bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthetic Lipoteichoic Acid fromStaphylococcus aureusIs a Potent Stimulus of Cytokine Release
Authors: Morath Siegfried , Stadelmaier Andreas , Geyer Armin , Schmidt Richard R. , Hartung Thomas
Published: July 2002

We recently purified lipoteichoic acid (LTA) from Staphylococcus aureus to more than 99% purity by a novel preparation method and deduced its structure with the first nuclear magnetic resonance (NMR) of a complete LTA. In contrast to Gram-negative lipopolysaccharides, this LTA requires the toll-like receptor (TLR)-2 and not TLR-4 for cytokine induction in monocytes and macrophages. To elucidate the structure–function relationships for LTA from S. aureus, the lipid anchor was prepared by either acidic hydrolysis of native LTA or chemical synthesis (gentiobiosyl-sn-dimyristoylglycerol). Next, a complete LTA molecule with six glycerophosphate units carrying four alanine plus one N-acetyl-glucosamine substituent was synthesized, which displayed the same potency to activate monocytes as native LTA. However, 100–1,000 times higher concentrations of the lipid anchor were required for cytokine induction. It is worthy to note that replacing d-alanine with l-alanine blunted the effect indicating stereoselective recognition. The structure identification of this synthesized and biologically active LTA was proven by NMR and matrix-assisted laser desorption-ionization mass spectrometry. We concluded that the lipid anchor, with its fatty acids, represents an integral part of the immunostimulatory activity of LTA, but requires additional structural components on the polyglycerophosphate backbone.
Spontaneous Transient Outward Currents Arise from Microdomains Where BK Channels Are Exposed to a Mean Ca2+ Concentration on the Order of 10 μM during a Ca2+ Spark
Authors: ZhuGe Ronghua , Fogarty Kevin E. , Tuft Richard A. , Walsh John V.
Published: July 2002

Ca2+ sparks are small, localized cytosolic Ca2+ transients due to Ca2+ release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca2+ sparks activate large conductance Ca2+-activated K+ channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca2+ to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g(STOC)), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca2+]s. The Ca2+ sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g(STOC) remained roughly constant from 20 to −40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca2+] on the order of 10 μM during a Ca2+ spark. The membrane area over which a concentration ≥10 μM is reached has an estimated radius of 150–300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca2+ current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.
Distinct Properties of CRAC and MIC Channels in RBL Cells
Authors: Kozak J. Ashot , Kerschbaum Hubert H. , Cahalan Michael D.
Published: July 2002

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca2+ release–activated Ca2+ (CRAC) channels open in response to passive Ca2+ store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg2+ exposes an outwardly rectifying current (Mg2+-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg2+, spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg2+. Removal of internal Mg2+ induced MIC current despite widely varying Ca2+ and EGTA levels, suggesting that Ca2+-store depletion is not involved in activation of MIC channels. Increasing internal Mg2+ from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg2+ and Cs+ carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg2+ blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.
Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase
Authors: Kaiser Brett K. , Zimmerman Zachary A. , Charbonneau Harry , Jackson Peter K.
Published: July 2002

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.
A Fourth Component of the Fission Yeast γ-Tubulin Complex, Alp16, Is Required for Cytoplasmic Microtubule Integrity and Becomes Indispensable When γ-Tubulin Function Is Compromised
Authors: Fujita Akiko , Vardy Leah , Garcia Miguel Angel , Toda Takashi
Published: July 2002

γ-Tubulin functions as a multiprotein complex, called the γ-tubulin complex (γ-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved γ-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16+, as a multicopy suppressor of temperature-sensitive alp6-719mutants. alp16+encodes a 759-amino-acid protein with two conserved regions found in all other members of γ-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16+is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225,alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with γ-tubulin and cosediments with the γ-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the γ-TuC between yeast and higher eukaryotes.
Critical Roles of Phosphorylation and Actin Binding Motifs, but Not the Central Proline-rich Region, for Ena/Vasodilator-stimulated Phosphoprotein (VASP) Function during Cell Migration
Authors: Loureiro Joseph J. , Rubinson Douglas A. , Bear James E. , Baltus Gretchen A. , Kwiatkowski Adam V. , Gertler Frank B.
Published: July 2002

