bluuebunny/crossref_metadata_2025 huggingface dataset:
The Phosphatidylinositol Transfer Protein Domain of Drosophila Retinal Degeneration B Protein Is Essential for Photoreceptor Cell Survival and Recovery from Light Stimulation
Authors: Milligan Scott C. , Alb James G. , Elagina Raya B. , Bankaitis Vytas A. , Hyde David R.
Published: July 2002

The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPα, a classical PI transfer protein, for RdgB's PITP domain (PITPα or PITPα-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPα-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPα-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.
Morphogenesis beyond Cytokinetic Arrest in Saccharomyces cerevisiae
Authors: Jiménez Javier , Cid Víctor J. , Cenamor Rosa , Yuste María , Molero Gloria , Nombela César , Sánchez Miguel
Published: July 2002

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.
Differential Regulation of β1 Integrins by Chemoattractants Regulates Neutrophil Migration through Fibrin
Authors: Loike John D. , Cao Long , Budhu Sadna , Marcantonio Eugene E. , Khoury Joseph El , Hoffman Stanley , Yednock Ted A. , Silverstein Samuel C.
Published: July 2002

Chemoattractants differ in their capacity to stimulate neutrophils to adhere to and to migrate through matrices containing fibrin. Formyl methionyl leucyl phenylalanine (fMLP) stimulates neutrophils to adhere closely to, but not to migrate into, fibrin gels. Leukotriene B4 (LTB4) stimulates neutrophils to adhere loosely to and to migrate through fibrin gels. We report that α5β1 integrins regulate the different migratory behaviors on fibrin gels of neutrophils in response to these chemoattractants. fMLP, but not LTB4, activated neutrophil β1 integrins, as measured by binding of mAb 15/7 to an activation epitope on the β1 integrins. Antibodies or peptides that block α5β1 integrins prevented fMLP-stimulated neutrophils from forming zones of close apposition on fibrin and reversed fMLP's inhibitory effect on neutrophil chemotaxis through fibrin. In contrast, neither peptides nor antibodies that block β1 integrins affected the capacity of LTB4-stimulated neutrophils to form zones of loose apposition or to migrate through fibrin gels. These results suggest that chemoattractants generate at least two different messages that direct neutrophils, and perhaps other leukocytes, to accumulate at specific anatomic sites: a general message that induces neutrophils to crawl and a specific message that prepares neutrophils to stop when they contact appropriate matrix proteins for activated β1 integrins.
Trafficking of an Acylated Cytosolic Protein: Newly Synthesized p56lck Travels to the Plasma Membrane via the Exocytic Pathway
Authors: Bijlmakers Marie-José J.E. , Marsh Mark
Published: July 2002

The Src-related tyrosine kinase p56lck (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse–chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30–45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.
Chimeric Forms of Furin and Tgn38 Are Transported from the Plasma Membrane to the Trans-Golgi Network via Distinct Endosomal Pathways
Authors: Mallet William G. , Maxfield Frederick R.
Published: July 2002

Furin and TGN38 are membrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N., W.G. Mallet, T.T. Soe, T.E. McGraw, and F.R. Maxfield. 1998. J. Cell Biol. 142:923–936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independent pathways for endosomal transport of proteins to the TGN from the plasma membrane.
Long-Term Culture of Purified Postnatal Oligodendrocyte Precursor Cells
Authors: Tang Dean G. , Tokumoto Yasuhito M. , Raff Martin C.
Published: July 2002

Oligodendrocytes myelinate axons in the vertebrate central nervous system (CNS). They develop from precursor cells (OPCs), some of which persist in the adult CNS. Adult OPCs differ in many of their properties from OPCs in the developing CNS. In this study we have purified OPCs from postnatal rat optic nerve and cultured them in serum-free medium containing platelet-derived growth factor (PDGF), the main mitogen for OPCs, but in the absence of thyroid hormone in order to inhibit their differentiation into oligodendrocytes. We find that many of the cells continue to proliferate for more than a year and progressively acquire a number of the characteristics of OPCs isolated from adult optic nerve. These findings suggest that OPCs have an intrinsic maturation program that progressively changes the cell's phenotype over many months. When we culture the postnatal OPCs in the same conditions but with the addition of basic fibroblast growth factor (bFGF), the cells acquire these mature characteristics much more slowly, suggesting that the combination of bFGF and PDGF, previously shown to inhibit OPC differentiation, also inhibits OPC maturation. The challenge now is to determine the molecular basis of such a protracted maturation program and how the program is restrained by bFGF.
Association of Yeast RNA Polymerase I with a Nucleolar Substructure Active in Rrna Synthesis and Processing
Authors: Fath Stephan , Milkereit Philipp , Podtelejnikov Alexandre V. , Bischler Nicolas , Schultz Patrick , Bier Mirko , Mann Matthias , Tschochner Herbert
Published: July 2002

