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Bacillus thuringiensis (Bt) produces different insecticidal proteins effective for pest control. Among them, Cry insecticidal proteins have been used in transgenic plants for the control of insect pests. However, evolution of resistance by insects endangers this technology. Previous work showed that the lepidopteran insect Plutella xylostella PxHsp90 chaperone enhanced the toxicity of Bt Cry1A protoxins by protecting them from degradation by the larval gut proteases and by enhancing binding of the protoxin to its receptors present in larval midgut cells. In this work, we show that PxHsp70 chaperone also protects Cry1Ab protoxin from gut proteases degradation, enhancing Cry1Ab toxicity. We also show that both PxHsp70 and PxHsp90 chaperones act cooperatively, increasing toxicity and the binding of Cry1Ab439D mutant, affected in binding to midgut receptors, to cadherin receptor. Also, insect chaperones recovered toxicity of Cry1Ac protein to a Cry1Ac-highly resistant P. xylostella population, NO-QAGE, that has a disruptive mutation in an ABCC2 transporter linked to Cry1Ac resistance. These data show that Bt hijacked an important cellular function for enhancing its infection capability, making use of insect cellular chaperones for enhancing Cry toxicity and for lowering the evolution of insect resistance to these toxins. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis (Bt) produces different insecticidal proteins effective for pest control. Among them, Cry insecticidal proteins have been used in transgenic plants for the control of insect pests. However, evolution of resistance by insects endangers this technology. Previous work showed that the lepidopteran insect Plutella xylostella PxHsp90 chaperone enhanced the toxicity of Bt Cry1A protoxins by protecting them from degradation by the larval gut proteases and by enhancing binding of the protoxin to its receptors present in larval midgut cells. In this work, we show that PxHsp70 chaperone also protects Cry1Ab protoxin from gut proteases degradation, enhancing Cry1Ab toxicity. We also show that both PxHsp70 and PxHsp90 chaperones act cooperatively, increasing toxicity and the binding of Cry1Ab439D mutant, affected in binding to midgut receptors, to cadherin receptor. Also, insect chaperones recovered toxicity of Cry1Ac protein to a Cry1Ac-highly resistant P. xylostella population, NO-QAGE, that has a disruptive mutation in an ABCC2 transporter linked to Cry1Ac resistance. These data show that Bt hijacked an important cellular function for enhancing its infection capability, making use of insect cellular chaperones for enhancing Cry toxicity and for lowering the evolution of insect resistance to these toxins.

Selected findings about Clostridioides difficile (formerly Clostridium difficile) toxins are presented in a narrative review. Starting with a personal view on research about G proteins, adenylyl cyclase, and ADP-ribosylating toxins in the laboratory of Günter Schultz in Heidelberg, milestones of C. difficile toxin research are presented with the focus on toxin B (TcdB), covering toxin structure, receptor binding, toxin up-take and refolding, the intracellular actions of TcdB, and the treatment of C. difficile infection. [ABSTRACT FROM AUTHOR]

Members of phylum Cnidaria are an ancient group of venomous animals and rely on a number of specialized tissues to produce toxins in order to fulfil a range of ecological roles including prey capture, defence against predators, digestion and aggressive encounters. However, limited comprehensive analyses of the evolution and expression of toxin genes currently exist for cnidarian species. In this study, we use genomic and transcriptomic sequencing data to examine gene copy number variation and selective pressure on toxin gene families in phylum Cnidaria. Additionally, we use quantitative RNA‐seq and mass spectrometry imaging to understand expression patterns and tissue localization of toxin production in sea anemones. Using genomic data, we demonstrate that the first large‐scale expansion and diversification of known toxin genes occurs in phylum Cnidaria, a process we also observe in other venomous lineages, which we refer to as convergent amplification. Our analyses of selective pressure on sea anemone toxin gene families reveal that purifying selection is the dominant mode of evolution for these genes and that phylogenetic inertia is an important determinant of toxin gene complement in this group. The gene expression and tissue localization data revealed that specific genes and proteins from toxin gene families show strong patterns of tissue and developmental‐phase specificity in sea anemones. Overall, convergent amplification and phylogenetic inertia have strongly influenced the distribution and evolution of the toxin complement observed in sea anemones, while the production of venoms with different compositions across tissues is related to the functional and ecological roles undertaken by each tissue type. [ABSTRACT FROM AUTHOR]

We have developed versatile methods toward the synthesis of a variety of piperidine/piperazine bridged isosteres of pridopidine. The compounds were assessed against the D2 receptor in agonist and antagonist modes and against the D4 receptor in agonist mode. hERG Binding and the ADME profiles were studied. [ABSTRACT FROM AUTHOR]

In the steady state, tumors harbor several populations of dendritic cells (DCs) and myeloid cells that are key regulators of the intratumoral immune environment. Among these cells, migratory CD103+ cross-presenting DCs are thought to be critical for tumor-specific CTL responses and tumor resistance. However, it is unclear whether this prominent role also extends to immunotherapy. We used a murine orthotopic mammary tumor model, as well as Clec9A-diphtheria toxin receptor mice that can be depleted of the specialized cross-presenting CD8α+ and CD103+ DC1 subsets, to investigate the role of these DCs in immunotherapy. Treatment with monosodium urate crystals and mycobacteria at the tumor site delayed tumor growth and required DC1s for efficacy. In contrast, treatment with poly I:C was equally effective regardless of DC1 depletion. Neither treatment affected myeloid-derived suppressor cell numbers in the spleen or tumor. Similar experiments using subcutaneous B16 melanoma tumors in BATF3-knockout mice confirmed that CD103+ DCs were not necessary for successful poly I:C immunotherapy. Nevertheless, adaptive immune responses were essential for the response to poly I:C, because mice depleted of CD8+ T cells or all DC subsets were unable to delay tumor growth. In vivo experiments showed that DC1 and DC2 subsets were able to take up tumor Ags, with DC2s making up the larger proportion of lymph node DCs carrying tumor material. Both DC subsets were able to cross-present OVA to OT-I T cells in vitro. Thus, immunotherapy with poly I:C enables multiple DC subsets to cross-present tumor Ag for effective antitumor immune responses. [ABSTRACT FROM AUTHOR]

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (rs = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients' disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression. [ABSTRACT FROM AUTHOR]

