bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic Association of Proteasomal Machinery with the Centrosome
Authors: Christian Wigley W. , Fabunmi Rosalind P. , Lee Min Goo , Marino Christopher R. , Muallem Shmuel , DeMartino George N. , Thomas Philip J.
Published: July 2002

Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with γ-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation.
Regulation of P21cip1 Expression by Growth Factors and the Extracellular Matrix Reveals a Role for Transient ERK Activity in G1 Phase
Authors: Bottazzi Maria Elena , Zhu Xiaoyun , Böhmer Ralph M. , Assoian Richard K.
Published: July 2002

We have examined the regulation of p21cip1 by soluble mitogens and cell anchorage as well as the relationship between the expression of p21cip1 and activation of the ERK subfamily of MAP kinases. We find that p21cip1 expression in G1 phase can be divided into two discrete phases: an initial induction that requires growth factors and the activation of ERK, and then a subsequent decline that is enhanced by cell anchorage in an ERK-independent manner. In contrast to the induction of cyclin D1, the induction of p21cip1 is mediated by transient ERK activity. Comparative studies with wild-type and p21cip1-null fibroblasts indicate that adhesion-dependent regulation of p21cip1 is important for proper control of cyclin E–cdk2 activity. These data lead to a model in which mitogens and anchorage act in a parallel fashion to regulate G1 phase expression of p21cip1. They also show that (a) growth factors and growth factor/extracellular matrix cooperation can have different roles in regulating G1 phase ERK activity and (b) both transient and sustained ERK signals have functionally significant roles in controlling cell cycle progression through G1 phase.
Reconstitution of Membrane Transport Powered by a Novel Dimeric Kinesin Motor of the Unc104/Kif1a Family Purified from Dictyostelium
Authors: Pollock Nira , de Hostos Eugenio L. , Turck Christoph W. , Vale Ronald D.
Published: July 2002

Motor-powered movement along microtubule tracks is important for membrane organization and trafficking. However, the molecular basis for membrane transport is poorly understood, in part because of the difficulty in reconstituting this process from purified components. Using video microscopic observation of organelle transport in vitro as an assay, we have purified two polypeptides (245 and 170 kD) from Dictyostelium extracts that independently reconstitute plus-end–directed membrane movement at in vivo velocities. Both polypeptides were found to be kinesin motors, and the 245-kD protein (DdUnc104) is a close relative of Caenorhabditis elegans Unc104 and mouse KIF1A, neuron-specific motors that deliver synaptic vesicle precursors to nerve terminals. A knockout of the DdUnc104 gene produces a pronounced defect in organelle transport in vivo and in the reconstituted assay. Interestingly, DdUnc104 functions as a dimeric motor, in contrast to other members of this kinesin subfamily, which are monomeric.
The Tripartite Type III Secreton of Shigella flexneri Inserts Ipab and Ipac into Host Membranes
Authors: Blocker Ariel , Gounon Pierre , Larquet Eric , Niebuhr Kirsten , Cabiaux Véronique , Parsot Claude , Sansonetti Philippe
Published: July 2002

Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-Å pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37°C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.
The Function of Plakophilin 1 in Desmosome Assembly and Actin Filament Organization
Authors: Hatzfeld Mechthild , Haffner Christof , Schulze Katrin , Vinzens Ute
Published: July 2002

Plakophilin 1, a member of the armadillo multigene family, is a protein with dual localization in the nucleus and in desmosomes. To elucidate its role in desmosome assembly and regulation, we have analyzed its localization and binding partners in vivo. When overexpressed in HaCaT keratinocytes, plakophilin 1 localized to the nucleus and to desmosomes, and dramatically enhanced the recruitment of desmosomal proteins to the plasma membrane. This effect was mediated by plakophilin 1's head domain, which interacted with desmoglein 1, desmoplakin, and keratins in the yeast two-hybrid system. Overexpression of the armadillo repeat domain induced a striking dominant negative phenotype with the formation of filopodia and long cellular protrusions, where plakophilin 1 colocalized with actin filaments. This phenotype was strictly dependent on a conserved motif in the center of the armadillo repeat domain. Our results demonstrate that plakophilin 1 contains two functionally distinct domains: the head domain, which could play a role in organizing the desmosomal plaque in suprabasal cells, and the armadillo repeat domain, which might be involved in regulating the dynamics of the actin cytoskeleton.
Phosphorylation by Cdc28 Activates the Cdc20-Dependent Activity of the Anaphase-Promoting Complex
Authors: Rudner Adam D. , Murray Andrew W.
Published: July 2002

