Reverse Chemical Ecology Suggests Putative Primate Pheromones.

Pheromonal communication is widespread among living organisms, but in apes and particularly in humans there is currently no strong evidence for such phenomenon. Among primates, lemurs use pheromones to communicate within members of the same species, whereas in some monkeys such capabilities seem to be lost. Chemical communication in humans appears to be impaired by the lack or malfunctioning of biochemical tools and anatomical structures mediating detection of pheromones. Here, we report on a pheromone-carrier protein (SAL) adopting a "reverse chemical ecology" approach to get insights on the structures of potential pheromones in a representative species of lemurs (Microcebus murinus) known to use pheromones, Old-World monkeys (Cercocebus atys) for which chemical communication has been observed, and humans (Homo sapiens), where pheromones and chemical communication are still questioned. We have expressed the SAL orthologous proteins of these primate species, after reconstructing the gene encoding the human SAL, which is disrupted due to a single base mutation preventing its translation into RNA. Ligand-binding experiments with the recombinant SALs revealed macrocyclic ketones and lactones as the best ligands for all three proteins, suggesting cyclopentadecanone, pentadecanolide, and closely related compounds as the best candidates for potential pheromones. Such hypothesis agrees with the presence of a chemical very similar to hexadecanolide in the gland secretions of Mandrillus sphinx, a species closely related to C. atys. Our results indicate that the function of this carrier protein has not changed much during evolution from lemurs to humans, although its physiological role has been certainly impaired in humans.

Cross-linking reactions in food proteins and proteomic approaches for their detection.

Various protein cross-linking reactions leading to molecular polymerization and covalent aggregates have been described in processed foods. They are an undesired side effect of processes designed to reduce bacterial load, extend shelf life, and modify technological properties, as well as being an expected result of treatments designed to modify raw material texture and function. Although the formation of these products is known to affect the sensory and technological properties of foods, the corresponding cross-linking reactions and resulting protein polymers have not yet undergone detailed molecular characterization. This is essential for describing how their generation can be related to food processing conditions and quality parameters. Due to the complex structure of cross-linked species, bottom-up proteomic procedures developed to characterize various amino acid modifications associated with food processing conditions currently offer a limited molecular description of bridged peptide structures. Recent progress in cross-linking mass spectrometry for the topological characterization of protein complexes has facilitated the development of various proteomic methods and bioinformatic tools for unveiling bridged species, which can now also be used for the detailed molecular characterization of polymeric cross-linked products in processed foods. We here examine their benefits and limitations in terms of evaluating cross-linked food proteins and propose future scenarios for application in foodomics. They offer potential for understanding the protein cross-linking formation mechanisms in processed foods, and how the inherent beneficial properties of treated foodstuffs can be preserved or enhanced.

Streptomyces coelicolor Vesicles: Many Molecules To Be Delivered.

Streptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-positive bacteria that undergoes a complex life cycle and produces a broad repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view; this was achieved by combining microscopic, physical and -omics analyses. Two main populations of MVs, with different sizes and cargos, were isolated and purified. S. coelicolor MV cargo was determined to be complex, containing different kinds of proteins and metabolites. In particular, a total of 166 proteins involved in cell metabolism/differentiation, molecular processing/transport, and stress response were identified in MVs, the latter functional class also being important for bacterial morpho-physiological differentiation. A subset of these proteins was protected from degradation following treatment of MVs with proteinase K, indicating their localization inside the vesicles. Moreover, S. coelicolor MVs contained an array of metabolites, such as antibiotics, vitamins, amino acids, and components of carbon metabolism. In conclusion, this analysis provides detailed information on S. coelicolor MVs under basal conditions and on their corresponding content, which may be useful in the near future to elucidate vesicle biogenesis and functions. IMPORTANCE Streptomycetes are widely distributed in nature and characterized by a complex life cycle that involves morphological differentiation. They are very relevant in industry because they produce about half of all clinically used antibiotics, as well as other important pharmaceutical products of natural origin. Streptomyces coelicolor is a model organism for the study of bacterial differentiation and bioactive molecule production. S. coelicolor produces extracellular vesicles that carry many molecules, such as proteins and metabolites, including antibiotics. The elucidation of S. coelicolor extracellular vesicle cargo will help us to understand different aspects of streptomycete physiology, such as cell communication during differentiation and response to environmental stimuli. Moreover, the capability of these vesicles for carrying different kinds of biomolecules opens up new biotechnological possibilities related to drug delivery. Indeed, decoding the molecular mechanisms involved in cargo selection may lead to the customization of extracellular vesicle content.

