bluuebunny/crossref_metadata_2025 huggingface dataset:
Fhit is a physiological target of the protein kinase Src
Authors: Pekarsky Yuri , Garrison Preston N. , Palamarchuk Alexey , Zanesi Nicola , Aqeilan Rami I. , Huebner Kay , Barnes Larry D. , Croce Carlo M.
Published: March 2004

The
FHIT
gene is a tumor suppressor that is frequently inactivated by genomic alterations at chromosomal region 3p14.2. In the last few years, a considerable amount of data describing inactivation of
FHIT
in a variety of human malignancies and demonstrating the tumor suppressor potential of Fhit have been reported. Despite the demonstration that
FHIT
functions as a tumor suppressor, the pathway through which Fhit induces apoptosis and inhibits growth of cancer cells is not known. Our data demonstrate that Fhit is a target of tyrosine phosphorylation by the Src protein kinase. We show that Src phosphorylates Y114 of Fhit
in vitro
and
in vivo
, providing insight into a biochemical pathway involved in Fhit signaling.
Structural basis for the function of a minimembrane protein subunit of yeast oligosaccharyltransferase
Authors: Zubkov Sergey , Lennarz William J. , Mohanty Smita
Published: March 2004

N-glycosylation of proteins is an essential, highly conserved modification reaction that occurs in all eukaryotes and some prokaryotes. This process is catalyzed by oligosaccharyltransferase (OT), a multisubunit enzyme localized in the endoplasmic reticulum. Complete loss of N-glycosylation is lethal in all organisms. In
Saccharomyces cerevisiae
, OT is composed of nine nonidentical membrane proteins. Here, we report the atomic structure of an OT subunit from
S. cerevisiae
, Ost4p. This unusually small membrane protein containing only 36 residues folds into a well formed, kinked helix in the model-membrane solvent system used in this study. The residues critical for the OT activity and the stability of Stt3p-Ost4p-Ost3p subcomplex are located in helix α2, the larger cytosolic half of this kinked helix. The residues known to disrupt Ost4p-Stt3p complex form a well defined ridge in the 3D structure. Taking together prior mutational studies and the NMR structure of Ost4p, we propose that in the OT complex Stt3p is packed against the α2-helix of Ost4p by using a “ridges-into-grooves” model, with Met-18, Leu-21, and Ile-24 as the packing interface on one face, whereas Ost3p is involved in interactions with Met-19, Thr-20, Ile-22, and Val-23 on the other face.
Repression of
dMyc
expression by Wingless promotes Rbf-induced G
1
arrest in the presumptive
Drosophila
wing margin
Authors: Duman-Scheel Molly , Johnston Laura A. , Du Wei
Published: March 2004

Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless (Wg) generates G
1
arrest in the presumptive
Drosophila
wing margin is examined in detail. Wg signaling promotes activity of the
Drosophila
retinoblastoma family (Rbf) protein, which is required for G
1
arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G
1
-S regulator
Drosophila myc
(
dmyc
). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G
1
-S progression. Thus, G
1
arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress
dmyc
expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in
Drosophila
. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G
1
progression, dMyc appears to regulate growth independently of the RBF/E2f pathway.
Spontaneous proliferation, a response of naïve CD4 T cells determined by the diversity of the memory cell repertoire
Authors: Min Booki , Foucras Gilles , Meier-Schellersheim Martin , Paul William E.
Published: March 2004

T cell numbers are maintained within narrow ranges
in vivo
. Introduction of naïve cells into lymphopenic environments results in proliferation and differentiation driven by the recognition of peptide/MHC complexes and by cytokine signaling. This process, often described as homeostatic proliferation, is here referred to as spontaneous proliferation. We show that, although the presence of memory CD4 T cells of broad repertoire efficiently inhibits proliferation/differentiation of naïve CD4 T cells, a memory population of similar size comprised of cells with a repertoire of limited diversity fails to do so, implying that cells of a given specificity prevent responses of cells of the same or related specificity. This finding suggests that the immune system has evolved mechanisms to attain a memory cell repertoire of great diversity independently of foreign antigens.
Defective development of splenic and epidermal CD4+dendritic cells in mice deficient for IFN regulatory factor-2
Authors: Ichikawa Eri , Hida Shigeaki , Omatsu Yoshiki , Shimoyama Susumu , Takahara Kazuhiko , Miyagawa Shinichi , Inaba Kayo , Taki Shinsuke
Published: March 2004

