Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.     

Role of pagL and lpxO in Bordetella bronchiseptica Lipid A Biosynthesis

PagL and LpxO are enzymes that modify lipid A. PagL is a 3-O deacylase that removes the primary acyl chain from the 3 position, and LpxO is an oxygenase that 2-hydroxylates specific acyl chains in the lipid A. pagL and lpxO homologues have been identified in the genome of Bordetella bronchiseptica, but in the current structure for B. bronchiseptica lipid A the 3 position is acylated and 2-OH acylation is not reported. We have investigated the role of B. bronchiseptica pagL and lpxO in lipid A biosynthesis. We report a different structure for wild-type (WT) B. bronchiseptica lipid A, including the presence of 2-OH-myristate, the presence of which is dependent on lpxO. We also demonstrate that the 3 position is not acylated in the major WT lipid A structures but that mutation of pagL results in the presence of 3-OH-decanoic acid at this position, suggesting that lipid A containing this acylation is synthesized but that PagL removes most of it from the mature lipid A. These data refine the structure of B. bronchiseptica lipid A and demonstrate that pagL and lpxO are involved in its biosynthesis.     

The role of bryozoans in fossil reefs—an example from the Middle Devonian of the Western Sahara

Stenolaemate bryozoans with their stable calcitic skeletons play a significant role in reef building. In the Middle Devonian Sabkhat Lafayrina reef complex (Western Sahara), bryozoans are abundant and diverse. Although they do not form part of the principal framework of reefs, bryozoans are involved significantly in reef growth, especially in the initial stage. In this way, bryozoans are important with respect to initiating reef growth. They contribute greatly to sediment stabilization, making it possible for principal reef builders to grow on hardened and stabilized substrates, and also play sediment-baffling and sediment-filling roles. The aim of this study is to document the diversity of bryozoans in a Middle Devonian reef complex and to estimate their potential for initiation and contribution to reef structures.     

THEORY AND MEASUREMENT OF VISUAL MECHANISMS : VII. THE FLICKER RESPONSE FUNCTION OUTSIDE THE FOVEA

The several parameters of the flicker response contour (F – log I) are considered as a function of wave-length composition (white, blue, and red) and light-time fraction, for an extra-foveal region (monocular, temporal retina). These data are compared with those secured for the same image area centrally fixated at the fovea. The systematic changes in the parameters are shown to be in rational relation to other relevant excitability data. Since for two retinal regions the primary contours are quite different, the systematic nature of the behavior of the parameters in the two cases is a real test of the power of the analysis proposed. Theoretical interpretation is required to deal with the properties of sets of performance contours under systematically varied conditions, and cannot rely simply on the comparison of (for example) two contours under the same arbitrary conditions at two retinal locations. In particular it is emphasized that a qualitative separation must be made of the two factors of (a) number of units and (b) the frequencies of their actions, before the wave-length problem can be dealt with effectively.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Four distinct chondrocyte populations in the fetal bovine growth plate: highest expression levels of PTH/PTHrP receptor,Indian hedgehog,and MMP-13 in hypertrophic chondrocytes and their suppression by PTH (1-34) and PTHrP (1-40)

Differentiation and growth of chondrocytes in fetal growth plates of vertebrate long bones and ribs appear to occur in a gradual, continuous manner between the resting zone through the proliferation zone, maturation zone, and upper and lower hypertrophic zones, with a continuous increase in cell size up to 10-fold of the volume of a resting chondrocyte. Here we provide evidence, however, that after centrifugation through a continuous Percoll gradient growth plate chondrocytes separate into four distinct cell populations (B1 to B4) which differ markedly in density, size, and gene expression. These populations collect in the absence of any phase borders in the gradient which might serve as concentration barriers. Fractions B1 and B2 contained the largest cells with the lowest buoyant density and showed the highest expression levels for type X collagen (Col X), but only the B1 population expressed high levels of matrix metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly smaller and expressed little Col X, while cells in fraction B4 were of similar size to cells in the resting zone without significant Col X expression. The highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR-1), and Indian hedgehog (Ihh) expression were also found in the hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as expected from in situ hybridization data on PTHR-1 expression in fetal rodent or chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal fragments PTH (1-34) or PTHrP (1-40) suppressed the expression of Col X and PTHR-1 by more than 50% and the expression of Ihh nearly completely. In contrast, the mid-regional PTH fragment PTH (28-48) and PTH (52-84) consistently stimulated the expression of PTHR-1 by 10-20% in fractions B1 to B3. These findings confirm the existence of distinct differentiation stages within chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp et al. (Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP fragments controlling the differentiation of proliferating to prehypertrophic chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic chondrocytes.     

