Phylogenetic Analyses Under Secondary Structure-Specific Substitution Models Outperform Traditional Approaches: Case Studies with Diploblast LSU

Many rDNA molecular phylogenetic studies result in trees that are incongruent to either alternative gene tree reconstructions and/or morphological assumptions. One reason for this outcome might be the application of suboptimal phylogenetic substitution models. While the most commonly implemented models describe the evolution of independently evolving characters fairly well, they do not account for character dependencies such as rRNA strands that form a helix in the ribosome. Such nonindependent sites require the use of models that take into account the coevolution of the complete nucleotide pair (doublet). We analyzed 28S rDNA (LSU) demosponge phylogenies using a “doublet” model for pairing sites (rRNA-helices) and compared our findings with the results of “standard” approaches using Bayes factors. We demonstrate that paired and unpaired sites of the same gene result in different reconstructions and that usage of a doublet model leads to more reliable demosponge trees. We show the influence of more sophisticated models on phylogenetic reconstructions of early-branching metazoans and the phylogenetic relationships of demosponge orders. [Reviewing Editor: Dr. Rasmus Nielsen]     

Viability of the encysted metacercariae of Echinostoma caproni judged by light microscopy versus chemical excystation

The purpose of this study was to determine the viability of encysted metacercariae of Echinostoma caproni stored in Locke's solution 1:1 at 4 C for 1-24 wk. Viability was judged by light microscopy (LM) based on morphological characteristics of the encysted metacercariae versus chemical excystation of the cysts in a trypsin-bile salts excystation medium. The percent viability was very similar under both methods of assessment at 4, 8, and 16 wk poststorage. At 1 and 24 wk poststorage, viability was found to be about 2x greater based on excystation than using LM. We concluded that LM alone underestimated the viability of cysts and that determination of cyst viability was more accurate under assessment by chemical excystation.     

Two distinct low-pH steps promote entry of vaccinia virus

Entry of vaccinia virus into cells occurs by an endosomal route as well as through the plasma membrane. Evidence for an endosomal pathway was based on findings that treatment at a pH of <6 of mature virions attached to the plasma membrane enhances entry, whereas inhibitors of endosomal acidification reduce entry. Inactivation of infectivity by low-pH treatment of virions prior to membrane attachment is characteristic of many viruses that use the endosomal route. Nevertheless, we show here that the exposure of unattached vaccinia virus virions to low pH at 37 degrees C did not alter their infectivity. Instead, such treatment stably activated virions as indicated by their accelerated entry upon subsequent addition to cells, as measured by reporter gene expression. Moreover, the rate of entry was not further enhanced by a second low-pH treatment following adsorption to the plasma membrane. However, the entry of virions activated prior to adsorption remained sensitive to inhibitors of endosomal acidification, whereas virions treated with low pH after adsorption were resistant. Activation of virions by low pH was closely mimicked by proteinase digestion, suggesting that the two treatments operate through a related mechanism. Although proteinase cleavage of the virion surface proteins D8 and A27 correlated with activation, mutant viruses constructed by individually deleting these genes did not exhibit an activated phenotype. We propose a two-step model of vaccinia virus entry through endosomes, in which activating or unmasking the fusion complex by low pH or by proteinase is rate limiting but does not eliminate a second low-pH step mediating membrane fusion.     

Proteome analysis of grape skins during ripening

The characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and beta-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process.     

Poly(I:C) and lipopolysaccharide innate sensing functions of circulating human myeloid dendritic cells are affected in vivo in hepatitis C virus-infected patients

The role of peripheral dendritic cells (DCs) in hepatitis C virus (HCV) infection is unclear. To determine if persistent infection exerts an inhibitory pressure on HCV-specific innate responses, we analyzed DC function in blood through quantification of cell-associated HCV RNA levels in conjunction with multiparametric flow cytometry analysis of pathogen recognition receptor-induced cytokine expression. Independently of the serum viral load, fluorescence-activated cell sorter-purified total DCs had a wide range of cell-associated HCV genomic RNA copy numbers (mean log(10), 5.0 per 10(6) cells; range, 4.3 to 5.8). Here we report that for viremic patients with high viral loads in their total DCs, the myeloid DC (MDC) subset displayed impaired expression of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) but normal IL-6 or chemokine CCL3 expression in response to poly(I:C) and lipopolysaccharide (LPS). IL-6-expressing cells from this subgroup of viremic patients demonstrated a significant increase (sixfold more) in TNF-alpha(-) IL-12(-) cell frequency compared to healthy donors (mean, 38.8% versus 6.5%; P < 0.0001), indicating a functional defect in a subpopulation of cytokine-producing MDCs ( approximately 6% of MDCs). Attenuation of poly(I:C) and LPS innate sensing was HCV RNA density dependent and did not correlate with viremia or deficits in circulating MDC frequencies in HCV-infected patients. Monocytes from these patients were functionally intact, responding normally on a per-cell basis following stimulation, independent of cell-associated HCV RNA levels. Taken together, these data indicate that detection of HCV genomic RNA in DCs and loss of function in the danger signal responsiveness of a small proportion of DCs in vivo are interrelated rather than independent phenomena.     

