bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Requirements for Bi-directional Movement of Phagosomes Along Microtubules
Authors: Blocker Ariel , Severin Fedor F. , Burkhardt Janis K. , Bingham James B. , Yu Hanry , Olivo Jean-Christophe , Schroer Trina A. , Hyman Anthony A. , Griffiths Gareth
Published: July 2002

Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubuledependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with “late” phagosomes moving threefold more frequently than “early” ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dyneinmediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.
In Vivo Evidence That the Stromelysin-3 Metalloproteinase Contributes in a Paracrine Manner to Epithelial Cell Malignancy
Authors: Masson Régis , Lefebvre Olivier , Noël Agnès , Fahime Mostapha El , Chenard Marie-Pierre , Wendling Corinne , Kebers Florence , LeMeur Marianne , Dierich Andrée , Foidart Jean-Michel , Basset Paul , Rio Marie-Christine
Published: July 2002

Stromelysin-3 (ST3; Basset, P., J.P. Bellocq, C. Wolf, I. Stoll, P. Hutin, J.M. Limacher, O.L. Podhajcer, M.P. Chenard, M.C. Rio, P. Chambon. 1990. Nature. 348:699–704) is a matrix metalloproteinase (MMP) expressed in mesenchymal cells located close to epithelial cells, during physiological and pathological tissue remodeling processes. In human carcinomas, high ST3 levels are associated with a poor clinical outcome, suggesting that ST3 plays a role during malignant processes. In this study we report the ST3 gene inactivation by homologous recombination. Although ST3 null mice (ST3−/−) were fertile and did not exhibit obvious alterations in appearance and behavior, the lack of ST3 altered malignant processes. Thus, the suppression of ST3 results in a decreased 7,12-dimethylbenzanthracene-induced tumorigenesis in ST3−/− mice. Moreover, ST3−/− fibroblasts have lost the capacity to promote implantation of MCF7 human malignant epithelial cells in nude mice (P < 0.008). Finally, we show that this ST3 paracrine function requires extracellular matrix (ECM)-associated growth factors. Altogether, these findings give evidence that ST3 promotes, in a paracrine manner, homing of malignant epithelial cells, a key process for both primary tumors and metastases. Therefore, ST3 represents an appropriate target for specific MMP inhibitor(s) in future therapeutical approaches directed against the stromal compartment of human carcinomas.
The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops
Authors: de Belle Ian , Cai Shutao , Kohwi-Shigematsu Terumi
Published: July 2002

Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1–1.1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island–containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear “halos” with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type–specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell–specific gene regulation.
Caspase Inhibition Extends the Commitment to Neuronal Death Beyond Cytochrome c Release to the Point of Mitochondrial Depolarization
Authors: Deshmukh Mohanish , Kuida Keisuke , Johnson Eugene M.
Published: July 2002

Nerve growth factor (NGF) deprivation induces a Bax-dependent, caspase-dependent programmed cell death in sympathetic neurons. We examined whether the release of cytochrome c was accompanied by the loss of mitochondrial membrane potential during sympathetic neuronal death. NGF- deprived, caspase inhibitor–treated mouse sympathetic neurons maintained mitochondrial membrane poten-tial for 25–30 h after releasing cytochrome c. NGF- deprived sympathetic neurons became committed to die, as measured by the inability of cells to be rescued by NGF readdition, at the time of cytochrome c release. In the presence of caspase inhibitor, however, this commitment to death was extended beyond the point of cytochrome c release, but only up to the subsequent point of mitochondrial membrane potential loss. Caspase-9 deficiency also arrested NGF-deprived sympathetic neurons after release of cytochrome c, and permitted these neurons to be rescued with NGF readdition. Commitment to death in the NGF-deprived, caspase- 9–deficient sympathetic neurons was also coincident with the loss of mitochondrial membrane potential. Thus, caspase inhibition extended commitment to death in trophic factor–deprived sympathetic neurons and allowed recovery of neurons arrested after the loss of cytochrome c, but not beyond the subsequent loss of mitochondrial membrane potential.
Characterization of Vertebrate Cohesin Complexes and Their Regulation in Prophase
Authors: Sumara Izabela , Vorlaufer Elisabeth , Gieffers Christian , Peters Beate H. , Peters Jan-Michael
Published: July 2002