Texturerkennung und texturreproduktion

Computer generated textures are used to examine the recognition of pictorial textures and their edges by supervised learning. Since the recognition of texture at a given spot of the image involves measurements within a certain neighborhood of this spot, misclassification occurs at the edge between different textures, as was shown in Part 1. The edge enhancement procedures do also not produce a sharp edge. Based on some psychooptical evidence, a propagation operator is utilized to define the edges. First, the textures are classified only in those regions where a high level of confidence exists, the rest of the picture being marked as unrecognized. Second, the unrecognized picture elements adjoining a recognized spot are given its class, thus enlarging the recognized area. This process is repeated a few times, the limit being the propagation of results over an area greater than the neighborhood within which the texture parameters were measured in the first instance. The procedure yields welldefined edges at the proper locations.     

Analysis of spiking synchrony in visual cortex reveals distinct types of top-down modulation signals for spatial and object-based attention

The activity of a border ownership selective (BOS) neuron indicates where a foreground object is located relative to its (classical) receptive field (RF). A population of BOS neurons thus provides an important component of perceptual grouping, the organization of the visual scene into objects. In previous theoretical work, it has been suggested that this grouping mechanism is implemented by a population of dedicated grouping (“G”) cells that integrate the activity of the distributed feature cells representing an object and, by feedback, modulate the same cells, thus making them border ownership selective. The feedback modulation by G cells is thought to also provide the mechanism for object-based attention. A recent modeling study showed that modulatory common feedback, implemented by synapses with N-methyl-D-aspartate (NMDA)-type glutamate receptors, accounts for the experimentally observed synchrony in spike trains of BOS neurons and the shape of cross-correlations between them, including its dependence on the attentional state. However, that study was limited to pairs of BOS neurons with consistent border ownership preferences, defined as two neurons tuned to respond to the same visual object, in which attention decreases synchrony. But attention has also been shown to increase synchrony in neurons with inconsistent border ownership selectivity. Here we extend the computational model from the previous study to fully understand these effects of attention. We postulate the existence of a second type of G-cell that represents spatial attention by modulating the activity of all BOS cells in a spatially defined area. Simulations of this model show that a combination of spatial and object-based mechanisms fully accounts for the observed pattern of synchrony between BOS neurons. Our results suggest that modulatory feedback from G-cells may underlie both spatial and object-based attention.     

Autophagy in stem cells

Autophagy is a highly conserved cellular process by which cytoplasmic components are sequestered in autophagosomes and delivered to lysosomes for degradation. As a major intracellular degradation and recycling pathway, autophagy is crucial for maintaining cellular homeostasis as well as remodeling during normal development, and dysfunctions in autophagy have been associated with a variety of pathologies including cancer, inflammatory bowel disease and neurodegenerative disease. Stem cells are unique in their ability to self-renew and differentiate into various cells in the body, which are important in development, tissue renewal and a range of disease processes. Therefore, it is predicted that autophagy would be crucial for the quality control mechanisms and maintenance of cellular homeostasis in various stem cells given their relatively long life in the organisms. In contrast to the extensive body of knowledge available for somatic cells, the role of autophagy in the maintenance and function of stem cells is only beginning to be revealed as a result of recent studies. Here we provide a comprehensive review of the current understanding of the mechanisms and regulation of autophagy in embryonic stem cells, several tissue stem cells (particularly hematopoietic stem cells), as well as a number of cancer stem cells. We discuss how recent studies of different knockout mice models have defined the roles of various autophagy genes and related pathways in the regulation of the maintenance, expansion and differentiation of various stem cells. We also highlight the many unanswered questions that will help to drive further research at the intersection of autophagy and stem cell biology in the near future.