bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes
Authors: Simons Mikael , Krämer Eva-Maria , Macchi Paolo , Rathke-Hartlieb Silvia , Trotter Jacqueline , Nave Klaus-Armin , Schulz Jörg B.
Published: July 2002

Duplications and overexpression of the proteolipid protein (PLP) gene are known to cause the dysmyelinating disorder Pelizaeus-Merzbacher disease (PMD). To understand the cellular response to overexpressed PLP in PMD, we have overexpressed PLP in BHK cells and primary cultures of oligodendrocytes with the Semliki Forest virus expression system. Overexpressed PLP was routed to late endosomes/lysosomes and caused a sequestration of cholesterol in these compartments. Similar results were seen in transgenic mice overexpressing PLP. With time, the endosomal/lysosomal accumulation of cholesterol and PLP led to an increase in the amount of detergent-insoluble cellular cholesterol and PLP. In addition, two fluorescent sphingolipids, BODIPY–lactosylceramide and –galactosylceramide, which under normal conditions are sorted to the Golgi apparatus, were missorted to perinuclear structures. This was also the case for the lipid raft marker glucosylphosphatidylinositol–yellow fluorescence protein, which under normal steady-state conditions is localized on the plasma membrane and to the Golgi complex. Taken together, we show that overexpression of PLP leads to the formation of endosomal/lysosomal accumulations of cholesterol and PLP, accompanied by the mistrafficking of raft components. We propose that these accumulations perturb the process of myelination and impair the viability of oligodendrocytes.
V(D)J Recombination: Modulation of RAG1 and RAG2 Cleavage Activity on 12/23 Substrates by Whole Cell Extract and DNA-bending Proteins
Authors: Sawchuk Dennis J. , Weis-Garcia Frances , Malik Sohail , Besmer Eva , Bustin Michael , Nussenzweig Michel C. , Cortes Patricia
Published: July 2002

Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23 rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1 and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.
CD19-Regulated Signaling Thresholds Control Peripheral Tolerance and Autoantibody Production in B Lymphocytes
Authors: Inaoki Makoto , Sato Shinichi , Weintraub Bennett C. , Goodnow Christopher C. , Tedder Thomas F.
Published: July 2002

The CD19 cell surface molecule regulates signal transduction events critical for B lymphocyte development and humoral immunity. Increasing the density of CD19 expression renders B lymphocytes hyper-responsive to transmembrane signals, and transgenic mice that overexpress CD19 have increased levels of autoantibodies. The role of CD19 in tolerance regulation and autoantibody generation was therefore examined by crossing mice that overexpress a human CD19 transgene with transgenic mice expressing a model autoantigen (soluble hen egg lysozyme, sHEL) and high-affinity HEL-specific IgMa and IgDa (IgHEL) antigen receptors. In this model of peripheral tolerance, B cells in sHEL/IgHEL double-transgenic mice are functionally anergic and do not produce autoantibodies. However, it was found that overexpression of CD19 in sHEL/IgHEL double-transgenic mice resulted in a breakdown of peripheral tolerance and the production of anti-HEL antibodies at levels similar to those observed in IgHEL mice lacking the sHEL autoantigen. Therefore, altered signaling thresholds due to CD19 overexpression resulted in the breakdown of peripheral tolerance. Thus, CD19 overexpression shifts the balance between tolerance and immunity to autoimmunity by augmenting antigen receptor signaling.
Role of CD8 in Aberrant Function of Cytotoxic T Lymphocytes
Authors: Kessler Benedikt , Hudrisier Denis , Cerottini Jean-Charles , Luescher Immanuel F.
Published: July 2002

Using H-2Kd-restricted photoprobe-specific cytotoxic T lymphocyte (CTL) clones, which permit assessment of T cell receptor (TCR)-ligand interactions by TCR photoaffinity labeling, we observed that the efficiency of antigen recognition by CTL was critically dependent on the half-life of TCR-ligand complexes. We show here that antigen recognition by CTL is essentially determined by the frequency of serial TCR engagement, except for very rapid dissociations, which resulted in aberrant TCR signaling and antagonism. Thus agonists that were efficiently recognized exhibited rapid TCR–ligand complex dissociation, and hence a high frequency of serial TCR engagement, whereas the opposite was true for weak agonists. Surprisingly, these differences were largely accounted for by the coreceptor CD8. While it was known that CD8 substantially decreases TCR–ligand complex dissociation, we observed in this study that this effect varied considerably among ligand variants, indicating that epitope modifications can alter the CD8 contribution to TCR-ligand binding, and hence the efficiency of antigen recognition by CTL.
Rapid Effector Function in CD8+ Memory T Cells
Authors: Lalvani Ajit , Brookes Roger , Hambleton Sophie , Britton Warwick J. , Hill Adrian V.S. , McMichael Andrew J.
Published: July 2002

