Signals of the Neuropilin-1-MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells.

The neuropilin-1 (NRP1)-MET signaling axis regulates the motility of individual endothelial cells (ECs). It is unknown how this signaling pathway affects the endothelial barrier in coherent ECs forming a tight monolayer. We hypothesized that it is involved both in modulation of the endothelial barrier and in EC activation. To investigate the role of NRP1-MET signaling in inflammatory processes (e.g., systemic inflammatory response syndrome [SIRS] or snakebite-induced SIRS-like conditions), we employed the C-type lectin-related protein rhodocetin-αß (RCαß) as a specific trigger of this signal axis in ECs in vitro. In coherent HUVECs, RCαß reinforced the actin cytoskeleton and increased cell stiffness, thus favoring vascular endothelial cadherin-mediated transmission of intercellular forces. Increased cell stiffness was associated with enhanced activation of RhoA and nuclear translocation of NF-κB. Simultaneously, RCαß-triggered signaling via the NRP1-MET axis increased EC monolayer permeability, induced transcription of proinflammatory genes such as ICAM-1 and, consequently, leukocyte tethering. The RCαß-induced transcriptome differed from that induced by hepatocyte growth factor, although in both cases the same tyrosine kinase, MET, was involved. This was due to RCαß-mediated recruitment of the MET coreceptor NRP1 and additional Rho-mediated activation of the actomyosin system. RCαß induced similar transcriptional and cellular changes if external shear forces were applied. These data highlight the modulatory role of NRP1 as MET coreceptor, and they explain how some snake venoms induce SIRS-like conditions. Additionally, this study demonstrates that inflammatory activation of coherent ECs is triggered by converging signals that are induced by NRP1-MET signaling and influenced by intercellular forces.

Cutting edge: the UNC93B1 tyrosine-based motif regulates trafficking and TLR responses via separate mechanisms.

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.

Production of tongue twisters by speakers with partial glossectomy.

Abstract A partial glossectomy can affect speech production. The goal of this study was to investigate the effect of the presence of a tumour as well as the glossectomy surgery on the patients' production of tongue twisters with the sounds [t] and [k]. Fifteen patients with tongue cancer and 10 healthy controls took part in the study. The outcome measures were the patients' speech acceptability, rate of errors, the time needed to produce the tongue twisters, pause duration between item repetitions and the tongue shape during the production of the consonants [t] and [k] before and after surgery. The patients' speech acceptability deteriorated after the surgery. Compared to controls, the patients' productions of the tongue twisters were slower but not more errorful. Following the surgery, their speed of production did not change, but the rate of errors was higher. Pause duration between items was longer in the patients than in the controls but did not increase from before to after surgery. Analysis of the patients' tongue shapes for the productions of [t] and [k] indicated a higher elevation following the surgery for the patients with flap reconstructions. The results demonstrated that the surgical resection of the tongue changed the error rate but not the speed of production for the patient. The differences in pause duration also indicate that the tumour and the surgical resection of the tongue may impact the phonological planning of the tongue twister.

Epsin N-terminal homology domains bind on opposite sides of two SNAREs.

SNARE proteins are crucial for membrane fusion in vesicular transport. To ensure efficient and accurate fusion, SNAREs need to be sorted into different budding vesicles. This process is usually regulated by specific recognition between SNAREs and their adaptor proteins. How different pairs of SNAREs and adaptors achieve their recognition is unclear. Here, we report the recognition between yeast SNARE Vti1p and its adaptor Ent3p derived from three crystal structures. Surprisingly, this yeast pair Vti1p/Ent3p interacts through a distinct binding site compared to their homologues vti1b/epsinR in mammals. An opposite surface on Vti1p_Habc domain binds to a conserved area on the epsin N-terminal homology (ENTH) domain of Ent3p. Two-hybrid, in vitro pull-down and in vivo experiments indicate this binding interface is important for correct localization of Vti1p in the cell. This previously undescribed discovery that a cargo and adaptor pair uses different binding sites across species suggests the diversity of SNARE-adaptor recognition in vesicular transport.

Dissecting Ent3p: the ENTH domain binds different SNAREs via distinct amino acid residues while the C-terminus is sufficient for retrograde transport from endosomes.