The Ena/vasodilator-stimulated phosphoprotein (VASP) protein family is implicated in the regulation of a number of actin-based cellular processes, including lamellipodial protrusion necessary for whole cell translocation. A growing body of evidence derived largely from in vitro biochemical experiments using purified proteins, cell-free extracts, and pathogen motility has begun to suggest various mechanistic roles for Ena/VASP proteins in the control of actin dynamics. Using complementation of phenotypes in Ena/VASP-deficient cells and overexpression in normal fibroblasts, we have assayed the function of a panel of mutants in one member of this family, Mena, by mutating highly conserved sequence elements found in this protein family. Surprisingly, deletion of sites required for binding of the actin monomer-binding protein profilin, a known ligand of Ena/VASP proteins, has no effect on the ability of Mena to regulate random cell motility. Our analysis revealed two features essential for Ena/VASP function in cell movement, cyclic nucleotide-dependent kinase phosphorylation sites and an F-actin binding motif. Interestingly, expression of the C-terminal EVH2 domain alone is sufficient to complement loss of Ena/VASP function in random cell motility.
Confidence measures for protein fold recognition
Authors: Sommer Ingolf , Zien Alexander , von Öhsen Niklas , Zimmer Ralf , Lengauer Thomas
Published: July 2002

Motivation: We present an extensive evaluation of different methods and criteria to detect remote homologs of a given protein sequence. We investigate two associated problems: first, to develop a sensitive searching method to identify possible candidates and, second, to assign a confidence to the putative candidates in order to select the best one.

For searching methods where the score distributions are known, p-values are used as confidence measure with great success. For the cases where such theoretical backing is absent, we propose empirical approximations to p-values for searching procedures.

Results: As a baseline, we review the performances of different methods for detecting remote protein folds (sequence alignment and threading, with and without sequence profiles, global and local). The analysis is performed on a large representative set of protein structures.

For fold recognition, we find that methods using sequence profiles generally perform better than methods using plain sequences, and that threading methods perform better than sequence alignment methods.

In order to assess the quality of the predictions made, we establish and compare several confidence measures, including raw scores, z-scores, raw score gaps, z-score gaps, and different methods of p-value estimation. We work our way from the theoretically well backed local scores towards more explorative global and threading scores.

The methods for assessing the statistical significance of predictions are compared using specificity--sensitivity plots. For local alignment techniques we find that p-value methods work best, albeit computationally cheaper methods such as those based on score gaps achieve similar performance. For global methods where no theory is available methods based on score gaps work best.

By using the score gap functions as the measure of confidence we improve the more powerful fold recognition methods for which p-values are unavailable.

Availability: The benchmark set is available upon request.

Contact: ingolf.sommer@gmd.de

* To whom correspondence should be addressed.
NETASA: neural network based prediction of solvent accessibility
Authors: Ahmad Shandar , Gromiha M. Michael
Published: July 2002

Motivation: Prediction of the tertiary structure of a protein from its amino acid sequence is one of the most important problems in molecular biology. The successful prediction of solvent accessibility will be very helpful to achieve this goal. In the present work, we have implemented a server, NETASA for predicting solvent accessibility of amino acids using our newly optimized neural network algorithm. Several new features in the neural network architecture and training method have been introduced, and the network learns faster to provide accuracy values, which are comparable or better than other methods of ASA prediction.

Results: Prediction in two and three state classification systems with several thresholds are provided. Our prediction method achieved the accuracy level upto 90% for training and 88% for test data sets. Three state prediction results provide a maximum 65% accuracy for training and 63% for the test data. Applicability of neural networks for ASA prediction has been confirmed with a larger data set and wider range of state thresholds. Salient differences between a linear and exponential network for ASA prediction have been analysed.

Availability: Online predictions are freely available at: http://www.netasa.org. Linux ix86 binaries of the program written for this work may be obtained by email from the corresponding author.

Contact: shandar@jamia.net

* To whom correspondence should be addressed at: RIKEN Tsukuba Institute, 3-1-1, Koyadai, Tsukuba 305 0074, Ibaraki, Japan.

† Present address: Computational Biology Research Center (CBRC), AIST 2-41-6 Aomi, Koto-ku, Tokyo 135-0064, Japan.
A novel approach to local reliability of sequence alignments
Authors: Schlosshauer Maximilian , Ohlsson Mattias
Published: July 2002

Motivation: The pairwise alignment of biological sequences obtained from an algorithm will in general contain both correct and incorrect parts. Hence, to allow for a valid interpretation of the alignment, the local trustworthiness of the alignment has to be quantified.

Results: We present a novel approach that attributes a reliability index to every pair of residues, including gapped regions, in the optimal alignment of two protein sequences. The method is based on a fuzzy recast of the dynamic programming algorithm for sequence alignment in terms of mean field annealing. An extensive evaluation with structural reference alignments not only shows that the probability for a pair of residues to be correctly aligned grows consistently with increasing reliability index, but moreover demonstrates that the value of the reliability index can directly be translated into an estimate of the probability for a correct alignment.

