bluuebunny/crossref_metadata_2025 huggingface dataset:
The NH2 Terminus of Titin Spans the Z-Disc: Its Interaction with a Novel 19-kD Ligand (T-cap) Is Required for Sarcomeric Integrity
Authors: Gregorio Carol C. , Trombitás Karoly , Centner Thomas , Kolmerer Bernhard , Stier Gunter , Kunke Kathleen , Suzuki Koichi , Obermayr Franz , Herrmann Bernhard , Granzier Henk , Sorimachi Hiroyuki , Labeit Siegfried
Published: July 2002

Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3–4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for α-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of α-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament–regulated sarcomeric assembly.
Analysis of Dynactin Subcomplexes Reveals a Novel Actin-Related Protein Associated with the Arp1 Minifilament Pointed End
Authors: Eckley D. Mark , Gill Steven R. , Melkonian Karin A. , Bingham James B. , Goodson Holly V. , Heuser John E. , Schroer Trina A.
Published: July 2002

The multisubunit protein, dynactin, is a critical component of the cytoplasmic dynein motor machinery. Dynactin contains two distinct structural domains: a projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo. Here, we use biochemical, ultrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynactin with recombinant dynamitin yields two assemblies: the actin-related protein, Arp1, minifilament and the p150Glued sidearm. Both contain dynamitin. Treatment of dynactin with the chaotropic salt, potassium iodide, completely depolymerizes the Arp1 minifilament to reveal multiple protein complexes that contain the remaining dynactin subunits. The shoulder/sidearm complex contains p150Glued, dynamitin, and p24 subunits and is ultrastructurally similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assembly that may link the shoulder/sidearm complex to the Arp1 minifilament. Pointed-end complex contains p62, p27, and p25 subunits, plus a novel actin-related protein, Arp11. p62, p27, and p25 contain predicted cargo-binding motifs, while the Arp11 sequence suggests a pointed-end capping activity. These isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.
The Yeast Nuclear Pore Complex
Authors: Rout Michael P. , Aitchison John D. , Suprapto Adisetyantari , Hjertaas Kelly , Zhao Yingming , Chait Brian T.
Published: July 2002

An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.
Assembly of the Dystrophin-Associated Protein Complex Does Not Require the Dystrophin Cooh-Terminal Domain
Authors: Crawford Gregory E. , Faulkner John A. , Crosbie Rachelle H. , Campbell Kevin P. , Froehner Stanley C. , Chamberlain Jeffrey S.
Published: July 2002

Dystrophin is a multidomain protein that links the actin cytoskeleton to laminin in the extracellular matrix through the dystrophin associated protein (DAP) complex. The COOH-terminal domain of dystrophin binds to two components of the DAP complex, syntrophin and dystrobrevin. To understand the role of syntrophin and dystrobrevin, we previously generated a series of transgenic mouse lines expressing dystrophins with deletions throughout the COOH-terminal domain. Each of these mice had normal muscle function and displayed normal localization of syntrophin and dystrobrevin. Since syntrophin and dystrobrevin bind to each other as well as to dystrophin, we have now generated a transgenic mouse deleted for the entire dystrophin COOH-terminal domain. Unexpectedly, this truncated dystrophin supported normal muscle function and assembly of the DAP complex. These results demonstrate that syntrophin and dystrobrevin functionally associate with the DAP complex in the absence of a direct link to dystrophin. We also observed that the DAP complexes in these different transgenic mouse strains were not identical. Instead, the DAP complexes contained varying ratios of syntrophin and dystrobrevin isoforms. These results suggest that alternative splicing of the dystrophin gene, which naturally generates COOH-terminal deletions in dystrophin, may function to regulate the isoform composition of the DAP complex.
Ectodomain Shedding of Epidermal Growth Factor Receptor Ligands Is Required for Keratinocyte Migration in Cutaneous Wound Healing
Authors: Tokumaru Sho , Higashiyama Shigeki , Endo Takeshi , Nakagawa Takatoshi , Miyagawa Jun-ichiro , Yamamori Katsumi , Hanakawa Yasushi , Ohmoto Hiroshi , Yoshino Kohichiro , Shirakata Yuji , Matsuzawa Yuji , Hashimoto Koji , Taniguchi Naoyuki
Published: July 2002

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)–ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR–immunoglobulin G-Fcγ fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.
Differential Expression of Lumican and Fibromodulin Regulate Collagen Fibrillogenesis in Developing Mouse Tendons
Authors: Ezura Yoichi , Chakravarti Shukti , Oldberg Åke , Chervoneva Inna , Birk David E.
Published: July 2002

Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.
A Novel Chromatin Protein, Distantly Related to Histone H2a, Is Largely Excluded from the Inactive X Chromosome
Authors: Chadwick Brian P. , Willard Huntington F.
Published: July 2002

Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope–tagged or green fluorescent protein–tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease–digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.
ZNF265—a novel spliceosomal protein able to induce alternative splicing
Authors: Adams David J. , van der Weyden Louise , Mayeda Akila , Stamm Stefan , Morris Brian J. , Rasko John E.J.
Published: July 2002

The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-β1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF35. Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265–EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain–containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery.
A Regulatory Role for TRAF1 in Antigen-induced Apoptosis of  T Cells
Authors: Speiser Daniel E. , Lee Soo Young , Wong Brian , Arron Joseph , Santana Angela , Kong Young-Yun , Ohashi Pamela S. , Choi Yongwon
Published: July 2002

Tumor necrosis factor receptor (TNFR)–associated factor 2 (TRAF2) and TRAF1 were found as components of the TNFR2 signaling complex, which exerts multiple biological effects on cells such as cell proliferation, cytokine production, and cell death. In the TNFR2-mediated signaling pathways, TRAF2 works as a mediator for activation signals such as NF-κB, but the role of TRAF1 has not been previously determined. Here we show in transgenic mice that TRAF1 overexpression inhibits antigen-induced apoptosis of CD8+ T lymphocytes. Our results demonstrate a biological role for TRAF1 as a regulator of apoptotic signals and also support the hypothesis that the combination of TRAF proteins in a given cell type determines distinct biological effects triggered by members of the TNF receptor superfamily.
Role of the Multiple T Cell Receptor (TCR)-ζ Chain Signaling Motifs in Selection of the T Cell Repertoire
Authors: Shores Elizabeth W. , Tran Tom , Grinberg Alexander , Sommers Connie L. , Shen Howard , Love Paul E.
Published: July 2002

Immature thymocytes undergo a selection process within the thymus based on their T cell antigen receptor (TCR) specificity that results either in their maturation into functionally competent, self-MHC–restricted T cells (positive selection) or their deletion (negative selection). The outcome of thymocyte selection is thought to be controlled by signals transduced by the TCR that vary in relation to the avidity of the TCR–ligand interaction. The TCR is composed of four distinct signal transducing subunits (CD3-γ, -δ, -ε, and ζ) that contain either one (CD3-γ, -δ, -ε) or three (-ζ) signaling motifs (ITAMs) within their intracytoplasmic domains. A possible function for multiple TCR ITAMs could be to amplify signals generated by the TCR during selection. To determine the importance of the multiple TCR-ζ chain ITAMs in thymocyte selection, transgenes encoding α/βTCRs with known specificity were bred into mice in which ζ chains lacking one or more ITAMs had been genetically substituted for endogenous ζ. A direct relationship was observed between the number of ζ chain ITAMs within the TCR complex and the efficiency of both positive and negative selection. These results reveal a role for multiple TCR ITAMs in thymocyte selection and identify a function for TCR signal amplification in formation of the T cell repertoire.
NKp44, a Novel Triggering Surface Molecule Specifically Expressed by Activated Natural Killer Cells, Is Involved in Non–Major Histocompatibility Complex–restricted Tumor Cell Lysis
Authors: Vitale Massimo , Bottino Cristina , Sivori Simona , Sanseverino Lorenza , Castriconi Roberta , Marcenaro Emanuela , Augugliaro Raffaella , Moretta Lorenzo , Moretta Alessandro
Published: July 2002