Budding yeast initiates anaphase by activating the Cdc20-dependent anaphase-promoting complex (APC). The mitotic activity of Cdc28 (Cdk1) is required to activate this form of the APC, and mutants that are impaired in mitotic Cdc28 function have difficulty leaving mitosis. This defect can be explained by a defect in APC phosphorylation, which depends on mitotic Cdc28 activity in vivo and can be catalyzed by purified Cdc28 in vitro. Mutating putative Cdc28 phosphorylation sites in three components of the APC, Cdc16, Cdc23, and Cdc27, makes the APC resistant to phosphorylation both in vivo and in vitro. The nonphosphorylatable APC has normal activity in G1, but its mitotic, Cdc20-dependent activity is compromised. These results show that Cdc28 activates the APC in budding yeast to trigger anaphase. Previous reports have shown that the budding yeast Cdc5 homologue, Plk, can also phosphorylate and activate the APC in vitro. We show that, like cdc28 mutants, cdc5 mutants affect APC phosphorylation in vivo. However, although Cdc5 can phosphorylate Cdc16 and Cdc27 in vitro, this in vitro phosphorylation does not occur on in vivo sites of phosphorylation.
Evidence That Dynamin-2 Functions as a Signal-Transducing Gtpase
Authors: Fish Kenneth N. , Schmid Sandra L. , Damke Hanna
Published: July 2002

The role of dynamin GTPases in the regulation of receptor-mediated endocytosis is well established. Here, we present new evidence that the ubiquitously expressed isoform dynamin-2 (dyn2) can also function in a signal transduction pathway(s). A ≤5-fold increase of dyn2 relative to endogenous levels activates the transcription factor p53 and induces apoptosis, as demonstrated by reduced cell proliferation, DNA fragmentation, and caspase-3 activation. Dyn2-triggered apoptosis occurs only in dividing cells and is p53 dependent. A mutant defective in GTP binding does not trigger apoptosis, indicating that increased levels of dyn2·GTP, rather than protein levels per se, are required to transduce signals that activate p53. A truncated dyn2 lacking the COOH-terminal proline/arginine-rich domain (PRD), which interacts with many SH3 domain-containing partners implicated in both endocytosis and signal transduction, triggers apoptosis even more potently than the wild-type. This observation provides additional support for the importance of the NH2-terminal GTPase domain for the apoptotic phenotype. All described effects are dyn2-specific because >200-fold overexpression of dyn1, the 70% identical neuronal isoform, has no effect. Our data suggest that dyn2 can act as a signal transducing GTPase affecting transcriptional regulation.
The Dynamics of Postmitotic Reassembly of the Nucleolus
Authors: Dundr Miroslav , Misteli Tom , Olson Mark O.J.
Published: July 2002

Mammalian cell nucleoli disassemble at the onset of M-phase and reassemble during telophase. Recent studies showed that partially processed preribosomal RNA (pre-rRNA) is preserved in association with processing components in the perichromosomal regions (PRs) and in particles called nucleolus-derived foci (NDF) during mitosis. Here, the dynamics of nucleolar reassembly were examined for the first time in living cells expressing fusions of the processing-related proteins fibrillarin, nucleolin, or B23 with green fluorescent protein (GFP). During telophase the NDF disappeared with a concomitant appearance of material in the reforming nuclei. Prenucleolar bodies (PNBs) appeared in nuclei in early telophase and gradually disappeared as nucleoli formed, strongly suggesting the transfer of PNB components to newly forming nucleoli. Fluorescence recovery after photobleaching (FRAP) showed that fibrillarin-GFP reassociates with the NDF and PNBs at rapid and similar rates. The reentry of processing complexes into telophase nuclei is suggested by the presence of pre-rRNA sequences in PNBs. Entry of specific proteins into the nucleolus approximately correlated with the timing of processing events. The mitotically preserved processing complexes may be essential for regulating the distribution of components to reassembling daughter cell nucleoli.
Nuclear Lamins a and B1
Authors: Moir Robert D. , Yoon Miri , Khuon Satya , Goldman Robert D.
Published: July 2002