A nanoarray planar waveguide biosensor was developed for the detection of tetrodotoxin (TTX). This technique, specifically designed for the first time for TTX, provided a compact versatile user friendly device that obtained a test result in ten minutes. The device consisted of nanoprinted toxin-conjugate arrays constructed in the manner of an indirect competitive immunoassay, for the analysis of puffer fish samples under high flow conditions. The applicability to natural samples was investigated through the study of matrix effects and toxin recovery. The biosensor enabled the detection of TTX from 0.4 to 3.29 μg g −1 puffer fish tissue. The sensitivity attained demonstrates this assay as a rapid and sensitive screening method to detect TTX in different species of puffer fish, well below the Japanese maximum permitted level (2 μg g −1 ) and the estimated safety level used in the EU and the US (0.8 μg g −1 ). Assay repeatability and reproducibility were assessed at 0.4 and 0.8 μg g −1 , showing relative standard deviation (RSD) values below 15% and toxin recovery within 85–115%. The appropriate correlation of data obtained from the biosensor compared to that reported by ELISA, RBA, SPR biosensor and LC–MS/MS for the analysis of 12 puffer fish samples, proved the feasibility and reliability of this immunosensor to support monitoring programs and research activities. [ABSTRACT FROM AUTHOR]

Cry toxins produced by Bacillus thuringiensis (Bt) are insecticidal proteins widely used in insect control. Recently, it was shown that ATP-binding cassette transporter proteins (ABC) such as ABCC2, ABCC3, ABCG1 and ABCA2 are implicated in the insecticidal action of Cry toxins as putative receptors. However, the transcriptional regulators involved in the expression of ABC transporter genes remain unknown. Sequence analysis of promoter regions of ABCC2 gene from Helicoverpa armigera and ABCC3 gene from Spodoptera litura Sl-HP cultured cells, revealed the potential participation of Forkhead box protein A (FOXA), a transcription factor that regulates the expression of genes through remodeling chromatin. To determine if FOXA was involved in regulating expression of ABCC2 and ABCC3 genes, the expression of FOXA, ABCC2 and ABCC3 was compared in Sl-HP cells that are sensitive to Cry1Ac toxin with those in S. frugiperda Sf9 cells that are not sensitive to the toxin. Expression levels of those genes were significantly higher in Sl-HP than in Sf9 cells. Transient expression of FOXA in Sf9 cells activated ABCC2 and ABCC3 transcription, which directly correlated with enhanced Cry1Ac-susceptibility in these cells. Silencing of FOXA gene expression by RNAi in H. armigera larvae resulted in a decreased expression of ABCC2 and ABCC3 without affecting expression of other Cry toxin receptor genes such as alkaline phosphatase , aminopeptidase or cadherin . Silencing of FOXA gene expression also resulted in a Cry1Ac-tolerant phenotype since lower mortality and higher pupation rate were observed in diet containing Cry1Ac protoxin in comparison with the control group. These results demonstrate that FOXA up-regulates expression of the Cry1Ac-toxin receptor ABCC2 and ABCC3 genes, and that lower FOXA expression correlates with tolerance to Cry toxin in cell lines and in lepidopteran larvae. [ABSTRACT FROM AUTHOR]

Clostridium difficile is the cause of antibiotics-associated diarrhea and pseudomembranous colitis. The pathogen produces three protein toxins: C. difficile toxins A (TcdA) and B (TcdB), and C. difficile transferase toxin (CDT). The single-chain toxins TcdA and TcdB are the main virulence factors. They bind to cell membrane receptors and are internalized. The N-terminal glucosyltransferase and autoprotease domains of the toxins translocate from low-pH endosomes into the cytosol. After activation by inositol hexakisphosphate (InsP6), the autoprotease cleaves and releases the glucosyltransferase domain into the cytosol, where GTP-binding proteins of the Rho/Ras family are mono- O-glucosylated and, thereby, inactivated. Inactivation of Rho proteins disturbs the organization of the cytoskeleton and affects multiple Rho-dependent cellular processes, including loss of epithelial barrier functions, induction of apoptosis, and inflammation. CDT, the third C. difficile toxin, is a binary actin-ADP-ribosylating toxin that causes depolymerization of actin, thereby inducing formation of the microtubule-based protrusions. Recent progress in understanding of the toxins' actions include insights into the toxin structures, their interaction with host cells, and functional consequences of their actions. [ABSTRACT FROM AUTHOR]

Information about the receptor-interaction region of Cry toxins, insecticidal proteins produced by Bacillus thuringiensis , is needed to elucidate the mode of action of Cry toxins and improve their toxicity through protein engineering. We analyzed the interaction sites on Cry1Aa with ABC transporter C2 (ABCC2), one of the most important Cry1A toxin receptors. A competitive binding assay revealed that the Bombyx mori ABCC2 (BmABCC2) Cry1A binding site was the same as the BtR175 binding site, suggesting that the loop region of Cry1Aa domain II is a binding site. Next, we constructed several domain II loop mutant toxins and tested their binding affinity in an SPR analysis, and also performed a cell swelling assay to evaluate receptor-mediated cytotoxicity. Our results indicate that the loop regions required for BtR175 and BmABCC2 binding and the regions important for cytotoxicity partially overlap. Our results also suggest that receptor binding is necessary but not sufficient for cytotoxicity. This is the first report showing the region of interaction between ABCC2 and Cry1Aa and the cytotoxicity-relevant properties of the Cry1Aa domain II loop region. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis israelensis (Bti) toxins are increasingly used for mosquito control, but little is known about the precise mode of action of each of these toxins, and how they interact to kill mosquito larvae. By using RNA sequencing, we investigated change in gene transcription level and polymorphism variations associated with resistance to each Bti Cry toxin and to the full Bti toxin mixture in the dengue vector Aedes aegypti . The up-regulation of genes related to chitin metabolism in all selected strain suggests a generalist, non-toxin-specific response to Bti selection in Aedes aegypti . Changes in the transcription level and/or protein sequences of several putative Cry toxin receptors (APNs, ALPs, α-amylases, glucoside hydrolases, ABC transporters) were specific to each Cry toxin. Selective sweeps associated with Cry4Aa resistance were detected in 2 ALP and 1 APN genes. The lack of selection of toxin-specific receptors in the Bti-selected strain supports the hypothesis that Cyt toxin acts as a receptor for Cry toxins in mosquitoes. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to simultaneously visualize the lateral distribution of different lipid classes in tissue sections, but the applicability of the method to real-life samples is often limited by ion suppression effects. In particular, the presence of abundant phosphatidylcholines (PCs) can reduce the ion yields for all other lipid species in positive ion mode measurements. Here, we used on-tissue treatment with buffer-free phospholipase C (PLC) to near-quantitatively degrade PCs in fresh-frozen tissue sections. The ion signal intensities of mono-, di-, and oligohexosylceramides were enhanced by up to 10-fold. In addition, visualization of Shiga toxin receptor globotriaosylceramide (Gb3Cer) in the kidneys of wild-type and a-galactosidase A-knockout (Fabry) mice was possible at about ten micrometer resolution. Importantly, the PLC treatment did not decrease the high lateral resolution of the MS imaging analysis. [ABSTRACT FROM AUTHOR]

