Antibody to Ricin A Chain Hinders Intracellular Routing of Toxin and Protects Cells Even after Toxin Has Been Internalized

Background

Mechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood.

Methodology/Principal Findings

We use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45–60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure.

Conclusions/Key Findings

We demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin’s entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs.     

Exploring the effects of phylogenetic uncertainty and consensus trees on stratigraphic consistency scores: a new program and a standardized method

The stratigraphic record of first appearances provides an independent source of data for evaluating and comparing phylogenetic hypotheses that include taxa with fossil histories. However, no standardized method exists for calculating these metrics for polytomous phylogenies, restricting their applicability. Previously proposed methods insufficiently deal with this problem because they skew or restrict the resulting scores. To resolve this issue, we propose a standardized method for treating polytomies when calculating these metrics: the Comprehensive Polytomy approach (ComPoly). This approach accurately describes how phylogenetic uncertainty, indicated by polytomies, affects stratigraphic consistency scores. We also present a new program suite (Assistance with Stratigraphic Consistency Calculations) that incorporates the ComPoly approach and simplifies the calculation of absolute temporal stratigraphic consistency metrics. This study also demonstrates that stratigraphic consistency scores calculated from strict consensus trees can be overly inclusive and those calculated from less‐than‐strict consensus trees inaccurately describe the phylogenetic signal present in the source most‐parsimonious trees (MPTs). Therefore, stratigraphic consistency scores should be calculated directly from the source MPTs whenever possible to ensure their accuracy. Finally, we offer recommendations for standardizing comparisons between molecular divergence dates and the stratigraphic record of first appearances, a promising new application of these methods. © The Willi Hennig Society 2010.     

Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation

In contrast to other Nox isoforms, the activity of Nox5 does not require the presence of accessory proteins and is entirely dependent on the elevation of intracellular calcium. Previous studies have shown that the EC(50) of Nox5 for calcium is relatively high and raises the question of whether Nox5 can be sufficiently activated in cells that do not experience extreme elevations of intracellular calcium. In the current study, we have identified a novel mechanism governing the activity of Nox5. Exposure of cells expressing Nox5 to phorbol 12-myristate 13-acetate (PMA) resulted in a slow and sustained increase in ROS, which was markedly different from the rapid response to ionomycin. PMA greatly potentiated the activity of Nox5 in response to low concentrations of ionomycin. The ability of PMA to increase Nox5 activity was abolished by calcium chelation and was a direct effect on enzyme activity, since PMA increased the calcium sensitivity of Nox5 in a cell-free assay. PMA stimulated the time-dependent phosphorylation of Nox5 on Thr(494) and Ser(498). Mutation of these residues to alanine abolished both PMA-dependent phosphorylation and calcium sensitization. Conversely, mutation of Thr(494) and Ser(498) to glutamic acid produced a gain of function mutant that had increased activity at low concentrations of ionomycin. Within the cell, Nox5 was detected in detergent-resistant microdomains of the endoplasmic reticulum. In summary, the phosphorylation of Nox5 at key residues facilitates enzyme activation at lower levels of intracellular calcium and may provide an avenue for enzyme activation in response to a greater variety of extracellular stimuli.     

Caught after the Act: a human A-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide

A/B-type metallocarboxypeptidases (MCPs) are among the most thoroughly studied proteolytic enzymes, and their catalytic mechanisms have been considered as prototypes even for several unrelated metalloprote(in)ase families. It has long been postulated that the nature of the side chains of at least five substrate residues, i.e., P4-P1', influence Km and kcat and that once the peptide or protein substrate is cleaved, both products remain in the first instance bound to the active-site cleft of the enzyme in a double-product complex. Structural details of binding of substrate to the nonprimed side of the cleft have largely relied on complexes with protein inhibitors and peptidomimetic small-molecule inhibitors that do not span the entire groove. In the former, the presence of N-terminal globular protein domains participating in large-scale interactions with the surface of the cognate catalytic domain outside the active-site cleft mostly conditions the way their C-terminal tails bind to the cleft. Accordingly, they may not be accurate models for a product complex. We hereby provide the structural details of a true cleaved double-product complex with a hexapeptide of an MCP engaged in prostate cancer, human carboxypeptidase A4, employing diffraction data to 1.6 A resolution (Rcryst and Rfree = 0.159 and 0.176, respectively). These studies provide detailed information about subsites S5-S1' and contribute to our knowledge of the cleavage mechanism, which is revisited in light of these new structural insights.     

