bluuebunny/crossref_metadata_2025 huggingface dataset:
Tomographic Evidence for Continuous Turnover of Golgi Cisternae inPichia pastoris
Authors: Mogelsvang Soren , Gomez-Ospina Natalia , Soderholm Jon , Glick Benjamin S. , Staehelin L. Andrew
Published: April 2003

The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure–function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking. The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding “matrix.” Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these “free-floating” TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack.
Regulation of Intermuscular Electrical Coupling by theCaenorhabditis elegansInnexininx-6
Authors: Li Shaolin , Dent Joseph A. , Roy Richard
Published: April 2003

The innexins represent a highly conserved protein family, the members of which make up the structural components of gap junctions in invertebrates. We have isolated and characterized a Caenorhabditis elegans gene inx-6 that encodes a new member of the innexin family required for the electrical coupling of pharyngeal muscles. inx-6(rr5) mutants complete embryogenesis without detectable abnormalities at restrictive temperature but fail to initiate postembryonic development after hatching. inx-6 is expressed in the pharynx at all larval stages, and an INX-6::GFP fusion protein showed a punctate expression pattern characteristic of gap junction proteins localized to plasma membrane plaques. Video recording and electropharyngeograms revealed that in inx-6(rr5) mutants the anterior pharyngeal (procorpus) muscles were electrically coupled to a lesser degree than the posterior metacorpus muscles, which caused a premature relaxation in the anterior pharynx and interfered with feeding. Dye-coupling experiments indicate that the gap junctions that link the procorpus to the metacorpus are functionally compromised in inx-6(rr5) mutants. We also show that another C. elegans innexin, EAT-5, can partially substitute for INX-6 function in vivo, underscoring their likely analogous function.
Intracellular Na+Controls Cell Surface Expression of Na,K-ATPase via a cAMP-independent PKA Pathway in Mammalian Kidney Collecting Duct Cells
Authors: Vinciguerra Manlio , Deschênes Georges , Hasler Udo , Mordasini David , Rousselot Martine , Doucet Alain , Vandewalle Alain , Martin Pierre-Yves , Féraille Eric
Published: April 2003

In the mammalian kidney the fine control of Na+reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+transport. In the cortical collecting duct (CCD), a rise in intracellular Na+concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K+-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.
Syntaxin 6 Regulates Glut4 Trafficking in 3T3-L1 Adipocytes
Authors: Perera H. Kumudu I. , Clarke Mairi , Morris Nicholas J. , Hong Wanjin , Chamberlain Luke H. , Gould Gwyn W.
Published: April 2003

Insulin stimulates the movement of glucose transporter-4 (Glut4)–containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.
Differential Requirement for the Nonhelical Tailpiece and the C Terminus of the Myosin Rod inCaenorhabditis elegansMuscle
Authors: Hoppe Pamela E. , Andrews Rebecca C. , Parikh Payal D.
Published: April 2003

Myosin heavy chain (MHC) is a large, multidomain protein important for both cellular structure and contraction. To examine the functional role of two C-terminal domains, the end of the coiled-coil rod and the nonhelical tailpiece, we have generated constructs in which residues within these domains are removed or mutated, and examined their behavior in Caenorhabditis elegans striated muscle. Genetic tests demonstrate that MHC lacking only tailpiece residues is competent to support the timely onset of embryonic contractions, and therefore viability, in animals lacking full-length MHC. Antibody staining experiments show that this truncated molecule localizes as wild type in early stages of development, but may be defective in processes important for thick filament organization later in embryogenesis. Ultrastructural analysis reveals thick filaments of normal morphology in disorganized arrangement, as well as occasional abnormal assemblages. In contrast, molecules in which the four terminal residues of the coiled coil are absent or mutated fail to rescue animals lacking endogenous MHC. Loss of these four residues is associated with delayed protein localization and delayed contractile function during early embryogenesis. Our results suggest that these two MHC domains, the rod and the tailpiece, are required for distinct steps during muscle development.
ADP-Ribosylation Factor/COPI-dependent Events at the Endoplasmic Reticulum-Golgi Interface Are Regulated by the Guanine Nucleotide Exchange Factor GBF1
Authors: García-Mata Rafael , Szul Tomasz , Alvarez Cecilia , Sztul Elizabeth
Published: April 2003

