Localization and Recycling of gp27 (hp24γ3): Complex Formation with Other p24 Family Members

We report here the characterization of gp27 (hp24γ3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15°C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24α2), p24 (hp24β1), and p23 (hp24δ1), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24γ4) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.

Double-level “orthogonal” dynamic combinatorial libraries on transition metal template

Dynamic combinatorial libraries are mixtures of compounds that exist in a dynamic equilibrium and can be driven to compositional self adaptation via selective binding of a specific assembly of certain components to a molecular target. We present here an extension of this initial concept to dynamic libraries that consists of two levels, the first formed by the coordination of terpyridine-based ligands to the transition metal template, and the second, by the imine formation with the aldehyde substituents on the terpyridine moieties. Dialdehyde 7 has been synthesized, converted into a variety of ligands, oxime ethers L11–L33 and acyl hydrazones L44–L77, and subsequently into corresponding cobalt complexes. A typical complex, Co(L22)22+ is shown to engage in rapid exchange with a competing ligand L11 and with another complex, Co(L22)22+ in 30% acetonitrile/water at pH 7.0 and 25°C. The exchange in the corresponding Co(III) complexes is shown to be much slower. Imine exchange in the acyl hydrazone complexes (L44–L77) is strongly controlled by pH and temperature. The two types of exchange, ligand and imine, can thus be used as independent equilibrium processes controlled by different types of external intervention, i.e., via oxidation/reduction of the metal template and/or change in the pH/temperature of the medium. The resulting double-level dynamic libraries are therefore named orthogonal, in similarity with the orthogonal protecting groups in organic synthesis. Sample libraries of this type have been synthesized and showed the complete expected set of components in electrospray ionization MS.

Human fatty acid synthase: Role of interdomain in the formation of catalytically active synthase dimer

The human and animal fatty acid synthases are dimers of two identical multifunctional proteins (Mr 272,000) arranged in an antiparallel configuration. This arrangement generates two active centers for fatty acid synthesis separated by interdomain (ID) regions and predicts that two appropriate halves of the monomer should be able to reconstitute an active fatty acid synthesizing center. This prediction was confirmed by the reconstitution of the synthase active center by using two heterologously expressed halves of the monomer protein. Each of these recombinant halves of synthase monomer contains half of the ID regions. We show here that the fatty acid synthase activity could not be reconstituted when the ID sequences present in the two recombinant halves are deleted, suggesting that these ID sequences are essential for fatty acid synthase dimer formation. Further, we confirm that the ID sequences are the only regions of fatty acid synthase monomers that showed significant dimer formation, by using the yeast two-hybrid system. These results are consistent with the proposal that the ID region, which has no known catalytic activity, associates readily and holds together the two dynamic active centers of the fatty acid synthase dimer, therefore playing an important role in the architecture of catalytically active fatty acid synthase.

Synaptic pattern formation during cellular recognition

Cell–cell recognition often requires the formation of a highly organized pattern of receptor proteins (a synapse) in the intercellular junction. Recent experiments [e.g., Monks, C. R. F., Freiberg, B. A., Kupfer, H., Sciaky, N. & Kupfer, A. (1998) Nature (London) 395, 82–86; Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M. & Dustin, M. L. (1999) Science 285, 221–227; and Davis, D. M., Chiu, I., Fassett, M., Cohen, G. B., Mandelboim, O. & Strominger, J. L. (1999) Proc. Natl. Acad. Sci. USA 96, 15062–15067] vividly demonstrate a complex evolution of cell shape and spatial receptor–ligand patterns (several microns in size) in the intercellular junction during immunological synapse formation. The current view is that this dynamic rearrangement of proteins into organized supramolecular activation clusters is driven primarily by active cytoskeletal processes [e.g., Dustin, M. L. & Cooper, J. A. (2000) Nat. Immunol. 1, 23–29; and Wulfing, C. & Davis, M. M. (1998) Science 282, 2266–2269]. Here, aided by a quantitative analysis of the relevant physico-chemical processes, we demonstrate that the essential characteristics of synaptic patterns observed in living cells can result from spontaneous self-assembly processes. Active cellular interventions are superimposed on these self-organizing tendencies and may also serve to regulate the spontaneous processes. We find that the protein binding/dissociation characteristics, protein mobilities, and membrane constraints measured in the cellular environment are delicately balanced such that the length and time scales of spontaneously evolving patterns are in near-quantitative agreement with observations for synapse formation between T cells and supported membranes [Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M. & Dustin, M. L. (1999) Science 285, 221–227]. The model we present provides a common way of analyzing immunological synapse formation in disparate systems (e.g., T cell/antigen-presenting cell junctions with different MHC-peptides, natural killer cells, etc.).

