BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

TGF-β family members play a relevant role in tumorigenic processes, including hepatocellular carcinoma (HCC), but a specific implication of the Bone Morphogenetic Protein (BMP) subfamily is still unknown. Although originally isolated from fetal liver, little is known about BMP9, a BMP family member, and its role in liver physiology and pathology. Our results show that BMP9 promotes growth in HCC cells, but not in immortalized human hepatocytes. In the liver cancer cell line HepG2, BMP9 triggers Smad1,5,8 phosphorylation and inhibitor of DNA binding 1 (Id1) expression up- regulation. Importantly, by using chemical inhibitors, ligand trap and gene silencing approaches we demonstrate that HepG2 cells autocrinely produce BMP9 that supports their proliferation and anchorage independent growth. Additionally, our data reveal that in HepG2 cells BMP9 triggers cell cycle progression, and strikingly, completely abolishes the increase in the percentage of apoptotic cells induced by long-term incubation in low serum. Collectively, our data unveil a dual role for BMP9, both promoting a proliferative response and exerting a remarkable anti-apoptotic function in HepG2 cells, which result in a robust BMP9 effect on liver cancer cell growth. Finally, we show that BMP9 expression is increased in 40% of human HCC tissues compared with normal human liver as revealed by immunohistochemistry analysis, suggesting that BMP9 signaling may be relevant during hepatocarcinogenesis in vivo. Our findings provide new clues for a better understanding of BMPs contribution, and in particular BMP9, in HCC pathogenesis that may result in the development of effective and targeted therapeutic interventions.     

PSSA-2, a Membrane-Spanning Phosphoprotein of Trypanosoma brucei,Is Required for Efficient Maturation of Infection

The coat of Trypanosoma brucei consists mainly of glycosylphosphatidylinositol-anchored proteins that are present in several million copies and are characteristic of defined stages of the life cycle. While these major components of the coats of bloodstream forms and procyclic (insect midgut) forms are well characterised, very little is known about less abundant stage-regulated surface proteins and their roles in infection and transmission. By creating epitope-tagged versions of procyclic-specific surface antigen 2 (PSSA-2) we demonstrated that it is a membrane-spanning protein that is expressed by several different life cycle stages in tsetse flies, but not by parasites in the mammalian bloodstream. In common with other membrane-spanning proteins in T. brucei, PSSA-2 requires its cytoplasmic domain in order to exit the endoplasmic reticulum. Correct localisation of PSSA-2 requires phosphorylation of a cytoplasmic threonine residue (T₃₀₅), a modification that depends on the presence of TbMAPK4. Mutation of T₃₀₅ to alanine (T₃₀₅A) has no effect on the localisation of the protein in cells that express wild type PSSA-2. In contrast, this protein is largely intracellular when expressed in a null mutant background. A variant with a T₃₀₅D mutation gives strong surface expression in both the wild type and null mutant, but slows growth of the cells, suggesting that it may function as a dominant negative mutant. The PSSA-2 null mutant exhibits no perceptible phenotype in culture and is fully competent at establishing midgut infections in tsetse, but is defective in colonising the salivary glands and the production of infectious metacyclic forms. Given the protein''s structure and the effects of mutation of T₃₀₅ on proliferation and localisation, we postulate that PSSA-2 might sense and transmit signals that contribute to the parasite''s decision to divide, differentiate or migrate.     

Variation of heartwood and sapwood in 18-year-old Eucalyptus globulus trees grown with different spacings

Heartwood and sapwood development was studied in 18-year-old Eucalyptus globulus trees from pulpwood plantations with different spacings (3 × 2, 3 × 3, 4 × 3, 4 × 4 and 4 × 5 m), on cross-sectional discs taken at breast height. The trees possessed a large proportion of heartwood, on average 60% of the wood cross-sectional surface. Spacing was a statistically significant source of variation of heartwood area, which ranged between 99 and 206 cm² for the closer (3 × 2) and wider (4 × 5) spacings, respectively. There was a positive and high statistical significant correlation between heartwood diameter and tree diameter (heartwood diameter = −0.272 + 0.616 dbh; r ² = 0.77; P < 0.001), and larger trees contained more heartwood regardless of spacing. Heartwood proportion in cross-section remained practically constant between spacings but increased with tree diameter class: 55.1, 62.2, 65.0 and 69.5% for diameter at breast height classes <15, 15–20, 20–25 and >25 cm, respectively. The sapwood width did not depend on tree diameter growth and remained practically constant at an average of 18 mm (range 15–21 mm), but sapwood area showed a good linear regression with tree diameter. Therefore, tree growth enhancement factors, such as wide spacings, will induce formation of larger heartwoods that can negatively impact raw-material quality for pulping. The increase in heartwood in relation with tree dimensions should therefore be taken into account when designing forest management guidelines.     

