bluuebunny/crossref_metadata_2025 huggingface dataset:
Clostridium perfringens Enterotoxin Fragment Removes Specific Claudins from Tight Junction Strands
Authors: Sonoda Noriyuki , Furuse Mikio , Sasaki Hiroyuki , Yonemura Shigenobu , Katahira Jun , Horiguchi Yasuhiko , Tsukita Shoichiro
Published: July 2002

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single ∼35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.
Rac Downregulates Rho Activity
Authors: Sander Eva E. , ten Klooster Jean P. , van Delft Sanne , van der Kammen Rob A. , Collard John G.
Published: July 2002

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.
Functional Cis-Heterodimers of N- and R-Cadherins
Authors: Shan Wei-Song , Tanaka Hidekazu , Phillips Greg R. , Arndt Kirsten , Yoshida Mika , Colman David R. , Shapiro Lawrence
Published: July 2002

Classical cadherins form parallel cis-dimers that emanate from a single cell surface. It is thought that the cis-dimeric form is active in cell–cell adhesion, whereas cadherin monomers are likely to be inactive. Currently, cis-dimers have been shown to exist only between cadherins of the same type. Here, we show the specific formation of cis-heterodimers between N- and R-cadherins. E-cadherin cannot participate in these complexes. Cells coexpressing N- and R-cadherins show homophilic adhesion in which these proteins coassociate at cell–cell interfaces. We performed site- directed mutagenesis studies, the results of which support the strand dimer model for cis-dimerization. Furthermore, we show that when N- and R-cadherins are coexpressed in neurons in vitro, the two cadherins colocalize at certain neural synapses, implying biological relevance for these complexes. The present study provides a novel paradigm for cadherin interaction whereby selective cis-heterodimer formation may generate new functional units to mediate cell–cell adhesion.
Membrane Recruitment of Aut7p in the Autophagy and Cytoplasm to Vacuole Targeting Pathways Requires Aut1p, Aut2p, and the Autophagy Conjugation Complex
Authors: Kim John , Huang Wei-Pang , Klionsky Daniel J.
Published: July 2002

Autophagy is a degradative pathway by which cells sequester nonessential, bulk cytosol into double-membrane vesicles (autophagosomes) and deliver them to the vacuole for recycling. Using this strategy, eukaryotic cells survive periods of nutritional starvation. Under nutrient-rich conditions, autophagy machinery is required for the delivery of a resident vacuolar hydrolase, aminopeptidase I, by the cytoplasm to vacuole targeting (Cvt) pathway. In both pathways, the vesicle formation process requires the function of the starvation-induced Aut7 protein, which is recruited from the cytosol to the forming Cvt vesicles and autophagosomes. The membrane binding of Aut7p represents an early step in vesicle formation. In this study, we identify several requirements for Aut7p membrane association. After synthesis in the cytosol, Aut7p is proteolytically cleaved in an Aut2p-dependent manner. While this novel processing event is essential for Aut7p membrane binding, Aut7p must undergo additional physical interactions with Aut1p and the autophagy (Apg) conjugation complex before recruitment to the membrane. Lack of these interactions results in a cytosolic distribution of Aut7p rather than localization to forming Cvt vesicles and autophagosomes. This study assigns a functional role for the Apg conjugation system as a mediator of Aut7p membrane recruitment. Further, we demonstrate that Aut1p, which physically interacts with components of the Apg conjugation complex and Aut7p, constitutes an additional factor required for Aut7p membrane recruitment. These findings define a series of steps that results in the modification of Aut7p and its subsequent binding to the sequestering transport vesicles of the autophagy and cytoplasm to vacuole targeting pathways.
The Nc1/Endostatin Domain ofCaenorhabditis elegansType Xviii Collagen Affects Cell Migration and Axon Guidance
Authors: Ackley Brian D. , Crew Jennifer R. , Elamaa Harri , Pihlajaniemi Tania , Kuo Calvin J. , Kramer James M.
Published: July 2002

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.
Glial Growth Factor/Neuregulin Inhibits Schwann Cell Myelination and Induces Demyelination
Authors: Zanazzi George , Einheber Steven , Westreich Richard , Hannocks Melanie-Jane , Bedell-Hogan Debra , Marchionni Mark A. , Salzer James L.
Published: July 2002