The Odorant-Binding Proteins of the Spider Mite Tetranychus urticae.

Spider mites are one of the major agricultural pests, feeding on a large variety of plants. As a contribution to understanding chemical communication in these arthropods, we have characterized a recently discovered class of odorant-binding proteins (OBPs) in Tetranychus urticae. As in other species of Chelicerata, the four OBPs of T. urticae contain six conserved cysteines paired in a pattern (C1-C6, C2-C3, C4-C5) differing from that of insect counterparts (C1-C3, C2-C5, C4-C6). Proteomic analysis uncovered a second family of OBPs, including twelve members that are likely to be unique to T. urticae. A three-dimensional model of TurtOBP1, built on the recent X-ray structure of Varroa destructor OBP1, shows protein folding different from that of insect OBPs, although with some common features. Ligand-binding experiments indicated some affinity to coniferyl aldehyde, but specific ligands may still need to be found among very large molecules, as suggested by the size of the binding pocket.

Dehydrogenase/reductase activity of human carbonyl reductase 1 with NADP(H) acting as a prosthetic group.

Carbonyl reductase 1 (CBR1) is an NADP-dependent enzyme that exerts a detoxifying role, which catalyses the transformation of carbonyl-containing compounds. The ability of CBR1 to act on adducts between glutathione and lipid peroxidation derived aldehydes has recently been reported. In the present study, exploiting mass spectrometry and fluorescence spectroscopy, evidence is shown that CBR1 is able to retain NADP(H) at the active site even after extensive dialysis, and that this retention may also occur when the enzyme is performing catalysis. This property, together with the multi-substrate specificity of CBR1 in both directions of red/ox reactions, generates inter-conversion red/ox cycles. This particular feature of CBR1, in the case of the transformation of 3-glutathionyl, 4-hydroxynonanal (GSHNE), which is a key substrate of the enzyme in detoxification, supports the disproportionation reaction of GSHNE without any apparent exchange of the cofactor with the solution. The importance of the cofactor as a prosthetic group for other dehydrogenases exerting a detoxification role is discussed.

Proteome Alterations in Equine Osteochondrotic Chondrocytes.

Osteochondrosis is a failure of the endochondral ossification that affects developing joints in humans and several animal species. It is a localized idiopathic joint disorder characterized by focal chondronecrosis and growing cartilage retention, which can lead to the formation of fissures, subchondral bone cysts, or intra-articular fragments. Osteochondrosis is a complex multifactorial disease associated with extracellular matrix alterations and failure in chondrocyte differentiation, mainly due to genetic, biochemical, and nutritional factors, as well as traumas. This study describes the main proteomic alterations occurring in chondrocytes isolated from osteochondrotic cartilage fragments. A comparative analysis performed on equine osteochondrotic and healthy chondrocytes showed 26 protein species as differentially represented. In particular, quantitative changes in the extracellular matrix, cytoskeletal and chaperone proteins, and in cell adhesion and signaling molecules were observed in osteochondrotic cells, compared to healthy controls. Functional group analysis annotated most of these proteins in "growth plate and cartilage development", while others were included in "glycolysis and gluconeogenesis", "positive regulation of protein import", "cell-cell adhesion mediator activity", and "mitochondrion nucleoid". These results may help to clarify some chondrocyte functional alterations that may play a significant role in determining the onset and progression of equine osteochondrosis and, being related, of human juvenile osteochondrosis.

An integrated proteomic and metabolomic study to evaluate the effect of nucleus-cytoplasm interaction in a diploid citrus cybrid between sweet orange and lemon.

