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Nisin is a class I polycyclic bacteriocin produced by the bacterium Lactococcus lactis, which is used extensively as a food additive to inhibit the growth of foodborne Gram-positive bacteria. Nisin also inhibits growth of Gram-negative bacteria when combined with membrane-disrupting chelators such as citric acid. To gain insight into nisin’s mode of action, we analyzed chemical–genetic interactions and identified nisin-sensitive Escherichia coli strains in the Keio library of knockout mutants. The most sensitive mutants fell into two main groups. The first group accords with the previously proposed mode of action based on studies with Gram-positive bacteria, whereby nisin interacts with factors involved in cell wall, membrane, envelope biogenesis. We identified an additional, novel mode of action for nisin based on the second group of sensitive mutants that involves cell cycle and DNA replication, recombination, and repair. Further analyses supported these two distinct modes of action.

Nisin is a class I polycyclic bacteriocin produced by the bacterium

The antimicrobial activity of chitosan has been acknowledged for more than 30 years and yet its mode-of-action remains ambiguous. We analyzed chemical-genetic interactions of low-molecular weight chitosan using a collection of ≈ 4600 S. cerevisiae deletion mutants and found that 31% of the 107 mutants most sensitive to chitosan had deletions of genes related primarily to functions involving protein synthesis. Disruption of protein synthesis by chitosan was substantiated by an in vivo β-galactosidase expression assay suggesting that this is a primary mode of antifungal action. Analysis of the yeast gene deletion array and secondary assays also indicate that chitosan has a minor membrane disruption effect - a leading model of chitosan antimicrobial activity.

Fixed-parameter tractability (FPT) techniques have recently been successful in solving NP-complete problem instances of practical importance which were too large to be solved with previous methods. In this paper, we show how to enhance this approach through the addition of parallelism, thereby allowing even larger problem instances to be solved in practice. More precisely, we demonstrate the potential of parallelism when applied to the bounded-tree search phase of FPT algorithms. We apply our methodology to the k-Vertex Cover problem which has important applications in, for example, the analysis of multiple sequence alignments for computational biochemistry. We have implemented our parallel FPT method for the k-Vertex Cover problem using C and the MPI communication library, and tested it on a 32-node Beowulf cluster. This is the first experimental examination of parallel FPT techniques. As part of our experiments, we solved larger instances of k-Vertex Cover than in any previously reported implementations. For example, our code can solve problem instances with k⩾400 in less than 1.5h.

The synapsins constitute a family of synaptic vesicle-associated phosphoproteins essential for regulating neurotransmitter release and synaptogenesis. The molecular mechanisms underlying the selective targeting of synapsin I to synaptic vesicles are thought to involve specific protein–protein interactions, while the high-affinity binding to the synaptic vesicle membrane may involve both protein–protein and protein–lipid interactions. The highly hydrophobic N-terminal region of the protein has been shown to bind with high affinity to the acidic phospholipids phosphatidylserine and phosphatidylinositol and to penetrate the hydrophobic core of the lipid bilayer. To precisely identify the domains of synapsin I which mediate the interaction with lipids, synapsin I was bound to liposomes containing the membrane-directed carbene-generating reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and subjected to photolysis. Isolation and N-terminal amino acid sequencing of 125I-labelled synapsin I peptides derived from CNBr cleavage indicated that three distinct regions in the highly conserved domain C of synapsin I insert into the hydrophobic core of the phospholipid bilayer. The boundaries of the regions encompass residues 166–192, 233–258 and 278–327 of bovine synapsin I. These regions are surface-exposed in the crystal structure of domain C of bovine synapsin I and are evolutionarily conserved among isoforms across species. The present data offer a molecular explanation for the high-affinity binding of synapsin I to phospholipid bilayers and synaptic vesicles.