During development, neuregulin-1 promotes Schwann cell proliferation and survival; its role in later events of Schwann cell differentiation, including myelination, is poorly understood. Accordingly, we have examined the effects of neuregulin-1 on myelination in neuron-Schwann cell cocultures. Glial growth factor (GGF), a neuregulin-1 isoform, significantly inhibited myelination by preventing axonal segregation and ensheathment. Basal lamina formation was not affected. Treatment of established myelinated cultures with GGF resulted in striking demyelination that frequently began at the paranodes and progressed to the internode. Demyelination was dose dependent and accompanied by dedifferentiation of Schwann cells to a promyelinating stage, as evidenced by reexpression of the transcription factor suppressed cAMP-inducible POU; a significant proportion of cells with extensive demyelination also proliferated. Two other Schwann cell mitogens, fibroblast growth factor-2 and transforming growth factor-β, inhibited myelination but did not cause demyelination, suggesting this effect is specific to the neuregulins. The neuregulin receptor proteins, erbB2 and erbB3, are expressed on ensheathing and myelinating Schwann cells and rapidly phosphorylated with GGF treatment. GGF treatment of myelinating cultures also induced phosphorylation of phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and a 120-kD protein. These results suggest that neuronal mitogens, including the neuregulins, may inhibit myelination during development and that activation of mitogen signaling pathways may contribute to the initial demyelination and subsequent Schwann cell proliferation observed in various pathologic conditions.
Circulating Skeletal Stem Cells
Authors: Kuznetsov Sergei A. , Mankani Mahesh H. , Gronthos Stan , Satomura Kazuhito , Bianco Paolo , Robey Pamela Gehron
Published: July 2002

We report the isolation of adherent, clonogenic, fibroblast-like cells with osteogenic and adipogenic potential from the blood of four mammalian species. These cells phenotypically resemble but are distinguishable from skeletal stem cells found in bone marrow (stromal stem cells, “mesenchymal stem cells”). The osteogenic potential of the blood-borne cells was proven by an in vivo transplantation assay in which either polyclonal or single colony–derived strains were transplanted into the subcutis of immunocompromised mice, and the donor origin of the fully differentiated bone cells was proven using species-specific probes. This is the first definitive proof of the existence of circulating skeletal stem cells in mammals.
The Alzheimer Amyloid Precursor Protein (APP) and Fe65, an APP-Binding Protein, Regulate Cell Movement
Authors: Sabo Shasta L. , Ikin Annat F. , Buxbaum Joseph D. , Greengard Paul
Published: July 2002

FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with β1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound–healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.
The concept of self-organization in cellular architecture
Authors: Misteli Tom
Published: July 2002

In vivo microscopy has recently revealed the dynamic nature of many cellular organelles. The dynamic properties of several cellular structures are consistent with a role for self-organization in their formation, maintenance, and function; therefore, self-organization might be a general principle in cellular organization.
Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis
Authors: Niethammer Philipp , Delling Markus , Sytnyk Vladimir , Dityatev Alexander , Fukami Kiyoko , Schachner Melitta
Published: July 2002

The neural cell adhesion molecule (NCAM) has been reported to stimulate neuritogenesis either via nonreceptor tyrosine kinases or fibroblast growth factor (FGF) receptor. Here we show that lipid raft association of NCAM is crucial for activation of the nonreceptor tyrosine kinase pathway and induction of neurite outgrowth. Transfection of hippocampal neurons of NCAM-deficient mice revealed that of the three major NCAM isoforms only NCAM140 can act as a homophilic receptor that induces neurite outgrowth. Disruption of NCAM140 raft association either by mutation of NCAM140 palmitoylation sites or by lipid raft destruction attenuates activation of the tyrosine focal adhesion kinase and extracellular signal–regulated kinase 1/2, completely blocking neurite outgrowth. Likewise, NCAM-triggered neurite outgrowth is also completely blocked by a specific FGF receptor inhibitor, indicating that cosignaling via raft-associated kinases and FGF receptor is essential for neuritogenesis.
The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion
Authors: Schwoebel Eric D. , Ho Thai H. , Moore Mary Shannon
Published: July 2002

Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.
The role of ARK in stress-induced apoptosis in Drosophila cells
Authors: Zimmermann Katja C. , Ricci Jean-Ehrland , Droin Nathalie M. , Green Douglas R.
Published: July 2002