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.
Anaphase-Promoting Complex/Cyclosome–Dependent Proteolysis of Human Cyclin a Starts at the Beginning of Mitosis and Is Not Subject to the Spindle Assembly Checkpoint
Authors: Geley Stephan , Kramer Edgar , Gieffers Christian , Gannon Julian , Peters Jan-Michael , Hunt Tim
Published: July 2002

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The “destruction box” (D-box) of cyclin A is 10–20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.
Identities of Sequestered Proteins in Aggregates from Cells with Induced Polyglutamine Expression
Authors: Suhr Steven T. , Senut Marie-Claude , Whitelegge Julian P. , Faull Kym F. , Cuizon Denise B. , Gage Fred H.
Published: July 2002

Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle–regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.
Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC–Hcf106 complex before Tha4-dependent transport
Authors: Cline Kenneth , Mori Hiroki
Published: July 2002

The thylakoid ΔpH-dependent pathway transports folded proteins with twin arginine–containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC–Hcf106 complex serves as receptor for specific binding of twin arginine–containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an ∼700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an ∼700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC–Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of ΔpH. These results indicate that precursor binding to the cpTatC–Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.
The Chemokine SDF-1 Is a Chemoattractant for Human CD34+ Hematopoietic Progenitor Cells and Provides a New Mechanism to Explain the Mobilization of CD34+ Progenitors to Peripheral Blood
Authors: Aiuti A. , Webb I.J. , Bleul C. , Springer T. , Gutierrez-Ramos J.C.
Published: July 2002

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38− or CD34+/DR−) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.
Targeted Disruption of the Chemokine Eotaxin Partially Reduces Antigen-induced Tissue Eosinophilia
Authors: Rothenberg Marc E. , MacLean James A. , Pearlman Eric , Luster Andrew D. , Leder Philip
Published: July 2002

The chemokines are a large group of chemotactic cytokines that regulate leukocyte trafficking and have recently been shown to inhibit human immunodeficiency virus entry into cells. Eotaxin is a C–C chemokine implicated in the recruitment of eosinophils in a variety of inflammatory disorders and, unlike all other eosinophil chemoattractants, is eosinophil specific. However, given the large number of chemoattractants that have activities on eosinophils, it is unclear whether eotaxin has an important role in vivo. Furthermore, it remains unclear why there is constitutive expression of eotaxin in healthy states in the absence of eosinophilic inflammation. To begin to determine the significance of eotaxin at baseline and during eosinophil-mediated disease processes, we have used targeted gene disruption to generate mice that are deficient in eotaxin. Such mice demonstrate that eotaxin enhances the magnitude of the early (but not late) eosinophil recruitment after antigen challenge in models of asthma and stromal keratitis. Surprisingly, a role for eotaxin in regulating the constitutive number of eosinophils in the peripheral circulation is also demonstrated. These results indicate a contributory role for eotaxin in the generation of peripheral blood and antigen-induced tissue eosinophilia.
Soluble Domain 1 of Platelet–Endothelial Cell Adhesion Molecule (PECAM) Is Sufficient to Block Transendothelial Migration In Vitro and In Vivo
Authors: Liao Fang , Ali Jahanara , Greene Tricia , Muller William A.
Published: July 2002

The inflammatory response involves sequential adhesive interactions between cell adhesion molecules of leukocytes and the endothelium. Unlike the several adhesive steps that precede it, transendothelial migration (diapedesis), the step in which leukocytes migrate between apposed endothelial cells, appears to involve primarily one adhesion molecule, platelet–endothelial cell adhesion molecule (PECAM, CD31). Therefore, we have focused on PECAM as a target for antiinflammatory therapy. We demonstrate that soluble chimeras made of the entire extracellular portion of PECAM, or of only the first immunoglobulin domain of PECAM, fused to the Fc portion of IgG, block diapedesis in vitro and in vivo. Furthermore, the truncated form of the PECAM-IgG chimera does not bind stably to its cellular ligand. This raises the possibility of selective anti-PECAM therapies that would not have the untoward opsonic or cell-activating properties of antibodies directed against PECAM.
A CASP-8 Mutation Recognized by Cytolytic T Lymphocytes on a Human Head and Neck Carcinoma
Authors: Mandruzzato Susanna , Brasseur Francis , Andry Guy , Boon Thierry , Bruggen Pierre van der
Published: July 2002