A novel ribonucleoprotein complex enriched in nucleolar proteins was purified from yeast extracts and constituents were identified by mass spectrometry. When isolated from rapidly growing cells, the assembly contained ribonucleic acid (RNA) polymerase (pol) I, and some of its transcription factors like TATA-binding protein (TBP), Rrn3p, Rrn5p, Rrn7p, and Reb1p along with rRNA processing factors, like Nop1p, Cbf5p, Nhp2p, and Rrp5p. The small nucleolar RNAs (snoRNAs) U3, U14, and MRP were also found to be associated with the complex, which supports accurate transcription, termination, and pseudouridylation of rRNA. Formation of the complex did not depend on pol I, and the complex could efficiently recruit exogenous pol I into active ribosomal DNA (rDNA) transcription units. Visualization of the complex by electron microscopy and immunogold labeling revealed a characteristic cluster-forming network of nonuniform size containing nucleolar proteins like Nop1p and Fpr3p and attached pol I. Our results support the idea that a functional nucleolar subdomain formed independently of the state of rDNA transcription may serve as a scaffold for coordinated rRNA synthesis and processing.
Chromosomal Addresses of the Cohesin Component Mcd1p
Authors: Laloraya Shikha , Guacci Vincent , Koshland Douglas
Published: July 2002

We identified the chromosomal addresses of a cohesin subunit, Mcd1p, in vivo by chromatin immunoprecipitation coupled with high resolution PCR-based chromosomal walking. The mapping of new Mcd1p-binding sites (cohesin-associated regions [CARs]) in single-copy sequences of several chromosomes establish their spacing (∼9 kb), their sequestration to intergenic regions, and their association with AT-rich sequences as general genomic properties of CARs. We show that cohesins are not excluded from telomere proximal regions, and the enrichment of cohesins at the centromere at mitosis reflects de novo loading. The average size of a CAR is 0.8–1.0 kb. They lie at the boundaries of transcriptionally silenced regions, suggesting they play a direct role in defining the silent chromatin domain. Finally, we identify CARs in tandem (rDNA) and interspersed repetitive DNA (Ty2 and subtelomeric repeats). Each 9-kb rDNA repeat has a single CAR proximal to the 5S gene. Thus, the periodicity of CARs in single-copy regions and the rDNA repeats is conserved. The presence and spacing of CARs in repetitive DNA has important implications for genomic stability and chromosome packaging/condensation.
Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival
Authors: Newman Bill , Gigout Laure I. , Sudre Laure , Grant Michael E. , Wallis Gillian A.
Published: July 2002

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.
Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis
Authors: Hoffmann Isabelle , Eugène Emmanuel , Nassif Xavier , Couraud Pierre-Olivier , Bourdoulous Sandrine
Published: July 2002

ErbB2 is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by heterodimerization with the other members of the family. We show here that type IV pilus–mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phosphorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Specific inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results provide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efficient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers.
Mapping the Active Site of CD59
Authors: Yu Jinghua , Abagyan Ruben , Dong Shanghong , Gilbert Alexander , Nussenzweig Victor , Tomlinson Stephen
Published: July 2002

CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a “hydrophobic strip” suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.
Atypical Disease after Bordetella pertussis Respiratory Infection of Mice with Targeted Disruptions of Interferon-γ Receptor or Immunoglobulin μ Chain Genes
Authors: Mahon Bernard P. , Sheahan Brian J. , Griffin Fiona , Murphy Geraldine , Mills Kingston H.G.
Published: July 2002