The molecular mechanisms of apoptosis are highly conserved throughout evolution. The homologs of genes essential for apoptosis in Caenorhabditis elegans and Drosophila melanogaster have been shown to be important for apoptosis in mammalian systems. Although a homologue for CED-4/apoptotic protease-activating factor (Apaf)-1 has been described in Drosophila, its exact function and the role of the mitochondrial pathway in its activation remain unclear. Here, we used the technique of RNA interference to dissect apoptotic signaling pathways in Drosophila cells. Inhibition of the Drosophila CED-4/Apaf-1–related killer (ARK) homologue resulted in pronounced inhibition of stress-induced apoptosis, whereas loss of ARK did not protect the cells from Reaper- or Grim-induced cell death. Reduction of DIAP1 induced rapid apoptosis in these cells, whereas the inhibition of DIAP2 expression did not but resulted in increased sensitivity to stress-induced apoptosis; apoptosis in both cases was prevented by inhibition of ARK expression. Cells in which cytochrome c expression was decreased underwent apoptosis induced by stress stimuli, Reaper or Grim. These results demonstrate the central role of ARK in stress-induced apoptosis, which appears to act independently of cytochrome c. Apoptosis induced by Reaper or Grim can proceed via a distinct pathway, independent of ARK.
Class I–restricted Cross-Presentation of Exogenous Self-Antigens Leads to Deletion of Autoreactive CD8+ T Cells
Authors: Kurts Christian , Kosaka Hiroshi , Carbone Francis R. , Miller Jacques F.A.P. , Heath William R.
Published: July 2002

In this report, we show that cross-presentation of self-antigens can lead to the peripheral deletion of autoreactive CD8+ T cells. We had previously shown that transfer of ovalbumin (OVA)-specific CD8+ T cells (OT-I cells) into rat insulin promoter–membrane-bound form of OVA transgenic mice, which express the model autoantigen OVA in the proximal tubular cells of the kidneys, the β cells of the pancreas, the thymus, and the testis of male mice, led to the activation of OT-I cells in the draining lymph nodes. This was due to class I–restricted cross-presentation of exogenous OVA on a bone marrow–derived antigen presenting cell (APC) population. Here, we show that adoptively transferred or thymically derived OT-I cells activated by cross-presentation are deleted from the peripheral pool of recirculating lymphocytes. Such deletion only required antigen recognition on a bone marrow–derived population, suggesting that cells of the professional APC class may be tolerogenic under these circumstances. Our results provide a mechanism by which the immune system can induce CD8+ T cell tolerance to autoantigens that are expressed outside the recirculation pathway of naive T cells.
Estrogen Inhibits Bone Resorption by Directly Inducing Apoptosis of the Bone-resorbing Osteoclasts
Authors: Kameda Takashi , Mano Hiroshi , Yuasa Tatsuhisa , Mori Yoshihisa , Miyazawa Koshi , Shiokawa Miho , Nakamaru Yukiya , Hiroi Emi , Hiura Kenji , Kameda Akira , Yang Na N. , Hakeda Yoshiyuki , Kumegawa Masayoshi
Published: July 2002

Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 β-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor– mediated mechanism.
Quantitative Analysis of the T Cell Repertoire Selected by a Single Peptide–Major Histocompatibility Complex
Authors: Gapin Laurent , Fukui Yoshinori , Kanellopoulos Jean , Sano Tetsuro , Casrouge Armanda , Malier Vanessa , Beaudoing Emmanuel , Gautheret Daniel , Claverie Jean-Michel , Sasazuki Takehiko , Kourilsky Philippe
Published: July 2002

The positive selection of CD4+ T cells requires the expression of major histocompatibility complex (MHC) class II molecules in the thymus, but the role of self-peptides complexed to class II molecules is still a matter of debate. Recently, it was observed that transgenic mice expressing a single peptide–MHC class II complex positively select significant numbers of diverse CD4+ T cells in the thymus. However, the number of selected T cell specificities has not been evaluated so far. Here, we have sequenced 700 junctional complementarity determining regions 3 (CDR3) from T cell receptors (TCRs) carrying Vβ11-Jβ1.1 or Vβ12-Jβ1.1 rearrangements. We found that a single peptide–MHC class II complex positively selects at least 105 different Vβ rearrangements. Our data yield a first evaluation of the size of the T cell repertoire. In addition, they provide evidence that the single Eα52-68–I-Ab complex skews the amino acid frequency in the TCR CDR3 loop of positively selected T cells. A detailed analysis of CDR3 sequences indicates that a fraction of the β chain repertoire bears the imprint of the selecting self-peptide.
Antiinflammatory Effects of CD95 Ligand (FasL)-induced Apoptosis
Authors: Gao Yakun , Herndon John M. , Zhang Hui , Griffith Thomas S. , Ferguson Thomas A.
Published: July 2002