Sucrose polyester (SPE), in the form of sucrose octaesters and sucrose hexaesters of palmitic (16:0), stearic (18:0), oleic (18:1cis), and linoleic (18:2cis) acids, have many uses. Applications include: a non-caloric fat substitute, detoxification agent, and oral contrast agent for human abdominal (MRI) magnetic resonance imaging. However, it has been shown that the ingestion of SPE was shown to generate a depletion of physiologically important lipidic vitamins and other lipophilic molecules. In order to better understand, at the molecular level, the type of interaction between SPE and lipid membrane, we have, first synthesized different type of labelled and non-labelled SPEs. Secondly, we have studied the effect of SPEs on multilamellar dispersions of dielaidoylphosphatidylethanolamine (DEPE) and dipalmitoylphosphocholine (DPPC) as a function of temperature, SPE composition and concentration. The effects of SPEs were studied by differential scanning calorimetry (DSC), X-ray diffraction, 2H and 31P NMR spectroscopy. At low concentration (1 mol%) all of the SPEs lowered the bilayer to the inverted hexagonal phase transition temperature of DEPE and induced the formation of a cubic phase in a composition dependent manner. At the same low concentration, SPEs in DPPC induce the formation of a non-bilayer phase as seen by 31P NMR. Order parameter measurements of DPPC-d62/SPE mixtures show that the SPE effect on the DPPC monolayer thickness is dependent on the SPE, concentration, chains length and saturation level. At higher concentration (or = 10 mol%) SPE are very potent DEPE bilayer to HII phase transition promoters, although at that concentration the SPE have lost the ability to form cubic phases. SPEs have profound effects on the phase behaviour of model membrane systems, and may be important to consider when developing current and potential industrial and medical applications.

The mechanism of inhibition of protein phosphatase-1 catalytic subunit (PP-1c) by recombinant DARPP-32 and synthetic peptides was studied. DARPP-32 was expressed in Escherichia coli as a non-fusion protein using a pEt-3a plasmid, purified to homogeneity and shown to have physicochemical properties similar to those of the protein purified from bovine brain. Recombinant DARPP-32 phosphorylated on threonine-34 by cAMP-dependent protein kinase inhibited PP-1c with an IC50 approximately 0.5 nM, comparable to that obtained with bovine DARPP-32. Non-phosphorylated DARPP-32, and mutated forms in which threonine-34 was replaced by an alanine or a glutamic acid, inhibited PP-1c with an IC50 approximately 1 microM. Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent Kd values of 1.2 and 0.3 microM, respectively, supporting the existence of an interaction between non-phosphorylated DARPP-32 and PP-1c that is increased by phosphorylation of DARPP-32 at threonine-34. These results suggest a model in which DARPP-32 interacts with PP-1c by at least two low affinity sites, the combination of which is responsible for the high affinity (nM) inhibition.

A number of amphiphiles which raise the bilayer to hexagonal phase transition temperature (TH) of phosphatidylethanolamine (PE) have been shown to inhibit viral fusion. In this study we have further evaluated the mechanism of this inhibition. Several anionic amphiphiles, including cholesterol sulfate, a component of mammalian plasma membranes, lower the final extent of Sendai virus fusion with both human erythrocyte ghosts and liposomes composed of PE and 5% of the ganglioside, GD1a. A cationic amphiphile slightly increased the final extent of fusion. The fusion rate constant is not greatly affected by the presence of as much as 20% cholesterol sulfate or other charged amphiphiles. The zwitterionic amphiphile, cholesterol phosphorylcholine has no effect on the final extent of fusion but it lowers the fusion rate constant. This amphiphile is potent in raising TH. The amphiphile cholesterol hemisuccinate (CHEMS) stabilizes the bilayer relative to the hexagonal phase at neutral pH, while at acidic pH the formation of the hexagonal phase is promoted. When CHEMS is added to vesicles of egg PE containing 5% GD1a, the rate of Sendai virus fusion is little affected at neutral pH but the rate is significantly enhanced at pH 5.0. These results demonstrate that viral fusion can be modulated, in part, by the tendency of the membrane to convert to the hexagonal phase.