Using a murine respiratory challenge model we have previously demonstrated a role for Th1 cells in natural immunity against Bordetella pertussis, but could not rule out a role for antibody. Here we have demonstrated that B. pertussis respiratory infection of mice with targeted disruptions of the genes for the IFN-γ receptor resulted in an atypical disseminated disease which was lethal in a proportion of animals, and was characterized by pyogranulomatous inflammation and postnecrotic scarring in the livers, mesenteric lymph nodes and kidneys. Viable virulent bacteria were detected in the blood and livers of diseased animals. An examination of the course of infection in the lung of IFN-γ receptor–deficient, IL-4–deficient and wild-type mice demonstrated that lack of functional IFN-γ or IL-4, cytokines that are considered to play major roles in regulating the development of Th1 and Th2 cells, respectively, did not affect the kinetics of bacterial elimination from the lung. In contrast, B cell–deficient mice developed a persistent infection and failed to clear the bacteria after aerosol inoculation. These findings demonstrate an absolute requirement for B cells or their products in the resolution of a primary infection with B. pertussis, but also define a critical role for IFN-γ in containing bacteria to the mucosal site of infection.
Preselection Thymocytes Are More Sensitive to T Cell Receptor Stimulation Than Mature T Cells
Authors: Davey Gayle M. , Schober Sonya L. , Endrizzi Bart T. , Dutcher Angela K. , Jameson Stephen C. , Hogquist Kristin A.
Published: July 2002

During T cell development, thymocytes which are tolerant to self-peptides but reactive to foreign peptides are selected. The current model for thymocyte selection proposes that self-peptide–major histocompatibility complex (MHC) complexes that bind the T cell receptor with low affinity will promote positive selection while those with high affinity will result in negative selection. Upon thymocyte maturation, such low affinity self-peptide–MHC ligands no longer provoke a response, but foreign peptides can incidentally be high affinity ligands and can therefore stimulate T cells. For this model to work, thymocytes must be more sensitive to ligand than mature T cells. Contrary to this expectation, several groups have shown that thymocytes are less responsive than mature T cells to anti-T cell receptor for antigen (TCR)/CD3 mAb stimulation. Additionally, the lower TCR levels on thymocytes, compared with T cells, would potentially correlate with decreased thymocyte sensitivity. Here we compared preselection thymocytes and mature T cells for early activation events in response to peptide–MHC ligands. Remarkably, the preselection thymocytes were more responsive than mature T cells when stimulated with low affinity peptide variants, while both populations responded equally well to the antigenic peptide. This directly demonstrates the increased sensitivity of thymocytes compared with T cells for TCR engagement by peptide–MHC complexes.
Chemokine Sequestration by Viral Chemoreceptors as a Novel Viral Escape Strategy: Withdrawal of Chemokines from the Environment of Cytomegalovirus-infected Cells
Authors: Bodaghi Bahram , Jones Thomas R. , Zipeto Donato , Vita Claudio , Sun Lei , Laurent Lysiane , Arenzana-Seisdedos Fernando , Virelizier Jean-Louis , Michelson Susan
Published: July 2002

Human cytomegalovirus (HCMV), a betaherpesvirus, has developed several ways to evade the immune system, notably downregulation of cell surface expression of major histocompatibility complex class I heavy chains. Here we report that HCMV has devised another means to compromise immune surveillance mechanisms. Extracellular accumulation of both constitutively produced monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor–superinduced RANTES (regulated on activation, normal T cell expressed and secreted) was downregulated in HCMV-infected fibroblasts in the absence of transcriptional repression or the expression of polyadenylated RNA for the cellular chemokine receptors CCR-1, CCR-3, and CCR-5. Competitive binding experiments demonstrated that HCMV-infected cells bind RANTES, MCP-1, macrophage inflammatory protein (MIP)-1β, and MCP-3, but not MCP-2, to the same receptor as does MIP-1α, which is not expressed in uninfected cells. HCMV encodes three proteins with homology to CC chemokine receptors: US27, US28, and UL33. Cells infected with HCMV mutants deleted of US28, or both US27 and US28 genes, failed to downregulate extracellular accumulation of either RANTES or MCP-1. In contrast, cells infected with a mutant deleted of US27 continues to bind and downregulate those chemokines. Depletion of chemokines from the culture medium was at least partially due to continuous internalization of extracellular chemokine, since exogenously added, biotinylated RANTES accumulated in HCMV-infected cells. Thus, HCMV can modify the chemokine environment of infected cells through intense sequestering of CC chemokines, mediated principally by expression of the US28-encoded chemokine receptor.
Secondary Rearrangements and Hypermutation Generate Sufficient B Cell Diversity to Mount Protective Antiviral Immunoglobulin Responses
Authors: López-Macías Constantino , Kalinke Ulrich , Cascalho Marilia , Wabl Matthias , Hengartner Hans , Zinkernagel Rolf M. , Lamarre Alain
Published: July 2002

Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of “quasimonoclonal” (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, ∼20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.
The Cysteine-Rich Regions of the Regulatory Domains of Raf and Protein Kinase C as Retinoid Receptors
Authors: Hoyos Beatrice , Imam Asiya , Chua Ramon , Swenson Christina , Tong Guo-Xia , Levi Ester , Noy Noa , Hämmerling Ulrich
Published: July 2002

Vitamin A and its biologically active derivatives, the retinoids, are recognized as key regulators of vertebrate development, cell growth, and differentiation. Although nuclear receptors have held the attention since their discovery a decade ago, we report here on serine/threonine kinases as a new class of retinoid receptors. The conserved cysteine-rich domain of the NH2-terminal regulatory domains of cRaf-1, as well as several select domains of the mammalian protein kinase C (PKC) isoforms α, δ, ζ, and μ, the Drosophila and yeast PKCs, were found to bind retinol with nanomolar affinity. The biological significance was revealed in the alternate redox activation pathway of these kinases. Retinol served as a cofactor to augment the activation of both cRaf and PKCα by reactive oxygen, whereas the classical receptor-mediated pathway was unaffected by the presence or absence of retinol. We propose that bound retinol, owing to its electron transfer capacity, functions as a tag to enable the efficient and directed redox activation of the cRaf and PKC families of kinases.
FAS Ligand Triggers Pulmonary Silicosis
Authors: Borges Valéria M. , Falcão Haroldo , Leite-Júnior José H. , Alvim Luciana , Teixeira Gerlinde P. , Russo Momtchilo , Nóbrega Alberto F. , Lopes Marcela F. , Rocco Patricia M. , Davidson Wendy F. , Linden Rafael , Yagita Hideo , Zin Walter A. , DosReis George A.
Published: July 2002

We investigated the role of Fas ligand in murine silicosis. Wild-type mice instilled with silica developed severe pulmonary inflammation, with local production of tumor necrosis factor (TNF)-α, and interstitial neutrophil and macrophage infiltration in the lungs. Strikingly, Fas ligand–deficient generalized lymphoproliferative disease mutant (gld) mice did not develop silicosis. The gld mice had markedly reduced neutrophil extravasation into bronchoalveolar space, and did not show increased TNF-α production, nor pulmonary inflammation. Bone marrow chimeras and local adoptive transfer demonstrated that wild-type, but not Fas ligand–deficient lung macrophages recruit neutrophils and initiate silicosis. Silica induced Fas ligand expression in lung macrophages in vitro and in vivo, and promoted Fas ligand–dependent macrophage apoptosis. Administration of neutralizing anti-Fas ligand antibody in vivo blocked induction of silicosis. Thus, Fas ligand plays a central role in induction of pulmonary silicosis.
Permeability Properties of Enac Selectivity Filter Mutants
Authors: Kellenberger Stephan , Auberson Muriel , Gautschi Ivan , Schneeberger Estelle , Schild Laurent
Published: July 2002

The epithelial Na+ channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na+ absorption. The amiloride-sensitive ENaC is highly selective for Na+ and Li+ ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the αS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K+ ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted αS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position α589, indicating that αS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na+ and Li+. These findings demonstrate the critical role of the pore size at the αS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the αS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the αS589 side chain is oriented toward the subunit–subunit interface and that substitution of αS589 by larger residues increases the pore diameter by adding extra volume at the subunit–subunit interface.
Virtual realities in Brobdingnag and Lilliput: gulfs and bridges between large and small publishers
Authors: Edmondson John
Published: July 2002

