bluuebunny/crossref_metadata_2025 huggingface dataset:
Bax-induced Cytochrome C Release from Mitochondria Is Independent of the Permeability Transition Pore but Highly Dependent on Mg2+ Ions
Authors: Eskes Robert , Antonsson Bruno , Osen-Sand Astrid , Montessuit Sylvie , Richter Christoph , Sadoul Rémy , Mazzei Gonzalo , Nichols Anthony , Martinou Jean-Claude
Published: July 2002

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A–sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive.
Identification of Kel1p, a Kelch Domain-containing Protein Involved in Cell Fusion and Morphology in Saccharomyces cerevisiae
Authors: Philips Jennifer , Herskowitz Ira
Published: July 2002

We showed previously that protein kinase C, which is required to maintain cell integrity, negatively regulates cell fusion (Philips, J., and I. Herskowitz. 1997. J. Cell Biol. 138:961–974). To identify additional genes involved in cell fusion, we looked for genes whose overexpression relieved the defect caused by activated alleles of Pkc1p. This strategy led to the identification of a novel gene, KEL1, which encodes a protein composed of two domains, one containing six kelch repeats, a motif initially described in the Drosophila protein Kelch (Xue, F., and L. Cooley. 1993. Cell. 72:681– 693), and another domain predicted to form coiled coils. Overexpression of KEL1 also suppressed the defect in cell fusion of spa2Δ and fps1Δ mutants. KEL2, which corresponds to ORF YGR238c, encodes a protein highly similar to Kel1p. Its overexpression also suppressed the mating defect associated with activated Pkc1p. Mutants lacking KEL1 exhibited a moderate defect in cell fusion that was exacerbated by activated alleles of Pkc1p or loss of FUS1, FUS2, or FPS1, but not by loss of SPA2. kel1Δ mutants form cells that are elongated and heterogeneous in shape, indicating that Kel1p is also required for proper morphology during vegetative growth. In contrast, kel2Δ mutants were not impaired in cell fusion or morphology. Both Kel1p and Kel2p localized to the site where cell fusion occurs during mating and to regions of polarized growth during vegetative growth. Coimmunoprecipitation and two-hybrid analyses indicated that Kel1p and Kel2p physically interact. We conclude that Kel1p has a role in cell morphogenesis and cell fusion and may antagonize the Pkc1p pathway.
Separation of Propulsive and Adhesive Traction Stresses in Locomoting Keratocytes
Authors: Oliver Tim , Dembo Micah , Jacobson Ken
Published: July 2002

Strong, actomyosin-dependent, pinching tractions in steadily locomoting (gliding) fish keratocytes revealed by traction imaging present a paradox, since only forces perpendicular to the direction of locomotion are apparent, leaving the actual propulsive forces unresolved. When keratocytes become transiently “stuck” by their trailing edge and adopt a fibroblast-like morphology, the tractions opposing locomotion are concentrated into the tail, leaving the active pinching and propulsive tractions clearly visible under the cell body. Stuck keratocytes can develop ∼1 mdyn (10,000 pN) total propulsive thrust, originating in the wings of the cell. The leading lamella develops no detectable propulsive traction, even when the cell pulls on its transient tail anchorage. The separation of propulsive and adhesive tractions in the stuck phenotype leads to a mechanically consistent hypothesis that resolves the traction paradox for gliding keratocytes: the propulsive tractions driving locomotion are normally canceled by adhesive tractions resisting locomotion, leaving only the pinching tractions as a resultant. The resolution of the traction pattern into its components specifies conditions to be met for models of cytoskeletal force production, such as the dynamic network contraction model (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G.G. Borisy. 1997. J. Cell Biol. 139:397–415). The traction pattern associated with cells undergoing sharp turns differs markedly from the normal pinching traction pattern, and can be accounted for by postulating an asymmetry in contractile activity of the opposed lateral wings of the cell.
Three-Dimensional Visualization of Transcription Sites and Their Association with Splicing Factor–Rich Nuclear Speckles
Authors: Wei Xiangyun , Somanathan Suryanarayan , Samarabandu Jagath , Berezney Ronald
Published: July 2002