Apoptosis is critical to homeostasis of multicellular organisms. In immune privileged sites such as the eye, CD95 ligand (FasL)-induced apoptosis controls dangerous inflammatory reactions that can cause blindness. Recently, we demonstrated that apoptotic cell death of inflammatory cells was a prerequisite for the induction of immune deviation after antigen presentation in the eye. In this report, we examine the mechanism by which this takes place. Our results show that Fas- mediated apoptosis of lymphoid cells leads to rapid production of interleukin (IL)-10 in these cells. The apoptotic cells containing IL-10 are responsible for the activation of immune deviation through interaction with antigen-presenting cells (APC). In support of this, we found that apoptotic cells from IL-10+/+ animals fed to APC in vitro promote Th2 cell differentiation, whereas apoptotic IL-10−/− cells, as well as nonapoptotic cells, favor Th1 induction. Thus, apoptotic cell death and tolerance are linked through the production of an antiinflammatory cytokine to prevent dangerous and unwanted immune responses that might compromise organ integrity.
CD1d-mediated Recognition of an α-Galactosylceramide by Natural Killer T Cells Is Highly Conserved through Mammalian Evolution
Authors: Brossay Laurent , Chioda Mariacristina , Burdin Nicolas , Koezuka Yasuhiko , Casorati Giulia , Dellabona Paolo , Kronenberg Mitchell
Published: July 2002

Natural killer (NK) T cells are a lymphocyte subset with a distinct surface phenotype, an invariant T cell receptor (TCR), and reactivity to CD1. Here we show that mouse NK T cells can recognize human CD1d as well as mouse CD1, and human NK T cells also recognize both CD1 homologues. The unprecedented degree of conservation of this T cell recognition system suggests that it is fundamentally important. Mouse or human CD1 molecules can present the glycolipid α-galactosylceramide (α-GalCer) to NK T cells from either species. Human T cells, preselected for invariant Vα24 TCR expression, uniformly recognize α-GalCer presented by either human CD1d or mouse CD1. In addition, culture of human peripheral blood cells with α-GalCer led to the dramatic expansion of NK T cells with an invariant (Vα24+) TCR and the release of large amounts of cytokines. Because invariant Vα14+ and Vα24+ NK T cells have been implicated both in the control of autoimmune disease and the response to tumors, our data suggest that α-GalCer could be a useful agent for modulating human immune responses by activation of the highly conserved NK T cell subset.
Growth Inhibition and Apoptosis Due to Restoration of E2A Activity in T Cell Acute Lymphoblastic Leukemia Cells
Authors: Park Steven T. , Nolan Garry P. , Sun Xiao-Hong
Published: July 2002

Two models have been proposed for the molecular mechanism by which the Tal1 oncogene causes T cell acute lymphoblastic leukemia (T-ALL). The activation model suggests that Tal1 as heterodimers with the E2A transcription factor activates the expression of oncogenes. The inhibition model postulates that Tal1 interferes with the tumor-suppressing function of E2A. In the Jurkat T cell line, originally derived from a patient with T-ALL, Tal1 is complexed with E2A proteins and the transcriptional activity of E2A is very low. When E2A activity was restored by expressing an E2A–Tal1 fusion protein, E-T/2, the Jurkat cells underwent growth arrest and subsequently apoptosis, thus supporting the inhibition model and suggesting that E2A loss may contribute to leukemic progression.
A Coordinated Change in Chemokine Responsiveness Guides Plasma Cell Movements
Authors: Hargreaves Diana C. , Hyman Paul L. , Lu Theresa T. , Ngo Vu N. , Bidgol Afshin , Suzuki Gen , Zou Yong-Rui , Littman Dan R. , Cyster Jason G.
Published: July 2002