Contact: mattias@thep.lu.se
BAOBAB: a Java editor for large phylogenetic trees
Authors: Dutheil J. , Galtier N.
Published: July 2002

Summary: BAOBAB is a Java user interface dedicated to viewing and editing large phylogenetic trees. Original features include: (i) a colour-mediated overview of magnified subtrees; (ii) copy/cut/paste of (sub)trees within or between windows; (iii) compressing/ uncompressing subtrees; and (iv) managing sequence files together with tree files.

Availability: http://www.univ-montp2.fr/~genetix/

Contact: julien.dutheil@ens-lyon.frgaltier@univ-montp2.fr

* To whom correspondence should be addressed.
Clustering of proximal sequence space for the identification of protein families
Authors: Abascal Federico , Valencia Alfonso
Published: July 2002

Motivation: The study of sequence space, and the deciphering of the structure of protein families and subfamilies, has up to now been required for work in comparative genomics and for the prediction of protein function. With the emergence of structural proteomics projects, it is becoming increasingly important to be able to select protein targets for structural studies that will appropriately cover the space of protein sequences, functions and genomic distribution.

These problems are the motivation for the development of methods for clustering protein sequences and building families of potentially orthologous sequences, such as those proposed here.

Results: First we developed a clustering strategy (Ncut algorithm) capable of forming groups of related sequences by assessing their pairwise relationships. The results presented for the ras super-family of proteins are similar to those produced by other clustering methods, but without the need for clustering the full sequence space. The Ncut clusters are then used as the input to a process of reconstruction of groups with equilibrated genomic composition formed by closely-related sequences. The results of applying this technique to the data set used in the construction of the COG database are very similar to those derived by the human experts responsible for this database.

Availability: The analysis of different systems, including the COG equivalent 21 genomes are available at http://www.pdg.cnb.uam.es/GenoClustering.html

Contact: valencia@cnb.uam.es

* To whom correspondence should be addressed.
Target space for structural genomics revisited
Authors: Liu Jinfeng , Rost Burkhard
Published: July 2002

Motivation: Structural genomics eventually aims at determining structures for all proteins. However, in the beginning experimentalists are likely to focus on globular proteins to achieve a rapid basic coverage of protein sequence space. How many proteins will structural genomics have to target? How many proteins will be excluded since we already have structural information for these or since they are not globular? We have to answer these questions in the context of our target selection for the North-East Structural Genomics Consortium (NESG).

Results: We estimated that structural information is available for about 6–38% of all proteins; 6% if we require high accuracy in comparative modelling, 38% if we are satisfied with having a rough idea about the fold. Excluding all regions that are not globular, we found that structural genomics may have to target about 48% of all proteins. This corresponded to a similar percentage of residues of the entire proteomes (52%). We explored a number of different strategies to cluster protein space in order to find the number of families representing these 48% of structurally unknown proteins. For the subset of all entirely sequenced eukaryotes, we found over 18 000 fragment clusters each of which may be a suitable target for structural genomics.

Availability: All data are available from the authors, most results are summarized at: http://cubic.bioc.columbia.edu/genomes/RES/2002_bioinformatics/

Contact: rost@columbia.edu

* To whom correspondence should be addressed.
Intranuclear degradation of nonsense codon‐containing mRNA
Authors: Bühler Marc , Wilkinson Miles F , Mühlemann Oliver
Published: July 2002

Most vertebrate mRNAs with premature termination codons (PTCs) are specifically recognized and degraded by a process referred to as nonsense‐mediated mRNA decay (NMD) while still associated with the nucleus. However, it is still a matter of debate whether PTCs can be identified by intranuclear scanning or only by ribosomes on the cytoplasmic side of the nuclear envelope. Here we show that inhibition of mRNA export by two independent approaches does not affect the downregulation of PTC‐containing T‐cell receptor β transcripts in the nuclear fraction of mammalian cells, providing strong evidence for intranuclear NMD. Our results are fully consistent with recently reported evidence for nuclear translation and suggest that an important biological role for nuclear ribosomes is the early elimination of nonsense mRNA during a pioneer round of translation.
Functional involvement of a novel Nedd4‐like ubiquitin ligase on retrovirus budding
Authors: Yasuda Jiro , Hunter Eric , Nakao Mitsuyoshi , Shida Hisatoshi
Published: July 2002

In this study, we have identified a novel Nedd4‐like ubiquitin ligase, BUL1, as the host factor involved in budding of type D retrovirus Mason‐Pfizer monkey virus (M‐PMV). Overexpression of BUL1 enhanced virus particle release, while a BUL1 mutant in which a W to G substitution was introduced into a WW domain, W791G, lost the ability to bind to the viral Gag protein and abolished its ability to mediate virus budding. In addition, a fragment of BUL1 containing only the WW domains inhibited virus budding in a dominant negative manner. These results, together with previous findings, indicate that the M‐PMV Gag L domain interacts with the BUL1 WW domain and that this interaction is essential for virus budding. Our observations provide new insights into the mechanism of virus budding, and could be useful in establishing new antiviral strategies targeted at progeny virus release from a host cell.
Death by necrosis
Authors: Syntichaki Popi , Tavernarakis Nektarios
Published: July 2002

