Rapid Assessment of Seed Removal and Frugivore Activity as a Tool for Monitoring the Health Status of Tropical Forests

Ecological processes in tropical forests are being affected at unprecedented rates by human activities. Yet, the continuity of ecological functions like seed dispersal is crucial for forest regeneration. It thus becomes increasingly urgent to be able to rapidly assess the health status of these processes in order to take appropriate management measures. We tested a method to rapidly evaluate seed removal rates on two animal‐dispersed tree species, Virola kwatae and V. michelii (Myristicaceae), at three sites in French Guiana with increasing levels of anthropogenic disturbance. We counted fallen fruits, fruit valves, and seeds of each focal fruiting tree in a single 1 m² quadrat, and calculated two indices: the proportion of seeds removed and the proportion of fruits opened by mammals. They both provide an indirect and rapid assessment of frugivore activity. Our results showed a significant decrease in the proportion of removed seeds (16%) and fruits opened (19%) at the most impacted site in comparison with the other two sites (79% for seeds, 60% and 35% for fruits). This testifies to an increased impoverishment of the primate and toucan communities at the disturbed sites. This standardized protocol provides fast information about the health status of the community of seed dispersers and predators and of their seed removal services. It is time‐ and cost‐effective and is not species‐specific, allowing comparisons among sites or over time. We suggest using it with the pantropical Myristicaceae and any other capsule‐producing family to rapidly assess the health status of seed removal processes across the tropics.     

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.     

Non-Microtubular Localizations of Microtubule-Associated Protein 6 (MAP6)

MAP6 proteins (MAP6s), which include MAP6-N (also called Stable Tubule Only Polypeptide, or STOP) and MAP6d1 (MAP6 domain-containing protein 1, also called STOP-Like protein 21 kD, or SL21), bind to and stabilize microtubules. MAP6 deletion in mice severely alters integrated brain functions and is associated with synaptic defects, suggesting that MAP6s may also have alternative cellular roles. MAP6s reportedly associate with the Golgi apparatus through palmitoylation of their N-terminal domain, and specific isoforms have been shown to bind actin. Here, we use heterologous systems to investigate several biochemical properties of MAP6 proteins. We demonstrate that the three N-terminal cysteines of MAP6d1 are palmitoylated by a subset of DHHC-type palmitoylating enzymes. Analysis of the subcellular localization of palmitoylated MAP6d1, including electron microscopic analysis, reveals possible localization to the Golgi and the plasma membrane but no association with the endoplasmic reticulum. Moreover, we observed localization of MAP6d1 to mitochondria, which requires the N-terminus of the protein but does not require palmitoylation. We show that endogenous MAP6d1 localized at mitochondria in mature mice neurons as well as at the outer membrane and in the intermembrane space of purified mouse mitochondria. Last, we found that MAP6d1 can multimerize via a microtubule-binding module. Interestingly, most of these properties of MAP6d1 are shared by MAP6-N. Together, these results describe several properties of MAP6 proteins, including their intercellular localization and multimerization activity, which may be relevant to neuronal differentiation and synaptic functions.     

Bmcc1s, a novel brain-isoform of Bmcc1, affects cell morphology by regulating MAP6/STOP functions

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology.     

Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.     

Glucose utilization of strains lacking PGI1 and expressing a transhydrogenase suggests differences in the pentose phosphate capacity among Saccharomyces cerevisiae strains

Saccharomyces cerevisiae strains lacking phosphoglucose isomerase (pgi1) cannot use the pentose phosphate (PP) pathway to oxidize glucose, which has been explained by the lack of mechanism for reoxidation of the NADPH surplus. Consistent with this, the defective growth on glucose of a ENYpgi1 strain can be partially restored by expressing the Escherichia coli transhydrogenase udhA. In this work it was found that growth of V5 (wine yeast-derived) and FY1679 (isogenic to S288C) pgi1 mutants is not rescued by expression of udhA. Moreover, the flux through the PP pathway of 11 S. cerevisiae strains from various origins was estimated, by calculating the ratio between the enzymatic activity of the G6PDH and HXK, placed at the glycolysis-PP pathway branch point. The results show that ENY.WA-1A exhibited the highest ratio (1.5-3-fold) and the highest G6PDH activity. Overexpression of ZWF1 encoding the G6PDH in V5pgi1udhA did not rescue growth on glucose, suggesting that steps downstream the G6PDH might limit the PP pathway in this strain. As a whole, these data highlight a great intraspecies diversity in the PP pathway capacity among S. cerevisiae strains and suggest that a low capacity may be the prime limiting factor in glucose oxidation through this pathway.     

GSK-3β inhibition by lithium confers resistance to chemotherapy-induced apoptosis through the repression of CD95 (Fas/APO-1) expression

Lithium exerts neuroprotective actions that involve the inhibition of glycogen synthase kinase-3β (GSK-3β). Otherwise, recent studies suggest that sustained GSK-3β inhibition is a hallmark of tumorigenesis. In this context, the present study was undertaken to examine whether lithium modulated cancer cell sensitivity to apoptosis induced by chemotherapy agents. We observed that, in different human cancer cell lines, lithium significantly reduced etoposide- and camptothecin-induced apoptosis. In HepG2 cells, lithium repressed drug induction of CD95 expression and clustering at the cell surface as well as caspase-8 activation. Lithium acted through deregulation of GSK-3β signaling since (1) it provoked a rapid and sustained phosphorylation of GSK-3β on the inhibitory serine 9 residue; (2) the GSK-3β inhibitor SB-415286 mimicked lithium effects by repressing drug-induced apoptosis and CD95 membrane expression; and (3) lithium promoted the disruption of nuclear GSK-3β/p53 complexes. Moreover, the overexpression of an inactivated GSK-3β mutant counteracted the stimulatory effects of etoposide and camptothecin on a luciferase reporter plasmid driven by a p53-responsive sequence from the CD95 gene. In conclusion, we provide the first evidence that lithium confers resistance to apoptosis in cancer cells through GSK-3β inhibition and subsequent repression of CD95 gene expression. Our study also highlights the concerted action of GSK-3β and p53 on CD95 gene expression.     

Variation of mucin distribution in the rat intestine, caecum and colon: effect of the bacterial flora.

The influence of the intestinal microflora on mucin types was studied in the small intestine, caecum and colon of conventional (CV) rats as compared to germ-free (GF) rats. A colorimetric method was used on purified water-soluble mucin extracted from mucosal scrapings and contents. Variations occurred between the three anatomical sites both in the mucosas and intestinal contents of GF rats. In CV rats, the presence of the bacterial flora led to different effects depending on the intestinal site: in the small intestinal mucosa, neutral and sulphomucins values were higher whereas sialomucin was much lower. Conversely, sialomucin was higher in the caecal and colonic mucosas and contents whereas sulphated mucins were decreased significantly in caecal contents and caecal and colonic mucosas. These variations in the contents may reflect the bacterial mucolytic activity and the effect of bacterial metabolites on the mucosa.