Segregation of Two Spectrin Isoforms: Polarized Membrane-binding Sites Direct Polarized Membrane Skeleton Assembly

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and β spectrin are recruited to sites of cell–cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (αβH), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and αβ spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, αβ spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, αβH spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell–cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

Equilibrium distributions of microsatellite repeat length resulting from a balance between slippage events and point mutations

We describe and test a Markov chain model of microsatellite evolution that can explain the different distributions of microsatellite lengths across different organisms and repeat motifs. Two key features of this model are the dependence of mutation rates on microsatellite length and a mutation process that includes both strand slippage and point mutation events. We compute the stationary distribution of allele lengths under this model and use it to fit DNA data for di-, tri-, and tetranucleotide repeats in humans, mice, fruit flies, and yeast. The best fit results lead to slippage rate estimates that are highest in mice, followed by humans, then yeast, and then fruit flies. Within each organism, the estimates are highest in di-, then tri-, and then tetranucleotide repeats. Our estimates are consistent with experimentally determined mutation rates from other studies. The results suggest that the different length distributions among organisms and repeat motifs can be explained by a simple difference in slippage rates and that selective constraints on length need not be imposed.

SRPDB (Signal Recognition Particle Database)

Signal recognition particle (SRP) is a stable cytoplasmic ribonucleoprotein complex that serves to translocate secretory proteins across membranes during translation. The SRP Database (SRPDB) provides compilations of SRP components, ordered alphabetically and phylogenetically. Alignments emphasize phylogenetically-supported base pairs in SRP RNA and conserved residues in the proteins. Data are provided in various formats including a column arrangement for improved access and simplified computational usability. Included are motifs for identification of new sequences, SRP RNA secondary structure diagrams, 3-D models and links to high-resolution structures. This release includes 11 new SRP RNA sequences (total of 129), two protein SRP9 sequences (total of seven), two protein SRP14 sequences (total of 10), two protein SRP19 sequences (total of 16), 10 new SRP54 (ffh) sequences (total of 66), two protein SRP68 sequences (total of seven) and two protein SRP72 sequences (total of nine). Seven sequences of the SRP receptor α-subunit and its FtsY homolog (total of 51) are new. Also considered are β-subunit of SRP receptor, Flhf, Hbsu, CaM kinase II and cpSRP43. Access to SRPDB is at http://psyche.uthct.edu/dbs/SRPDB/SRPDB.html and the European mirror http://www.medkem.gu.se/dbs/SRPDB/SRPDB.html

The Cellular Level of PR500, a Protein Complex Related to the 19S Regulatory Particle of the Proteasome, Is Regulated in Response to Stresses in Plants

In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of ∼800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the “lid” subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.

Multifaceted Physiological Response Allows Yeast to Adapt to the Loss of the Signal Recognition Particle-dependent Protein-targeting PathwayD⃞

Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions. We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway. Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression. Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed. Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes. Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes. Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus. Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.

Assembly and transport of a premessenger RNP particle

Salivary gland cells in the larvae of the dipteran Chironomus tentans offer unique possibilities to visualize the assembly and nucleocytoplasmic transport of a specific transcription product. Each nucleus harbors four giant polytene chromosomes, whose transcription sites are expanded, or puffed. On chromosome IV, there are two puffs of exceptional size, Balbiani ring (BR) 1 and BR 2. A BR gene is 35–40 kb, contains four short introns, and encodes a 1-MDa salivary polypeptide. The BR transcript is packed with proteins into a ribonucleoprotein (RNP) fibril that is folded into a compact ring-like structure. The completed RNP particle is released into the nucleoplasm and transported to the nuclear pore, where the RNP fibril is gradually unfolded and passes through the pore. On the cytoplasmic side, the exiting extended RNP fibril becomes engaged in protein synthesis and the ensuing polysome is anchored to the endoplasmic reticulum. Several of the BR particle proteins have been characterized, and their fate during the assembly and transport of the BR particle has been elucidated. The proteins studied are all added cotranscriptionally to the pre-mRNA molecule. The various proteins behave differently during RNA transport, and the flow pattern of each protein is related to the particular function of the protein. Because the cotranscriptional assembly of the pre-mRNP particle involves proteins functioning in the nucleus as well as proteins functioning in the cytoplasm, it is concluded that the fate of the mRNA molecule is determined to a considerable extent already at the gene level.

