bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosome instability contributes to loss of heterozygosity in mice lacking p53
Authors: Shao Changshun , Deng Li , Henegariu Octavian , Liang Li , Stambrook Peter J. , Tischfield Jay A.
Published: July 2002

The p53 tumor suppressor protein participates in multiple cellular processes including cell cycle checkpoints and programmed cell death. In cell lines, loss of p53 function is associated with increased genetic instability including aneuploidy, gene amplification, and point mutation. Although similar genetic instability often accompanies the progression of malignancy in tumors, its role in tumor initiation in normal cells is not clear. To study whether or not loss of p53 leads to genetic instability in normal cells
in vivo
, we have examined mechanisms of loss of heterozygosity (LOH) at the
Aprt
(adenine phosphoribsyltransferase) and flanking loci in normal fibroblasts and T lymphocytes of
p53
-deficient mice. Somatic cell variants that arose
in vivo
as a consequence of genetic or epigenetic alterations abolishing
Aprt
function were selected and expanded
in vitro
by virtue of their resistance to 2,6-diaminopurine (DAP). We observed that
p53
null mice produced about three times as many DAP-resistant fibroblast colonies than wild-type mice, but the frequency of DAP-resistant T lymphocyte colonies was not significantly changed. Mitotic recombination, but not point mutation, partly accounted for the increase in the frequency of DAP-resistant fibroblasts. Most significantly, chromosome loss/duplication and interstitial deletion, which were extremely rare events in the wild-type mice, represented a significant proportion of LOH events in both fibroblasts and T lymphocytes of
p53
null mice. Also, increased interstitial deletion was observed in fibroblasts of
p53
heterozygous mice. These data suggest that increased genetic variation, including chromosome instability, starts at the initiation stage of tumorigenesis when functional p53 is absent or reduced.
Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis
Authors: Schwarte-Waldhoff Irmgard , Volpert Olga V. , Bouck Noël P. , Sipos Bence , Hahn Stephan A. , Klein-Scory Susanne , Lüttges Jutta , Klöppel Günter , Graeven Ulrich , Eilert-Micus Christina , Hintelmann Annette , Schmiegel Wolff
Published: July 2002

Smad4/
DPC4
(deleted in pancreatic carcinoma, locus 4) is a tumor suppressor gene lost at high frequency in cancers of the pancreas and other gastrointestinal organs.
Smad4
encodes a key intracellular messenger in the transforming growth factor β (TGF-β) signaling cascade. TGF-β is a potent inhibitor of the growth of epithelial cells; thus, it has been assumed that loss of Smad4 during tumor progression relieves this inhibition. Herein, we show that restoration of Smad4 to human pancreatic carcinoma cells suppressed tumor formation
in vivo
, yet it did not restore sensitivity to TGF-β. Rather, Smad4 restoration influenced angiogenesis, decreasing expression of vascular endothelial growth factor and increasing expression of thrombospondin-1. In contrast to the parental cell line and to control transfectants that produced rapidly growing tumors
in vivo
, Smad4 revertants induced small nonprogressive tumors with reduced vascular density. These data define the control of an angiogenic switch as an alternative, previously unknown mechanism of tumor suppression for Smad4 and identify the angiogenic mediators vascular endothelial growth factor and thrombospondin-1 as key target genes.
Requirement for the
lp
A1
lysophosphatidic acid receptor gene in normal suckling behavior
Authors: Contos James J. A. , Fukushima Nobuyuki , Weiner Joshua A. , Kaushal Dhruv , Chun Jerold
Published: July 2002

Although extracellular application of lysophosphatidic acid (LPA)
has been extensively documented to produce a variety of cellular
responses through a family of specific G protein-coupled receptors, the
in vivo
organismal role of LPA signaling remains largely
unknown. The first identified LPA receptor gene,
lp
A1
/
vzg-1
/
edg-2
,
was previously shown to have remarkably enriched embryonic expression
in the cerebral cortex and dorsal olfactory bulb and postnatal
expression in myelinating glia including Schwann cells. Here, we show
that targeted deletion of
lp
A1
results in
approximately 50% neonatal lethality, impaired suckling in neonatal
pups, and loss of LPA responsivity in embryonic cerebral cortical
neuroblasts with survivors showing reduced size, craniofacial
dysmorphism, and increased apoptosis in sciatic nerve Schwann
cells. The suckling defect was responsible for the death among
lp
A1
(−/−)
neonates and the
stunted growth of survivors. Impaired suckling behavior was
attributable to defective olfaction, which is likely related to
developmental abnormalities in olfactory bulb and/or cerebral cortex.
Our results provide evidence that endogenous
lysophospholipid signaling requires an
lp
receptor gene
and indicate that LPA signaling through the LP
A1
receptor
is required for normal development of an inborn, neonatal behavior.
National Awakening and National Consciousness in Belarus
Authors: Marples David R.
Published: July 2002