Transcription sites are detected by labeling nascent transcripts with BrUTP in permeabilized 3T3 mouse fibroblasts followed by laser scanning confocal microscopy. Inhibition and enzyme digestion studies confirm that the labeled sites are from RNA transcripts and that RNA polymerase I (RP I) and II (RP II) are responsible for nucleolar and extranucleolar transcription, respectively. An average of 2,000 sites are detected per nucleus with over 90% in the extranucleolar compartment where they are arranged in clusters and three-dimensional networklike arrays. The number of transcription sites, their three-dimensional organization and arrangement into functional zones (Wei et al. 1998) is strikingly maintained after extraction for nuclear matrix. Significant levels of total RP II mediated transcription sites (45%) were associated with splicing factor–rich nuclear speckles even though the speckles occupied <10% of the total extranucleolar space. Moreover, the vast majority of nuclear speckles (>90%) had moderate to high levels of associated transcription activity. Transcription sites were found along the periphery as well as inside the speckles themselves. These spatial relations were confirmed in optical sections through individual speckles and after in vivo labeling of nascent transcripts. Our results demonstrate that nuclear speckles and their surrounding regions are major sites of RP II-mediated transcription in the cell nucleus, and support the view that both speckle- and nonspeckle-associated regions of the nucleus contain sites for the coordination of transcription and splicing processes.
Kinesin-II Is Required for Axonal Transport of Choline Acetyltransferase in Drosophila
Authors: Ray Krishanu , Perez Sharon E. , Yang Zhaohuai , Xu Jenny , Ritchings Bruce W. , Steller Hermann , Goldstein Lawrence S.B.
Published: July 2002

KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport.
Nsec1 Binds a Closed Conformation of Syntaxin1a
Authors: Yang Bin , Steegmaier Martin , Gonzalez Lino C. , Scheller Richard H.
Published: July 2002

The Sec1 family of proteins is proposed to function in vesicle trafficking by forming complexes with target membrane SNAREs (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptors) of the syntaxin family. Here, we demonstrate, by using in vitro binding assays, nondenaturing gel electrophoresis, and specific neurotoxin treatment, that the interaction of syntaxin1A with the core SNARE components, SNAP-25 (synaptosome-associated protein of 25 kD) and VAMP2 (vesicle-associated membrane protein 2), precludes the interaction with nSec1 (also called Munc18 and rbSec1). Inversely, association of nSec1 and syntaxin1A prevents assembly of the ternary SNARE complex. Furthermore, using chemical cross-linking of rat brain membranes, we identified nSec1 complexes containing syntaxin1A, but not SNAP-25 or VAMP2. These results support the hypothesis that Sec1 proteins function as syntaxin chaperons during vesicle docking, priming, and membrane fusion.
Direct Involvement of Yeast Type I Myosins in Cdc42-Dependent Actin Polymerization
Authors: Lechler Terry , Shevchenko Anna , Shevchenko Andrej , Li Rong
Published: July 2002

The generation of cortical actin filaments is necessary for processes such as cell motility and cell polarization. Several recent studies have demonstrated that Wiskott-Aldrich syndrome protein (WASP) family proteins and the actin-related protein (Arp) 2/3 complex are key factors in the nucleation of actin filaments in diverse eukaryotic organisms. To identify other factors involved in this process, we have isolated proteins that bind to Bee1p/Las17p, the yeast WASP-like protein, by affinity chromatography and mass spectroscopic analysis. The yeast type I myosins, Myo3p and Myo5p, have both been identified as Bee1p-interacting proteins. Like Bee1p, these myosins are essential for cortical actin assembly as assayed by in vitro reconstitution of actin nucleation sites in permeabilized yeast cells. Analysis using this assay further demonstrated that the motor activity of these myosins is required for the polymerization step, and that actin polymerization depends on phosphorylation of myosin motor domain by p21-activated kinases (PAKs), downstream effectors of the small guanosine triphosphatase, Cdc42p. The type I myosins also interact with the Arp2/3 complex through a sequence at the end of the tail domain homologous to the Arp2/3-activating region of WASP-like proteins. Combined deletions of the Arp2/3-interacting domains of Bee1p and the type I myosins abolish actin nucleation sites at the cortex, suggesting that these proteins function redundantly in the activation of the Arp2/3 complex.
Integrin Dynamics and Matrix Assembly
Authors: Pankov Roumen , Cukierman Edna , Katz Ben-Zion , Matsumoto Kazue , Lin Diane C. , Lin Shin , Hahn Cornelia , Yamada Kenneth M.
Published: July 2002

