The sensitivity of eggs of Echinococcus multilocularis to environmental and controlled laboratory conditions was tested. Egg material was exposed and the infectivity was subsequently monitored by in vitro activation and by oral infection of the natural host, Microtus arvalis. To study the impact of environmental conditions in an endemic area of south-western Germany, eggs were sealed into bags of nylon mesh and exposed to the natural climate during various seasons. The maximal survival time of eggs was 240 days in an experiment performed in autumn and winter and 78 days in summer. A study of the tenacity of eggs under laboratory conditions revealed a high sensitivity to elevated temperatures and to desiccation. At 45 degrees C and 85-95% relative humidity the infectivity was lost after 3 h as well as after 4 h exposure to 43 degrees C suspended in water. Exposure to 27% relative humidity at 25 degrees C as well as exposure to 15% relative humidity at 43 degrees C resulted in a total loss of infectivity within 48 and 2 h, respectively. Temperatures of 4 degrees C and of -18 degrees C were well tolerated (478 days and 240 days survival, respectively), whereas exposure to -83 degrees C and to -196 degrees C quickly killed off the eggs (within 48 h and 20 h, respectively). Eggs of E. multilocularis were not killed off by exposure to various commercially available disinfectants applied according to the manufacturers' instructions and by exposure for 24 h to low concentrations of ethanol. Irradiation with 40 krad. from a 137Caesium source prevented the development of metacestodes but allowed seroconversion of infected rodents.

Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P 2 ]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathrin coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domaincontaining accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in lamprey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the "side sites" of the AP2 α-and β-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrinmediated SV recycling in synapses.endocytosis | synapse | scaffolding proteins | appendage | synaptojanin S ynaptic vesicles (SVs), following their activity-dependent exocytic fusion with the presynaptic plasma membrane, are recycled by compensatory endocytosis at the periactive zone (1-3), largely via clathrin-mediated reinternalization of fully fused SV membrane (4). Clathrin-coated pit (CCP) formation (5) proceeds through the assembly of endocytic proteins at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P 2 ]-enriched membrane sites (6, 7). A key factor in the assembly pathway is the heterotetrameric adaptor complex AP2, whose α-and β2-appendage domains act as major recruitment platforms for accessory proteins (6, 7), regulating distinct steps within the pathway. Despite our extensive knowledge regarding the endocytic interactome, we know comparably little about the structural components within the periactive zone that scaffold the endocytic process, thereby allowing the high fidelity of SV recycling. Such spatiotemporal control is needed to coordinate SV cargo protein sorting with coat recruitment (8) and to restrain membrane fission and uncoating until membrane deformation and CCP assembly are completed. Moreover, stabilizing scaffolds may aid coupling of SV exo-and endocytosis (1, 3). The Drosophila multidomain protein Dap160, an ortholog of mammalian intersectin, has been postulated to act as an endocytic scaffold of the periactive zone (9-11), although its precise role in SV recycling in mammalian nerve terminals remains largely unclear (12).Here we show that intersectin 1 scaffolds the endocytic process by directly associating with AP2. Acute perturbation of intersectin-AP2 complex formation blocks the onset of SV recycling. Moreover, association of the SH3A-B l...

Aiming at the identification of the key bitter peptides in fermented foods, a new approach, coined "sensoproteomics", was developed and applied to fresh cheese samples differing in bitter taste intensity. By means of MPLC fractionation of the water-soluble cheese extracts in combination with taste dilution analysis, complex fractions with intense bitter taste were located and then screened by UPLC−MS/MS for the entire repertoire of ∼1600 candidate peptides, extracted from a literature meta-analysis on dairy products, by using a total of 120 selected reaction monitoring methods computed in silico. A total of 340 out of the 1600 peptides were found in the cheese samples, among which 17 peptides were identified as candidate bitter peptides by considering only peptides that were located in the bitter-tasting MPLC fractions (signal-to-noise ratio: ≥10) with a fold-change of ≥3 when comparing the less bitter to the more bitter cheese sample and that were validated by comparison with the synthetic reference peptides. While EIVPNS[phos]VEQK (α s1 -CN 70−78 ) and INTIASGEPT (κ-CN 122−131 ) did not exhibit any bitter taste up to 2000 μmol/L, 15 of the 17 target peptides showed bitter taste thresholds ranging from 30 (ARHPHPHLSFM, κ-CN 96−106 ) to 690 μmol/L (IQKEDVPS, α s1 -CN 81−88 ). Finally, quantitative peptide analysis followed by calculation of dose-overthreshold factors revealed a primary contribution of MAPKHKEMPFPKYPVEPF (β-CN 102−119 ) and ARHPHPHLSFM (κ-CN 96−106 ) to the perceived bitter taste of the fresh cheese samples. Finally, the evolution of the bitter peptides throughout two different fresh cheese manufacturing processes was quantitatively recorded.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the I␥-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P 2 metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKI␥-p90 associates with both the and ␤2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI␥-p90 tail binds to a cognate recognition site on the sandwich subdomain of the ␤2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKI␥-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI␥ tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2␤ and AP-2. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI␥-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI␥-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P 2 synthesis during clathrin-mediated SV endocytosis.Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) 5 plays a key role in a variety of cell physiological pathways including but not limited to CME, neurotransmitter release (31) and SV cycling, phagocytosis, cell signaling and proliferation, and regulation of the actin cytoskeleton, as well as nuclear functions (1-7). In mammals these diverse roles of PI(4,5)P 2 are regulated by only three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) isozymes. Although PIPKI␣ and -␤ are ubiquitous proteins, the type I␥ isozyme is most highly expressed in the brain where it localizes to synaptic sites (8). PIPKI␥ is also present at focal adhesion sites in non-neuronal cells (9, 10).An overwhelming body of data implicates PI(4,5)P 2 in CME and in the exo-/endocytic cycling of SV membranes (2-4). Recruitment of clathrin adaptors, including epsins, CALM/ AP180, Dab2, HIP1/1R, and AP-2, to the plasma membrane strictly requires the presence of PI(4,5)P 2 , which associates directly with cognate recognition motifs and domains within these proteins. AP-2 is a heterotetrameric complex composed of ␣, ␤2, 2, and 2 subunits that fold into a largely ␣-helical solenoid (termed the "core") with two appendage domains joined to the core by long flexible linkers (11). Membrane...

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