Dendritic cells (DCs) play important roles in the initiation and regulation of immune responses. Although several subsets of DCs were identified according to their expression of surface molecules such as CD4, CD8, and CD11b, the regulatory mechanism for the development and homeostasis of these DC subsets remains unclear. Here we show that mice lacking IFN regulatory factor-2 (IRF-2-/-mice) exhibited a marked and selective defect in splenic CD4+CD11b+DCs, instead of CD8α+CD11b-DCs that were reported to be missing in mice lacking the related transcription factor IRF-8. Furthermore, the numbers of epidermal Langerhans cells in IRF-2-/-mice were reduced at least in part because of the lack of the CD4+CD11b+subset. Studies with radiation bone marrow chimeras as well asin vitroretrovirus-mediated gene transduction showed that IRF-2 was required cell-autonomously for the development of myeloid-related DCs. Notably, these abnormalities in DCs diminished in mice lacking both IRF-2 and the IFN-α/β receptor, indicating that IRF-2 acted through negatively regulating IFN-α/β signals. In contrast, natural killer cells still showed developmental arrest in these double mutant mice, indicating that the mode of action of IRF-2 for CD4+DC development is distinct from that for natural killer cell development. Our current findings thus pointed to a previously unknown unique cell-type-selective multimode function of IRF-2 in the regulation of lymphohematopoiesis.
Coordinate enhancers share common organizational features in the
Drosophila
genome
Authors: Erives Albert , Levine Michael
Published: March 2004

The evolution of animal diversity depends on changes in the regulation of a relatively fixed set of protein-coding genes. To understand how these changes might arise, we examined the organization of shared sequence motifs in four coordinately regulated neurogenic enhancers that direct similar patterns of gene expression in the early
Drosophila
embryo. All four enhancers possess similar arrangements of a subset of putative regulatory elements. These shared features were used to identify a neurogenic enhancer in the distantly related
Anopheles
genome. We suggest that the constrained organization of metazoan enhancers may be essential for their ability to produce precise patterns of gene expression during development. Organized binding sites should facilitate the identification of regulatory codes that link primary DNA sequence information with predicted patterns of gene activity.
Large Diameter of Palytoxin-induced Na/K Pump Channels and Modulation of Palytoxin Interaction by Na/K Pump Ligands
Authors: Artigas Pablo , Gadsby David C.
Published: March 2004

Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na+-containing (K+-free) external and internal solutions, lowest for K+-containing (Na+-free) external and internal solutions, and intermediate for the mixed external Na+/internal K+, and external K+/internal Na+ conditions; with Na+ solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na+ solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained ∼30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine+, suggesting that the narrowest section of the pore must be ∼7.5 Å wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.
Overexpressed Cavβ3 Inhibits N-type (Cav2.2) Calcium Channel Currents through a Hyperpolarizing Shift of “Ultra-slow” and “Closed-state” Inactivation
Authors: Yasuda Takahiro , Lewis Richard J. , Adams David J.
Published: March 2004

It has been shown that β auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of β3 subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed β3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at “physiological” holding potentials (HPs) of −60 and −80 mV. At a HP of −80 mV, coinjection of various concentrations (0–12.5 ng) of the β3 with Cav2.2α1 and α2δ enhanced the maximum conductance of expressed channels at lower β3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The β3-induced current suppression was reversed at a HP of −120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba2+ (40 mM) as a charge carrier also largely diminished the effect of β3 at −80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and β3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel β3 effect. Steady-state inactivation curves revealed that N-type channels exhibited “closed-state” inactivation without β3, and that β3 caused an ∼40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately −85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of β3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at −80 mV. Similar “ultra-slow” inactivation was observed for N-type channels without β3. Thus, β3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting “ultra-slow” and “closed-state” inactivation properties.
RYR2 Proteins Contribute to the Formation of Ca2+ Sparks in Smooth Muscle
Authors: Ji Guangju , Feldman Morris E. , Greene Kai Su , Sorrentino Vincenzo , Xin Hong-Bo , Kotlikoff Michael I.
Published: March 2004

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.
Differential Effects of Tyrosine Kinase Inhibitors on Volume-sensitive Chloride Current in Human Atrial Myocytes
Authors: Du Xin-Ling , Gao Zhan , Lau Chu-Pak , Chiu Shui-Wah , Tse Hung-Fat , Baumgarten Clive M. , Li Gui-Rong
Published: March 2004

