bluuebunny/crossref_metadata_2025 huggingface dataset:
Nup84, A Novel Nucleoporin That Is Associated With CAN/Nup214 on the Cytoplasmic Face of the Nuclear Pore Complex
Authors: Bastos Ricardo , Pouplana Lluis Ribas de , Enarson Mark , Bodoor Khaldon , Burke Brian
Published: July 2002

The short filaments extending from the cytoplasmic face of nuclear pore complexes are thought to contain docking sites for nuclear import substrates. One component of these filaments is the large O-linked glycoprotein CAN/Nup214. Immunoprecipitation studies carried out under nondenaturing conditions, and using a variety of antibodies, reveal a novel nonglycosylated nucleoporin, Nup84, that is tightly associated with CAN/Nup214. Consistent with such an association, Nup84 is found to be exposed on the cytoplasmic face of the nuclear pore complex. cDNA sequence analyses indicate that Nup84 contains neither the GLFG nor the XFXFG repeats that are a characteristic of a number of other nuclear pore complex proteins. Secondary structure predictions, however, suggest that Nup84 contains a coiled–coil COOH-terminal domain, a conclusion supported by the observation of significant sequence similarity between this region of the molecule and various members of the tropomyosin family. Mutagenesis and expression studies indicate that the putative coiled–coil domain is required for association with the cytoplasmic face of the nuclear pore complex, whereas it is the NH2-terminal region of Nup84 that contains the site of interaction with CAN/Nup214. These findings suggest a model in which Nup84 may function in the attachment of CAN/Nup214 to the central framework of the nuclear pore complex. In this way, Nup84 could play a central role in the organization of the interface between the pore complex and the cytoplasm.
Delayed Retraction of Filopodia in Gelsolin Null Mice
Authors: Lu Mei , Witke Walter , Kwiatkowski David J. , Kosik Kenneth S.
Published: July 2002

Growth cones extend dynamic protrusions called filopodia and lamellipodia as exploratory probes that signal the direction of neurite growth. Gelsolin, as an actin filament-severing protein, may serve an important role in the rapid shape changes associated with growth cone structures. In wild-type (wt) hippocampal neurons, antibodies against gelsolin labeled the neurite shaft and growth cone. The behavior of filopodia in cultured hippocampal neurons from embryonic day 17 wt and gelsolin null (Gsn−) mice (Witke, W., A.H. Sharpe, J.H. Hartwig, T. Azuma, T.P. Stossel, and D.J. Kwiatkowski. 1995. Cell. 81:41–51.) was recorded with time-lapse video microscopy. The number of filopodia along the neurites was significantly greater in Gsn− mice and gave the neurites a studded appearance. Dynamic studies suggested that most of these filopodia were formed from the region of the growth cone and remained as protrusions from the newly consolidated shaft after the growth cone advanced. Histories of individual filopodia in Gsn− mice revealed elongation rates that did not differ from controls but an impaired retraction phase that probably accounted for the increased number of filopodia long the neutrite shaft. Gelsolin appears to function in the initiation of filopodial retraction and in its smooth progression.
Homotypic Lysosome Fusion in Macrophages: Analysis Using an In Vitro Assay
Authors: Ward Diane M. , Leslie Jonathan D. , Kaplan Jerry
Published: July 2002