Niger-Congo speaking populations and the formation of the Brazilian gene pool: mtDNA and Y-chromosome data

We analyzed sequence variation in the mitochondrial DNA (mtDNA) hypervariable segment I (HVS-I) from 201 Black individuals from two Brazilian cities (Rio de Janeiro and Porto Alegre), and compared these data with published information from 21 African populations. A subset of 187 males of the sample was also characterized for 30 Y-chromosome biallelic polymorphisms, and the data were compared with those from 48 African populations. The mtDNA data indicated that respectively 69% and 82% of the matrilineages found in Rio de Janeiro and Porto Alegre originated from West-Central/Southeast Africa. These estimates are in close agreement with historical records which indicated that most of the Brazilian slaves who arrived in Rio de Janeiro were from West-Central Africa. In contrast to mtDNA, Y-chromosome haplogroup analysis did not allow discrimination between places of origin in West or West-Central Africa. Thus, when comparing these two major African regions, there seems to be higher genetic structure with mtDNA than with Y-chromosome data.     

Biosorption of nickel,cobalt, zinc and copper ions by Serratia marcescens strain 16 in mono and multimetallic systems

Biodegradation - The metallurgical industry is one of the main sources of heavy metal pollution, which represents a severe threat to life. Metals can be removed from aqueous solutions by using...     

Disentangling the intertwined genetic bases of root and shoot growth in Arabidopsis

Root growth and architecture are major components of plant nutrient and water use efficiencies and these traits are the matter of extensive genetic analysis in several crop species. Because root growth relies on exported assimilate from the shoot, and changes in assimilate supply are known to alter root architecture, we hypothesized (i) that the genetic bases of root growth could be intertwined with the genetic bases of shoot growth and (ii) that the link could be either positive, with alleles favouring shoot growth also favouring root growth, or negative, because of competition for assimilates. We tested these hypotheses using a quantitative genetics approach in the model species Arabidopsis thaliana and the Bay-0 × Shahdara recombinant inbred lines population. In accordance with our hypothesis, root and shoot growth traits were strongly correlated and most root growth quantitative trait loci (QTLs) colocalized with shoot growth QTLs with positive alleles originating from either the same or the opposite parent. In order to identify regions that could be responsible for root growth independently of the shoot, we generated new variables either based on root to shoot ratios, residuals of root to shoot correlations or coordinates of principal component analysis. These variables showed high heritability allowing genetic analysis. They essentially all yielded similar results pointing towards two regions involved in the root--shoot balance. Using Heterogeneous Inbred Families (a kind of near-isogenic lines), we validated part of the QTLs present in these two regions for different traits. Our study thus highlights the difficulty of disentangling intertwined genetic bases of root and shoot growth and shows that this difficulty can be overcome by using simple statistical tools.     

Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.     

Gnaphalium teydeum and Gnaphalium luteo-album: two taxa of the Canary Islands with different genetic histories

Gnaphalium teydeum and G. luteo-album (Asteraceae) are two closely congeneric taxa native to the Canary Islands. While G. luteo-album is widespread in the Macaronesian Region, G. teydeum is endemic to the island of Tenerife and considered endangered by IUCN. Using the RAPD technique this study investigated the level and apportionment of genetic diversity of these taxa, trying to solve a taxonomic dispute related to G. teydeum. Based on the 102 DNA fragments generated by 11 primers, a high level of genetic differentiation was found between the taxa (F _ST  = 0.366), with G. luteo-album showing levels of genetic variability (P = 100%; H = 0.246) higher than those found in G. teydeum (P = 75.5%; H = 0.173). UPGMA dendrogram and Bayesian cluster analysis clearly separated populations from both the species. Overall, results show that although morphological differentiation between G. teydeum and G. luteo-album is not strong, they show marked molecular divergence, supporting the current taxonomic status.