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (ICl.vol) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO4−3). ICl.vol evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC50 = 22.4 μM); 100 μM genistein stimulated ICl.vol by 122.4 ± 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 150 μM) and tamoxifen (20 μM), blockers of ICl.vol. Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl− current in 1T. In contrast to the stimulatory effects of genistein, 100 μM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited ICl.vol by 38.2 ± 4.9% and 40.9 ± 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter ICl.vol. In addition, the PTP inhibitor VO4−3 (1 mM) reduced ICl.vol by 53.5 ± 4.5% (IC50 = 249.6 μM). Pretreatment with VO4−3 antagonized genistein-induced augmentation and A23- or A25-induced suppression of ICl.vol. Furthermore, the selective Src-family PTK inhibitor PP2 (5 μM) stimulated ICl.vol, mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 μM) reduced ICl.vol, mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO4−3. The results suggest that ICl.vol is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on ICl.vol, and multiple target proteins are likely to be involved.
Activation of Mammalian Unfolded Protein Response Is Compatible with the Quality Control System Operating in the Endoplasmic Reticulum
Authors: Nadanaka Satomi , Yoshida Hiderou , Kano Fumi , Murata Masayuki , Mori Kazutoshi
Published: March 2004

Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway. The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER. However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus. This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway. We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER. Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER.
Ubiquitin-mediated Targeting of a Mutant Plasma Membrane ATPase, Pma1-7, to the Endosomal/Vacuolar System in Yeast
Authors: Pizzirusso Maddalena , Chang Amy
Published: March 2004

Pma1-7 is a mutant plasma membrane ATPase that is impaired in targeting to the cell surface at 37°C and is delivered instead to the endosomal/vacuolar pathway for degradation. We have proposed that Pma1-7 is a substrate for a Golgibased quality control mechanism. By contrast with wild-type Pma1, Pma1-7 is ubiquitinated. Ubiquitination and endosomal targeting of Pma1-7 is dependent on the Rsp5-Bul1-Bul2 ubiquitin ligase protein complex but not the transmembrane ubiquitin ligase Tul1. Analysis of Pma1-7 ubiquitination in mutants blocked in protein transport at various steps of the secretory pathway suggests that ubiquitination occurs after ER exit but before endosomal entry. In the absence of ubiquitination in rsp5-1 cells, Pma1-7 is delivered to the cell surface and remains stable. Nevertheless, Pma1-7 remains impaired in association with detergent-insoluble glycolipid-enriched complexes in rsp5-1 cells, suggesting that ubiquitination is not the cause of Pma1-7 exclusion from rafts. In vps1 cells in which protein transport into the endosomal pathway is blocked, Pma1-7 is routed to the cell surface. On arrival at the plasma membrane in vps1 cells, Pma1-7 remains stable and its ubiquitination disappears, suggesting deubiquitination activity at the cell surface. We suggest that Pma1-7 sorting and fate are regulated by ubiquitination.
PtdIns-specific MPR Pathway Association of a Novel WD40 Repeat Protein, WIPI49
Authors: Jeffries Tim R. , Dove Stephen K. , Michell Robert H. , Parker Peter J.
Published: March 2004

WIPI49 is a member of a previously undescribed family of WD40-repeat proteins that we demonstrate binds 3-phosphorylated phosphoinositides. Immunofluorescent imaging indicates that WIPI49 is localized to both trans-Golgi and endosomal membranes, organelles between which it traffics in a microtubule-dependent manner. Live cell imaging establishes that WIPI49 traffics through the same set of endosomal membranes as that followed by the mannose-6-phosphate receptor (MPR), and consistent with this, WIPI49 is enriched in clathrin-coated vesicles. Ectopic expression of wild-type WIPI49 disrupts the proper functioning of this MPR pathway, whereas expression of a double point mutant (R221,222AWIPI49) unable to bind phosphoinositides does not disrupt this pathway. Finally, suppression of WIPI49 expression through RNAi, demonstrates that its presence is required for normal endosomal organization and distribution of the CI-MPR. We conclude that WIPI49 is a novel regulatory component of the endosomal and MPR pathway and that this role is dependent upon the PI-binding properties of its WD40 domain.
Rabenosyn-5 and EHD1 Interact and Sequentially Regulate Protein Recycling to the Plasma Membrane
Authors: Naslavsky Naava , Boehm Markus , Backlund Peter S. , Caplan Steve
Published: March 2004

