bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic Toxicity and Carcinogenic Potential of Tris-(1,3-Dichloro-2-propyl) Phosphate in Sprague-Dawley Rat
Authors: Freudenthal Ralph I. , Henrich Richard T.
Published: July 2002

The Flammable Fabrics Act of 1953 and its amendments established a need for flame-resistant fabrics. Tris-(1,3-dichloro-2-propyl) phosphate (TDCPP) was briefly used in apparel fabrics to assist in the compliance with federal flammability standards, and continues to be used as a flame retardant in flexible polyurethane foams. A chronic toxicity and carcinogenicity bioassay was conducted in Sprague-Dawley rats to determine the toxicologic and carcinogenic potential of TDCPP after repeated exposure. Four groups of animals, each consisting of 60 male and 60 female rats, received via their diet a daily dose of either 0, 5, 20, or 80 mg TDCPP per kg body weight for up to 24 months. Diets were adjusted after each weekly (first 13 weeks) or biweekly (weeks 14 through 104) body weight and food consumption measurement to achieve and maintain the indicated doses. Ten animals per sex were taken from each group for interim sacrifice at the end of the 12th month. Body weights, food consumption, clinical signs, and hematological and clinical chemistry parameters were measured periodically, and ophthalmoscopic examinations were conducted on all animals. After complete postmortem examination of all animals, microscopic examination of all tissues was conducted for the control and high-dose animals. Liver, kidneys, testes, and adrenal glands were examined from all animals. Mortality was significantly higher and body weights were significantly lower in the high-dose group when compared to control animals. Certain hematology parameters, such as hemoglobin, hematocrit, and total erythrocyte values, were decreased in the high-dose animals. Ophthalmoscope examination revealed no treatment-related changes. Microscopic examination revealed a higher incidence of benign neoplasms and non-neoplastic alterations in several organs of the mid and high dose animals. The no-observed-adverse-effect level (NOAEL) for chronic toxicity and neoplastic activity was the dietary dose of 5 mg/kg/day.
In Vitro and In Vivo Inhibition of Rat Brain Nitric Oxide Synthase Activity by Phencyclidine
Authors: Desaiah D. , Pande M. , Vig P. J. S. , Cameron J. A. , Ali S. F.
Published: July 2002

Phencyclidine (PCP) is a widely abused psychoactive drug that perturbs many neurotransmitter systems studied to date. Nitric oxide (NO) has been established as a neuronal messenger and its rapid diffusibility across cell membranes makes NO an extensive and versatile messenger in brain development and functioning. The present study was initiated to investigate the effect of PCP on rat brain nitric oxide synthase (NOS) activity both in vitro and in vivo. Brain cytosolic fractions from normal rats were used for in vitro and in vivo studies. The rats were treated with a single dose of PCP (10 mg/kg, intraperitoneally); the brains were removed at 0, 1, 2, 6, and 12 hours after PCP treatment and the cytosolic fractions were prepared by homogenization and centrifugation. NOS activity was assessed by quantifying the release of [3H]-citrulline from [3H]-arginine. PCP significantly inhibited rat brain NOS in vitro in a concentration (0.05–2 mM)-dependent manner. The kinetic evaluation of arginine, NADPH, and Ca2+ activation of NOS revealed that PCP (0.5 mM) inhibited NOS activity competitively with respect to arginine and NADPH and noncompetitively inhibited with respect to Ca2+. PCP also caused a time-dependent reduction of brain NOS activity in vivo as early as 1 hour after treatment. Even after 12 hours of PCP treatment, NOS activity did not reverse to its normal level as compared to the control group, suggesting sequestration and persistence of the drug in the central nervous system. These results suggest that inhibition of brain NOS by PCP might be one of the mechanisms through which PCP causes neurotoxicity.
Conversational Cohesion Patterns In Men And Women With Alzheimer'S Disease: A Longitudinal Study
Authors: Ripich Danielle N. , Carpenter Brian D. , Ziol Elaine W.
Published: July 2002