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor αvβ3 remains within focal contacts, the fibronectin receptor α5β1 translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 ± 0.7 μm/h and is independent of cell migration. It is induced by ligation of α5β1 integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied α5β1 integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating α5β1 integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.
Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis
Authors: Mancini Marie , Machamer Carolyn E. , Roy Sophie , Nicholson Donald W. , Thornberry Nancy A. , Casciola-Rosen Livia A. , Rosen Antony
Published: July 2002

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors.
The Condensin Complex Governs Chromosome Condensation and Mitotic Transmission of Rdna
Authors: Freeman Lita , Aragon-Alcaide Luis , Strunnikov Alexander
Published: July 2002

We have characterized five genes encoding condensin components in Saccharomyces cerevisiae. All genes are essential for cell viability and encode proteins that form a complex in vivo. We characterized new mutant alleles of the genes encoding the core subunits of this complex, smc2-8 and smc4-1. Both SMC2 and SMC4 are essential for chromosome transmission in anaphase. Mutations in these genes cause defects in establishing condensation of unique (chromosome VIII arm) and repetitive (rDNA) regions of the genome but do not impair sister chromatid cohesion. In vivo localization of Smc4p fused to green fluorescent protein showed that, unexpectedly, in S. cerevisiae the condensin complex concentrates in the rDNA region at the G2/M phase of the cell cycle. rDNA segregation in mitosis is delayed and/or stalled in smc2 and smc4 mutants, compared with separation of pericentromeric and distal arm regions. Mitotic transmission of chromosome III carrying the rDNA translocation is impaired in smc2 and smc4 mutants. Thus, the condensin complex in S. cerevisiae has a specialized function in mitotic segregation of the rDNA locus. Chromatin immunoprecipitation (ChIP) analysis revealed that condensin is physically associated with rDNA in vivo. Thus, the rDNA array is the first identified set of DNA sequences specifically bound by condensin in vivo. The biological role of higher-order chromosome structure in S. cerevisiae is discussed.
Dynacortin, a Genetic Link between Equatorial Contractility and Global Shape Control Discovered by Library Complementation of a Dictyostelium discoideum Cytokinesis Mutant
Authors: Robinson Douglas N. , Spudich James A.
Published: July 2002

We have developed a system for performing interaction genetics in Dictyostelium discoideum that uses a cDNA library complementation/multicopy suppression strategy. Chemically mutagenized cells were screened for cytokinesis-deficient mutants and one mutant was subjected to library complementation. Isolates of four different genes were recovered as modifiers of this strain's cytokinesis defect. These include the cleavage furrow protein cortexillin I, a novel protein we named dynacortin, an ezrin-radixin-moesin-family protein, and coronin. The cortexillin I locus and transcript were found to be disrupted in the strain, identifying it as the affected gene. Dynacortin is localized partly to the cell cortex and becomes enriched in protrusive regions, a localization pattern that is similar to coronin and partly dependent on RacE. During cytokinesis, dynacortin is found in the cortex and is somewhat enriched at the poles. Furthermore, it appears to be reduced in the cleavage furrow. The genetic interactions and the cellular distributions of the proteins suggest a hypothesis for cytokinesis in which the contraction of the medial ring is a function of spatially restricted cortexillin I and myosin II and globally distributed dynacortin, coronin, and RacE.
Replication Origins in XenopusEgg Extract Are 5–15 Kilobases Apart and Are Activated in Clusters That Fire at Different Times
Authors: Blow J. Julian , Gillespie Peter J. , Francis Dennis , Jackson Dean A.
Published: July 2002

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [3H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5–15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5–10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.
Rab27a Regulates the Peripheral Distribution of Melanosomes in Melanocytes
Authors: Hume Alistair N. , Collinson Lucy M. , Rapak Andrzej , Gomes Anita Q. , Hopkins Colin R. , Seabra Miguel C.
Published: July 2002

Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Rab27a, a protein implicated in several diseases, including Griscelli syndrome, choroideremia, and the Hermansky-Pudlak syndrome mouse model, gunmetal. We studied endogenous Rab27a and overexpressed enhanced GFP-Rab27a fusion protein in several cultured melanocyte and melanoma-derived cell lines. In pigmented cells, we observed that Rab27a decorates melanosomes, whereas in nonpigmented cells Rab27a colocalizes with melanosome-resident proteins. When dominant interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. This phenotype is similar to that observed by others in melanocytes derived from the ashen and dilute mutant mice, which bear mutations in the Rab27a and MyoVa loci, respectively. We also found that myosinVa coimmunoprecipitates with Rab27a in extracts from melanocytes and that both Rab27a and myosinVa colocalize on the cytoplasmic face of peripheral melanosomes in wild-type melanocytes. However, the amount of myosinVa in melanosomes from Rab27a-deficient ashen melanocytes is greatly reduced. These results, together with recent data implicating myosinVa in the peripheral capture of melanosomes, suggest that Rab27a is necessary for the recruitment of myosinVa, so allowing the peripheral retention of melanosomes in melanocytes.
Δn89β-Catenin Induces Precocious Development, Differentiation, and Neoplasia in Mammary Gland
Authors: Imbert Alexandra , Eelkema Rachel , Jordan Sara , Feiner Helen , Cowin Pamela
Published: July 2002

