A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons

A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3' to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3' and 5' conserved regions of the DNA element have been determined. A 59-base sequence element, found at the junctions of inserted DNA sequences and the conserved 3' segment, is also present at this location in the R46 sequence. A copy of one half of this 59-base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site-specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5' conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site-specific integration functions (the integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.     

Cyclooxygenase inhibition during allergic sensitization increases STAT6-independent primary and memory Th2 responses

Immune sensitization and memory generation are required for the development of allergic inflammation. Our previous studies demonstrate that the cyclooxygenase (COX) metabolic pathway is actively involved in allergic responses and COX inhibition increases allergic airway inflammation in a STAT6-independent fashion. To test the hypothesis that COX inhibition augments allergic inflammation by enhancing immune sensitization and memory, we sensitized STAT6 knockout mice with an i.p. injection of OVA with aluminum hydroxide as an adjuvant and treated the mice with the COX inhibitor indomethacin or vehicle for analyses of the primary and memory immune responses. We found that COX inhibition during immune sensitization, but not the allergic challenge phase, was necessary and sufficient to increase allergic inflammation. COX inhibition during sensitization increased the numbers of mature dendritic cells and activated CD4 T cells in the spleen and augmented OVA-specific IL-5 and IL-13 responses of the splenic CD4 T cells at day 5 after sensitization. COX inhibition during sensitization also augmented allergic Th2 response to OVA challenge 90 days after the sensitization. Therefore, COX inhibition during allergic sensitization augments allergic responses by enhancing Th2 cell activation and memory generation and the proallergic effect is STAT6-independent. These findings provide a mechanistic explanation for the increased allergic inflammation previously shown in the mice treated with COX inhibitors and in COX-deficient mice and suggest that use of COX-inhibiting drugs during initial allergen exposure may increase the risk of developing allergic responses.     

The role of “forensic” dendrochronology in the conservation of alerce (Fitzroya cupressoides ((Molina) Johnston)) forests in Chile

Dendrochronological techniques have proved to be an effective tool in the investigation of illegal cutting of Fitzroya because it is illegal to trade and cut wood coming from living individuals or from trees that were killed after 1976 either by natural or anthropogenic causes. Accurate cross-dating of death or burn dates provides strong evidence for prosecution in the legal enforcement of the ban and an important defense in the protection of this and other endangered species. Some examples illustrate the application of these dating techniques and discuss potential difficulties.     

Neuroblastoma Tyrosine Kinase Signaling Networks Involve FYN and LYN in Endosomes and Lipid Rafts

Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.     

Natural alternatives to in-feed antibiotics in pig production: can immunomodulators play a role?

As a result of the European ban of in-feed growth-promoting antibiotics, new strategies are being developed to increase the resistance to disease in farm animals. In pig production, this is of particular importance during the weaning transition when piglets are subjected to major stressful events, making them highly sensitive to digestive disorders. At this time, the development of both innate and adaptive immunity at the mucosal surface is critical in preventing the potential harmful effects of intestinal pathogenic agents. Strategies aiming at stimulating natural host defences through the use of substances able to modulate immune functions have gained increasing interest in animal research, and different bioactive components a priori sharing those properties have been the subject of in vivo nutritional investigations in pig. Among these, yeast derivates (β-glucans and mannans) are able to interact with immune cells, particularly phagocytic cells. However, studies where they have been fed to pigs have shown inconsistent results, suggesting that their ability to target the sensitive immune cells through the oral route is questionable. The plant extracts, which would benefit from a positive image in the public opinion, have also been tested. However, due to a lack of data on the bioactive components of particular plants and the large diversity of species, it has proved difficult to prepare extracts of equivalent potency and thus, the literature on their influence on pig immunity remains inconclusive. In considering piglet immunity and health benefits, the most promising results to date have been obtained with spray-dried animal plasma, whose positive effects would be provided by specific antibodies and non-specific competition of some plasma components with bacteria for intestinal receptors. The major positive effect of spray-dried animal plasma is in reducing the infiltration of gut-associated lymphoid tissue by immune cells, which is likely to be the result of a decreased colonisation by potentially harmful bacteria. This review also highlights the limitations of some of the published in vivo studies on the immunomodulatory activity of certain feed additives. Among those, the lack of standardisation of extracts and the heterogeneity of piglet-rearing conditions (e.g. exposure to pathogens) are likely the most limiting.     

A structural model for the catalytic cycle of Ca(2+)-ATPase

Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule.     

Actions and interactions of growth hormone and insulin-like growth factor-II: body and organ growth of transgenic mice

To characterize long-term actions and interactions of growth hormone (GH) and insulin-like growth factor-II (IGF-II) on postnatal body and organ growth, hemizygous phosphoenolpyruvate carboxykinase (PEPCK)-human IGF-II transgenic mice were crossed with hemizygous PEPCK-bovine GH transgenic mice. The latter are characterized by two-fold increased serum levels of IGF-I and exhibit markedly increased body, skeletal and organ growth. Four different genetic groups were obtained: mice harbouring the IGF-II transgene (I), the bGH transgene (B), or both transgenes (IB), and non- transgenic controls (C). These groups of mice have previously been studied for circulating IGF-I levels (Wolf et al., 1995a), whereas the present study deals with body and organ growth. Growth curves (week 3 to 12) were estimated by regression with linear and quadratic components of age on body weight and exhibited significantly (p < 0.001) greater linear coefficients in B and IB than in I and C mice. The linear coefficients of male I and C mice were significantly (p < 0.001) greater than those of their female counterparts, whereas this sex-related difference was absent in the bGH transgenic groups. The weights of internal organs as well as the weights of abdominal fat, skin and carcass were recorded from 3.5- to 8- month-old mice. In addition, organ weight-to-body weight-ratios (relative organ weights) were calculated. Except for the weight of abdominal fat, absolute organ weights were as a rule significantly greater in B and IB than in I and C mice. IGF-II overproduction as a tendency increased the weights of kidneys, adrenal glands, pancreas and uterus both in the absence and presence of the bGH transgene. Analysis of relative organ weights demonstrated significant (p < 0.05) effects of elevated IGF- II on the relative growth of kidneys (males and females) and adrenal glands (females), confirming our previous report on organ growth of PEPCK-IGF-II transgenic mice. In females, IGF-II and GH overproduction were additive in stimulating the growth of spleen and uterus, providing evidence for tissue-specific postnatal growth promoting effects by IGF-II in the presence of elevated IGF-I