Glucose uptake and its effect on gene expression in prochlorococcus

The marine cyanobacteria Prochlorococcus have been considered photoautotrophic microorganisms, although the utilization of exogenous sugars has never been specifically addressed in them. We studied glucose uptake in different high irradiance- and low irradiance-adapted Prochlorococcus strains, as well as the effect of glucose addition on the expression of several glucose-related genes. Glucose uptake was measured by adding radiolabelled glucose to Prochlorococcus cultures, followed by flow cytometry coupled with cell sorting in order to separate Prochlorococcus cells from bacterial contaminants. Sorted cells were recovered by filtration and their radioactivity measured. The expression, after glucose addition, of several genes (involved in glucose metabolism, and in nitrogen assimilation and its regulation) was determined in the low irradiance-adapted Prochlorococcus SS120 strain by semi-quantitative real time RT-PCR, using the rnpB gene as internal control. Our results demonstrate for the first time that the Prochlorococcus strains studied in this work take up glucose at significant rates even at concentrations close to those found in the oceans, and also exclude the possibility of this uptake being carried out by eventual bacterial contaminants, since only Prochlorococcus cells were used for radioactivity measurements. Besides, we show that the expression of a number of genes involved in glucose utilization (namely zwf, gnd and dld, encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and lactate dehydrogenase, respectively) is strongly increased upon glucose addition to cultures of the SS120 strain. This fact, taken together with the magnitude of the glucose uptake, clearly indicates the physiological importance of the phenomenon. Given the significant contribution of Prochlorococcus to the global primary production, these findings have strong implications for the understanding of the phytoplankton role in the carbon cycle in nature. Besides, the ability of assimilating carbon molecules could provide additional hints to comprehend the ecological success of Prochlorococcus.     

The Gammaherpesvirus m2 protein manipulates the Fyn/Vav pathway through a multidocking mechanism of assembly

To establish latent infections in B-cells, gammaherpesviruses express proteins in the infected B-cells of the host that spuriously activate signalling pathways located downstream of the B-cell receptor. One such protein is M2, a murine gammaherpesvirus 68-encoded molecule that activates the Vav1/Rac1 pathway via the formation of trimolecular complexes with Scr family members. Previous reports have shown that the formation of this heteromolecular complex involves interactions between a proline rich region of M2 and the Vav1 and Fyn SH3 domains. Here, we show that the optimal association of these proteins requires a second structural motif encompassing two tyrosine residues (Tyr120 and 129). These residues are inducibly phosphorylated by Fyn in non-hematopoietic cells and constitutively phosphorylated in B-cells. We also demonstrate that the phosphorylation of Tyr120 creates specific docking sites for the SH2 domains of both Vav1 and Fyn, a condition sine qua non for the optimal association of these two signalling proteins in vivo. Interestingly, signaling experiments indicate that the expression of M2 in B-cells promotes the tyrosine phosphorylation of Vav1 and additional signaling proteins, a biological process that requires the integrity of both the M2 phosphotyrosine and proline rich region motifs. By infecting mice with viruses mutated in the m2 locus, we show that the integrity of each of these two M2 docking motifs is essential for the early steps of murine gammaherpesvirus-68 latency. Taken together, these results indicate that the M2 phosphotyrosine motif and the previously described M2 proline rich region work in a concerted manner to manipulate the signaling machinery of the host B-cell.     

Seed dispersal anachronisms: rethinking the fruits extinct megafauna ate

Background

Some neotropical, fleshy-fruited plants have fruits structurally similar to paleotropical fruits dispersed by megafauna (mammals >10³ kg), yet these dispersers were extinct in South America 10–15 Kyr BP. Anachronic dispersal systems are best explained by interactions with extinct animals and show impaired dispersal resulting in altered seed dispersal dynamics.

Methodology/Principal Findings

We introduce an operational definition of megafaunal fruits and perform a comparative analysis of 103 Neotropical fruit species fitting this dispersal mode. We define two megafaunal fruit types based on previous analyses of elephant fruits: fruits 4–10 cm in diameter with up to five large seeds, and fruits >10 cm diameter with numerous small seeds. Megafaunal fruits are well represented in unrelated families such as Sapotaceae, Fabaceae, Solanaceae, Apocynaceae, Malvaceae, Caryocaraceae, and Arecaceae and combine an overbuilt design (large fruit mass and size) with either a single or few (<3 seeds) extremely large seeds or many small seeds (usually >100 seeds). Within-family and within-genus contrasts between megafaunal and non-megafaunal groups of species indicate a marked difference in fruit diameter and fruit mass but less so for individual seed mass, with a significant trend for megafaunal fruits to have larger seeds and seediness.

Conclusions/Significance

Megafaunal fruits allow plants to circumvent the trade-off between seed size and dispersal by relying on frugivores able to disperse enormous seed loads over long-distances. Present-day seed dispersal by scatter-hoarding rodents, introduced livestock, runoff, flooding, gravity, and human-mediated dispersal allowed survival of megafauna-dependent fruit species after extinction of the major seed dispersers. Megafauna extinction had several potential consequences, such as a scale shift reducing the seed dispersal distances, increasingly clumped spatial patterns, reduced geographic ranges and limited genetic variation and increased among-population structuring. These effects could be extended to other plant species dispersed by large vertebrates in present-day, defaunated communities.     

Insights into the structure-function relationships of pneumococcal cell wall lysozymes, LytC and Cpl-1

The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 degrees C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 degrees C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 +/- 0.5 sites/monomer) behave as equivalent (Kd = 2.7 +/- 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 degrees C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 degrees C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile.     

Receptor activator of nuclear factor-kappaB ligand (RANKL) as a novel prognostic marker in prostate carcinoma

Combined immunodetection of parathyroid hormone-related protein (PTHrP) and receptor activator of NF-kappaB ligand (RANKL) has shown to successfully distinguish poorly- and well-differentiated prostate carcinoma (PCa). In the present study, we aimed to assess whether immunohistochemical evaluation of these factors, and also osteoprotegerin (OPG) and Ki67, in radical prostatectomy specimens can predict biochemical recurrence. Fifty nine PCa cases undergoing radical prostatectomy between 1995 and 1998, without history of neoadjuvant hormonal therapy, were studied. Preoperative serum prostate-specific antigen (PSA), Gleason-sum score, pathologic stage, perineural invasion, seminal vesicle involvement, and positive surgical margins were assessed in these patients. Biochemical recurrence, defined by PSA > 0.4 ng/mL at 90 days or later after prostatectomy, occurred in 32/59 patients. In these patients, positivity for OPG and RANKL in the tumoral epithelium was higher than in those patients with no biochemical recurrence. Using univariate analysis, Gleason-sum score, surgical margins, and seminal vesicle involvement, as well as OPG and RANKL immunostaining (using a score value corresponding to moderate staining as cut-off) were significant predictors of biochemical recurrence (p<0.05). Using the multivariate Cox model, among the evaluated factors only RANKL expression (hazard ratio 11.6; p <0.001) was an independent prognostic indicator. Our findings suggest that immunohistochemical evaluation of RANKL in the primary tumor is a potential risk factor in PCa patients.