The nature of the CD8+ T cells that underlie antiviral protective immunological memory in vivo is unclear. We have characterized peptide-specific CD8+ T lymphocytes directly ex vivo from peripheral blood in humans with past exposure to influenza virus, using single cell interferon γ (IFN-γ) release as a measure of effector function. In individuals in the memory state with respect to influenza virus infection, unrestimulated antigen-specific CD8+ T cells displayed IFN-γ release within 6 h of antigen contact, identifying a population of memory CD8+ T cells that exhibit effector function without needing to divide and differentiate over several days. We have quantified circulating CD8+ effector T cells specific for six different MHC class I–restricted influenza virus epitopes. Enumeration of these CD8+ T cells gives frequencies of peptide-specific T cells that correlate with, but are in general severalfold higher than, CTL precursor frequencies derived from limiting dilution analysis, indicating that this novel population of memory CD8+ T cells has hitherto been undetected by standard means. The phenotype of these cells, which persist at a low frequency long after recovery from an acute viral infection, suggests that they play a role in protective immunological memory.
Interferon (IFN) Consensus Sequence-binding Protein, a Transcription Factor of the IFN Regulatory Factor Family, Regulates Immune Responses In Vivo through Control of Interleukin 12 Expression
Authors: Giese Nathalia A. , Gabriele Lucia , Doherty T. Mark , Klinman Dennis M. , Tadesse-Heath Lekidelu , Contursi Christina , Epstein Suzanne L. , Morse Herbert C.
Published: July 2002

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a chronic myelogenous leukemia-like syndrome and mount impaired responses to certain viral and bacterial infections. To gain a mechanistic understanding of the contributions of ICSBP to humoral and cellular immunity, we characterized the responses of control and ICSBP−/− mice to infection with influenza A (flu) and Leishmania major (L. major). Mice of both genotypes survived infections with flu, but differed markedly in the isotype distribution of antiflu antibodies. In sera of normal mice, immunoglobulin (Ig)G2a antibodies were dominant over IgG1 antibodies, a pattern indicative of a T helper cell type 1 (Th1)-driven response. In sera of ICSBP−/− mice, however, IgG1 antibodies dominated over IgG2a antibodies, a pattern indicative of a Th2-driven response. The dominance of IgG1 and IgE over IgG2a was detected in the sera of uninfected mice as well. A seeming Th2 bias of ICSBP-deficient mice was also uncovered in their inability to control infection with L. major, where resistance is known to be dependent on IL-12 and IFN-γ as components of a Th1 response. Infected ICSBP-deficient mice developed fulminant, disseminated leishmaniasis as a result of failure to mount a Th1-mediated curative response, although T cells remained capable of secreting IFN-γ and macrophages of producing nitric oxide. Compromised Th1 differentiation in ICSBP−/− mice could not be attributed to hyporesponsiveness of CD4+ T cells to interleukin (IL)-12; however, the ability of uninfected and infected ICSBP-deficient mice to produce IL-12 was markedly impaired. This indicates that ICSBP is a deciding factor in Th responses governing humoral and cellular immunity through its role in regulating IL-12 expression.
The Sequential Role of Lymphotoxin and B Cells in the Development of Splenic Follicles
Authors: Gonzalez Mercedes , Mackay Fabienne , Browning Jeffrey L. , Kosco-Vilbois Marie H. , Noelle Randolph J.
Published: July 2002

The transfer of lymphocytes into severe combined immunodeficiency (SCID) mice induces a series of histological changes in the spleen, including the appearance of mature follicular dendritic cells (FDCs). Studies were undertaken to clarify the role of lymphotoxin (LT) in this process. The results show that SCID mice have a small and partially differentiated white pulp containing marginal zone and interdigitating dendritic cells, but lacking FDCs. Transferred spleen cells can segregate into T and B cell areas shortly after their injection to SCID mice. This ability is dependent on signaling through LT-β receptor (LT-βR), since blocking ligand–receptor interaction in recipient SCID mice ablates the capacity of the transferred cells to segregate. A week after lymphocyte transfer, host-derived FDCs appeared in the reconstituted SCID mice. This induction of FDCs is dependent on LT-βR signaling by B cells since LT-α−/− B cells are incapable of inducing development of FDCs in SCID mice, even after cotransfer of LT-α+/+ T cells. Therefore, LT plays at least two discrete roles in splenic organization. First, it appears that LT induces the differentiation of the white pulp to create sites for lymphocyte segregation. Second, LT expression by B cells drives the maturation of FDCs and the organization of B cell follicles.
Interferon γ–independent Rejection of Interleukin 12–transduced Carcinoma Cells Requires CD4+ T Cells and Granulocyte/Macrophage Colony–stimulating Factor
Authors: Zilocchi Chiara , Stoppacciaro Antonella , Chiodoni Claudia , Parenza Mariella , Terrazzini Nadia , Colombo Mario P.
Published: July 2002

