Munc18a controls SNARE assembly through its interaction with the syntaxin N-peptide

Sec1/Munc18-like (SM) proteins functionally interact with SNARE proteins in vesicular fusion. Despite their high sequence conservation, structurally disparate binding modes for SM proteins with syntaxins have been observed. Several SM proteins appear to bind only to a short peptide present at the N terminus of syntaxin, designated the N-peptide, while Munc18a binds to a 'closed' conformation formed by the remaining portion of syntaxin 1a. Here, we show that the syntaxin 16 N-peptide binds to the SM protein Vps45, but the remainder of syntaxin 16 strongly enhances the affinity of the interaction. Likewise, the N-peptide of syntaxin 1a serves as a second binding site in the Munc18a/syntaxin 1a complex. When the syntaxin 1a N-peptide is bound to Munc18a, SNARE complex formation is blocked. Removal of the N-peptide enables binding of syntaxin 1a to its partner SNARE SNAP-25, while still bound to Munc18a. This suggests that Munc18a controls the accessibility of syntaxin 1a to its partners, a role that might be common to all SM proteins.     

Integrating neuromorphic action-oriented perceptual inputs to generate a navigation behaviour for a robot

We use neural networks with pointer map architectures to provide simple attentional processing in a robotic task. A pointer map comprises a map of neurons that encode a stimulus. Besides global feedback inhibition, the map receives feedback excitation via a small group of pointer neurons that encode the location of a salient stimulus on the map as a vectorial representation. The pointer neurons are able to apply selective processing to a particular region of the network. The robot uses these properties to manoeuver in relation to an attended object. We implemented a controller composed of two pointer maps, and a motor map. The first pointer map reports the direction of a salient obstacle in a one-dimensional map of distance derived from infrared sensors. The second pointer map reports the direction to potential obstacles in a two-dimensional edge-enhanced image derived from a forward looking CCD-camera. These outputs are applied to a motor map, where they bias the motor control signals issued to the robots wheels, according to navigational intentions.     

Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis

The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation.     

In-Vivo Real-Time Control of Protein Expression from Endogenous and Synthetic Gene Networks

We describe an innovative experimental and computational approach to control the expression of a protein in a population of yeast cells. We designed a simple control algorithm to automatically regulate the administration of inducer molecules to the cells by comparing the actual protein expression level in the cell population with the desired expression level. We then built an automated platform based on a microfluidic device, a time-lapse microscopy apparatus, and a set of motorized syringes, all controlled by a computer. We tested the platform to force yeast cells to express a desired fixed, or time-varying, amount of a reporter protein over thousands of minutes. The computer automatically switched the type of sugar administered to the cells, its concentration and its duration, according to the control algorithm. Our approach can be used to control expression of any protein, fused to a fluorescent reporter, provided that an external molecule known to (indirectly) affect its promoter activity is available.     

Elimination of suppressor T-cells in mice undergoing a graft-versus-host reaction expressed by increased response to polyvinylpyrrolidone (PVP).

Mice undergoing a mild GVHR exhibited an enhanced response to PVP considered to be independent of T-helper function and simultaneously, a decreased response to SRBC, known to be T-cell dependent. Such mice had a reduced number of T cells in their spleens expressed by a lower reactivity to T-lectins PHA and Con A and by a decrease in the number of cells killed by anti θ serum. These mice did not show, however, a substantial change in the activity of B lymphocytes contained in their spleens, since the PFC and the mitogenic response to LPS, as well as the number of immunoglobulin bearing cells were similar to that of controls. These results suggest that the enhanced response to PVP in mice undergoing a mild GVHR is a result of the elimination of a certain T-cell population which seems to regulate the immune response to this antigen.     