After culture in interleukin (IL)-2, natural killer (NK) cells acquire an increased capability of mediating non–major histocompatibility complex (MHC)–restricted tumor cell lysis. This may reflect, at least in part, the de novo expression by NK cells of triggering receptors involved in cytolysis. In this study we identified a novel 44-kD surface molecule (NKp44) that is absent in freshly isolated peripheral blood lymphocytes but is progressively expressed by all NK cells in vitro after culture in IL-2. Different from other markers of cell activation such as CD69 or VLA.2, NKp44 is absent in activated T lymphocytes or T cell clones. Since NKp44 was not detected in any of the other cell lineages analyzed, it appears as the first marker specific for activated human NK cells. Monoclonal antibody (mAb)–mediated cross-linking of NKp44 in cloned NK cells resulted in strong activation of target cell lysis in a redirected killing assay. This data indicated that NKp44 can mediate triggering of NK cell cytotoxicity. mAb-mediated masking of NKp44 resulted in partial inhibition of cytolytic activity against certain (FcγR-negative) NK-susceptible target cells. This inhibition was greatly increased by the simultaneous masking of p46, another recently identified NK-specific triggering surface molecule. These data strongly suggest that NKp44 functions as a triggering receptor selectively expressed by activated NK cells that, together with p46, may be involved in the process of non-MHC-restricted lysis. Finally, we show that p46 and NKp44 are coupled to the intracytoplasmic transduction machinery via the association with CD3ζ or KARAP/DAP12, respectively; these associated molecules are tyrosine phosphorylated upon NK cell stimulation.
CD3 Ligation on Immature Thymocytes Generates Antagonist-like Signals Appropriate for CD8 Lineage Commitment, Independently of  T Cell Receptor Specificity
Authors: Albert Basson M. , Bommhardt Ursula , Cole Michael S. , Tso J. Yun , Zamoyska Rose
Published: July 2002

The signals that direct differentiation of T cells to the CD4 or CD8 lineages in the thymus remain poorly understood. Although it has been relatively easy to direct differentiation of CD4 single positive (CD4+) cells using combinations of antibodies and pharmacological agents that mimic receptor engagements, equivalent stimuli do not induce efficient maturation of CD8+ cells. Here we report that, irrespective of the MHC-restriction specificity of the TCR, differentiation of mature CD8+ thymocytes can be induced by ligation of CD3 polypeptides on immature thymocytes with a F(ab′)2 reagent (CD3fos-F(ab′)2). The tyrosine phosphorylation patterns stimulated by CD3fos-F(ab′)2 have been shown to resemble those delivered to mature T cells by antagonist peptides, which are known to direct positive selection of CD8+ cells, and we can show that this reagent exhibits potent antagonistic-like activity for primary T cell responses. Our results suggest a distinction in the signals that specify lineage commitment in the thymus. We present a model of thymocyte differentiation that proposes that the relative balance of signals delivered by TCR engagement and by p56lck activation is responsible for directing commitment to the CD8 or CD4 lineages.
Disturbed CD4+ T Cell Homeostasis and In Vitro HIV-1 Susceptibility in Transgenic Mice Expressing T Cell Line–tropic HIV-1 Receptors
Authors: Sawada Shinichiro , Gowrishankar Kavitha , Kitamura Rui , Suzuki Misao , Suzuki Gen , Tahara Satoko , Koito Atsushi
Published: July 2002

T cell line–tropic (T-tropic) HIV type 1 strains enter cells by interacting with the cell-surface molecules CD4 and CXCR4. We have generated transgenic mice predominantly expressing human CD4 and CXCR4 on their CD4-positive T lymphocytes (CD4+ T cells). Their primary thymocytes are susceptible to T-tropic but not to macrophage-tropic HIV-1 infection in vitro, albeit with a viral antigen production less efficient than human peripheral blood mononuclear cells. Interestingly, even without HIV infection, transgenic mice display a CD4+ T cell depletion profile of peripheral blood reminiscent of that seen in AIDS patients. We demonstrate that CD4+ T cell trafficking in transgenic mice is biased toward bone marrow essentially due to CXCR4 overexpression, resulting in the severe loss of CD4+ T cells from circulating blood. Our data suggest that CXCR4 plays an important role in lymphocyte trafficking through tissues, especially between peripheral blood and bone marrow, participating in the regulation of lymphocyte homeostasis in these compartments. Based on these findings, we propose a hypothetical model in which the dual function of CXCR4 in HIV-1 infection and in lymphocyte trafficking may cooperatively induce progressive HIV-1 infection and CD4+ T cell decline in patients.
MD-2, a Molecule that Confers Lipopolysaccharide Responsiveness on Toll-like Receptor 4
Authors: Shimazu Rintaro , Akashi Sachiko , Ogata Hirotaka , Nagai Yoshinori , Fukudome Kenji , Miyake Kensuke , Kimoto Masao
Published: July 2002