A frequent assertion about the recent events and pervasive mood in Belarus—the apparent efforts to reunite with Russia, the virtual denial of a Belarusian identity by a Russophone president, official nostalgia for the time of the former Soviet Union— is that national consciousness is somehow retarded or delayed, and national development is lagging considerably behind that of its neighboring states, Lithuania and Ukraine. This article seeks to address the question of national self-awareness in Belarus from three angles: those of demography, culture, and language. Was development of the republic in the Soviet period different from that of the other republics, and is that development responsible for what has been described as the “national nihilism” of today? Is that mood likely to change with a new generation of Belarusians? How far is President Alyaksander Lukashenka, the first president of Belarus, who was elected in July 1994, responsible for the present situation and how far is he a symptom of the notable lack of self-assertion of Belarusians?
Deductive Reasoning with Prose Passages: Effects of Age, Inference Form, Prior Knowledge, and Reading Skill
Authors: Franks Bridget A.
Published: July 2002

This study applied knowledge about inference-making from the deductive reasoning literature to the drawing of specific inferences from prose passages. It explored the effects of age, inference form, prior knowledge, and reading skill on inferential comprehension. In Experiment 1, fourth-grade, seventh-grade, and college students read three prose passages, each containing six inferential questions based on premises expressed in the passages. Premise information was either true, false, or neutral with regard to subjects’ prior knowledge. To answer the questions correctly, subjects were required to make deductive inferences with six different inference forms. Content (true, false, or neutral) and form interacted differently depending on the age of subjects, but content affected performance with at least some forms for all age groups. When reasoning with conditional forms, subjects’ use of more advanced reasoning patterns with true content decreased with false and neutral content, where less advanced reasoning patterns were shown. In Experiment 2, the relationships among reading skill, inference form, and content were explored with seventh-grade and college students. For college students, reading skill had a positive main effect, but did not interact with form or content. For seventh-graders, skilled readers were better able than less skilled readers to reason from false and neutral premises with determinate inference forms.
How Focus of Interest in Pictures Changes with Age: A Cross-cultural Comparison
Authors: Jolley Richard P. , Zhi Zhang , Thomas Glyn V.
Published: July 2002

British and Chinese participants ranging from 4 years of age to adult were presented with sets of drawings of everyday objects, and asked to match two out of three. The drawings could be matched on colour, subject matter, or visual metaphor. In both cultures there was a significant progression from matching on colour to subject matter, and then from matching on subject matter to metaphor. These age-related differences in the selected basis for matching may reflect age-related changes in focus of interest, and provide experimental data that is consistent with Parsons’ (1987) claims towards the development of understanding about art. The findings of broadly similar age differences in Chinese as well as British children suggest that this pattern of development is not culture-specific. Chinese children, however, showed an earlier and more pronounced progression to matching on metaphor than did the British children, which is hard to reconcile with previous suggestions (see, for example, Parsons, 1987; Winner, 1989) that a progression of interest beyond subject matter may not take place in Eastern cultures. The training Chinese children receive in monitoring detail in pictures and in Chinese characters may facilitate attention to the graphic devices that communicate metaphorical messages.
Needle and syringe provision and disposal in an Australian regional centre
Authors: MILLER PETER G.
Published: July 2002