To investigate the role of β-catenin in mammary gland development and neoplasia, we expressed a stabilized, transcriptionally active form of β-catenin lacking the NH2-terminal 89 amino acids (ΔN89β-catenin) under the control of the mouse mammary tumor virus long terminal repeat. Our results show that ΔN89β-catenin induces precocious lobuloalveolar development and differentiation in the mammary glands of both male and female mice. Virgin ΔN89β-catenin mammary glands resemble those found in wild-type (wt) pregnant mice and inappropriately express cyclin D1 mRNA. In contrast to wt mammary glands, which resume a virgin appearance after cessation of lactation, transgenic mammary glands involute to a midpregnant status. All transgenic females develop multiple aggressive adenocarcinomas early in life. Surprisingly, the ΔN89β-catenin phenotype differs from those elicited by overexpression of Wnt genes in this gland. In particular, ΔN89β-catenin has no effect on ductal side branching. This suggests that Wnt induction of ductal branching involves additional downstream effectors or modulators.
Role of the F-Box Protein Skp2 in Adhesion-Dependent Cell Cycle Progression
Authors: Carrano Andrea C. , Pagano Michele
Published: July 2002

Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells. In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27. We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCFSkp2 ubiquitin ligase complex. Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM. Conversely, the expression of the other three subunits of the SCFSkp2 complex is independent of cell anchorage. Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation. Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division. The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells. Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation.
Regulation of the Bfa1p–Bub2p complex at spindle pole bodies by the cell cycle phosphatase Cdc14p
Authors: Pereira Gislene , Manson Claire , Grindlay Joan , Schiebel Elmar
Published: July 2002

The budding yeast mitotic exit network (MEN) is a GTPase-driven signal transduction cascade that controls the release of the phosphatase Cdc14p from the nucleolus in anaphase and thereby drives mitotic exit. We show that Cdc14p is partially released from the nucleolus in early anaphase independent of the action of the MEN components Cdc15p, Dbf2p, and Tem1p. Upon release, Cdc14p binds to the spindle pole body (SPB) via association with the Bfa1p–Bub2p GTPase activating protein complex, which is known to regulate the activity of the G protein Tem1p. Cdc14p also interacts with this GTPase. The association of the MEN component Mob1p with the SPB acts as a marker of MEN activation. The simultaneous binding of Cdc14p and Mob1p to the SPB in early anaphase suggests that Cdc14p initially activates the MEN. In a second, later step, which coincides with mitotic exit, Cdc14p reactivates the Bfa1p–Bub2p complex by dephosphorylating Bfa1p. This inactivates the MEN and displaces Mob1p from SPBs. These data indicate that Cdc14p activates the MEN in early anaphase but later inactivates it through Bfa1p dephosphorylation and so restricts MEN activity to a short period in anaphase.
Macrophage-dependent Apoptosis of CD4+ T Lymphocytes from HIV-infected Individuals Is Mediated by FasL and Tumor Necrosis Factor
Authors: Badley Andrew D. , Dockrell David , Simpson Margaret , Schut Ron , Lynch David H. , Leibson Paul , Paya Carlos V.
Published: July 2002

Apoptosis of bystander uninfected CD4+ T lymphocytes by neighboring HIV-infected cells is observed in cell culture and in lymphoid tissue of HIV-infected individuals. This study addresses whether antigen-presenting cells such as human macrophages mediate apoptosis of CD4+ T cells from HIV-infected individuals. Uninfected human macrophages, and to a larger degree, HIV-infected macrophages mediate apoptosis of T cells from HIV-infected, but not from uninfected control individuals. This macrophage-dependent killing targets CD4+, but not CD8+ T lymphocytes from HIV-infected individuals, and direct contact between macrophages and lymphocytes is required. Additional analyses indicated that the apoptosis-inducing ligands, FasL and tumor necrosis factor (TNF), mediate this macrophage-induced apoptosis of CD4+ T cells. These results support a role for macrophage-associated FasL and TNF in the selective depletion of CD4+ T cells in HIV-infected individuals.
Identification of Tyrosinase-related Protein 2 as a Tumor Rejection Antigen for the B16 Melanoma
Authors: Bloom Matthew B. , Perry-Lalley Donna , Robbins Paul F. , Li Yong , El-Gamil Mona , Rosenberg Steven A. , Yang James C.
Published: July 2002

Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinaserelated protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.
The Enigmatic Plasmacytoid T Cells Develop into Dendritic Cells with Interleukin (IL)-3 and CD40-Ligand
Authors: Grouard Géraldine , Rissoan Marie-Clotilde , Filgueira Luis , Durand Isabelle , Banchereau Jacques , Liu Yong-Jun
Published: July 2002

A subset of CD4+CD11c−CD3− blood cells was recently shown to develop into dendritic cells when cultured with monocyte conditioned medium. Here, we demonstrate that CD4+ CD11c−CD3− cells, isolated from tonsils, correspond to the so-called plasmacytoid T cells, an obscure cell type that has long been observed by pathologists within secondary lymphoid tissues. They express CD45RA, but not markers specific for known lymphoid- or myeloid-derived cell types. They undergo rapid apoptosis in culture, unless rescued by IL-3. Further addition of CD40-ligand results in their differentiation into dendritic cells that express low levels of myeloid antigens CD13 and CD33.
Single Cell Analysis Reveals Regulated Hierarchical T Cell Antigen Receptor Signaling Thresholds and Intraclonal Heterogeneity for Individual Cytokine Responses of CD4+ T Cells
Authors: Itoh Yasushi , Germain Ronald N.
Published: July 2002

T cell receptor (TCR) recognition of peptide–major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4+ cell population. Low concentrations of TCR ligand elicit only interferon-γ (IFN-γ) production. Increasing ligand recruits more cells into the IFN-γ+ pool, increases IFN-γ produced per cell, and also elicits IL-2, but only from cells already making IFN-γ. Most cells producing only IFN-γ show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-γ synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-γ/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-γ and not IL-2, and the amount of IFN-γ exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.
Prostaglandin E2 and Tumor Necrosis Factor α Cooperate to Activate Human Dendritic Cells: Synergistic Activation of Interleukin 12 Production
Authors: Rieser Claudia , Böck Günther , Klocker Helmut , Bartsch Georg , Thurnher Martin
Published: July 2002

Interleukin (IL)-12 is a proinflammatory cytokine that contributes to innate resistance and to the development of antigen-specific T cell responses. Among other effects, prostaglandin E2 (PGE2) inhibits the production of IL-12 by macrophages activated with lipopolysaccharide (LPS). Here we investigated the effects of PGE2 on human dendritic cells (DCs) which develop in the presence of GM-CSF and IL-4. We demonstrate that in the absence of LPS, PGE2 dose dependently stimulated the production of IL-12 by DCs. Although PGE2 alone stimulated the production of low amounts of IL-12 only, it synergized with tumor necrosis factor (TNF)-α to induce high levels of IL-12 production by DCs. Addition of TNF-α in the absence of PGE2 had no effect on IL-12 production. Conversely, in the presence of LPS, PGE2 inhibited IL-12 production by DCs in a dose-dependent manner. The combination of PGE2 and TNF-α efficiently silenced mannose receptor–mediated endocytosis in DCs and readily induced neo-expression of the CD83 antigen. In addition, the expression of various surface antigens such as major histocompatibility complex class I and II, adhesion, as well as costimulatory molecules was upregulated by this treatment. The effects of PGE2 on IL-12 synthesis and CD83 expression could be mimicked by dibutyryl-cAMP and forskolin, indicating that they were due to the intracellular elevation of cAMP levels. DC treated with PGE2 and TNF-α were most potent in stimulating allogeneic T cell proliferation. Our data demonstrate that PGE2 contributes to the maturation of human DCs and that PGE2 can be a potent enhancer of IL-12 production by human DCs.
Mice Deficient in Nuclear Factor (NF)-κB/p52 Present with Defects in Humoral Responses, Germinal Center Reactions, and Splenic Microarchitecture
Authors: Franzoso Guido , Carlson Louise , Poljak Ljiljana , Shores Elizabeth W. , Epstein Suzanne , Leonardi Antonio , Grinberg Alex , Tran Tom , Scharton-Kersten Tanya , Anver Miriam , Love Paul , Brown Keith , Siebenlist Ulrich
Published: July 2002