Toll-like receptor 4 (TLR4) is a mammalian homologue of Drosophila Toll, a leucine-rich repeat molecule that can trigger innate responses against pathogens. The TLR4 gene has recently been shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS). TLR4 may be a long-sought receptor for LPS. However, transfection of TLR4 does not confer LPS responsiveness on a recipient cell line, suggesting a requirement for an additional molecule. Here, we report that a novel molecule, MD-2, is requisite for LPS signaling of TLR4. MD-2 is physically associated with TLR4 on the cell surface and confers responsiveness to LPS. MD-2 is thus a link between TLR4 and LPS signaling. Identification of this new receptor complex has potential implications for understanding host defense, as well as pathophysiologic, mechanisms.
Receptor Revision of Immunoglobulin Heavy Chain Variable Region Genes in Normal Human B Lymphocytes
Authors: Wilson Patrick C. , Wilson Kenneth , Liu Yong-Jun , Banchereau Jacques , Pascual Virginia , Capra J. Donald
Published: July 2002

Contrary to the general precepts of the clonal selection theory, several recent studies have provided evidence for the secondary rearrangement of immunoglobulin (Ig) genes in peripheral lymphoid tissues. These analyses typically used transgenic mouse models and have only detected secondary recombination of Ig light chain genes. Although Ig heavy chain variable region (VH) genes encode a substantial element of antibody combining site specificity, there is scant evidence for VH gene rearrangement in the periphery, leaving the physiological importance of peripheral recombination questionable. The extensive somatic mutations and clonality of the IgD+Strictly-IgM−CD38+ human tonsillar B cell subpopulation have now allowed detection of the first clear examples of receptor revision of human VH genes. The revised VDJ genes contain “hybrid” VH gene segments consisting of portions from two separate germline VH genes, a phenomenon previously only detected due to the pressures of a transgenic system.
Expression of Bcl-XL Restores Cell Survival, but Not Proliferation and Effector Differentiation, in Cd28-Deficient T Lymphocytes
Authors: Dahl A. Maria , Klein Christoph , Andres Pietro G. , London Cheryl A. , Lodge Michael P. , Mulligan Richard C. , Abbas Abul K.
Published: July 2002

Lymphocytes deficient in the T cell costimulatory molecule CD28 exhibit defects in cell survival, clonal expansion, and differentiation into effector cells. It is known that CD28-mediated signaling results in the upregulation of the Bcl family member Bcl-XL. To investigate the role that Bcl-XL plays in the various functions of CD28, we expressed Bcl-XL in CD28-deficient primary T lymphocytes using retrovirus-mediated gene transfer. T cells were activated in vitro and infected with Bcl-XL or control retroviruses; this method allows gene expression in activated, cycling cells. Expression of Bcl-XL in naive T cells was achieved by reconstitution of the immune system of lethally irradiated recipient mice with retrovirus-infected purified bone marrow stem cells from CD28−/− or wild-type donor mice. Our studies demonstrate that Bcl-XL prolongs the survival of CD28−/− T cells but does not restore normal proliferation or effector cell development. These results indicate that the various functions of CD28 can be dissociated, and provide an experimental approach for testing the roles of downstream signals in the functions of cellular receptors such as CD28.
Protein Kinase D
Authors: Matthews Sharon A. , Rozengurt Enrique , Cantrell Doreen
Published: July 2002

Protein kinase Cs (PKCs) are activated by antigen receptors in lymphocytes, but little is known about proximal targets for PKCs in antigen receptor–mediated responses. In this report, we define a role for diacylglycerol-regulated PKC isoforms in controlling the activity of the serine/threonine kinase protein kinase D (PKD; also known as PKCμ) in T cells, B cells, and mast cells. Antigen receptor activation of PKD is a rapid and sustained response that can be seen in T cells activated via the T cell antigen receptor, B cells activated via the B cell antigen receptor, and in mast cells triggered via the high-affinity receptor for IgE (FcεR1). Herein, we show that antigen receptor activation of PKD requires the activity of classical/novel PKCs. Moreover, PKC activity is sufficient to bypass the requirement for antigen receptor signals in the induction of PKD activity. These biochemical and genetic studies establish a role for antigen receptor–regulated PKC enzymes in the control of PKD activity. Regulation of PKD activity through upstream PKCs reveals a signaling network that exists between different members of the PKC superfamily of kinases that can operate to amplify and disseminate antigen receptor signals generated at the plasma membrane.
Neonatal Tumor Necrosis Factor α Promotes Diabetes in Nonobese Diabetic Mice by Cd154-Independent Antigen Presentation to Cd8+ T Cells
Authors: Green E. Allison , Wong F. Susan , Eshima Koji , Mora Conchi , Flavell Richard A.
Published: July 2002

