bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of the Functional Domain Organization of the Importin α Nuclear Import Factor
Authors: Herold Andrea , Truant Ray , Wiegand Heather , Cullen Bryan R.
Published: July 2002

Although importin α (Imp α) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin β nuclear import factor, little is known about the functional domains present in Imp α, with the exception that importin β binding is known to map close to the Imp α NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp α COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp α nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp α nuclear export. Analysis of sequences within Imp α that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp α. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp α may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp α with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.
Roles of Rho-associated Kinase in Cytokinesis; Mutations in Rho-associated Kinase Phosphorylation Sites Impair Cytokinetic Segregation of Glial Filaments
Authors: Yasui Yoshihiro , Amano Mutsuki , Nagata Koh-ichi , Inagaki Naoyuki , Nakamura Hideo , Saya Hideyuki , Kaibuchi Kozo , Inagaki Masaki
Published: July 2002

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.
Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2
Authors: Fraschini Roberta , Formenti Elisa , Lucchini Giovanna , Piatti Simonetta
Published: July 2002

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.
Degraded Collagen Fragments Promote Rapid Disassembly of Smooth Muscle Focal Adhesions That Correlates with Cleavage of Pp125FAK, Paxillin, and Talin
Authors: Carragher Neil O. , Levkau Bodo , Ross Russell , Raines Elaine W.
Published: July 2002

Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125FAK), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125FAK is a substrate for both calpain I– and II–mediated processing. Mapping of the proteolytic cleavage fragments of pp125FAK predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125FAK to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125FAK with the cytoskeletal fraction, while pp125FAK cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125FAK cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125FAK, paxillin, and talin and dissolution of the focal adhesion complex.
Visualization of Head–Head Interactions in the Inhibited State of Smooth Muscle Myosin
Authors: Wendt Thomas , Taylor Dianne , Messier Terri , Trybus Kathleen M. , Taylor Kenneth A.
Published: July 2002

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.
Role of Cofilin in Epidermal Growth Factor–Stimulated Actin Polymerization and Lamellipod Protrusion
Authors: Chan Amanda Y. , Bailly Maryse , Zebda Noureddine , Segall Jeffrey E. , Condeelis John S.
Published: July 2002

Stimulation of metastatic MTLn3 cells with epidermal growth factor (EGF) causes a rapid and transient increase in actin nucleation activity resulting from the appearance of free barbed ends at the extreme leading edge of extending lamellipods. To investigate the role of cofilin in EGF-stimulated actin polymerization and lamellipod extension in MTLn3 cells, we examined in detail the temporal and spatial distribution of cofilin relative to free barbed ends and characterized the actin dynamics by measuring the changes in the number of actin filaments. EGF stimulation triggers a transient increase in cofilin in the leading edge near the membrane, which is precisely cotemporal with the appearance of free barbed ends there. A deoxyribonuclease I binding assay shows that the number of filaments per cell increases by 1.5-fold after EGF stimulation. Detection of pointed ends in situ using deoxyribonuclease I binding demonstrates that this increase in the number of pointed ends is confined to the leading edge compartment, and does not occur within stress fibers or in the general cytoplasm. Using a light microscope severing assay, cofilin's severing activity was observed directly in cell extracts and shown to be activated after stimulation of the cells with EGF. Microinjection of function-blocking antibodies against cofilin inhibits the appearance of free barbed ends at the leading edge and lamellipod protrusion after EGF stimulation. These results support a model in which EGF stimulation recruits cofilin to the leading edge where its severing activity is activated, leading to the generation of short actin filaments with free barbed ends that participate in the nucleation of actin polymerization.
Induction of Caveolae in the Apical Plasma Membrane of Madin-Darby Canine Kidney Cells
Authors: Verkade Paul , Harder Thomas , Lafont Frank , Simons Kai
Published: July 2002

In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929–942), only small (∼100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly (∼10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits.

Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.
Ankyrin–Tiam1 Interaction Promotes Rac1 Signaling and Metastatic Breast Tumor Cell Invasion and Migration
Authors: Bourguignon Lilly Y.W. , Zhu Hongbo , Shao Lijun , Chen Yue Wei
Published: July 2002

Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11–amino acid sequence between amino acids 717 and 727 of Tiam1 (717GEGTDAVKRS727L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1).