In order to be infectious, viruses must possess a mechanism allowing their nucleic acid to pass through the plasma membrane of the target cells. Two such mechanisms have been described for en- veloped viruses, both requiring membrane fusion. One path is by direct fusion of the viral membrane with the plasma membrane of the target cell, while the other path requires prior endocytosis of the virus through coated pits, followed by fusion of the viral membrane with an endosomal membrane of the host cell. The latter mechanism requires a lowering of the pH, while the former can take place at physio- logical pH. In the present study, we have chosen measles virus as an example of a virus that fuses to the plasma membrane and influenza virus as one requiring endosomal acidification. There has been much work attempting to eluci- date the mechanism of viral fusion to target mem- branes. Through experiments of proteolytic cleav- age, mutagenesis, and reconstitution of viral proteins into virosomes, it is well established that the F pro- tein of measles and the

Background High throughput yeast functional genomics experiments are revealing associations among tens to hundreds of genes using numerous experimental conditions. To fully understand how the identified genes might be involved in the observed system, it is essential to consider the widest range of biological annotation possible. Biologists often start their search by collating the annotation provided for each protein within databases such as the Saccharomyces Genome Database, manually comparing them for similar features, and empirically assessing their significance. Such tasks can be automated, and more precise calculations of the significance can be determined using established probability measures. Results We developed Yeast Features, an intuitive online tool to help establish the significance of finding a diverse set of shared features among a collection of yeast proteins. A total of 18,786 features from the Saccharomyces Genome Database are considered, including annotation based on the Gene Ontology’s molecular function, biological process and cellular compartment, as well as conserved domains, protein-protein and genetic interactions, complexes, metabolic pathways, phenotypes and publications. The significance of shared features is estimated using a hypergeometric probability, but novel options exist to improve the significance by adding background knowledge of the experimental system. For instance, increased statistical significance is achieved in gene deletion experiments because interactions with essential genes will never be observed. We further demonstrate the utility by suggesting the functional roles of the indirect targets of an aminoglycoside with a known mechanism of action, and also the targets of an herbal extract with a previously unknown mode of action. The identification of shared functional features may also be used to propose novel roles for proteins of unknown function, including a role in protein synthesis for YKL075C. Conclusions Yeast Features (YF) is an easy to use web-based application (http://software.dumontierlab.com/yeastfeatures/) which can identify and prioritize features that are shared among a set of yeast proteins. This approach is shown to be valuable in the analysis of complex data sets, in which the extracted associations revealed significant functional relationships among the gene products.

Cholesterol sulfate is a component of several biological membranes. In erythrocytes, cholesterol sulfate inhibits hypotonic hemolysis, while in sperm, it can decrease fertilization efficiency. We have found cholesterol sulfate to be a potent inhibitor of Sendai virus fusion to both human erythrocyte and liposomal membranes. Cholesterol sulfate also raises the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine as demonstrated by differential scanning calorimetry and 31P nuclear magnetic resonance spectrometry. Although hexagonal phase structures are not readily found in biological membranes, there is a correlation between the effects of membrane additives on bilayer/non-bilayer equilibria and membrane stabilization. It is proposed that the ability of cholesterol sulfate to alter the physical properties of membranes contributes to its stabilization of biological membranes and the inhibition of membrane fusion.

Synapsins are membrane-associated proteins that cover the surface of synaptic vesicles and are responsible for maintaining a pool of neurotransmitter-loaded vesicles for use during neuronal activity. We have used atomic force microscopy (AFM) to study the interaction of synapsin I with negatively charged lipid domains in phase-separated supported lipid bilayers prepared from mixtures of phosphatidylcholines (PCs) and phosphatidylserines (PSs). The results indicate a mixture of electrostatic binding to anionic PS-rich domains as well as some nonspecific binding to the PC phase. Interestingly, both protein binding and scanning with synapsin-coated AFM tips can be used to visualize charged lipid domains that cannot be detected by topography alone.