Interaction between bacterial toxins and cellular surface receptors is an important component of the host-pathogen interaction. Anthrax toxin protective antigen (PA) binds to the cell surface receptor, enters the cell through receptor-mediated endocytosis, and forms a pore on the endosomal membrane that translocates toxin enzymes into the cytosol of the host cell. As the major receptor for anthrax toxin in vivo, anthrax toxin receptor 2 (ANTXR2) plays an essential role in anthrax toxin action by providing the toxin with a high-affinity binding anchor on the cell membrane and a path of entry into the host cell. ANTXR2 also acts as a molecular clamp by shifting the pH threshold of PA pore formation to a more acidic pH range, which prevents premature pore formation at neutral pH before the toxin reaches the designated intracellular location. Most recent studies have suggested that the disulfide bond in the immunoglobulin (Ig)-like domain of ANTXR2 plays an essential role in anthrax toxin action. Here we will review the roles of ANTXR2 in anthrax toxin action, with an emphasis on newly updated knowledge. [ABSTRACT FROM AUTHOR]

The IARC Monographs are a series of scientific reviews that identify environmental factors that can increase the risk of cancer in humans. In its first part, the principles and procedures of the IARC Monographs evaluations are summarized. In a second part, we present the most recent IARC evaluation of polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs), performed in February 2013: PCBs and dioxin-like PCBs were both classified into group 1 'carcinogens,' while PBBs were evaluated as 'probably carcinogenic to humans' (group 2A). Noteworthy is that the relative contributions of different PCB congeners to the carcinogenicity of PCB mixtures are not known. The use of mechanistic data for the classification into a higher category is discussed in the context of the history of the consecutive evaluations of several related polychlorinated compounds. [ABSTRACT FROM AUTHOR]

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A 4 (LXA 4 ), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA 4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA 4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA 4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA 4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA 4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA 4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. [ABSTRACT FROM AUTHOR]

Insect resistance threatens sustainability of insecticides based on Cry proteins from the bacterium Bacillus thuringiensis (Bt). Since high levels of resistance to Cry proteins involve alterations in Cry-binding midgut receptors, their identification is needed to develop resistance management strategies. Through Illumina sequencing we generated a transcriptome containing 16,161 annotated unigenes for the Oriental leafworm ( Spodoptera litura ). Transcriptome mining identified 6 contigs with identity to reported lepidopteran Cry toxin receptors. Using PCR we confirmed their expression during the larval stage and compared their quantitative expression in larvae from susceptible and a field-derived Cry1Ca resistant strain of S . litura . Among reduced transcript levels detected for most tested contigs in the Cry1Ca-resistant S . litura larvae, the most dramatic reduction (up to 99%) was detected for alkaline phosphatase contigs. This study significantly expands S . litura transcriptomic resources and provides preliminary identification of putative receptor genes with altered expression in S . litura resistant to Cry1Ca toxin. [ABSTRACT FROM AUTHOR]

It is well known that angiogenesis is linked to fibrotic processes in fibroproliferative diseases, but insights into pathophysiological processes are limited, due to lack of understanding of molecular mechanisms controlling endothelial and fibroblastic homeostasis. We demonstrate here that the matrix receptor anthrax toxin receptor 1 (ANTXR1), also known as tumor endothelial marker 8 (TEM8), is an essential component of these mechanisms. Loss of TEM8 function in mice causes reduced synthesis of endothelial basement membrane components and hyperproliferative and leaky blood vessels in skin. In addition, endothelial cell alterations in mutants are almost identical to those of endothelial cells in infantile hemangioma lesions, including activated VEGF receptor signaling in endothelial cells, increased expression of the downstream targets VEGF and CXCL12, and increased numbers of macrophages and mast cells. In contrast, loss of TEM8 in fibroblasts leads to increased rates of synthesis of fiber-forming collagens, resulting in progressive fibrosis in skin and other organs. Compromised interactions between TEM8-deficient endothelial and fibroblastic cells cause dramatic reduction in the activity of the matrix-degrading enzyme MMP2. In addition to insights into mechanisms of connective tissue homeostasis, our data provide molecular explanations for vascular and connective tissue abnormalities in GAPO syndrome, caused by loss-of-function mutations in ANTXR1 . Furthermore, the loss of MMP2 activity suggests that fibrotic skin abnormalities in GAPO syndrome are, in part, the consequence of pathophysiological mechanisms underlying syndromes (NAO, Torg and Winchester) with multicentric skin nodulosis and osteolysis caused by homozygous loss-of-function mutations in MMP2. [ABSTRACT FROM AUTHOR]

In 2011, a large outbreak of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) infections occurred in northern Germany, which mainly affected adults. Out of 3842 patients, 104 experienced a complicated course comprising hemolytic uremic syndrome and neurological complications, including cognitive impairment, aphasia, seizures and coma. T2 hyperintensities on magnet resonance imaging (MRI) bilateral in the thalami and in the dorsal pons were found suggestive of a metabolic toxic effect. Five of the 104 patients died because of toxic heart failure. In the present study, the post-mortem neuropathological findings of the five EHEC patients are described. Histological investigation of 13 brain regions (frontal, temporal, occipital cortex, corpora mammillaria, thalamus, frontal operculum, corona radiata, gyrus angularis, pons, medulla oblongata, cerebellar vermis and cerebellar hemisphere) showed no thrombosis, ischemic changes or fresh infarctions. Further, no changes were found in electron microscopy. In comparison with five age-matched controls, slightly increased activation of microglia and a higher neuronal expression of interleukin-1β and of Shiga toxin receptor CD77/globotriaosylceramide 3 was observed. The findings were confirmed by Western blot analyses. It is suggested that CD77/globotriaosylceramide upregulation may be a consequence to Shiga toxin exposure, whereas increased interleukin-1β expression may point to activation of inflammatory cascades. [ABSTRACT FROM AUTHOR]