Using an Escherichia coli–derived calmodulin-binding peptide (CBP)–tagged recombinant Tiam1 (amino acids 393–728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393–738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1–ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)–tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1–ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin–Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.
Regulation of Microtubule Dynamics and Myogenic Differentiation by Murf, a Striated Muscle Ring-Finger Protein
Authors: Spencer Jeffrey A. , Eliazer Susan , Ilaria Robert L. , Richardson James A. , Olson Eric N.
Published: July 2002

The RING-finger domain is a novel zinc-binding Cys-His protein motif found in a growing number of proteins involved in signal transduction, ubiquitination, gene transcription, differentiation, and morphogenesis. We describe a novel muscle-specific RING-finger protein (MURF) expressed specifically in cardiac and skeletal muscle cells throughout pre- and postnatal mouse development. MURF belongs to the RING-B-box-coiled-coil subclass of RING-finger proteins, characterized by an NH2-terminal RING-finger followed by a zinc-finger domain (B-box) and a leucine-rich coiled-coil domain. Expression of MURF is required for skeletal myoblast differentiation and myotube fusion. The leucine-rich coiled-coil domain of MURF mediates association with microtubules, whereas the RING-finger domain is required for microtubule stabilization and an additional region is required for homo-oligomerization. Expression of MURF establishes a cellular microtubule network that is resistant to microtubule depolymerization induced by alkaloids, cold and calcium. These results identify MURF as a myogenic regulator of the microtubule network of striated muscle cells and reveal a link between microtubule organization and myogenesis.
A regulatory cascade involving retinoic acid, Cbfa1, and matrix metalloproteinases is coupled to the development of a process of perichondrial invasion and osteogenic differentiation during bone formation
Authors: Jiménez Maria J.G. , Balbín Milagros , Alvarez Jesús , Komori Toshihisa , Bianco Paolo , Holmbeck Kenn , Birkedal-Hansen Henning , López José M. , López-Otín Carlos
Published: July 2002

Tissue-remodeling processes are largely mediated by members of the matrix metalloproteinase (MMP) family of endopeptidases whose expression is strictly controlled both spatially and temporally. In this article, we have examined the molecular mechanisms that could contribute to modulate the expression of MMPs like collagenase-3 and MT1-MMP during bone formation. We have found that all-trans retinoic acid (RA), which usually downregulates MMPs, strongly induces collagenase-3 expression in cultures of embryonic metatarsal cartilage rudiments and in chondrocytic cells. This effect is dose and time dependent, requires the de novo synthesis of proteins, and is mediated by RAR-RXR heterodimers. Analysis of the signal transduction mechanisms underlying the upregulating effect of RA on collagenase-3 expression demonstrated that this factor acts through a signaling pathway involving p38 mitogen-activated protein kinase. RA treatment of chondrocytic cells also induces the production of MT1-MMP, a membrane-bound metalloproteinase essential for skeletal formation, which participates in a proteolytic cascade with collagenase-3. The production of these MMPs is concomitant with the development of an RA-induced differentiation program characterized by formation of a mineralized bone matrix, downregulation of chondrocyte markers like type II collagen, and upregulation of osteoblastic markers such as osteocalcin. These effects are attenuated in metatarsal rudiments in which RA induces the invasion of perichondrial osteogenic cells from the perichondrium into the cartilage rudiment. RA treatment also resulted in the upregulation of Cbfa1, a transcription factor responsible for collagenase-3 and osteocalcin induction in osteoblastic cells. The dynamics of Cbfa1, MMPs, and osteocalcin expression is consistent with the fact that these genes could be part of a regulatory cascade initiated by RA and leading to the induction of Cbfa1, which in turn would upregulate the expression of some of their target genes like collagenase-3 and osteocalcin.
Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult
Authors: Schweizer Ulrich , Gunnersen Jennifer , Karch Christoph , Wiese Stefan , Holtmann Bettina , Takeda Kiyoshi , Akira Shizuo , Sendtner Michael
Published: July 2002

Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-β and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L–Cre; Stat3flox/KO mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.
Twinfilin is required for actin-dependent developmental processes in Drosophila
Authors: Wahlström Gudrun , Vartiainen Maria , Yamamoto Lumi , Mattila Pieta K. , Lappalainen Pekka , Heino Tapio I.
Published: July 2002

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer–binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.
Regulation of  T Cell Receptor δ Gene Rearrangement by CBF/PEBP2
Authors: Lauzurica Pilar , Zhong Xiao-Ping , Krangel Michael S. , Roberts Joseph L.
Published: July 2002

We have analyzed transgenic mice carrying versions of a human T cell receptor (TCR)-δ gene minilocus to study the developmental control of  VDJ (variable/diversity/joining) recombination. Previous data indicated that a 1.4-kb DNA fragment carrying the TCR-δ enhancer (Eδ) efficiently activates minilocus VDJ recombination in vivo. We tested whether the transcription factor CBF/PEBP2 plays an important role in the ability of Eδ to activate VDJ recombination by analyzing VDJ recombination in mice carrying a minilocus in which the δE3 element of Eδ includes a mutated CBF/PEBP2 binding site. The enhancer-dependent VD to J step of minilocus rearrangement was dramatically inhibited in three of four transgenic lines, arguing that the binding of CBF/PEBP2 plays a role in modulating local accessibility to the VDJ recombinase in vivo. Because mutation of the δE3 binding site for the transcription factor c-Myb had previously established a similar role for c-Myb, and because a 60-bp fragment of Eδ carrying δE3 and δE4 binding sites for CBF/PEBP2, c-Myb, and GATA-3 displays significant enhancer activity in transient transfection experiments, we tested whether this fragment of Eδ is sufficient to activate VDJ recombination in vivo. This fragment failed to efficiently activate the enhancerdependent VD to J step of minilocus rearrangement in all three transgenic lines examined, indicating that the binding of CBF/PEBP2 and c-Myb to their cognate sites within Eδ, although necessary, is not sufficient for the activation of VDJ recombination by Eδ. These results imply that CBF/PEBP2 and c-Myb collaborate with additional factors that bind elsewhere within Eδ to modulate local accessibility to the VDJ recombinase in vivo.
Regulation of L-Selectin–mediated Rolling through Receptor Dimerization
Authors: Li Xuan , Steeber Douglas A. , Tang Mimi L.K. , Farrar Michael A. , Perlmutter Roger M. , Tedder Thomas F.
Published: July 2002

L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin were induced using the coumermycin–GyrB dimerization strategy for cross-linking L-selectin cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin- induced L-selectin dimerization resulted in an approximately fourfold increase in binding of phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand, comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by ∼700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand-binding function of other cell-surface receptors.
Signal Transducer and Activator of Transcription 6 Is Essential in the Induction of Contact Hypersensitivity
Authors: Yokozeki Hiroo , Ghoreishi Mehran , Takagawa Shinsuke , Takayama Kaoru , Satoh Takahiro , Katayama Ichiro , Takeda Kiyoshi , Akira Shizuo , Nishioka Kiyoshi
Published: July 2002

Contact hypersensitivity (CHS) is thought to be mainly associated with the activation of T helper type 1 (Th1) cells. However, there is also evidence that Th2 cells or Th2 cytokines play a role in the development of CHS. To analyze the functional contribution of Th2 cytokines interleukin (IL)-4 and IL-13, signal transducer and activator of transcription 6 (STAT6)-deficient (STAT6−/−) and wild-type (wt) control C57BL/6 mice were contact sensitized with 5% 2,4,6-trinitrochlorobenzene (TNCB), 0.5% 2,4-dinitrofluorobenzene, or 5% 4-ethoxyl methylene-2-phenyl-2-oxazolin-5-one, and any skin reactions were examined. Ear swelling was significantly reduced with a delayed peak response in STAT6−/− mice compared with wt mice.