Neonatal islet-specific expression of tumor necrosis factor (TNF)-α in nonobese diabetic mice promotes diabetes by provoking islet-infiltrating antigen-presenting cells to present islet peptides to autoreactive T cells. Here we show that TNF-α promotes autoaggression of both effector CD4+ and CD8+ T cells. Whereas CD8+ T cells are critical for diabetes progression, CD4+ T cells play a lesser role. TNF-α–mediated diabetes development was not dependent on CD154–CD40 signals or activated CD4+ T cells. Instead, it appears that TNF-α can promote cross-presentation of islet antigen to CD8+ T cells using a unique CD40–CD154-independent pathway. These data provide new insights into the mechanisms by which inflammatory stimuli can bypass CD154–CD40 immune regulatory signals and cause activation of autoreactive T cells.
Promyelocytic Leukemia Protein (Pml) and Daxx Participate in a Novel Nuclear Pathway for Apoptosis
Authors: Zhong Sue , Salomoni Paolo , Ronchetti Simona , Guo Ailan , Ruggero Davide , Pandolfi Pier Paolo
Published: July 2002

The promyelocytic leukemia protein (PML) gene of acute promyelocytic leukemia (APL) encodes a cell growth and tumor suppressor essential for multiple apoptotic signals. Daxx was identified as a molecule important for the cytoplasmic transduction of the Fas proapoptotic stimulus. Here, we show that upon mitogenic activation of mature splenic lymphocytes, Daxx is dramatically upregulated and accumulates in the PML nuclear body (NB) where PML and Daxx physically interact. In the absence of PML, Daxx acquires a dispersed nuclear pattern, and activation-induced cell death of splenocytes is profoundly impaired. PML inactivation results in the complete abrogation of the Daxx proapoptotic ability. In APL cells, Daxx is delocalized from the NB. Upon retinoic acid treatment, which induces disease remission in APL, Daxx relocalizes to the PML NBs. These results indicate that PML and Daxx cooperate in a novel NB-dependent pathway for apoptosis and shed new light in the role of PML in tumor suppression.
Analysis of Successful Immune Responses in Persons Infected with Hepatitis C Virus
Authors: Lechner Franziska , Wong David K.H. , Dunbar P. Rod , Chapman Roger , Chung Raymond T. , Dohrenwend Paul , Robbins Gregory , Phillips Rodney , Klenerman Paul , Walker Bruce D.
Published: July 2002

Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-γ, a “stunned” phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection.
Antimicrobial Actions of the Nadph Phagocyte Oxidase and Inducible Nitric Oxide Synthase in Experimental Salmonellosis. I. Effects on Microbial Killing by Activated Peritoneal Macrophages in Vitro
Authors: Vazquez-Torres Andrés , Jones-Carson Jessica , Mastroeni Pietro , Ischiropoulos Harry , Fang Ferric C.
Published: July 2002

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox−/−, iNOS−/−, and doubly immunodeficient phox−/−iNOS−/− mice. The respiratory burst and NO radical (NO·) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase–dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O2·−), hydrogen peroxide (H2O2), and peroxynitrite (ONOO−) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon γ appears to augment antibacterial activity predominantly by enhancing NO· production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.
The μ Switch Region Tandem Repeats Are Important, but Not Required, for Antibody Class Switch Recombination
Authors: Luby Thomas M. , Schrader Carol E. , Stavnezer Janet , Selsing Erik
Published: July 2002

Class switch DNA recombinations change the constant (C) region of the antibody heavy (H) chain expressed by a B cell and thereby change the antibody effector function. Unusual tandemly repeated sequence elements located upstream of H chain gene exons have long been thought to be important in the targeting and/or mechanism of the switch recombination process. We have deleted the entire switch tandem repeat element (Sμ) from the murine μ H chain gene. We find that the Sμ tandem repeats are not required for class switching in the mouse immunoglobulin H-chain locus, although the efficiency of switching is clearly reduced. Our data demonstrate that sequences outside of the Sμ tandem repeats must be capable of directing the class switch mechanism. The maintenance of the highly repeated Sμ element during evolution appears to reflect selection for a highly efficient switching process rather than selection for a required sequence element.
Protective Role of Raf-1 in Salmonella-Induced Macrophage Apoptosis
Authors: Jesenberger Veronika , Procyk Katarzyna J. , Rüth Jochen , Schreiber Martin , Theussl Hans-Christian , Wagner Erwin F. , Baccarini Manuela
Published: July 2002