The ENTH (epsin N-terminal homology) domain protein Ent3p and the ANTH [AP (adaptor protein)-180 N-terminal homology] domain protein Ent5p serve as partially redundant adaptors in vesicle budding from the TGN (trans-Golgi network) in Saccharomyces cerevisiae. They interact with phosphoinositides, clathrin, adaptor proteins and cargo such as chitin synthase Chs3p and SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors). In the present study, we show that ent3Δent5Δ cells displayed defects in cell separation and bud site selection. Ent3p and Ent5p were also involved in retrograde transport from early endosomes to the TGN because GFP (green fluorescent protein)-Snc1p shifted from a plasma membrane to an intracellular localization in ent3Δent5Δ cells. The C-terminal part of Ent3p was sufficient to restore retrograde transport from early endosomes to the TGN in ent3Δent5Δ cells. In contrast, the ENTH domain and the C-terminus were required for transport from the TGN to late endosomes, demonstrating that both functions are distinct. The ENTH domain of Ent3p is known to bind the N-terminal domains of the SNAREs Vti1p, Pep12p and Syn8p, which are required for fusion with late endosomes. The interaction surface between the Ent3p-related mammalian epsinR and vti1b is known. In the present paper, we show that Vti1p bound to the homologous surface patch of Ent3p. Pep12p and Syn8p interacted with the same surface area of Ent3p. However, different amino acid residues in Ent3p were crucial for the interaction with these SNAREs in two-hybrid assays. This provides the necessary flexibility to bind three SNAREs with little sequence homology but maintains the specificity of the interaction.

Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens.

BACKGROUND: Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. RESULTS: Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. CONCLUSION: The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

TVP23 interacts genetically with the yeast SNARE VTI1 and functions in retrograde transport from the early endosome to the late Golgi.

SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins contribute to specific recognition between transport vesicles and target membranes and are required for fusion of membranes. The SNARE Vti1p is required for several transport steps between late Golgi, endosomes and the vacuole in the yeast Saccharomyces cerevisiae. Here, we identified the late Golgi membrane protein TVP23 as a multicopy suppressor of the growth defect in vti1-2 cells. By contrast, the growth defect in vti1-11 cells was not suppressed by TVP23 overexpression. Deletion of TVP23 aggravated the growth defect in vti1-2 cells. Genetic interactions between TVP23 and vti1-2 were not found in transport from the late Golgi via the late endosome to the vacuole or in transport from the Golgi directly to the vacuole. These results suggest that Tvp23p is not involved in forward transport from the late Golgi. Therefore retrograde traffic to the late Golgi was analysed. vti1-2 cells accumulated GFP (green fluorescent protein)-Snc1p within the cell, indicating that retrograde transport from the early endosome to the late Golgi was defective in these cells. Deletion of TVP23 in vti1-2 cells resulted in a synthetic defect in GFP-Snc1p recycling, whereas tvp23Delta cells had a slight defect. These results indicate that Tvp23p performs a partially redundant function in retrograde transport from the early endosome to the late Golgi. This transport step was unaffected in vti1-11 cells, providing an explanation for the allele-specific multicopy suppression by TVP23.

ENTH domain proteins are cargo adaptors for multiple SNARE proteins at the TGN endosome.

ENTH and ANTH domain proteins are involved in budding of clathrin-coated vesicles. SNAREs are fusogenic proteins that function in the targeting and fusion of transport vesicles. In mammalian and yeast cells, ENTH domain proteins (epsinR and Ent3p) interact with SNAREs of the vti1 family (Vti1b or Vti1p). This interaction indicates that ENTH proteins could function in cargo sorting, which prompted us to search for additional SNAREs as potential cargo for Ent3p and epsinR. We carried out specific yeast two-hybrid assays, which identified interactions between epsinR and the mammalian late endosomal SNAREs syntaxin 7 and syntaxin 8 as well as between Ent3p and the endosomal SNAREs Pep12p and Syn8p from yeast. Lack of Ent3p affected the trafficking of Pep12p. Ent3p binding to Pep12p required the FSD late endosomal sorting signal in Pep12p. Inactivation of the sorting signal had a similar effect to removal of Ent3p on Pep12p stability indicating that Ent3p acts as a cargo adaptor for Pep12p by binding to the sorting signal. As Vti1p, Pep12p and Syn8p participate in a SNARE complex whereas Vti1b, syntaxin 7 and syntaxin 8 are mammalian SNARE partners, we propose that ENTH domain proteins at the TGN-endosome are cargo adaptors for these endosomal SNAREs.

Extracellular calcium deficiency and ryanodine inhibit Eimeria tenella sporozoite invasion in vitro.

An in vitro assay system with Eimeria tenella sporozoites was used to study the effects of extracellular calcium and active agents affecting the invasion of parasites into host cells. At concentrations of 900 microM Ca(2+) and less the invasion rates were distinctly decreased. Ryanodine, a herbal alkaloid known for binding to internal Ca(2+) channels (ryanodine receptors), showed an inhibitory effect on E. tenella sporozoite invasion. Preincubation tests and staining with a fluorescent derivative of ryanodine assured that the compound bound specifically to the sporozoites and affected them rather than the host cells.