EHD1 has been implicated in the recycling of internalized proteins to the plasma membrane. However, the mechanism by which EHD1 mediates recycling and its relationship to Rab-family–controlled events has yet to be established. To investigate further the mode of EHD1 action, we sought to identify novel interacting partners. GST-EHD1 was used as bait to isolate a ∼120-kDa species from bovine and murine brain cytosol, which was identified by mass spectrometry as the divalent Rab4/Rab5 effector Rabenosyn-5. We mapped the sites of interaction to the EH domain of EHD1, and the first two of five NPF motifs of Rabenosyn-5. Immunofluorescence microscopy studies revealed that EHD1 and Rabenosyn-5 partially colocalize to vesicular and tubular structures in vivo. To address the functional roles of EHD1 and Rabenosyn-5, we first demonstrated that RNA interference (RNAi) dramatically reduced the level of expression of each protein, either individually or in combination. Depletion of either EHD1 or Rabenosyn-5 delayed the recycling of transferrin and major histocompatibility complex class I to the plasma membrane. However, whereas depletion of EHD1 caused the accumulation of internalized cargo in a compact juxtanuclear compartment, Rabenosyn-5-RNAi caused its retention within a dispersed peripheral compartment. Simultaneous RNAi depletion of both proteins resulted in a similar phenotype to that observed with Rabenosyn-5-RNAi alone, suggesting that Rabenosyn-5 acts before EHD1 in the regulation of endocytic recycling. Our studies suggest that Rabenosyn-5 and EHD1 act sequentially in the transport of proteins from early endosomes to the endosomal recycling compartment and back to the plasma membrane.
Cytoplasmic Dynein–Dynactin Complex Is Required for Spermatid Growth but Not Axoneme Assembly inDrosophila
Authors: Ghosh-Roy Anindya , Kulkarni Madhura , Kumar Vikash , Shirolikar Seema , Ray Krishanu
Published: March 2004

Spermatids derived from a single gonial cell remain interconnected within a cyst and elongate by synchronized growth inside the testis in Drosophila. Cylindrical spectrin-rich elongation cones form at their distal ends during the growth. The mechanism underlying this process is poorly understood. We found that developing sperm tails were abnormally coiled at the growing ends inside the cysts in the Drosophila Dynein light chain 1 (ddlc1) hemizygous mutant testis. A quantitative assay showed that average number of elongation cones was reduced, they were increasingly deformed, and average cyst lengths were shortened in ddlc1 hemizygous testes. These phenotypes were further enhanced by additional partial reduction of Dhc64C and Glued and rescued by Myc-PIN/LC8 expression in the gonial cells in ddlc1 backgrounds. Furthermore, DDLC1, DHC, and GLUED were enriched at the distal ends of growing spermatids. Finally, ultrastructure analysis of ddlc1 testes revealed abnormally formed interspermatid membrane, but the 9 + 2 microtubule organization, the radial spoke structures, and the Dynein arms of the axoneme were normal. Together, these findings suggest that axoneme assembly and spermatid growth involve independent mechanisms in Drosophila and DDLC1 interacts with the Dynein–Dynactin complex at the distal ends of spermatids to maintain the spectrin cytoskeleton assembly and cell growth.
HIV-1 Tat Enters T Cells Using Coated Pits before Translocating from Acidified Endosomes and Eliciting Biological Responses
Authors: Vendeville Agnès , Rayne Fabienne , Bonhoure Anne , Bettache Nadir , Montcourrier Philippe , Beaumelle Bruno
Published: March 2004

The HIV-1 Tat protein is secreted by infected cells. Extracellular Tat can affect bystander uninfected T cells and induce numerous biological responses such as apoptosis and cytokine secretion. Tat is likely involved in several immune disorders during AIDS. Nevertheless, it is not known whether Tat triggers cell responses directly upon binding to signaling receptors at the plasma membrane or after delivery to the cytosol. The pathway that enables Tat to reach the cytosol is also unclear. Here we visualized Tat within T-cell–coated pits and endosomes. Moreover, inhibitors of clathrin/AP-2–mediated uptake such as chlorpromazine, activated RhoA, or dominant-negative mutants of Eps15, intersectin, dynamin, or rab5 impaired Tat delivery to the cytosol by preventing its endocytosis. Molecules neutralizing low endosomal pH or Hsp90 inhibitors abolished Tat entry at a later stage by blocking its endosomal translocation, as directly shown using a cell-free translocation assay. Finally, endosomal pH neutralization prevented Tat from inducing T-cell responses such as NF-κB activation, apoptosis, and interleukin secretion, indicating that cytosolic delivery is required for Tat signaling. Hence, Tat enters T cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH–induced and Hsp90-assisted endosomal translocation. Cell responses are then induced from the cytosol.
Measuring the Size of Biological Nanostructures with Spatially Modulated Illumination Microscopy
Authors: Martin Sonya , Failla Antonio Virgilio , Spöri Udo , Cremer Christoph , Pombo Ana
Published: March 2004

Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads (∼40–100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of ∼70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.
Modulation of Microtubule Dynamics by Tau in Living Cells: Implications for Development and Neurodegeneration
Authors: Bunker Janis M. , Wilson Leslie , Jordan Mary Ann , Feinstein Stuart C.
Published: March 2004