A histological analysis revealed that the infiltration of both eosinophils and neutrophils in the skin challenged after 24 h in STAT6−/− mice decreased substantially compared with that in wt mice. The expression of Th2 cytokines (IL-4, IL-5) in TNCB-challenged skin tissues and the supernatants from T cells stimulated by 2,4,6-trinitrobenzene sulfonate–modified spleen cells, as well as the immunoglobulin (Ig)E and IgG1 response after challenge, were also profoundly reduced in STAT6−/− mice, whereas the expression of interferon γ was the same in STAT6−/− and wt mice after challenge. Furthermore, adoptive transfer experiments revealed that STAT6−/− mice induced CHS after injection of lymph node cells obtained from sensitized wt mice. Our data suggest that the STAT6 signal plays a critical role in the induction phase of CHS.
Prostaglandin D2 Selectively Induces Chemotaxis in T Helper Type 2 Cells, Eosinophils, and Basophils via Seven-Transmembrane Receptor Crth2
Authors: Hirai Hiroyuki , Tanaka Kazuya , Yoshie Osamu , Ogawa Kazuyuki , Kenmotsu Kazumi , Takamori Yasushi , Ichimasa Michiko , Sugamura Kazuo , Nakamura Masataka , Takano Shoichi , Nagata Kinya
Published: July 2002

Prostaglandin (PG)D2, which has long been implicated in allergic diseases, is currently considered to elicit its biological actions through the DP receptor (DP). Involvement of DP in the formation of allergic asthma was recently demonstrated with DP-deficient mice. However, proinflammatory functions of PGD2 cannot be explained by DP alone. We show here that a seven-transmembrane receptor, CRTH2, which is preferentially expressed in T helper type 2 (Th2) cells, eosinophils, and basophils in humans, serves as the novel receptor for PGD2. In response to PGD2, CRTH2 induces intracellular Ca2+ mobilization and chemotaxis in Th2 cells in a Gαi-dependent manner. In addition, CRTH2, but not DP, mediates PGD2-dependent cell migration of blood eosinophils and basophils. Thus, PGD2 is likely involved in multiple aspects of allergic inflammation through its dual receptor systems, DP and CRTH2.
On the singular values of Weber modular functions
Authors: Yui Noriko , Zagier Don
Published: July 2002

The minimal polynomials of the singular values of the classical Weber modular functions give far simpler defining polynomials for the class fields of imaginary quadratic fields than the minimal polynomials of singular moduli of level 1. We describe computations of these polynomials and give conjectural formulas describing the prime decomposition of their resultants and discriminants, extending the formulas of Gross-Zagier for the level 1 case.
Algebraic families of nonzero elements of Shafarevich-Tate groups
Authors: Colliot-Thélène Jean-Louis , Poonen Bjorn
Published: July 2002

Principal homogeneous spaces under an abelian variety defined over a number field

k
k

may have rational points in all completions of the number field without having rational points over

k
k

. Such principal homogeneous spaces represent the nonzero elements of the Shafarevich-Tate group of the abelian variety. We produce nontrivial, one-parameter families of such principal homogeneous spaces. The total space of these families are counterexamples to the Hasse principle. We show these may be accounted for by the Brauer-Manin obstruction.
Mutation ofYCS4, a Budding Yeast Condensin Subunit, Affects Mitotic and Nonmitotic Chromosome Behavior
Authors: Bhalla Needhi , Biggins Sue , Murray Andrew W.
Published: July 2002