ANTXR 1 and 2, also known as TEM8 and CMG2, are two type I membrane proteins, which have been extensively studied for their role as anthrax toxin receptors, but with a still elusive physiological function. Here we have analyzed the importance of N-glycosylation on folding, trafficking and ligand binding of these closely related proteins. We find that TEM8 has a stringent dependence on N-glycosylation. The presence of at least one glycan on each of its two extracellular domains, the vWA and Ig-like domains, is indeed necessary for efficient trafficking to the cell surface. In the absence of any N-linked glycans, TEM8 fails to fold correctly and is recognized by the ER quality control machinery. Expression of N-glycosylation mutants reveals that CMG2 is less vulnerable to sugar loss. The absence of N-linked glycans in one of the extracellular domains indeed has little impact on folding, trafficking or receptor function of the wild type protein expressed in tissue culture cells. N-glycans do, however, seem required in primary fibroblasts from human patients. Here, the presence of N-linked sugars increases the tolerance to mutations in cmg2 causing the rare genetic disease Hyaline Fibromatosis Syndrome. It thus appears that CMG2 glycosylation provides a buffer towards genetic variation by promoting folding of the protein in the ER lumen. [ABSTRACT FROM AUTHOR]

While bacterial effectors are often directly introduced into eukaryotic target cells by various types of injection machines, toxins enter the cytosol of host cells from endosomal compartments or after retrograde transport via Golgi from the ER. A first crucial step of toxin–host interaction is receptor binding. Using optimized protocols and new methods novel toxin receptors have been identified, including metalloprotease ADAM 10 for Staphylococcus aureus α-toxin, laminin receptor Lu/BCAM for Escherichia coli cytotoxic necrotizing factor CNF1, lipolysis stimulated lipoprotein receptor (LSR) for Clostridium difficile transferase CDT and low-density lipoprotein receptor-related protein (LRP) 1 for Clostridium perfringens TpeL toxin. [ABSTRACT FROM AUTHOR]

Arsenic exposure has been linked to an increased incidence of atherosclerosis. Previously, we have shown in vitro and in vivo that arsenic inhibits transcriptional activation of the liver X receptors (LXRs), key regulators of lipid homeostasis. Therefore, we evaluated the role of LXRα in arsenic-induced atherosclerosis using the apoE−/− mouse model. Indeed, deletion of LXRα protected apoE−/− mice against the proatherogenic effects of arsenic. We have previously shown that arsenic changes the plaque composition in apoE−/− mice. Arsenic decreased collagen content in the apoE−/− model, and we have observed the same diminution in LXRα−/−apoE−/− mice. However, the collagen-producing smooth muscle cells (SMCs) were decreased in apoE−/−, but increased in LXRα−/−apoE−/−. Although transcriptional activation of collagen remained the same in SMC from both genotypes, arsenic-exposed LXRα−/−apoE−/− plaques had increased matrix metalloproteinase activity compared with both control LXRα−/−apoE−/− and apoE−/−, which could be responsible for both the decrease in plaque collagen and the SMC invasion. In addition, arsenic increased plaque lipid accumulation in both genotypes. However, macrophages, the cells known to retain lipid within the plaque, were unchanged in arsenic-exposed apoE−/− mice, but decreased in LXRα−/−apoE−/−. We confirmed in vitro that these cells retained more lipid following arsenic exposure and are more sensitive to apoptosis than apoE−/−. Mice lacking LXRα are resistant to arsenic-enhanced atherosclerosis, but arsenic-exposed LXRα−/−apoE−/− mice still present a different plaque composition pattern than the arsenic-exposed apoE−/− mice. [ABSTRACT FROM AUTHOR]

Cell ablation technology is useful for studying specific cell lineages in a developing organ in vivo. Herein, we established a novel anti- Mü llerian hormone (AMH)-toxin receptor-mediated cell knockout (Treck) mouse line, in which the diphtheria toxin (DT) receptor was specifically activated in Sertoli and granulosa cells in postnatal testes and ovaries respectively. In the postnatal testes of Amh-Treck transgenic (Tg) male mice, DT injection induced a specific loss of the Sertoli cells in a dose-dependent manner, as well as the specific degeneration of granulosa cells in the primary and secondary follicles caused by DTinjection in Tg females. In the testes with depletion of Sertoli cell, germ cells appeared to survive for only several days after DT treatment and rapidly underwent cell degeneration, which led to the accumulation of a large amount of cell debris within the seminiferous tubules by day 10 after DT treatment. Transplantation of exogenous healthy Sertoli cells following DT treatment rescued the germ cell loss in the transplantation sites of the seminiferous epithelia, leading to a partial recovery of the spermatogenesis. These results provide not only in vivo evidence of the crucial role of Sertoli cells in the maintenance of germ cells, but also show that the Amh-Treck Tg line is a useful in vivo model of the function of the supporting cell lineage in developing mammalian gonads. [ABSTRACT FROM AUTHOR]

Staphyococcus aureus frequently causes recurrent skin and soft-tissue infection (SSTI). In the pediatric population, elevated serum antibody targeting S. aureus α-toxin is correlated with a reduced incidence of recurrent SSTI. Using a novel model of recurrent SSTI, we demonstrated that expression of α-toxin during primary infection increases the severity of recurrent disease. Antagonism of α-toxin by either a dominant-negative toxin mutant or a small molecule inhibitor of the toxin receptor ADAM10 during primary infection reduces reinfection abscess severity. Early neutralization of α-toxin activity during S. aureus SSTI therefore offers a new therapeutic strategy to mitigate primary and recurrent disease. [ABSTRACT FROM AUTHOR]

Abstract: Composition of mamba's venom is quite atypical and characterized by the presence of a large diversity of three-finger fold toxins (3FTx) interacting with various enzymes, receptors and ion channels. In particular, 3FTx from mambas display the unique property to interact with class A GPCRs, sometimes with a high affinity and selectivity. A screening of five of these toxins (MT1, MT3, MT7, ρ-Da1a and ρ-Da1b) on 29 different subtypes of bioaminergic receptors, using competition binding experiments, highlights the diversity of their pharmacological profiles. These toxins may display either absolute selectivity for one receptor subtype or a polypharmacological property for various bioaminergic receptors. Nevertheless, adrenoceptor is the main receptor family targeted by these toxins. Furthermore, a new receptor target was identified for 3FTx and toxins in general, the ρ-Da1b interacting competitively with the human dopamine D3 receptor in the micromolar range. This result expands the diversity of GPCRs targeted by toxins and more generally highlights the multipotent interacting property of 3FTx. Phylogenic analyzes of these toxins show that muscarinic, adrenergic and dopaminergic toxins may be pooled in one family called aminergic toxins, this family coming probably from a specific radiation of ligands present in mamba venoms. [Copyright &y& Elsevier]