We analyzed the ability of interferon (IFN)-γ knockout mice (GKO) to reject a colon carcinoma transduced with interleukin (IL)-12 genes (C26/IL-12). Although the absence of IFN-γ impaired the early response and reduced the time to tumor onset in GKO mice, the overall tumor take rate was similar to that of BALB/c mice. In GKO mice, C26/IL-12 tumors had a reduced number of infiltrating leukocytes, especially CD8 and natural killer cells. Analysis of the tumor site, draining nodes, and spleens of GKO mice revealed reduced expression of IFN- inducible protein 10 and monokine induced by γ-IFN. Despite these defects, GKO mice that rejected C26/IL-12 tumor, and mice that were primed in vivo with irradiated C26/IL-12 cells, showed the same cytotoxic T lymphocyte activity but higher production of granulocyte/macrophage colony–stimulating factor (GM-CSF) as compared with control BALB/c mice. Treatment with monoclonal antibodies against GM-CSF abrogated tumor regression in GKO but not in BALB/c mice. CD4 T lymphocytes, which proved unnecessary or suppressive during rejection of C26/IL-12 cells in BALB/c mice, were required for tumor rejection in GKO mice. CD4 T cell depletion was coupled with a decline in GM-CSF expression by lymphocytes infiltrating the tumors or in the draining nodes, and with the reduction and disappearance of granulocytes and CD8 T cells, respectively, in tumor nodules. These results suggest that GM-CSF can substitute for IFN-γ in maintaining the CD8–polymorphonuclear leukocyte cross-talk that is a hallmark of tumor rejection.
Efficient Peripheral Clonal Elimination of B Lymphocytes in MRL/lpr Mice Bearing Autoantibody Transgenes
Authors: Kench Jennifer A. , Russell David M. , Nemazee David
Published: July 2002

Peripheral B cell tolerance was studied in mice of the autoimmune-prone, Fas-deficient MRL/ lpr.H-2d genetic background by introducing a transgene that directs expression of membrane-bound H-2Kb antigen to liver and kidney (MT-Kb) and a second transgene encoding antibody reactive with this antigen (3-83μδ, anti-Kk,b). Control immunoglobulin transgenic (Ig-Tg) MRL/lpr.H-2d mice lacking the Kb antigen had large numbers of splenic and lymph node B cells bearing the transgene-encoded specificity, whereas B cells of the double transgenic (Dbl-Tg) MRL/lpr.H-2d mice were deleted as efficiently as in Dbl-Tg mice of a nonautoimmune B10.D2 genetic background. In spite of the severely restricted peripheral B cell repertoire of the Ig-Tg MRL/lpr.H-2d mice, and notwithstanding deletion of the autospecific B cell population in the Dbl-Tg MRL/lpr.H-2d mice, both types of mice developed lymphoproliferation and exhibited elevated levels of IgG anti-chromatin autoantibodies. Interestingly, Dbl-Tg MRL/lpr.H-2d mice had a shorter lifespan than Ig-Tg MRL/lpr.H-2d mice, apparently as an indirect result of their relative B cell lymphopenia. These data suggest that in MRL/lpr mice peripheral B cell tolerance is not globally defective, but that certain B cells with receptors specific for nuclear antigens are regulated differently than are cells reactive to membrane autoantigens.
The Final Maturation of at Least Some Single-Positive Cd4hiThymocytes Does Not Require T Cell Receptor–Major Histocompatibility Complex Contact
Authors: Dyall Ruben , Nikolić-Z̆ugić Janko
Published: July 2002

The majority (∼70%) of postselection CD4+ single-positive (SP) thymocytes are CD8loCD4hi. These cells express very low levels of CD8, undetectable by flow cytofluorimetric (FCM) analysis, but sufficiently high to allow purification by panning. Unlike the fully mature CD8−CD4hi thymocytes, which account for the remaining ∼30% of the SP CD4+ thymocytes, CD8loCD4hi cells are functionally immature and short-lived unless they receive an unidentified maturation signal from the thymus. In this study, we tested the hypothesis that this signal is provided by a T cell receptor (TCR)–major histocompatibility complex (MHC) class II interaction. Using intrathymic transfer, we show that the immature CD8loCD4hi cells could complete their intrathymic maturation and populate the peripheral lymphoid organs in the absence of MHC class II (and class I) molecules. Furthermore, in mice devoid of class II (and class I) molecules, the progeny of CD8loCD4hi cells was long-lived and functionally reactive to allogeneic class II molecules, although their numbers in the spleen and the mesenteric lymph node were ∼40–50% lower than those in class II+ mice 5 mo after transfer. Control experiments demonstrated that the surviving cells did not originate from the contaminating mature thymocytes. These results demonstrate that the final maturation, proliferation, and peripheral survival (up to 5 mo) of at least some postselection CD4+ SP cells do not require the TCR–MHC class II interaction. They also indicate that the TCR–MHC class II interaction(s) required for the intrathymic development of long-lived CD4+ SP cells occurs before the CD4hi SP stage of development.
Thrombospondin-1 Is Downregulated by Anoxia and Suppresses Tumorigenicity of Human Glioblastoma Cells
Authors: Tenan Mirna , Fulci Giulia , Albertoni Michele , Diserens Annie-Claire , Hamou Marie-France , El Atifi-Borel Michèle , Feige Jean-Jacques , Pepper Michael S. , Van Meir Erwin G.
Published: July 2002

Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, results from a disruption of the balance between stimulatory and inhibitory factors. Here, we show that anoxia reduces expression of thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, in glioblastoma cells. This suggests that reduced oxygen tension can promote angiogenesis not only by stimulating the production of inducers, such as vascular endothelial growth factor, but also by reducing the production of inhibitors. This downregulation may significantly contribute to glioblastoma development, since we show that an increase in TSP-1 expression is sufficient to strongly suppress glioblastoma cell tumorigenicity in vivo.
Signaling via β2 Integrins Triggers Neutrophil-Dependent Alteration in Endothelial Barrier Function
Authors: Gautam Narinder , Herwald Heiko , Hedqvist Per , Lindbom Lennart
Published: July 2002

