Stable Agrobacterium-mediated transformation of embryogenic tissues from Pinus pinaster Portuguese genotypes

Protocols for genetic transformation of maritime pine (Pinus pinaster Sol. ex Aiton) embryogenic tissues were developed using the Agrobacterium C58pMP90/pPCV6NFGUS. This is the first report of Agrobacterium-mediated T-DNA integration in P. pinaster confirmed by Southern blot analysis. The omission of casein hydrolysate from culture medium during cocultivation and subsequent subculture was crucial to control Agrobacterium growth. Two different transformation protocols were compared: (1) bacterial drops were spread over embryogenic clumps; (2) a mixture of bacterial and embryogenic cell suspensions was plated on filter paper. The highest frequency of transformation (22 independent transformed lines per g fresh weight, for embryogenic clone 31/668/00) was obtained with Protocol 2. The same basic procedure allowed transformation of embryogenic cell suspensions, which was dependent on subculture age. From 52 hygromycin-resistant independent lines obtained, 47 showed stable uidA gene expression and were PCR-positive for uidA gene and 42 for hpt gene. No residual Agrobacterium was detected in the transformed lines. Transgene integration was achieved using both protocols, as confirmed by Southern hybridization. From 38 (90%) transformed lines successfully cryopreserved and recovered, 71% regrown replicates have maintained the frequency of cell aggregates and early-formed embryos with uidA expression. Maturation of 44 transformed lines gave rise to 3 mature somatic embryos, each one coming from a different transformed line. Our results show the high potential of Protocol 2 for application to different culture systems.     

The design and synthesis of novel alpha-ketoamide-based p38 MAP kinase inhibitors

We have identified a novel series of potent p38 MAP kinase inhibitors through structure-based design which due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME and in vivo PK studies show these compounds to have drug-like characteristics which could result in the development of an oral treatment for inflammatory conditions.     

Discovery of a potent and selective c-Kit inhibitor for the treatment of inflammatory diseases

A potent and selective c-Kit inhibitor 20 was identified through a structure–activity relationship study. In an in vivo mouse model of mast cell activation, 20 blocked the SCF-induced histamine release with an EC₅₀ of 26 nM.     

The surprising evolutionary history of South American deer

To clarify the systematic relationships and evolutionary history of South American deer, we conducted a comprehensive phylogenetic analysis using representative species of all of the genera of Neotropical deer. Our results revealed high levels of molecular and cytogenetic divergence between groups of morphologically similar species of brockets (Mazama), and suggest a polyphyletic origin. At least eight ancestral forms of deer invaded South America during the late Pliocene (2.5-3 MYA), and members of the red brockets had an independent early explosive diversification soon after their ancestor arrived there, giving rise to a number of morphologically cryptic species.     

Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function

A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated alpha1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated alpha1-antitrypsin in the ER. In hepatoma cells, shift of mutated alpha1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated alpha1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway.     

Dehydroepiandrosterone modulates antioxidant enzymes and Akt signaling in healthy Wistar rat hearts

Dehydroepiandrosterone (DHEA) is an endogenous steroid synthesized mainly in the adrenal cortex. It is known that DHEA is a precursor of sex steroids and that part of its effects depends on its conversion to estrogens and androgens. Sex steroids exert profound and controversial effects on cardiovascular function. Exogenous DHEA can exert a dual effect, antioxidant or prooxidant, depending on the dose and on the tissue specificity [1,2] (F. Celebi, I. Yilmaz, H. Aksoy, M. Gümüs, S. Taysi, D. Oren, Dehydroepiandrosterone prevents oxidative injury in obstructive jaundice in rats, J. Int. Med. Res. 32 (4) (2004) 400-405; S.K. Kim, R.F. Novak, The role of intracellular signaling in insulin-mediated regulation of drug metabolizing enzyme gene and protein expression, Pharmacol. Ther. 113 (1) (2007) 88-120). Akt signaling pathway is one of the anti-proliferative mechanisms of DHEA (Y. Jiang, T. Miyazaki, A. Honda, T. Hirayama, S. Yoshida, N. Tanaka, Y. Matsuzaki, Apoptosis and inhibition of the phosphatidylinositol 3-kinase/Akt signaling pathway in the anti-proliferative actions of dehydroepiandrosterone, J. Gastroenterol. 40 (5) (2005) 490-497). Heart homogenates were prepared to quantify lipid peroxidation (LPO), concentration of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), 4-hydroxy-2-nonenal (HNE) and p-Akt/Akt ratio, and the activities of those antioxidant enzymes. When administrated to male Wistar rats in short-term (6 or 24h) intraperitoneally, DHEA produced significant differences in some parameters of oxidative stress in rat hearts among the distinct doses (1, 10, and 50mg/kg) used. The results here presented, regarding 6 and 24h oxidative stress status, have shown that DHEA injections promoted a prooxidant answer in healthy Wistar rat hearts.     

Evaluation of ultra- and nanofiltration for refining soluble products from rice husk xylan

Liquors from water treatments of rice husks (containing soluble xylan-derived products) were processed with NF and UF membranes for concentrating and removing both monosaccharides and non-saccharide compounds. Among the commercial membranes assayed, the best results were achieved with the 4 kDa polymeric tubular ESP04 (PCI Membranes), and the 1 kDa ceramic monolithic Kerasep Nano (Novasep). Several trade-offs were identified both in membrane selection and in operating conditions. The ESP04 polymeric membrane provided the best fractionation, but lower recovery under comparable experimental conditions, while its fluxes were about half of those of the ceramic Kerasep Nano membrane. Increase in transmembrane pressure resulted in improved product recovery, at the expense of a lower purity. Additional data on product refining by coupling membrane processing with extraction and ion exchange are provided.     

Structural analysis of FAD synthetase from <Emphasis Type="Italic">Corynebacterium ammoniagenes</Emphasis>

Background  

The prokaryotic FAD synthetase family - a group of bifunctional enzymes that catalyse riboflavin phosphorylation and FMN adenylylation within a single polypeptide chain- was analysed in terms of sequence and structure.     

Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research

Brown algae share several important features with land plants, such as their photoautotrophic nature and their cellulose-containing wall, but the two groups are distantly related from an evolutionary point of view. The heterokont phylum, to which the brown algae belong, is a eukaryotic crown group that is phylogenetically distinct not only from the green lineage, but also from the red algae and the opisthokont phylum (fungi and animals). As a result of this independent evolutionary history, the brown algae exhibit many novel features and, moreover, have evolved complex multicellular development independently of the other major groups already mentioned. In 2004, a consortium of laboratories, including the Station Biologique in Roscoff and Genoscope, initiated a project to sequence the genome of Ectocarpus siliculosus, a small filamentous brown alga that is found in temperate, coastal environments throughout the globe. The E. siliculosus genome, which is currently being annotated, is expected to be the first completely characterized genome of a multicellular alga. In this review we look back over two centuries of work on this brown alga and highlight the advances that have led to the choice of E. siliculosus as a genomic and genetic model organism for the brown algae.