The aim of this study was to investigate the provision and disposal of needles and syringes in Geelong, Victoria, supplying a comparison of needles provided to needles returned through needle and syringe programmes (NSPs), needles disposed of in ‘Sharpsafe’ bins and needles discarded. This study combined quantitative data obtained from a number of sources and qualitative data obtained from 60 interviews. An average of 18,337 (SD = 2796) per month were dispensed from NSPs and an average of 5576 needles are returned to NSP sites in Geelong per month. For the current year, 450 needles and syringes per month were placed in publicly located disposal bins. On average there were 19 callouts to discarded needles per month in the Geelong region and these needles constituted 0.38% of the monthly average of needles provided through NSPs. It was concluded that, while discarded needles are of major community concern, the vast majority of needles and syringes provided in this regional centre are not discarded. Findings are discussed in relation to the current laws surrounding the possession of injecting equipment.
Distinct effects of paradoxical sleep deprivation and cocaine administration on sexual behavior in male rats
Authors: ANDERSEN MONICA L. , TUFIK SERGIO
Published: July 2002

Abstract As there are data showing enhanced sexual activity in paradoxical sleep deprived (PSD) rats, and cocaine (COC) is a drug of abuse known to also increase the sexual drive, the present study aimed to assess sexual behavior in animals submitted to PSD and/or COC administration. The data revealed that PSD followed by COC administration produced the largest percentage of animals having erection and ejaculation (100% and 60%) and differ statistically from the control groups. The PSD group that received saline produced 50% erections and 20% ejaculations whereas COC administration induced 10% erections and 10% ejaculations. Sexual activity was absent in control animals (saline injection). These findings are due probably to PSD causing supersensitivity of dopamine (DA) receptors exacerbating the cocaine actions on dopaminergic pathways inducing penile erection and ejaculation.
Multiple Determinants Direct the Orientation of Signal–Anchor Proteins: The Topogenic Role of the Hydrophobic Signal Domain
Authors: Wahlberg Johanna M. , Spiess Martin
Published: July 2002

The orientation of signal–anchor proteins in the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar, membrane-spanning domain and is influenced by the folding properties of the NH2-terminal sequence. However, these features are not generally sufficient to ensure a unique topology. The topogenic role of the hydrophobic signal domain was studied in vivo by expressing mutants of the asialoglycoprotein receptor subunit H1 in COS-7 cells. By replacing the 19-residue transmembrane segment of wild-type and mutant H1 by stretches of 7–25 leucine residues, we found that the length and hydrophobicity of the apolar sequence significantly affected protein orientation. Translocation of the NH2 terminus was favored by long, hydrophobic sequences and translocation of the COOH terminus by short ones. The topogenic contributions of the transmembrane domain, the flanking charges, and a hydrophilic NH2-terminal portion were additive. In combination these determinants were sufficient to achieve unique membrane insertion in either orientation.
The Zinc-Finger Protein Slug Causes Desmosome Dissociation, an Initial and Necessary Step for Growth Factor–induced Epithelial–Mesenchymal Transition
Authors: Savagner Pierre , Yamada Kenneth M. , Thiery Jean Paul
Published: July 2002

Epithelial–mesenchymal transition (EMT) is an essential morphogenetic process during embryonic development. It can be induced in vitro by hepatocyte growth factor/scatter factor (HGF/SF), or by FGF-1 in our NBT-II cell model for EMT. We tested for a central role in EMT of a zinc-finger protein called Slug. Slug mRNA and protein levels were increased transiently in FGF-1–treated NBT-II cells. Transient or stable transfection of Slug cDNA in NBT-II cells resulted in a striking disappearance of the desmosomal markers desmoplakin and desmoglein from cell–cell contact areas, mimicking the initial steps of FGF-1 or HGF/SF- induced EMT. Stable transfectant cells expressed Slug protein and were less epithelial, with increased cell spreading and cell–cell separation in subconfluent cultures. Interestingly, NBT-II cells transfected with antisense Slug cDNA were able to resist EMT induction by FGF-1 or even HGF/SF. This antisense effect was suppressed by retransfection with Slug sense cDNA. Our results indicate that Slug induces the first phase of growth factor–induced EMT, including desmosome dissociation, cell spreading, and initiation of cell separation. Moreover, the antisense inhibition experiments suggest that Slug is also necessary for EMT.
IQGAP1, a Rac- and Cdc42-binding Protein, Directly Binds and Cross-links Microfilaments
Authors: Bashour Anne-Marie , Fullerton Aaron T. , Hart Matthew J. , Bloom George S.
Published: July 2002

Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two ∼190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D–sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.
Acid-induced Dissociation of VacA, the Helicobacter pylori Vacuolating Cytotoxin, Reveals Its Pattern of Assembly
Authors: Cover Timothy L. , Hanson Phyllis I. , Heuser John E.
Published: July 2002

In this study, we describe the ultrastructural changes associated with acid activation of Helicobacter pylori vacuolating cytotoxin (VacA). Purified VacA molecules imaged by deep-etch electron microscopy form ∼30-nm hexagonal “flowers,” each composed of an ∼15-nm central ring surrounded by six ∼6-nm globular “petals.” Upon exposure to acidic pH, these oligomeric flowers dissociate into collections of up to 12 teardrop-shaped subunits, each measuring ∼6 × 14 nm. Correspondingly, glycerol density gradient centrifugation shows that at neutral pH VacA sediments at ∼22 S, whereas at acidic pH it dissociates and sediments at ∼5 S. Immunoblot and EM analysis of the 5-S material demonstrates that it represents ∼90-kD monomers with 6 × 14–nm “teardrop” morphology. These data indicate that the intact VacA oligomer consists of 12 ∼90-kD subunits assembled into two interlocked six-membered arrays, overlap of which gives rise to the flower-like appearance. Support for this interpretation comes from EM identification of small numbers of relatively “flat” oligomers composed of six teardrop-shaped subunits, interpreted to be halves of the complete flower. These flat forms adsorb to mica in two different orientations, corresponding to hexameric surfaces that are either exposed or sandwiched inside the dodecamer, respectively. This view of VacA structure differs from a previous model in which the flowers were interpreted to be single layers of six monomers and the flat forms were thought to be proteolysed flowers. Since acidification has been shown to potentiate the cytotoxic effects of VacA, the present results suggest that physical disassembly of the VacA oligomer is an important feature of its activation.
Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis
Authors: Bi Guo-Qiang , Morris Robert L. , Liao Guochun , Alderton Janet M. , Scholey Jonathan M. , Steinhardt Richard A.
Published: July 2002

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin– dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.
Maintenance of Calcium Homeostasis in the Endoplasmic Reticulum by Bcl-2
Authors: He Huiling , Lam Minh , McCormick Thomas S. , Distelhorst Clark W.
Published: July 2002

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca2+) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca2+ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca2+-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca2+ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca2+ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca2+ pool in untreated cells when extracellular Ca2+ is low. However, low extracellular Ca2+ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca2+ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca2+ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.
A Vacuolar v–t-SNARE Complex, the Predominant Form In Vivo and on Isolated Vacuoles, Is Disassembled and Activated for Docking and Fusion
Authors: Ungermann Christian , Nichols Benjamin J. , Pelham Hugh R.B. , Wickner William
Published: July 2002

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [α-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v–t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v–t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v–t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, α-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.
Mannose 6–Phosphate Receptors Are Sorted from Immature Secretory Granules via Adaptor Protein AP-1, Clathrin, and Syntaxin 6–positive Vesicles
Authors: Klumperman Judith , Kuliawat Regina , Griffith Janice M. , Geuze Hans J. , Arvan Peter
Published: July 2002

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic β cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6–phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by ∼90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in β cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595–608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261–1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR–ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.
Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates
Authors: Sakai Yasuyoshi , Koller Antonius , Rangell Linda K. , Keller Gilbert A. , Subramani Suresh
Published: July 2002

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.
Evidence for a Role of CLIP-170 in the Establishment of Metaphase Chromosome Alignment
Authors: Dujardin Denis , Wacker U. Irene , Moreau Anne , Schroer Trina A. , Rickard Janet E. , De Mey Jan R.
Published: July 2002

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.
Ganglioside Structure Dictates Signal Transduction by Cholera Toxin and Association with Caveolae-like Membrane Domains in Polarized Epithelia
Authors: Wolf Anne A. , Jobling Michael G. , Wimer-Mackin Susan , Ferguson-Maltzman Margaret , Madara James L. , Holmes Randall K. , Lencer Wayne I.
Published: July 2002