Cells suffer necrotic death when exposed to extreme environmental conditions, adverse and excessive stimuli, or when deleterious mutations are encoded in their genetic material. Unlike apoptosis, which involves a highly regulated and elaborate network of biochemical events and cascades, necrosis has been considered generally to be a chaotic decadence process that effects the inexorable demise of cells otherwise not destined to die. This grim prospect is now slowly being overturned, mostly by exciting new findings in two simple model organisms, Caenorhabditis elegans and Drosophila melanogaster. Despite the wide spectrum of necrosis‐initiating conditions, evidence is accumulating that execution of necrotic or neurodegenerative cell death may be carried out by a finite common set of mechanisms.
Estimating Aggregate Policy Reform Effects: New Baselines for Registration, Participation, and Representation
Authors: Timpone Richard J.
Published: July 2002

Statistical models are often extended to explore the aggregate impact of policy reforms. After discussing these techniques and the incorporation of prediction uncertainty, this article examines the effects of registration reform in an analytic framework that explicitly considers the two stages that defined electoral participation throughout the 20th century in the United States—registration and then voting. Using selection bias techniques, the effects of counterfactual registration reform conditions are explored on the aggregate level of participation and the nature of representation in the electoral process. These offer a richer baseline of the impact of policy changes than previous work by directly exploring the expected level of dropoff in going to the polls by new registrants. Results indicate that the dropoff between registration and voting would be expected to increase as more individuals become registered. In addition, while turnout due to reforms among projected “new registrants” shows potentially larger biases than those among existing registrants, because of the different bases of registration the changes would still lead to a modest reduction in the disparity between actual group sizes and their role in elections.
Testing for Necessary and/or Sufficient Causation: Which Cases Are Relevant?
Authors: Seawright Jason
Published: July 2002

Previous researchers have argued that necessary and/or sufficient causes should be tested through research designs that consider only cases with limited combinations of scores on the independent and the dependent variables. I explore the utility for causal inference of the design proposed by these authors, as compared to an “All Cases Design.” I find that, if researchers define the population carefully and appropriately, each case in the population contributes to causal inference and is therefore useful. Previous authors reject this claim on the basis of a view that holds constant the marginal distribution of either the dependent or the independent variable across the working and the alternate hypotheses. I argue that this restriction is not generally appropriate, and hence, an analysis that samples from the entire population is logically defensible. I also argue that this design is more statistically efficient. A reanalysis of two well-known studies demonstrates that sampling from all cases in the relevant population produces greater confidence in the hypothesis than sampling only from cases that experience the outcome.
The Reverend and the Ravens: Comment on Seawright
Authors: Clarke Kevin A.
Published: July 2002

The purpose of this Comment is to put the current debate regarding testing necessary conditions into perspective and to point out a particularly troubling aspect of the “all cases” research design (Seawright 2002).

Prior to the recent spate of books and articles in the social sciences (Ragin 1987; Dion 1988; Braumoeller and Goertz 2000), the debate over the testing or the confirmation of necessary conditions took place in the philosophical literature, mainly in terms of Hempel's (1945) paradox of the ravens. In what follows, I briefly review Hempel's paradox and the Bayesian solution to it. I argue that Seawright's account, while Bayesian in nature, relies on an assumption that no Bayesian would be willing to make.
Watching Your Posterior: Comment on Seawright
Authors: Braumoeller Bear F. , Goertz Gary
Published: July 2002

In “Testing for Necessary and/or Sufficient Causation,” Jason Seawright (2002) presents an intriguing new Bayesian technique for testing theories that posit necessary conditions. His technique differs in substantial ways from those presented in separate articles by Dion (1998), Ragin (1987), and ourselves (2000) and constitutes a fundamental critique of those procedures. Having criticized quite a few procedures and theorists in our own article, we can hardly complain; rather, we recognize the constructive spirit of the endeavor and, in that same spirit, would like to offer a rejoinder.
Plants in a cold climate
Authors: Smallwood Maggie , Bowles Dianna J.
Published: July 2002

Plants are able to survive prolonged exposure to sub–zero temperatures; this ability is enhanced by pre–exposure to low, but above–zero temperatures. This process, known as cold acclimation, is briefly reviewed from the perception of cold, through transduction of the low–temperature signal to functional analysis of cold–induced gene products. The stresses that freezing of apoplastic water imposes on plant cells is considered and what is understood about the mechanisms that plants use to combat those stresses discussed, with particular emphasis on the role of the extracellular matrix.
Survival mechanisms in Antarctic lakes
Authors: Laybourn-Parry Johanna
Published: July 2002