Distinct desmocollin isoforms occur in the same desmosomes and show reciprocally graded distributions in bovine nasal epidermis.

The adhesive core of the desmosome is composed of cadherin-like glycoproteins of two families, desmocollins and desmogleins. Three isoforms of each are expressed in a tissue-specific and developmentally regulated pattern. In bovine nasal epidermis, the three desmocollin (Dsc) isoforms are expressed in overlapping domains; Dsc3 expression is strongest in the basal layer, while Dsc2 and Dsc1 are strongly expressed in the suprabasal layers. Herein we have investigated whether different isoforms are assembled into the same or distinct desmosomes by performing double immunogold labeling using isoform-specific antibodies directed against Dsc1 and Dsc3. The results show that individual desmosomes harbor both isoforms in regions where their expression territories overlap. Quantification showed that the ratio of the proteins in each desmosome altered gradually from basal to immediately suprabasal and upper suprabasal layers, labeling for Dsc1 increasing and Dsc3 decreasing. Thus desmosomes are constantly modified as cells move up the epidermis, with continuing turnover of the desmosomal glycoproteins. Statistical analysis of the quantitative data showed a possible relationship between the distributions of the two isoforms. This gradual change in desmosomal composition may constitute a vertical adhesive gradient within the epidermis, having important consequences for cell positioning and differentiation.

Cellular stress inhibits transposition of the yeast retrovirus-like element Ty3 by a ubiquitin-dependent block of virus-like particle formation.

Many stress proteins and their cognates function as molecular chaperones or as components of proteolytic systems. Viral infection can stimulate synthesis of stress proteins and particular associations of viral and stress proteins have been documented. However, demonstrations of functions for stress proteins in viral life cycles are few. We have initiated an investigation of the roles of stress proteins in eukaryotic viral life cycles using as a model the Ty3 retrovirus-like element of Saccharomyces cerevisiae. During stress, Ty3 transposition is inhibited; Ty3 DNA is not synthesized and, although precursor proteins are detected, mature Ty3 proteins and virus-like particles (VLPs) do not accumulate. The same phenotype is observed in the constitutively stressed ssa1 ssa2 mutant, which lacks two cytoplasmic members of the hsp70 family of chaperones. Ty3 VLPs preformed under nonstress conditions are degraded more rapidly if cells are shifted from 30 degrees C to 37 degrees C. These results suggest that Ty3 VLPs are destroyed by cellular stress proteins. Elevated expression of the yeast UBP3 gene, which encodes a protease that removes ubiquitin from proteins, allows mature Ty3 proteins and VLPs to accumulate in the ssa1 ssa2 mutant, suggesting that, at least under stress conditions, ubiquitination plays a role in regulating Ty3 transposition.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

"Rattlesnake" structure of a filamentous plant RNA virus built of two capsid proteins.

Elongated particles of simple RNA viruses of plants are composed of an RNA molecule coated with numerous identical capsid protein subunits to form a regular helical structure, of which tobacco mosaic virus is the archetype. Filamentous particles of the closterovirus beet yellow virus (BYV) reportedly contain approximately 4000 identical 22-kDa (p22) capsid protein subunits. The BYV genome encodes a 24-kDa protein (p24) that is structurally related to the p22. We searched for the p24 in BYV particles by using immunoelectron microscopy with specific antibodies against the recombinant p24 protein and its N-terminal peptide. A 75-nm segment at one end of the 1370-nm filamentous viral particle was found to be consistently labeled with both types of antibodies, thus indicating that p24 is indeed the second capsid protein and that the closterovirus particle, unlike those of other plant viruses with helical symmetry, has a "rattlesnake" rather than uniform structure.

