Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles. Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate. To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen. We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential. In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained. The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a Cterminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER. DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes. Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.Membrane-bound compartments in eukaryotic cells can fuse directly as shown for the endoplasmic reticulum (ER) 1 and mitotic Golgi fragments as well as endosomal and lysosomal compartments (homotypic fusion; see Ref. 1). However, vectorial transport between distinct compartments mainly involves small coated vesicles whose formation from the donor membrane is mediated by proteinaceous coats, either COPI, COPII, or clathrin. After uncoating, vesicles fuse selectively with an acceptor membrane (heterotypic fusion; see Ref.2). Both homotypic and heterotypic fusion events rely on specific attachment reactions to guarantee that only appropriate membranes can mix. The membrane attachment itself consists of two steps, tethering and docking, involving different sets of proteins (3, 4).Tethering factors are peripherally membrane-associated protein complexes consisting of up to 10 different subunits, which share little sequence similarity.The subsequent docking stage involves specific sets of membrane-anchored proteins, so-called SNARE proteins (SNARE is soluble NSF (for N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) (5-7). SNAREs are inserted into the membrane either by a C-terminal transmembrane domain or through lipid moieties attached to C-terminal cysteine residues. In contrast to the tethering factors, all known SNARE proteins are members of either of three protein families: the syntaxins, the synaptobrevins or VAMPs, and the SNAP-25 family members. To induce membrane fusion, SNARE proteins from apposed membranes must interact in trans. The formation of a stable four-helix bundle may generate enough energy to promote mixing of the lipid bilayer (8 -10).Lipid mixing experiments using SNARE complexes reconstituted into lipid bilayer vesicl...

The third edition of Introduction to Discrete Event Systems is available as both a print copy and an e-book with hyperlinking capability. We hope the readers will appreciate this new option.The third edition is a "superset" of the second one, with new material added principally in Chaps. 1, 2, 3, 5, 10, and 11. These additions are based on our teaching of discrete event systems courses at Boston University and at the University of Michigan, and they reflect active research trends in discrete event systems since the publication of the second edition. The additions consist of the inclusion of new topics as well as more thorough coverage of existing topics. For the benefit of readers familiar with the second edition, the main changes are summarized as follows.Ä Chapter 1: additional examples of discrete event systems and more discussion on modeling.Ä Chapter 2: new sections on opacity properties, labeled transitions systems, and formal verification and temporal logic; enhanced treatment of verification of diagnosability and codiagnosability properties, state space refinement, and strict subautomata; additional end-of-chapter problems.Ä Chapter 3: new sections on state-based and liveness specifications, marking in specifications, maximal controllable and observable sublanguages, and marking supervisors; expanded treatment of control under partial observation, including state partition automata, supremal normal and controllable sublanguage, inf imal observable and controllable superlanguage, and safe supervisors; more detailed treatment of verification of coobservability in decentralized control and safe decentralized supervision; additional end-of-chapter problems.Ä Chapter 5: new section on event diagnosis.Ä Chapter 10: updated section on discrete event simulation languages.Ä Chapter 11: updated sections on extensions of IPA and on concurrent estimation.While end-of-chapter references have been updated to reflect the new material included, we emphasize once again that these sections serve primarily as starting points for additional readings. The literature in discrete event systems is now vast and diverse, reflecting the growth in this f ield in the last 30 years.vii Once again, we sincerely thank our colleagues, students, and readers for their constructive feedback over the last 12 years. We have tried to account for their comments in this third edition, but obviously our coverage of the growing field of discrete event systems is still very much incomplete. Nevertheless, we hope this book will continue to serve as a comprehensive introduction to the important class of dynamical systems known as discrete event systems. Several additional resources as well as software tools are mentioned throughout the book, although we have avoided explicit listings of URLs, since these tend to change frequently; however, the desired resources should be easily located by web searches.Finally, it is a pleasure to acknowledge the leadership of Melissa Fearon and Wayne Wheeler at Springer throughout the course of this project.Boston, USA

An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity over time. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO2 incubator at 37 degrees C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/ day. Depending on the lid membrane thickness,pH drifts at ambient CO2 levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60 degrees C yielded a 10-fold reduced water vapour flux of 1.75 g/day/ dm 2 through PDMS lids as compared with 18.69 g/day/dm 2 with conventional Petri dishes. Using such PDMS lids,about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these,the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions.

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