In Antarctic lakes, organisms are confronted by continuous low temperatures as well as a poor light climate and nutrient limitation. Such extreme environments support truncated food webs with no fish, few metazoans and a dominance of microbial plankton. The key to success lies in entering the short Antarctic summer with actively growing populations. In many cases, the most successful organisms continue to function throughout the year. The few crustacean zooplankton remain active in the winter months, surviving on endogenous energy reserves and, in some cases, continuing development. Among the Protozoa, mixotrophy is an important nutritional strategy. In the extreme lakes of the McMurdo Dry Valleys, planktonic cryptophytes are forced to sustain a mixotrophic strategy and cannot survive by photosynthesis alone. The dependence on ingesting bacteria varies seasonally and with depth in the water column. In the Vestfold Hills,Pyramimonas, which dominates the plankton of some of the saline lakes, also resorts to mixotrophy, but does become entirely photosynthetic at mid–summer. Mixotrophic ciliates are also common and the entirely photosynthetic ciliateMesodinium rubrumhas a widespread distribution in the saline lakes of the Vestfold Hills, where it attains high concentrations. Bacteria continue to grow all year, showing cycles that appear to be related to the availability of dissolved organic carbon. In saline lakes, bacteria experience sub–zero temperatures for long periods of the year and have developed biochemical adaptations that include anti–freeze proteins, changes in the concentrations of polyunsaturated fatty acids in their membranes and suites of low–temperature enzymes.
Cryoprotectin: a plant lipid–transfer protein homologue that stabilizes membranes during freezing
Authors: Hincha Dirk K.
Published: July 2002

Plants from temperate and cold climates are able to increase their freezing tolerance during exposure to low non–freezing temperatures. It has been shown that several genes are induced in a coordinated manner during this process of cold acclimation. The functional role of most of the corresponding cold–regulated proteins is not yet known. We summarize our knowledge of those cold–regulated proteins that are able to stabilize membranes during a freeze–thaw cycle. Special emphasis is placed on cryoprotectin, a lipid–transfer protein homologue that was isolated from cold–acclimated cabbage leaves and that protects isolated chloroplast thylakoid membranes from freeze–thaw damage.
The mechanics of cell fate determination in petals
Authors: Martin Cathie , Bhatt Kiran , Baumann Kim , Jin Hailing , Zachgo Sabine , Roberts Keith , Schwarz-Sommer Zsuzsanna , Glover Beverley , Perez-Rodrigues Maria
Published: July 2002

The epidermal cells of petals of many species are specialized, having a pronounced conical shape. A transcription factor, MIXTA, is required for the formation of conical cells inAntirrhinum majus; in shoot epidermal cells of several species, expression of this gene is necessary and sufficient to promote conical cell formation. Ectopic expression has also shownMIXTAto be able to promote the formation of multicellular trichomes, indicating that conical cells and multicellular trichomes share elements of a common developmental pathway. Formation of conical cells or trichomes is also mutually exclusive with stomatal formation. InAntirrhinum,MIXTAnormally only promotes conical cell formation on the inner epidermal layer of the petals. Its restricted action in cell fate determination results from its specific expression pattern. Expression ofMIXTA, in turn, requires the activity of B–function genes, and biochemical evidence suggests that the products ofDEFICIENS,GLOBOSAandSEPALLATA–related genes directly activate MIXTA expression late in petal development, after the completion of cell division in the petal epidermis.

AMIXTA–like gene,AmMYBML1, is also expressed in petals.AmMYBML1expression is high early in petal development. This gene may direct the formation of trichomes in petals.

In specifying the fates of different cell types in petals, regulatory genes likeMIXTAmay have been duplicated. Changes in the timing and spatial localization of expression then provides similar regulatory genes which specify different cell fates.
Building a hair: tip growth inArabidopsis thalianaroot hairs
Authors: Carol Rachel J. , Dolan Liam
Published: July 2002

TheArabidopsis thalianaroot hair is used as a model for studying tip growth in plants. We review recent advances, made using physiological and genetic approaches, which give rise to different, yet compatible, current views of the establishment and maintenance of tip growth in epidermal cells. For example, an active calcium influx channel localized at the tip ofArabidopsisroot hairs has been identified by patch–clamp measurements. Actin has been visualizedin vivoinArabidopsisroot hairs by using a green–fluorescent–protein–talin reporter and shown to form a dense mesh in the apex of the growing tip. Thekojakgene, which encodes a protein similar to the catalytic subunit of cellulose synthase, is needed in the first stages of hair growth. A role forLRX1, a leucine–rich repeat extensin, in determining the morphology of the cell wall of root hairs has been established using reverse genetics. The new information can be integrated into a general and more advanced view of how these specialized plant cells grow.
Signal transduction leading to low–temperature tolerance in
Arabidopsis thaliana
Authors: Knight Marc R.
Published: July 2002