Antibody-secreting plasma cells are nonrecirculatory and lodge in splenic red pulp, lymph node medullary cords, and bone marrow. The factors that regulate plasma cell localization are poorly defined. Here we demonstrate that, compared with their B cell precursors, plasma cells exhibit increased chemotactic sensitivity to the CXCR4 ligand CXCL12. At the same time, they downregulate CXCR5 and CCR7 and have reduced responsiveness to the B and T zone chemokines CXCL13, CCL19, and CCL21. We demonstrate that CXCL12 is expressed within splenic red pulp and lymph node medullary cords as well as in bone marrow. In chimeric mice reconstituted with CXCR4-deficient fetal liver cells, plasma cells are mislocalized in the spleen, found in elevated numbers in blood, and fail to accumulate normally in the bone marrow. Our findings indicate that as B cells differentiate into plasma cells they undergo a coordinated change in chemokine responsiveness that regulates their movements in secondary lymphoid organs and promotes lodgment within the bone marrow.
C-Rel Regulates Interleukin 12 P70 Expression in Cd8+ Dendritic Cells by Specifically Inducing p35 Gene Transcription
Authors: Grumont Raelene , Hochrein Hubertus , O'Keeffe Meredith , Gugasyan Raffi , White Christine , Caminschi Irina , Cook Wendy , Gerondakis Steve
Published: July 2002

Interleukin 12 (IL-12) is a 70-kD proinflammatory cytokine produced by antigen presenting cells that is essential for the induction of T helper type 1 development. It comprises 35-kD (p35) and 40-kD (p40) polypeptides encoded by separate genes that are induced by a range of stimuli that include lipopolysaccharide (LPS), DNA, and CD40 ligand. To date, the regulation of IL-12 expression at the transcriptional level has mainly been examined in macrophages and restricted almost exclusively to the p40 gene. Here we show that in CD8+ dendritic cells, major producers of IL-12 p70, the Rel/nuclear factor (NF)-κB signaling pathway is necessary for the induction of IL-12 in response to microbial stimuli. In contrast to macrophages which require c-Rel for p40 transcription, in CD8+ dendritic cells, the induced expression of p35 rather than p40 by inactivated Staphylococcus aureus, DNA, or LPS is c-Rel dependent and regulated directly by c-Rel complexes binding to the p35 promoter. This data establishes the IL-12 p35 gene as a new target of c-Rel and shows that the regulation of IL-12 p70 expression at the transcriptional level by Rel/NF-κB is controlled through both the p35 and p40 genes in a cell type–specific fashion.
Complementary Dendritic Cell–activating Function of CD8+ and CD4+ T Cells
Authors: Mailliard Robbie B. , Egawa Shinichi , Cai Quan , Kalinska Anna , Bykovskaya Svetlana N. , Lotze Michael T. , Kapsenberg Martien L. , Storkus Walter J. , Kalinski Pawel
Published: July 2002

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of “T helper (Th)” signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood– or peripheral blood–isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-γ at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I–presented epitopes by antigen-specific CD8+ T cells results in the TNF-α– and IFN-γ–dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I–restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I–presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections.
The CD8+ Dendritic Cell Subset Selectively Endocytoses Dying Cells in Culture and In Vivo
Authors: Iyoda Tomonori , Shimoyama Susumu , Liu Kang , Omatsu Yoshiki , Akiyama Yuji , Maeda Yasuhiro , Takahara Kazuhiko , Steinman Ralph M. , Inaba Kayo
Published: July 2002

Dendritic cells (DCs) are able in tissue culture to phagocytose and present antigens derived from infected, malignant, and allogeneic cells. Here we show directly that DCs in situ take up these types of cells after fluorescent labeling with carboxyfluorescein succinimidyl ester (CFSE) and injection into mice. The injected cells include syngeneic splenocytes and tumor cell lines, induced to undergo apoptosis ex vivo by exposure to osmotic shock, and allogeneic B cells killed by NK cells in situ. The CFSE-labeled cells in each case are actively endocytosed by DCs in vivo, but only the CD8+ subset. After uptake, all of the phagocytic CD8+ DCs can form major histocompatibility complex class II–peptide complexes, as detected with a monoclonal antibody specific for these complexes. The CD8+ DCs also selectively present cell-associated antigens to both CD4+ and CD8+ T cells. Similar events take place with cultured DCs; CD8+ DCs again selectively take up and present dying cells. In contrast, both CD8+ and CD8− DCs phagocytose latex particles in culture, and both DC subsets present soluble ovalbumin captured in vivo. Therefore CD8+ DCs are specialized to capture dying cells, and this helps to explain their selective ability to cross present cellular antigens to both CD4+ and CD8+ T cells.
Subunit Stoichiometry of Human Muscle Chloride Channels
Authors: Fahlke Christoph , Knittle Timothy , Gurnett Christina A. , Campbell Kevin P. , George Alfred L.
Published: July 2002