ADP-ribosylation factor (ARF) mediated recruitment of COPI to membranes plays a central role in transport between the endoplasmic reticulum (ER) and the Golgi. The activation of ARFs is mediated by guanine nucleotide exchange factors (GEFs). Although several ARF-GEFs have been identified, the transport steps in which they function are still poorly understood. Here we report that GBF1, a member of the Sec7-domain family of GEFs, is responsible for the regulation of COPI-mediated events at the ER-Golgi interface. We show that GBF1 is essential for the formation, differentiation, and translocation of pre-Golgi intermediates and for the maintenance of Golgi integrity. We also show that the formation of transport-competent ER-to-Golgi intermediates proceeds in two stages: first, a COPI-independent event leads to the formation of an unstable compartment, which is rapidly reabsorbed in the absence of GBF1 activity. Second, the association of GBF1 with this compartment allows COPI recruitment and leads to its maturation into transport intermediates. The recruitment of GBF1 to this compartment is specifically inhibited by brefeldin A. Our findings imply that the continuous recruitment of GBF1 to spatially differentiated membrane domains is required for sustained membrane remodeling that underlies membrane traffic and Golgi biogenesis.
Involvement of the Endoplasmic Reticulum in Peroxisome Formation
Authors: Geuze Hans J. , Murk Jean Luc , Stroobants An K. , Griffith Janice M. , Kleijmeer Monique J. , Koster Abraham J. , Verkleij Arie J. , Distel Ben , Tabak Henk F.
Published: April 2003

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.
Actin Recovery and Bud Emergence in Osmotically Stressed Cells Requires the Conserved Actin Interacting Mitogen-activated Protein Kinase Kinase Kinase Ssk2p/MTK1 and the Scaffold Protein Spa2p
Authors: Yuzyuk Tatiana , Amberg David C.
Published: April 2003

Osmotic stress causes actin cytoskeleton disassembly, a cell cycle arrest, and activation of the high osmolarity growth mitogen-activated protein kinase pathway. A previous study showed that Ssk2p, a mitogen-activated protein kinase kinase kinase of the high osmolarity growth pathway, promotes actin cytoskeleton recovery to the neck of late cell cycle, osmotically stressed yeast cells. Data presented herein examined the role of Ssk2p in actin recovery early in the cell cycle. We found that actin recovery at all stages of the cell cycle is not controlled by Ssk1p, the known activator of Ssk2p, but required a polarized distribution of Ssk2p as well as its actin-interacting and kinase activity. Stress-induced localization of Ssk2p to the neck required the septin Shs1p, whereas localization to the bud cortex depended on the polarity scaffold protein Spa2p. spa2Δ cells, like ssk2Δ cells, were defective for actin recovery from osmotic stress. These spa2Δ defects could be suppressed by overexpression of catalytically active Ssk2p. Furthermore, Spa2p could be precipitated by GST-Ssk2p from extracts of osmotically stressed cells. The Ssk2p mediated actin recovery pathway seems to be conserved; MTK1, a human mitogen-activated protein kinase kinase kinase of the p38 stress response pathway and Ssk2p homolog, was also able to localize at polarized growth sites, form a complex with actin and Spa2p, and complement actin recovery defects in osmotically stressed ssk2Δ and spa2Δ yeast cells. We hypothesize that osmotic stress-induced actin disassembly leads to the formation of an Ssk2p–actin complex and the polarized localization of Ssk2p. Polarized Ssk2p associates with the scaffold protein Spa2p in the bud and Shs1p in the neck, allowing Ssk2p to regulate substrates involved in polarized actin assembly.
Three-dimensional Organization of Basal Bodies from Wild-Type and δ-Tubulin Deletion Strains ofChlamydomonas reinhardtii
Authors: O'Toole Eileen T. , Giddings Thomas H. , McIntosh J. Richard , Dutcher Susan K.
Published: April 2003

Improved methods of specimen preparation and dual-axis electron tomography have been used to study the structure and organization of basal bodies in the unicellular alga Chlamydomonas reinhardtii. Novel structures have been found in both wild type and strains with mutations that affect specific tubulin isoforms. Previous studies have shown that strains lacking δ-tubulin fail to assemble the C-tubule of the basal body. Tomographic reconstructions of basal bodies from the δ-tubulin deletion mutant uni3-1 have confirmed that basal bodies contain mostly doublet microtubules. Our methods now show that the stellate fibers, which are present only in the transition zone of wild-type cells, repeat within the core of uni3-1 basal bodies. The distal striated fiber is incomplete in this mutant, rootlet microtubules can be misplaced, and multiflagellate cells have been observed. A suppressor of uni3-1, designated tua2-6, contains a mutation in α-tubulin. tua2-6; uni3-1 cells build both flagella, yet they retain defects in basal body structure and in rootlet microtubule positioning. These data suggest that the presence of specific tubulin isoforms in Chlamydomonas directly affects the assembly and function of both basal bodies and basal body-associated structures.
Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells
Authors: Nyasae Lydia K. , Hubbard Ann L. , Tuma Pamela L.
Published: April 2003