Activation of polymorphonuclear leukocytes (PMNs) and adhesion to the endothelial lining is a major cause of edema formation. Although known to be dependent on the function of β2 integrins (CD11/CD18), the precise mechanisms by which adherent PMNs may impair endothelial barrier capacity remain unclear. Here, the role of transmembrane signaling by β2 integrins in PMN-induced alterations in tight junctional permeability of cultured endothelial cell (EC) monolayers was investigated. PMN activation, in the absence of proinflammatory stimuli, was accomplished through antibody cross-linking of CD11b/CD18, mimicking adhesion-dependent receptor engagement. CD18 cross-linking in PMNs added to the EC monolayer provoked a prompt increase in EC permeability that coincided with a rise in EC cytosolic free Ca2+ and rearrangement of actin filaments, events similar to those evoked by chemoattractant PMN activation. Cell-free supernatant obtained after CD18 cross-linking in suspended PMNs triggered an EC response indistinguishable from that induced by direct PMN activation, and caused clear-cut venular plasma leakage when added to the hamster cheek pouch in vivo preparation. The PMN-evoked EC response was specific to β2 integrin engagement inasmuch as antibody cross-linking of l-selectin or CD44 was without effect on EC function. Our data demonstrate a causal link between outside-in signaling by β2 integrins and the capacity of PMNs to induce alterations in vascular permeability, and suggest a paracrine mechanism that involves PMN-derived cationic protein(s) in the cellular crosstalk between PMNs and ECs.
Cd40-Independent Pathways of T Cell Help for Priming of Cd8+ Cytotoxic T Lymphocytes
Authors: Lu Zhengbin , Yuan Lingxian , Zhou Xianzheng , Sotomayor Eduardo , Levitsky Hyam I. , Pardoll Drew M.
Published: July 2002

In many cases, induction of CD8+ CTL responses requires CD4+ T cell help. Recently, it has been shown that a dominant pathway of CD4+ help is via antigen-presenting cell (APC) activation through engagement of CD40 by CD40 ligand on CD4+ T cells. To further study this three cell interaction, we established an in vitro system using dendritic cells (DCs) as APCs and influenza hemagglutinin (HA) class I and II peptide–specific T cell antigen receptor transgenic T cells as cytotoxic T lymphocyte precursors and CD4+ T helper cells, respectively. We found that CD4+ T cells can provide potent help for DCs to activate CD8+ T cells when antigen is provided in the form of either cell lysate, recombinant protein, or synthetic peptides. Surprisingly, this help is completely independent of CD40. Moreover, CD40-independent CD4+ help can be documented in vivo. Finally, we show that CD40-independent T cell help is delivered through both sensitization of DCs and direct CD4+–CD8+ T cell communication via lymphokines. Therefore, we conclude that CD4+ help comprises at least three components: CD40-dependent DC sensitization, CD40-independent DC sensitization, and direct lymphokine-dependent CD4+–CD8+ T cell communication.
T Cell Receptor Complementarity Determining Region 3 Length Analysis Reveals the Absence of a Characteristic Public T Cell Repertoire in Neonatal Tolerance
Authors: Maverakis Emanual , Beech Jonathan T. , Wilson Stephen S. , Quinn Anthony , Pedersen Brian , Sercarz Eli E.
Published: July 2002

All adult BALB/c mice immunized with hen egg white lysozyme (HEL) or its dominant determinant, peptide (p)106–116, mount a T cell response using a “public” Vβ8.2Jβ1.5 T cell clone. Neonatal exposure to tolerance-inducing doses of antigen can drastically diminish responsiveness in the draining lymph nodes but not in the spleens of animals challenged as adults with the cognate antigen. To determine the role of T cell deletion or anergy within the mechanisms of observed neonatal “tolerance,” we treated neonatal BALB/c mice with HEL and directly followed the characteristic public clone using complementarity determining region 3 length T cell repertoire analysis. Our results confirm that despite intraperitoneal injection of neonates with a high dose of HEL emulsified in incomplete Freund's adjuvant, a strong splenic proliferative response to HEL was observed upon recall. However, the adult splenic T cell response of these neonatally treated mice lacked the usual Vβ8.2Jβ1.5 public clone characteristic of HEL-primed BALB/c mice. After challenge with HEL–complete Freund's adjuvant as adults, immunoglobulin (Ig)G2a isotype antibody was drastically reduced, and IgG1 was found to be the predominant anti-HEL IgG isotype expressed, indicating a deviation of cytokine response toward T helper type 2. 5-wk-old mice, nasally instilled with tolerogenic doses of HEL p106–116, also showed significant inhibition of this public T cell expansion. These results demonstrate that during neonatal and adult nasal tolerance induction, deletion/anergy removes the public clone, exposing a response of similar specificity but that is characterized by the T helper type 2 phenotype and a splenic residence.
Mast Cells Are Essential for Early Onset and Severe Disease in a Murine Model of Multiple Sclerosis
Authors: Secor Virginia H. , Secor W. Evan , Gutekunst Claire-Anne , Brown Melissa A.
Published: July 2002