This article argues that the trend towards larger and larger publishing conglomerates is happening in parallel with apparently paradoxical changes in attitudes and approaches towards work and the organization of work. These changes, include for example remote working and the increasingly powerful potential of ‘outsourcing’. Looking at a publishing environment now populated mainly by very large and very small companies, the article suggests a symbiotic relationship between these giants and pygmies. It then looks at major characteristics of small publishers and how such companies can exploit the advantages of smallness to survive and succeed. The specific case of IP Publishing Ltd is used to illustrate some options for small publishers, including their opportunity to operate as ‘virtual’ companies. Finally, the article suggests that small companies can be useful to large corporations as indicators of change.
Book reviews
Authors: Williamson Alex
Published: July 2002

The Effective Editor's Handbook, By Barbara Horn, Leatherhead: Pira International 1997, 176 pp £20.00, ISBN: 1 85802 186 3
Electronic publishing: non‐commercial alternatives
Authors: Rowland Fytton
Published: July 2002

A considerable variety of new non‐commercial models for the electronic publication of quality‐controlled scholarly articles have been suggested and in some cases implemented over the last ten years. These models are connected, but vary in various parameters. This paper briefly describes them, and speculates that the most promising model is one where the modest initial costs are paid by the author's institution, quality control is imposed by editorial boards responsible to learned societies, and the journal is published in print at a subscription but published electronically free of charge.
Journals Pricing
Authors: Adler Kraig , Olsen Wally
Published: July 2002

A significant new study has been conducted at Cornell University. The Mann Library surveyed several hundred titles in Agriculture and Biology, in order to analyse their prices (per page and per thousand characters) over time, comparing university presses, commercial publishers, society publishers and government agencies. They aimed to develop a methodology, using Optical Character Recognition (OCR) technology, which could be used elsewhere, and to use their findings to examine the patterns of article ownership and pricing of published scientific research.
Distortion theorems for higher order Schwarzian derivatives of univalent functions
Authors: Schippers Eric
Published: July 2002

Let

S

~

\tilde {\mathcal {S}}

denote the class of functions which are univalent and holomorphic on the unit disc. We derive a simple differential equation for the Loewner flow of the Schwarzian derivative of a given

f
∈

S

~

f \in \tilde {\mathcal {S}}

. This is used to prove bounds on higher order Schwarzian derivatives which are sharp for the Koebe function. As well we prove some two-point distortion theorems for the higher order Schwarzians in terms of the hyperbolic metric.
Rees algebras of ideals with low codimension
Authors: Morey Susan , Ulrich Bernd
Published: July 2002

For certain grade two perfect ideals, there is an expected description of the equations of the Rees algebra. In this paper, the Cohen–Macaulayness of the Rees algebra, numerical invariants of the ideal, and a condition on the minors of a presentation matrix of the ideal are shown to be related to the equations having this form.
A classification of trigonometrical thin sets and their interrelations
Authors: Elias Peter
Published: July 2002

We introduce a uniform way of classifying thin sets of harmonic analysis related to absolute convergence of trigonometric series. This classification covers classical classes

(

D

,

P

D

,

A

,

N

0

,

N

)

(\mathcal {D},\mathcal {P}\mathcal {D},\mathcal {A}, \mathcal {N}_0,\mathcal {N})

and yields two new ones (

B

0

\mathcal {B}_0

and

B

)

\mathcal {B})

. We study interrelation between these classes concerning combinatorial structure of thin sets.
Numerical solution of parabolic integro-differential equations by the discontinuous Galerkin method
Authors: Larsson Stig , Thomée Vidar , Wahlbin Lars
Published: July 2002

The numerical solution of a parabolic equation with memory is considered. The equation is first discretized in time by means of the discontinuous Galerkin method with piecewise constant or piecewise linear approximating functions. The analysis presented allows variable time steps which, as will be shown, can then efficiently be selected to match singularities in the solution induced by singularities in the kernel of the memory term or by nonsmooth initial data. The combination with finite element discretization in space is also studied.
Dynamics of Cytoskeletal Proteins during Fcγ Receptor-mediated Phagocytosis in Macrophages
Authors: Diakonova Maria , Bokoch Gary , Swanson Joel A.
Published: July 2002