Lysosomes are dynamic structures capable of fusing with endosomes as well as other lysosomes. We examined the biochemical requirements for homotypic lysosome fusion in vitro using lysosomes obtained from rabbit alveolar macrophages or the cultured macrophage-like cell line, J774E. The in vitro assay measures the formation of a biotinylated HRP–avidin conjugate, in which biotinylated HRP and avidin were accumulated in lysosomes by receptor-mediated endocytosis. We determined that lysosome fusion in vitro was time- and temperature-dependent and required ATP and an N-ethylmaleimide (NEM)-sensitive factor from cytosol. The NEM-sensitive factor was NSF as purified recombinant NSF could completely replace cytosol in the fusion assay whereas a dominant-negative mutant NSF inhibited fusion. Fusion in vitro was extensive; up to 30% of purified macrophage lysosomes were capable of self-fusion. Addition of GTPγs to the in vitro assay inhibited fusion in a concentration-dependent manner. Purified GDP-dissociation inhibitor inhibited homotypic lysosome fusion suggesting the involvement of rabs. Fusion was also inhibited by the heterotrimeric G protein activator mastoparan, but not by its inactive analogue Mas-17. Pertussis toxin, a Gαi activator, inhibited in vitro lysosome fusion whereas cholera toxin, a Gαs activator did not inhibit the fusion reaction. Addition of agents that either promoted or disrupted microtubule function had little effect on either the extent or rate of lysosome fusion. The high value of homotypic fusion was supported by in vivo experiments examining lysosome fusion in heterokaryons formed between cells containing fluorescently labeled lysosomes. In both macrophages and J774E cells, almost complete mixing of the lysosome labels was observed within 1–3 h of UV sendai-mediated cell fusion. These studies provide a model system for identifying the components required for lysosome fusion.
XMAP310: A Xenopus Rescue-promoting Factor Localized to the Mitotic Spindle
Authors: Andersen Søren S.L. , Karsenti Eric
Published: July 2002

To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289–1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5–10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985–993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation.
A Novel Fission Yeast Gene, tht1+, Is Required for the Fusion of Nuclear Envelopes during Karyogamy
Authors: Tange Yoshie , Horio Tetsuya , Shimanuki Mizuki , Ding Da-Qiao , Hiraoka Yasushi , Niwa Osami
Published: July 2002

We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1+ gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1+ gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei.
LFA-1–mediated Adhesion Is Regulated by Cytoskeletal Restraint and by a Ca2+-dependent Protease, Calpain
Authors: Stewart Mairi P. , McDowall Alison , Hogg Nancy
Published: July 2002

The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues. Using lymphocyte function-associated antigen-1 (LFA-1) on T cells as a model, we have investigated adhesion to ligand intercellular adhesion molecule-1 induced by the Ca2+ mobilizers, ionomycin, 2,5-di-t-butylhydroquinone, and thapsigargin, and the well studied stimulators such as phorbol ester and cross-linking of the antigen-specific T cell receptor (TCR)– CD3 complex. We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca2+-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane. We propose that methods of leukocyte stimulation that cause Ca2+ fluxes induce LFA-1 adhesion by regulation of calpain activity. These findings suggest a mechanism whereby engagement of the TCR could promote adhesion strengthening at an early stage of interaction with an antigen-presenting cell.
A Class VI Unconventional Myosin Is Associated with a Homologue of a Microtubule-binding Protein, Cytoplasmic Linker Protein–170, in Neurons and at the Posterior Pole of Drosophila Embryos
Authors: Lantz Valerie A. , Miller Kathryn G.
Published: July 2002

Abstract. Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for cellular organization and function. We report here on molecules that may mediate coordination between the actin and microtubule cytoskeletons. We have identified a 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F unconventional myosin. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue identified of cytoplasmic linker protein (CLIP)–170, a protein that links endocytic vesicles to microtubules. We have named this protein D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, leads us to speculate that these two proteins may functionally link the actin and microtubule cytoskeletons.
Intracellular Localization of Phosphatidylinositide 3-kinase and Insulin Receptor Substrate-1 in Adipocytes: Potential Involvement of a Membrane Skeleton
Authors: Clark Sharon F. , Martin Sally , Carozzi Amanda J. , Hill Michelle M. , James David E.
Published: July 2002

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1–PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.
In Polarized MDCK Cells Basolateral Vesicles Arise from Clathrin-γ-adaptin–coated Domains on Endosomal Tubules
Authors: Futter C.E. , Gibson A. , Allchin E.H. , Maxwell S. , Ruddock L.J. , Odorizzi G. , Domingo D. , Trowbridge I.S. , Hopkins C.R.
Published: July 2002

Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices. These buds generate vesicles that carry TR to the basolateral border. The lattices contain γ-adaptin and are dispersed by treatment with brefeldin A (BFA). Since BFA treatment abrogates the vectorial trafficking of TR in polarized MDCK cells, we propose that the clathrin-coated domains of the endosome tubules contain the polarized sorting mechanism responsible for their preferential basolateral distribution.
Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment
Authors: Bryant Nia J. , Piper Robert C. , Weisman Lois S. , Stevens Tom H.
Published: July 2002

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.
The Intermediate Filament Protein Peripherin Is the Specific Interaction Partner of Mouse BPAG1-n (Dystonin) in Neurons
Authors: Leung Conrad L. , Sun Dongming , Liem Ronald K.H.
Published: July 2002

The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655–665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.
Extensive but Coordinated Reorganization of the Membrane Skeleton in Myofibers of Dystrophic (mdx) Mice
Authors: Williams McRae W. , Bloch Robert J.
Published: July 2002

We used immunofluorescence techniques and confocal imaging to study the organization of the membrane skeleton of skeletal muscle fibers of mdx mice, which lack dystrophin. β-Spectrin is normally found at the sarcolemma in costameres, a rectilinear array of longitudinal strands and elements overlying Z and M lines. However, in the skeletal muscle of mdx mice, β-spectrin tends to be absent from the sarcolemma over M lines and the longitudinal strands may be disrupted or missing. Other proteins of the membrane and associated cytoskeleton, including syntrophin, β-dystroglycan, vinculin, and Na,K-ATPase are also concentrated in costameres, in control myofibers, and mdx muscle. They also distribute into the same altered sarcolemmal arrays that contain β-spectrin. Utrophin, which is expressed in mdx muscle, also codistributes with β-spectrin at the mutant sarcolemma. By contrast, the distribution of structural and intracellular membrane proteins, including α-actinin, the Ca-ATPase and dihydropyridine receptors, is not affected, even at sites close to the sarcolemma. Our results suggest that in myofibers of the mdx mouse, the membrane- associated cytoskeleton, but not the nearby myoplasm, undergoes widespread coordinated changes in organization. These changes may contribute to the fragility of the sarcolemma of dystrophic muscle.
A Myristoylated Calcium-binding Protein that Preferentially Interacts with the Alzheimer's Disease Presenilin 2 Protein
Authors: Stabler Stacy M. , Ostrowski Lisa L. , Janicki Susan M. , Monteiro Mervyn J.
Published: July 2002

It is well established that mutations in the presenilin 1 and 2 genes cause the majority of early onset Alzheimer's disease (AD). However, our understanding of the cellular functions of the proteins they encode remains rudimentary. Knowledge of proteins with which the presenilins interact should lead to a better understanding of presenilin function in normal and disease states. We report here the identification of a calcium-binding protein, calmyrin, that interacts preferentially with presenilin 2 (PS2). Calmyrin is myristoylated, membrane-associated, and colocalizes with PS2 when the two proteins are overexpressed in HeLa cells. Yeast two-hybrid liquid assays, affinity chromatography, and coimmunoprecipitation experiments confirm binding between PS2 and calmyrin. Functionally, calmyrin and PS2 increase cell death when cotransfected into HeLa cells. These results allude to several provocative possibilities for a dynamic role of calmyrin in signaling, cell death, and AD.
Nucleolar Localization of Human Methionyl–Trna Synthetase and Its Role in Ribosomal RNA Synthesis
Authors: Ko Young-Gyu , Kang Young-Sun , Kim Eun-Kyoung , Park Sang Gyu , Kim Sunghoon
Published: July 2002