In addition to their well characterized role in allergic inflammation, recent data confirm that mast cells play a more extensive role in a variety of immune responses. However, their contribution to autoimmune and neurologic disease processes has not been investigated. Experimental allergic encephalomyelitis (EAE) and its human disease counterpart, multiple sclerosis, are considered to be CD4+ T cell–mediated autoimmune diseases affecting the central nervous system. Several lines of indirect evidence suggest that mast cells could also play a role in the pathogenesis of both the human and murine disease. Using a myelin oligodendrocyte glycoprotein (MOG)-induced model of acute EAE, we show that mast cell–deficient W/Wv mice exhibit significantly reduced disease incidence, delayed disease onset, and decreased mean clinical scores when compared with their wild-type congenic littermates. No differences were observed in MOG-specific T and B cell responses between the two groups, indicating that a global T or B cell defect is not present in W/Wv animals. Reconstitution of the mast cell population in W/Wv mice restores induction of early and severe disease to wild-type levels, suggesting that mast cells are critical for the full manifestation of disease. These data provide a new mechanism for immune destruction in EAE and indicate that mast cells play a broader role in neurologic inflammation.
Cytotoxic T Lymphocyte Antigen 4 and CD28 Modulate Cell Surface Raft Expression in Their Regulation of T Cell Function
Authors: Martin Margarita , Schneider Helga , Azouz Abdallah , Rudd Christopher E.
Published: July 2002

Coreceptors CD28 and cytotoxic T lymphocyte antigen (CTLA)-4 have opposing effects on TcR/CD3 activation of T cells. While CD28 enhances and CTLA-4 inhibits activation, the underlying molecular basis of these effects has yet to be established. In this context, ganglioside and cholesterol enriched membrane microdomains (rafts, GEMs) serve as centers of signaling in T cells. Although CD28 can promote TcR/raft colocalization, evidence is lacking on whether the surface expression of membrane rafts can be targeted by CTLA-4 in its modulation of T cell responses. In this study, we demonstrate that both CD28 and CTLA-4 profoundly alter the surface expression of membrane rafts during T cell activation. While CD28 increased expression and the number of peripheral T cells induced to express surface rafts in response to TcR ligation, CTLA-4 potently inhibited both TcR and TcR × CD28 induced raft expression on the surface of T cells. Consistent with this, CD28 increased the presence of the linker of activated T cells (LAT) in purified membrane rafts, while CTLA-4 coligation effectively blocked this increase. Further, the reversal of the CTLA-4 block with CD3/CD28 ligation was accompanied by an increase in surface raft expression and associated LAT. Our observations demonstrate for the first time that CTLA-4 targets the release of rafts to the surface of T cells, and provides a mechanism for the opposing effects of CD28 and CTLA-4 on costimulation.
Unique Chemotactic Response Profile and Specific Expression of Chemokine Receptors Ccr4 and Ccr8 by Cd4+Cd25+ Regulatory T Cells
Authors: Iellem Andrea , Mariani Margherita , Lang Rosmarie , Recalde Helios , Panina-Bordignon Paola , Sinigaglia Francesco , D'Ambrosio Daniele
Published: July 2002

Chemokines dictate regional trafficking of functionally distinct T cell subsets. In rodents and humans, a unique subset of CD4+CD25+ cytotoxic T lymphocyte antigen (CTLA)-4+ regulatory T cells (Treg) has been proposed to control peripheral tolerance. However, the molecular basis of immune suppression and the trafficking properties of Treg cells are still unknown. Here, we determined the chemotactic response profile and chemokine receptor expression of human blood-borne CD4+CD25+ Treg cells. These Treg cells were found to vigorously respond to several inflammatory and lymphoid chemokines. Treg cells specifically express the chemokine receptors CCR4 and CCR8 and represent a major subset of circulating CD4+ T cells responding to the chemokines macrophage-derived chemokine (MDC)/CCL22, thymus and activation-regulated chemokine (TARC)/CCL17, I-309/CCL1, and to the virokine vMIP-I (ligands of CCR4 and CCR8). Blood-borne CD4+ T cells that migrate in response to CCL1 and CCL22 exhibit a reduced alloproliferative response, dependent on the increased frequency of Treg cells in the migrated population. Importantly, mature dendritic cells preferentially attract Treg cells among circulating CD4+ T cells, by secretion of CCR4 ligands CCL17 and CCL22. Overall, these results suggest that CCR4 and/or CCR8 may guide Treg cells to sites of antigen presentation in secondary lymphoid tissues and inflamed areas to attenuate T cell activation.
Early Intermediates in the Transport Cycle of the Neuronal Excitatory Amino Acid Carrier Eaac1
Authors: Watzke Natalie , Bamberg Ernst , Grewer Christof
Published: July 2002

Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EAAC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The molecular nature of these charge movements is poorly understood at present and, therefore, was studied in this report in detail by using the technique of laser-pulse photolysis of caged glutamate providing a 100-μs time resolution. In the inward transport mode, the deactivation of the transient component of the glutamate-induced coupled transport current exhibits two exponential components. Similar results were obtained when restricting EAAC1 to Na+ translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na+-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge movement and of the steady-state EAAC1 properties, we developed a kinetic model that is based on sequential binding of Na+ and glutamate to their extracellular binding sites on EAAC1 explaining our results. In this model, at least one Na+ ion and thereafter glutamate rapidly bind to the transporter initiating a slower, electroneutral structural change that makes EAAC1 competent for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, electrogenic translocation reaction to expose the substrate and ion binding sites to the cytoplasm.
Volume-regulated Anion Channels Serve as an Auto/Paracrine Nucleotide Release Pathway in Aortic Endothelial Cells
Authors: Hisadome Kazunari , Koyama Tetsuya , Kimura Chiwaka , Droogmans Guy , Ito Yushi , Oike Masahiro
Published: July 2002