The neural microtubule-associated protein tau binds to and stabilizes microtubules. Because of alternative mRNA splicing, tau is expressed with either 3 or 4 C-terminal repeats. Two observations indicate that differences between these tau isoforms are functionally important. First, the pattern of tau isoform expression is tightly regulated during development. Second, mutation-induced changes in tau RNA splicing cause neuronal cell death and dementia simply by altering the isoform expression ratio. To investigate whether 3- and 4-repeat tau differentially regulate microtubule behavior in cells, we microinjected physiological levels of these two isoforms into EGFP-tubulin–expressing cultured MCF7 cells and measured the effects on the dynamic instability behavior of individual microtubules by time-lapse microscopy. Both isoforms suppressed microtubule dynamics, though to different extents. Specifically, 4-repeat tau reduced the rate and extent of both growing and shortening events. In contrast, 3-repeat tau stabilized most dynamic parameters about threefold less potently than 4-repeat tau and had only a minimal ability to suppress shortening events. These differences provide a mechanistic rationale for the developmental shift in tau isoform expression and are consistent with a loss-of-function model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal cell death and dementia.
Assessing the Probability That a Positive Report is False: An Approach for Molecular Epidemiology Studies
Authors: Wacholder Sholom , Chanock Stephen , Garcia-Closas Montserrat , El ghormli Laure , Rothman Nathaniel
Published: March 2004

Too many reports of associations between genetic variants and common cancer sites and other complex diseases are false positives. A major reason for this unfortunate situation is the strategy of declaring statistical significance based on a P value alone, particularly, any P value below .05. The false positive report probability (FPRP), the probability of no true association between a genetic variant and disease given a statistically significant finding, depends not only on the observed P value but also on both the prior probability that the association between the genetic variant and the disease is real and the statistical power of the test. In this commentary, we show how to assess the FPRP and how to use it to decide whether a finding is deserving of attention or “noteworthy.” We show how this approach can lead to improvements in the design, analysis, and interpretation of molecular epidemiology studies. Our proposal can help investigators, editors, and readers of research articles to protect themselves from overinterpreting statistically significant findings that are not likely to signify a true association. An FPRP-based criterion for deciding whether to call a finding noteworthy formalizes the process already used informally by investigators—that is, tempering enthusiasm for remarkable study findings with considerations of plausibility.
Role of Unc51.1 and its binding partners in CNS axon outgrowth
Authors: Tomoda Toshifumi , Kim Jee Hae , Zhan Caixin , Hatten Mary E.
Published: March 2004

Previous studies showed that the serine/threonine kinase Unc51.1 is one of the earliest genes in neuronal differentiation and is required for granule cell axon formation. To examine the mechanism of Unc51.1 regulation of axon extension, we have identified two direct binding partners. The first, SynGAP, a negative regulator of Ras, is expressed within axons and growth cones of developing granule cells. Overexpression of SynGAP blocks neurite outgrowth by a mechanism that involves Ras-like GTPase cascade. The second binding partner is a PDZ domain-containing scaffolding protein, Syntenin, that binds Rab5 GTPase, the activity of which is attenuated by SynGAP. Thus, our results demonstrate that the Unc51.1-containing protein complex governs axon formation via Ras-like GTPase signaling and through regulation of the Rab5-mediated endocytic pathways within developing axons.
Vestibular defects in head-tilt mice result from mutations inNox3, encoding an NADPH oxidase
Authors: Paffenholz Rainer , Bergstrom Rebecca A. , Pasutto Francesca , Wabnitz Philipp , Munroe Robert J. , Jagla Wolfgang , Heinzmann Ulrich , Marquardt Andreas , Bareiss Armin , Laufs Jürgen , Russ Andreas , Stumm Gabriele , Schimenti John C. , Bergstrom David E.
Published: March 2004

The vestibular system of the inner ear is responsible for the perception of motion and gravity. Key elements of this organ are otoconia, tiny biomineral particles in the utricle and the saccule. In response to gravity or linear acceleration, otoconia deflect the stereocilia of the hair cells, thus transducing kinetic movements into sensorineural action potentials. Here, we present an allelic series of mutations at the otoconia-deficient head tilt (het) locus, affecting the gene for NADPH oxidase 3 (Nox3). This series of mutations identifies for the first time a protein with a clear enzymatic function as indispensable for otoconia morphogenesis.
Phosphorylation of RCC1 in mitosis is essential for producing a high RanGTP concentration on chromosomes and for spindle assembly in mammalian cells
Authors: Li Hoi-Yeung , Zheng Yixian
Published: March 2004