The budding yeast YCS4 gene encodes a conserved regulatory subunit of the condensin complex. We isolated an allele of this gene in a screen for mutants defective in sister chromatid separation or segregation. The phenotype of the ycs4-1mutant is similar to topoisomerase II mutants and distinct from theesp1-1 mutant: the topological resolution of sister chromatids is compromised in ycs4-1 despite normal removal of cohesins from mitotic chromosomes. Consistent with a role in sister separation, YCS4 function is required to localize DNA topoisomerase I and II to chromosomes. Unlike its homologs inXenopus and fission yeast, Ycs4p is associated with chromatin throughout the cell cycle; the only change in localization occurs during anaphase when the protein is enriched at the nucleolus. This relocalization may reveal the specific challenge that segregation of the transcriptionally hyperactive, repetitive array of rDNA genes can present during mitosis. Indeed, segregation of the nucleolus is abnormal in ycs4-1 at the nonpermissive temperature. Interrepeat recombination in the rDNA array is specifically elevated inycs4-1 at the permissive temperature, suggesting that the Ycs4p plays a role at the array aside from its segregation. Furthermore, ycs4-1 is defective in silencing at the mating type loci at the permissive temperature. Taken together, our data suggest that there are mitotic as well as nonmitotic chromosomal abnormalities associated with loss of condensin function in budding yeast.
Oral bioequivalence of three ciprofloxacin formulations following single-dose administration: 500 mg tablet compared with 500 mg/10 mL or 500 mg/5 mL suspension and the effect of food on the absorption of ciprofloxacin oral suspension
Authors: Shah Anita , Liu Ming-Chung , Vaughan David , Heller Allen H.
Published: July 2002

The oral bioequivalence and tolerability of two ciprofloxacin formulations (tablet and suspension) and the effect of food on the absorption of ciprofloxacin oral suspension were investigated. Sixty-eight young, healthy male subjects participated in two separate, randomized, crossover studies. In study 1, ciprofloxacin as a single 500 mg tablet or as 500 mg/10 mL oral suspension was administered in a fasted state on day 1. In study 2, subjects participated in a three-way crossover study in which ciprofloxacin suspension was administered as 500 mg/ 10 mL in a fasted state, or 500 mg/10 mL with food, or 500 mg/5 mL in a fasted state. Plasma ciprofloxacin concentrations were measured by high-performance liquid chromatography. Standard pharmacokinetic parameters were estimated using non-compartmental methods. In study 1, geometric mean Cmax values of ciprofloxacin following the single 500 mg tablet and 500 mg/10 mL suspension doses were 2.36 and 2.18 mg/L, respectively; corresponding geometric mean tmax values were 1.1 and 1.6 h, respectively. Geometric mean AUC 0–∞ values were 12.0 and 11.8 mg⋅h/L, respectively. In study 2, geometric least squares mean Cmax values following ciprofloxacin 500 mg/10 mL and 500 mg/5 mL suspension during fasted conditions were 1.54 and 1.59 mg/L, respectively. Corresponding geometric least squares mean AUC 0–∞ values were 7.3 and 8.0 mg⋅h/L. Administration of ciprofloxacin 500 mg/10 mL suspension, in either a fasted or fed state, was not associated with significant changes in Cmax (1.54 mg/L for fasted vs 1.37 mg/L for fed) or AUC 0–∞ values (7.28 mg·h/L for fasted vs 8.19 mg·h/L for fed). Each ciprofloxacin formulation was well tolerated for the duration of each study. These studies demonstrated bioequivalence between ciprofloxacin 500 mg tablet and two strengths of ciprofloxacin suspension (500 mg/10 mL and 500 mg/5 mL). Bioavailability was unaltered by food.
Hydromagnetic stability of current-induced flow in a small gap between concentric rotating cylinders
Authors: Chang Min Hsing , Chen Ch'o–Kuang
Published: July 2002

The stability analysis of the flow of a viscous electrically conducting fluid between concentric rotating cylinders in the presence of an axial magnetic field is extended to the case where the primary flow includes a pressure gradient acting in the azimuthal direction. The pressure gradient is produced electromagnetically by the interaction of a superimposed radial current and the uniform axial magnetic field. The assumption of small gap approximation is made and the governing equations with respect to both axisymmetric and non–axisymmetric three–dimensional disturbances are derived and solved by a direct numerical procedure. A parametric study covering wide ranges of
Q
, the Hartmann number which represents the strength of axial magnetic field, and β, a parameter characterizing the ratio of current induced and rotation velocities, is conducted for weakly conducting cylinders and the situation of practical interest where the outer cylinder is stationary and the inner cylinder is rotating. The area where the onset mode is non–axisymmetric is shown in the plane β,
Q
). It is found that the most stable state occurs approximately along a critical curve (β + 4.3)
Q
2
+ 56250(β + 3.75) = 0 and the critical axial wavenumber always has discontinuity when the parameters
Q
and β cross this curve. The critical mode transition of the onset of instability will be demonstrated in detail and results for the critical wavenumber and the critical Taylor number are presented. The corresponding values of the radial current density required for the appearance of secondary flow are also determined.
Mechanics and models of the myosin motor
Authors: Huxley A. F.
Published: July 2002