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca2+ oscillations and NO production. They did not, however, affect Ca2+ oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a “permeating blocker model,” in which ATP binds with an affinity of 1.0 ± 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl− hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.
Cohen-Macaulayness of tangent cones
Authors: Arslan Feza
Published: July 2002

We give a criterion for checking the Cohen-Macaulayness of the tangent cone of a monomial curve by using the Gröbner basis. For a family of monomial curves, we give the full description of the defining ideal of the curve and its tangent cone at the origin. By using this family of curves and their extended versions to higher dimensions, we prove that the minimal number of generators of a Cohen-Macaulay tangent cone of a monomial curve in an affine

l
l

-space can be arbitrarily large for

l
≥
4

l \geq 4

contrary to the

l
=
3

l=3

case shown by Robbiano and Valla. We also determine the Hilbert series of the associated graded ring of this family of curves and their extended versions.
The number of lattice points in alcoves and the exponents of the finite Weyl groups
Authors: Suter Ruedi
Published: July 2002

We count lattice points in certain rational simplices associated with an irreducible finite Weyl group

W
W

and observe that these numbers are linked to the exponents of

W
W

.
The McKay correspondence as an equivalence of derived categories
Authors: Bridgeland Tom , King Alastair , Reid Miles
Published: July 2002

Let

G
G

be a finite group of automorphisms of a nonsingular three-dimensional complex variety

M
M

, whose canonical bundle

ω
M

\omega _M

is locally trivial as a

G
G

-sheaf. We prove that the Hilbert scheme

Y
=
G

Y = G

-

Hilb
⁡
M

\operatorname {Hilb}M

parametrising

G
G

-clusters in

M
M

is a crepant resolution of

X
=
M

/

G

X=M/G

and that there is a derived equivalence (Fourier–Mukai transform) between coherent sheaves on

Y
Y

and coherent 𝐺-sheaves

on

M
M

. This identifies the K theory of

Y
Y

with the equivariant K theory of

M
M

, and thus generalises the classical McKay correspondence. Some higher-dimensional extensions are possible.
Family algebras
Authors: Kirillov A.
Published: July 2002

A new class of associative algebras is introduced and studied. These algebras are related to simple complex Lie algebras (or root systems). Roughly speaking, they are finite dimensional approximations to the enveloping algebra

U
(

g

)

U(\mathfrak {g})

viewed as a module over its center. It seems that several important questions on semisimple algebras and their representations can be formulated, studied and sometimes solved in terms of our algebras. Here we only start this program and hope that it will be continued and developed.
Weyl modules for classical and quantum affine algebras
Authors: Chari Vyjayanthi , Pressley Andrew
Published: July 2002

We introduce and study the notion of a Weyl module for the classical affine algebras, these modules are universal finite-dimensional highest weight modules. We conjecture that the modules are the classical limit of a family of irreducible modules of the quantum affine algebra, and prove the conjecture in the case of

s

l
2

sl_2

. The conjecture implies also that the Weyl modules are the classical limits of the standard modules introduced by Nakajima and further studied by Varagnolo and Vasserot.
Non-linear optimization of biochemical pathways: applications to metabolic engineering and parameter estimation.
Authors: Mendes P , Kell D
Published: July 2002

MOTIVATION: The simulation of biochemical kinetic systems is a powerful approach that can be used for: (i) checking the consistency of a postulated model with a set of experimental measurements, (ii) answering 'what if?' questions and (iii) exploring possible behaviours of a model. Here we describe a generic approach to combine numerical optimization methods with biochemical kinetic simulations, which is suitable for use in the rational design of improved metabolic pathways with industrial significance (metabolic engineering) and for solving the inverse problem of metabolic pathways, i.e. the estimation of parameters from measured variables. RESULTS: We discuss the suitability of various optimization methods, focusing especially on their ability or otherwise to find global optima. We recommend that a suite of diverse optimization methods should be available in simulation software as no single one performs best for all problems. We describe how we have implemented such a simulation-optimization strategy in the biochemical kinetics simulator Gepasi and present examples of its application. AVAILABILITY: The new version of Gepasi (3.20), incorporating the methodology described here, is available on the Internet at http://gepasi.dbs.aber.ac.uk/softw/Gepasi. html. CONTACT: prm@aber.ac.uk
Local UV‐induced DNA damage in cell nuclei results in local transcription inhibition
Authors: Moné Martijn J , Volker Marcel , Nikaido Osamu , Mullenders Leon H F , van Zeeland Albert A , Verschure Pernette J , Manders Erik M M , van Driel Roel
Published: July 2002