Voltage-gated Cl− channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl− channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl− channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.
Stoichiometry of Recombinant N-Methyl-d-Aspartate Receptor Channels Inferred from Single-channel Current Patterns
Authors: Premkumar Louis S. , Auerbach Anthony
Published: July 2002

Single-channel currents were recorded from mouse NR1-NR2B (ζ-ε2) receptors containing mixtures of wild-type and mutant subunits expressed in Xenopus oocytes. Mutant subunits had an asparagine-to-glutamine (N-to-Q) mutation at the N0 site of the M2 segment (NR1:598, NR2B:589). Receptors with pure N or Q NR1 and NR2 subunits generated single-channel currents with distinctive current patterns. Based on main and sublevel amplitudes, occupancy probabilities, and lifetimes, four patterns of current were identified, corresponding to receptors with the following subunit compositions (NR1/NR2): N/N, N/Q, Q/N, and Q/Q. Only one current pattern was apparent for each composition. When a mixture of N and Q NR2 subunits was coexpressed with pure mutant NR1 subunits, three single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as Q/N receptors. The third, novel pattern presumably arose from hybrid receptors having both N and Q NR2 subunits. When a mixture of N and Q NR1 subunits was coexpressed with pure mutant NR2 subunits, six single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as N/Q receptors. The four novel patterns presumably arose from hybrid receptors having both N and Q NR1 subunits. The relative frequency of NR1 hybrid receptor current patterns depended on the relative amounts of Q and N subunits that were injected into the oocytes. The number of hybrid receptor patterns suggests that there are two NR2 subunits per receptor and is consistent with either three or five NR1 subunits per receptor, depending on whether or not the order of mutant and wild-type subunits influences the current pattern. When considered in relation to other studies, the most straightforward interpretation of the results is that N-methyl-d-aspartate receptors are pentamers composed of three NR1 and two NR2 subunits.
Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium
Authors: Hirschberg Birgit , Maylie James , Adelman John P. , Marrion Neil V.
Published: July 2002

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.
Regulation of Organelle Acidity
Authors: Grabe Michael , Oster George
Published: July 2002

Intracellular organelles have characteristic pH ranges that are set and maintained by a balance between ion pumps, leaks, and internal ionic equilibria. Previously, a thermodynamic study by Rybak et al. (Rybak, S., F. Lanni, and R. Murphy. 1997. Biophys. J. 73:674–687) identified the key elements involved in pH regulation; however, recent experiments show that cellular compartments are not in thermodynamic equilibrium. We present here a nonequilibrium model of lumenal acidification based on the interplay of ion pumps and channels, the physical properties of the lumenal matrix, and the organelle geometry. The model successfully predicts experimentally measured steady-state and transient pH values and membrane potentials. We conclude that morphological differences among organelles are insufficient to explain the wide range of pHs present in the cell. Using sensitivity analysis, we quantified the influence of pH regulatory elements on the dynamics of acidification. We found that V-ATPase proton pump and proton leak densities are the two parameters that most strongly influence resting pH. Additionally, we modeled the pH response of the Golgi complex to varying external solutions, and our findings suggest that the membrane is permeable to more than one dominant counter ion. From this data, we determined a Golgi complex proton permeability of 8.1 × 10−6 cm/s. Furthermore, we analyzed the early-to-late transition in the endosomal pathway where Na,K-ATPases have been shown to limit acidification by an entire pH unit. Our model supports the role of the Na,K-ATPase in regulating endosomal pH by affecting the membrane potential. However, experimental data can only be reproduced by (1) positing the existence of a hypothetical voltage-gated chloride channel or (2) that newly formed vesicles have especially high potassium concentrations and small chloride conductance.
A recurrence/transience result for circle packings
Authors: McCaughan Gareth
Published: July 2002

It is known that any infinite simplicial complex homeomorphic to the plane and satisfying a couple of other conditions is the nerve of a circle packing of either the plane or the disc (and not of both). We prove that such a complex is the nerve of a packing of the plane or the disc according as the simple random walk on its 1-skeleton is recurrent or transient, and discuss some applications. We also prove a criterion for transience of simple random walk on the 1-skeleton of a triangulation of the plane, in terms of average degrees of suitable sets of vertices.
A sweep-plane algorithm for generating random tuples in simple polytopes
Authors: Leydold Josef , Hörmann Wolfgang
Published: July 2002