Low-resolution spectroscopy and spectral energy distributions of selected sources towards σ Orionis

Aims. We study in detail nine sources in the direction of the young σ Orionis cluster, which is considered to be a unique site for studying stellar and substellar formation. The nine sources were selected because of their peculiar properties, such as extremely-red infrared colours or excessively strong Hα emission for their blue optical colours. Methods. We acquired high-quality, low-resolution spectroscopy (R ∼ 500) of the nine targets with ALFOSC at the Nordic Optical Telescope. We also re-analysed [24]-band photometry from MIPS/Spitzer and compiled the highest quality photometric dataset available at the ViJHK_s passbands and the four IRAC/Spitzer channels, for constructing accurate spectral energy distributions between 0.55 and 24 μm. Results. The nine targets were classified into: one Herbig Ae/Be star with a scattering edge-on disc; two G-type stars; one X-ray flaring, early-M, young star with chromospheric Hα emission; one very low-mass, accreting, young spectroscopic binary; two young objects at the brown-dwarf boundary with the characteristics of classical T Tauri stars; and two emission-line galaxies, one undergoing star formation, and another whose spectral energy distribution is dominated by an active galactic nucleus. We also discovered three infrared sources associated with overdensities in a cold cloud of the cluster centre. Conclusions. Low-resolution spectroscopy and spectral energy distributions are a vital tool for measuring the physical properties and evolution of young stars and candidates in the σ Orionis cluster.

Handling of Uncertainty in Life Cycle Inventory by Correlated Multivariate Lognormal Distributions: Application to the Design of Supply Chain Networks

Poster presented in the 24th European Symposium on Computer Aided Process Engineering (ESCAPE 24), Budapest, Hungary, June 15-18, 2014.

Handling of Uncertainty in Life Cycle Inventory by Correlated Multivariate Lognormal Distributions: Application to the Design of Supply Chain Networks

In this work, we analyze the effect of incorporating life cycle inventory (LCI) uncertainty on the multi-objective optimization of chemical supply chains (SC) considering simultaneously their economic and environmental performance. To this end, we present a stochastic multi-scenario mixed-integer linear programming (MILP) coupled with a two-step transformation scenario generation algorithm with the unique feature of providing scenarios where the LCI random variables are correlated and each one of them has the desired lognormal marginal distribution. The environmental performance is quantified following life cycle assessment (LCA) principles, which are represented in the model formulation through standard algebraic equations. The capabilities of our approach are illustrated through a case study of a petrochemical supply chain. We show that the stochastic solution improves the economic performance of the SC in comparison with the deterministic one at any level of the environmental impact, and moreover the correlation among environmental burdens provides more realistic scenarios for the decision making process.

An adaptive algorithm for clustering cumulative probability distribution functions using the Kolmogorov–Smirnov two-sample test

This paper proposes an adaptive algorithm for clustering cumulative probability distribution functions (c.p.d.f.) of a continuous random variable, observed in different populations, into the minimum homogeneous clusters, making no parametric assumptions about the c.p.d.f.’s. The distance function for clustering c.p.d.f.’s that is proposed is based on the Kolmogorov–Smirnov two sample statistic. This test is able to detect differences in position, dispersion or shape of the c.p.d.f.’s. In our context, this statistic allows us to cluster the recorded data with a homogeneity criterion based on the whole distribution of each data set, and to decide whether it is necessary to add more clusters or not. In this sense, the proposed algorithm is adaptive as it automatically increases the number of clusters only as necessary; therefore, there is no need to fix in advance the number of clusters. The output of the algorithm are the common c.p.d.f. of all observed data in the cluster (the centroid) and, for each cluster, the Kolmogorov–Smirnov statistic between the centroid and the most distant c.p.d.f. The proposed algorithm has been used for a large data set of solar global irradiation spectra distributions. The results obtained enable to reduce all the information of more than 270,000 c.p.d.f.’s in only 6 different clusters that correspond to 6 different c.p.d.f.’s.