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.     

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.     

The question of virginity testing in Turkey

Pre-marital sex for a woman is regarded as wrong in my country. As a result, it is socially forbidden for a woman to engage in this act. In order to present a woman as a virgin on her marriage day, she is subjected to pressure, and put under control both by her family and societal norms. However, a man is free and never made to suffer any of the above. A woman found to be a virgin on her first night of marriage is seen as a normal person while one suspected to have lost her virginity is made to undergo a series of medical examinations to bring clarity to her situation.     

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.     

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.     

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.     

Methodology to select a set of urban sustainability indicators to measure the state of the city,and performance assessment

This paper proposes a methodology for establishing a set of sustainability indicators as a baseline to measure the state of a city, and after appropriate actions are taken, to assess performance, to determine if changes point to improvements regarding goals. The methodology starts with choosing a procedure for identification, scope, evaluation, and level of indicators to build the initial dataset. As a second step, produces a reduced set of indicators, complying with all criteria imposed, and selected under the condition of extracting the maximum amount of information from the initial dataset.     

Chronic dietary fat and cholesterol inhibit the normal postprandial stimulation of plasma cholesterol metabolism

The response of parameters of plasma cholesterol metabolism was studied in baboons adapted either to a low-fat, low-cholesterol diet or a high-fat, high-cholesterol diet. Animals adapted to the low-fat diet responded to a single low-fat or high-fat meal, as do normal humans, by a stimulation of cholesterol transport from blood cells to plasma, a stimulation of esterification of cholesterol, and a stimulation of cholesteryl ester transfer to very low and low density lipoproteins. While fasting rates of esterification and transfer increased as a result of diet-induced hypercholesterolemia, the postprandial response was reversed, so that postprandial metabolism was characterized by a movement of cholesterol from plasma to blood cells, an inhibition of cholesterol esterification, and a net transfer of cholesteryl esters from VLDL and LDL to HDL. These data indicate that the effects of postprandial lipemia on plasma cholesterol metabolism critically depend upon fasting plasma cholesterol levels.     

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.     

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.     

Fuzzy molecular surfaces.

Surface area of a macromolecule, accessible to a solvent, is defined and calculated, taking into account the probabilistic character of atomic positions due to the high frequency atomic vibrations. For a given a space point, we consider a probability of the event, that this point is covered by a macromolecule. A volume is defined as a space integral of this probability field. The envelope, accessible to a solvent molecule center, becomes fuzzy, existing only in a probabilistic sense. The accessible area is defined as a derivative of the envelope volume with respect to the probe size.     

Scaling the physiological effects of exposure to radiofrequency electromagnetic radiation: consequences of body size

We have demonstrated that a comparative analysis of the physiological effects of exposure of laboratory mammals to radiofrequency electromagnetic radiation (RFR) may be useful in predicting exposure thresholds for humans if the effect is assumed to be due only to heating of tissue. The threshold specific absorption rate (SAR) necessary to affect a thermoregulatory parameter shows an inverse and linear relationship to body mass. The inverse relationship between threshold SAR and body mass is attributed to a surface area: body mass relationship. In comparison to small mammals, relatively large mammals have a reduced capacity to dissipate an internal heat load passively, and are therefore physiologically more sensitive to RFR exposure. The threshold for a thermoregulatory response depends on the type of response measured, species, ambient temperature, etc. By extrapolation, it can be shown that a SAR of only 0.2-0.4 W/kg is required to promote a thermoregulatory response in a mammal with a body mass of 70 kg (e.g. weight of adult human). The specific absorption rate bioeffects data collected from laboratory mammals can be related by means of a simple power formula: threshold SAR (W/kg) = aMb, where M is body mass in kg, a is a constant and b is equal to approximately -0.5. Through this equation we have illustrated that a threshold SAR measured in a species weighing 100 g would be 10 times greater than that of a species weighing 10 000 g. Accordingly, a relatively low SAR that is physiologically ineffective in small mammals may be stressful to larger species.     

Locomotory modes in the larva and pupa of Chironomus plumosus (Diptera, Chironomidae)

The locomotory kinematics of Chironomus plumosus larvae and pupae were investigated in order to determine how different locomotory techniques may be related to (a) possible underlying patterns of muscle activation and (b) the particular lifestyles and behaviours of these juvenile stages. Larvae display three independent modes of motile activity: swimming, crawling and whole-body respiratory undulation. Swimming and respiratory undulation involve the use of metachronal waves of body bending which travel in a head-to-tail direction. Whereas swimming is produced by side-to-side flexures of the whole body, respiratory undulation employs a sinusoidal wave. Crawling appears to result from an independent programme of muscle activation. Instead of a longitudinally transmitting metachronal wave of body flexure, a simultaneous arching of the body, combined with the alternating use of the abdominal and prothoracic pseudopods as anchorage points, produces a form of locomotion analogous to caterpillar-looping. Larval swimming has a set speed and rhythm and is an 'all-or-nothing' locomotory manoeuvre, but the neural programme controlling larval crawling is adaptable; switching from a less to a more slippery substrate resulted in a shorter, faster stepping pattern. The pupa displays two swimming modes, somersaulting and eel-like whole-body undulation, the former being principally a brief, escape manoeuvre, the latter being a faster form of locomotion employed to deliver the pupa to the surface prior to adult emergence. Comparison with the pupa of the culicid Culex pipiens shows that this insect also uses the somersault mechanism but at a higher cycle frequency which produces a faster swimming speed. This appears to be related to differences in lifestyle; the surface-living culicid pupa is exposed to greater predator threat than the bottom-dwelling chironomid pupa, and consequently needs a faster escape.     