Spindle assembly is subject to the regulatory controls of both the cell-cycle machinery and the Ran-signaling pathway. An important question is how the two regulatory pathways communicate with each other to achieve coordinated regulation in mitosis. We show here that Cdc2 kinase phosphorylates the serines located in or near the nuclear localization signal (NLS) of human RCC1, the nucleotide exchange factor for Ran. This phosphorylation is necessary for RCC1 to generate RanGTP on mitotic chromosomes in mammalian cells, which in turn is required for spindle assembly and chromosome segregation. Moreover, phosphorylation of the NLS of RCC1 is required to prevent the binding of importin α and β to RCC1, thereby allowing RCC1 to couple RanGTP production to chromosome binding. These findings reveal that the cell-cycle machinery directly regulates the Ran-signaling pathway by placing a high RanGTP concentration on the mitotic chromosome in mammalian cells.
Specificity of microRNA target selection in translational repression
Authors: Doench John G. , Sharp Phillip A.
Published: March 2004

MicroRNAs (miRNAs) are a class of noncoding RNAs found in organisms as evolutionarily distant as plants and mammals, yet most of the mRNAs they regulate are unknown. Here we show that the ability of an miRNA to translationally repress a target mRNA is largely dictated by the free energy of binding of the first eight nucleotides in the 5′ region of the miRNA. However, G:U wobble base-pairing in this region interferes with activity beyond that predicted on the basis of thermodynamic stability. Furthermore, an mRNA can be simultaneously repressed by more than one miRNA species. The level of repression achieved is dependent on both the amount of mRNA and the amount of available miRNA complexes. Thus, predicted miRNA:mRNA interactions must be viewed in the context of other potential interactions and cellular conditions.
Calcium–lead fluoro-vanadinite apatites. I. Disequilibrium structures
Authors: Dong ZhiLi , White T. J.
Published: March 2004

The synthetic vanadinites (Pb
x
Ca10−x
)(VO4)6F2δ, 1 < x < 9, adopt a P63/m apatite structure with 9.7590 (1) ≤ a ≤ 10.1179 (1) Å and 7.0434 (3) ≤ c ≤ 7.4021 (1) Å. The partitioning of calcium and lead over the A
I(4f) and A
II(6h) positions is nonstoichiometric with lead preferentially entering the larger A
II site. High-resolution electron microscopy showed that samples annealed for 10 h at 1073 K are in disequilibrium with calcium- and lead-rich microdomains co-existing at unit-cell scales. For (Pb5Ca5)(VO4)6F2δ, sintering in excess of 2 weeks is required for the metals to order macroscopically. As annealing progresses, c/a, the partitioning coefficient k
Pb(A
I/A
II) and the A
IO6 metaprism twist angle (φ) adjust cooperatively to enlarge the apatite channel, and thereby accommodate higher lead content. These results demonstrate that φ is a sensitive measure of disequilibrium and a useful device for monitoring changes in apatite topology as a function of composition.
Hydrogen-bonded adducts of triphenylsilanol with diamines: a finite ten-molecule aggregate, and chain, sheet and framework structures built from O—H...O, O—H...N and C—H...π(arene) hydrogen bonds
Authors: Turkington David E. , Lough Alan J. , Ferguson George , Glidewell Christopher
Published: March 2004

The structures of five hydrogen-bonded adducts of triphenylsilanol, Ph3SiOH, with diamines have been determined. In the 4:1 adduct formed with 1,4-diazabicyclo[2.2.2]octane, (Ph3SiOH)4·C6H12N2 (I) (triclinic, P\bar 1, Z′ = 2), there are two independent five-component aggregates built from O—H...N and O—H...O hydrogen bonds, in one of which the diamine exhibits orientational disorder: these two aggregates are linked into a cyclic ten-molecule unit by means of two independent C—H...π(arene) hydrogen bonds. The 4:1 adduct formed with 1,2-bis(4-pyridyl)ethene, (Ph3SiOH)4·C12H10N2 (II) (triclinic, P\bar 1, Z′ = 0.5), forms a similar five-component aggregate which lies across a centre of inversion: a single C—H...π(arene) hydrogen bond links these aggregates into a molecular ladder. With N,N′-dimethylpiperazine, triphenylsilanol forms a 2:1 adduct, (Ph3SiOH)2·C6H14N2 (III) (monoclinic, P21/c, Z′ = 0.5), in which a three-component aggregate built from O—H...N hydrogen bonds lies across a centre of inversion: two independent C—H...π(arene) hydrogen bonds link these aggregates into sheets. In the hydrated 2:1 adduct formed with 1,2-bis(4′-bipyridyl)ethane, (Ph3SiOH)2·C12H12N2·H2O (IV) (triclinic, P\bar 1, Z′ = 1), a combination of two independent O—H...O and two independent N—H...O hydrogen bonds links the five molecular components into a centrosymmetric eight-molecule aggregate, and six independent C—H...π(arene) hydrogen bonds link these chains into a continuous three-dimensional framework structure. In the dihydrated 2:1 adduct formed with 4,4′-bipyridyl, (Ph3SiOH)2·C10H8N2·(H2O)2 (V) (triclinic, P\bar 1, Z′ = 1), a combination of four independent O—H...O and two independent N—H...O hydrogen bonds links the five molecular components into a chain of rings, and four independent C—H...π(arene) hydrogen bonds link these chains into a continuous three-dimensional framework structure.
Two tautomers in one crystal: 4(5)-nitro-5(4)-methoxyimidazole
Authors: Kubicki Maciej
Published: March 2004