Human aminoacyl–tRNA synthetases (ARSs) are normally located in cytoplasm and are involved in protein synthesis. In the present work, we found that human methionyl–tRNA synthetase (MRS) was translocated to nucleolus in proliferative cells, but disappeared in quiescent cells. The nucleolar localization of MRS was triggered by various growth factors such as insulin, PDGF, and EGF. The presence of MRS in nucleoli depended on the integrity of RNA and the activity of RNA polymerase I in the nucleolus. The ribosomal RNA synthesis was specifically decreased by the treatment of anti-MRS antibody as determined by nuclear run-on assay and immunostaining with anti-Br antibody after incorporating Br-UTP into nascent RNA. Thus, human MRS plays a role in the biogenesis of rRNA in nucleoli, while it is catalytically involved in protein synthesis in cytoplasm.
Erv41p and Erv46p
Authors: Otte Stefan , Belden William J. , Heidtman Matthew , Liu Jay , Jensen Ole N. , Barlowe Charles
Published: July 2002

Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity. The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Δ strain, and Erv41p was not detected in an erv46Δ strain. When the erv41Δ or ev46Δ alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains. A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p–Erv46p complex influences the membrane fusion stage of transport.
Implication of a novel multiprotein Dam1p complex in outer kinetochore function
Authors: Cheeseman Iain M. , Brew Christine , Wolyniak Michael , Desai Arshad , Anderson Scott , Muster Nemone , Yates John R. , Huffaker Tim C. , Drubin David G. , Barnes Georjana
Published: July 2002

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.
CCR6, a CC Chemokine Receptor that Interacts with Macrophage Inflammatory Protein 3α and Is Highly Expressed in Human Dendritic Cells
Authors: Greaves David R. , Wang Wei , Dairaghi Daniel J. , Dieu Marie Caroline , Saint-Vis Blandine de , Franz-Bacon Karin , Rossi Devora , Caux Christophe , McClanahan Terrill , Gordon Siamon , Zlotnik Albert , Schall Thomas J.
Published: July 2002

Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3α. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection.
Two Novel Routes of Transporter Associated with Antigen Processing (TAP)-independent Major Histocompatibility Complex Class I Antigen Processing
Authors: Snyder Heidi Link , Bačík Igor , Bennink Jack R. , Kearns Grainne , Behrens Timothy W. , Bächi Thomas , Orlowski Marian , Yewdell Jonathan W.
Published: July 2002

Jaw1 is an endoplasmic reticulum (ER) resident protein representative of a class of proteins post translationally inserted into membranes via a type II membrane anchor (cytosolic NH2 domain, lumenal COOH domain) in a translocon-independent manner. We found that Jaw1 can efficiently deliver a COOH-terminal antigenic peptide to class I molecules in transporter associated with antigen processing (TAP)-deficient cells or cells in which TAP is inactivated by the ICP47 protein. Peptide delivery mediated by Jaw1 to class I molecules was equal or better than that mediated by the adenovirus E3/19K glycoprotein signal sequence, and was sufficient to enable cytofluorographic detection of newly recruited thermostabile class I molecules at the surface of TAP-deficient cells. Deletion of the transmembrane region retargeted Jaw1 from the ER to the cytosol, and severely, although incompletely, abrogated its TAP-independent peptide carrier activity. Use of different protease inhibitors revealed the involvement of a nonproteasomal protease in the TAP-independent activity of cytosolic Jaw1. These findings demonstrate two novel TAP-independent routes of antigen processing; one based on highly efficient peptide liberation from the COOH terminus of membrane proteins in the ER, the other on delivery of a cytosolic protein to the ER by an unknown route.
Spontaneous Follicular Exclusion of SHP1-deficient B Cells Is Conditional on the Presence of Competitor Wild-type B Cells
Authors: Schmidt Kerstin N. , Hsu Christopher W. , Griffin Courtney T. , Goodnow Christopher C. , Cyster Jason G.
Published: July 2002