Stability of the Transthyretin Molecule as a Key Factor in the Interaction with A-Beta Peptide - Relevance in Alzheimer's Disease

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/A-Beta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4-(3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.     

The crystal structure of the dimeric colicin M immunity protein displays a 3D domain swap

Bacteriocins are proteins secreted by many bacterial cells to kill related bacteria of the same niche. To avoid their own suicide through reuptake of secreted bacteriocins, these bacteria protect themselves by co-expression of immunity proteins in the compartment of colicin destination. In Escherichia coli the colicin M (Cma) is inactivated by the interaction with the Cma immunity protein (Cmi). We have crystallized and solved the structure of Cmi at a resolution of 1.95? by the recently developed ab initio phasing program ARCIMBOLDO. The monomeric structure of the mature 10kDa protein comprises a long N-terminal α-helix and a four-stranded C-terminal β-sheet. Dimerization of this fold is mediated by an extended interface of hydrogen bond interactions between the α-helix and the four-stranded β-sheet of the symmetry related molecule. Two intermolecular disulfide bridges covalently connect this dimer to further lock this complex. The Cmi protein resembles an example of a 3D domain swapping being stalled through physical linkage. The dimer is a highly charged complex with a significant surplus of negative charges presumably responsible for interactions with Cma. Dimerization of Cmi was also demonstrated to occur in vivo. Although the Cmi-Cma complex is unique among bacteria, the general fold of Cmi is representative for a class of YebF-like proteins which are known to be secreted into the external medium by some Gram-negative bacteria.     

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells.     

Soil seed bank in a patchy vegetation of coastal sandy plains in southeastern Brazil

Large seed banks have been found in tropical dry forests and also in habitats with high seasonality in rainfall. However, patchily structured vegetation could induce great spatial variation in the seed bank. We characterized the seed bank in a patchy vegetation of restinga, a common type of coastal vegetation found in the Atlantic forest biome. We also evaluated whether there is any spatial variation between the litter and soil layer, bare sand, and the edge and center of vegetation patches with distinct species dominance. We found 104 seeds/m² in the seed bank using a 5‐cm‐depth sampling. Seven out of 16 species found in the restinga seed bank germinated; two of these were found in the early stages of vegetation patches. We found a higher number of seeds at the edge than in the center of vegetation patches. However, there were no significant differences in the number of seeds in the seed bank between the litter and soil layer, and between vegetation patches with distinct species dominance. Bare sandy soils had lower seed bank densities than vegetation patches. A small seed bank size might be explained by the low proportion of seeds from herbaceous and woody species, which are pioneers in the Atlantic forest. However, seed bank might play an important role in the early stages of the successional process, due to the occurrence of the few species that are able to colonize new young vegetation patches.     

The effects of drying on sediment nitrogen content in a Mediterranean intermittent stream: a microcosms study

Mediterranean climates predispose aquatic systems to both flood and drought periods, therefore, stream sediments may be exposed to desiccation periods. Changes in oxygen concentrations and sediment water content influence the biotic processes implicated in nitrogen dynamics. The objectives of this study were to identify (1) the changes of inorganic nitrogen in stream sediments during the transition from wet to dry conditions, and (2) the underlying processes in N dynamics and its regulation. Extractable sediment NO₃ ⁻-N and NH₄ ⁺-N, organic matter and extractable organic carbon content were assessed during natural desiccation in microcosms with sediments from an intermittent Mediterranean stream. In agreement with our initial hypothesis, our results showed how the NO₃ ⁻-N content of the sediment was enhanced during the first 10 days of sediment drying, whereas NH₄ ⁺-N was lost by 14 days post-drying. During the first 10 days, sediment desiccation seemed to stimulate the net N-mineralization and net nitrification from sediments. Afterwards, the extractable NO₃ ⁻-N concentration sharply dropped, which may be attributed to lower ammonium-oxidation rates as ammonium and organic matter are depleted, and to an increase in NO₃ ⁻-N consumption by microbial populations. Denitrification was inhibited, with a significant decrease as % water-filled pore space lowered. We hypothesize that the sediment inorganic N content enhanced during sediment desiccation could be released as part of the N pulse observed after sediment rewetting. However, the stream N availability after rewetting dried sediments would differ depending on desiccation period duration.