The case of prototropic annular tautomerism in an imidazole derivative has been found. The crystal structure contains a 50:50 mixture of two tautomers: 4-nitro-5-methoxyimidazole and 5-nitro-4-methoxyimidazole. The X-ray experiment actually shows the superposition of these compounds; it appears as if the structure is centrosymmetric and the N—H hydrogen atoms are disordered over two ring N atoms. Owing to the hydrogen-bond pattern, the values of their site occupation factors have to be exactly equal to 1/2. The molecules are connected into a three-dimensional network by means of N—H...N and C—H...O hydrogen bonds.
The surprisingly elusive crystal structure of sodium metabisulfite
Authors: Carter Kay L. , Siddiquee Tasneem A. , Murphy Kristen L. , Bennett Dennis W.
Published: March 2004

The crystal structure of Na2S2O5, a simple and common ionic compound, is reported here for the first time. The crystals form non-merohedral twins, with the twin domains related by a twofold axis which bisects the angle between the a and c axes of each unit cell. The structure was determined from a single-crystal fragment of a twinned crystal that had undergone cleavage along the twin boundary. In addition to the problems associated with twinning, space-group determination proved difficult as well, with the structure initially determined in the P21 space group appearing to be disordered with two rotational conformers of the metabisulfite ion occupying equivalent sites in the lattice. An analysis at low temperature provided new weak reflections which were inconsistent with the original unit cell, but indexed to the correct unit cell, allowing for space group and crystal structure determination. The apparent structure, which appeared disordered in P21, seems to have resulted from an apparently fortuitous superposition of two conformationally inequivalent S2O_5^{2-} anions in the asymmetric unit of the correct structure in the P21/n space group. The metabisulfite ions in this structure do not adopt the Cs
geometry observed in previously determined crystal structures containing S2O_5^{2-}. The structures of both ions in the asymmetric unit are effectively conformational mirror images of one another with two of the O atoms on each S atom in the ion approaching an eclipsed geometry. This observation provides further evidence that the structures of sulfur-oxy anions in the solid state are dictated mainly by interionic coulombic forces rather than by intraionic bonding interactions
Pressure and temperature effects on the degree of symmetry and chirality of the molecular building blocks of low quartz
Authors: Yogev-Einot Dina , Avnir David
Published: March 2004

We establish quantitative correlations between pressure and temperature (PT) changes, and the degree of symmetry and of chirality of the main molecular building blocks of low quartz that these PT changes induce. The distortion from ideal tetrahedral symmetry, from helicity (deviation from C
2 symmetry), and the level of chirality are evaluated quantitatively using the continuous-symmetry and chirality-measures approach. These measures are global and reflect all changes in bond angles and bond lengths. The specific molecular building blocks analyzed are the SiO4 elementary building block (which is found to be chiral!), the Si(OSi)4 unit, the second-shell SiSi4 tetrahedron [composed of the five Si atoms of Si(OSi)4] and the four-tetrahedra helix fragment, —O(SiO3)4—. The temperature and pressure effects on symmetry and chirality were found to mirror each other in all building blocks. By employing this quantitative approach to symmetry and chirality we were able to combine the pressure effects and temperature effects into a unified picture. Furthermore, the global nature of the symmetry measure allows the comparison of the behavior of isostructural materials such as germania and quartz. For these crystals it has been shown that the symmetry/chirality behavior of germania at low pressures is a predictor for the behavior of these structural properties in quartz at higher pressures. Finally, given that the rigid SiO4 unit undergoes only minor structural changes, it has been a useful observation that the symmetry/chirality of the small SiSi4 tetrahedron is a very sensitive probe for the symmetry and chirality changes in quartz as a whole.
Structures of piperazine, piperidine and morpholine
Authors: Parkin Andrew , Oswald Iain D. H. , Parsons Simon
Published: March 2004