Of the antigens recognized on human tumors by autologous cytolytic T lymphocytes, all those defined thus far have been identified on melanoma or renal cell carcinoma. We report here the identification of an antigen recognized by autologous cytolytic T lymphocytes on a human squamous cell carcinoma of the oral cavity. The antigen is encoded by a mutated form of the CASP-8 gene. This gene, also named FLICE or MACH, codes for protease caspase-8, which is required for induction of apoptosis through the Fas receptor and tumor necrosis factor receptor-1. The mutation, which was found in the tumor cells but not in the normal cells of the patient, modifies the stop codon and adds an Alu repeat to the coding region, thereby lengthening the protein by 88 amino acids. The ability of the altered protein to trigger apoptosis appears to be reduced relative to the normal caspase-8. The antigenic peptide is a nonamer presented by HLA-B*3503. The five last amino acids are encoded by the extension of the reading frame caused by the mutation. This, together with previous observations of CDK4 and β-catenin mutations, suggests that a significant fraction of the point mutations generating a tumor antigen also play a role in the tumoral transformation or progression.
Specific Activation of the Cysteine Protease CPP32 during the Negative Selection of T Cells in the Thymus
Authors: Alam Antoine , Braun Michel Y. , Hartgers Franca , Lesage Sylvie , Cohen Luchino , Hugo Patrice , Denis François , Sékaly Rafick-Pierre
Published: July 2002

Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide–MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti–CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.
The Effect of Class II Major Histocompatibility Complex Expression on Adherence of Helicobacter pylori and Induction of Apoptosis in Gastric Epithelial Cells: A Mechanism for T Helper Cell Type 1–mediated Damage
Authors: Fan Xuejun , Crowe Sheila E. , Behar Simon , Gunasena Harshani , Ye Gang , Haeberle Helene , Van Houten Nancy , Gourley William K. , Ernst Peter B. , Reyes Victor E.
Published: July 2002

Helicobacter pylori infection is associated with gastric epithelial damage, including apoptosis, ulceration, and cancer. Although bacterial factors and the host response are believed to contribute to gastric disease, no receptor has been identified that explains how the bacteria attach and signal the host cell to undergo apoptosis. Using H. pylori as “bait” to capture receptor proteins in solubilized membranes of gastric epithelial cells, class II major histocompatibility complex (MHC) molecules were identified as a possible receptor. Signaling through class II MHC molecules leading to the induction of apoptosis was confirmed using cross-linking IgM antibodies to surface class II MHC molecules. Moreover, binding of H. pylori and the induction of apoptosis were inhibited by antibodies recognizing class II MHC. Since type 1 T helper cells are present during infection and produce interferon (IFN)-γ, which increases class II MHC expression, gastric epithelial cell lines were exposed to H. pylori in the presence or absence of IFN-γ. IFN-γ increased the attachment of the bacteria as well as the induction of apoptosis in gastric epithelial cells. In contrast to MHC II–negative cell lines, H. pylori induced apoptosis in cells expressing class II MHC molecules constitutively or after gene transfection. These data describe a novel receptor for H. pylori and provide a mechanism by which bacteria and the host response interact in the pathogenesis of gastric epithelial cell damage.
CD28-independent, TRAF2-dependent Costimulation of Resting T Cells by 4-1BB Ligand
Authors: Saoulli Katina , Lee Soo Young , Cannons Jennifer L. , Yeh Wen Chen , Santana Angela , Goldstein Marni D. , Bangia Naveen , DeBenedette Mark A. , Mak Tak W. , Choi Yongwon , Watts Tania H.
Published: July 2002

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28− T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor–associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase–4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.
Promiscuous Presentation and Recognition of Nucleosomal Autoepitopes in Lupus: Role of Autoimmune T Cell Receptor α Chain
Authors: Shi Yan , Kaliyaperumal Arunan , Lu Liangjun , Southwood Scott , Sette Alessandro , Michaels Marissa A. , Datta Syamal K.
Published: July 2002