Background: Despite the intensive use of Bacillus thuringiensis israelensis (Bti) toxins for mosquito control, little is known about the long term effect of exposure to this cocktail of toxins on target mosquito populations. In contrast to the many cases of resistance to Bacillus thuringiensis Cry toxins observed in other insects, there is no evidence so far for Bti resistance evolution in field mosquito populations. High fitness costs measured in a Bti selected mosquito laboratory strain suggest that evolving resistance to Bti is costly. The aim of the present study was to identify transcription level and polymorphism variations associated with resistance to Bti toxins in the dengue vector Aedes aegypti. We used RNA sequencing (RNA-seq) for comparing a laboratory-selected strain showing elevated resistance to Bti toxins and its parental non-selected susceptible strain. As the resistant strain displayed two marked larval development phenotypes (slow and normal), each phenotype was analyzed separately in order to evidence potential links between resistance mechanisms and mosquito life-history traits. Results: A total of 12,458 genes were detected of which 844 were differentially transcribed between the resistant and susceptible strains. Polymorphism analysis revealed a total of 68,541 SNPs of which 12,571 SNPs exhibited more than 40% frequency difference between the resistant and susceptible strains, affecting 2,953 genes. Bti resistance is associated with changes in the transcription level of enzymes involved in detoxification and chitin metabolism. Among previously described Bti-toxin receptors, four alkaline phosphatases (ALPs) were differentially transcribed between resistant and susceptible larvae, and non-synonymous changes affected the protein sequence of one cadherin, six aminopeptidases (APNs) and four á-amylases. Other putative Cry receptors located in lipid rafts, such as flotillin and glycoside hydrolases, were under-transcribed and/or contained non-synonymous substitutions. Finally, immunity-related genes showed contrasted transcription and polymorphisms patterns between the two developmental resistant phenotypes, suggesting the existence of trade-offs between Bti-resistance, life-history traits and immunity. Conclusions: The present study is the first to analyze the whole transcriptome of Bti-resistant mosquitoes by RNA-seq, shedding light on the importance of studying both transcription levels and sequence polymorphism variations to get a comprehensive view of insecticide resistance. [ABSTRACT FROM AUTHOR]

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax. [ABSTRACT FROM AUTHOR]

Large glycosylating toxins are major virulence factors of various species of pathogenic Clostridia. Prototypes are Clostridium difficile toxins A and B, which cause antibiotics-associated diarrhea and pseudomembranous colitis. The current model of the toxins' action suggests that receptor binding is mediated by a C-terminal domain of combined repetitive oligopeptides (CROP). This model is challenged by the glycosylating Clostridium perfringens large cytotoxin (TpeL toxin) that is devoid of the CROP domain but still intoxicates cells. Using a haploid genetic screen, we identified LDL receptor-related protein 1 (LRP1) as a host cell receptor for the TpeL toxin. LRP1-deficient cells are not able to take up TpeL and are not intoxicated. Expression of cluster IV of LRP1 is sufficient to rescue toxin uptake in these cells. By plasmon resonance spectroscopy, a KD value of 23 nM was determined for binding of TpeL to LRP1 cluster IV. The C terminus of TpeL (residues 1335-1779) represents the receptor-binding domain (RBD) of the toxin. RBD-like regions are conserved in all other clostridial glycosylating toxins preceding their CROP domain. CROP-deficient C. difficile toxin B is toxic to cells, depending on the RBD-like region (residues 1349-1811) but does not interact with LRP1. Our data indicate the presence of a second, CROP-independent receptor-binding domain in clostridial glycosylating toxins and suggest a two-receptor model for the cellular uptake of clostridial glycosylating toxins. [ABSTRACT FROM AUTHOR]

Anthrax toxin protective antigen (PA) binds cellular receptors and self-assembles into oligomeric prepores. A prepore converts to a protein translocating pore after it has been transported to an endosome where the low pH triggers formation of a membrane-spanning β-barrel channel. Formation of this channel occurs after some PA-receptor contacts are broken to allow pore formation, while others are retained to preserve receptor association. The interaction between PA and anthrax toxin receptor 1 (ANTXR1) is weaker than its interaction with ANTXR2 such that the pH threshold of ANTXR1-mediated pore formation is higher by 1 pH unit. Here we examine receptor-specific differences in toxin binding and pore formation by mutating PA residue G342 that selectively abuts ANTXR2. Mutation of G342 to valine, leucine, isoleucine, or tryptophan increased the amount of PA bound to ANTXR1-expressing cells and decreased the amount of PA bound to ANTXR2-expressing cells. The more conservative G342A mutation did not affect the level of binding to ANTXR2, but ANTXR2-bound PA-G342A prepores exhibited a pH threshold higher than that of wild-type prepores. Mixtures of wild-type PA and PA-G342A were functional in toxicity assays, and the pH threshold of ANTXR2-mediated pore formation was dictated by the relative amounts of the two proteins in the hetero-oligomers. These results suggest that PA subunits within an oligomer do not have to be triggered simultaneously for a productive membrane insertion event to occur. [ABSTRACT FROM AUTHOR]

Background Spiders have evolved pharmacologically complex venoms that serve to rapidly subdue prey and deter predators. The major toxic factors in most spider venoms are small, disulfide-rich peptides. While there is abundant evidence that snake venoms evolved by recruitment of genes encoding normal body proteins followed by extensive gene duplication accompanied by explosive structural and functional diversification, the evolutionary trajectory of spidervenom peptides is less clear. Results Here we present evidence of a spider-toxin superfamily encoding a high degree of sequence and functional diversity that has evolved via accelerated duplication and diversification of a single ancestral gene. The peptides within this toxin superfamily are translated as prepropeptides that are posttranslationally processed to yield the mature toxin. The Nterminal signal sequence, as well as the protease recognition site at the junction of the propeptide and mature toxin are conserved, whereas the remainder of the propeptide and mature toxin sequences are variable. All toxin transcripts within this superfamily exhibit a striking cysteine codon bias. We show that different pharmacological classes of toxins within this peptide superfamily evolved under different evolutionary selection pressures. Conclusions Overall, this study reinforces the hypothesis that spiders use a combinatorial peptide library strategy to evolve a complex cocktail of peptide toxins that target neuronal receptors and ion channels in prey and predators. We show that the ω-hexatoxins that target insect voltagegated calcium channels evolved under the influence of positive Darwinian selection in an episodic fashion, whereas the κ-hexatoxins that target insect calcium-activated potassium channels appear to be under negative selection. A majority of the diversifying sites in the ω- hexatoxins are concentrated on the molecular surface of the toxins, thereby facilitating neofunctionalisation leading to new toxin pharmacology. [ABSTRACT FROM AUTHOR]