KEY MESSAGE: Our results provide a comprehensive overview how the alloplasmic condition might lead to a significant improvement in citrus plant breeding, developing varieties more adaptable to a wide range of conditions. Citrus cybrids resulting from somatic hybridization hold great potential in plant improvement. They represent effective products resulting from the transfer of organelle-encoded traits into cultivated varieties. In these cases, the plant coordinated array of physiological, biochemical, and molecular functions remains the result of integration among different signals, which derive from the compartmentalized genomes of nucleus, plastids and mitochondria. To dissect the effects of genome rearrangement into cybrids, a multidisciplinary study was conducted on a diploid cybrid (C2N), resulting from a breeding program aimed to improve interesting agronomical traits for lemon, the parental cultivars 'Valencia' sweet orange (V) and 'femminello' lemon (F), and the corresponding somatic allotetraploid hybrid (V + F). In particular, a differential proteomic analysis, based on 2D-DIGE and MS procedures, was carried out on leaf proteomes of C2N, V, F and V + F, using the C2N proteome as pivotal condition. This investigation revealed differentially represented protein patterns that can be associated with genome rearrangement and cell compartment interplay. Interestingly, most of the up-regulated proteins in the cybrid are involved in crucial biological processes such as photosynthesis, energy production and stress tolerance response. The cybrid differential proteome pattern was concomitant with a general increase of leaf gas exchange and content of volatile organic compounds, highlighting a stimulation of specific pathways that can be related to observed plant performances. Our results contribute to a better understanding how the alloplasmic condition might lead to a substantial improvement in plant breeding, opening new opportunities to develop varieties more adaptable to a wide range of conditions.

Pirin: A novel redox-sensitive modulator of primary and secondary metabolism in Streptomyces.

Pirins are evolutionarily conserved iron-containing proteins that are found in all kingdoms of life, and have been implicated in diverse molecular processes, mostly associated with cellular stress. In the present study, we started from the evidence that the insertional inactivation of pirin-like gene SAM23877_RS18305 (pirA) by ΦC31 Att/Int system-based vectors in spiramycin-producing strain Streptomyces ambofaciens ATCC 23877 resulted in marked effects on central carbon and energy metabolism gene expression, high sensitivity to oxidative injury and repression of polyketide antibiotic production. By using integrated transcriptomic, proteomic and metabolite profiling, together with genetic complementation, we here show that most of these effects could be traced to the inability of the pirA-defective strain to modulate beta-oxidation pathway, leading to an unbalanced supply of precursor monomers for polyketide biosynthesis. Indeed, in silico protein-protein interaction modeling and in vitro experimental validation allowed us to demonstrate that PirA is a novel redox-sensitive negative modulator of very long-chain acyl-CoA dehydrogenase, which catalyzes the first committed step of the beta-oxidation pathway.

Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity.

The proteome of liver biopsies from human obese (O) subjects has been compared to those of nonobese (NO) subjects using two-dimensional gel electrophoresis (2-DE). Differentially represented proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF) and nanoflow-liquid chromatography coupled to electrospray-tandem mass spectrometry (nLC-ESI-MS/MS). Overall, 61 gene products common to all of the liver biopsies were identified within 65 spots, among which 25 ones were differently represented between O and NO subjects. In particular, over-representation of short-chain acyl-CoA dehydrogenase, Δ(3,5)-Δ(2,4)dienoyl-CoA isomerase, acetyl-CoA acetyltransferase, glyoxylate reductase/hydroxypyruvate reductase, fructose-biphosphate aldolase B, peroxiredoxin I, protein DJ-1, catalase, α- and ß-hemoglobin subunits, 3-mercaptopyruvate S-transferase, calreticulin, aminoacylase 1, phenazine biosynthesis-like domain-containing protein and a form of fatty acid-binding protein, together with downrepresentation of glutamate dehydrogenase, glutathione S-transferase A1, S-adenosylmethionine synthase 1A and a form of apolipoprotein A-I, was associated with the obesity condition. Some of these metabolic enzymes and antioxidant proteins have already been identified as putative diagnostic markers of liver dysfunction in animal models of steatosis or obesity, suggesting additional investigations on their role in these syndromes. Their differential representation in human liver was suggestive of their consideration as obesity human biomarkers and for the development of novel antiobesity drugs.

Elucidating the molecular physiology of lantibiotic NAI-107 production in Microbispora ATCC-PTA-5024.