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951–962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (Kd ≈ 5 nM), only CT elicited a cAMP-dependent Cl− secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.
The SNARE Machinery Is Involved in Apical Plasma Membrane Trafficking in MDCK Cells
Authors: Low Seng Hui , Chapin Steven J. , Wimmer Christian , Whiteheart Sidney W. , Kömüves László G. , Mostov Keith E. , Weimbs Thomas
Published: July 2002

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide–sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against α-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of N-ethyl maleimide–sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23, and α-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism that is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing specificity to polarized membrane traffic.
The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein
Authors: Lin Ping , Le-Niculescu Helen , Hofmeister Robert , Michael McCaffery J. , Jin Mingjie , Hennemann Hanjo , McQuistan Tammie , De Vries Luc , Farquhar Marilyn Gist
Published: July 2002

We have identified CALNUC, an EF-hand, Ca2+-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gαi3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gαi3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca2+-binding protein detected by 45Ca2+-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.
Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase
Authors: Rieder Conly L. , Cole Richard W.
Published: July 2002

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.
Changes in Organization of Crithidia fasciculata Kinetoplast DNA Replication Proteins during the Cell Cycle
Authors: Johnson Catharine E. , Englund Paul T.
Published: July 2002

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicircles. We investigated cell cycle–dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein–dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase β colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase β is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern. These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.
Fusogenic Domains of Golgi Membranes Are Sequestered into Specialized Regions of the Stack that Can Be Released by Mechanical Fragmentation
Authors: Dominguez Michel , Fazel Ali , Dahan Sophie , Lovell Jacque , Hermo Louis , Claude Alejandro , Melançon Paul , Bergeron J.J.M.
Published: July 2002

A well-characterized cell-free assay that reconstitutes Golgi transport is shown to require physically fragmented Golgi fractions for maximal activity. A Golgi fraction containing large, highly stacked flattened cisternae associated with coatomer-rich components was inactive in the intra-Golgi transport assay. In contrast, more fragmented hepatic Golgi fractions of lower purity were highly active in this assay. Control experiments ruled out defects in glycosylation, the presence of excess coatomer or inhibitory factors, as well as the lack or consumption of limiting diffusible factors as responsible for the lower activity of intact Golgi fractions. Neither Brefeldin A treatment, preincubation with KCl (that completely removed associated coatomer) or preincubation with imidazole buffers that caused unstacking, activated stacked fractions for transport. Only physical fragmentation promoted recovery of Golgi fractions active for transport in vitro. Rate-zonal centrifugation partially separated smaller transport-active Golgi fragments with a unique v-SNARE pattern, away from the bulk of Golgi-derived elements identified by their morphology and content of Golgi marker enzymes (N-acetyl glucosaminyl and galactosyl transferase activities). These fragments released during activation likely represent intra-Golgi continuities involved in maintaining the dynamic redistribution of resident enzymes during rapid anterograde transport of secretory cargo through the Golgi in vivo.
Mitotic Regulators Govern Progress through Steps in the Centrosome Duplication Cycle
Authors: Vidwans Smruti J. , Wong Mei Lie , O'Farrell Patrick H.
Published: July 2002

Centrosome duplication is marked by discrete changes in centriole structure that occur in lockstep with cell cycle transitions. We show that mitotic regulators govern steps in centriole replication in Drosophila embryos. Cdc25string, the expression of which initiates mitosis, is required for completion of daughter centriole assembly. Cdc20fizzy, which is required for the metaphase-anaphase transition, is required for timely disengagement of mother and daughter centrioles. Stabilization of mitotic cyclins, which prevents exit from mitosis, blocks assembly of new daughter centrioles. Common regulation of the nuclear and centrosome cycles by mitotic regulators may ensure precise duplication of the centrosome.
Intracellular Ca2+ and Ca2+/Calmodulin-Dependent Kinase II Mediate Acute Potentiation of Neurotransmitter Release by Neurotrophin-3
Authors: He Xiang-ping , Yang Feng , Xie Zuo-ping , Lu Bai
Published: July 2002

Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve–muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca2+ influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca2+ influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca2+. Depletion of intracellular Ca2+ stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca2+/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca2+ release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII.
Observing Fcεri Signaling from the Inside of the Mast Cell Membrane
Authors: Wilson Bridget S. , Pfeiffer Janet R. , Oliver Janet M.
Published: July 2002