Highlights: [•] The ectodomain of human anthrax toxin receptor 2 (R318) was insoluble in Escherichia coli. [•] R318 was cloned into a pCOLD-TF vector to produce a TF-R318 fusion protein. [•] TF-R318 was produced as a soluble protein in Origami B cells at 16°C. [•] R318 was purified into homogeneity through a series of chromatography. [•] Purified R318 was functional in binding and mediating anthrax toxin pore formation. [ABSTRACT FROM AUTHOR]

Highlights: [•] Staphylococcus aureus pore-forming toxins (PFTs) use different proteinaceous receptors to target host cells. [•] Receptors explain cell-type and species tropisms exhibited by S. aureus toxins. [•] Human-specific receptors underscore the limitations of animal models in PFT research. [ABSTRACT FROM AUTHOR]

This review gives a glimpse of probiotic role in human life and how it affects the individual. Probiotics have been studied as an alternative to antibiotic therapy. The term "probiotics" comes from the Greek word "pro bios" meaning "for life" opposed to "antibiotics," which means "against life." As the research is progressing, new approaches to treat diseases are being developed such as prebiotics and synbiotics. Probiotics work in our body through various modes of action, namely, production of inhibitory substances, stimulation of immunity, affecting host gene expression, blocking of adhesion sites, competition for nutrients, and degradation of toxin receptors. The various criteria employed to select strains for probiotics are acid-bile stability, adhesion stability, antimicrobial activity, viability and stability during processing and storage. Research has proved the therapeutic effects of probiotics in diseases such as diarrhea, rotavirus diarrhea, irritable bowel syndrome, Helicobacter pylori infection, hepatic encephalopathy, celiac disease, and hyperoxaluria. Probiotics have prophylactic effects in diseases such as pouchitis and ulcerative colitis that come under inlammatory bowel disease, antibiotic-induced diarrhea, necrotizing enterocolitis, ventilator-associated pneumonia, dental decay, periodontal infection, halitosis, constipation, gastrointestinal infections, and colon cancer. Probiotics also help in xenobiotic metabolism, lactose intolerance, cholesterol reduction, sucrase-isomaltase deiciency, stress, and hypertension. Further research is needed to prove the eficacy of probiotics in case of radiation-induced diarrhea, HIV/AIDS diarrhea, Crohn's disease, nonalcoholic fatty liver disease, and allergy. [ABSTRACT FROM AUTHOR]

Sea anemone venoms have become a rich source of peptide toxins which are invaluable tools for studying the structure and functions of ion channels. In this work, BcsTx3, a toxin found in the venom of a Bunodosoma caissarum (population captured at the Saint Peter and Saint Paul Archipelago, Brazil) was purified and biochemically and pharmacologically characterized. The pharmacological effects were studied on 12 different subtypes of voltage-gated potassium channels (KV1.1-KV1.6; KV2.1; KV3.1; KV4.2; KV4.3; hERG and Shaker IR) and three cloned voltage-gated sodium channel isoforms (NaV1.2, NaV1.4 and BgNaV1.1) expressed in Xenopus laevis oocytes. BcsTx3 shows a high affinity for Drosophila Shaker IR channels over rKv1.2, hKv1.3 and rKv1.6, and is not active on NaV channels. Biochemical characterization reveals that BcsTx3 is a 50 amino acid peptide crosslinked by four disulfide bridges, and sequence comparison allowed BcsTx3 to be classified as a novel type of sea anemone toxin acting on KV channels. Moreover, putative toxins homologous to BcsTx3 from two additional actiniarian species suggest an ancient origin of this newly discovered toxin family. [ABSTRACT FROM AUTHOR]

Highlights: [•] We re-analysed a toxicogenomic study of 4 isoproterenol doses, using multivariate statistics. [•] We focused on target gene sets for cellular stress-response mechanisms and toxin receptors. [•] Significant, co-regulated, expression changes in target genes were detected at low doses. [•] The target-gene approach is toxicologically-relevant, with high statistical sensitivity. [•] Expression data, combined within sets, was used to find benchmark doses for risk assessment. [Copyright &y& Elsevier]

Cancer stem-like cells are thought to contribute to tumor recurrence. The anthrax toxin receptor 1 (ANTXR1) has been identified as a functional biomarker of normal stem cells and breast cancer stem-like cells. Primary stem cell-enriched basal cells (CD49f+/ EpCAM-/Lin-) expressed higher levels of ANTXR1 compared with mature luminal cells. CD49f+/EpCAM-, CD44+/EpCAM-, CD44+/CD24-, or ALDEFLUOR-positive subpopulations of breast cancer cells were enriched for ANTXR1 expression. CD44+/CD24-/ANTXR1+ cells displayed enhanced self-renewal as measured by mammosphere assay compared with CD44+/CD24+/ ANTXR1- cells. Activation of ANTXR1 by its natural ligand C5A, a fragment of collagen VI α3, increased stem cell self-renewal in mammosphere assays and Wnt signaling including the expression of the Wnt receptor--lipoprotein receptor-related protein 6 (LRP6), phosphorylation of GSK3α/β, and elevated expression of Wnt target genes. RNAi-mediated silencing of ANTXR1 enhanced the expression of luminal-enriched genes but diminished Wnt signaling including reduced LRP6 and ZEB1 expression, self-renewal, invasion, tumorigenicity, and metastasis. ANTXR1 silencing also reduced the expression of HSPA1A, which is overexpressed in metastatic breast cancer stem cells. Analysis of public databases revealed ANTXR1 amplification in medullary breast carcinoma and overexpression in estrogen receptor-negative breast cancers with the worst outcome. Furthermore, ANTXR1 is among the 10% most overexpressed genes in breast cancer and is coexpressed with collagen VI. Thus, ANTXR1:C5A interactions bridge a network of collagen cleavage and remodeling in the tumor microenvironment, linking it to a stemness signaling network that drives metastatic progression. [ABSTRACT FROM AUTHOR]