Particle ingestion by phagocytosis results from sequential rearrangements of the actin cytoskeleton and overlying membrane. To assemble a chronology of molecular events during phagosome formation and to examine the contributions of phosphoinositide 3-kinase (PI 3-kinase) to these dynamics, a method was developed for synchronizing Fcγ receptor-mediated phagocytosis by murine macrophages. Erythrocytes opsonized with complement component C3bi were bound to macrophages at 37°C, a condition that does not favor particle phagocytosis. Addition of soluble anti-erythrocyte IgG resulted in rapid opsonization of the bound erythrocytes, followed by their immediate internalization via phagocytosis. Cellular content of F-actin, as measured by binding of rhodamine-phalloidin, increased transiently during phagocytosis, and this increase was not diminished by inhibitors of PI 3-kinase. Immunofluorescence localization of myosins in macrophages fixed at various times during phagocytosis indicated that myosins II and IXb were concentrated in early phagosomes, myosin IC increased later, and myosin V appeared after phagosome closure. Other cytoskeletal proteins showed similar variations in the timing of their appearance in phagosomes. The PI 3-kinase inhibitor wortmannin did not change the dynamics of PI 3-kinase or ezrin localization but prevented the loss of PAK1 from phagosomes. These results suggest that PI 3-kinase deactivates PAK1, and that this may be needed for phagosome closure.
Tournaments as a Worker Discipline Device
Authors: Lin Chung‐Cheng , Yang C. C.
Published: July 2002

This article explores the macroeconomic implications of tournaments as a worker discipline device in the vein of shirking models. It is shown that, if the full exploitation of tournaments is feasible, there will be no involuntary unemployment. Thus, as far as the elimination of involuntary unemployment is concerned, tournaments can serve as a perfect substitute for the so‐called “employment fee” or “bonding” scheme. An immediate corollary of this finding is that the involuntary nature of unemployment must ultimately rely upon impediments that hinder the full exploitation of tournaments.
TIF‐IA, the factor mediating growth‐dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p
Authors: Bodem Jochen , Dobreva Gergana , Hoffmann‐Rohrer Urs , Iben Sebastian , Zentgraf Hanswalter , Delius Hajo , Vingron Martin , Grummt Ingrid
Published: July 2002

Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth‐dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF‐IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide‐treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF‐IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF‐IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth‐arrested cells in vitro.
The GAGA factor of Drosophila interacts with SAP18, a Sin3‐associated polypeptide
Authors: Espinás Maria Lluïsa , Canudas Sílvia , Fanti Laura , Pimpinelli Sergio , Casanova Jordi , Azorín Fernando
Published: July 2002

SAP18, a polypeptide associated with the Sin3–HDAC co‐repressor complex, was identified in a yeast two‐hybrid screen as capable of interacting with the Drosophila GAGA factor. The interaction was confirmed in vitro by glutathione S‐transferase pull‐down assays using recombinant proteins and crude SL2 nuclear extracts. The first 245 residues of GAGA, including the POZ domain, are necessary and sufficient to bind dSAP18. In polytene chromosomes, dSAP18 and GAGA co‐localize at a few discrete sites and, in particular, at the bithorax complex where GAGA binds some silenced polycomb response elements. When the dSAP18 dose is reduced, flies heterozygous for the GAGA mutation Trl67 show the homeotic transformation of segment A6 into A5, indicating that GAGA–dSAP18 interaction contributes to the functional regulation of the iab‐6 element of the bithorax complex. These results suggest that, through recruitment of the Sin3–HDAC complex, GAGA might contribute to the regulation of homeotic gene expression.
In-vitro susceptibility of 1982 respiratory tract pathogens and 1921 urinary tract pathogens against 19 antimicrobial agents: a Canadian multicentre study
Authors:   The Canadian Antimicrobial Study Group, Blondeau Joseph M. , Yaschuk Y. , Suter M. , Vaughan David
Published: July 2002

A total of 3903 pathogens from 48 Canadian medical centres were tested against 19 antimicrobial agents. Five agents showed activity against ≥90%; cefoperazone, 91%; ticarcillin/clavulanate, 92%; ceftazidime and imipenem, 93% each). Nine agents had ≥90% activity against Enterobacteriaceae from respiratory tract infection (cefotaxime and ticarcillin/clavulanate, 90% each; aztreonam, ceftizoxime and ceftriaxone, 91% each; ceftazidime, 93%; ciprofloxacin, 97%; imipenem and netilmicin, 98% each). Similarly, five agents had activity against ≥90% of all 1921 urinary tract pathogens tested (ciprofloxacin and ticarcillin/clavulanate, 90% each; cefoperazone and netilmicin, 91% each; imipenem, 99%). Nine agents had ≥95% activity against Enterobacteriaceae from urinary tract infection (ciprofloxacin, 95%; cefotetan, 97%; aztreonam, cefotaxime, ceftazidime, ceftizoxime, ceftriaxone and netilmicin, 98% each; imipenem, 99%). Seventeen agents had activity against ≥ 95% of Staphylococcus aureus strains. Susceptibility of Pseudomonasaeruginosa isolates ranged from 2% to 91%.
Comparative susceptibility to penicillin and quinolones of 1385 Streptococcus pneumoniae isolates
Authors:   and the Austrian Bacterial Surveillance Network, Buxbaum A. , Straschil U. , Moser C. , Graninger W. , Georgopoulos A.
Published: July 2002