We have determined the membrane topography of the high-affinity IgE receptor, FcεRI, and its associated tyrosine kinases, Lyn and Syk, by immunogold labeling and transmission electron microscopic (TEM) analysis of membrane sheets prepared from RBL-2H3 mast cells. The method of Sanan and Anderson (Sanan, D.A., and R.G.W. Anderson. 1991. J. Histochem. Cytochem. 39:1017–1024) was modified to generate membrane sheets from the dorsal surface of RBL-2H3 cells. Signaling molecules were localized on the cytoplasmic face of these native membranes by immunogold labeling and high-resolution TEM analysis. In unstimulated cells, the majority of gold particles marking both FcεRI and Lyn are distributed as small clusters (2–9 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of FcεRI clusters contain Lyn. In contrast, there is essentially no FcεRI-Syk colocalization in resting cells. 2 min after FcεRI cross-linking, ∼10% of Lyn colocalizes with small and medium-sized FcεRI clusters (up to 20 gold particles), whereas ∼16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20–100 gold particles) that form on characteristically osmiophilic membrane patches. While Lyn is excluded, Syk is dramatically recruited into these larger aggregates. The clathrin-coated pits that internalize cross-linked receptors bud from membrane adjacent to the Syk-containing receptor complexes. The sequential association of FcεRI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of FcεRI signaling.
Cyclin E as a Coactivator of the Androgen Receptor
Authors: Yamamoto Ayako , Hashimoto Yoshihiro , Kohri Kenjiro , Ogata Etsuro , Kato Shige-aki , Ikeda Kyoji , Nakanishi Makoto
Published: July 2002

Androgens play an important role in the growth of prostate cancer, but the molecular mechanism that underlies development of resistance to antiandrogen therapy remains unknown. Cyclin E has now been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of its ligand dihydrotestosterone. The enhancement of AR activity by cyclin E was resistant to inhibition by the antiandrogen 5-hydroxyflutamide. Cyclin E was shown to bind directly to the COOH terminus portion of the AB domain of the AR, and to enhance its AF-1 transactivation function. These results suggest that cyclin E functions as a coactivator of the AR, and that aberrant expression of cyclin E in tumors may contribute to persistent activation of AR function, even during androgen ablation therapy.
Cell to Cell Communication in Response to Mechanical Stress via Bilateral Release of Atp and Utp in Polarized Epithelia
Authors: Homolya László , Steinberg Thomas H. , Boucher Richard C.
Published: July 2002

Airway epithelia are positioned at the interface between the body and the environment, and generate complex signaling responses to inhaled toxins and other stresses. Luminal mechanical stimulation of airway epithelial cells produces a propagating wave of elevated intracellular Ca2+ that coordinates components of the integrated epithelial stress response. In polarized airway epithelia, this response has been attributed to IP3 permeation through gap junctions. Using a combination of approaches, including enzymes that destroy extracellular nucleotides, purinergic receptor desensitization, and airway cells deficient in purinoceptors, we demonstrated that Ca2+ waves induced by luminal mechanical stimulation in polarized airway epithelia were initiated by the release of the 5′ nucleotides, ATP and UTP, across both apical and basolateral membranes. The nucleotides released into the extracellular compartment interacted with purinoceptors at both membranes to trigger Ca2+ mobilization. Physiologically, apical membrane nucleotide-release coordinates airway mucociliary clearance responses (mucin and salt, water secretion, increased ciliary beat frequency), whereas basolateral release constitutes a paracrine mechanism by which mechanical stresses signal adjacent cells not only within the epithelium, but other cell types (nerves, inflammatory cells) in the submucosa. Nucleotide-release ipsilateral and contralateral to the surface stimulated constitutes a unique mechanism by which epithelia coordinate local and distant airway defense responses to mechanical stimuli.
Regulated Interactions between Dynamin and the Actin-Binding Protein Cortactin Modulate Cell Shape
Authors: McNiven Mark A. , Kim Leung , Krueger Eugene W. , Orth James D. , Cao Hong , Wong Tai Wai
Published: July 2002

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin–SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortΔSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)ΔPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.