The use of cohesion devices in conversations was examined in 60 individuals (31 women, 29 men) with early to midstage Alzheimer's disease (AD) and 47 non‐demented elderly (NE) (27 women, 20 men). AD individuals produced more referent errors than NE, although AD and NE subjects otherwise demonstrated similar use of cohesion devices. AD women showed more frequent use of two elliptical devices (clause omission and word omission) compared with AD men and NE men and women. A subset of 23 AD subjects (13 women, 10 men) who completed four data collections (entry, 6, 12 and 18 months) was followed longitudinally. They demonstrated a significant decline in the number of ellipses and conjunctions at 18 months post‐entry. As AD subjects produced fewer and shorter utterances across time, their use of all cohesive devices declined. No gender differences were found over time. Although referencing errors differentiated early to midstage AD from NE, conversational discourse tasks alone may have limited clinical value to assess and monitor communication competence.
Mechanism of Free Radical Production in Exhaustive Exercise in Humans and Rats; Role of Xanthine Oxidase and Protection by Allopurinol
Authors: Viña José , Gimeno Amparo , Sastre Juan , Desco Carmen , Asensi Miguel , Pallardó Federico V. , Cuesta Andrés , Ferrero José Antonio , Terada Lance S. , Repine John E.
Published: July 2002

Exhaustive exercise generates free radicals. However, the source of this oxidative damage remains controversial. The aim of this paper was to study further the mechanism of exercise‐induced production of free radicals. Testing the hypothesis that xanthine oxidase contributes to the production of free radicals during exercise, we found not only that exercise caused an increase in blood xanthine oxidase activity in rats but also that inhibiting xanthine oxidase with allopurinol prevented exercise‐induced oxidation of glutathione in both rats and in humans. Furthermore, inhibiting xanthine oxidase prevented the increases in the plasma activity of cytosolic enzymes (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase) seen after exhaustive exercise. Our results provide evidence that xanthine oxidase is responsible for the free radical production and tissue damage during exhaustive exercise. These findings also suggest that mitochondria play a minor role as a source of free radicals during exhaustive physical exercise.
Brain Myosin V Is a Synaptic Vesicle-associated Motor Protein: Evidence for a Ca2+-dependent Interaction with the Synaptobrevin–Synaptophysin Complex
Authors: Prekeris Rytis , Terrian David M.
Published: July 2002

Brain myosin V is a member of a widely distributed class of unconventional myosins that may be of central importance to organelle trafficking in all eukaryotic cells. Molecular constituents that target this molecular motor to organelles have not been previously identified. Using a combination of immunopurification, extraction, cross-linking, and coprecipitation assays, we demonstrate that the tail domain of brain myosin V forms a stable complex with the synaptic vesicle membrane proteins, synaptobrevin II and synaptophysin. While myosin V was principally bound to synaptic vesicles during rest, this putative transport complex was promptly disassembled upon the depolarization-induced entry of Ca2+ into intact nerve endings. Coimmunoprecipitation assays further indicate that Ca2+ disrupts the in vitro binding of synaptobrevin II to synaptophysin in the presence but not in the absence of Mg2+. We conclude that hydrophilic forces reversibly couple the myosin V tail to a biochemically defined class of organelles in brain nerve terminals.
Supervillin (p205): A Novel Membrane-associated, F-Actin–binding Protein in the Villin/Gelsolin Superfamily
Authors: Pestonjamasp Kersi N. , Pope Robert K. , Wulfkuhle Julia D. , Luna Elizabeth J.
Published: July 2002

Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE–purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell–cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions “rings.” At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, “supervillin.” We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.
Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts
Authors: Kouranov Andrei , Schnell Danny J.
Published: July 2002

We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.
Essential Role of the Disulfide-bonded Loop of Chromogranin B for Sorting to Secretory Granules Is Revealed by Expression of a Deletion Mutant in the Absence of Endogenous Granin Synthesis
Authors: Krömer Andreas , Glombik Michael M. , Huttner Wieland B. , Gerdes Hans-Hermann
Published: July 2002