Antibiotic resistance among Streptococcus pneumoniae isolates has spread rapidly throughout the world since the first description of a strain with diminished susceptibility to penicillin in Australia in 1967. A total of 1385 strains of S. pneumoniae, collected in several centres throughout Austria, were assessed for their sensitivity to moxifloxacin, trovafloxacin, ciprofloxacin, ofloxacin, levofloxacin and lomefloxacin. The MICs were determined using the agar dilution method, according to NCCLS guidelines. Both moxifloxacin and trovafloxacin showed good anti-pneumococcal activity in terms of MIC 50 (both 0.125 mg/L) and MIC 90 (both 0.25 mg/L). Less active, but with similar activity to each other, were ciprofloxacin and levofloxacin, each with an MIC 50 of 1 mg/L and an MIC 90 of 2 mg/L. Ofloxacin showed only moderate activity (MIC 50, 1 mg/L; MIC 90, 2 mg/L) and lomefloxacin was the least active compound (MIC 50, 4 mg/L; MIC 90, 8 mg/L). Both moxifloxacin and trovafloxacin at a concentration of ≤ 0.5 mg/L inhibited all of the S. pneumoniae strains tested.
Accessing Population Health Information through Interactive Systems: Lessons Learned and Future Directions
Authors: Friedman Daniel J. , Anderka Marlene , Krieger James W. , Land Garland , Solet David
Published: July 2002

In the mid-1990s, several state and county public health departments implemented interactive software systems that provided easy access to public health-related data for local boards of health, other public health agencies, health care providers, community groups, and other interested members of the public. Based on their experiences with two well-established state interactive systems and one well-established county system, the authors summarize lessons that could prove useful to state and local public health agencies interested in developing new interactive systems or adapting existing ones. The article addresses issues such as: basing interactive systems on a broad definition of health, designing systems to incorporate user preferences, moving from data warehouses to information warehouses, and fostering prevention communities. Finally, the article provides recommendations to assist federal, state, and local public health agencies in developing the next generation of interactive data access systems.
Neuronal nitric oxide synthase (NOS) regulates the expression of inducible NOS in rat small intestine via modulation of nuclear factor kappa B
Authors: QU XIAO‐WU , WANG HAO , DE PLAEN ISABELLE G. , ROZENFELD RANNA A. , HSUEH WEI
Published: July 2002

We previously reported that neuronal nitric oxide synthase (nNOS) is the predominant NOS in the intestine. Inducible NOS (iNOS), an enzyme involved in the inflammatory response, is regulated by cytokines via the transcriptional factor NF‐κB. We examined a new mechanism of intestinal iNOS regulation with respect to the role of nNOS and its effect on NF‐κB. Young Sprague‐Dawley rats were treated for 4 days with 1) saline, 2) 7‐nitroindazole (7‐NI, specific nNOS inhibitor), 3) 7‐NI + pyrrolidine dithiocarbam‐ate (PDTC, NF‐κB inhibitor), or 4) PDTC. Intestinal iNOS mRNA, NF‐κB activity, and the tissue content of the regulatory IκBα were examined. We found that 7‐NI‐treated animals had higher intestinal NF‐κB (p50‐p65) activity, lower IκBα content, and increased intestinal iNOS mRNA, iNOS protein, and iNOS activity compared with controls. All of these changes were abolished when PDTC was given together with 7‐NI. PDTC alone had no effect. 7‐NI induces a delayed increase in intestinal myeloperoxidase activity (after elevation in NF‐κB and iNOS), which could be abrogated by PDTC. We conclude that in normal rat small intestine, nNOS suppresses the gene expression of iNOS through NF‐κB down‐regulation and that nNOS suppression leads to IκBα degradation, NF‐κB activation, and iNOS expression.—Qu, X.‐w., Wang, H., De Plaen, I. G., Rozenfeld, R. A., Hsueh, W. Neuronal nitric oxide synthase (NOS) regulates the expression of inducible NOS in rat small intestine via modulation of nuclear factor kappa B. FASEB J. 15, 439‐446 (2001)
The control of specificity in guard cell signal transduction
Authors: Hetherington Alistair M. , Gray Julie E. , Leckie Calum P. , McAinsh Martin R. , Ng Carl , Pical Christophe , Priestley Alistair J. , Staxén Irina , Webb Alex A. R.
Published: July 2002

