets are structurally related with the existence of a conserved catalytic triad (Cys-His-Asp). In addition, it has been reported that factor XIII transglutaminase activity could be regulated by nitric oxide (NO), in particular S-nitrosothiols (RSNO). We thus tested whether NAT1 could be a target of S-nitrosothiols. We show here that human NAT1 is reversibly inactivated by S-nitrosothiols such as SNAP (S-nitroso-N-acetyl-DL-penicillamine). A second-order rate constant for the inactivation of NAT1 by SNAP was determined (k(inact)=270M(-1)min(-1)) and shown to be in the same range of values reported for other enzymes. The inhibition of NAT1 by S-nitrosothiols was reversed by dithiothreitol and reduced glutathione, but not by ascorbate. As reported for some reactive cysteine-containing enzymes, our results suggest that inactivation of NAT1 by S-nitrosothiols is due to direct attack of the highly reactive cysteine residue in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between these NO-derived oxidants and NAT1. Finally, our findings suggest that, in addition to the polymorphic-dependent variation of NAT1 activity, NO-derived oxidants, in particular S-nitrosothiols, could also regulate NAT1 activity.     

Predicting the conformational states of cyclic tetrapeptides

Biologically active cyclic tetrapeptides, usually found among fungi metabolites, exhibit phytotoxic or cytostatic activities that are likely to be governed by specific conformations adopted in solution. For conformational studies and drug design, there is a strong interest in using fast and reliable methods to determine correctly the conformational population of cyclotetrapeptides. We show here that standard molecular mechanics computational approach gives satisfactory results. The method was validated step by step by experimental data either obtained after synthesis and NMR analysis, or found in the literature. The cyclo(Gly)(4), cyclo(Ala)(4), cyclo(Sar)(4), and cyclo(SarGly)(2) peptides were used to evaluate the prediction of the peptide backbone conformation, and the detailed conformational analysis of tentoxin, a natural phytotoxic cyclotetrapeptide in which N-alkylated peptide bonds alternate with regular secondary ones, was used to validate the computation of conformers proportions. From the knowledge of an initial cyclic primary structure and of the D or L configuration of the amino acids, we show that it is possible to determine the exact orientation of carbonyl groups and to predict the nature of conformers present in solution. The proportion of each conformer can be inferred from a statistical thermodynamics approach by using the potential energy values of each conformer, computed by molecular mechanics methods with the TRIPOS force field, which allowed us to account for the solvent. The solvent contribution was processed by two different methods according to the nature of the interactions: whether through the dielectric constant introduced in the electrostatic potential, when interaction with solute molecules are weak or negligible, or through the computation of free energy of solvation using the algorithm SILVERWARE for solvents explicitly interacting with the solute. When applied to tentoxin, this conformational analysis yielded results in very good agreement with the experimental data reported by Pinet et al. (Biopolymers, 1995, Vol. 36, pp. 135-152), on both the nature of existing conformers and their relative proportions, whatever the nature of the considered solvent.     

Low-level iron-dependent mutants of Listeria monocytogenes and their virulence in macrophages

Listeria monocytogenes is an opportunistic intracellular pathogen capable of growth that requires iron for growth within phagocytic cells and virulence expression. In the presence of an appropriate concentration tropolone, an iron-chelating agent, growth of L. monocytogenes is completely inhibited. However, this inhibition can be relieved by addition of dopamine, norepinephrine, or ferric citrate. By selection on streptonigrin medium supplemented with tropolone and norepinephrine, we have obtained two spontaneous mutants, Lm-8 and Lm-15, with the same iron dependence but lower iron dependence than the wild-type Lm-B38. The association between iron requirement and virulence of the two mutants and the wild type was studied in the J774 macrophage cell line. One hour after phagocytosis by the J774 macrophage cell line, the two mutants and the parental strain displayed no difference in the number of phagocytosed bacteria. Twenty-four hours after phagocytosis, the number of bacteria within the surviving macrophages was identical for the wild strain and the two clones. However, only 40% of macrophage cells infected with Lm-8 and 90% of those infected with Lm-15 were alive after 24 h in comparison with macrophage cells infected with the parental strain Lm-B38. These data demonstrate that there is no direct correlation between iron requirement and virulence of L. monocytogenes in the J774 macrophage cell line.     

An assessment of ADAMs in bone cells: absence of TACE activity prevents osteoclast recruitment and the formation of the marrow cavity in developing long bones

ADAMs (A Disintegrin And Metalloprotease domain) are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Since such events are critical for bone resorption and osteoclast recruitment, we investigated whether they require ADAMs. We report here which ADAMs we have identified in bone cells, as well as our analysis of the generation, migration and resorptive activity of osteoclasts in developing metatarsals of mouse embryos lacking catalytically active ADAM 17 [TNFalpha converting enzyme (TACE)]. The absence of TACE activity still allowed the generation of cells showing an osteoclastic phenotype, but prevented their migration into the core of the diaphysis and the subsequent formation of marrow cavity. This suggests a role of TACE in the recruitment of osteoclasts to future resorption sites.