BACKGROUND: The filamentous actinomycete Microbispora ATCC-PTA-5024 produces the lantibiotic NAI-107, which is an antibiotic peptide effective against multidrug-resistant Gram-positive bacteria. In actinomycetes, antibiotic production is often associated with a physiological differentiation program controlled by a complex regulatory and metabolic network that may be elucidated by the integration of genomic, proteomic and bioinformatic tools. Accordingly, an extensive evaluation of the proteomic changes associated with NAI-107 production was performed on Microbispora ATCC-PTA-5024 by combining two-dimensional difference in gel electrophoresis, mass spectrometry and gene ontology approaches. RESULTS: Microbispora ATCC-PTA-5024 cultivations in a complex medium were characterized by stages of biomass accumulation (A) followed by biomass yield decline (D). NAI-107 production started at 90 h (A stage), reached a maximum at 140 h (D stage) and decreased thereafter. To reveal patterns of differentially represented proteins associated with NAI-107 production onset and maintenance, differential proteomic analyses were carried-out on biomass samples collected: i) before (66 h) and during (90 h) NAI-107 production at A stage; ii) during three time-points (117, 140, and 162 h) at D stage characterized by different profiles of NAI-107 yield accumulation (117 and 140 h) and decrement (162 h). Regulatory, metabolic and unknown-function proteins, were identified and functionally clustered, revealing that nutritional signals, regulatory cascades and primary metabolism shift-down trigger the accumulation of protein components involved in nitrogen and phosphate metabolism, cell wall biosynthesis/maturation, lipid metabolism, osmotic stress response, multi-drug resistance, and NAI-107 transport. The stimulating role on physiological differentiation of a TetR-like regulator, originally identified in this study, was confirmed by the construction of an over-expressing strain. Finally, the possible role of cellular response to membrane stability alterations and of multi-drug resistance ABC transporters as additional self-resistance mechanisms toward the lantibiotic was confirmed by proteomic and confocal microscopy experiments on a Microbispora ATCC-PTA-5024 lantibiotic-null producer strain which was exposed to an externally-added amount of NAI-107 during growth. CONCLUSION: This study provides a net contribution to the elucidation of the regulatory, metabolic and molecular patterns controlling physiological differentiation in Microbispora ATCC-PTA-5024, supporting the relevance of proteomics in revealing protein players of antibiotic biosynthesis in actinomycetes.

Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk, Patella caerulea.

A magnesium-dependent cysteinyl-glycine hydrolyzing enzyme from the gastropod mollusk Patella caerulea was purified to electrophoretic homogeneity through a simple and rapid purification protocol. The molecular masses of the native protein and the subunit suggest that the enzyme has a homohexameric structure. Structural data in combination with kinetic parameters determined with Cys-Gly and compared with Leu-Gly as a substrate, indicate that the purified enzyme is a member of the peptidase family M17. The finding that an enzyme of the peptidase family M17 is responsible also in mollusks for the breakdown of Cys-Gly confirms the important role of this peptidase family in the glutathione metabolism.

Flavonoid Interaction with a Chitinase from Grape Berry Skin: Protein Identification and Modulation of the Enzymatic Activity.

In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab) was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR) class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

Non-enzymatic glycation and glycoxidation protein products in foods and diseases: an interconnected, complex scenario fully open to innovative proteomic studies.

The Maillard reaction includes a complex network of processes affecting food and biopharmaceutical products; it also occurs in living organisms and has been strictly related to cell aging, to the pathogenesis of several (chronic) diseases, such as diabetes, uremia, cataract, liver cirrhosis and various neurodegenerative pathologies, as well as to peritoneal dialysis treatment. Dozens of compounds are involved in this process, among which a number of protein-adducted derivatives that have been simplistically defined as early, intermediate and advanced glycation end-products. In the last decade, various bottom-up proteomic approaches have been successfully used for the identification of glycation/glycoxidation protein targets as well as for the characterization of the corresponding adducts, including assignment of the modified amino acids. This article provides an updated overview of the mass spectrometry-based procedures developed to this purpose, emphasizing their partial limits with respect to current proteomic approaches for the analysis of other post-translational modifications. These limitations are mainly related to the concomitant sheer diversity, chemical complexity, and variable abundance of the various derivatives to be characterized. Some challenges to scientists are finally proposed for future proteomic investigations to solve main drawbacks in this research field.

Tryptophan promotes morphological and physiological differentiation in Streptomyces coelicolor.

