Marta Pérez

Carbohydrate Hydrogen‐Bonding Cooperativity − Intramolecular Hydrogen Bonds and Their Cooperative Effect on Intermolecular Processes − Binding to a Hydrogen‐Bond Acceptor Molecule

The high hydroxy (OH) group content in carbohydrates makes the study of carbohydrate OH···XH and OH···X H-bond energetics fundamental to understanding of carbohydrate recognition. There is, however, a relative lack of knowledge concerning the factors that allow a carbohydrate to participate in recognition events stabilised by intermolecular H bonds. We therefore present here a systematic study on the factors that determine the formation of a well-defined intramolecular H-bonding network between carbohydrate hydroxy groups, and its cooperative or anti-cooperative influence on selected intermolecular processes mediated by H bonds. With this in mind, we first determined the H-bonding networks of a series of carbohydrate derivatives − monoalcohols, 1,2- and 1,3-diols and amidoalcohols − by 1H NMR and FT-IR spectroscopy. The hydroxy groups of these compounds showed different abilities to form intramolecular H bonds, depending on their relative positions and configurations on the pyranose ring, and on the nature of the adjacent functional groups. It has also been shown that both the directionality and strength of the intramolecular H-bonding network of a carbohydrate govern the formation of cooperative or anti-cooperative H-bond centres, with consequent repercussions on the thermodynamics of the intermolecular H-bonding interactions of the carbohydrate in question. From this study, some general rules for the prediction of the intramolecular H-bonding network characteristics of a given carbohydrate and its influence on the energetics of intended intermolecular recognition processes have been inferred. The results presented here give a new perspective over understanding of the role of the H-bonding interactions in carbohydrate recognition and have fundamental implications for the rational design of glycoconjugates incorporating H-bonding motifs with geometrical and electronic complementarity to given receptor molecules.

DNA demethylation and decrease on free polyamines is associated with the embryogenic capacity of Pinus nigra Arn. cell culture

Embryogenic cell lines initiated from immature zygotic embryos of Pinus nigra Arn. ssp. Austriaca were characterized in terms of macromorphological traits (colour, bipolar structures formation, germination ability) and their embryogenic potential was defined as high, medium or null. Quantification of global genomic DNA methylation revealed the existence of specific DNA methylation levels for the determinated embryogenic potentials. The line considered as effectively embryogenic, i.e., with the ability of develop the whole embryogenic program and producing plants, showed the lowest methylation levels. There was also proved the existence of an inverse relationship between total contents of free PAs and embryogenic potential, being the highest contents of free putrescine and spermidine in the non-embryogenic line and the lowest in the effectively embryogenic one. Relationships among DNA methylation levels, profiles of free individual polyamine contents and embryogenic potentials based on the ability to produce well-formed somatic embryos with effective plant conversion are discussed.

Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl β-1,6 glucosamine specific antibody production using biofilm-embedded bacteria

Staphylococcus aureus vaccines based on bacterins surrounded by slime, surface polysaccharides coupled to protein carriers and polysaccharides embedded in liposomes administered together with non-biofilm bacterins confer protection against mastitis. However, it remains unknown whether protective antibodies are directed to slime-associated known exopolysaccharides and could be produced in the absence of bacterin immunizations. Here, a sheep mastitis vaccination study was carried out using bacterins, crude bacterial extracts or a purified exopolysaccharide from biofilm bacteria delivered in different vehicles. This polysaccharide reacted specifically with antibodies to poly-N-acetyl-beta-1,6-glucosamine (PNAG) and not with antibodies to other capsular antigens or bacterial components. Following intra-mammary challenge with biofilm-producing bacteria, antibody production against the polysaccharide, milk bacterial counts and mastitis lesions were determined. Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula. Thus, bacterins from strong biofilm bacteria, rather than purified polysaccharide, are proposed as a cost-efficient vaccination against S. aureus ruminant mastitis.

DNA methylation during sexual embryogenesis and implications on the induction of somatic embryogenesis in Castanea sativa Miller.

