Enzymatic synthesis of 3'-hydroxyacetaminophen catalyzed by tyrosinase

3'-Hydroxyacetaminophen, a catechol metabolite of N-acetyl-p-aminophenol (acetaminophen) and N-acetyl-m-aminophenol (a structural analogue of acetaminophen and considered as a possible alternative because it is not hepatotoxic), is enzymatically synthesized for the first time using mushroom tyrosinase. Although reported to be weakly hepatotoxic in vivo, this catechol derivative of acetaminophen is not commercially available. This compound was obtained from its monophenolic precursor, acetaminophen, using the enzyme tyrosinase in the presence of an excess of ascorbic acid, thus reducing back the o-quinone product of catalytic activity to the catechol acetaminophen derivative. A mathematical model of the system is proposed, which is also applicable to the tyrosinase-mediated synthesis of any o-diphenolic compound from its corresponding monophenol. This synthesis procedure is continuous, easy to perform and control, and adaptable to a bioreactor with the immobilized enzyme for industrial purposes in a nonpolluting way.     

Self-incompatibility genotypes in almond re-evaluated by PCR,stylar ribonucleases,sequencing analysis and controlled pollinations

As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbí, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S₅ and S₁₀ alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S₅ allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S₂₅ This new S allele, previously reported to be S₁₀, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S₂S₂₅), Ardechoise (S₁S₂₅), Desmayo Largueta (S₁S₂₅), Ferrastar (S₂S₂₅) and Gabaix (S₁₀S₂₅). The S alleles of Garbí, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.     

Home range of greater and lesser rhea in Argentina : relevance to conservation

In this work we report results of radiotracking studies on the movements and home range sizes of two near-threatened species, the greater rhea (Rhea americana) and the lesser rhea (Pterocnemia pennata pennata) in relation to different land use regimes. We radiomonitored greater and lesser rheas for 3 years in their respective habitats: the Pampas and the Patagonia regions. We chose two study areas in each habitat with similar agricultural activities and different hunting control. We did not find significant differences in movements and home range size between study areas of each species. This suggests that disturbance caused by human presence in the areas did not affect rhea spacing behaviors. Moreover, lesser rheas showed larger home range and movements than greater rheas, showing that the home range size is not an immutable property of body mass, and that abundance and distribution of food appears to be the main factor that influences the movements and home range size of these birds.     

Validation of a constraint-based model of <Emphasis Type="Italic">Pichia pastoris</Emphasis> metabolism under data scarcity

Background  

Constraint-based models enable structured cellular representations in which intracellular kinetics are circumvented. These models, combined with experimental data, are useful analytical tools to estimate the state exhibited (the phenotype) by the cells at given pseudo-steady conditions.     

Fine structure of the branchial epithelium in the teleost Oreochromis niloticus

We have studied the gill epithelium of Oreochromis niloticus using transmission electron microscopy with the particular interested relationship between cell morphology and osmotic, immunoregulatory, or other non‐regulatory functions of the gill. Pavement cells covered the filament epithelium and lamellae of gills, with filament pavement cells showing distinct features from lamellar pavement cells. The superficial layer of the filament epithelium was formed by osmoregulatory elements, the columnar mitochondria‐rich, mucous and support cells, as well as by their precursors. Light mitochondria‐rich cells were located next to lamellae. They exhibited an apical crypt with microvilli and horizontal small dense rod‐like vesicles, sealed by tight junctions to pavement cells. Dark mitochondria‐rich cells had long dense rod‐like vesicles and a small apical opening sealed by tight junctions to pavement cells. The deep layer of the filament epithelium was formed by a network of undifferentiated cells, containing neuroepithelial and myoepithelial cells, macrophage and eosinophil‐like cells and their precursors, as well as precursors of mucous cells. The lateral‐basal surface was coated by myoepithelial cells and a basal lamina. The lamellar blood lacunae was lined by pillar cells and surrounded by a basal lamina and pericytes. The data presented here support the existence of two distinct types of pavement cells, mitochondria‐rich cells, and mitochondria‐rich cells precursors, a structural role for support cells, a common origin for pavement cells and support cells, a paracrine function for neuroepithelial cells in the superficial layer, and the control of the lamellar capillary base by endocrine and contractile cells. Data further suggest that the filament superficial layer is involved in gill osmoregulation, that may interact, through pale mitochondria‐rich cells, with the deep layer and lamellae, whereas the deep layer, through immune and neuroendocrine systems, acts in the regeneration and defense of the tissue. J. Morphol. 2010. © 2010 Wiley‐Liss, Inc.     

Effect of the sesterterpene-type metabolites, ophiobolins A and B, on zygomycetes fungi

Ophiobolins are sesterterpene-type phytotoxins produced by fungi belonging mainly to the genus Bipolaris. In this study, the antifungal effect of ophiobolins A and B on different zygomycetes has been examined. Depending on the zygomycete tested, MIC values of 3.175-50 μg mL(-1) were found for ophiobolin A and 25-50 μg mL(-1) for ophiobolin B. Ophiobolin A inhibited sporangiospore germination of Mucor circinelloides and caused morphological changes; the fungus formed degenerated, thick or swollen cells with septa. Cytoplasm effusions from the damaged cells were also observed. Fluorescence microscopy after annexin and propidium iodide staining of the treated cells suggested that the drug induced an apoptosis-like cell death process in the fungus.     

Solution Structure of Human Growth Arrest and DNA Damage 45α (Gadd45α) and Its Interactions with Proliferating Cell Nuclear Antigen (PCNA) and Aurora A Kinase

Gadd45α is a nuclear protein encoded by a DNA damage-inducible gene. Through its interactions with other proteins, Gadd45α participates in the regulation of DNA repair, cell cycle, cell proliferation, and apoptosis. The NMR structure of human Gadd45α has been determined and shows an α/β fold with two long disordered and flexible regions at the N terminus and one of the loops. Human Gadd45α is predominantly monomeric in solution but exists in equilibrium with dimers and other oligomers whose population increases with protein concentration. NMR analysis shows that Aurora A interacts through its N-terminal domain with a region of human Gadd45α encompassing the site of dimerization, suggesting that the oligomerization of Gadd45α could be a regulatory mechanism to modulate its interactions with Aurora A, and possibly with other proteins too. However, Gadd45α appears to interact only weakly with PCNA through its flexible loop, in contrast with previous and contradictory reports.     

The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

Background

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12).

Methodology/Principal Findings

In this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-α, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-α, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with ≥50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-α are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs.

Conclusions/Significance

The HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-α release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen.     

Assessment of survival during starvation ofEscherichia coli andKlebsiella pneumoniae in artificial urine: analysis of the kinetics of colony formation

A kinetic model of colony formation was proposed by Hattori, based on a count of the colonies that appear on a plate in successive short intervals of time. In this model, three parameters (,t _r and N) are defined, which reflect the ability of a bacterium to yield colonies and allow us to described the dynamics of bacterial populations in soil and ofE. coli at different growth phases. In this paper we report a reparametrization of the kinetic model of colony formation, with the aim of facilitating more accurate calculation of andt _r. Moreover, we observed that during the starvation ofE. coli andK. pneumoniae in urine, can be used to assess survival, since this parameter clearly decreases during starvation. Retardation time values (t _r) were similar inE. coli andK. pneumoniae throughout the starvation experimental period.     

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.