The synapsins (I, II, and III) comprise a family of peripheral membrane proteins that are involved in both regulation of neurotransmitter release and synaptogenesis. Synapsins are concentrated at presynaptic nerve terminals and are associated with the cytoplasmic surface of synaptic vesicles. Membrane-binding of synapsins involves interaction with both protein and lipid components of synaptic vesicles. Synapsin I binds rapidly and with high affinity to liposomes containing anionic lipids. The binding of bovine synapsin I to liposomes was studied using fluoresceinphosphatidyl-ethanolamine (FPE) to measure membrane electrostatic potential. Synapsin binding to liposomes caused a rapid increase in FPE fluorescence, indicating an increase in positive charge at the membrane surface. Synapsin I binding to monolayers resulted in a substantial increase in monolayer surface pressure. At higher initial surface pressures, the synapsin-induced increase in monolayer surface pressure is dependent on the presence of anionic lipids in the monolayer. Synapsin I also induced rapid aggregation of liposomes, but did not induce leakage of entrapped carboxyfluorescein, while other aggregation-inducing agents promoted extensive leakage. These results are in agreement with the presence of amphipathic stretches of amino acids in synapsin I that exhibit both electrostatic and hydrophobic interactions with membranes, and offer a molecular explanation for the high affinity binding of synapsin I to liposomes and for stabilization of membranes by synapsin I.

Synapsin I is a neuronal phosphoprotein involved in the localization and stabilization of synaptic vesicles. Recently, synapsin I has been detected in several non-neuronal cell lines, but its function in these cells is unclear. To determine the localization of synapsin I in non-neuronal cells, it was transiently expressed in HeLa and NIH/3T3 cells as an enhanced green fluorescent protein fusion protein. Synapsin I-enhanced green fluorescent protein colocalized with F-actin in both cell lines, particularly with microspikes and membrane ruffles. It did not colocalize with microtubules or vimentin and it did not cause major alterations in cytoskeletal organization. Synapsin Ia-enhanced green fluorescent protein colocalized with microtubule bundles in taxol-treated HeLa cells and with F-actin spots at the plasma membrane in cells treated with cytochalasin B. It did not noticeably affect F-actin reassembly following drug removal. Synapsin Ia-enhanced green fluorescent protein remained colocalized with F-actin in cells treated with nocodazole, and it did not affect reassembly of microtubules following drug removal. These results demonstrate that synapsin I interacts with F-actin in non-neuronal cells and suggest that synapsin I may have a role in regions where actin is highly dynamic.

Fixed-parameter tractability (FPT) techniques have recently been successful in solving NP-complete problem instances of practical importance which were too large to be solved with previous methods. In this paper we show how to enhance this approach through the addition of parallelism, thereby allowing even larger problem instances to be solved in practice. More precisely, we demonstrate the potential of parallelism when applied to the bounded-tree search phase of FPT algorithms. We apply our methodology to the k-VERTEX COVER problem which has important applications, e.g., in multiple sequence alignments for computational biochemistry. We have implemented our parallel FPT method for the k-VERTEX COVER problem using C and the MPI communication library, and tested it on a PC cluster. This is the first experimental examination of parallel FPT techniques. We have tested our parallel k-VERTEX COVER method on protein sequences obtained from the National Center for Biotechnology Information. As part of our experiments, we solved larger instances of k-VERTEX COVER than in any previously reported implementations. For example, our code can solve problem instances with k /spl ges/ 400 in less than 1.5 hours. Since our parallel FPT algorithm requires only very little communication between processors, we expect our method to also perform well on Grids.

This paper presents a parallel version of CLUSTAL W, called pCLUSTAL. In contrast to the commercial SGI parallel Clustal, which requires an expensive shared memory SGI multiprocessor, pCLUSTAL can be run on a range of distributed and shared memory parallel machines, from high-end parallel multiprocessors (e.g. Sunfire 6800, IBM SP2, etc.) to PC clusters, to simple networks of workstations. We have implemented pCLUSTAL using C and the MPI communication library, and tested it on a PC cluster. Our experimental evaluation shows that our pCLUSTAL code achieves similar or better speedup on a distributed memory PC clusters than the commercial SGI parallel Clustal on a shared memory SGI multiprocessor.