T cells specific for nucleosomal autoepitopes are selectively expanded in lupus mice and these Th cells drive autoimmune B cells to produce pathogenic antinuclear antibodies. We transfected the TCR-α and -β chain genes of a representative, pathogenic autoantibody-inducing Th clone specific for the nucleosomal core histone peptide H471–94 into TCR-negative recipient cells. Although the autoimmune TCRs were originally derived from SNF1 (I-Ad/q) mice, the transfectants could recognize the nucleosomal autoepitope presented by APC-bearing I-A molecules of all haplotypes tested, as well as human DR molecules. Competition assays indicated that the autoepitopes bound to the MHC class II groove. Most remarkably, MHC-unrestricted recognition of the nucleosomal peptide epitope was conferred by the lupus TCR-α chain even when it paired with a TCR-β chain of irrelevant specificity. Several other disease-relevant Th clones and splenic T cells of lupus mice had similar properties. The TCR-α chains of these murine lupus Th clones shared related motifs and charged residues in their CDRs, and similar motifs were apparent even in TCR-α chains of human lupus Th clones. The lupus TCR-α chains probably contact the nucleosomal peptide complexed with MHC with relatively high affinity/avidity to sustain TCR signaling, because CD4 coreceptor was not required for promiscuous recognition. Indeed, pathogenic autoantibody-inducing, CD4-negative, TCR-αβ+ Th cells are expanded in systemic lupus erythematosus. These results have implications regarding thymic selection and peripheral expansion of nucleosome-specific T cells in lupus. They also suggest that universally tolerogenic epitopes could be designed for therapy of lupus patients with diverse HLA alleles. We propose to designate nucleosomes and other antigens bearing universal epitopes “Pantigens” (for promiscuous antigens).
Extent of Laminin-5 Assembly and Secretion Effect Junctional Epidermolysis Bullosa Phenotype
Authors: Matsui Chihiro , Pereira Phyllis , Wang C. Kathy , Nelson Charlotte F. , Kutzkey Timothy , Lanigan Caroline , Woodley David , Morohashi Masaaki , Welsh Elizabeth A. , Hoeffler Warren K.
Published: July 2002

Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin blistering disease with both lethal and nonlethal forms, with most patients shown to have defects in laminin-5. We analyzed the location of mutations, gene expression levels, and protein chain assembly of the laminin-5 heterotrimer in six JEB patients to determine how the type of genetic lesion influences the pathophysiology of JEB. Mutations within laminin-5 genes were diversely located, with the most severe forms of JEB correlating best with premature termination codons, rather than mapping to any particular protein domain. In all six JEB patients, the laminin-5 assembly intermediates we observed were as predicted by our previous work indicating that the α3β3γ2 heterotrimer assembles intracellularly via a β3γ2 heterodimer intermediate. Since assembly precedes secretion, mutations that disrupt protein–protein interactions needed for assembly are predicted to limit the secretion of laminin-5, and likely to interfere with function. However, our data indicate that typically the most severe mutations diminish mRNA stability, and serve as functional null alleles that block chain assembly by resulting in either a deficiency (in the nonlethal mitis variety) or a complete absence (in lethal Herlitz-JEB) of one of the chains needed for laminin-5 heterotrimer assembly.
Activated Human T Cells, B Cells, and Monocytes Produce Brain-derived Neurotrophic Factor In Vitro and in Inflammatory Brain Lesions: A Neuroprotective Role of Inflammation?
Authors: Kerschensteiner Martin , Gallmeier Eike , Behrens Lüder , Leal Vivian Vargas , Misgeld Thomas , Klinkert Wolfgang E.F. , Kolbeck Roland , Hoppe Edmund , Oropeza-Wekerle Rosa-Laura , Bartke Ilse , Stadelmann Christine , Lassmann Hans , Wekerle Hartmut , Hohlfeld Reinhard
Published: July 2002

Brain-derived neurotrophic factor (BDNF) has potent effects on neuronal survival and plasticity during development and after injury. In the nervous system, neurons are considered the major cellular source of BDNF. We demonstrate here that in addition, activated human T cells, B cells, and monocytes secrete bioactive BDNF in vitro. Notably, in T helper (Th)1- and Th2-type CD4+ T cell lines specific for myelin autoantigens such as myelin basic protein or myelin oligodendrocyte glycoprotein, BDNF production is increased upon antigen stimulation. The BDNF secreted by immune cells is bioactive, as it supports neuronal survival in vitro. Using anti-BDNF monoclonal antibody and polyclonal antiserum, BDNF immunoreactivity is demonstrable in inflammatory infiltrates in the brain of patients with acute disseminated encephalitis and multiple sclerosis. The results raise the possibility that in the nervous system, inflammatory infiltrates have a neuroprotective effect, which may limit the success of nonselective immunotherapies.
Small, Clonally Variant Antigens Expressed on the Surface of the Plasmodium falciparum–Infected Erythrocyte Are Encoded by the rif Gene Family and Are the Target of Human Immune Responses
Authors: Fernandez Victor , Hommel Marcel , Chen Qijun , Hagblom Per , Wahlgren Mats
Published: July 2002