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Δ cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Δ cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.

Diffusion and formation of microtubule asters: physical processes versus biochemical regulation.

Microtubule asters forming the mitotic spindle are assembled around two centrosomes through the process of dynamic instability in which microtubules alternate between growing and shrinking states. By modifying the dynamics of this assembly process, cell cycle enzymes, such as cdc2 cyclin kinases, regulate length distributions in the asters. It is believed that the same enzymes control the number of assembled microtubules by changing the "nucleating activity" of the centrosomes. Here we show that assembly of microtubule asters may be strongly altered by effects connected with diffusion of tubulin monomers. Theoretical analysis of a simple model describing assembly of microtubule asters clearly shows the existence of a region surrounding the centrosome depleted in GTP tubulin. The number of assembled microtubules may in some cases be limited by this depletion effect rather than by the number of available nucleation sites on the centrosome.

Segregation band formation in Al-Si die castings

Banded defects are often found in high-pressure die castings. These bands can contain segregation, porosity, and/or tears, and changing casting conditions and alloy are known to change the position and make-up of the bands. Due to the complex, dynamic nature of the high-pressure die-casting (HPDC) process, it is very difficult to study the effect of individual parameters on band formation. In the work presented here, bands of segregation similar to those found in cold-chamber HPDC aluminum alloys were found in laboratory gravity die castings. Samples were cast with a range of fraction solids from 0 to 0.3 and the effect of die temperature and external solid fraction on segregation bands was investigated. The results are considered with reference to the theological properties of the filling semisolid metal and a formation mechanism for bands is proposed by considering flow past a solidifying immobile wall layer.

Dynamic and regulated association of caveolin with lipid bodies: Modulation of lipid body motility and function by a dominant negative mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cav(DGV)) to study LB formation and to examine its effect on LB function. We now show that the cav(DGV) mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

On the equilibrium and dynamic behavior of alcohol vapors in activated carbon

As alcohol molecules such as methanol and ethanol have both polar and non-polar groups, their adsorption behavior is governed by the contributions of dispersion interaction (alkyl group) and hydrogen bonding (OH group). In this paper, the adsorption behavior of alcohol molecules and its effect on transport processes are elucidated. From the total permeability (B-T) of alcohol molecules in activated carbon, an adsorption mechanism is proposed, describing well the experimental data, by taking combination effects of clustering, entering micropores, layering and pore filling processes. Unlike the case of non-polar compounds, it was found that at low pressures there are two rises in the BT of alcohol molecules in activated carbon. The first rise is due to the major contribution of surface diffusion to the transport (which is the case of non-polar molecules) and the second one may be associated with cluster formation at the edge of micropores and entering micropores when the clusters are sufficiently large enough to induce a dispersive energy. In addition the clusters formed may enhance surface diffusion at low pressures and hinder gas phase diffusion and flow in meso/macropores. (c) 2006 Elsevier Ltd. All fights reserved.

Mechanisms of transfer film formation on data recording heads

The increasing demand for high capacity data storage requires decreasing the head-to-tape gap and reducing the track width. A problem very often encountered is the development of adhesive debris on the heads at low humidity and high temperatures that can lead to an increase of space between the head and media, and thus a decrease in the playback signal. The influence of stains on the playback signal of reading heads is studied using RAW (Read After Write) tests and their influence on the wear of the heads by using indentation technique. The playback signal has been found to vary and the errors to increase as stains form a patchy pattern and grow in size to form a continuous layer. The indentation technique shows that stains reduce the wear rate of the heads. In addition, the wear tends to be more pronounced at the leading edge of the head compared to the trailing one. Chemical analysis of the stains using ferrite samples in conjunction with MP (metal particulate) tapes shows that stains contain iron particles and polymeric binder transferred from the MP tape. The chemical anchors in the binder used to grip the iron particles now react with the ferrite surface to create strong chemical bonds. At high humidity, a thin layer of iron oxyhydroxide forms on the surface of the ferrite. This soft material increases the wear rate and so reduces the amount of stain present on the heads. The stability of the binder under high humidity and under high temperature as well as the chemical reactions that might occur on the ferrite poles of the heads influences the dynamic behaviour of stains. A model of stain formation taking into account the channels of binder degradation and evolution upon different environmental conditions is proposed.