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH2-terminal disulfide– bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Δcys-hCgB) lacking the 22–amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Δcys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Δcys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus–mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.
Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins
Authors: Huang Da Wei , Fanti Laura , Pak Daniel T.S. , Botchan Michael R. , Pimpinelli Sergio , Kellum Rebecca
Published: July 2002

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.
Regulated Targeting of BAX to Mitochondria
Authors: Goping Ing Swie , Gross Atan , Lavoie Josée N. , Nguyen Mai , Jemmerson Ronald , Roth Kevin , Korsmeyer Stanley J. , Shore Gordon C.
Published: July 2002

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, our results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting.
Membrane Fusion Mediated by Baculovirus gp64 Involves Assembly of Stable gp64 Trimers into Multiprotein Aggregates
Authors: Markovic Ingrid , Pulyaeva Helena , Sokoloff Alexander , Chernomordik Leonid V.
Published: July 2002

The baculovirus fusogenic activity depends on the low pH conformation of virally-encoded trimeric glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved in gp64 fusion machine. First, dithiothreitol (DTT)- based reduction of intersubunit disulfides was found to reversibly inhibit fusion, as assayed by fluorescent probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE. Thus, stable (i.e., with intact intersubunit disulfides) gp64 trimers, rather than independent monomers, drive fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes were stabilized by cell–surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable gp64 trimer. Although DTT-destabilized trimers were still capable of assuming the low pH conformation, they failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in timing, and was dependent on (a) low pH application, (b) stable gp64 trimers, and (c) cell–cell contacts, suggests that such multimeric complexes represent a fusion machine.
Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein
Authors: Kuroda Shun'ichi , Nakagawa Noritaka , Tokunaga Chiharu , Tatematsu Kenji , Tanizawa Katsuyuki
Published: July 2002

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.
Direct Interaction of Pericentrin with Cytoplasmic Dynein Light Intermediate Chain Contributes to Mitotic Spindle Organization
Authors: Purohit Aruna , Tynan Sharon H. , Vallee Richard , Doxsey Stephen J.
Published: July 2002

Pericentrin is a conserved protein of the centrosome involved in microtubule organization. To better understand pericentrin function, we overexpressed the protein in somatic cells and assayed for changes in the composition and function of mitotic spindles and spindle poles. Spindles in pericentrin-overexpressing cells were disorganized and mispositioned, and chromosomes were misaligned and missegregated during cell division, giving rise to aneuploid cells. We unexpectedly found that levels of the molecular motor cytoplasmic dynein were dramatically reduced at spindle poles. Cytoplasmic dynein was diminished at kinetochores also, and the dynein-mediated organization of the Golgi complex was disrupted. Dynein coimmunoprecipitated with overexpressed pericentrin, suggesting that the motor was sequestered in the cytoplasm and was prevented from associating with its cellular targets. Immunoprecipitation of endogenous pericentrin also pulled down cytoplasmic dynein in untransfected cells. To define the basis for this interaction, pericentrin was coexpressed with cytoplasmic dynein heavy (DHCs), intermediate (DICs), and light intermediate (LICs) chains, and the dynamitin and p150Glued subunits of dynactin. Only the LICs coimmunoprecipitated with pericentrin. These results provide the first physiological role for LIC, and they suggest that a pericentrin–dynein interaction in vivo contributes to the assembly, organization, and function of centrosomes and mitotic spindles.
ER to Golgi Transport
Authors: Alvarez Cecilia , Fujita Hideaki , Hubbard Ann , Sztul Elizabeth
Published: July 2002

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti–p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca2+-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15°C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.
Bifurcation of Cell Migratory and Proliferative Signaling by the Adaptor Protein Shc
Authors: Collins Lila R. , Ricketts William A. , Yeh Linda , Cheresh David
Published: July 2002