In striated muscles, shortening comes about by the sliding movement of thick filaments, composed mostly of myosin, relative to thin filaments, composed mostly of actin. This is brought about by cyclic action of ‘cross–bridges’ composed of the heads of myosin molecules projecting from a thick filament, which attach to an adjacent thin filament, exert force for a limited time and detach, and then repeat this cycle further along the filament. The requisite energy is provided by the hydrolysis of a molecule of adenosine triphosphate to the diphosphate and inorganic phosphate, the steps of this reaction being coupled to mechanical events within the cross–bridge. The nature of these events is discussed. There is good evidence that one of them is a change in the angle of tilt of a ‘lever arm’ relative to the ‘catalytic domain’ of the myosin head which binds to the actin filament. It is suggested here that this event is superposed on a slower, temperature–sensitive change in the orientation of the catalytic domain on the actin filament. Many uncertainties remain.
Single–motor mechanics and models of the myosin motor
Authors: Yanagida T. , Esaki S. , Iwane A. Hikikoshi , Inoue Y. , Ishijima A. , Kitamura K. , Tanaka H. , Tokunaga M.
Published: July 2002

Recent progress in single–molecule detection techniques is remarkable. These techniques have allowed the accurate determination of myosin–head–induced displacements and how mechanical cycles are coupled to ATP hydrolysis, by measuring individual mechanical events and chemical events of actomyosin directly at the single–molecule level. Here we review our recent work in which we have made detailed measurements of myosin step size and mechanochemical coupling, and propose a model of the myosin motor.
Mass spectrometric analysis of a UV‐cross‐linked protein–DNA complex: Tryptophans 54 and 88 ofE. coliSSB cross‐link to DNA
Authors: Steen Hanno , Petersen Jørgen , Mann Matthias , Jensen Ole N.
Published: July 2002

Protein–nucleic acid complexes are commonly studied by photochemical cross‐linking. UV‐induced cross‐linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross‐linked amino acids and thereby idey the nucleic acid binding site. Mass spectrometry is becoming increasingly popular for characterization of purified peptide–nucleic acid heteroconjugates derived from UV cross‐linked protein–nucleic acid complexes. The efficiency of mass spectrometry‐based methods is, however, hampered by the contrasting physico‐chemical properties of nucleic acid and peptide entities present in such heteroconjugates. Sample preparation of the peptide–nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry‐based analytical procedure. This study demonstrates the performance of four different MS‐based strategies to characterizeE. colisingle‐stranded DNA binding protein (SSB) that was UV‐cross‐linked to a 5‐iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis. Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross‐linked peptide–nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis. Detailed characterization of the peptidic component of two different peptide–DNA heteroconjugates was accomplished by matrix‐assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan‐54 and tryptophan‐88 as candidate cross‐linked residues. Sequencing of those peptide–DNA heteroconjugates by nanoelectrospray quadrupole time‐of‐flight tandem mass spectrometry ideied tryptophan‐54 and tryptophan‐88 as the sites of cross‐linking. Although the UV‐cross‐linking yield of the protein–DNA complex did not exceed 15%, less than 100 pmole of SSB protein was required for detailed structural analysis by mass spectrometry.
Proliferation and the advantage of longer‐lived radionuclides in radioimmunotherapy
Authors: Howell Roger W. , Goddu S. Murty , Rao Dandamudi V.
Published: July 2002