Using min-sum loopy belief propagation for decentralised supply chain formation

Modern business trends such as agile manufacturing and virtual corporations require high levels of flexibility and responsiveness to consumer demand, and require the ability to quickly and efficiently select trading partners. Automated computational techniques for supply chain formation have the potential to provide significant advantages in terms of speed and efficiency over the traditional manual approach to partner selection. Automated supply chain formation is the process of determining the participants within a supply chain and the terms of the exchanges made between these participants. In this thesis we present an automated technique for supply chain formation based upon the min-sum loopy belief propagation algorithm (LBP). LBP is a decentralised and distributed message-passing algorithm which allows participants to share their beliefs about the optimal structure of the supply chain based upon their costs, capabilities and requirements. We propose a novel framework for the application of LBP to the existing state-of-the-art case of the decentralised supply chain formation problem, and extend this framework to allow for application to further novel and established problem cases. Specifically, the contributions made by this thesis are: • A novel framework to allow for the application of LBP to the decentralised supply chain formation scenario investigated using the current state-of-the-art approach. Our experimental analysis indicates that LBP is able to match or outperform this approach for the vast majority of problem instances tested. • A new solution goal for supply chain formation in which economically motivated producers aim to maximise their profits by intelligently altering their profit margins. We propose a rational pricing strategy that allows producers to earn significantly greater profits than a comparable LBP-based profitmaking approach. • An LBP-based framework which allows the algorithm to be used to solve supply chain formation problems in which goods are exchanged in multiple units, a first for a fully decentralised technique. As well as multiple-unit exchanges, we also model in this scenario realistic constraints such as factory capacities and input-to-output ratios. LBP continues to be able to match or outperform an extended version of the existing state-of-the-art approach in this scenario. • Introduction of a dynamic supply chain formation scenario in which participants are able to alter their properties or to enter or leave the process at any time. Our results suggest that LBP is able to deal easily with individual occurences of these alterations and that performance degrades gracefully when they occur in larger numbers.

Can an episodic approach to social identification account for identification as interaction between individual and collective identity work in an increasingly flexible and transient identity formation context?

A review of available literature suggests that social identification exists at the interface between individual and collective identity work. This poster proposes that it is the interaction between these two processes that leads a person to define themselves in terms of their membership of a particular social group. The poster suggests that identity work undertaken by the group (or ‘the creation of identities as widely understood signs with a set of rules and conventions for their use’, Schwalbe & Mason-Schrock, 1996, p.115), can be used by a person to inform their own individual identity work and, from this, the extent of alignment between their identity and the perceived identity of the group. In stable or internally-structured groups collective identity work may simply take the form of communication and preservation of dominant collective identities. However, in unstable, new or transitional groups, interaction between individual and collective identity work may be more dynamic, as both collective and individual identities are simultaneously codified, enacted and refined. To develop an understanding of social identification that is applicable in both stable and transitional social groups, it is useful to consider recent proposals that identification may occur cyclically as a series of discrete episodes (Ashforth, Harrison & Corley, 2008). This poster draws on the literature to present these suggestions in greater detail, outlining propositions for social identification that are relevant to transient as well as stable identity formation, supported by suggestion of how episodes of social identification may lead to a person identifying with a group.

Modular construction of dynamic nucleodendrimers

Isoguanosine-containing dendritic small molecules self-assemble into decameric nucleodendrimers as observed by 1D NMR spectroscopy, 2D DOSY, and mass spectrometry. In particular, apolar building blocks readily form pentameric structures in acetonitrile while the presence of alkali metals promotes the formation of stable decameric assemblies with a preference for cesium ions. Remarkably, co-incubation of guanosine and isoguanosine-containing nucleodendrons results in the formation of decameric structures in absence of added salts. Further analysis of the mixture indicated that guanosine derivatives facilitate the formation, but are not involved in decameric structures; a process reminiscent of molecular crowding. This molecular system provides a powerful canvas for the rapid and modular assembly of polyfunctional dendritic macromolecules. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Using the max-sum algorithm for supply chain emergence in dynamic multiunit environments

Supply chain formation (SCF) is the process of determining the set of participants and exchange relationships within a network with the goal of setting up a supply chain that meets some predefined social objective. Many proposed solutions for the SCF problem rely on centralized computation, which presents a single point of failure and can also lead to problems with scalability. Decentralized techniques that aid supply chain emergence offer a more robust and scalable approach by allowing participants to deliberate between themselves about the structure of the optimal supply chain. Current decentralized supply chain emergence mechanisms are only able to deal with simplistic scenarios in which goods are produced and traded in single units only and without taking into account production capacities or input-output ratios other than 1:1. In this paper, we demonstrate the performance of a graphical inference technique, max-sum loopy belief propagation (LBP), in a complex multiunit unit supply chain emergence scenario which models additional constraints such as production capacities and input-to-output ratios. We also provide results demonstrating the performance of LBP in dynamic environments, where the properties and composition of participants are altered as the algorithm is running. Our results suggest that max-sum LBP produces consistently strong solutions on a variety of network structures in a multiunit problem scenario, and that performance tends not to be affected by on-the-fly changes to the properties or composition of participants.