Engagement of antigen receptors on mature B lymphocytes is known to block cell entry into lymphoid follicles and promote accumulation in T cell zones, yet the molecular basis for this change in cell distribution is not understood. Previous studies have shown that follicular exclusion requires a threshold level of antigen receptor engagement combined with occupancy of follicles by B cells without equivalent receptor engagement. The possibility has been raised that follicular composition affects B cell positioning by altering the amount of available antigen and the degree of receptor occupancy. Here we show that follicular composition affects migration of mature B cells under conditions that are independent of antigen receptor occupancy. B cells deficient in the negative regulatory protein tyrosine phosphatase, SHP1, which have elevated intracellular signaling by the B cell receptor, are shown to accumulate in the T zone in the absence of their specific antigen. Follicular exclusion of SHP1–deficient B cells was found to be conditional on the presence of excess B cells that lack elevated intracellular signaling, and was not due to a failure of SHP-1–deficient cells to mature and express the follicle-homing chemokine receptor Burkitt's lymphoma receptor 1. These findings strongly suggest that signals that are negatively regulated by SHP1 promote B cell localization in T cell zones by reducing competitiveness for follicular entry, and provide further evidence that follicular composition influences the positioning of antigen-engaged B cells.
Requirement of SH2-containing Protein Tyrosine Phosphatases SHP-1 and SHP-2 for Paired Immunoglobulin-like Receptor B (PIR-B)–mediated Inhibitory Signal
Authors: Maeda Akito , Kurosaki Mari , Ono Masao , Takai Toshiyuki , Kurosaki Tomohiro
Published: July 2002

Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B–mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5′-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B–mediated inhibition requires redundant functions of SHP-1 and SHP-2.
GrpL, a Grb2-related Adaptor Protein, Interacts with SLP-76 to Regulate Nuclear Factor of Activated T Cell Activation
Authors: Law Che-Leung , Ewings Maria K. , Chaudhary Preet M. , Solow Sasha A. , Yun Theodore J. , Marshall Aaron J. , Hood Leroy , Clark Edward A.
Published: July 2002

Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain–containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL). Expression of GrpL is restricted to hematopoietic tissues, and it is distinguished from Grb2 by having a proline-rich region. GrpL can be coimmunoprecipitated with SLP-76 but not with Sos1 or Sos2 from Jurkat cell lysates. In contrast, Grb2 can be coimmunoprecipitated with Sos1 and Sos2 but not with SLP-76. Moreover, tyrosine-phosphorylated LAT/pp36/38 in detergent lysates prepared from anti-CD3 stimulated T cells associated with Grb2 but not GrpL. These data reveal the presence of distinct complexes involving GrpL and Grb2 in T cells. A functional role of the GrpL–SLP-76 complex is suggested by the ability of GrpL to act alone or in concert with SLP-76 to augment NF-AT activation in Jurkat T cells.
Telomere Fluorescence Measurements in Granulocytes and T Lymphocyte Subsets Point to a High Turnover of Hematopoietic Stem Cells and Memory T Cells in Early Childhood
Authors: Rufer Nathalie , Brümmendorf Tim H. , Kolvraa Steen , Bischoff Claus , Christensen Kaare , Wadsworth Louis , Schulzer Michael , Lansdorp Peter M.
Published: July 2002

To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.
Trapping of Organic Blockers by Closing of Voltage-dependent K+ Channels
Authors: Holmgren Miguel , Smith Paula L. , Yellen Gary
Published: July 2002

Small organic molecules, like quaternary ammonium compounds, have long been used to probe both the permeation and gating of voltage-dependent K+ channels. For most K+ channels, intracellularly applied quaternary ammonium (QA) compounds such as tetraethylammonium (TEA) and decyltriethylammonium (C10) behave primarily as open channel blockers: they can enter the channel only when it is open, and they must dissociate before the channel can close. In some cases, it is possible to force the channel to close with a QA blocker still bound, with the result that the blocker is “trapped.” Armstrong (J. Gen. Physiol. 58:413–437) found that at very negative voltages, squid axon K+ channels exhibited a slow phase of recovery from QA blockade consistent with such trapping. In our studies on the cloned Shaker channel, we find that wild-type channels can trap neither TEA nor C10, but channels with a point mutation in S6 can trap either compound very efficiently. The trapping occurs with very little change in the energetics of channel gating, suggesting that in these channels the gate may function as a trap door or hinged lid that occludes access from the intracellular solution to the blocker site and to the narrow ion-selective pore.
Dual Regulation of Calcium Mobilization by Inositol 1,4,5-Trisphosphate in a Living Cell
Authors: Tertyshnikova Svetlana , Fein Alan
Published: July 2002