Prediction-based structured variable selection through the receiver operating characteristic curves

In many clinical settings, a commonly encountered problem is to assess accuracy of a screening test for early detection of a disease. In these applications, predictive performance of the test is of interest. Variable selection may be useful in designing a medical test. An example is a research study conducted to design a new screening test by selecting variables from an existing screener with a hierarchical structure among variables: there are several root questions followed by their stem questions. The stem questions will only be asked after a subject has answered the root question. It is therefore unreasonable to select a model that only contains stem variables but not its root variable. In this work, we propose methods to perform variable selection with structured variables when predictive accuracy of a diagnostic test is the main concern of the analysis. We take a linear combination of individual variables to form a combined test. We then maximize a direct summary measure of the predictive performance of the test, the area under a receiver operating characteristic curve (AUC of an ROC), subject to a penalty function to control for overfitting. Since maximizing empirical AUC of the ROC of a combined test is a complicated nonconvex problem (Pepe, Cai, and Longton, 2006, Biometrics62, 221-229), we explore the connection between the empirical AUC and a support vector machine (SVM). We cast the problem of maximizing predictive performance of a combined test as a penalized SVM problem and apply a reparametrization to impose the hierarchical structure among variables. We also describe a penalized logistic regression variable selection procedure for structured variables and compare it with the ROC-based approaches. We use simulation studies based on real data to examine performance of the proposed methods. Finally we apply developed methods to design a structured screener to be used in primary care clinics to refer potentially psychotic patients for further specialty diagnostics and treatment.     

Levinthal's question revisited, and answered

Attempts to answer the Levinthal question "How proteins fold to give such a unique structure" are discussed. In the first part of this article, we focus on a few reasons as to why the solution to the protein-folding problem (PFP) has been elusive for a very long time. One is a result of the misinterpretation of Anfinsen's Thermodynamic hypothesis which led to the conclusion that the native structure of a protein must be at a global minimum of the Gibbs energy. The second is the result of the adherence to the hydrophobic paradigm, and at the same time ignoring a whole repertoire of hydrophilic effects. It is argued that switching from a target-based to a caused-based approach, and adopting the hydrophilic paradigm leads straightforwardly to a simple answer to Levinthal's question, as well as to a solution of the PFP.     

A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region

The mechanisms involved in the targeting of proteins to different cytosolic compartments are still largely unknown. In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus. GAD65 is expressed in neurons and in pancreatic beta-cells, where it is concentrated in the Golgi complex region and in proximity to GABA- containing vesicles. GAD65, but not the similar isoform GAD67 which has a more diffuse cytosolic distribution, is palmitoylated within its first 100 amino acids (a.a.). We have previously demonstrated that the domain corresponding to a.a. 1-83 of GAD65 is required for the targeting of GAD65 to the Golgi complex region. Here we show that this domain is sufficient to target an unrelated protein, beta- galactosidase, to the same region. Site-directed mutagenesis of all the putative acceptor sites for thiopalmitoylation within this domain did not abolish targeting of GAD65 to the Golgi complex region. The replacement of a.a. 1-29 of GAD67 with the corresponding a.a. 1-27 of GAD65 was sufficient to target the otherwise soluble GAD67 to the Golgi complex region. Conversely, the replacement of a.a. 1-27 of GAD65 with a.a. 1-29 of GAD67 resulted in a GAD65 protein that had a diffuse cytosolic distribution and was primarily hydrophilic, suggesting that targeting to the Golgi complex region is required for palmitoylation of GAD65. We propose that the domain corresponding to a.a. 1-27 of GAD65, contains a signal required for the targeting of GAD65 to the Golgi complex region.     

Structure-function relationship of myotoxin a using peptide fragments.

Myotoxin a, a small basic polypeptide isolated from the venom of prairie rattlesnake (Crotalus viridis viridis), has been shown to bind to sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The attachment of myotoxin a to Ca(2+)-ATPase is believed to cause uncoupling of the calcium pump. In order to further elucidate which portion of myotoxin a is important for the uncoupling action, five peptides were synthesized and two peptide fragments were obtained by chemical cleavage. These peptides correspond to discrete portions of the primary sequence of myotoxin a. The peptides are equivalent to the primary sequence of myotoxin a from 1 to 16 residues, 7 to 22 residues, 13 to 28 residues, 19 to 34 residues, and 25 to 42 residues. Chemically produced fragments are equivalent to 1 to 28 residues and 29 to 42 residues of myotoxin a. Peptides of the sequences "YKQCHKKGGHCFPKEK" and "LGKMDCRWKWKCCKKGSG" of myotoxin a inhibited 45Ca uptake into isolated SR and bound to Ca(2+)-ATPase. The same peptides caused weak skeletal muscle vacuolization similar to that caused by native myotoxin a and increased serum creatine kinase activity. The active peptides correspond to the N-terminal and C-terminal portions of myotoxin a. The inactive or less active peptides have sequences which correspond to the middle sequence of myotoxin a. From this study, both the N-terminal and the C-terminal regions of primary sequence of myotoxin a are required to express myotoxin a's biological activity.     