Cytokines and extracellular matrix proteins initiate signaling cascades that regulate cell migration and proliferation. Evidence is provided that the adaptor protein Shc can differentially regulate these processes. Specifically, under growth factor–limiting conditions, Shc stimulates haptotactic cell migration without affecting anchorage-dependent proliferation. However, when growth factors are present, Shc no longer influences cell migration; rather, Shc is crucial for DNA synthesis. Mutational analysis of Shc demonstrates that, while tyrosine phosphorylation is required for both DNA synthesis and cell migration, the switch in Shc signaling is associated with differential use of Shc's phosphotyrosine interacting domains; the PTB domain regulates haptotaxis, while the SH2 domain is selectively required for proliferation.
The
UNC-112
Gene in Caenorhabditis elegansEncodes a Novel Component of Cell–Matrix Adhesion Structures Required for Integrin Localization in the Muscle Cell Membrane
Authors: Rogalski Teresa M. , Mullen Gregory P. , Gilbert Mary M. , Williams Benjamin D. , Moerman Donald G.
Published: July 2002

Embryos homozygous for mutations in the unc-52, pat-2, pat-3, and unc-112 genes of C. elegans exhibit a similar Pat phenotype. Myosin and actin are not organized into sarcomeres in the body wall muscle cells of these mutants, and dense body and M-line components fail to assemble. The unc-52 (perlecan), pat-2 (α-integrin), and pat-3 (β-integrin) genes encode ECM or transmembrane proteins found at the cell–matrix adhesion sites of both dense bodies and M-lines. This study describes the identification of the unc-112 gene product, a novel, membrane-associated, intracellular protein that colocalizes with integrin at cell–matrix adhesion complexes. The 720–amino acid UNC-112 protein is homologous to Mig-2, a human protein of unknown function. These two proteins share a region of homology with talin and members of the FERM superfamily of proteins.

We have determined that a functional UNC-112::GFP fusion protein colocalizes with PAT-3/β-integrin in both adult and embryonic body wall muscle. We also have determined that UNC-112 is required to organize PAT-3/β-integrin after it is integrated into the basal cell membrane, but is not required to organize UNC-52/perlecan in the basement membrane, nor for DEB-1/vinculin to localize with PAT-3/β-integrin. Furthermore, UNC-112 requires the presence of UNC-52/perlecan and PAT-3/β-integrin, but not DEB-1/vinculin to become localized to the muscle cell membrane.
Visualization of Trna Movements on theEscherichia coli70s Ribosome during the Elongation Cycle
Authors: Agrawal Rajendra K. , Spahn Christian M.T. , Penczek Pawel , Grassucci Robert A. , Nierhaus Knud H. , Frank Joachim
Published: July 2002

Three-dimensional cryomaps have been reconstructed for tRNA–ribosome complexes in pre- and posttranslocational states at 17-Å resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNAfMet-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon–anticodon interaction at the E site appears feasible.
The Reduction of Chromosome Number in Meiosis Is Determined by Properties Built into the Chromosomes
Authors: Paliulis Leocadia V. , Nicklas R. Bruce
Published: July 2002

In meiosis I, two chromatids move to each spindle pole. Then, in meiosis II, the two are distributed, one to each future gamete. This requires that meiosis I chromosomes attach to the spindle differently than meiosis II chromosomes and that they regulate chromosome cohesion differently. We investigated whether the information that dictates the division type of the chromosome comes from the whole cell, the spindle, or the chromosome itself. Also, we determined when chromosomes can switch from meiosis I behavior to meiosis II behavior. We used a micromanipulation needle to fuse grasshopper spermatocytes in meiosis I to spermatocytes in meiosis II, and to move chromosomes from one spindle to the other. Chromosomes placed on spindles of a different meiotic division always behaved as they would have on their native spindle; e.g., a meiosis I chromosome attached to a meiosis II spindle in its normal fashion and sister chromatids moved together to the same spindle pole. We also showed that meiosis I chromosomes become competent meiosis II chromosomes in anaphase of meiosis I, but not before. The patterns for attachment to the spindle and regulation of cohesion are built into the chromosome itself. These results suggest that regulation of chromosome cohesion may be linked to differences in the arrangement of kinetochores in the two meiotic divisions.
Characterization of the Signal That Directs Tom20 to the Mitochondrial Outer Membrane
Authors: Kanaji Sachiko , Iwahashi Jun , Kida Yuichiro , Sakaguchi Masao , Mihara Katsuyoshi
Published: July 2002

Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH2-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)–induced translation arrest and photo–cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH2-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.
An Essential Role for the Substrate-Binding Region of Hsp40s in Saccharomyces cerevisiae
Authors: Johnson Jill L. , Craig Elizabeth A.
Published: July 2002

In addition to regulating the ATPase cycle of Hsp70, a second critical role of Hsp40s has been proposed based on in vitro studies: binding to denatured protein substrates, followed by their presentation to Hsp70 for folding. However, the biological importance of this model is challenged by the fact that deletion of the substrate-binding domain of either of the two major Hsp40s of the yeast cytosol, Ydj1 and Sis1, leads to no severe defects, as long as regions necessary for Hsp70 interaction are retained. As an in vivo test of this model, requirements for viability were examined in a strain having deletions of both Hsp40 genes. Despite limited sequence similarity, the substrate-binding domain of either Sis1 or Ydj1 allowed cell growth, indicating they share overlapping essential functions. Furthermore, the substrate-binding domain must function in cis with a functional Hsp70-interacting domain. We conclude that the ability of cytosolic Hsp40s to bind unfolded protein substrates is an essential function in vivo.
Desmoglein Isoform Distribution Affects Stratum Corneum Structure and Function
Authors: Elias Peter M. , Matsuyoshi Norihisa , Wu Hong , Lin Chenyan , Wang Zhi Hong , Brown Barbara E. , Stanley John R.
Published: July 2002

Desmogleins are desmosomal cadherins that mediate cell–cell adhesion. In stratified squamous epithelia there are two major isoforms of desmoglein, 1 and 3, with different distributions in epidermis and mucous membrane. Since either desmoglein isoform alone can mediate adhesion, the reason for their differential distribution is not known. To address this issue, we engineered transgenic mice with desmoglein 3 under the control of the involucrin promoter. These mice expressed desmoglein 3 with the same distribution in epidermis as found in normal oral mucous membranes, while expression of other major differentiation molecules was unchanged. Although the nucleated epidermis appeared normal, the epidermal stratum corneum was abnormal with gross scaling, and a lamellar histology resembling that of normal mucous membrane. The mice died shortly after birth with severe dehydration, suggesting excessive transepidermal water loss, which was confirmed by in vitro and in vivo measurement. Ultrastructure of the stratum corneum showed premature loss of cohesion of corneocytes. This dysadhesion of corneocytes and its contribution to increased transepidermal water loss was confirmed by tape stripping. These data demonstrate that differential expression of desmoglein isoforms affects the major function of epidermis, the permeability barrier, by altering the structure of the stratum corneum.
Mistargeting of B-Type Lamins at the End of Mitosis
Authors: Steen Rikke L. , Collas Philippe
Published: July 2002

We previously showed that targeting of protein phosphatase 1 (PP1) to the nuclear envelope (NE) by the A-kinase anchoring protein, AKAP149, correlates with nuclear assembly of B-type lamins in vitro. We demonstrate here that failure of AKAP149-mediated assembly of B-type lamins into the nuclear lamina at the end of mitosis is followed by apoptosis, and induces expression of the gene encoding A-type lamins in cells that normally do not express lamins A/C. In HeLa cells, inhibition of PP1 association with the NE mediated by a peptide containing the PP1-binding domain of AKAP149 results in failure of B-type lamins to assemble, and in their rapid caspase-dependent proteolysis. However, assembly of lamins A/C is not affected. Nonetheless, apoptosis follows within hours of nuclear reformation after mitosis. In lymphoid KE37 cells, which do not express lamins A/C, inhibition of B-type lamin assembly triggers rapid synthesis and nuclear assembly of both lamins A and C before apoptosis takes place. The results indicate that nuclear assembly of B-type lamins is essential for cell survival. They also suggest that mistargeting of B-type lamins at the end of mitosis elicits a tentative rescue process to assemble a nuclear lamina in lymphoid cells that normally do not express lamins A/C.
Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans
Authors: Oegema Karen , Desai Arshad , Rybina Sonja , Kirkham Matthew , Hyman Anthony A.
Published: July 2002