Changes in cytosolic free calcium ([Ca2+]i) often take the form of a sustained response or repetitive oscillations. The frequency and amplitude of [Ca2+]i oscillations are essential for the selective stimulation of gene expression and for enzyme activation. However, the mechanism that determines whether [Ca2+]i oscillates at a particular frequency or becomes a sustained response is poorly understood. We find that [Ca2+]i oscillations in rat megakaryocytes, as in other cells, results from a Ca2+-dependent inhibition of inositol 1,4,5-trisphosphate (IP3)–induced Ca2+ release. Moreover, we find that this inhibition becomes progressively less effective with higher IP3 concentrations. We suggest that disinhibition, by increasing IP3 concentration, of Ca2+-dependent inhibition is a common mechanism for the regulation of [Ca2+]i oscillations in cells containing IP3-sensitive Ca2+ stores.
A note on stability of the Douglas splitting method
Authors: Hundsdorfer Willem
Published: July 2002

In this note some stability results are derived for the Douglas splitting method. The relevance of the theoretical results is tested for an advection-reaction equation.
A census of cusped hyperbolic 3-manifolds
Authors: Callahan Patrick , Hildebrand Martin , Weeks Jeffrey
Published: July 2002

The census provides a basic collection of noncompact hyperbolic 3-manifolds of finite volume. It contains descriptions of all hyperbolic 3-manifolds obtained by gluing the faces of at most seven ideal tetrahedra. Additionally, various geometric and topological invariants are calculated for these manifolds. The findings are summarized and a listing of all manifolds appears in the microfiche supplement.
On non-Spechtianness of the variety of associative rings that satisfy the identity 𝑥³²=0
Authors: Grishin A.
Published: July 2002

In this paper we construct examples of

T
T

-spaces and

T
T

-ideals over a field of characteristic 2, which do not have the finite basis property.
Symmetric polynomials and 𝑈_{𝑞}(̂𝑠𝑙₂)
Authors: Jing Naihuan
Published: July 2002

We study the explicit formula of Lusztig’s integral forms of the level one quantum affine algebraUq(sl^2)U_q(\widehat {sl}_2)in the endomorphism ring of symmetric functions in infinitely many variables tensored with the group algebra ofZ\mathbb Z. Schur functions are realized as certain orthonormal basis vectors in the vertex representation associated to the standard Heisenberg algebra. In this picture the Littlewood-Richardson rule is expressed by integral formulas, and is used to define the action of Lusztig’sZ[q,q−1]\mathbb Z[q, q^{-1}]-form ofUq(sl^2)U_q(\widehat {sl}_2)on Schur polynomials. As a result theZ[q,q−1]\mathbb Z[q, q^{-1}]-lattice of Schur functions tensored with the group algebra contains Lusztig’s integral lattice.
On square-integrable representations of classical 𝑝-adic groups II
Authors: Jantzen Chris
Published: July 2002

In this paper, we continue our study of non-supercuspidal discrete series for the classical groups

S
p
(
2
n
,
F
)

Sp(2n,F)

,

S
O
(
2
n
+
1
,
F
)

SO(2n+1,F)

, where

F
F

is

p
p

-adic.
New scoring schemes for protein fold recognition based on Voronoi contacts.
Authors: Zimmer R , W√∂hler M , Thiele R
Published: July 2002