Stomatal guard cells have proven to be an attractive system for dissecting the mechanisms of stimulus–response coupling in plants. In this review we focus on the intracellular signal transduction pathways by which extracellular signals bring about closure and opening of the stomatal pore. It is proposed that guard cell signal transduction pathways may be organized into functional arrays or signalling cassettes that contain elements common to a number of converging signalling pathways. The purpose of these signalling cassettes may be to funnel extracellular signals down onto the ion transporters that control the fluxes of ions that underlie stomatal movements. Evidence is emerging that specificity in guard cell signalling may be, in part, encoded in complex spatio–temporal patterns of increases in the concentration of cytosolic–free calcium ([Ca2+]cyt). It is suggested that oscillations in [Ca2+]cytmay generate calcium signatures that encode information concerning the stimulus type and strength. New evidence is presented that suggests that these calcium signatures may integrate information when many stimuli are present.
The frog neuromuscular junction revisited after quick–freezing–freeze–drying: ultrastructure, immunogold labelling and high resolution calcium mapping
Authors: Pezzati R. , Grohovaz F.
Published: July 2002

Until now, most ultrastructural studies on the neuromuscular junction have been carried out on samples first exposed to chemical treatments—with fixatives and/or dehydration agents—that are known to induce, or to be inadequate to prevent, artefactual changes of the native state. We report here on the potential of a physical approach to the preparation of samples that combines quick–freezing and freeze–drying (with or without exposure to OsO
4
vapours) followed by direct embedding of the samples in various resins. Thin sections from physically processed frog neuromuscular junctions, when compared to their chemically fixed counterparts, exhibit an overall excellent preservation, with the organelles retaining their native density and shape. These preparations were also investigated by electron spectroscopic imaging and electron energy loss spectroscopy, obtaining high resolution maps of native total calcium distribution within the nerve terminal. Finally, thin sections from analogously processed, however unfixed, preparations embedded in Lowicryl, were immunogold labelled before exposure to OsO
4
. Nerve–muscle preparations treated this way exhibited adequate preservation of ultrastructure and revealed the distribution of synaptophysin with high sensitivity and resolution. In conclusion, we provide an overview of the potential of the quick–freezing–freeze–drying approach in the study of the neuromuscular junction function.
Temporal and mosaic distribution of large ganglion cells in the retina of a daggertooth aulopiform deep–sea fish (
Anotopterus pharao
)
Authors: Uemura M. , Somiya H. , Moku M. , Kawaguchi K.
Published: July 2002

The daggertooth Anotopteruspharao (Aulopiformes: Anotopteridae) is a large, piscivorous predator that lives within the epipelagic zone at night. In this species, the distribution of retinal ganglion cells has been examined. An isodensity contour map of ganglion cells shows that the cells concentrate in a slightly ventral region of the temporal retina. The region of high ganglion cell density contains 4.07 × 10
3
cells mm
−2
, and the resulting visual acuity is 3.5 cycles deg
−1
. Outside the area centralis, conspicuously large ganglion cells (LGCs) are observed in the temporal margin of the retina. The LGCs are regularly arrayed, and displaced into the inner plexiform layer. Thick dendrites extend into the outer part (sublamina a) of the inner plexiform layer. In the retinal whole mount, the total number of LGCs is 1590 (90.7cm specimen), and the mean size of the LGCs is about four times larger than that of the ordinary ganglion cells. The morphological appearance of the LGCs was similar to the off–type alpha cells of the cat retina. The function of these distinctive LGCs is discussed in relation to specific head–up feeding behaviour.