In our previous study we used the linear‐quadratic model [J. Nucl. Med. 35, 1861 (1994)] to confirm our initial finding, based on the time‐dose‐fractionation model [J. Nucl. Med. 34, 1801 (1993)], that longer‐lived radionuclides (e.g., ) can offer a substantial therapeutic advantage over the shorter‐lived radionuclides presently used in radioimmunotherapy (e.g., ). The original calculations using the linear‐quadratic (LQ) model did not account for proliferation of the tumor and critical bone marrow tissues. It has been suggested that inclusion of a proliferation term in the LQ model can have a substantial impact on the biologically effective dose (BED). With this in mind, we have reexamined the therapeutic efficacy of longer versus short‐lived radionuclides using the LQ model replete with proliferation terms for tumor and bone marrow. Relative advantage factors (RAF), which quantify the overall therapeutic advantage of a long‐lived compared to short‐lived radionuclide, were calculated accordingly. While the extrapolated initial dose rate required to achieve a given BED can be affected by the inclusion of proliferation terms for both the tumor and marrow, the relative advantage factors for the longer‐lived radionuclides were not significantly affected. Longer‐lived radionuclides such as and are about three times more therapeutically effective than the shorter‐lived which is currently used in RIT. In other words, for a given therapeutic effect in the tumor, a longer‐lived radionuclide can result in a lower deleterious effect to the bone marrow than a short‐lived radionuclide. Given that bone marrow is generally considered to be the dose‐limiting organ, these results have important implications for radioimmunotherapy.
Analysis of acoustic communication by ants
Authors: Hickling Robert , Brown Richard L.
Published: July 2002

An analysis is presented of acoustic communication by ants, based on near-field theory and on data obtained from the black imported fire ant Solenopsis richteri and other sources. Generally ant stridulatory sounds are barely audible, but they occur continuously in ant colonies. Because ants appear unresponsive to airborne sound, myrmecologists have concluded that stridulatory signals are transmitted through the substrate. However, transmission through the substrate is unlikely, for reasons given in the paper. Apparently ants communicate mainly through the air, and the acoustic receptors are hairlike sensilla on the antennae that respond to particle sound velocity. This may seem inconsistent with the fact that ants are unresponsive to airborne sound (on a scale of meters), but the inconsistency can be resolved if acoustic communication occurs within the near field, on a scale of about 100 mm. In the near field, the particle sound velocity is significantly enhanced and has a steep gradient. These features can be used to exclude extraneous sound, and to determine the direction and distance of a near-field source. Additionally, we observed that the tracheal air sacs of S. richteri can expand within the gaster, possibly amplifying the radiation of stridulatory sound.
Experiments with a flow-through thermoacoustic refrigerator
Authors: Reid R. S. , Swift G. W.
Published: July 2002

Deliberate superposition of steady flow parallel to the thermoacoustic oscillations in a stack is used to cool the steady flow as it passes through the stack. The loudspeaker-driven refrigerator described here cools a flowing helium–argon mixture from 35 to 27 °C and provides additional cooling power at a traditional cold heat exchanger. The dependence of the stack’s temperature profile and of the additional cooling power on the steady flow rate are in agreement with a simple theory based on augmentation of the well-known thermoacoustic momentum, continuity, and energy equations with a single new term in the energy equation that describes the steady flow.
Estimation of viscoelastic shear properties of vocal-fold tissues based on time–temperature superposition
Authors: Chan Roger W.
Published: July 2002

Empirical data on the viscoelastic shear properties of human vocal-fold mucosa (cover) were recently reported at relatively low frequency (0.01–15 Hz). For the data to become relevant to voice production, attempts have been made to parametrize and extrapolate the data to higher frequencies using constitutive modeling [Chan and Titze, J. Acoust. Soc. Am. 107, 565–580 (2000)]. This study investigated the feasibility of an alternative approach for data extrapolation, namely the principle of time–temperature superposition (TTS). TTS is a hybrid theoretical–empirical approach widely used by rheologists to estimate the viscoelastic properties of polymeric systems at time or frequency scales not readily accessible experimentally. It is based on the observation that for many polymers, the molecular configurational changes that occur in a given time scale at a low temperature correspond to those that occur in a shorter time scale at a higher temperature. Using a rotational rheometer, the elastic shear modulus (G′) and viscous shear modulus (G″) of vocal-fold cover (superficial layer of lamina propria) tissue samples were measured at 0.01–15 Hz at relatively low temperatures (5°–37 °C). Data were empirically shifted according to TTS, yielding composite “master curves” for predicting the magnitude of the shear moduli at higher frequencies at 37 °C. Results showed that TTS may be a feasible approach for estimating the viscoelastic shear properties of vocal-fold tissues at frequencies of phonation (on the order of 100–1000 Hz).
Ultrasonic measurement of the sound velocity and attenuation in some polymers
Authors: Moustafa Aly H. A.
Published: July 2002