We examined the role that lipid rafts play in regulating apical protein trafficking in polarized hepatic cells. Rafts are postulated to form in the trans-Golgi network where they recruit newly synthesized apical residents and mediate their direct transport to the apical plasma membrane. In hepatocytes, single transmembrane and glycolipid-anchored apical proteins take the “indirect” route. They are transported from the trans-Golgi to the basolateral plasma membrane where they are endocytosed and transcytosed to the apical surface. Do rafts sort hepatic apical proteins along this circuitous pathway? We took two approaches to answer this question. First, we determined the detergent solubility of selected apical proteins and where in the biosynthetic pathway insolubility was acquired. Second, we used pharmacological agents to deplete raft components and assessed their effects on basolateral-to-apical transcytosis. We found that cholesterol and glycosphingolipids are required for delivery from basolateral early endosomes to the subapical compartment. In contrast, fluid phase uptake and clathrin-mediated internalization of recycling receptors were only mildly impaired. Apical protein solubility did not correlate with raft depletion or impaired transcytosis, suggesting other factors contribute to apical protein insolubility. Examination of apical proteins in Fao cells also revealed that raft-dependent sorting does not require the polarized cell context.
TheSaccharomyces cerevisiaeCalponin/Transgelin Homolog Scp1 Functions with Fimbrin to Regulate Stability and Organization of the Actin Cytoskeleton
Authors: Goodman Anya , Goode Bruce L. , Matsudaira Paul , Fink Gerald R.
Published: April 2003

Calponins and transgelins are members of a conserved family of actin-associated proteins widely expressed from yeast to humans. Although a role for calponin in muscle cells has been described, the biochemical activities and in vivo functions of nonmuscle calponins and transgelins are largely unknown. Herein, we have used genetic and biochemical analyses to characterize the budding yeast member of this family, Scp1, which most closely resembles transgelin and contains one calponin homology (CH) domain. We show that Scp1 is a novel component of yeast cortical actin patches and shares in vivo functions and biochemical activities with Sac6/fimbrin, the one other actin patch component that contains CH domains. Purified Scp1 binds directly to filamentous actin, cross-links actin filaments, and stabilizes filaments against disassembly. Sequences in Scp1 sufficient for actin binding and cross-linking reside in its carboxy terminus, outside the CH domain. Overexpression of SCP1 suppresses sac6Δ defects, and deletion of SCP1 enhances sac6Δ defects. Together, these data show that Scp1 and Sac6/fimbrin cooperate to stabilize and organize the yeast actin cytoskeleton.
β-Catenin Regulation during the Cell Cycle: Implications in G2/M and Apoptosis
Authors: Olmeda David , Castel Susanna , Vilaró Senén , Cano Amparo
Published: April 2003

β-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. β-Catenin signaling has been proposed to act as inducer of cell proliferation in different tumors. However, in some developmental contexts and cell systems β-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of β-Catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous β-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase–synchronized epithelial cells. β-Catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of β-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not β-catenin, is detected in M phase. Interestingly, overexpression of a stable form of β-catenin, or inhibition of endogenous β-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for β-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.
Keratin 20 Helps Maintain Intermediate Filament Organization in Intestinal Epithelia
Authors: Zhou Qin , Toivola Diana M. , Feng Ningguo , Greenberg Harry B. , Franke Werner W. , Omary M. Bishr
Published: April 2003

Of the >20 epithelial keratins, keratin 20 (K20) has an unusual distribution and is poorly studied. We began to address K20 function, by expressing human wild-type and Arg80→His (R80H) genomic (18 kb) and cDNA K20 in cells and mice. Arg80 of K20 is conserved in most keratins, and its mutation in epidermal keratins causes several skin diseases. R80H but not wild-type K20 generates disrupted keratin filaments in transfected cells. Transgenic mice that overexpress K20 R80H have collapsed filaments in small intestinal villus regions, when expressed at moderate levels, whereas wild-type K20-overexpressing mice have normal keratin networks. Overexpressed K20 maintains its normal distribution in several tissues, but not in the pancreas and stomach, without causing any tissue abnormalities. Hence, K20 pancreatic and gastric expression is regulated outside the 18-kb region. Cross-breeding of wild-type or R80H K20 mice with mice that overexpress wild-type K18 or K18 that is mutated at the conserved K20 Arg80-equivalent residue show that K20 plays an additive and compensatory role with K18 in maintaining keratin filament organization in the intestine. Our data suggest the presence of unique regulatory domains for pancreatic and gastric K20 expression and support a significant role for K20 in maintaining keratin filaments in intestinal epithelia.
Java editor for biological pathways
Authors: Trost Elmar , Hackl Hubert , Maurer Michael , Trajanoski Zlatko
Published: April 2003