p52 is a subunit of nuclear factor (NF)-κB transcription factors, most closely related to p50. Previously, we have shown that p52, but not p50 homodimers can form transactivating complexes when associated with Bcl-3, an unusual member of the IκB family. To determine nonredundant physiologic roles of p52, we generated mice deficient in p52. Null mutant mice were impaired in their ability to generate antibodies to T-dependent antigens, consistent with an absence of B cell follicles and follicular dendritic cell networks in secondary lymphoid organs, and an inability to form germinal centers. Furthermore, the splenic marginal zone was disrupted. These phenotypes are largely overlapping with those observed in Bcl-3 knockout animals, but distinct from those of p50 knockouts, supporting the notion of a physiologically relevant complex of p52 homodimers and Bcl-3. Adoptive transfer experiments further suggest that such a complex may be critical in accessory cell functions during antigen-specific immune reactions. Possible roles of p52 and Bcl-3 are discussed that may underlie the oncogenic potential of these proteins, as evidenced by recurrent chromosomal translocations of their genes in lymphoid tumors.
HIV-1 Directly Kills CD4+ T Cells by a Fas-independent Mechanism
Authors: Gandhi Rajesh T. , Chen Benjamin K. , Straus Stephen E. , Dale Janet K. , Lenardo Michael J. , Baltimore David
Published: July 2002

The mechanism by which HIV-1 induces CD4+ T cell death is not known. A fundamental issue is whether HIV-1 primarily induces direct killing of infected cells or indirectly causes death of uninfected bystander cells. This question was studied using a reporter virus system in which infected cells are marked with the cell surface protein placental alkaline phosphatase (PLAP). Infection by HIV-PLAP of peripheral blood mononuclear cells (PBMCs) and T cell lines leads to rapid depletion of CD4+ T cells and induction of apoptosis. The great majority of HIV-induced T cell death in vitro involves direct loss of infected cells rather than indirect effects on uninfected bystander cells. Because of its proposed role in HIV-induced cell death, we also examined the Fas (CD95/Apo1) pathway in killing of T cells by HIV-1. Infected PBMCs or CEM cells display no increase in surface Fas relative to uninfected cells. In addition, HIV-1 kills CEM and Jurkat T cells in the presence of a caspase inhibitor that completely blocks Fas-mediated apoptosis. HIV-1 also depletes CD4+ T cells in PBMCs from patients who have a genetically defective Fas pathway. These results suggest that HIV-1 induces direct apoptosis of infected cells and kills T cells by a Fas-independent mechanism.
Urokinase Receptor (CD87) Regulates Leukocyte Recruitment via β2 Integrins In Vivo
Authors: May Andreas E. , Kanse Sandip M. , Lund Leif R. , Gisler Roland H. , Imhof Beat A. , Preissner Klaus T.
Published: July 2002

The urokinase receptor (CD87; uPAR) is found in close association with β2 integrins on leukocytes. We studied the functional consequence of this association for leukocyte adhesion and migration. In vivo, the β2 integrin–dependent recruitment of leukocytes to the inflamed peritoneum of uPAR-deficient mice was significantly reduced as compared with wild-type animals. In vitro, β2 integrin–mediated adhesion of leukocytes to endothelium was lost upon removal of uPAR from the leukocyte surface by phosphatidyl-inositol–specific phospholipase C. Leukocyte adhesion was reconstituted when soluble intact uPAR, but not a truncated form lacking the uPA-binding domain, was allowed to reassociate with the cell surface. uPAR ligation with a monoclonal antibody induced adhesion of monocytic cells and neutrophils to vascular endothelium by six- to eightfold, whereas ligation with inactivated uPA significantly reduced cell-to-cell adhesion irrespective of the β2 integrin–stimulating pathway. These data indicate that β2 integrin–mediated leukocyte–endothelial cell interactions and recruitment to inflamed areas require the presence of uPAR and define a new phenotype for uPAR-deficient mice. Moreover, uPAR ligation differentially modulates leukocyte adhesion to endothelium and provides novel targets for therapeutic strategies in inflammation-related vascular pathologies.
Adenosine Diphosphate (ADP)-Ribosylation of the Guanosine Triphosphatase (GTPase) Rho in Resting Peripheral Blood Human T Lymphocytes Results in Pseudopodial Extension and the Inhibition of  T Cell Activation
Authors: Woodside Darren G. , Wooten David K. , McIntyre Bradley W.
Published: July 2002