Disease severity in Plasmodium falciparum infections is a direct consequence of the parasite's efficient evasion of the defense mechanisms of the human host. To date, one parasite-derived molecule, the antigenically variant adhesin P. falciparum erythrocyte membrane protein 1 (PfEMP1), is known to be transported to the infected erythrocyte (pRBC) surface, where it mediates binding to different host receptors. Here we report that multiple additional proteins are expressed by the parasite at the pRBC surface, including a large cluster of clonally variant antigens of 30–45 kD. We have found these antigens to be identical to the rifins, predicted polypeptides encoded by the rif multigene family. These parasite products, formerly called rosettins after their identification in rosetting parasites, are prominently expressed by fresh isolates of P. falciparum. Rifins are immunogenic in natural infections and strain-specifically recognized by human immune sera in immunoprecipitation of surface-labeled pRBC extracts. Furthermore, human immune sera agglutinate pRBCs digested with trypsin at conditions such that radioiodinated PfEMP1 polypeptides are not detected but rifins are detected, suggesting the presence of epitopes in rifins targeted by agglutinating antibodies. When analyzed by two-dimensional electrophoresis, the rifins resolved into several isoforms in the pI range of 5.5–6.5, indicating molecular microheterogeneity, an additional potential novel source of antigenic diversity in P. falciparum. Prominent polypeptides of 20, 22, 76–80, 140, and 170 kD were also detected on the surfaces of pRBCs bearing in vitro–propagated or field-isolated parasites. In this report, we describe the rifins, the second family of clonally variant antigens known to be displayed by P. falciparum on the surface of the infected erythrocyte.
In Breast Carcinoma Tissue, Immature Dendritic Cells Reside within the Tumor, Whereas Mature Dendritic Cells Are Located in Peritumoral Areas
Authors: Bell Diana , Chomarat Pascale , Broyles Denise , Netto George , Harb Ghada Moumneh , Lebecque Serge , Valladeau Jenny , Davoust Jean , Palucka Karolina A. , Banchereau Jacques
Published: July 2002

We have analyzed the presence of immature and mature dendritic cells (DCs) within adenocarcinoma of the breast using immunohistochemistry. Immature DCs were defined by expression of CD1a-, Langerin-, and intracellular major histocompatibility complex class II–rich vesicles. Mature DCs were defined by expression of CD83 and DC-Lamp. Breast carcinoma cells were defined by morphology and/or cytokeratin expression. We demonstrate two levels of heterogeneity of DCs infiltrating breast carcinoma tissue: (a) immature CD1a+ DCs, mostly of the Langerhans cell type (Langerin+), were retained within the tumor bed in 32/32 samples and (b) mature DCs, CD83+DC-Lamp+, present in 20/32 samples, are confined to peritumoral areas. The high numbers of immature DCs found in the tumor may be best explained by high levels of macrophage inflammatory protein 3α expression by virtually all tumor cells. Confirming the immature/mature DC compartmentalization pattern, in vitro–generated immature DCs adhere to the tumor cells, whereas mature DCs adhere selectively to peritumoral areas. In some cases, T cells are clustering around the mature DCs in peritumoral areas, thus resembling the DC–T cell clusters of secondary lymphoid organs, which are characteristic of ongoing immune reactions.
Transport of Bacterial Lipopolysaccharide to the Golgi Apparatus
Authors: Thieblemont Nathalie , Wright Samuel D.
Published: July 2002