The two tandem phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domains of the Fe65 protein interact with the intracellular region of the Alzheimer's beta-amyloid precursor protein (APP). This interaction, previously demonstrated in vitro and in the yeast two hybrid system, also takes place in vivo in mammalian cells, as demonstrated here by anti-Fe65 co-immunoprecipitation experiments. This interaction differs from that occurring between other PID/PTB domain-containing proteins, such as Shc and insulin receptor substrate 1, and activated growth factor receptors as follows: (i) the Fe65-APP interaction is phosphorylation-independent; (ii) the region of the APP intracellular domain involved in the binding is larger than that of the growth factor receptor necessary for the formation of the complex with Shc; and (iii) despite a significant similarity the carboxyl-terminal regions of PID/PTB of Fe65 and of Shc are not functionally interchangeable in terms of binding cognate ligands. A role for Fe65 in the pathogenesis of familial Alzheimer's disease is suggested by the finding that mutant APP, responsible for some cases of familial Alzheimer's disease, shows an altered in vivo interaction with Fe65.

We have studied the effects of gangliosides on the polymorphic behaviour of phosphatidylethanolamines. The ganglioside GD1a promotes the formation of phases which give rise to isotropic 31P-NMR resonance lines, particularly in the temperature range of the L alpha to HII transition. In addition, higher mol fractions of ganglioside raise the L alpha to HII phase transition temperature. Our results demonstrate that small mol fractions of gangliosides can have profound effects on the molecular organization of phosphatidylethanolamines.

Cholesterol sulphate is a potent stabilizer of membrane bilayer structure in both dielaidoylphosphatidylethanolamine and egg phosphatidylethanolamine model membranes, however, the addition of calcium abolishes this bilayer stabilization. Calcium also induces fusion and leakage of egg phosphatidylethanolamine large unilamellar vesicles containing cholesterol sulphate, but has no effect on fusion or leakage of egg phosphatidylcholine large unilamellar vesicles containing cholesterol sulphate. With egg phosphatidylethanoiamine liposomes, the initial rate, and extent of fusion, at constant calcium concentration, vary inversely with the mol percentage of cholesterol sulphate present in the vesicle membrane. The interaction of calcium and cholesterol sulphate, which causes membrane destabilization and fusion in phosphatidylethanolamine containing model systems, may play a role in the acrosome reaction in human sperm.

Background Identification of protein interaction networks has received considerable attention in the post-genomic era. The currently available biochemical approaches used to detect protein-protein interactions are all time and labour intensive. Consequently there is a growing need for the development of computational tools that are capable of effectively identifying such interactions. Results Here we explain the development and implementation of a novel Protein-Protein Interaction Prediction Engine termed PIPE. This tool is capable of predicting protein-protein interactions for any target pair of the yeast Saccharomyces cerevisiae proteins from their primary structure and without the need for any additional information or predictions about the proteins. PIPE showed a sensitivity of 61% for detecting any yeast protein interaction with 89% specificity and an overall accuracy of 75%. This rate of success is comparable to those associated with the most commonly used biochemical techniques. Using PIPE, we identified a novel interaction between YGL227W (vid30) and YMR135C (gid8) yeast proteins. This lead us to the identification of a novel yeast complex that here we term vid30 complex (vid30c). The observed interaction was confirmed by tandem affinity purification (TAP tag), verifying the ability of PIPE to predict novel protein-protein interactions. We then used PIPE analysis to investigate the internal architecture of vid30c. It appeared from PIPE analysis that vid30c may consist of a core and a secondary component. Generation of yeast gene deletion strains combined with TAP tagging analysis indicated that the deletion of a member of the core component interfered with the formation of vid30c, however, deletion of a member of the secondary component had little effect (if any) on the formation of vid30c. Also, PIPE can be used to analyse yeast proteins for which TAP tagging fails, thereby allowing us to predict protein interactions that are not included in genome-wide yeast TAP tagging projects. Conclusion PIPE analysis can predict yeast protein-protein interactions. Also, PIPE analysis can be used to study the internal architecture of yeast protein complexes. The data also suggests that a finite set of short polypeptide signals seem to be responsible for the majority of the yeast protein-protein interactions.