The transmembrane helix of the Escherichia coli division protein FtsI localizes to the septal ring

FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of about a dozen division proteins that localize to the septal ring. FtsI comprises a short amino-terminal cytoplasmic domain, a single transmembrane helix (TMH), and a large periplasmic domain that encodes the catalytic (transpeptidase) activity. We show here that a 26-amino-acid fragment of FtsI is sufficient to direct green fluorescent protein to the septal ring in cells depleted of wild-type FtsI. This fragment extends from W22 to V47 and corresponds to the TMH. This is a remarkable finding because it is unusual [corrected] for a TMH to target a protein to a site more specific than the membrane. Alanine-scanning mutagenesis of the TMH identified several residues important for septal localization. These residues cluster on one side of an alpha-helix, which we propose interacts directly with another division protein to recruit FtsI to the septal ring.     

Quantitative measures of organization for multiagent systems

A set of "information theoretic" measures has been developed to quantify the degree of constraint inherent in the organization of a multiagent system. Separate measures can be provided to quantify spatial organization, trophic organization and, more generally, the overall structure of interactions. The additive character of these quantities allows them to be distributed in various fashions among species and places in a way that allows one to assign an "Importance Index" to those taxa and places. In addition, a measure to gauge the degree of adaptation of a species to a particular environment is proffered. The proposed measures allow one to formulate the following hypotheses in quantitative fashion: (1). that any disturbance of an ecosystem at a location associated with a high spatial Importance Index will exert a greater impact on the population dynamics than will a similar disturbance aimed at a place where the values of these indexes are lower; (2). that any disturbance in an ecosystem affecting a particular species with high individual Importance Indexes will cause a greater impact on the overall population dynamics than will a disturbance aimed at a species with a lower values of these indexes; (3). that the ascendancy of evolving system has a propensity to increase. The precise quantitative formulation of these hypothesis would permit them to be tested via multiagent simulation. Estimating the probablities pertaining to these hypotheses presents a number of problems that merit discussion.     

High-resolution genetic mapping of complex traits.

Positional cloning requires high-resolution genetic mapping. To plan a positional cloning project, one needs to know how many informative meioses will be required to narrow the search for a disease gene to an acceptably small region. For a simple Mendelian trait studied with linkage analysis, the answer is straightforward. In this paper, we address the situation of a complex trait studied with affected-relative-pair methods. We derive mathematical formulas for the size of an appropriate confidence region, as a function of the relative risk attributable to the gene. Using these results, we provide graphs showing the number of relative pairs required to narrow the gene hunt to an interval of a given size. For example, we show that localizing a gene to 1 cM requires a median of 200 sib pairs for a locus causing a fivefold increased risk to an offspring and 700 sib pairs for a locus causing a twofold increased risk. We discuss the implications of these results for the positional cloning of genes underlying complex traits.     

ASSORTATIVE MATING AND THE ADAPTIVE LANDSCAPE

The ability of a population to shift from one adaptive peak to another was examined for a two-locus model with different degrees of assortative mating, selection, and linkage. As expected, if the proportion of the population that mates assortatively increases, so does its ability to shift to a new peak. Assortative mating affects this process by allowing the mean fitness of a population to increase monotonically as it passes through intermediate gene frequencies on the way to a new, higher, homozygotic peak. Similarly, if the height of the new peak increases or selection against intermediates becomes less severe, the population becomes more likely to shift to a new peak. Close linkage also helps the shift to a new adaptive peak and acts similarly to assortative mating, but it is not necessary for such a shift as was previously thought. When a population shifts to a new peak, the number of generations required is significantly less than that needed to return to the original peak when that happens. The short period of time required may be an explanation for rapid changes in the geological record. Under extremely high degrees of assortative mating, the shift takes longer, presumably because of the difficulty of breaking up less favored allelic combinations.     