Calcium is used by most cells to convert external signals into biochemical events within the cytosol. To detect the effects of cold stress, a gene encoding apoaequorin has been introduced into various cell types that, in the presence of coelenterazine, allows calcium levels to be monitored by the emission of blue light. All cell types respond to the cold by elevating calcium. This event is rapid after cold stress but then shows a slower kinetic response. The magnitude is dependent on both the rate and the final temperature to which cooling occurs. It would appear that calcium is transferred both from outside the cell and from the vacuole into the cytosol. The more rapid phase involves the former and the slower phase the latter. By studying mutant plants, it has been identified that an increase in intracellular calcium can activate the expression of transcription factors that control the subsequent transcription of a whole battery of genes which must be switched on to provide cold tolerance in the plant.
The Wilms tumor suppressor WT1 regulates early gonad development by activation ofSf1
Authors: Wilhelm Dagmar , Englert Christoph
Published: July 2002

In mammals, several genes including the Wilms tumor suppressor geneWt1, the Lim homeobox geneLhx9, and the gene encoding steroidogenic factor 1 (Sf1) have been implicated in the development of the indifferent gonad prior to sexual differentiation. Interactions among these genes have not yet been elucidated. Using biochemical and genetic experiments, we demonstrate here that WT1 and LHX9 function as direct activators of theSf1gene. Interestingly, only the −KTS form of WT1 is able to bind to and transactivate theSf1promoter. This observation is consistent with differential roles for the −KTS and +KTS variants of WT1 which have been postulated on the basis of human disorders such as the Frasier syndrome. Our data suggest a pathway in which the products of theWt1andLhx9genes activate expression ofSf1and thus mediate early gonadogenesis.
Transcription factor complex formation and chromatin fine structure alterations at the murine c-fms (CSF-1 receptor) locus during maturation of myeloid precursor cells
Authors: Tagoh Hiromi , Himes Roy , Clarke Deborah , Leenen Pieter J.M. , Riggs Arthur D. , Hume David , Bonifer Constanze
Published: July 2002

Expression of the gene for the macrophage colony stimulating factor receptor (CSF-1R), c-fms, has been viewed as a hallmark of the commitment of multipotent precursor cells to macrophages. Lineage-restricted expression of the gene is controlled by conserved elements in the proximal promoter and within the first intron. To investigate the developmental regulation of c-fms at the level of chromatin structure, we developed an in vitro system to examine the maturation of multipotent myeloid precursor cells into mature macrophages. The dynamics of chromatin fine structure alterations and transcription factor occupancy at the c-fms promoter and intronic enhancer was examined by in vivo DMS and UV-footprinting. We show that the c-fms gene is already transcribed at low levels in early myeloid precursors on which no CSF-1R surface expression can be detected. At this stage of myelopoiesis, the formation of transcription factor complexes on the promoter was complete. By contrast, occupancy of the enhancer was acutely regulated during macrophage differentiation. Our data show that cell-intrinsic differentiation decisions at the c-fms locus precede the appearance of c-fms on the cell surface. They also suggest that complex lineage-specific enhancers such as the c-fms intronic enhancer regulate local chromatin structure through the coordinated assembly and disassembly of distinct transcription factor complexes.
In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato
Authors: Mishra Shravan Kumar , Tripp Joanna , Winkelhaus Sybille , Tschiersch Bettina , Theres Klaus , Nover Lutz , Scharf Klaus-Dieter
Published: July 2002

We generated transgenic tomato plants with altered expression of heat stress transcription factor HsfA1. Plants with 10-fold overexpression of HsfA1 (OE plants) were characterized by a singleHsfA1 transgene cassette, whereas plants harboring a tandem inverted repeat of the cassette showed cosuppression (CS plants) by posttranscriptional silencing of the HsfA1 gene connected with formation of small interfering RNAs. Under normal growth conditions, major developmental parameters were similar for wild-type (WT), OE, and CS plants. However, CS plants and fruits were extremely sensitive to elevated temperatures, because heat stress-induced synthesis of chaperones and Hsfs was strongly reduced or lacking. Despite the complexity of the plant Hsf family with at least 17 members in tomato, HsfA1 has a unique function as master regulator for induced thermotolerance. Using transient reporter assays with mesophyll protoplasts from WT tomato, we demonstrated that plasmid-encoded HsfA1 and HsfA2 were well expressed. However, in CS protoplasts the cosuppression phenomenon was faithfully reproduced. Only transformation with HsfA2 expression plasmid led to normal expression of the transcription factor and reporter gene activation, whereas even high amounts of HsfA1 expression plasmids were silenced. Thermotolerance in CS protoplasts was restored by plasmid-borne HsfA2, resulting in expression of chaperones, thermoprotection of firefly luciferase, and assembly of heat stress granules.
Spo13 protects meiotic cohesin at centromeres in meiosis I
Authors: Shonn Marion A. , McCarroll Robert , Murray Andrew W.
Published: July 2002

