A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans

BACKGROUND: Both animals and plants respond rapidly to pathogens by inducing the expression of defense-related genes. Whether such an inducible system of innate immunity is present in the model nematode Caenorhabditis elegans is currently an open question. Among conserved signaling pathways important for innate immunity, the Toll pathway is the best characterized. In Drosophila, this pathway also has an essential developmental role. C. elegans possesses structural homologs of components of this pathway, and this observation raises the possibility that a Toll pathway might also function in nematodes to trigger defense mechanisms or to control development. RESULTS: We have generated and characterized deletion mutants for four genes supposed to function in a nematode Toll signaling pathway. These genes are tol-1, trf-1, pik-1, and ikb-1 and are homologous to the Drosophila melanogaster Toll, dTraf, pelle, and cactus genes, respectively. Of these four genes, only tol-1 is required for nematode development. None of them are important for the resistance of C. elegans to a number of pathogens. On the other hand, C. elegans is capable of distinguishing different bacterial species and has a tendency to avoid certain pathogens, including Serratia marcescens. The tol-1 mutants are defective in their avoidance of pathogenic S. marcescens, although other chemosensory behaviors are wild type. CONCLUSIONS: In C. elegans, tol-1 is important for development and pathogen recognition, as is Toll in Drosophila, but remarkably for the latter r?le, it functions in the context of a behavioral mechanism that keeps worms away from potential danger.     

Assessment of the environmental fate of the biological control agent of fire blight, Pseudomonas fluorescens EPS62e, on apple by culture and real-time PCR methods

The colonization of apple blossoms and leaves by Pseudomonas fluorescens EPS62e was monitored in greenhouse and field trials using cultivable cell counting and real-time PCR. The real-time PCR provided a specific quantitative method for the detection of strain EPS62e. The detection level was around 10(2) cells g (fresh weight)(-1) and the standard curve was linear within a 5-log range. EPS62e actively colonized flowers reaching values from 10(7) to 10(8) cells per blossom. In apple flowers, no significant differences were observed between population levels obtained by real-time PCR and plating, suggesting that viable but nonculturable (VBNC) cells and residual nondegraded DNA were not present. In contrast, on apple leaves, where cultivable populations of EPS62e decreased with time, significant differences were observed between real-time PCR and plating. These differences indicate the presence of VBNC cells or nondegraded DNA after cell death. Therefore, the EPS62e population was under optimal conditions during the colonization of flowers but it was stressed and poorly survived on leaves. It was concluded that for monitoring this biological control agent, the combined use of cultivable cell count and real-time PCR is necessary.     

Long-Term Effects of RU24722 on Tyrosine Hydroxylase of the Rat Brain

The effects of RU24722 (14,15-dihydro-20,21-dinoreburnamine-14-ol) on tyrosine hydroxylase in central catecholaminergic neurons were studied in rats treated with different quantities of the molecule, and a time course was done for the minimal dose that gave the maximal effect. RU24722 induced increases in tyrosine hydroxylase activities and specific protein content in noradrenergic cells of the locus ceruleus and decreased all these parameters in dopaminergic neurons of the substantia nigra and ventral tegmental area. The results pointed out that the specific activity of newly synthesized tyrosine hydroxylase in the loci cerulei was potentially greater but was not expressed "in vivo" except 7 days after injection. The phenotypic specificity and the time course pattern of the action could be considered as a consequence of an induction mechanism. The comparison of long-term change in tyrosine hydroxylase values after piperoxane, RU24722, clonidine, and combined RU24722-clonidine treatment demonstrated that an activation during a few hours did not induce tyrosine hydroxylase in central noradrenergic neurons. Clonidine antagonized the activating effect of RU24722 following its injection but did not affect its long-term induction properties.     