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical “kinetochore null” phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A–containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.
Nectin
Authors: Mizoguchi Akira , Nakanishi Hiroyuki , Kimura Kazushi , Matsubara Kaho , Ozaki-Kuroda Kumi , Katata Tatsuo , Honda Tomoyuki , Kiyohara Yoshimoto , Heo Kyun , Higashi Mikito , Tsutsumi Tomonari , Sonoda Satomi , Ide Chizuka , Takai Yoshimi
Published: July 2002

The nectin–afadin system is a novel cell–cell adhesion system that organizes adherens junctions cooperatively with the cadherin–catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament–binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin–afadin system colocalizes with the cadherin–catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin–afadin system in the formation of synapses.
A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization
Authors: Cau Julien , Faure Sandrine , Comps Michel , Delsert Claude , Morin Nathalie
Published: July 2002

Coordination of the different cytoskeleton networks in the cell is of central importance for morphogenesis, organelle transport, and motility. The Rho family proteins are well characterized for their effects on the actin cytoskeleton, but increasing evidence indicates that they may also control microtubule (MT) dynamics. Here, we demonstrate that a novel Cdc42/Rac effector, X-p21-activated kinase (PAK)5, colocalizes and binds to both the actin and MT networks and that its subcellular localization is regulated during cell cycle progression. In transfected cells, X-PAK5 promotes the formation of stabilized MTs that are associated in bundles and interferes with MTs dynamics, slowing both the elongation and shrinkage rates and inducing long paused periods. X-PAK5 subcellular localization is regulated tightly, since coexpression with active Rac or Cdc42 induces its shuttling to actin-rich structures. Thus, X-PAK5 is a novel MT-associated protein that may communicate between the actin and MT networks during cellular responses to environmental conditions.
Regulation of nucleocytoplasmic trafficking by cell adhesion receptors and the cytoskeleton
Authors: Aplin Andrew E. , Juliano R.L.
Published: July 2002

It has become widely accepted that adhesion receptors can either directly activate, or significantly modulate, many of the signaling cascades initiated by circulating growth factors. An interesting recent development is the realization that adhesion receptors and their cytoskeletal partners can regulate the trafficking of signaling proteins between the cytoplasm and nucleus. Cell adhesion molecule control of nucleocytoplasmic trafficking allows adhesion to influence many cell decisions, and highlights the diversity of nuclear import and export mechanisms.
KIFC3, a microtubule minus end–directed motor for the apical transport of annexin XIIIb–associated Triton-insoluble membranes
Authors: Noda Yasuko , Okada Yasushi , Saito Nobuhito , Setou Mitsutoshi , Xu Ying , Zhang Zheizeng , Hirokawa Nobutaka
Published: July 2002

We have identified and characterized a COOH-terminal motor domain–type kinesin superfamily protein (KIFC), KIFC3, in the kidney. KIFC3 is a minus end–directed microtubule motor protein, therefore it accumulates in regions where minus ends of microtubules assemble. In polarized epithelial cells, KIFC3 is localized on membrane organelles immediately beneath the apical plasma membrane of renal tubular epithelial cells in vivo and polarized MDCK II cells in vitro. Flotation assay, coupled with detergent extraction, demonstrated that KIFC3 is associated with Triton X-100–insoluble membrane organelles, and that it overlaps with apically transported TGN-derived vesicles. This was confirmed by immunoprecipitation and by GST pulldown experiments showing the specific colocalization of KIFC3 and annexin XIIIb, a previously characterized membrane protein for apically transported vesicles (Lafont, F., S. Lecat, P. Verkade, and K. Simons. 1998. J. Cell Biol. 142:1413–1427). Furthermore, we proved that the apical transport of both influenza hemagglutinin and annexin XIIIb was partially inhibited or accelerated by overexpression of motor-domainless (dominant negative) or full-length KIFC3, respectively. Absence of cytoplasmic dynein on these annexin XIIIb–associated vesicles and distinct distribution of the two motors on the EM level verified the existence of KIFC3-driven transport in epithelial cells.
The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p
Authors: Castillo Andrea R. , Meehl Janet B. , Morgan Garry , Schutz-Geschwender Amy , Winey Mark
Published: July 2002