Summary: A visual Java-based tool for drawing and annotating biological pathways was developed. This tool integrates the possibilities of charting elements with different attributes (size, color, labels), drawing connections between elements in distinct characteristics (color, structure, width, arrows), as well as adding links to molecular biology databases, promoter sequences, information on the function of the genes or gene products, and references. It is easy to use and system independent. The result of the editing process is a PNG (portable network graphics) file for the images and XML (extended markup language) file for the appropriate links.

Availability: http://genome.tugraz.at

Contact: zlatko.trajanoski@tugraz.at

* To whom correspondence should be addressed.
Interpreting peptide mass spectra by VEMS
Authors: Matthiesen R. , Lundsgaard M. , Welinder K.G. , Bauw G.
Published: April 2003

Most existing Mass Spectra (MS) analysis programs are automatic and provide limited opportunity for editing during the interpretation. Furthermore, they rely entirely on publicly available databases for interpretation. VEMS (Virtual Expert Mass Spectrometrist) is a program for interactive analysis of peptide MS/MS spectra imported in text file format. Peaks are annotated, the monoisotopic peaks retained, and the b-and y-ion series identified in an interactive manner. The called peptide sequence is searched against a local protein database for sequence identity and peptide mass. The report compares the calculated and the experimental mass spectrum of the called peptide. The program package includes four accessory programs. VEMStrans creates protein databases in FASTA format from EST or cDNA sequence files. VEMSdata creates a virtual peptide database from FASTA files. VEMSdist displays the distribution of masses up to 5000 Da. VEMSmaldi searches singly charged peptide masses against the local database.

Availability: http://www.bio.auc.dk/~rm/sub/VEMS.htm

Contact: rm@bio.auc.dk

* To whom correspondence should be addressed.
Phylogenetic Web Profiler
Authors: Wong Philip , Kolesov Grigory , Frishman Dmitrij , Houry Walid A.
Published: April 2003

Summary: Phylogenetic Web Profiler (PWP) is a web-based service designed to perform phylogenetic profiling of proteins against genomes. The current version offers a selection of 63 completed genomes and available plasmids as annotated in the PEDANT genome database. Unlike currently available applications, this tool offers several choices of ortholog prediction parameters including E-value cutoff, percent length difference tolerance, and annotation similarity. Additional features include tight integration with the PEDANT database and tools to analyze properties of predicted proteins. PWP should prove very useful for the analysis of functional-linkage between proteins.

Availability: The PWP server is available at http://pedant.gsf.de/pwp/. The PWP functional predictions are also offered as part of the PEDANT genome analysis server (http://pedant.gsf.de).

Contact: walid.houry@utoronto.ca

* To whom correspondence should be addressed.
PGAGENE: integrating quantitative gene-specific results from the
NHLBI Programs for Genomic Applications
Authors: Lee Kyungjoon , Kohane Isaac S. , Butte Atul J.
Published: April 2003

Summary: PGAGENE is a web-based gene-specific genomic data search engine, which allows users to search over 5.9 million pieces of collective genetic and genomic data from the NHLBI supported Programs for Genomic Applications. This data includes microarray measurements, SNPs, and mutations, and data may be found using symbols, parts of gene names or products, Affymetrix probe IDs, GenBank accession numbers, UniGene IDs, dbSNP IDs, and others. The PGAGENE indexing agent periodically maps all publicly available gene-specific PGA data onto LocusLink using dynamically generated cross-referencing tables.

Availability: http://pgagene.chip.org

Contact: atul_butte@harvard.edu

* To whom correspondence should be addressed.
GAAS: Gene Array Analyzer Software for management, analysis and
visualization of gene expression data
Authors: Masseroli M. , Cerveri P. , Pelicci P.G. , Alcalay M.
Published: April 2003

Summary: GAAS, Gene Array Analyzer Software supports multi-user efficient management and suitable analyses of large amounts of gene expression data across replicated experiments. Its management framework handles input data generated by different technologies. A multi-user environment allows each user to store his/her own data visualization scheme, analysis parameters used, values and formats of the output data. The analysis engine performs: background and spot quality evaluation, data normalization, differential gene expression analyses in single and multiple replica experiments. Results of expression profiles can be interactively navigated through graphical interfaces and stored into output databases.