MOTIVATION: The genome projects produce a wealth of protein sequences. Theoretical methods to predict possible structures and functions are needed for screening purposes, large-scale comparisons and in-depth analysis to identify worthwhile targets for further experimental research. Sequence-structure alignment is a basic tool for the identification of model folds for protein sequences and the construction of crude structural models. Empirical contact potentials (potentials of mean force) are used to optimize and evaluate such alignments. RESULTS: We propose new scoring schemes based on a contact definition derived from Voronoi decompositions of the three-dimensional coordinates of protein structures. We demonstrate that Voronoi potentials are superior to pure distance-based contact potentials with respect to recognition rate and significance for native folds. Moreover, the scoring scheme has the potential to provide a reasonable balance of detail and ion such that it is also useful for the recognition of distantly related (both homologous and non-homologous) proteins. This is demonstrated here on a set of structural alignments showing much better correspondence of native and model scores for the Voronoi potentials as compared to conventional distance-based potentials. AVAILABILITY: The potentials are made available via the program system ToPLign (URL: http://cartan.gmd.de/ToPLign.html). CONTACT: Ralf.Zimmer,Ralf.Thiele@gmd.de
AN ECONOMIC ANALYSIS OF ASPECTS OF PETROLEUM AND MILITARY SECURITY IN THE PERSIAN GULF
Authors: Chapman Duane , Khanna Neha
Published: July 2002

Geologic estimates of remaining global petroleum resources place about 50% in the Persian Gulf. Production costs are estimated at $5 per barrel there and $15 per barrel in the North Sea and Alaska. According to mathematical results derived from depletion theory, the present value of economic rent from oil is on the order of $20 trillion. This article uses game theory to explain the $15–$20 per barrel price band that existed from 1986 to 1999. New economic forces have displaced this previously stable pattern; a new price range of $23–$30 is emerging. International trade in petroleum and conventional weapons are analyzed with econometric methods; the occurrence of nuclear weapons capability in the Persian Gulf region is explored.
Clinical effectiveness of levofloxacin in patients with acute purulent exacerbations of chronic bronchitis: the relationship with in-vitro activity
Authors: Davies B. I. , Maesen F. P. V.
Published: July 2002

The objective of this randomized, double-blind study was to compare the clinical efficacy of levofloxacin at two different dosages with that of cefuroxime axetil in patients with acute purulent exacerbations of chronic bronchitis and, in particular, to assess the impact of the susceptibility to levofloxacin on the clinical findings. In total, 124 evaluable patients were treated for 7 days with oral levofloxacin 250 mg or 500 mg od, or cefuroxime axetil 250 mg bd. Sputum cultures were monitored pre-treatment, and at 1 and 7 days after the end of treatment. The susceptibility of Streptococcus pneumoniae isolates was tested by agar dilution in Columbia blood agar and by disc diffusion, but all other isolates were tested solely by the disc diffusion method. A greater number of infections were eradicated by levofloxacin than by cefuroxime axetil: infections were eradicated in 68% of patients receiving the 500 mg dosage and in 63% of those taking 250 mg levofloxacin, whereas the eradication rate with the comparator drug was much lower (48%). Against all pre-treatment S. pneumoniae isolates ( n = 39), the MICs of levofloxacin were between 0.25 and 2 mg/L (geometric mean 0.95 mg/L), similar to those of the post-treatment strains ( n = 32; mean 1.11 mg/L). All except one of the S. pneumoniae isolates were susceptible to penicillin G (MIC ≤ 0.06 mg/L), and the remaining isolate was inhibited by 0.5 mg/L of penicillin G, but was fully susceptible to levofloxacin. Some pre-treatment strains of Pseudomonas aeruginosa were resistant to levofloxacin, but many more resistant strains were encountered afterwards. All strains of Moraxella catarrhalis and Haemophilus influenzae were highly susceptible to levofloxacin in the disc diffusion tests. All the antimicrobial agents used in the study were well tolerated: only two patients discontinued treatment because of adverse drug effects. The results of this study indicated that, although there were some failures in patients with S. pneumoniae and P. aeruginosa infections, resistance to levofloxacin did not emerge rapidly among strains of S. pneumoniae during therapy with levofloxacin, and that natural resistance among pneumococci, H. influenzae and M. catarrhalis was rare.
Locomotor mimicry inHeliconiusbutterflies: contrast analyses of flight morphology and kinematics
Authors: Srygley Robert B.
Published: July 2002