In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.
Molecular Chaperones in the Yeast Endoplasmic Reticulum Maintain the Solubility of Proteins for Retrotranslocation and Degradation
Authors: Nishikawa Shuh-ichi , Fewell Sheara W. , Kato Yoshihito , Brodsky Jeffrey L. , Endo Toshiya
Published: July 2002

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro–α-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain–containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.
Physiological regulation of β-catenin stability by Tcf3 and CK1ϵ
Authors: Lee Ethan , Salic Adrian , Kirschner Marc W.
Published: July 2002

The wnt pathway regulates the steady state level of β-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit β-catenin turnover via its competition with axin and adenomatous polyposis for β-catenin binding. A mutant of β-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of β-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between β-catenin and Tcf3 stimulate β-catenin degradation, indicating this interaction normally plays an important role in regulating β-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1ϵ, and phosphorylation of Tcf3 by CKIϵ stimulates its binding to β-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1ϵ to inhibit β-catenin degradation, whereas CKI-7, an inhibitor of CK1ϵ, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1ϵ stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1ϵ can modulate wnt signaling in vivo by regulating both the β-catenin-Tcf3 and the GBP-dsh interfaces.
MuSK induces in vivo acetylcholine receptor clusters in a ligand-independent manner
Authors: Sander Andreas , Hesser Boris A. , Witzemann Veit
Published: July 2002

Muscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of ϵ-subunit–specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.
Collagen-induced Arthritis Is Reduced in 5-Lipoxygenase-activating Protein-deficient Mice
Authors: Griffiths Richard J. , Smith MaryAlice , Roach Marsha L. , Stock Jeffrey L. , Stam Ethan J. , Milici A.J. , Scampoli Deborah N. , Eskra James D. , Byrum Robert S. , Koller Beverly H. , McNeish John D.
Published: July 2002

Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.
Virus-specific CD8+ T Lymphocytes Downregulate T Helper Cell Type 2 Cytokine Secretion and Pulmonary Eosinophilia during Experimental Murine Respiratory Syncytial Virus Infection
Authors: Srikiatkhachorn Anon , Braciale Thomas J.
Published: July 2002

T lymphocytes play a pivotal role in the immune response during viral infections. In a murine model of experimental respiratory syncytial virus (RSV) infection, mice sensitized to either of the two major glycoproteins of RSV develop distinct patterns of cytokine secretion and lung inflammation upon subsequent RSV infection. Mice sensitized to RSV-G (attachment) glycoprotein exhibit a strong interleukin (IL)-4 and IL-5 response and develop pulmonary eosinophilia, whereas mice sensitized to RSV-F (fusion) glycoprotein develop a predominantly T helper cell (Th)1 response and pulmonary inflammation characterized by mononuclear cell infiltration. In this study, we examined the potential role of virus-specific CD8+ T cytolytic T cells on the differentiation and activation of functionally distinct CD4+ T cells specific to these viral glycoproteins. Mice primed with recombinant vaccinia virus expressing RSV-F glycoprotein mounted a strong RSV-specific, MHC class I–restricted cytolytic response, whereas priming with recombinant vaccinia virus expressing RSV-G glycoprotein failed to elicit any detectable cytolytic response. Priming for a RSV-specific CD8+ T cell response, either with a recombinant vaccinia virus expressing RSV-G glycoprotein in which a strong CD8+ T cell epitope from RSV-M2 (matrix) protein has been inserted or with a combination of vaccinia virus expressing the matrix protein and the RSV-G glycoprotein, suppressed the eosinophil recruitment into the lungs of these mice upon subsequent challenge with RSV. This reduction in pulmonary eosinophilia correlated with the suppression of Th2 type cytokine production. The importance of CD8+ T cells in this process was further supported by the results in CD8+ T cell deficient, β2 microglobulin KO mice. In these mice, priming to RSV-F glycoprotein (which in normal mice primed for a strong cytolytic response and a pulmonary infiltrate consisting primarily of mononuclear cells on RSV challenge) resulted in the development of marked pulmonary eosinophilia that was not seen in mice with an intact CD8+ T cell compartment. These results indicate that CD8+ T cells may play an important role in the regulation of the differentiation and activation of Th2 CD4+ T cells as well as the recruitment of eosinophils into the lungs during RSV infection.
Peripheral T Cell Survival Requires Continual Ligation of the T Cell Receptor to Major Histocompatibility Complex–Encoded Molecules
Authors: Kirberg Jörg , Berns Anton , Boehmer Harald von
Published: July 2002