Availability: http://www.medinfopoli.polimi.it/GAAS/

Contact: masseroli@biomed.polimi.it

Supplementary information: http://www.medinfopoli.polimi.it/GAAS/

* To whom correspondence should be addressed.
A segment alignment approach to protein comparison
Authors: Ye Yuzhen , Jaroszewski Lukasz , Li Weizhong , Godzik Adam
Published: April 2003

Motivation: Local structure segments (LSSs) are small structural units shared by unrelated proteins. They are extensively used in protein structure comparison, and predicted LSSs (PLSSs) are used very successfully in ab initio folding simulations. However, predicted or real LSSs are rarely exploited by protein sequence comparison programs that are based on position-by-position alignments.

Results: We developed a SEgment Alignment algorithm (SEA) to compare proteins described as a collection of predicted local structure segments (PLSSs), which is equivalent to an unweighted graph (network). Any specific structure, real or predicted corresponds to a specific path in this network. SEA then uses a network matching approach to find two most similar paths in networks representing two proteins. SEA explores the uncertainty and diversity of predicted local structure information to search for a globally optimal solution. It simultaneously solves two related problems: the alignment of two proteins and the local structure prediction for each of them. On a benchmark of protein pairs with low sequence similarity, we show that application of the SEA algorithm improves alignment quality as compared to FFAS profile-profile alignment, and in some cases SEA alignments can match the structural alignments, a feat previously impossible for any sequence based alignment methods.

Availability: SEA is freely available for academic users on a web server http://ffas.ljcrf.edu/sea.

Supplementary information: http://ffas.ljcrf.edu/sea

Contact: adam@burnham.org

* To whom correspondence should be addressed. Program in Bioinformatics and Biological Complexity, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Client–Server environment for high-performance gene expression data analysis
Authors: Sturn Alexander , Mlecnik Bernhard , Pieler Roland , Rainer Johannes , Truskaller Thomas , Trajanoski Zlatko
Published: April 2003

Summary: We have developed a platform independent, flexible and scalable Java environment for high-performance large-scale gene expression data analysis, which integrates various computational intensive hierarchical and non-hierarchical clustering algorithms. The environment includes a powerful client for data preparation and results visualization, an application server for computation and an additional administration tool. The package is available free of charge for academic and non-profit institutions.

Availability: http://genome.tugraz.at/Software

Contact: zlatko.trajanoski@tugraz.at

* To whom correspondence should be addressed.
Evaluation of annotation strategies using an entire genome sequence
Authors: Iliopoulos Ioannis , Tsoka Sophia , Andrade Miguel A. , Enright Anton J. , Carroll Mark , Poullet Patrick , Promponas Vassilis , Liakopoulos Theodore , Palaios Giorgos , Pasquier Claude , Hamodrakas Stavros , Tamames Javier , Yagnik Asutosh T. , Tramontano Anna , Devos Damien , Blaschke Christian , Valencia Alfonso , Brett David , Martin David , Leroy Christophe , Rigoutsos Isidore , Sander Chris , Ouzounis Christos A.
Published: April 2003

Motivation: Genome-wide functional annotation either by manual or automatic means has raised considerable concerns regarding the accuracy of assignments and the reproducibility of methodologies. In addition, a performance evaluation of automated systems that attempt to tackle sequence analyses rapidly and reproducibly is generally missing. In order to quantify the accuracy and reproducibility of function assignments on a genome-wide scale, we have re-annotated the entire genome sequence of Chlamydia trachomatis (serovar D), in a collaborative manner.

Results: We have encoded all annotations in a structured format to allow further comparison and data exchange and have used a scale that records the different levels of potential annotation errors according to their propensity to propagate in the database due to transitive function assignments. We conclude that genome annotation may entail a considerable amount of errors, ranging from simple typographical errors to complex sequence analysis problems. The most surprising result of this comparative study is that automatic systems might perform as well as the teams of experts annotating genome sequences.

Availability and supplementary information: http://www.ebi.ac.uk/research/cgg/annotation/cteval/

Contact: ouzounis@ebi.ac.uk

* To whom correspondence should be addressed.