Müllerian mimicry is a mutualism involving the evolutionary convergence of colour patterns of prey on a warning signal to predators. Behavioural mimicry presumably adds complexity to the signal and makes it more difficult for Batesian mimics to parasitize it. To date, no one has quantified behavioural mimicry in Müllerian mimicry groups. However, morphological similarities among members of mimicry groups suggested that pitching oscillations of the body and wing–beat frequency (WBF) might converge with colour pattern. I compared the morphology and kinematics of fourHeliconiusspecies, which comprised two mimicry pairs. Because the mimics arose from two distinct lineages, the relative contributions of mimicry and phylogeny to variation in the species' morphologies and kinematics were examined. The positions of the centre of body mass and centre of wing mass and wing shape diverged among species within lineages, and converged among species within mimicry groups. WBF converged within mimicry groups, and it was coupled with body pitching frequency. However, body–pitching frequency was too variable to distinguish mimicry groups. Convergence in WBF may be due, at least in part, to biomechanical consequences of similarities in wing length, wing shape or the centre of wing mass among co–mimics. Nevertheless, convergence in WBF among passion–vine butterflies serves as the first evidence of behavioural mimicry in a mutualistic context.
Balancing on a narrow ridge: biomechanics and control
Authors: Otten E.
Published: July 2002

The balance of standing humans is usually explained by the inverted pendulum model. The subject invokes a horizontal ground–reaction force in this model and controls it by changing the location of the centre of pressure under the foot or feet. In experiments I showed that humans are able to stand on a ridge of only a few millimetres wide on one foot for a few minutes. In the present paper I investigate whether the inverted pendulum model is able to explain this achievement. I found that the centre of mass of the subjects sways beyond the surface of support, rendering the inverted pendulum model inadequate. Using inverse simulations of the dynamics of the human body, I found that hip–joint moments of the stance leg are used to vary the horizontal component of the ground–reaction force. This force brings the centre of mass back over the surface of support. The subjects generate moments of force at the hip–joint of the swing leg, at the shoulder–joints and at the neck. These moments work in conjunction with a hip strategy of the stance leg to limit the angular acceleration of the head–arm–trunk complex. The synchrony of the variation in moments suggests that subjects use a motor programme rather than long latency reflexes.
Analysis of the 5S RNA Pool in Arabidopsis thaliana: RNAs Are Heterogeneous and Only Two of the Genomic 5S Loci Produce Mature 5S RNA
Authors: Cloix Catherine , Tutois Sylvie , Yukawa Yasushi , Mathieu Olivier , Cuvillier Claudine , Espagnol Marie-Claude , Picard Georges , Tourmente Sylvette
Published: July 2002

One major 5S RNA, 120 bases long, was revealed by an analysis of mature 5S RNA from tissues, developmental stages, and polysomes inArabidopsis thaliana. Minor 5S RNA were also found, varying from the major one by one or two base substitutions; 5S rDNA units from each 5S array of the Arabidopsis genome were isolated by PCR using CIC yeast artificial chromosomes (YACs) mapped on the different loci. By using a comparison of the 5S DNA and RNA sequences, we could show that both major and minor 5S transcripts come from only two of the genomic 5S loci: chromosome 4 and chromosome 5 major block. Other 5S loci are either not transcribed or produce rapidly degraded 5S transcripts. Analysis of the 5′- and 3′-DNA flanking sequence has permitted the definition of specific signatures for each 5S rDNA array.

[EMBL accession nos: AF330825-AF331032;AF335777-AF335873.]