Addition of lipopolysaccharide (LPS) to cells in the form of LPS–soluble (s)CD14 complexes induces strong cellular responses. During this process, LPS is delivered from sCD14 to the plasma membrane, and the cell-associated LPS is then rapidly transported to an intracellular site. This transport appears to be important for certain cellular responses to LPS, as drugs that block transport also inhibit signaling and cells from LPS-hyporesponsive C3H/HeJ mice fail to exhibit this transport. To identify the intracellular destination of fluorescently labeled LPS after its delivery from sCD14 into cells, we have made simultaneous observations of different organelles using fluorescent vital dyes or probes. Endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus were labeled using Texas red (TR)–dextran, LysoTracker™ Red DND-99, DiOC6(3), and boron dipyrromethane (BODIPY)–ceramide, respectively. After 30 min, LPS did not colocalize with endosomes, lysosomes, or endoplasmic reticulum in polymorphonuclear leukocytes, although some LPS-positive vesicles overlapped with the endosomal marker, fluorescent dextran. On the other hand, LPS did appear to colocalize with two markers of the Golgi apparatus, BODIPY–ceramide and TRITC (tetramethylrhodamine isothiocyanate)–labeled cholera toxin B subunit. We further confirmed the localization of LPS in the Golgi apparatus using an epithelial cell line, HeLa, which responds to LPS–sCD14 complexes in a CD14-dependent fashion: BODIPY–LPS was internalized and colocalized with fluorescently labeled Golgi apparatus probes in live HeLa cells. Morphological disruption of the Golgi apparatus in brefeldin A–treated HeLa cells caused intracellular redistribution of fluorescent LPS. These results are consistent with the Golgi apparatus being the primary delivery site of monomeric LPS.
Self-Recognition of Cd1 by γ/δ T Cells
Authors: Spada Franca M. , Grant Ethan P. , Peters Peter J. , Sugita Masahiko , Melián Augustín , Leslie David S. , Lee Hoi K. , van Donselaar Elly , Hanson Dennis A. , Krensky Alan M. , Majdic Otto , Porcelli Steven A. , Morita Craig T. , Brenner Michael B.
Published: July 2002

The specificity of immunoglobulins and α/β T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of γ/δ antigen receptors that share characteristics of both immunoglobulins and α/β TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue γ/δ T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive γ/δ T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the γ/δ TCR. Importantly, all CD1c-reactive γ/δ T cells express Vδ1 TCRs, the TCR expressed by most tissue γ/δ T cells. Recognition by this tissue pool of γ/δ T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.
Interleukin (Il)-4 Is a Major Regulatory Cytokine Governing Bioactive IL-12 Production by Mouse and Human Dendritic Cells
Authors: Hochrein Hubertus , O'Keeffe Meredith , Luft Thomas , Vandenabeele Stéphane , Grumont Raelene J. , Maraskovsky Eugene , Shortman Ken
Published: July 2002

Interleukin (IL)-12 may be secreted as a bioactive T helper type 1 (Th1) cell–inducing heterodimer, as a monomer, or as an antagonistic homodimer. We analyzed the IL-12 produced by mouse splenic dendritic cells (DCs), human thymic DCs, and cultured human monocyte-derived DCs. IL-12 production required both a microbial or T cell–derived stimulus and an appropriate cytokine milieu. The different IL-12 forms were differentially regulated by the cytokines present rather than the stimulus used. IL-4 alone or together with granulocyte/macrophage colony-stimulating factor or interferon γ effectively enhanced the production of the bioactive heterodimer and selectively reduced the antagonistic homodimer of IL-12. Therefore, IL-4, the major Th2-driving cytokine, provides a negative feedback causing DCs to produce the major Th1-inducing cytokine, bioactive IL-12.
Ex Vivo Isolation and Characterization of Cd4+Cd25+ T Cells with Regulatory Properties from Human Blood
Authors: Dieckmann Detlef , Plottner Heidi , Berchtold Susanne , Berger Thomas , Schuler Gerold
Published: July 2002

It has been known for years that rodents harbor a unique population of CD4+CD25+ “professional” regulatory/suppressor T cells that is crucial for the prevention of spontaneous autoimmune diseases. Here we demonstrate that CD4+CD25+CD45RO+ T cells (mean 6% of CD4+ T cells) are present in the blood of adult healthy volunteers. In contrast to previous reports, these CD4+CD25+ T cells do not constitute conventional memory cells but rather regulatory cells exhibiting properties identical to their rodent counterparts. Cytotoxic T lymphocyte–associated antigen (CTLA)-4 (CD152), for example, which is essential for the in vivo suppressive activity of CD4+CD25+ T cells, was constitutively expressed, and remained strongly upregulated after stimulation. The cells were nonproliferative to stimulation via their T cell receptor for antigen, but the anergic state was partially reversed by interleukin (IL)-2 and IL-15. Upon stimulation with allogeneic (but not syngeneic) mature dendritic cells or platebound anti-CD3 plus anti-CD28 the CD4+CD25+ T cells released IL-10, and in coculture experiments suppressed the activation and proliferation of CD4+ and CD8+ T cells. Suppression proved IL-10 independent, yet contact dependent as in the mouse. The identification of regulatory CD4+CD25+ T cells has important implications for the study of tolerance in man, notably in the context of autoimmunity, transplantation, and cancer.
Modulation of LIGHT-HVEM Costimulation Prolongs Cardiac Allograft Survival
Authors: Ye Qunrui , Fraser Christopher C. , Gao Wei , Wang Liqing , Busfield Samantha J. , Wang Chichung , Qiu Yubin , Coyle Anthony J. , Gutierrez-Ramos Jose-Carlos , Hancock Wayne W.
Published: July 2002