The crystal structures of piperazine, piperidine and morpholine have been determined at 150 K. All three structures are characterized by the formation of NH...N hydrogen-bonded chains. In piperazine these are linked to form sheets, but the chains are shifted so that the molecules interleave. In morpholine there are in addition weak CH...O interactions. Topological analyses show that these three structures are closely related to that of cyclohexane-II, which can be described in terms of a pseudo-cubic close-packed array of molecules in a familiar ABC layered arrangement. While the positions of the molecules within each layer are similar, hydrogen bonding occurs between the ABC layers and in order to accommodate this the molecules are rotated relative to those in cyclohexane-II. Piperidine and morpholine also adopt layered structures, with hydrogen-bonding or CH...O interactions between the layers. In these cases, however, the layering more resembles a hexagonal close-packed arrangement.
Crystal structure of the supramolecular linear polymer formed by the self-assembly of mono-6-deoxy-6-adamantylamide-β-cyclodextrin
Authors: Tellini Victor Hugo Soto , Jover Aida , Galantini Luciano , Meijide Francisco , Tato José Vázquez
Published: March 2004

Mono-6-deoxy-6-adamantylamide-β-cyclodextrin–dimethylformamide–15H2O, C53H85NO35·C3H7NO·15H2O, crystallizes in the orthorhombic space group P212121. The adamantyl group is inserted into the cyclodextrin cavity of the adjacent molecule, entering by the side of the secondary hydroxy rim, thus forming a supramolecular linear polymer by self-assembly. Adjacent macrocycles are linked into columns by hydrogen bonds involving the nearest glucose residues, and the structure is further stabilized by their involvement in hydrogen bonding with water molecules which reside in channels surrounding the polymer columns, thus acting as bridges between the cyclodextrin units. The centroid of the adamantyl group lies below the plane formed by the seven glycosidic O atoms of the host cyclodextrin, excluding water molecules from the secondary side of β-cyclodextrin (β-CD). Between the adamantyl group and the primary hydroxy rim of the cyclodextrin cavity lies a dimethylformamide molecule, which shields the hydrophobic adamantyl group from the primary hydroxy rim of its carrying β-CD and excludes water molecules from the primary side of the β-CD cavity.
Space group Cc: an update
Authors: Marsh Richard E.
Published: March 2004

A recent survey of the Cambridge Structural Database, CSD [Allen (2002). Acta Cryst. B58, 380–388], shows that the percentage of incorrect assignments of the space group Cc has remained at about 10% since the last survey in 1997.
Measuring alliance value and risk
Authors: Beth Stanek Mary
Published: March 2004

Alliances have been the focus of research studies often because they offer an attractive alternative to traditional arrangements. With a global economy, rapid product cycles, capital constraints and advances in technology, no one firm has all the capability to maintain and grow market share. Factors such as these are leading firms to consider different forms of partnerships including alliances. Contemporary alliance research is often segmented by types of arrangements, durations, returns, failures and best practices. Studies that evaluate and rank specific alliance criteria are somewhat limited. To explore specific alliance risk and value variables a study was conducted at a large manufacturing organization with extensive alliance history. A questionnaire was developed and distributed that contained both ordinal and scale rankings of specific alliance value and risk criteria. The aggregate responses were prioritized and a weight was assigned allowing for quantitative assessment for both alliance value and risk. The information was later converted into an alliance value/risk check sheet to be used to rank multiple projects.
Managing human capital
Authors: Lewis Jeff , Wright Philip C. , Geroy Gary D.
Published: March 2004

One of the keys to success in the digital economy is the appropriate management of human capital. Because of sweeping technological changes, old behaviors of human capital management may not be suitable in the digital domain. Traditional businesses will need to evolve and radically transition themselves into an electronic business mode. This study implements a rigorous qualitative case study methodology to examine an electronic business corporate entrepreneurship team within a large, traditional business. Faced with the need to work at “Web speed”, this group had to adapt quickly, both to complete its task on time and to master a steep learning curve. This process is examined systematically, in order to examine the major research question: “What are the changes necessary to support the transition of a new venture creation team as it moves from the traditional domain into the digital economy domain?” Through in‐depth interviews, this study examines the transition process, drawing conclusions that may have far‐reaching ramifications for both practice and theory in human capital management.
A matter of style: reconciling Henri and Henry
Authors: Lamond David
Published: March 2004

During the twentieth century, much of the discussion about managerial behaviour centred on the difference between management functions and manager roles, with much of the debate centring on “Who is right, Mintzberg or Fayol?” Reports on a study, involving 523 Australian managers, which suggests both are right – Fayol gave us management as we would like it to be and Mintzberg gave us management as it is. In doing so, promulgates a set of new constructions of managerial behaviour – preferred managerial style (management as we would like it to be) and enacted managerial style (management as it is). Taken together, we now have available to us a more integrated theoretical base for research on management and managerial behaviour, and a measure that can be used to progress the required research.