From anthesis to mature seed formation, burrs from cross-pollinated adult Castanea sativa Miller trees were characterized and seven developmental stages defined based on macro and micromorphological traits. In order to get an insight into the involvement of epigenetic mechanisms in sexual embryogenesis and to define somatic embryogenesis induction capability, global DNA methylation and the somatic embryogenic competence were quantified. On cross-pollinated trees once fertilization takes place, at least one ovule per ovary becomes dominant, and transient DNA demethylation occurs coinciding with the start of the sexual embryogenic programme. Unfertilized ovules from the same cluster, which maintain their prior size, increase their methylation level and undergo degeneration. These results were validated using non-cross-pollinated trees and the asynchrony of flower receptivity. When testing in vitro somatic embryogenesis response of isolated dominant ovules and axes from zygotic embryos under cross-pollinated conditions, the highest competence was found for reaching seed maturity. Thus, a “developmental window” of somatic embryogenesis in chestnut has been characterized. It includes from fertilization to embryo maturity, and a transient decrease in methylation is necessary after fertilization for the development of the somatic embryogenesis response.

Tartrolon D, a Cytotoxic Macrodiolide from the Marine-Derived Actinomycete Streptomyces sp. MDG-04-17-069

Exploration of marine-derived actinomycetes as a source of antitumor compounds has led to the isolation of a new member of the tartrolon series, tartrolon D (4). This new compound was obtained from Streptomyces sp. MDG-04-17-069 fermentation broths and displayed strong cytotoxic activity against three human tumor cell lines. Additionally, the known compound ikarugamycin (5) was also found in the culture broths of the same microorganism. The structure of this new tartrolon was established by a combination of spectroscopic techniques (1D and 2D NMR, HRMS, and UV) as well as by comparison with published data for similar compounds.

IN VITRO DEVELOPMENT OF STAPHYLOCOCCUS AUREUS BIOFILMS USING SLIME-PRODUCING VARIANTS AND ATP-BIOLUMINESCENCE FOR AUTOMATED BACTERIAL QUANTIFICATION

In this work, a method was developed to establish Staphylococcus aureus biofilms on 96-well plates and automatically quantify viable cells within these biofilms by ATP-bioluminescence. Different strains were compared for biofilm formation. Cells from slime producing (SP) strain variants were more adherent (p < 0.001) and therefore more suitable for biofilm formation than non-slime producing original isolates. To compare biofilm support surfaces, SP biofilms were formed for 6, 24 and 48 h on 96-well polystyrene plates, containing wells coated with gelatin, poly-L-lysine or pre-treated for tissue culture and uncoated wells. Tissue culture-treated wells enhanced biofilm formation, allowing the highest growth (p < 0.001) in well-established biofilms (24 or 48 h old). For ATP quantification, the efficacy of different ATP extractants was compared: dimethyl sulphoxide (DMSO), trichloroacetic acid (TCA), a commercially available releasing reagent(R) (RR) and lysostaphin. A greater inhibitory effect on the ATP detection (p < 0.01), a more variable light emission (variation coefficient >/=50% vs. <19%, respectively) and a lower extraction efficiency (p < 0.05) were found in the case of TCA or lysostaphin in relation to RR or DMSO. DMSO was found preferable in relation to RR (upper detection limits 2.3 x 10(9) and 2 x 10(8) CFU/mL respectively) for bacterial ATP extraction from biofilms with high bacterial density. DMSO extracted ATP within seconds, light emission being stable for 6 h. The method developed allows automated viability determination of biofilm cells using bioluminescence and simultaneous study of factors affecting this viability (culture media, antibiotic types, antimicrobial concentrations, support surfaces and biofilm ages). It may be of use in bacteriological and antimicrobial research.