LIGHT (TNFSF14), a tumor necrosis factor superfamily member expressed by activated T cells, binds to herpes virus entry mediator (HVEM) which is constitutively expressed by T cells and costimulates T cell activation in a CD28-independent manner. Given interest in regulating the effector functions of T cells in vivo, we examined the role of LIGHT-HVEM costimulation in a murine cardiac allograft rejection model. Normal hearts lacked LIGHT or HVEM mRNA expression, but allografts showed strong expression of both genes from day 3 after transplant, and in situ hybridization and immunohistology-localized LIGHT and HVEM to infiltrating leukocytes. To test the importance of LIGHT expression on allograft survival, we generated LIGHT−/− mice by homologous recombination. The mean survival of fully major histocompatibility complex–mismatched vascularized cardiac allografts in LIGHT−/− mice (10 days, P < 0.05) or cyclosporine A (CsA)-treated LIGHT+/+ mice (10 days, P < 0.05) was only slightly prolonged compared with LIGHT+/+ mice (7 days). However, mean allograft survival in CsA-treated LIGHT−/− allograft recipients (30 days) was considerably enhanced (P < 0.001) compared with the 10 days of mean survival in either untreated LIGHT−/− mice or CsA-treated LIGHT+/+ controls. Molecular analyzes showed that the beneficial effects of targeting of LIGHT in CsA-treated recipients were accompanied by decreased intragraft expression of interferon (IFN)-γ, plus IFN-γ–induced chemokine, inducible protein-10, and its receptor, CXCR3. Treatment of LIGHT+/+ allograft recipients with HVEM-Ig plus CsA also enhanced mean allograft survival (21 days) versus wild-type controls receiving HVEM-Ig (mean of 7 days) or CsA alone (P < 0.001). Our data suggest that T cell to T cell–mediated LIGHT/HVEM-dependent costimulation is a significant component of the host response leading to cardiac allograft rejection.
The Research Assessment Exercise: its effect on scholarly journal publishing
Authors: Walford Leo
Published: July 2002

The Research Assessment Exercise (RAE) plays a major part in academic life in the UK. One of its chief measures of ‘research performance’ relates to the publication of articles in refereed scholarly journals. This paper examines the effect of the RAE on the journal publishing system, looking at the communities of authors, editors, and publishers.
On the Brylinski-Kostant filtration
Authors: Joseph Anthony , Letzter Gail , Zelikson Shmuel
Published: July 2002

Let

g

\mathfrak g

be a semisimple Lie algebra and

V
V

a finite dimensional simple

g

\mathfrak g

module. The Brylinski-Kostant (simply, BK) filtration on weight spaces of

V
V

is defined by applying powers of a principle nilpotent element. It leads to a

q
q

-character of

V
V

. Through a result of B. Kostant the BK filtration of the zero weight space is determined by the so-called generalized exponents of

g

\mathfrak g

. Later R. K. Brylinski calculated the BK filtration on dominant weights of

V
V

assuming a vanishing result for cohomology later established by B. Broer. The result could be expressed in terms of

q
q

polynomials introduced by G. Lusztig. In the present work, Verma module maps are used to determine the BK filtration for all weights. To do this several filtrations are introduced and compared, a key point being the graded injectivity of the ring of differential operators on the open Bruhat cell viewed as a

g

\mathfrak g

module under diagonal action. This replaces cohomological vanishing and thereby Brylinski’s result is given a new proof. The calculation for non-dominant weights uses the fact that the corresponding graded ring is a domain as well as a positivity result of G. Lusztig which ensures that there are no accidental cancellations. This method allows one to compare the BK filtrations in adjacent chambers.