In the absence of Spo13, budding yeast cells complete a single meiotic division during which sister chromatids often separate. We investigated the function of Spo13 by following chromosomes tagged with green fluorescent protein. The occurrence of a single division inspo13Δ homozygous diploids depends on the spindle checkpoint. Eliminating the checkpoint accelerates meiosis I in spo13Δcells and allows them to undergo two divisions in which sister chromatids often separate in meiosis I and segregate randomly in meiosis II. Overexpression of Spo13 and the meiosis-specific cohesin Rec8 in mitotic cells prevents separation of sister chromatids despite destruction of Pds1 and activation of Esp1. This phenotype depends on the combined overexpression of both proteins and mimics one aspect of meiosis I chromosome behavior. Overexpressing the mitotic cohesin, Scc1/Mcd1, does not substitute for Rec8, suggesting that the combined actions of Spo13 and Rec8 are important for preventing sister centromere separation in meiosis I.
FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor
Authors: Lando David , Peet Daniel J. , Gorman Jeffrey J. , Whelan Dean A. , Whitelaw Murray L. , Bruick Richard K.
Published: July 2002

Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF transcriptional activity is suppressed under normoxic conditions by hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators. Here we show that the protein FIH-1, previously shown to interact with HIF, is an asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O2 to modify its substrate. Together with the recently discovered prolyl hydroxylases that regulate HIF stability, this class of oxygen-dependent enzymes comprises critical regulatory components of the hypoxic response pathway.
Inhibition of apoptosis by ATFx: a novel role for a member of the ATF/CREB family of mammalian bZIP transcription factors
Authors: Persengiev Stephan P. , Devireddy Laxminarayana R. , Green Michael R.
Published: July 2002

The mammalian ATF/CREB family of transcription factors comprises a large group of basic-region leucine zipper (bZIP) proteins whose members mediate diverse transcriptional regulatory functions. Here we report that expression of a specific mouse ATF gene,ATFx, is down-regulated in a variety of cells undergoing apoptosis following growth factor deprivation. When stably expressed in an interleukin 3 (IL-3)-dependent cell line, ATFx suppresses apoptosis resulting from cytokine deprivation. Conversely, a dominant-negative ATFx mutant induces apoptosis of cells cultured in the presence of growth factors. We also show that 24p3, a secreted lipocalin that induces apoptosis when added to hematopoietic cells, represses ATFx expression. However, constitutive expression ofATFx renders cells resistant to 24p3-mediated apoptosis. Collectively, our results indicate that ATFx is an anti-apoptotic factor, a novel role for an ATF protein.
Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E
Authors: Fingar Diane C. , Salama Sofie , Tsou Christina , Harlow Ed , Blenis John
Published: July 2002

The coordinated action of cell cycle progression and cell growth (an increase in cell size and cell mass) is critical for sustained cellular proliferation, yet the biochemical signals that control cell growth are poorly defined, particularly in mammalian systems. We find that cell growth and cell cycle progression are separable processes in mammalian cells and that growth to appropriate cell size requires mTOR- and PI3K-dependent signals. Expression of a rapamycin-resistant mutant of mTOR rescues the reduced cell size phenotype induced by rapamycin in a kinase-dependent manner, showing the evolutionarily conserved role of mTOR in control of cell growth. Expression of S6K1 mutants that possess partial rapamycin-resistant activity or overexpression of eIF4E individually and additively partially rescues the rapamycin-induced decrease in cell size. In the absence of rapamycin, overexpression of S6K1 or eIF4E increases cell size, and, when coexpressed, they cooperate to increase cell size further. Expression of a phosphorylation site-defective mutant of 4EBP1 that constitutively binds the eIF4E–Cap complex to inhibit translation initiation reduces cell size and blocks eIF4E effects on cell size. These data show that mTOR signals downstream to at least two independent targets, S6K1 and 4EBP1/eIF4E, that function in translational control to regulate mammalian cell size.
A nonsense mutation in the fibrillin-1 gene of a Marfan syndrome patient induces NMD and disrupts an exonic splicing enhancer
Authors: Caputi Massimo , Kendzior Raymond J. , Beemon Karen L.
Published: July 2002