Scrape loading Clostridium botulinum C3 exoenzyme into primary peripheral blood human T lymphocytes (PB T cells) efficiently adenosine diphosphate (ADP)-ribosylates and thus inactivates the guanosine triphosphatase (GTPase) Rho. Basal adhesion of PB T cells to the β1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn++ (1 mM) affected. Whereas untreated PB T cells adherent to Fn remain spherical, C3-treated PB T cells extend F-actin–containing pseudopodia. Inactivation of Rho delayed the kinetics of PMA-dependent PB T cell homotypic aggregation, a process involving integrin αLβ2. Although C3 treatment of PB T cells did not prevent adhesion to the β1 integrin substrate Fn, it did inhibit β1 integrin/CD3-mediated costimulation of proliferation. Analysis of intracellular cytokine production at the single cell level demonstrated that ADP-ribosylation of Rho inhibited β1 integrin/ CD3 and CD28/CD3 costimulation of IL-2 production within 6 h of activation. Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatment. Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchitecture, and functional Rho is required for very early events regulating costimulation of IL-2 production in PB T cells.
Characterization of Gene Expression in Resting and Activated Mast Cells
Authors: Chen Huaxian , Centola Michael , Altschul Stephen F. , Metzger Henry
Published: July 2002

To characterize gene expression in activated mast cells more comprehensively than heretofore, we surveyed the changes in genetic transcripts by the method of serial analysis of gene expression in the RBL-2H3 line of rat mast cells before and after they were stimulated through their receptors with high affinity for immunoglobulin E (FcεRI). A total of 40,759 transcripts derived from 11,300 genes were analyzed. Among the diverse genes that had not been previously associated with mast cells and that were constitutively expressed were those for the cytokine macrophage migration inhibitory factor neurohormone receptors such as growth hormone- releasing factor and melatonin and components of the exocytotic machinery. In addition, several dozen transcripts were differentially expressed in response to antigen-induced clustering of the FcεRI. Included among these were the genes for preprorelaxin, mitogen-activated protein kinase kinase 3, and the dual specificity protein phosphatase, rVH6. Significantly, the majority of genes differentially expressed in this well-studied model of mast cell activation have not been identified before this analysis.
Structural Evidence of  T Cell Xeno-reactivity in the Absence of Molecular Mimicry
Authors: Zhao Rui , Loftus Douglas J. , Appella Ettore , Collins Edward J.
Published: July 2002

The T cell receptor (TCR), from a xeno-reactive murine cytotoxic T lymphocyte clone AHIII12.2, recognizes murine H-2Db complexed with peptide p1027 (FAPGVFPYM), as well as human HLA-A2.1 complexed with peptide p1049 (ALWGFFPVL). A commonly proposed model (the molecular mimicry model) used to explain TCR cross-reactivity suggests that the molecular surfaces of the recognized complexes are similar in shape, charge, or both, in spite of the primary sequence differences. To examine the mechanism of xeno-reactivity of AHIII12.2, we have determined the crystal structures of A2/p1049 and Db/p1027 to 2.5 Å and 2.8 Å resolution, respectively. The crystal structures show that the TCR footprint regions of the two class I complexes are significantly different in shape and charge. We propose that rather than simple molecular mimicry, unpredictable arrays of common and differential contacts on the two class I complexes are used for their recognition by the same TCR.
Effect of Factor XIII on Endothelial Barrier Function
Authors: Noll Thomas , Wozniak Gernold , McCarson Karin , Hajimohammad Amir , Metzner Hubert J. , Inserte Javier , Kummer Wolfgang , Hehrlein Friedrich Wilhelm , Piper Hans Michael
Published: July 2002