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     

Dynamin: possible mechanism of "Pinchase" action.

Dynamin is a GTPase playing an essential role in ubiquitous intra cellular processes involving separation of vesicles from plasma membranes and membranes of cellular compartments. Recent experimental progress (. Cell. 93:1021-1029;. Cell. 94:131-141) has made it possible to attempt to understand the action of dynamin in physical terms. Dynamin molecules are shown to bind to a lipid membrane, to self-assemble into a helicoidal structure constricting the membrane into a tubule, and, as a result of GTP hydrolysis, to mediate fission of this tubule (). In a similar way, dynamin is supposed to mediate fission of a neck connecting an endocytic bud and the plasma membrane, i.e., to complete endocytosis. We suggest a mechanism of this "pinchase" action of dynamin. We propose that, as a result of GTP hydrolysis, dynamin undergoes a conformational change manifested in growth of the pitch of the dynamin helix. We show that this gives rise to a dramatic change of shape of the tubular membrane constricted inside the helix, resulting in a local tightening of the tubule, which is supposed to promote its fission. We treat this model in terms of competing elasticities of the dynamin helix and the tubular membrane and discuss the predictions of the model in relation to the previous views on the mechanism of dynamin action.     

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.     

Novel 3'-ribonuclease and 3'-phosphatase activities of the bacterial non-homologous end-joining protein, DNA ligase D

Pseudomonas aeruginosa DNA ligase D (PaeLigD) exemplifies a family of bacterial DNA end-joining proteins that consist of a ligase domain fused to a polymerase domain and a putative nuclease module. The LigD polymerase preferentially adds single ribonucleotides at blunt DNA ends and, as we show here, is also capable of adding up to 4 ribonucleotides to a DNA primer-template. We report that PaeLigD has an intrinsic ability to resect the short tract of 3'-ribonucleotides of a primer-template substrate to the point at which the primer strand has a single 3'-ribonucleotide remaining. The failure to digest beyond this point reflects a requirement for a 2'-OH group on the penultimate nucleoside of the primer strand. Replacing the 2'-OH by a 2'-F, 2'-NH2, 2'-OCH3, or 2'-H abolishes the resection reaction. The ribonucleotide resection activity resides within a 187-amino acid N-terminal nuclease domain and is the result of at least two component steps: (i) the 3'-terminal nucleoside is first removed to yield a primer strand with a ribonucleoside 3'-PO4 terminus, and (ii) the 3'-PO4 is hydrolyzed to a 3'-OH. The 3'-ribonuclease and 3'-phosphatase activities are both dependent on a divalent cation, specifically manganese. PaeLigD preferentially remodels the 3'-ends of a duplex primer-template substrate rather than a single strand of identical composition, and it prefers DNA primer strands containing a short 3'-ribonucleotide tract to an all-RNA primer. The nuclease domain of PaeLigD and its bacterial homologs has no apparent structural or mechanistic similarity to previously characterized nucleases. Thus, we surmise that it exemplifies a novel phosphoesterase family, defined in part by conserved residues Asp-50, Arg-52, and His-84, which are essential for the 3'-ribonuclease and 3'-phosphatase reactions.     

Multicultural society and everyday cultural racism: second generation of Ethiopian Jews in Israel's ‘crisis of modernization’

Abstract

The core of this article sets out to examine the extent to which a multicultural society can prevent cultural racism, which, like multiculturalism, is by definition based on a culture of diversity and separation. The ‘first modernity’ was organized along national lines, with a centralist state that opted to create an essentialist and uncontested national identity. Immigrants, especially those who came from ‘third world’ countries, were expected to undergo a process of assimilation, and to integrate into the dominant culture by relinquishing their particular past and tradition. Multiculturalism, which emerged historically as a criticism of that perspective, aims at creating a kaleidoscope of associations and cultural communities, which inevitably presents a challenge to the one ‘truth’ of the nation-state with the argument that this ‘truth’ favours some groups over others. Within the multicultural model, identity politics of various groups is perceived as a means to achieve recognition, acceptance, respect and even public affirmation of differences. However, do multicultural society and identity-related differences provide a solution to cultural racism as well?

Investigating the second generation of the Ethiopian Jews, who migrated to Israel during its transformation from ethno-national republicanism to a neo-liberal, multicultural society, can help answer this question. By presenting their patterns of association, character of protest activities and the newly formed hybrid identity that Ethiopian youth have developed as a means to liberate themselves from a discriminating reality, and by examining the Others’ reaction to that challenge, this article uncovers certain mechanisms and methods of action through which a multicultural society, having a thin and mild version of multiculturalism, does not diminish cultural racism, particularly its everyday non-institutional version, but in fact augments it.