Angel Cebolla

The mitotic inhibitor ccs52 is required for endoreduplication and ploidy‐dependent cell enlargement in plants

Plant organs develop mostly post‐embryonically from persistent or newly formed meristems. After cell division arrest, differentiation frequently involves endoreduplication and cell enlargement. Factors controlling transition from mitotic cycles to differentiation programmes have not been identified yet in plants. Here we describe ccs52, a plant homologue of APC activators involved in mitotic cyclin degradation. The ccs52 cDNA clones were isolated from Medicago sativa root nodules, which exhibit the highest degree of endopolyploidy in this plant. ccs52 represents a small multigenic family and appears to be conserved in plants. Overexpression of ccs52 in yeast triggered mitotic cyclin degradation, cell division arrest, endoreduplication and cell enlargement. In Medicago, enhanced expression of ccs52 was found in differentiating cells undergoing endoreduplication. In transgenic M.truncatula plants, overexpression of the ccs52 gene in the antisense orientation resulted in partial suppression of ccs52 expression and decreased the number of endocycles and the volume of the largest cells. Thus, the ccs52 product may switch proliferating cells to differentiation programmes which, in the case of endocycles, result in cell size increments.

Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules

In Medicago nodules, endoreduplication cycles and ploidy-dependent cell enlargement occur during the differentiation of bacteroid-containing nitrogen-fixing symbiotic cells. These events are accompanied by the expression of ccs52A, a plant ortholog of the yeast and animal cdh1/srw1/fzr genes, acting as a substrate-specific activator of the anaphase-promoting complex (APC) ubiquitin ligase. Because CCS52A is involved in the transition of mitotic cycles to endoreduplication cycles, we investigated the importance of somatic endoploidy and the role of the M. truncatula ccs52A gene in symbiotic cell differentiation. Transcription analysis and ccs52A promoter–driven β-glucuronidase activity in transgenic plants showed that ccs52A was dispensable for the mitotic cycles and nodule primordium formation, whereas it was induced before nodule differentiation. The CCS52A protein was present in the nucleus of endoreduplication-competent cells, indicating that it may activate APC constitutively during the endoreduplication cycles. Downregulation of ccs52A in transgenic M. truncatula plants drastically affected nodule development, resulting in lower ploidy, reduced cell size, inefficient invasion, and the maturation of symbiotic cells, accompanied by early senescence and finally the death of both the bacterium and plant cells. Thus, ccs52A expression is essential for the formation of large highly polyploid symbiotic cells, and endoreduplication is an integral part of normal nodule development.

Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide

BACKGROUND Celiac disease is an immune-mediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Immunotoxic gluten peptides that are recalcitrant to degradation of digestive enzymes appear to trigger celiac syndromes. A 33-mer peptide from alpha-2 gliadin has been identified as a principal contributor to gluten immunotoxicity. A gluten-free diet is the usual first therapy for celiac disease patients; therefore, the characterization and quantification of the toxic portion of the gluten in foodstuffs is crucial to avoid celiac damage. OBJECTIVE We aimed to develop immunologic assays as a novel food analysis tool for measuring cereal fractions that are immunotoxic to celiac disease patients. DESIGN The design focused on the production of monoclonal antibodies against the gliadin 33-mer peptide and the development of enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis with the use of novel antibodies. RESULTS A sandwich ELISA method showed a detection limit for wheat, barley, and rye of <1 ppm prolamine. However, the method required a sample that was > or =1 order of magnitude greater for the detection of low-toxic oats, and there was no signal with the safe cereals maize and rice. A competitive ELISA method was also developed for detection of the toxic peptide in hydrolyzed food, which had a detection limit of <0.5 ppm gliadin. CONCLUSIONS Both ELISAs designed for use with the toxic gliadin 33-mer peptide suggested a high correlation between the presence of the peptide and the amount of cereal that was toxic to celiac disease patients. The sensitivity was significantly higher than that of equivalent methods recognizing other gluten epitopes.

Diversity in oat potential immunogenicity: basis for the selection of oat varieties with no toxicity in coeliac disease

Background and aims Coeliac disease (CD) is triggered by an abnormal reaction to gluten. Peptides resulting from partially digested gluten of wheat, barley or rye cause inflammation of the small intestinal mucosa. Previous contradictory studies suggest that oats may trigger the abnormal immunological response in patients with CD. Monoclonal antibodies (moAbs) against the main immunotoxic 33-mer peptide (A1 and G12) react strongly against wheat, barley and rye but have less reactivity against oats. The stated aim of this study is to test whether this observed reactivity could be related to the potential toxicity of oats for patients with CD. Methods In the present study, different oat varieties, controlled for their purity and by their distinct protein pattern, were used to examine differences in moAb G12 recognition by ELISA and western blot. Immunogenicity of oat varieties was determined by 33-mer concentration, T cell proliferation and interferon γ production. Results Three groups of oat cultivars reacting differently against moAb G12 could be distinguished: a group with considerable affinity, a group showing slight reactivity and a third with no detectable reactivity. The immunogenicity of the three types of oats as well as that of a positive and negative control was determined with isolated peripheral blood mononuclear T cells from patients with CD by measurement of cell proliferation and interferon γ release. A direct correlation of the reactivity with G12 and the immunogenicity of the different prolamins was observed. Conclusions The results showed that the reactivity of the moAb G12 is proportional to the potential immunotoxicity of the cereal cultivar. These differences may explain the different clinical responses observed in patients suffering from CD and open up a means to identify immunologically safe oat cultivars, which could be used to enrich a gluten-free diet.

Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide.

Background and Aims Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied. Methods Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin. Results The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease. Conclusions The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.

Modulation of gene expression through chromosomal positioning in Escherichia coli

Variations in expression of the nah genes of the NAH7 (naphthalene biodegradation) plasmid of Pseudomonas putida when placed in different chromosomal locations in Escherichia coli have been studied by employing a collection of hybrid mini-T5 transposons bearing lacZ fusions to the Psal promoter, along with the cognate regulatory gene nahR. Insertions of Psal-lacZ reporters in the proximity of the chromosomal origin of replication, oriC, increased accumulation of beta-galactosidase in vivo. Position-dependent changes in expression of the reporter product could not be associated with local variations of the supercoiling in the DNA region, as revealed by probing the chromosome with mobile gyrB-lacZ elements. Such variations in beta-galactosidase activity (and, therefore, the expression of catabolic genes) seemed, instead, to be linked to the increase in gene dosage associated with regions close to oriC, and not to local variations in chromosome structure. The tolerance of strains to the selection markers borne by the transposons also varied in parallel with the changes in LacZ levels. The role of chromosomal positioning as a mechanism for the outcome of adaptation phenotypes is discussed.

Effector Specificity Mutants of the Transcriptional Activator NahR of Naphthalene Degrading Pseudomonas Define Protein Sites Involved in Binding of Aromatic Inducers

This work reports a genetic analysis of the interactions between NahR, the LysR-type regulator of the NAH operons for biodegradation of naphthalene in Pseudomonas, and its aromatic effectors. Six mutants encoding NahR variants responsive to salicylate analogs such as benzoate, which is not an inducer for the wild type regulator, were isolated with a polymerase chain reaction-based saturation mutagenesis protocol. Most mutants displaying a specific change of effector profile bore single amino acid substitutions within a short protein segment of 60 residues located at the central portion of the NahR sequence. Some of the protein variants exhibited an increased affinity for salicylate and also for otherwise suboptimal effectors, with apparent Ks′ values 5-100-fold lower than those of the wild type NahR protein. In addition, all mutants were activated by inducers bearing novel substituents at positions 1 or 2 of the aromatic ring and displayed also an enhanced tolerance to changes at positions 3 and 4. Correlation between mutations in NahR and the structures of the new effectors suggested that protein sites Met116, Arg132, Asn169, and Arg248 are involved in effector recognition and binding during the earlier steps of the process leading to transcriptional activation of cognate NAH promoters.

Monitoring of gluten-free diet compliance in celiac patients by assessment of gliadin 33-mer equivalent epitopes in feces

Background: Certain immunotoxic peptides from gluten are resistant to gastrointestinal digestion and can interact with celiac-patient factors to trigger an immunologic response. A gluten-free diet (GFD) is the only effective treatment for celiac disease (CD), and its compliance should be monitored to avoid cumulative damage. However, practical methods to monitor diet compliance and to detect the origin of an outbreak of celiac clinical symptoms are not available. Objective: We assessed the capacity to determine the gluten ingestion and monitor GFD compliance in celiac patients by the detection of gluten and gliadin 33-mer equivalent peptidic epitopes (33EPs) in human feces. Design: Fecal samples were obtained from healthy subjects, celiac patients, and subjects with other intestinal pathologies with different diet conditions. Gluten and 33EPs were analyzed by using immunochromatography and competitive ELISA with a highly sensitive antigliadin 33-mer monoclonal antibody. Results: The resistance of a significant part of 33EPs to gastrointestinal digestion was shown in vitro and in vivo. We were able to detect gluten peptides in feces of healthy individuals after consumption of a normal gluten-containing diet, after consumption of a GFD combined with controlled ingestion of a fixed amount of gluten, and after ingestion of <100 mg gluten/d. These methods also allowed us to detect GFD infringement in CD patients. Conclusions: Gluten-derived peptides could be sensitively detected in human feces in positive correlation with the amount of gluten intake. These techniques may serve to show GFD compliance or infringement and be used in clinical research in strategies to eliminate gluten immunotoxic peptides during digestion. This trial was registered at clinicaltrials.gov as NCT01478867.

Two Classes of the Cdh1-Type Activators of the Anaphase-Promoting Complex in Plants: Novel Functional Domains and Distinct Regulation

The Cdc20 and Cdh1/Fzr proteins are the substrate-specific activators of the anaphase-promoting complex (APC). In Medicago truncatula, the MtCcs52A and MtCcs52B proteins represent two subgroups of the Cdh1-type activators, which display differences in their cell cycle regulation, structure, and function. The ccs52A transcripts are present in all phases of the cell cycle. By contrast, expression of ccs52B is restricted to late G2-phase and M-phase, and its induced overexpression in BY2 cells inhibited mitosis. MtCcs52A is active in Schizosaccharomyces pombe and binds to the S. pombe APC, whereas MtCcs52B does not because of differences in the N-terminal region. We identified a new functional domain, the Cdh1-specific motif conserved in the Cdh1 proteins that, in addition to the C-box and the terminal Ile and Arg residues, was essential for the activity and required for efficient binding to the APC. Moreover, we demonstrate that cyclin-dependent kinase phosphorylation sites adjacent to the C-box may regulate the interaction with the APC. In the different plant organs, the expression of Mtccs52A and Mtccs52B displayed differences and indicated the involvement of the APC in differentiation processes.

In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20–150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine–converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.