The effect of 2,3-seco-5 alpha-cholestan-2,3-dioic acid on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine is markedly dependent on pH. Above pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid raises the temperature of this transition, i.e., it stabilizes the bilayer phase. At pH 6.56 there is little effect of this sterol derivative on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. However, below pH 6.56, the 2,3-seco-5 alpha-cholestan-2,3-dioic acid markedly lowers the temperature of this transition. The promotion of hexagonal phase formation increases both with increasing mol fraction of this sterol derivative and with lower pH, particularly in the range between pH 6.56 and pH 5.0. Below about pH 6, 2,3-seco-5 alpha-cholestan-2,3-dioic acid also induces vesicle fusion as measured both by lipid mixing as well as by mixing of aqueous contents. For these assays vesicles made of phosphatidylethanolamine (made from egg phosphatidylcholine) and extruded through 0.2 micron pore membranes were used. At higher concentrations or at lower pH the 2,3-seco-5 alpha-cholestan-2,3-dioic acid induces some leakage of the contents of these vesicles. Nevertheless, with vesicles containing only 2 weight% sterol derivative, it was possible to demonstrate substantial mixing of aqueous contents of the vesicles over the pH range 3.5 to 5.5. Several of the properties of 2,3-seco-5 alpha-cholestan-2,3-dioic acid indicate that this compound may be useful in sensitizing vesicles to acid-induced fusion for the purpose of endocytic drug delivery.

A number of substances affect the activity of protein kinase C. Among uncharged and zwitterionic compounds, those which activate protein kinase C also lower the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine while substances which inhibit protein kinase C raise this transition temperature. Using this criteria, we have identified 3 beta-chloro-5-cholestene, 5 beta-cholan-24-ol and eicosane as new protein kinase C activators and have shown that Z-Ser-Leu-NH2, Z-Gly-Leu-NH2, Z-Tyr-Leu-NH2, cyclosporin A and cholestan-3 beta, 5 alpha, 6 beta-triol are protein kinase C inhibitors.

Amantadine and tromantadine are agents used against influenza and herpes infections, respectively. Tromantadine raises the bilayer to hexagonal phase transition temperature of synthetic phosphatidylethanolamines and is less disruptive to phospholipid packing. Tromantadine acts similar to cyclosporin A, previously demonstrated to inhibit viral-induced cell-cell fusion. We suggest the balance between the hydrophobic and hydrophilic group sizes would allow tromantadine to prevent membrane fusion more than amantadine and thus inhibit infection by viruses such as Herpes, which fuse with the plasma membrane. Study of agents which stabilize the bilayer phase of membranes may lead to efficacious inhibitors of viral infections requiring cell fusion events.

The phase behavior of mixtures of cholesterol or epicholesterol with phosphatidylethanolamine was studied by differential scanning calorimetry and by X-ray diffraction. Discrete domains of cholesterol are detected by X-ray diffraction in the L alpha phase of phosphatidylethanolamine from egg yolk and synthetic dielaidoylphosphatidylethanolamine beginning at mole fractions of 0.35-0.4 cholesterol. Separate domains of crystalline epicholesterol can also be detected in the L alpha phase of dielaidoylphosphatidylethanolamine by X-ray diffraction at as little as 0.16 mole fraction of epicholesterol. This is a result of poor miscibility of the epicholesterol with dielaidoylphosphatidylethanolamine. Epicholesterol does not alter the L beta----L alpha transition or bilayer spacing. Epicholesterol also has little effect on the diameter of the cylinders in the hexagonal phase. Formation of the inverted hexagonal phase is facilitated by addition of small amounts of cholesterol (mole fraction less than 0.2) in both egg phosphatidylethanolamine and dielaidoylphosphatidylethanolamine. However, at higher mole fractions of cholesterol, the stability of the liquid-crystalline phase is found to increase markedly for dielaidoylphosphatidylethanolamine but not for egg phosphatidylethanolamine, indicating the importance of the structure of the acyl chains in controlling the relative stability of the lamellar and nonlamellar phases in these systems. In contrast to cholesterol, epicholesterol markedly lowers the L alpha----HII phase transition temperature at low mole fraction of sterol. This result demonstrates the importance of the orientation and motional properties of an additive in determining the L alpha----HII transition temperature.