The effect of factor XIII on endothelial barrier function was studied in a model of cultured monolayers of porcine aortic endothelial cells and saline-perfused rat hearts. The thrombin-activated plasma factor XIII (1 U/ml) reduced albumin permeability of endothelial monolayers within 20 min by 30 ± 7% (basal value of 5.9 ± 0.4 × 10−6 cm/s), whereas the nonactivated plasma factor XIII had no effect. Reduction of permeability to the same extent, i.e., by 34 ± 9% could be obtained with the thrombin-activated A subunit of factor XIII (1 U/ml), whereas the iodoacetamide-inactivated A subunit as well as the B subunit had no effect on permeability. Endothelial monolayers exposed to the activated factor XIII A exhibited immunoreactive deposition of itself at interfaces of adjacent cells; however, these were not found on exposure to nonactivated factor XIII A or factor XIII B. Hyperpermeability induced by metabolic inhibition (1 mM potassium cyanide plus 1 mM 2-deoxy-d-glucose) was prevented in the presence of the activated factor XIII A. Likewise, the increase in myocardial water content in ischemic-reperfused rat hearts was prevented in its presence. This study shows that activated factor XIII reduces endothelial permeability. It can prevent the loss of endothelial barrier function under conditions of energy depletion. Its effect seems related to a modification of the paracellular passageways in endothelial monolayers.
Lymphocyte Migration in Lymphocyte Function-associated Antigen (LFA)-1–deficient Mice
Authors: Berlin-Rufenach Cornelia , Otto Florian , Mathies Meg , Westermann Juergen , Owen Michael J. , Hamann Alf , Hogg Nancy
Published: July 2002

Using lymphocyte function-associated antigen (LFA)-1−/− mice, we have examined the role of LFA-1 and other integrins in the recirculation of lymphocytes. LFA-1 has a key role in migration to peripheral lymph nodes (pLNs), and influences migration into other LNs. Second, the α4 integrins, α4β7 and α4β1, have a hitherto unrecognized ability to compensate for the lack of LFA-1 in migration to pLNs. These findings are confirmed using normal mice and blocking LFA-1 and α4 monoclonal antibodies. Unexpectedly, vascular cell adhesion molecule (VCAM)-1, which is essential in inflammatory responses, serves as the ligand for the α4 integrins on pLN high endothelial venules. VCAM-1 also participates in trafficking into mesenteric LNs and Peyer's patch nodes where mucosal addressin cell adhesion molecule 1 (MAdCAM-1), the α4β7-specific ligand, dominates. Both α4β1, interacting with ligand VCAM-1, and also LFA-1 participate in substantial lymphocyte recirculation through bone marrow. These observations suggest that organ-specific adhesion receptor usage in mature lymphocyte recirculation is not as rigidly adhered to as previously considered, and that the same basic sets of adhesion receptors are used in both lymphocyte homing and inflammatory responses.
Dependence of T Cell Antigen Recognition on the Dimensions of an Accessory Receptor–Ligand Complex
Authors: Wild Martin K. , Cambiaggi Anna , Brown Marion H. , Davies Elizabeth A. , Ohno Hiroshi , Saito Takashi , van der Merwe P. Anton
Published: July 2002

The T cell antigen receptor (TCR) and its ligand peptide–major histocompatibility complex (MHC) are small (∼7 nm) compared with other abundant cell surface molecules such as integrins, CD43, and CD45 (23–50 nm). We have proposed that molecules at the T cell/antigen-presenting cell (APC) interface segregate according to size, with small “accessory” molecules (e.g., CD2, CD4, CD8, CD28, and CD154) contributing to the formation of a close-contact zone, within which the TCR engages peptide–MHC, and from which large molecules are excluded (Davis, S.J., and P.A. van der Merwe. 1996. Immunol. Today. 17:177–187). One prediction of this model is that increasing the size of these small accessory molecules will disrupt their function. Here, we test this prediction by varying the dimensions of the CD2 ligand, CD48, and examining how this affects T cell antigen recognition. Although the interaction of CD2 on T cells with wild-type or shortened forms of CD48 on APCs enhances T cell antigen recognition, the interaction of CD2 with elongated forms of CD48 is strongly inhibitory. Further experiments indicated that elongation of the CD2/CD48 complex inhibited TCR engagement of peptide–MHC, presumably by preventing the formation of sufficiently intimate contacts at the T cell/APC interface. These findings demonstrate the importance of small size in CD2/CD48 function, and support the hypothesis that T cell antigen recognition requires segregation of cell surface molecules according to size.
N-Formylpeptides Induce Two Distinct Concentration Optima for Mouse Neutrophil Chemotaxis by Differential Interaction with Two N-Formylpeptide Receptor (Fpr) Subtypes
Authors: Hartt Jennifer K. , Barish Grant , Murphy Philip M. , Gao Ji-Liang
Published: July 2002

The N-formylpeptide receptor (FPR) is a G protein–coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N-formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC50s, ∼5 μM for calcium flux and chemotaxis, were ∼100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.