The molecular mechanisms regulating tryptophan biosynthesis in actinomycetes are poorly understood; similarly, the possible roles of tryptophan in the differentiation program of microorganism life-cycle are still underexplored. To unveil the possible regulatory effect of this amino acid on gene expression, an integrated study based on quantitative teverse transcription-PCR (qRT-PCR) and proteomic approaches was performed on the actinomycete model Streptomyces coelicolor. Comparative analyses on the microorganism growth in a minimal medium with or without tryptophan supplementation showed that biosynthetic trp gene expression in S. coelicolor is not subjected to a negative regulation by the presence of the end product. Conversely, tryptophan specifically induces the transcription of trp genes present in the biosynthetic gene cluster of the calcium-dependent antibiotic (CDA), a lipopeptide containing D- and L-tryptophan residues. In addition, tryptophan stimulates the transcription of the CDA gene cluster regulator cdaR and, coherently, CDA production. Surprisingly, tryptophan also promotes the production of actinorhodin, another antibiotic that does not contain this amino acid in its structure. Combined 2D-DIGE and nano liquid chromatography electrospray linear ion trap tandem mass spectrometry (LC-ESI-LIT-MS/MS) analyses revealed that tryptophan exerts a growth-stage-dependent global effect on S. coelicolor proteome, stimulating anabolic pathways and promoting the accumulation of key factors associated with morphological and physiological differentiation at the late growth stages. Phenotypic observations by scanning electron microscopy and spore production assays demonstrated an increased sporulation in the presence of tryptophan. Transcriptional analysis of catabolic genes kynA and kynB suggested that the actinomycete also uses tryptophan as a carbon and nitrogen source. In conclusion, this study originally provides the molecular basis underlying the stimulatory effect of tryptophan on the production of antibiotics and morphological development program of this actinomycete.

Temporal analysis of poplar woody root response to bending stress.

Temperate perennial woody plants use different environmental signals to coordinate their growth and development in relation to seasonal changes. Preliminary evidences suggest that, even during dormancy, plants maintain effective metabolic activities and molecular mechanisms ensuring them an eventual recording of mechanical loads during winter times. Despite their great importance for productivity and survival, plant biology investigations have poorly characterized the root growth cycle and its response to environmental stresses. In this study, we describe the proteomic changes occurring over the time in poplar root either in the absence or in response to a bending stress; corresponding expression of cell cycle regulator and auxin transporter genes was also evaluated by reverse transcription polymerase chain reaction analysis. Our results confirm previous evidences on the effect of the bending stress on the anticipation of root growth resumption, providing additional insights on a temporal modulation of various plant metabolic processes involved in dormancy break, growth resumption and stress response in the bent root; these events seem related to the differential compression and tension force distribution occurring over the plant taproot.

Albumen and Yolk Plasma Peptidomics for the Identification and Quantitation of Bioactive Molecules and the Quality Control of Hen Egg Products.

Hen eggs and the corresponding food products are essential components of human diet. In addition to supplying basic nutrients, they contain functional peptides that are released in vivo within the intact raw material following physiological proteolytic events affecting specific proteins or derive from technological processing of albumen and yolk fractions as a result of the dedicated use of proteases from plant and microbial sources. Besides their potential importance for functional applications, peptides released under physiological conditions in intact egg can be used as markers of product storage and deterioration. Therefore, characterization and quantitation of peptides in egg and egg-derived products can be used to implement evaluation of potential bioactivities as well as to assess food product qualitative characteristics. Here, we provide dedicated information on extraction, identification, and quantitative analysis of peptides from albumen and yolk plasma; nano-liquid chromatography-mass spectrometry combined with bioinformatic analysis of resulting raw data by different software tools allowed to assign molecules based on database searching and to evaluate their relative quantity in different samples.

Integrated omics approach reveals the molecular pathways activated in tomato by Kocuria rhizophila, a soil plant growth-promoting bacterium.