Invasive Salmonella induces macrophage apoptosis via the activation of caspase-1 by the bacterial protein SipB. Here we show that infection of macrophages with Salmonella causes the activation and degradation of Raf-1, an important intermediate in macrophage proliferation and activation. Raf-1 degradation is SipB- and caspase-1–dependent, and is prevented by proteasome inhibitors. To study the functional significance of Raf-1 in this process, the c-raf-1 gene was inactivated by Cre-loxP–mediated recombination in vivo. Macrophages lacking c-raf-1 are hypersensitive towards pathogen-induced apoptosis. Surprisingly, activation of the antiapoptotic mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) and nuclear factor κB pathways is normal in Raf-1–deficient macrophages, and mitochondrial fragility is not increased. Instead, pathogen-mediated activation of caspase-1 is enhanced selectively, implying that Raf-1 antagonizes stimulus-induced caspase-1 activation and apoptosis.
Autonomous Maturation of α/β T Lineage Cells in the Absence of Cooh-Terminal Src Kinase (Csk)
Authors: Schmedt Christian , Tarakhovsky Alexander
Published: July 2002

The deletion of COOH-terminal Src kinase (Csk), a negative regulator of Src family protein tyrosine kinases (PTKs), in immature thymocytes results in the development of α/β T lineage cells in T cell receptor (TCR) β-deficient or recombination activating gene (rag)-1–deficient mice. The function of Csk as a repressor of Lck and Fyn activity suggests activation of these PTKs is solely responsible for the phenotype observed in csk-deficient T lineage cells. We provide genetic evidence for this notion as α/β T cell development is blocked in lck−/−fyn−/− csk-deficient mice. It remains unclear whether activation of Lck and Fyn in the absence of Csk uncouples α/β T cell development entirely from engagement of surface-expressed receptors. We show that in mice expressing the α/β TCR on csk-deficient thymocytes, positive selection is biased towards the CD4 lineage and does not require the presence of major histocompatibility complex (MHC) class I and II. Furthermore, the introduction of an MHC class I–restricted transgenic TCR into a csk-deficient background results in the development of mainly CD4 T cells carrying the transgenic TCR both in selecting and nonselecting MHC background. Thus, TCR–MHC interactions have no impact on positive selection and commitment to the CD4 lineage in the absence of Csk. However, TCR-mediated negative selection of csk-deficient, TCR transgenic cells is normal. These data suggest a differential involvement of the Csk-mediated regulation of Src family PTKs in positive and negative selection of developing thymocytes.
Intercellular Transfer and Supramolecular Organization of Human Leukocyte Antigen C at Inhibitory Natural Killer Cell Immune Synapses
Authors: Carlin Leo M. , Eleme Konstantina , McCann Fiona E. , Davis Daniel M.
Published: July 2002

After accumulation of target cell human leukocyte antigen (HLA)-C at inhibitory natural killer (NK) cell immune synapses, some HLA-C transfers from target cells to NK cell plasma membranes and cytoplasm. This unexpected intercellular transfer of HLA-C is dependent on NK receptor recognition, since HLA-Cw6 or -Cw4 but not -Cw3 transfer to an NK transfectant expressing killer Ig-like receptor (KIR)2DL1. Strikingly, live-cell time-lapse laser scanning confocal microscopy shows vesicles containing target cell green fluorescent protein–tagged HLA-C migrating away from immune synapses into NK cells. Unlike clustering of HLA-C at the immune synapse, intercellular transfer of HLA-C is dependent on NK cell ATP, but not target cell ATP. However, the intercellular transfer of HLA-C is not dependent on active polymerization of the actin cytoskeleton. In addition, different arrangements of HLA-C are seen at inhibitory NK immune synapses, and these alter as NK synapses mature, but in a fashion distinct from that seen upon T cell activation.
Delimiting the Binding Site for Quaternary Ammonium Lidocaine Derivatives in the Acetylcholine Receptor Channel
Authors: Pascual Juan M. , Karlin Arthur
Published: July 2002