The sound velocity and attenuation in perspex (poly methyl methacrylate) and in reson (hydroxyl terminated polybutadiene) are determined as functions of frequency and temperature using ultrasonic pulse technique. The importance of these materials comes from the need to examine their impedance match with water and to measure their attenuation coefficients over an extended range of frequency. A pair of transducers was placed in a small tank full of water and used to transmit and receive a 2.5-MHz pulse. The temperature of the water was varied using a temperature controlled heater (or cooler). The signal was captured with and without the material sample being inserted in the transmit/receive water path, using a digital storage oscilloscope (DSO), and then processed on a PC to obtain its FFT and to compute and plot the variation of the velocity of sound in the sample material as well as its absorption coefficient. The accuracy of the method was first checked and proved to be quite good by obtaining results for perspex and comparing them with the known published data. The measurements were then repeated for reson. A frequency range of ∼1–5 MHz was covered. The lowest considered temperature was 10 °C and the largest was 40 °C.
A liquid wedge as generating technique for Lamb and Rayleigh waves
Authors: Briers R. , Leroy O. , Shkerdin G.
Published: July 2002

A model is developed to describe the excitation of Lamb and Rayleigh waves by a Gaussian beam incident on a liquid/air/solid corner. Analytical expressions for the displacement fields of the generated modes are derived by applying a mode theory that accounts for the contribution of the reflected waves. Numerical computations illustrate the optimum conditions under which the strongest stimulation of Lamb and Rayleigh waves may be expected.
Visualization of the energy flux in an ensonified fluid-loaded elastic sphere
Authors: Dean Cleon E. , Braselton James P.
Published: July 2002

The energy flux in an ensonified fluid-loaded elastic sphere is visualized by its Poynting vector field in both its instantaneous real and time-averaged complex forms. The instantaneous Poynting vector field is shown in both freeze-frame and animated versions. The instantaneous Poynting vector field can be used to follow the propagation of elastodynamic waves in the elastic sphere. The complex Poynting vector field, which is a time-averaged representation of the energy flow, is shown in terms of separate real and imaginary parts. The real part of the complex Poynting vector field shows the traveling wave component of the elastodynamic wave in the ensonified sphere. The imaginary part of the complex Poynting vector field shows the standing wave part of the elastodynamic wave in the ensonified sphere. The patterns formed by the Poynting vector field, in both its instantaneous and the real and imaginary parts of the complex form, can be identified with the various modes of the ensonified sphere and used to explain the physical processes going on inside the ensonified sphere.
Mean force on a small sphere in a sound field in a viscous fluid
Authors: Danilov S. D. , Mironov M. A.
Published: July 2002

A mean force exerted on a small rigid sphere by a sound wave in a viscous fluid is calculated. The force is expressed as a sum of drag force coming from the external steady flow existing in the absence of the sphere and contributions that are cross products of velocity and velocity derivatives of the incident field. Because of the drag force and an acoustic streaming generated near the sphere, the mean force does not coincide with the acoustic radiation pressure, i.e., the mean momentum flux carried by the sound field through any surface enclosing the sphere. If the sphere radius R is considerably smaller than the viscous wave penetration depth δ, the drag force can give the leading-order contribution (in powers of δ/R) to the mean force and the latter can then be directed against the radiation pressure. In another limit, δ≪R, the drag force and acoustic streaming play a minor role, and the mean force reduces to the radiation pressure, which can be expressed through source strengths of the scattered sound field. The effect of viscosity can then be significant only if the incident wave is locally plane traveling.