A sweep-plane algorithm of Lawrence for convex polytope computation is adapted to generate random tuples on simple polytopes. In our method an affine hyperplane is swept through the given polytope until a random fraction (sampled from a proper univariate distribution) of the volume of the polytope is covered. Then the intersection of the plane with the polytope is a simple polytope with smaller dimension. In the second part we apply this method to construct a black-box algorithm for log-concave and

T
T

-concave multivariate distributions by means of transformed density rejection.
Calculation of values of 𝐿-functions associated to elliptic curves
Authors: Akiyama Shigeki , Tanigawa Yoshio
Published: July 2002

We calculated numerically the values of

L
L

-functions of four typical elliptic curves in the critical strip in the range

Im
(
s
)
≤
400

\text {Im}(s)\leq 400

. We found that all the non-trivial zeros in this range lie on the critical line

Re
(
s
)
=
1

\text {Re}(s)=1

and are simple except the one at

s
=
1

s=1

. The method we employed in this paper is the approximate functional equation with incomplete gamma functions in the coefficients. For incomplete gamma functions, we continued them holomorphically to the right half plane

Re
(
s
)
>
0

\text {Re}(s)>0

, which enables us to calculate for large

Im
(
s
)

\text {Im}(s)

. Furthermore we remark that a relation exists between Sato-Tate conjecture and the generalized Riemann Hypothesis.
Birbeck Granules Are Subdomains of Endosomal Recycling Compartment in Human Epidermal Langerhans Cells, Which Form Where Langerin Accumulates
Authors: Mc Dermott Ray , Ziylan Umit , Spehner Danièle , Bausinger Huguette , Lipsker Dan , Mommaas Mieke , Cazenave Jean-Pierre , Raposo Graça , Goud Bruno , de la Salle Henri , Salamero Jean , Hanau Daniel
Published: July 2002

Birbeck granules are unusual rod-shaped structures specific to epidermal Langerhans cells, whose origin and function remain undetermined. We investigated the intracellular location and fate of Langerin, a protein implicated in Birbeck granule biogenesis, in human epidermal Langerhans cells. In the steady state, Langerin is predominantly found in the endosomal recycling compartment and in Birbeck granules. Langerin internalizes by classical receptor-mediated endocytosis and the first Birbeck granules accessible to endocytosed Langerin are those connected to recycling endosomes in the pericentriolar area, where Langerin accumulates. Drug-induced inhibition of endocytosis results in the appearance of abundant open-ended Birbeck granule-like structures appended to the plasma membrane, whereas inhibition of recycling induces Birbeck granules to merge with a tubular endosomal network. In mature Langerhans cells, Langerin traffic is abolished and the loss of internal Langerin is associated with a concomitant depletion of Birbeck granules. Our results demonstrate an exchange of Langerin between early endosomal compartments and the plasma membrane, with dynamic retention in the endosomal recycling compartment. They show that Birbeck granules are not endocytotic structures, rather they are subdomains of the endosomal recycling compartment that form where Langerin accumulates. Finally, our results implicate ADP-ribosylation factor proteins in Langerin trafficking and the exchange between Birbeck granules and other endosomal membranes.
A heuristic managing errors for DNA sequencing
Authors: Błażewicz Jacek , Formanowicz Piotr , Guinand Frederic , Kasprzak Marta
Published: July 2002

Motivation: A new heuristic algorithm for solving DNA sequencing by hybridization problem with positive and negative errors.

Results: A heuristic algorithm providing better solutions than algorithms known from the literature based on tabu search method.

Contact: blazewic@sol.put.poznan.pl

* To whom correspondence should be addressed.
RETHINKING RESTRICTIONS ON PLAYER MOBILITY IN MAJOR LEAGUE BASEBALL
Authors: Maxcy Joel G.
Published: July 2002

This article considers the possibility that transactions costs and asymmetric income effects have altered the distribution of talent in the aftermath of institutional changes in Major League Baseball. This contradicts the invariance principle of Coase's theorem. Empirical evidence tracking player movement and competitive balance over the past 50 years conclusively supports these stands. Free agency, when permitted to proceed with limited intereference, has increased the rate that productive players transfer and has also improved some measures of competitive balance. Competitive balance is also shown to have improved with the implementation of the draft. Policy prescriptions calling for increased restrictions on player mobility are ill advised.
Current and future management of infections due to methicillin-resistant staphylococci infections: the role of quinupristin/dalfopristin
Authors: Péchère J. C.
Published: July 2002