The triethylammonium QX-314 and the trimethylammonium QX-222 are lidocaine derivatives that act as open-channel blockers of the acetylcholine (ACh) receptor. When bound, these blockers should occlude some of the residues lining the channel. Eight residues in the second membrane-spanning segment (M2) of the mouse-muscle α subunit were mutated one at a time to cysteine and expressed together with wild-type β, γ, and δ subunits in Xenopus oocytes. The rate constant for the reaction of each substituted cysteine with 2-aminoethyl methanethiosulfonate (MTSEA) was determined from the time course of the irreversible effect of MTSEA on the ACh-induced current. The reactions were carried out in the presence and absence of ACh and in the presence and absence of QX-314 and QX-222. These blockers had no effect on the reactions in the absence of ACh. In the presence of ACh, both blockers retarded the reaction of extracellularly applied MTSEA with cysteine substituted for residues from αVal255, one third of the distance in from the extracellular end of M2, to αGlu241, flanking the intracellular end of M2, but not with cysteine substituted for αLeu258 or αGlu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for αLeu258 and αGlu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect αL258C and αE262C against reaction with MTSEA in the presence of ACh implies that protection of the other residues was due to occlusion of the channel and not to the promotion of a less reactive state from a remote site. Given the 12-Å overall length of the blockers and the α-helical conformation of M2 in the open state, the binding site for both blockers extends from αVal255 down to αSer248.
Energetic and Spatial Parameters for Gating of the Bacterial Large Conductance Mechanosensitive Channel, MscL
Authors: Sukharev Sergei I. , Sigurdson Wade J. , Kung Ching , Sachs Frederick
Published: July 2002

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was ΔE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (ΔA), the free energy, TΔA, would correspond to ΔA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest ΔA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to ΔA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.
Possessions and the Extended Self
Authors: Belk Russell W.
Published: July 2002

Our possessions are a major contributor to and reflection of our identities. A variety of evidence is presented supporting this simple and compelling premise. Related streams of research are identified and drawn upon in developing this concept and implications are derived for consumer behavior. Because the construct of extended self involves consumer behavior rather than buyer behavior, it appears to be a much richer construct than previous formulations positing a relationship between self-concept and consumer brand choice.
On the distribution singular values of Toeplitz matrices
Authors: Dostanić Milutin
Published: July 2002

We prove a second order formula concerning distribution of singular values of Toeplitz matrices in some cases when conditions of the H. Widom Theorem are not satisfied.
Sphere-preserving maps in inversive geometry
Authors: Beardon A. , Minda D.
Published: July 2002

We give an extensive discussion of sphere-preserving maps defined on subdomains of Euclideannn-space, and their relationship to Möbius maps and to the preservation of cross-ratios. In the casen=2n=2(the complex plane) we also relate these ideas to the solutions of certain functional equations.
Convergence of the Poincaré series for some classical Schottky groups
Authors: Mityushev Vladimir
Published: July 2002

The Poincaréθ2\theta _2-series for a multiply connected circular region can be either convergent or divergent absolutely. In this paper we prove a uniform convergence result for such a region.
Preconditioners for nonconforming discretizations
Authors: Oswald Peter
Published: July 2002

We prove an abstract norm equivalence for a two-level method, which allows us to reduce the construction of preconditioners for nonconforming finite element discretizations to known cases of conforming elements.
On Wendt’s determinant
Authors: Helou Charles
Published: July 2002

Wendt’s determinant of ordermmis the circulant determinantWmW_{m}whose(i,j)(i,j)-th entry is the binomial coefficient(m|i−j|)\binom m{|i-j|}, for1≤i,j≤m1\leq i,j\leq m. We give a formula forWmW_{m}, whenmmis even not divisible by 6, in terms of the discriminant of a polynomialTm+1T_{m+1}, with rational coefficients, associated to(X+1)m+1−Xm+1−1(X+1)^{m+1}-X^{m+1}-1. In particular, whenm=p−1m=p-1whereppis a prime≡−1(mod6)\equiv -1 (mod 6), this yields a factorization ofWp−1W_{p-1}involving a Fermat quotient, a power ofppand the 6-th power of an integer.
The approximation power of moving least-squares
Authors: Levin David
Published: July 2002

A general method for near-best approximations to functionals on

R

d

\mathbb {R}^d

, using scattered-data information is discussed. The method is actually the moving least-squares method, presented by the Backus-Gilbert approach. It is shown that the method works very well for interpolation, smoothing and derivatives’ approximations. For the interpolation problem this approach gives Mclain’s method. The method is near-best in the sense that the local error is bounded in terms of the error of a local best polynomial approximation. The interpolation approximation in