UV‐induced DNA damage causes cells to repress RNA synthesis and to initiate nucleotide excision repair (NER). NER and transcription are intimately linked processes. Evidence has been presented that, in addition to damaged genes, undamaged loci are transcriptionally inhibited. We investigated whether RNA synthesis from undamaged genes is affected by the presence of UV damage elsewhere in the same nucleus, using a novel technique to UV irradiate only part of a nucleus. We show that the basal transcription/repair factor TFIIH is recruited to the damaged nuclear area, partially depleting the undamaged nuclear area. Remarkably, this sequestration has no effect on RNA synthesis. This result was obtained for cells that are able to carry out NER and for cells deficient in NER. We conclude that cross talk between NER and transcription occurs only over short distances in nuclei of living cells.
Production of Near-Isogenic Lines and Marked Monosomic Lines in Common Wheat (Triticum aestivum) cv. Chinese Spring
Authors: Tsujimoto H.
Published: July 2002

Sixteen near-isogenic lines (NILs) carrying a marker gene were produced by the recurrent backcrossing method in the genetic background of common wheat (Triticum aestivum) cv. Chinese Spring (CS). Three genes from alien species showed segregation distortion. In NILs carrying a marker gene of rye (Secale cereale) or Aegilops caudata, the alien chromosome segments were detected by fluorescence in situ hybridization (FISH). The NILs were grown with replications and the effect of marker genes on plant morphology in the genetic background of CS was investigated. These NILs were further crossed with the corresponding monosomics of CS and 13 monosomic lines whose monosome carries a respective marker gene were established and named “marked monosomics.” Many of the marked monosomics were distinguishable from the disomic NILs because of the different dosage effect of the genes. The NILs are utilized for studies on gene isolation or gene regulation. Marked monosomics are useful not only for monosomic analysis but also for production of homologous chromosome substitution lines because chromosome observation is not required.
Spatial characteristics to calcium signalling; the calcium wave as a basic unit in plant cell calcium signalling
Authors: Malhó Rui , Moutinho Ana , van der Luit Arnold , Trewavas Anthony J.
Published: July 2002

Many signals that modify plant cell growth and development initiate changes in cytoplasmic Ca2+. The subsequent movement of Ca2+in the cytoplasm is thought to take place via waves of free Ca2+. These waves may be initiated at defined regions of the cell and movement requires release from a reticulated endoplasmic reticulum and the vacuole. The mechanism of wave propagation is outlined and the possible basis of repetitive reticulum wave formation, Ca2+oscillations and capacitative Ca2+signalling is discussed. Evidence for the presence of Ca2+waves in plant cells is outlined, and from studies on raphides it is suggested that the capabilities for capacitative Ca2+signalling are also present. The paper finishes with an outline of the possible interrelation between Ca2+waves and organelles and describes the intercellular movement of Ca2+waves and the relevance of such information communication to plant development.
Termite assemblages, forest disturbance and greenhouse gas fluxes in Sabah, East Malaysia
Authors: Eggleton P. , Homathevi R. , Jones D. T. , MacDonald J. A. , Jeeva D. , Bignell D. E. , Davies R. G. , Maryati M.
Published: July 2002

A synthesis is presented of sampling work conducted under a UK government–funded Darwin Initiative grant undertaken predominantly within the Danum Valley Conservation Area (DVCA), Sabah, East Malaysia. The project concerned the assemblage structure, gas physiology and landscape gas fluxes of termites in pristine and two ages of secondary, dipterocarp forest. The DVCA termite fauna is typical of the Sunda region, dominated by
Termes
–group soil–feeders and Nasutitermitinae. Selective logging appears to have relatively little effect on termite assemblages, although soil–feeding termites may be moderately affected by this level of disturbance. Species composition changes, but to a small extent when considered against the background level of compositional differences within the Sunda region. Physiologically the assemblage is very like others that have been studied, although there are some species that do not fit on the expected body size–metabolic rate curve. As elsewhere, soil–feeders and soil–wood interface–feeders tend to produce more methane. As with the termite assemblage characteristics, gross gas and energy fluxes do not differ significantly between logged and unlogged sites. Although gross methane fluxes are high, all the soils at DVCA were methane sinks, suggesting that methane oxidation by methanotrophic bacteria was a more important process than methane production by gut archaea. This implies that methane production by termites in South–East Asia is not contributing significantly to the observed increase in levels of methane production worldwide. Biomass density, species richness, clade complement and energy flow were much lower at DVCA than at a directly comparable site in southern Cameroon. This is probably due to the different biogeographical histories of the areas.
The immune responses to bacterial antigens encountered in vivo at mucosal surfaces
Authors: Dougan Gordon , Ghaem–Maghami Marjan , Pickard Derek , Frankel Gad , Douce Gill , Clare Simon , Dunstan Sarah , Simmons Cameron
Published: July 2002

Mammals have evolved a sophisticated immune system for handling antigens encountered at their mucosal surfaces. The way in which mucosally delivered antigens are handled influences our ability to design effective mucosal vaccines. Live attenuated derivatives of pathogens are one route towards the development of mucosal vaccines. However, some molecules, described as mucosal immunogens, are inherently immunogenic at mucosal surfaces. Studies on mucosal immunogens may facilitate the identification of common characteristics that contribute to mucosal immunogenicity and aid the development of novel, non–living mucosal vaccines and immunostimulators.
The relationships of vascular plants
Authors: Kenrick P.
Published: July 2002