† INA-EKETA, GR-57001 Thessaloniki, Greece

‡ Computational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

§ Aetion Technologies LLC, Worthington, OH 43085, USA

¶ Institut Curie, F-75248 Paris, France

∥ CNRS, UMR6543, F-06108 Nice, France

** Alma Bioinformatics, E-28760 Madrid, Spain

†† Cap Gemini Ernst & Young, London SW1X 7LX, UK

‡‡ Univ. of Rome ‘La Sapienza’, I-00185 Rome, Italy

§§ MWG-Biotech AG, Ebersberg, D-85560 Berlin, Germany

¶¶ Wellcome Trust Biocentre, Univ. of Dundee, Dundee DD1 5HN, UK
Perfect phylogeny haplotyper: haplotype inferral using a tree model
Authors: Chung Ren Hua , Gusfield Dan
Published: April 2003

Summary: We have developed an efficient program, the Perfect Phylogeny Haplotyper (PPH) that takes in unphased population genotype data, and determines if that data can be explained by haplotype pairs that could have evolved on a perfect phylogeny.

Availability: Executable code for four common platforms is available at: http://wwwcsif.cs.ucdavis.edu/~gusfield

Contact: gusfield@cs.ucdavis.edu

* To whom correspondence should be addressed.
Directed indices for exploring gene expression data
Authors: LeBlanc Michael , Kooperberg Charles , Grogan Thomas M. , Miller Thomas P.
Published: April 2003

Motivation: Large expression studies with clinical outcome data are becoming available for analysis. An important goal is to identify genes or clusters of genes where expression is related to patient outcome. While clustering methods are useful data exploration tools, they do not directly allow one to relate the expression data to clinical outcome. Alternatively, methods which rank genes based on their univariate significance do not incorporate gene function or relationships to genes that have been previously identified. In addition, after sifting through potentially thousands of genes, summary estimates (e.g. regression coefficients or error rates) algorithms should address the potentially large bias introduced by gene selection.

Results: We developed a gene index technique that generalizes methods that rank genes by their univariate associations to patient outcome. Genes are ordered based on simultaneously linking their expression both to patient outcome and to a specific gene of interest. The technique can also be used to suggest profiles of gene expression related to patient outcome. A cross-validation method is shown to be important for reducing bias due to adaptive gene selection. The methods are illustrated on a recently collected gene expression data set based on 160 patients with diffuse large cell lymphoma (DLCL).

Availability: A program written in the R language implementing the gene index can be obtained at http://www.crab.org/papers/

Contact: mikel@crab.org

* To whom correspondence should be addressed.
The First Oral Rotavirus Vaccine, 1998–1999: Estimates of Uptake from the National Immunization Survey
Authors: Smith Philip J. , Schwartz Ben , Mokdad Ali , Bloch Alan B. , McCauley Mary , Murphy Trudy V.
Published: April 2003

Objective.

On August 31, 1998, the rhesus-human reassortant rotavirus vaccine (RRV-TV) was licensed for use in the U.S. During the next nine months, 15 cases of intussusception were reported among infants who received the vaccine. Case-control and cohort studies showed a significantly increased risk of developing intussusception within one week of receiving the vaccine; subsequent ecologic studies did not. In this study, the authors used data on RRV-TV vaccination rates from the National Immunization Survey (NIS) to estimate state and national RRV-TV uptake rates and factors associated with receiving RRV-TV. These estimates are a key component in evaluating published ecologic studies designed to investigate the relationship between receipt of the vaccine and intussusception.

Methods.

The authors analyzed NIS data for children ages 19 to 35 months who were eligible to receive RRV-TV between September 1998 and July 1999. The authors estimated vaccine coverage and the number of doses administered by state, NIS sampling quarter, and birth cohort, and analyzed demographic and socioeconomic variables to evaluate their relationship with receiving RRV-TV.

Results.

It was estimated that approximately 1 million doses of RRV-TV were administered to 504,585 (±61,854) children, 13.4% (±1.6%) of children who were eligible. The estimated number of doses administered and the vaccination coverage rate varied greatly from state to state. Children living in households with higher socioeconomic conditions were more likely to receive the vaccine.

Conclusion.

Ecologic studies had a limited ability to detect a significant increase in the population incidence rate of intussusception that could be attributed to RRV-TV because populations in these studies consisted primarily of children who did not receive the vaccine. The example from RRV-TV demonstrates some of the challenges of assessing the magnitude of the association between a vaccine and an uncommon or rare adverse event.
Completeness of Pediatric TB Reporting in New York City
Authors: San Gabriel Pablo , Saiman Lisa , Kaye Katherine , Silin Muriel , Onorato Ida , Schulte Joann
Published: April 2003

Objectives.