R

d

\mathbb {R}^d

is shown to be a

C
∞

C^\infty

function, and an approximation order result is proven for quasi-uniform sets of data points.
Zeroes of zeta functions and symmetry
Authors: Katz Nicholas , Sarnak Peter
Published: July 2002

Hilbert and Polya suggested that there might be a natural spectral interpretation of the zeroes of the Riemann Zeta function. While at the time there was little evidence for this, today the evidence is quite convincing. Firstly, there are the “function field” analogues, that is zeta functions of curves over finite fields and their generalizations. For these a spectral interpretation for their zeroes exists in terms of eigenvalues of Frobenius on cohomology. Secondly, the developments, both theoretical and numerical, on the local spacing distributions between the high zeroes of the zeta function and its generalizations give striking evidence for such a spectral connection. Moreover, the low-lying zeroes of various families of zeta functions follow laws for the eigenvalue distributions of members of the classical groups. In this paper we review these developments. In order to present the material fluently, we do not proceed in chronological order of discovery. Also, in concentrating entirely on the subject matter of the title, we are ignoring the standard body of important work that has been done on the zeta function andLL-functions.
Optimization, relaxation and Young measures
Authors: Pedregal Pablo
Published: July 2002

We review the use of Young measures in analyzing relaxed and generalized formulations for typical problems of optimization including variational principles, optimal control problems, models in materials science, optimal design problems and nonlocal optimization problems.
Strong shift equivalence theory and the shift equivalence problem
Authors: Wagoner J.
Published: July 2002

This paper discusses strong shift equivalence and counterexamples to the long standing Shift Equivalence Problem in symbolic dynamics. We also discuss how strong shift equivalence theory is closely related to areas of mathematics outside dynamics such as algebraic K-theory, cyclic homology, and topological quantum field theory.
The number of Reidemeister moves needed for unknotting
Authors: Hass Joel , Lagarias Jeffrey
Published: July 2002

There is a positive constantc1c_1such that for any diagramD\mathcal {D}representing the unknot, there is a sequence of at most2c1n2^{c_1 n}Reidemeister moves that will convert it to a trivial knot diagram, wherennis the number of crossings inD\mathcal {D}. A similar result holds for elementary moves on a polygonal knotKKembedded in the 1-skeleton of the interior of a compact, orientable, triangulatedPLPL3-manifoldMM. There is a positive constantc2c_2such that for eacht≥1t \geq 1, ifMMconsists oftttetrahedra andKKis unknotted, then there is a sequence of at most2c2t2^{c_2 t}elementary moves inMMwhich transformsKKto a triangle contained inside one tetrahedron ofMM. We obtain explicit values forc1c_1andc2c_2.
On the equivariant 𝐾-theory of the nilpotent cone
Authors: Ostrik Viktor
Published: July 2002

In this note we construct a “Kazhdan-Lusztig type” basis in equivariantKK-theory of the nilpotent cone of a simple algebraic groupGG. This basis conjecturally is very close to the basis of thisKK-group consisting of irreducible bundles on nilpotent orbits. As a consequence we get a natural (conjectural) construction of Lusztig’s bijection between dominant weights and pairs {nilpotent orbitO\mathcal O, irreducibleGG-bundle onO\mathcal O}.
The enveloping algebra of the Lie superalgebra 𝑜𝑠𝑝(1,2𝑟)
Authors: Musson Ian
Published: July 2002

Letg\mathfrak {g}be the Lie superalgebraosp(1,2r)osp(1,2r)andU(g)U(\mathfrak {g})the enveloping algebra ofg\mathfrak {g}. In this paper we obtain a description of the set of primitive ideals PrimU(g)U(\mathfrak {g})as an ordered set. We also obtain the multiplicities of composition factors of Verma modules overU(g)U(\mathfrak {g}), and of simple highest weight modules forU(g)U(\mathfrak {g})when regarded as aU(g0)U(\mathfrak {g}_{0})-module by restriction.
The five exceptional simple Lie superalgebras of vector fields and their fourteen regradings
Authors: Shchepochkina Irina
Published: July 2002

The five simple exceptional complex Lie superalgebras of vector fields are described. One of them,fas\mathfrak {fas}, is new; the other four are explicitly described for the first time. All nonisomorphic maximal subalgebras of finite codimension of these Lie superalgebras, i.e., allotherrealizations of these Lie superalgebras as Lie superalgebras of vector fields, are also described; there are 14 of them altogether. All of the exceptional Lie superalgebras are obtained with the help of the Cartan prolongation or a generalized prolongation.