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.     

The morphology proteins Mdm12/Mmm1 function in the major beta-barrel assembly pathway of mitochondria

The beta-barrel proteins of mitochondria are synthesized on cytosolic ribosomes. The proteins are imported by the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been assumed that the SAM(core) complex with the subunits Sam35, Sam37 and Sam50 represents the last import stage common to all beta-barrel proteins, followed by splitting in a Tom40-specific route and a route for other beta-barrel proteins. We have identified new components of the beta-barrel assembly machinery and show that the major beta-barrel pathway extends beyond SAM(core). Mdm12/Mmm1 function after SAM(core) yet before splitting of the major pathway. Mdm12/Mmm1 have been known for their role in maintenance of mitochondrial morphology but we reveal assembly of beta-barrel proteins as their primary function. Moreover, Mdm10, which functions in the Tom40-specific route, can associate with SAM(core) as well as Mdm12/Mmm1 to form distinct assembly complexes, indicating a dynamic exchange between the machineries governing mitochondrial beta-barrel assembly. We conclude that assembly of mitochondrial beta-barrel proteins represents a major function of the morphology proteins Mdm12/Mmm1.     

Tps1 regulates the pentose phosphate pathway, nitrogen metabolism and fungal virulence

Trehalose fulfils a wide variety of functions in cells, acting as a stress protectant, storage carbohydrate and compatible solute. Recent evidence, however, indicates that trehalose metabolism may exert important regulatory roles in the development of multicellular eukaryotes. Here, we show that in the plant pathogenic fungus Magnaporthe grisea trehalose-6-phosphate (T6P) synthase (Tps1) is responsible for regulating the pentose phosphate pathway, intracellular levels of NADPH and fungal virulence. Tps1 integrates glucose-6-phosphate (G6P) metabolism with nitrogen source utilisation, and thereby regulates the activity of nitrate reductase. Activity of Tps1 requires an associated regulator protein Tps3, which is also necessary for pathogenicity. Tps1 controls expression of the nitrogen metabolite repressor gene, NMR1, and is required for expression of virulence-associated genes. Functional analysis of Tps1 indicates that its regulatory functions are associated with binding of G6P, but independent of Tps1 catalytic activity. Taken together, these results demonstrate that Tps1 is a central regulator for integration of carbon and nitrogen metabolism, and plays a pivotal role in the establishment of plant disease.     

LAT and NTAL mediate immunoglobulin E-induced sustained extracellular signal-regulated kinase activation critical for mast cell survival

Immunoglobulin E (IgE) induces mast cell survival in the absence of antigen (Ag) through the high-affinity IgE receptor, Fcepsilon receptor I (FcepsilonRI). Although we have shown that protein tyrosine kinase Syk and sustained extracellular signal-regulated kinase (Erk) activation are required for IgE-induced mast cell survival, how Syk couples with sustained Erk activation is still unclear. Here, we report that the transmembrane adaptors LAT and NTAL are phosphorylated slowly upon IgE stimulation and that sustained but not transient Erk activation induced by IgE was inhibited in LAT(-/-) NTAL(-/-) bone marrow-derived mast cells (BMMCs). IgE-induced survival requires Ras activation, and both were impaired in LAT(-/-) NTAL(-/-) BMMCs. Sos was preferentially required for FcepsilonRI signals by IgE rather than IgE plus Ag. Survival impaired in LAT(-/-) NTAL(-/-) BMMCs was restored to levels comparable to those of the wild type by membrane-targeted Sos, which bypasses the Grb2-mediated membrane recruitment of Sos. The IgE-induced survival of BMMCs lacking Gads, an adaptor critical for the formation of the LAT-SLP-76-phospholipase Cgamma (PLCgamma) complex, was observed to be normal. IgE stimulation induced the membrane retention of Grb2-green fluorescent protein fusion proteins in wild-type but not LAT(-/-) NTAL(-/-) BMMCs. These results suggest that LAT and NTAL contribute to the maintenance of Erk activation and survival through the membrane retention of the Ras-activating complex Grb2-Sos and, further, that the LAT-Gads-SLP-76-PLCgamma and LAT/NTAL-Grb2-Sos pathways are differentially required for degranulation and survival, respectively.