The rise in the number of multidrug-resistant Gram-positive bacteria that has occurred in recent years has resulted in the development of infections that are difficult to treat, and also in severely restricted treatment options. In particular, the incidence of methicillin-resistant Staphylococcus aureus (MRSA) has increased, with strains shown to cause up to 21% of skin infections and 59.6% of nosocomial pneumonia. Recently, strains of S. aureus with reduced susceptibility to vancomycin (glycopeptide-intermediate S. aureus or GISA) are causing great concern, particularly as vancomycin has been the agent of choice in the treatment of infection caused by MRSA. GISA has been identified in Japan, the USA and Europe. New agents that have anti-MRSA activity are now being investigated. These include the novel streptogramin, quinupristin/dalfopristin. This report examines the activity of quinupristin/dalfopristin against strains of S. aureus and coagulase-negative staphylococci, including multidrug-resistant MRSA and GISA.
Preface
Authors: Drake Susan , Wood Martin
Published: July 2002

Infections with the α-herpesviruses, herpes simplex virus (HSV) and varicella-zoster virus (VZV) are common and are often considered trivial dermatological conditions. They should more appropriately be considered as chronic infections of the sensory nervous system. Reactivation of either virus results in significant discomfort and psychosocial distress, problems that could be minimized by appropriate therapy. Furthermore, the public health impact of genital HSV infection should not be underestimated. These aspects were explored at a symposium held at the Eighth Congress of the European Academy of Dermatology and Venereology, held in Amsterdam in September 1999, the proceedings of which form the basis for this supplement.

Four important aspects of HSV and VZV infections are addressed. The first is the true significance to the patient of pain persisting after acute herpes zoster. In spite of the availability of oral antiviral agents, appropriate use of which can reduce the duration of such pain, there are still barriers to early diagnosis and prompt institution of effective therapy. The second contribution discusses the importance of diagnosing genital HSV infection, both in terms of reducing morbidity by appropriate treatment of the symptomatic individual and, in public health terms, to reduce transmission of the virus. The authors recognize both the advantages in using type-specific serology in truly asymptomatic patients as well as any concomitant costs in terms of psychological distress to the individual or their partner. The pathogenesis, epidemiology, diverse clinical presentations, psychosocial impact and management of facial herpes simplex in general are discussed in the third manuscript. The final paper provides an in-depth review of the available data on HSV infection and its prevention in the context of facial resurfacing in cosmetic surgery. Further ideas for improved outcome are considered in this setting, where the natural expectation of excellent results is high. Awareness of the importance and extent of each of these issues should improve the management of many patients.

We acknowledge with gratitude the support of Glaxo Wellcome in the production of this publication.
Base–compositional biases and the bat problem. III. The question of microchiropteran monophyly
Authors: Hutcheon James M. , Kirsch John A.W. , Pettigrew John D.
Published: July 2002

Using single–copy DNA hybridization, we carried out a whole genome study of 16 bats (from ten families) and five outgroups (two primates and one each dermopteran, scandentian, and marsupial). Three of the bat species represented as many families of Rhinolophoidea, and these always associated with the two representatives of Pteropodidae. All other microchiropterans, however, formed a monophyletic unit displaying interrelationships largely in accord with current opinion. Thus noctilionoids comprised one clade, while vespertilionids, emballonurids, and molossids comprised three others, successively more closely related in that sequence. The unexpected position of rhinolophoids may be due either to the high AT bias they share with pteropodids, or it may be phylogenetically authentic. Reanalysis of the data with varying combinations of the five outgroups does not indicate a rooting problem, and the inclusion of many bat lineages divided at varying levels similarly discounts long branch attraction as an explanation for the pteropodid–rhinolophoid association. If rhinolophoids are indeed specially related to pteropodids, many synapomorphies of Microchiroptera are called into question, not least the unitary evolution of echolocation (although this feature may simply have been lost in pteropodids). Further, a rhinolophoid–pteropodid relationship — if true — has serious implications for the classification of bats. Finally, among the outgroups, an apparent sister–group relation of Dermoptera and Primates suggests that flying lemurs do not represent the ancestors of some or all bats; yet, insofar as gliding of the type implemented in dermopterans is an appropriate model for the evolution of powered mammalian flying, the position ofCynocephalusin our tree indirectly strengthens the argument that true flight could have evolved more than once among bats.