Immunohistochemical evidence for neuronal and non-neuronal synthesis of GABA in the rat subcommissural organ

In the past few years, several studies have demonstrated in the rat subcommissural organ the presence of nerve endings and modified ependymocytes showing an uptake of [³H]GABA. The present work was performed to demonstrate in this cerebral zone the possibility of a GABA synthesis by the immunohistochemical localization of glutamate decarboxylase (GAD). GAD-positive reaction was detected with unlabelled antibody-enzyme peroxidase anti-peroxidase. Some nerve terminals containing either clear round vesicles, or sometimes clear round vesicles and some large granular vesicles, exhibited a positive staining. These terminals could belong to GABAergic inputs in the subcommissural organ. The few reactive terminals containing some granular vesicles could be related to the serotoninergic input as suggested previously (Gamrani et al., 1981). Several ependymocytes of this structure contained GAD-like positive reaction; these cells are also capable of taking up [³H]GABA (Gamrani et al., 1981) and present neuronal properties with regard to GABA. However, the presence in their cytoplasm of enolase, a specific glial marker, related them to glial elements. The presence of GABA in these ependymocytes suggests a modulating function of GABA on the secretory activity of the subcommissural organ.     

The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids

Rhizobia synthesize mono- N -acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation ( nod ) genes, including the nodABC genes present in all rhizobial species. The N -acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti , an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (ω-1) hydroxylated fatty acid, whereas in Rhizobium tropici , a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non-polar deletion of nodA , and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N -acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.     

Resistance to osmotic stress of horse spermatozoa: the role of ionic pumps and their relationship to cryopreservation success

To study the resistance of horse spermatozoa against hyperosmotic stress, cells were incubated in solutions of 600 to 4000 mOsm(undisturbed media). Then, semen was immediately placed into an iso-osmotic solution (disrupted media). Incubation in undisturbed media decreased sperm viability in an osmolarity- and temperature-dependent manner. Viability was further decreased in disrupted media, with the effect dependent upon the initial osmolarity of the media and on the temperature. Treatment with ouabain or amiloride impaired the resistance of horse spermatozoa to hyperosmotic stress. Very few correlations were strong between viability after hyperosomotic stress and quality parameters of fresh and frozen-thawed horse semen. The results indicate that the usefulness of resistance to hyperosmotic stress in assessing frozen-thawed semen quality is compromised, since other factors are involved in the resistance to freezing-thawing. Both Na (+)K (+) ATP-ase and the Na (+)H (+) antiporter act in the resistance to hyperosmotic stress in horse spermatozoa.     

Identification and characterization of a Pantoea citrea gene encoding glucose dehydrogenase that is essential for causing pink disease of pineapple.

Pantoea citrea, a member of the family Enterobacteriaceae, causes pink disease of pineapple, whose symptom is characterized by the formation of pink to brown discolorations of the infected portions of the pineapple fruit cylinder upon canning. Molecular genetic approaches were applied to elucidate the mechanism responsible for this fruit discoloration. A P. citrea mutant strain, CMC6, defective in its ability to cause pink disease and fruit discoloration, was generated by nitrosoguanidine mutagenesis. A DNA fragment that restored these activities was isolated by screening a genomic cosmid library of P. citrea. A large open reading frame of 2,361 bp, identified by nucleotide sequencing of a subclone of the complementing DNA, showed high similarities to identified genes encoding glucose dehydrogenase (GDH) in Escherichia coli, Acinetobacter calcoaceticus, and Gluconobacter oxydans. The predicted amino acid sequence of GDH of P. citrea was identical to known GDHs in these bacteria by 54, 44, and 34%, respectively. GDH of P. citrea has a predicted molecular mass of 86.2 kDa, contains a conserved binding domain for the cofactor pyrroloquinoline quinone, and possesses GDH activity as demonstrated by biochemical assay. GDH is the key branch point enzyme leading to the biosynthesis of gluconate, which in turn serves as the substrate leading to the formation of 2-ketogluconate, 2,5-diketogluconate, 6-phosphogluconate, and 2-keto-6-phosphogluconate. Addition of gluconate to CMC6 restores the juice- and fruit-discoloring activity. Although the pigments formed by heating (or canning) have not been identified, it is clear that GDH is one of the enzymes required for pigment formation leading to pink disease.