Recent phylogenetic research indicates that vascular plants evolved from bryophyte–like ancestors and that this involved extensive modifications to the life cycle. These conclusions are supported by a range of systematic data, including gene sequences, as well as evidence from comparative morphology and the fossil record. Within vascular plants, there is compelling evidence for two major clades, which have been termed lycophytes (clubmosses) and euphyllophytes (seed plants, ferns, horsetails). The implications of recent phylogenetic work are discussed with reference to life cycle evolution and the interpretation of stratigraphic inconsistencies in the early fossil record of land plants. Life cycles are shown to have passed through an isomorphic phase in the early stages of vascular plant evolution. Thus, the gametophyte generation of all living vascular plants is the product of massive morphological reduction. Phylogenetic research corroborates earlier suggestions of a major representational bias in the early fossil record. Megafossils document a sequence of appearance of groups that is at odds with that predicted by cladogram topology. It is argued here that the pattern of appearance and diversification of plant megafossils owes more to changing geological conditions than to rapid biological diversification.
Global spectral–kinetic analysis of room temperature chlorophyll
a
fluorescence from light-harvesting antenna mutants of barley
Authors: Gilmore Adam M. , Itoh Shigeru , Govindjee
Published: July 2002

This study presents a novel measurement, and simulation, of the time–resolved room temperature chlorophyll
a
fluorescence emission spectra from leaves of the barley wild–type and chlorophyll–
b
–deficient chlorina (
clo
)
f2
and
f104
mutants. The primary data were collected with a streak–camera–based picosecond–pulsed fluorometer that simultaneously records the spectral distribution and time dependence of the fluorescence decay. A new global spectral–kinetic analysis programme method, termed the double convolution integral (DCI) method, was developed to convolve the exciting laser pulse shape with a multimodal–distributed decay profile function that is again convolved with the spectral emission band amplitude functions. We report several key results obtained by the simultaneous spectral–kinetic acquisition and DCI methods. First, under conditions of dark–level fluorescence, when photosystem II (PS II) photochemistry is at a maximum at room temperature, both the
clo f2
and
clo f104
mutants exhibit very similar PS II spectral–decay contours as the wild–type (
wt
), with the main band centred around 685 nm. Second, dark–level fluorescence is strongly influenced beyond 700 nm by broad emission bands from PS I, and its associated antennae proteins, which exhibit much more rapid decay kinetics and strong integrated amplitudes. In particular a 705–720 nm band is present in all three samples, with a 710nm band predominating in the
clo f2
leaves. When the PS II photochemistry becomes inhibited, maximizing the fluorescence yield, both the
clo f104
mutant and the
wt
exhibit lifetime increases for their major distribution modes from the minimal 250–500 ps range to the maximal 1500–2500 ps range for both the 685 nm and 740 nm bands. The
clo f2
mutant, however, exhibits several unique spectral–kinetic properties, attributed to its unique PS I antennae and thylakoid structure, indicating changes in both PS II fluorescence reabsorption and PS II to PS I energy transfer pathways compared to the
wt
and
clo f104
. Photoprotective energy dissipation mediated by the xanthophyll cycle pigments and the PsbS protein was uninhibited in the
clo f104
mutant but, as commonly reported in the literature, significantly inhibited in the
clo f2
; the inhibited energy dissipation is partly attributed to its thylakoid structure and PS II to PS I energy transfer properties. It is concluded that it is imperative with steady–state fluorometers, especially for
in vivo
studies of PS II efficiency or photoprotective energy dissipation, to quantify the influence of the PS I spectral emission.
The LN54 Radiation Hybrid Map of Zebrafish Expressed Sequences
Authors: Hukriede Neil , Fisher Dan , Epstein Jonathan , Joly Lucille , Tellis Patricia , Zhou Yi , Barbazuk Brad , Cox Kristine , Fenton-Noriega Laura , Hersey Candace , Miles Jennifer , Sheng Xiaoming , Song Anhua , Waterman Rick , Johnson Stephen L. , Dawid Igor B. , Chevrette Mario , Zon Leonard I. , McPherson John , Ekker Marc
Published: July 2002

To increase the density of a gene map of the zebrafish, Danio rerio, we have placed 3119 expressed sequence tags (ESTs) and cDNA sequences on the LN54 radiation hybrid (RH) panel. The ESTs and genes mapped here join 748 SSLp markers and 459 previously mapped genes and ESTs, bringing the total number of markers on the LN54 RH panel to 4226. Addition of these new markers brings the total LN54 map size to 14,372 cR, with 118 kb/cR. The distribution of ESTs according to linkage groups shows relatively little variation (minimum, 73; maximum, 201). This observation, combined with a relatively uniform size for zebrafish chromosomes, as previously indicated by karyotyping, indicates that there are no especially gene-rich or gene-poor chromosomes in this species. We developed an algorithm to provide a semiautomatic method for the selection of additional framework markers for the LN54 map. This algorithm increased the total number of framework markers to 1150 and permitted the mapping of a high percentage of sequences that could not be placed on a previous version of the LN54 map. The increased concentration of expressed sequences on the LN54 map of the zebrafish genome will facilitate the molecular characterization of mutations in this species.