Saccharomyces cerevisiae MPS1 encodes an essential protein kinase that has roles in spindle pole body (SPB) duplication and the spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identification of a unique conditional allele, mps1–8, that is defective in SPB duplication but not the spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identified the gene encoding the integral SPB component SPC42. Additional analysis revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunofluorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication.
Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein–ubiquitin conjugates
Authors: Bishop Naomi , Horman Alistair , Woodman Philip
Published: July 2002

There is increasing evidence that ubiquitination of receptors provides an important endosomal sorting signal. Here we report that mammalian class E vacuolar protein-sorting (vps) proteins recognize ubiquitin. Both tumor susceptibility gene 101 (TSG101)/human VPS (hVPS)28 and hepatocyte growth factor receptor substrate (Hrs) cytosolic complexes bind ubiquitin-agarose. TSG101 and hVPS28 are localized to endosomes that contain internalized EGF receptor and label strongly for ubiquitinated proteins. Microinjection of anti-hVPS28 specifically retards EGF degradation and leads to endosomal accumulation of ubiquitin–protein conjugates. Likewise, depletion of TSG101 impairs EGF trafficking and causes dramatic relocalization of ubiquitin to endocytic compartments. Similar defects are found in cells overexpressing Hrs, further emphasizing the links between class E protein function, receptor trafficking, and endosomal ubiquitination.
ARF-GAP–mediated interaction between the ER-Golgi v-SNAREs and the COPI coat
Authors: Rein Ulrike , Andag Uwe , Duden Rainer , Schmitt Hans Dieter , Spang Anne
Published: July 2002

In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)–GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.
Building an efficient factory
Authors: Huang Sui
Published: July 2002

The subnucleolar structure that is involved in rDNA transcription has been controversial. A report by Koberna et al. (2002)(this issue, page 743) adds significant weight toward the idea that dense fibrillar components (DFCs)**Abbreviations used in this paper: DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; Pol I, polymerase I. and fibrillar center (FC)/DFC borders are the sites of pre-rRNA synthesis.
Pathogenesis of an Infectious Mononucleosis-like Disease Induced by a Murine γ-Herpesvirus: Role for a Viral Superantigen?
Authors: Tripp Ralph A. , Hamilton-Easton Ann Marie , Cardin Rhonda D. , Nguyen Phuong , Behm Frederick G. , Woodland David L. , Doherty Peter C. , Blackman Marcia A.
Published: July 2002

The murine γ-herpesvirus 68 has many similarities to EBV, and induces a syndrome comparable to infectious mononucleosis (IM). The frequency of activated CD8+ T cells (CD62Llo) in the peripheral blood increased greater than fourfold by 21 d after infection of C57BL/6J (H-2b) mice, and remained high for at least a further month. The spectrum of T cell receptor usage was greatly skewed, with as many as 75% of the CD8+ T cells in the blood expressing a Vβ4+ phenotype. Interestingly, the Vβ4 dominance was also seen, to varying extents, in H-2k, H-2d, H-2u, and H-2q strains of mice. In addition, although CD4 depletion from day 11 had no effect on the Vβ4 bias of the T cells, the Vβ4+CD8+ expansion was absent in H-2IAb–deficient congenic mice. However, the numbers of cycling cells in the CD4 antibody–depleted mice and mice that are CD4 deficient as a consequence of the deletion of MHC class II, were generally lower. The findings suggest that the IM-like disease is driven both by cytokines provided by CD4+ T cells and by a viral superantigen presented by MHC class II glycoproteins to Vβ4+CD8+ T cells.