Highlights: [•] Novel transgenic mouse models for T1D were established by TRECK method. [•] They were established on C57BL/6 and SCID background. [•] DT administration resulted severe ablation of pancreatic β cells within 3days. [•] Hyperglycemia of SCID-Tg mice is cured by allogeneic and xenogeneic transplantation. [•] These Tg mice are highly useful for diabetogenic research. [ABSTRACT FROM AUTHOR]

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen ( PA) with one of three reported cell surface toxin receptors ( ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 ( TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon ( IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection. [ABSTRACT FROM AUTHOR]

Abstract: Opossums and pitvipers are sympatric throughout most of the New World, but trophic relationships between these speciose clades have only recently attracted the attention of researchers. Although it is now known that some venom-resistant opossums prey on pitvipers, a review of the literature on diets shows that some Neotropical pitvipers prey on opossums. Interestingly, some pitviper species prey on opossums known or suspected to be venom resistant. If venom resistance and venom potency are coevolved traits, then these observations suggest that alternative outcomes may result in role-switching between victims and exploiters. Because molecular antagonists (e.g., venom toxins and toxin-neutralizing serum proteins) that could mediate such outcomes have been plausibly identified, this system is a potentially fruitful field for evolutionary research. [Copyright &y& Elsevier]

Abstract: Bacillus thuringiensis subsp. aizawai strain HD133, known by its effectiveness against Spodoptera species, produces many insecticidal proteins including Cry1Ab, Cry1Ca and Cry1Da. In the present study, the insecticidal activity of Cry1Da against Spodoptera littoralis was investigated. It showed toxicity with an LC50 of 224.4ng/cm2 with 95% confidence limits of (178.61–270.19) and an LC90 of 467.77ng/cm2 with 95% confidence limits of (392.89–542.65). The midgut histopathology of Cry1Da fed larvae showed vesicle formation in the apical region, vacuolization and destruction of epithelial cells. Biotinylated-activated Cry1Da toxin bound protein of about 65kDa on blots of S. littoralis brush border membrane preparations. This putative receptor differs in molecular size from those recognized by Cry1C and Vip3A which are active against this polyphagous insect. This difference in midgut receptors strongly supports the use of Cry1Da as insecticidal agent, particularly in case of Cry and/or Vip-resistance management. [Copyright &y& Elsevier]

Abstract: In an effort to study the mode of action of Cry11Ba, we identified toxin binding proteins in Anopheles gambiae larval midgut and investigated their receptor roles. Previously, an aminopeptidase (AgAPN2) and an alkaline phosphatase (AgALP1) were identified as receptors for Cry11Ba toxin in A. gambiae. However, an A. gambiae cadherin (AgCad1) that bound Cry11Ba with low affinity (K d = 766 nM) did not support a receptor role of AgCad1 for Cry11Ba. Here, we studied a second A. gambiae cadherin (AgCad2) that shares 14% identity to AgCad1. Immunohistochemical study showed that the protein is localized on A. gambiae larval midgut apical membranes. Its cDNA was cloned and the protein was analyzed as a transmembrane protein containing 14 cadherin repeats. An Escherichia coli expressed CR14MPED fragment of AgCad2 bound Cry11Ba with high affinity (K d = 11.8 nM), blocked Cry11Ba binding to A. gambiae brush border vesicles and reduced Cry11Ba toxicity in bioassays. Its binding to Cry11Ba could be completely competed off by AgCad1, but only partially competed by AgALP1. The results are evidence that AgCad2 may function as a receptor for Cry11Ba in A. gambiae larvae. [Copyright &y& Elsevier]

Oriented mitosis is essential during tissue morphogenesis. The Wnt/planar cell polarity (Wnt/PCP) pathway orients mitosis in a number of developmental systems, including dorsal epiblast cell divisions along the animal-vegetal (A-V) axis during zebrafish gastrulation. How Wnt signalling orients the mitotic plane is, however, unknown. Here we show that, in dorsal epiblast cells, anthrax toxin receptor 2a (Antxr2a) accumulates in a polarized cortical cap, which is aligned with the embryonic A-V axis and forecasts the division plane. Filamentous actin (F-actin) also forms an A-V polarized cap, which depends on Wnt/PCP and its effectors RhoA and Rock2. Antxr2a is recruited to the cap by interacting with actin. Antxr2a also interacts with RhoA and together they activate the diaphanous-related formin zDia2. Mechanistically, Antxr2a functions as a Wnt-dependent polarized determinant, which, through the action of RhoA and zDia2, exerts torque on the spindle to align it with the A-V axis. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis bacteria are insect pathogens that produce different Cry and Cyt toxins to kill their hosts. Here we review the group of three-domain Cry (3d- Cry) toxins. Expression of these 3d- Cry toxins in transgenic crops has contributed to efficient control of insect pests and a reduction in the use of chemical insecticides. The mode of action of 3d- Cry toxins involves sequential interactions with several insect midgut proteins that facilitate the formation of an oligomeric structure and induce its insertion into the membrane, forming a pore that kills midgut cells. We review recent progress in our understanding of the mechanism of action of these Cry toxins and focus our attention on the different mechanisms of resistance that insects have evolved to counter their action, such as mutations in cadherin, APN and ABC transporter genes. Activity of Cry1 AMod toxins, which are able to form toxin oligomers in the absence of receptors, against different resistant populations, including those affected in the ABC transporter and the role of dominant negative mutants as antitoxins, supports the hypothesis that toxin oligomerization is a limiting step in the Cry insecticidal activity. Knowledge of the action of 3d- Cry toxin and the resistance mechanisms to these toxins will set the basis for a rational design of novel toxins to overcome insect resistance, extending the useful lifespan of Cry toxins in insect control programs. [ABSTRACT FROM AUTHOR]

Abstract: Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3–4-fold decrease in the IC50 dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity. [Copyright &y& Elsevier]

Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na v s) at two pharmacologically distinct sites. Alpha toxins bind at receptor site-3 and inhibit channel inactivation, whereas beta toxins bind at receptor site-4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and ability to compete for the receptor sites at Na v subtypes. To elucidate the toxin-channel surface of interaction at both receptor sites and clarify the molecular basis of varying toxin preferences, an efficient bacterial system for their expression in recombinant form was established. Mutagenesis accompanied by toxicity, binding and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography uncovered a bipartite bioactive surface in toxin representatives of all pharmacological groups. Exchange of external loops between the mammalian brain channel rNa v 1.2a and the insect channel DmNa v 1 highlighted channel regions involved in the varying sensitivity to assorted toxins. In parallel, thorough mutagenesis of channel external loops illuminated points of putative interaction with the toxins. Amino acid substitutions at external loops S1–S2 and S3–S4 of the voltage sensor module in domain II of rNa v 1.2a had prominent impact on the activity of the beta-toxin Css4 (from Centruroides suffusus suffusus ), and substitutions at external loops S1–S2 and S3–S4 of the voltage sensor module in domain IV affected the activity of the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus ). Rosetta modeling of toxin-Na v interaction using the voltage sensor module of the potassium channel as template raises commonalities in the way alpha and beta toxins interact with the channel. Css4 interacts with rNa v 1.2a at a crevice between S1–S2 and S3–S4 transmembrane segments in domain II, while Lqh2 interacts with rNa v 1.2a at a crevice between S1–S2 and S3–S4 transmembrane segments in domain IV. Double-mutant cycle analysis and dissociation assays employing a battery of Lqh2 mutants against rNa v 1.2a mutants identified the docking orientation of alpha toxins at the channel external surface of the Gating-module in domain IV. The other point of interaction between the toxin and the channel has not yet been defined and may involve channel residues of either the Pore-module or the Gating-module. [ABSTRACT FROM AUTHOR]

Plasmodium infections trigger strong innate and acquired immune responses, which can lead to severe complications, including the most feared and often fatal cerebral malaria (CM). To begin to dissect the roles of different dendritic cell (DC) subsets in Plasmodium-induced pathology, we have generated a transgenic strain, Clec9A-diphtiieria toxin receptor that allows us to ablate in vivo Clec9A+ DCs. Specifically, we have analyzed the in vivo contribution of this DC subset in an experimental CM model using Plasmodium berghei, and we provide strong evidence that the absence of this DC subset resulted in complete resistance to experimental CM. This was accompanied with dramatic reduction of brain CD8+ T cells, and those few cerebral CD8+ T cells present had a less activated phenotype, unlike their wildtype counterparts that expressed IFN-7 and especially granzyme B. This almost complete absence of local cellular responses was also associated with reduced parasite load in the brain. The Journal of Immunology, 2012, 189: 1128-1132. [ABSTRACT FROM AUTHOR]

Tumor endothelial marker 8 (TEM8; ANTXR1) is one of two anthrax toxin receptors; the other is capillary morphogenesis gene 2 protein (CMG2; ANTXR2). TEM8 shows enhanced expression in certain tumor endothelia, and is thought to be a player in tumor vasculature formation. However, a comprehensive expression profile of individual TEM8 variants in normal or cancerous tissues is lacking. In this work we carried out an extensive analysis of all splice variants of human TEM8 in 12 digestive tissues, and 8 each fetal and adult tissues, 6 of them cognate pairs. Using variant-specific primers, we first ascertained the status of full-length transcripts by nested PCR. We then carried out quantitative analysis of each transcript by real-time PCR. Three splice variants of TEM8 were reported before, two single-pass integral membrane forms (V1 and V2) and one secreted (V3). Our analysis has revealed two new variants, one encoding a membrane-bound form of the receptor and the other secreted, which we have designated V4 and V5, respectively. All tissues had V1, V2, V3, and V4, but only prostate had V5. Real-time PCR revealed that all variants are present at different levels in various tissues. V3 appeared the most abundant of all. To ascertain its functionality for anthrax toxin, we expressed the newly identified form V4 in a receptornegative host cell, and included V1 and V2 for comparison. Cytotoxicity, toxin binding, and internalization assays showed V4 to be as efficient a receptor as V1 and V2. [ABSTRACT FROM AUTHOR]

Anthrax Toxin Receptor proteins function as receptors for anthrax toxin, however physiological activity remains unclear. To evaluate the biological role of Antxr2, we generated Antxr2-/- mice. Antxr2-/- mice were viable, however Antxr2 is required for parturition in young females and for preserving fertility in older female mice. Histological analysis of the uterus and cervix revealed aberrant deposition of extracellular matrix proteins such as type I collagen, type VI collagen and fibronectin. A marked disruption of both the circular and longitudinal myometrial cell layers was evident in Antxr2-/- mice. These changes progressed as the mice aged, resulting in a thickened, collagen dense, acellular stroma and the disappearance of normal uterine architecture. To investigate the molecular mechanism underlying the uterine fibrosis we performed immunoblotting for MMP2 using uterine lysates and zymography using conditioned medium from Antxr2-/- mouse embryonic fibroblasts and found reduced levels of activated MMP2 in both. This prompted us to investigate MT1- MMP status, as MMP2 processing is regulated by MT1-MMP. We found MT1-MMP activity, as measured by MMP2 processing and activation, was enhanced by expression of either ANTXR1 or ANTXR2. We identified an ANTXR2/MT1-MMP complex and demonstrated that MT1-MMP activity is dependent on ANTXR2 expression levels in cells. Thus, we have discovered that ANTXR1 and ANTXR2 function as positive regulators of MT1-MMP activity. [ABSTRACT FROM AUTHOR]

Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream. [ABSTRACT FROM AUTHOR]

Muscarinic toxin 7 (MT7) is a mamba venom peptide that binds selectively to the M1 muscarinic acetylcholine receptor. We have previously shown that the second (ECL2) and third (ECL3) extracellular loops of the M1 receptor are critically involved in binding the peptide. In this study we used a mutagenesis approach on the M5 subtype of the receptor family to find out if this possesses a similar structural architecture in terms of toxin binding as the M1 receptor. An M5 receptor construct (M5-E175Y184E474), mutated at the formerly deciphered critical residues on ECL2 and 3, gained the ability to bind MT7, but with rather low affinity as determined in a functional assay (apparent Ki = 24 nM; apparent Ki for M1 = 0.5 nM). After screening for different domains and residues, we found a specific residue (P179 to L in M5) in the middle portion of ECL2 that was necessary for high affinity binding of MT7 (M5-EL179YE, apparent Ki = 0.5 nM). Mutation of P179 to A confirmed a role for the leucine side chain in the binding of MT7. Together the results reveal new binding interactions between receptors and the MT7 peptide and strengthen the hypothesis that ECL2 sequence is of utmost importance for MT binding to muscarinic receptors. [ABSTRACT FROM AUTHOR]