Development and hormonal regulation of mast cells in the Harderian gland of Syrian hamsters

SummaryThe morphological features and relative number of mast cells per mm2 were studied in the Harderian glands of male and female Syrian hamsters (Mesocricetus auratus) under different experimental conditions. The structural and ultrastructural characteristics of Harderian mast cells corresponded to those of connective tissue mast cells. The Harderian glands from female hamsters contained more mast cells than those of male hamsters. A subcutaneous implant of testosterone (2 mg/24 mg beeswax) resulted in a rapid decrease in the number of recognizable mast cells 6 h after the implantation. Neither orchidectomy nor ovariectomy significantly altered the relative number of mast cells. However, the daily subcutaneous injection of 20 IU of human chorionic gonadotropin during 20 days resulted in a significant decrease of identifiable mast cells. The administration of another steroid such as progesterone or the induction of states of hypo- and hyperthyroidism did not alter the distribution of mast cells in the Harderian glands of female Syrian hamsters.

Antitumor actinopyranones produced by Streptomyces albus POR-04-15-053 isolated from a marine sediment.

Four new antitumor pyranones, PM050511 (1), PM050463 (2), PM060054 (3), and PM060431 (4), were isolated from the cell extract of the marine-derived Streptomyces albus POR-04-15-053. Their structures were elucidated by a combination of spectroscopic methods, mainly 1D and 2D NMR and HRESIMS. They consist of an α-methoxy-γ-pyrone ring containing a highly substituted tetraene side chain glycosylated at C-10 in the case of 1 and 4. Compounds 1 and 4 displayed strong cytotoxicity against three human tumor cell lines with GI₅₀ values in the submicromolar range, whereas 2 showed subnanomolar activity as an inhibitor of EGFR-MAPK-AP1-mediated mitogenic signaling, causing inhibition of EGF-mediated AP1 trans-activation and EGF-mediated ERK activation and slight inhibition of EGF-mediated JNK activation. Taken together, these results suggest that members of the pyranone family of compounds could be developed as potential antitumor agents.

Tyramine biosynthesis is transcriptionally induced at low pH and improves the fitness of Enterococcus faecalis in acidic environments

Enterococcus faecalis is a commensal bacterium of the human gut that requires the ability to pass through the stomach and therefore cope with low pH. E. faecalis has also been identified as one of the major tyramine producers in fermented food products, where they also encounter acidic environments. In the present work, we have constructed a non-tyramine-producing mutant to study the role of the tyramine biosynthetic pathway, which converts tyrosine to tyramine via amino acid decarboxylation. Wild-type strain showed higher survival in a system that mimics gastrointestinal stress, indicating that the tyramine biosynthetic pathway has a role in acid resistance. Transcriptional analyses of the E. faecalis V583 tyrosine decarboxylase cluster showed that an acidic pH, together with substrate availability, induces its expression and therefore the production of tyramine. The protective role of the tyramine pathway under acidic conditions appears to be exerted through the maintenance of the cytosolic pH. Tyramine production should be considered important in the adaptability of E. faecalis to acidic environments, such as fermented dairy foods, and to survive passage through the human gastrointestinal tract.

The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis

Enterococci are considered mainly responsible for the undesirable accumulation of the biogenic amines tyramine and putrescine in cheeses. The biosynthesis of tyramine and putrescine has been described as a species trait in Enterococcus faecalis. Tyramine is formed by the decarboxylation of the amino acid tyrosine, by the tyrosine decarboxylase (TDC) route encoded in the tdc cluster. Putrescine is formed from agmatine by the agmatine deiminase (AGDI) pathway encoded in the agdi cluster. These biosynthesis routes have been independently studied, tyrosine and agmatine transcriptionally regulate the tdc and agdi clusters. The objective of the present work is to study the possible co-regulation among TDC and AGDI pathways in E. faecalis. In the presence of agmatine, a positive correlation between putrescine biosynthesis and the tyrosine concentration was found. Transcriptome studies showed that tyrosine induces the transcription of putrescine biosynthesis genes and up-regulates pathways involved in cell growth. The tyrosine modulation over AGDI route was not observed in the mutant Δtdc strain. Fluorescence analyses using gfp as reporter protein revealed PaguB (the promoter of agdi catabolic genes) was induced by tyrosine in the wild-type but not in the mutant strain, confirming that tdc cluster was involved in the tyrosine induction of putrescine biosynthesis. This study also suggests that AguR (the transcriptional regulator of agdi) was implicated in interaction among the two clusters.