Accurate surveillance of tuberculosis (TB) in children is critical because such cases represent recent transmission, but surveillance is difficult as only 10% to 50% of cases are culture-confirmed. Hospital-based sources were used to develop alternative surveillance to assess completeness of reporting for pediatric TB in northern Manhattan and Harlem from 1993 through 1995.

Methods.

Alternative surveillance sources included ICD-9-CM hospital discharge codes for active TB and gastric aspirate reports. Cases identified by alternative surveillance were compared with cases previously reported to the New York City Department of Health (NYC DOH).

Results.

Alternative surveillance detected 25 cases of possible pediatric TB, of which four (16%) had never been reported to the NYC DOH and three (12%) had been reported as suspect cases, but had not fulfilled the criteria for a reportable case of pediatric TB. Of these seven newly counted cases, three were detected by ICD-9-CM codes, three by a gastric aspirate log book, and one by both. In contrast, 13 other cases had been reported to the NYC DOH, but were undetected by our alternative surveillance; eight of these could be verified with available medical records. Thus, the demographic and clinical characteristics of the 25 detected and the eight undetected cases with available medical records were evaluated in this study.

Conclusions.

Alternative surveillance proved effective, was complementary to the NYC DOH surveillance efforts, and increased the number of pediatric TB cases identified during the study period by 21%.
Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis
Authors: Moshkov Igor E. , Mur Luis A.J. , Novikova Galina V. , Smith Aileen R. , Hall Michael A.
Published: April 2003

Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In theetr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 andAra3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In theetr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; inctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction.
Phloem-Localizing Sulfate Transporter, Sultr1;3, Mediates Re-Distribution of Sulfur from Source to Sink Organs in Arabidopsis
Authors: Yoshimoto Naoko , Inoue Eri , Saito Kazuki , Yamaya Tomoyuki , Takahashi Hideki
Published: April 2003

For the effective recycling of nutrients, vascular plants transport pooled inorganic ions and metabolites through the sieve tube. A novel sulfate transporter gene, Sultr1;3, was identified as an essential member contributing to this process for redistribution of sulfur source in Arabidopsis.Sultr1;3 belonged to the family of high-affinity sulfate transporters, and was able to complement the yeast sulfate transporter mutant. The fusion protein of Sultr1;3 and green fluorescent protein was expressed by theSultr1;3 promoter in transgenic plants, which revealed phloem-specific expression ofSultr1;3 in Arabidopsis. Sultr1;3-green fluorescent protein was found in the sieve element-companion cell complexes of the phloem in cotyledons and roots. Limitation of external sulfate caused accumulation of Sultr1;3mRNA both in leaves and roots. Movement of 35S-labeled sulfate from cotyledons to the sink organs was restricted in the T-DNA insertion mutant of Sultr1;3. These results provide evidence that Sultr1;3 transporter plays an important role in loading of sulfate to the sieve tube, initiating the source-to-sink translocation of sulfur nutrient in Arabidopsis.
Tolerance of Mannitol-Accumulating Transgenic Wheat to Water Stress and Salinity
Authors: Abebe Tilahun , Guenzi Arron C. , Martin Bjorn , Cushman John C.
Published: April 2003

Previous work with model transgenic plants has demonstrated that cellular accumulation of mannitol can alleviate abiotic stress. Here, we show that ectopic expression of the mtlD gene for the biosynthesis of mannitol in wheat improves tolerance to water stress and salinity. Wheat (Triticum aestivum L. cv Bobwhite) was transformed with the mtlD gene of Escherichia coli. Tolerance to water stress and salinity was evaluated using calli and T2 plants transformed with (+mtlD) or without (−mtlD) mtlD. Calli were exposed to −1.0 MPa of polyethylene glycol 8,000 or 100 mm NaCl. T2plants were stressed by withholding water or by adding 150 mm NaCl to the nutrient medium. Fresh weight of −mtlD calli was reduced by 40% in the presence of polyethylene glycol and 37% under NaCl stress. Growth of +mtlD calli was not affected by stress. In −mtlD plants, fresh weight, dry weight, plant height, and flag leaf length were reduced by 70%, 56%, 40%, and 45% compared with 40%, 8%, 18%, and 29%, respectively, in +mtlD plants. Salt stress reduced shoot fresh weight, dry weight, plant height, and flag leaf length by 77%, 73%, 25%, and 36% in −mtlD plants, respectively, compared with 50%, 30%, 12%, and 20% in +mtlD plants. However, the amount of mannitol accumulated in the callus and mature fifth leaf (1.7–3.7 μmol g−1 fresh weight in the callus and 0.6–2.0 μmol g−1 fresh weight in the leaf) was too small to protect against stress through osmotic adjustment. We conclude that the improved growth performance of mannitol-accumulating calli and mature leaves was due to other stress-protective functions of mannitol, although this study cannot rule out possible osmotic effects in growing regions of the plant.
Suppression of a Vegetative MADS Box Gene of Potato Activates Axillary Meristem Development
Authors: Rosin Faye M. , Hart Jennifer K. , Van Onckelen Harry , Hannapel David J.
Published: April 2003