Chemotactic responses of Rhodobacter sphaeroides in the absence of apparent adaptation

Rhodobacter sphaeroides, which lacks methyl accepting chemotaxis proteins, showed a strong response to gradients of either pyruvate or propionate. If cells were placed in a saturating background of pyruvate they no longer responded to a gradient of propionate but they still responded to potassium or ammonia. This demonstrates that pyruvate saturated the response to another carbon source, but not to other classes of compound. The total movement of cells in a pyruvate background was maintained at a high level relative to a buffer control, indicating an apparent lack of adaptation to saturating pyruvate. The response of R. sphaeroides to a saturating background of pyruvate was weak in cells grown on limiting ammonia although these cells showed a strong response to ammonia. These data suggest that cells show a strong response to the class of compound that currently limits motility. Two hypotheses to explain these results are presented. Firstly, cells show a chemotactic response to a gradient of the limiting compound until saturated by it, they then respond to a gradient of the new compound that has then become limiting. The chemotactic response is the result of a decrease in stopping frequency as cells move up a gradient and an increase as they move down. Secondly, the behavioural response may have two components, a short term chemotactic response and a long term excitation of motility.Abbreviations MCP methyl accepting chemotaxis protein     

Rock-scissors-paper and the survival of the weakest.

In the children's game of rock-scissors-paper, players each choose one of three strategies. A rock beats a pair of scissors, scissors beat a sheet of paper and paper beats a rock, so the strategies form a competitive cycle. Although cycles in competitive ability appear to be reasonably rare among terrestrial plants, they are common among marine sessile organisms and have been reported in other contexts. Here we consider a system with three species in a competitive loop and show that this simple ecology exhibits two counter-intuitive phenomena. First, the species that is least competitive is expected to have the largest population and, where there are oscillations in a finite population, to be the least likely to die out. As a consequence an apparent weakening of a species leads to an increase in its population. Second, evolution favours the most competitive individuals within a species, which leads to a decline in its population. This is analogous to the tragedy of the commons, but here, rather than leading to a collapse, the 'tragedy' acts to maintain diversity.     

A system for coupled multiple-column separation of proteins

Purification of proteins is commonly a multiple-step process involving size exclusion, ion exchange, affinity, hydrophobic, and other modes of chromatography. In an effort to circumvent the laborious process of collecting the solutes from each column and reintroducing them onto a second column, a valving system is described that directs the samples eluted from a high-performance liquid chromatographic column through a detector with a high-pressure cell into either a second column or into storage loops of a multiloop value. This multiloop value is referred to as a high-pressure fraction collector. After development of the first column is complete, a second solvent can be directed to the second column or high-pressure fraction collector to elute the solutes back through the detector and onto any other column in the system. The process of eluting a sample from a column through a single detector and directing it to the high-pressure fraction collector or any other column in the system may be repeated a number of times. Such valving systems make it possible to chromatograph a single protein component on two or three columns in a short time.     

A Slow Rotary Shaker for Embedding in Viscous Media

Embedding vessels are carried by a metal tray which has a tilt of about 20° to the horizontal plane. The tray is supported by a shaft attached perpendicular to its bottom. The shaft passes loosely through a hole in a rigid plate and is attached by a ball joint to a crank, which is driven at about 20 rev/min by a geared-down electric motor. Thus a combined tilting and rotary motion is imparted to the tray to produce a continuous flow of the embedding medium about the specimens.     

Intrinsic disorder: signaling via highly specific but short-lived association

Association between signaling proteins and their cellular targets is generally thought to be highly specific (implicating a high association constant, K(a)) and, at the same time, transient or short-lived (corresponding to a high dissociation rate constant, k(d)). However, a combination of high K(a) and high k(d) would lead to a high association rate constant (k(a) = K(a)k(d)), which poses a problem because there is a limit to which k(a) can be increased, set by the diffusional approach to form the complex. In this Opinion article, I propose that having the signaling protein disordered before binding to the target provides a way out of this quandary. The intrinsic disorder of the signaling protein would decrease K(a) without sacrificing the specificity of the complex, and thus would allow k(d) to be increased to a range appropriate for signaling.