A nonsense mutation in the fibrillin-1 (FBN1) gene of a Marfan syndrome (MFS) patient induces in-frame exon skipping ofFBN1 exon 51. We present evidence, based on both in vivo and in vitro experiments, that the skipping of this exon is due to the disruption of an SC35-dependent splicing enhancer within exon 51. In addition, this nonsense mutation induces nonsense-mediated decay (NMD), which degrades the normally spliced mRNA in the patient's cells. In contrast to NMD, skipping of FBN1 exon 51 does not require translation.
Fission yeast CENP-B homologs nucleate centromeric heterochromatin by promoting heterochromatin-specific histone tail modifications
Authors: Nakagawa Hiromi , Lee Joon-Kyu , Hurwitz Jerard , Allshire Robin C. , Nakayama Jun-ichi , Grewal Shiv I.S. , Tanaka Katsunori , Murakami Yota
Published: July 2002

Heterochromatin is a functionally important chromosomal component, especially at centromeres. In fission yeast, conserved heterochromatin-specific modifications of the histone H3 tail, involving deacetylation of Lys 9 and Lys 14 and subsequent methylation of Lys 9, promote the recruitment of a heterochromatin protein, Swi6, a homolog of the Drosophila heterochromatin protein 1. However, the primary determinants of the positioning of heterochromatin are still unclear. The fission yeast proteins Abp1, Cbh1, and Cbh2 are homologs of the human protein CENP-B that bind to centromeric α-satellite DNA and associate with centromeric heterochromatin. We show that the CENP-B homologs are functionally redundant at centromeres, and that Abp1 binds specifically to centromeric heterochromatin. In the absence of Abp1 or Cbh1, the centromeric association of Swi6 is diminished, resulting in a decrease in silencing of the region. CENP-B-homolog double disruptants show a synergistic reduction of Swi6 at centromeric heterochromatin, indicating that the three proteins are functionally redundant in the recruitment of Swi6. Furthermore, using chromatin immunoprecipitation assays, we show that disruption of CENP-B homologs causes a decrease in heterochromatin-specific modifications of histone H3. These results indicate that the CENP-B homologs act as site-specific nucleation factors for the formation of centromeric heterochromatin by heterochromatin-specific modifications of histone tails.
Pathway Processor: A Tool for Integrating Whole-Genome Expression Results into Metabolic Networks
Authors: Grosu Paul , Townsend Jeffrey P. , Hartl Daniel L. , Cavalieri Duccio
Published: July 2002

We have developed a new tool to visualize expression data on metabolic pathways and to evaluate which metabolic pathways are most affected by transcriptional changes in whole-genome expression experiments. Using the Fisher Exact Test, the method scores biochemical pathways according to the probability that as many or more genes in a pathway would be significantly altered in a given experiment by chance alone. This method has been validated on diauxic shift experiments and reproduces well known effects of carbon source on yeast metabolism. The analysis is implemented with Pathway Analyzer, one of the tools of Pathway Processor, a new statistical package for the analysis of whole-genome expression data. Results from multiple experiments can be compared, reducing the analysis from the full set of individual genes to a limited number of pathways of interest. The pathways are visualized with OpenDX, an open-source visualization software package, and the relationship between genes in the pathways can be examined in detail using Expression Mapper, the second program of the package. This program features a graphical output displaying differences in expression on metabolic charts of the biochemical pathways to which the open reading frames are assigned.

[Supplementary materials are available athttp://www.cgr.harvard.edu/cavalieri/pp.html andhttp://www.genome.org.]
Alu-Containing Exons are Alternatively Spliced
Authors: Sorek Rotem , Ast Gil , Graur Dan
Published: July 2002

Alu repetitive elements are found in ∼1.4 million copies in the human genome, comprising more than one-tenth of it. Numerous studies describe exonizations of Alu elements, that is, splicing-mediated insertions of parts of Alu sequences into mature mRNAs. To study the connection between the exonization ofAlu elements and alternative splicing, we used a database of ESTs and cDNAs aligned to the human genome. We compiled two exon sets, one of 1176 alternatively spliced internal exons, and another of 4151 constitutively spliced internal exons. Sixty one alternatively spliced internal exons (5.2%) had a significant BLAST hit to an Alu sequence, but none of the constitutively spliced internal exons had such a hit. The vast majority (84%) of theAlu-containing exons that appeared within the coding region of mRNAs caused a frame-shift or a premature termination codon.Alu-containing exons were included in transcripts at lower frequencies than alternatively spliced exons that do not contain anAlu sequence. These results indicate that internal exons that contain an Alu sequence are predominantly, if not exclusively, alternatively spliced. Presumably, evolutionary events that cause a constitutive insertion of an Alu sequence into an mRNA are deleterious and selected against.