Potato MADS box 1 (POTM1) is a member of the SQUAMOSA-like family of plant MADS box genes isolated from an early stage tuber cDNA library. The RNA ofPOTM1 is most abundant in vegetative meristems of potato (Solanum tuberosum), accumulating specifically in the tunica and corpus layers of the meristem, the procambium, the lamina of new leaves, and newly formed axillary meristems. Transgenic lines with reduced levels of POTM1 mRNA exhibited decreased apical dominance accompanied by a compact growth habit and a reduction in leaf size. Suppression lines produced truncated shoot clusters from stem buds and, in a model system, exhibited enhanced axillary bud growth instead of producing a tuber. This enhanced axillary bud growth was not the result of increased axillary bud formation. Tuber yields were reduced and rooting of cuttings was strongly inhibited inPOTM1 suppression lines. Both starch accumulation and the activation of cell division occurred in specific regions of the vegetative meristems of the POTM1 transgenic lines. Cytokinin levels in axillary buds of a transgenic suppression line increased 2- to 3-fold. These results imply that POTM1mediates the control of axillary bud development by regulating cell growth in vegetative meristems.
Expression and Function of Cell Wall-Bound Cationic Peroxidase in Asparagus Somatic Embryogenesis
Authors: Takeda Hiroyuki , Kotake Toshihisa , Nakagawa Naoki , Sakurai Naoki , Nevins Donald J.
Published: April 2003

Cultured asparagus (Asparagus officinalis L. cv Y6) cells induced to regenerate into whole plants through somatic embryogenesis secreted a 38-kD protein into cell walls. The full-length cDNA sequence of this protein (Asparagus officinalisperoxidase 1 [AoPOX1]) determined by reverse transcriptase-polymerase chain reaction showed similarity with plant peroxidases.AoPOX1 transcripts were particularly abundant during early somatic embryogenesis. To evaluate the in vivo function of AoPOX1 protein, purified recombinant AoPOX1 protein was reacted with a series of phenolic substrates. The AoPOX1 protein was effective in the metabolism of feruloyl (o-methoxyphenol)-substituted substrates, including coniferyl alcohol. The reaction product of coniferyl alcohol was fractionated and subjected to gas chromatography-mass spectrometry analysis and 1H-nuclear magnetic resonance analysis, indicating that the oxidation product of coniferyl alcohol in the presence of AoPOX1 was dehydrodiconiferyl alcohol. The concentration of dehydrodiconiferyl alcohol in the cultured medium of the somatic embryos was in the range of 10−8  m. Functions of the AoPOX1 protein in the cell differentiation are discussed.
Seasonal and Perennial Changes in the Distribution of Water in the Sapwood of Conifers in a Sub-Frigid Zone
Authors: Utsumi Yasuhiro , Sano Yuzou , Funada Ryo , Ohtani Jun , Fujikawa Seizo
Published: April 2003

An analysis was made of progressive changes in patterns of cavitation in the sapwood of three species of conifer (Larix kaempferi, Abies sachalinensis, and Picea jezoensis) that were growing in a sub-frigid zone. In all three conifers, all tracheids of the newly forming outermost annual ring were filled with water or cytoplasm during the period from May to August. However, many tracheids in the transition zone from earlywood to latewood lost water in September, presumably through drought-induced cavitation. Cavitated tracheids tended to be continuously distributed in a tangential direction. Subsequently, some earlywood tracheids of the outermost annual ring lost water during the period from January to March. This was associated with freeze-thaw cycles. In the second and third annual rings from the cambium of all three conifers, the lumina of most tracheids in the transition zone from earlywood to latewood contained no water. In contrast, some latewood tracheids near the annual ring boundary and many earlywood tracheids retained water in their lumina. The third annual ring had more cavitated tracheids than the second annual ring. Our observations indicated that cavitation progressed gradually in the tracheids of the conifers and that they were never refilled once cavitation had occurred. The region involved in water transport in conifers did not include the entire sapwood and differed among annual rings.