Plant microbial biostimulants application has become a promising and eco-friendly agricultural strategy to improve crop yields, reducing chemical inputs for more sustainable cropping systems. The soil dwelling bacterium Kocuria rhizophila was previously characterized as Plant Growth Promoting Bacteria (PGPB) for its multiple PGP traits, such as indole-3-acetic acid production, phosphate solubilization capability and salt and drought stress tolerance. Here, we evaluated by a multi-omics approach, the PGP activity of K. rhizophila on tomato, revealing the molecular pathways by which it promotes plant growth. Transcriptomic analysis showed several up-regulated genes mainly related to amino acid metabolism, cell wall organization, lipid and secondary metabolism, together with a modulation in the DNA methylation profile, after PGPB inoculation. In agreement, proteins involved in photosynthesis, cell division, and plant growth were highly accumulated by K. rhizophila. Furthermore, "amino acid and peptides", "monosaccharides", and "TCA" classes of metabolites resulted the most affected by PGPB treatment, as well as dopamine, a catecholamine neurotransmitter mediating plant growth through S-adenosylmethionine decarboxylase (SAMDC), a gene enhancing the vegetative growth, up-regulated in tomato by K. rhizophila treatment. Interestingly, eight gene modules well correlated with differentially accumulated proteins (DAPs) and metabolites (DAMs), among which two modules showed the highest correlation with nine proteins, including a nucleoside diphosphate kinase, and cytosolic ascorbate peroxidase, as well as with several amino acids and metabolites involved in TCA cycle. Overall, our findings highlighted that sugars and amino acids, energy regulators, involved in tomato plant growth, were strongly modulated by the K. rhizophila-plant interaction.

Characterization of carbonic anhydrase IX interactome reveals proteins assisting its nuclear localization in hypoxic cells.

Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface.

Transcriptomic and proteomic analysis of a compatible tomato-aphid interaction reveals a predominant salicylic acid-dependent plant response.

BACKGROUND: Aphids are among the most destructive pests in temperate climates, causing significant damage on several crops including tomato. We carried out a transcriptomic and proteomic study to get insights into the molecular mechanisms and dynamics of the tomato response to the Macrosyphum euphorbiae aphid. RESULTS: The time course analysis of aphid infestation indicated a complex, dynamic pattern of gene expression. Several biological functions were affected and genes related to the stress and defence response were the most represented. The Gene Ontology categories of the differentially expressed genes (899) and identified proteins (57) indicated that the tomato response is characterized by an increased oxidative stress accompanied by the production of proteins involved in the detoxification of oxygen radicals. Aphids elicit a defense reaction based on the cross-communication of different hormone-related signaling pathways such as those related to the salicylic acid (SA), jasmonic acid (JA), ethylene and brassinosteroids. Among them, the SA-signaling pathway and stress-responsive SA-dependent genes play a dominant role. Furthermore, tomato response is characterized by a reduced accumulation of photosynthetic proteins and a modification of the expression of various cell wall related genes. CONCLUSIONS: Our work allowed a more comprehensive understanding of the signaling events and the defense dynamics of the tomato response to aphids in a compatible interaction and, based on experimental data, a model of the tomato-aphid molecular interaction was proposed. Considering the rapid advancement of tomato genomics, this information will be important for the development of new protection strategies.

Cladosporol a stimulates G1-phase arrest of the cell cycle by up-regulation of p21(waf1/cip1) expression in human colon carcinoma HT-29 cells.

Cladosporols, purified and characterized as secondary metabolites from Cladosporium tenuissimum, display an antifungal activity. In this study, we tested the antiproliferative properties of cladosporol A, the main isoform of this metabolite family, against human cancer cell lines. By assessing cell viability, we found that cladosporol A inhibits the growth of various human colon cancers derived cell lines (HT-29, SW480, and CaCo-2) in a time- and concentration-dependent manner, specifically of HT-29 cells. The reduced cell proliferation was due to a G1-phase arrest, as assessed by fluorescence activated cell sorting analysis on synchronized HT-29 cells, and was associated with an early and robust over-expression of p21(waf1/cip1) , the well-known cyclin-dependent kinases inhibitor. This suggests that the drug may play a role in the control of cancer cell proliferation. Consistently, cyclin D1, cyclin E, CDK2, and CDK4 proteins were reduced and histone H1-associated CDK2 kinase activity inhibited. In addition to p21(waf1/cip1) , exposure to 20 µM cladosporol A caused a simultaneous increase of pERK and pJNK, suggesting that this drug activates a circuit that integrates cell cycle regulation and the signaling pathways both involved in the inhibition of cell proliferation. Finally, we showed that the increase of p21(waf1/cip1) expression was generated by a Sp1-dependent p53-independent stimulation of its gene transcription as mutagenesis of the Sp1 binding sites located in the p21 proximal promoter abolished induction. To our knowledge, this is the first report showing that cladosporol A inhibits colon cancer cell proliferation by modulating p21(waf1/cip1) expression.