In the thymus, T cells are selected according to their T cell receptor (TCR) specificity. After positive selection, mature cells are exported from primary lymphoid organs to seed the secondary lymphoid tissue. An important question is whether survival of mature T cells is an intrinsic property or requires continuous survival signals, i.e., engagement of the TCR by major histocompatibility complex (MHC) molecules in the periphery, perhaps in a similar way as occurring during thymic positive selection. To address this issue we used recombination-activating gene (Rag)-deficient H-2b mice expressing a transgenic TCR restricted by I-Ed class II MHC molecules. After engraftment with Rag−/− H-2d fetal thymi, CD4+8− peripheral T cells emerged. These cells were isolated and transferred into immunodeficient hosts of H-2b or H-2d haplotype, some of the latter being common cytokine receptor γ chain deficient to exclude rejection of H-2b donor cells by host natural killer cells. Our results show that in the absence, but not in the presence, of selecting MHC molecules, peripheral mature T cells are short lived and disappear within 7 wk, indicating that continuous contact of the TCR with selecting MHC molecules is required for survival of T cells.
Lung Epithelial Cells Are a Major Site of Murine Gammaherpesvirus Persistence
Authors: Stewart James P. , Usherwood Edward J. , Ross Alan , Dyson Heather , Nash Tony
Published: July 2002

It is currently believed that latently infected, resting B lymphocytes are central to gammaherpesvirus persistence, whereas mucosal epithelial cells are considered nonessential. We have readdressed the question of nonlymphoid persistence using murine gammaherpesvirus 68 (MHV-68). To dissect lymphoid from nonlymphoid persistence, we used μMT transgenic mice that are defective in B cells. MHV-68 DNA persisted in the lungs of intact and B cell–deficient mice. Both episomal and linear forms of the virus genome were present in lungs, implying the presence of both latency and productive replication. In situ hybridization for virus tRNA transcripts revealed latent MHV-68 in pulmonary epithelial cells. Infectious virus was recovered from the lungs of μMT mice after T cell depletion, showing that the persisting virus DNA was reactivatable. Finally, using adoptive transfer of B cells into B cell–deficient mice, it was shown that virus persisting in lungs seeded splenic B cells, and virus resident in the spleen seeded the lungs. These results show that mucosal epithelia can act as a nonlymphoid reservoir for gammaherpesvirus persistence, and that there is a two-way movement of virus between lymphoid and nonlymphoid compartments during persistence.
Engraftment of Bone Marrow from Severe Combined Immunodeficient (SCID) Mice Reverses the Reproductive Deficits in Natural Killer Cell–deficient tgε26 Mice
Authors: Guimond Marie-Josée , Wang Baoping , Croy B. Anne
Published: July 2002

A large, transient population of natural killer (NK) cells appears in the murine uterine mesometrial triangle during pregnancy. Depletion of uterine (u) NK cells, recently achieved using gene-ablated and transgenic mice, results in pathology. Pregnancies from matings of homozygous NK and T cell–deficient tgε26 mice have <1% of normal uNK cell frequency, no development of an implantation site–associated metrial gland, and an edematous decidua with vascular pathology that includes abnormally high vessel walls/lumens ratios. Fetal loss of 64% occurs midgestation and placentae are small. None of these features are seen in pregnant T cell–deficient mice. To confirm the role of the NK cell deficiency in these reproductive deficits, transplantation of tgε26 females was undertaken using bone marrow from B and T cell–deficient scid/scid donors. Engrafted pregnant females have restoration of the uNK cell population, induced metrial gland differentiation, reduced anomalies in the decidua and decidual blood vessels, increased placental sizes, and restoration of fetal viability at all gestational days studied (days 10, 12, and 14). Thus, uNK cells appear to have critical functions in pregnancy that promote decidual health, the appropriate vascularization of implantation sites, and placental size.
Crucial Role of Tumor Necrosis Factor Receptor 1 Expression on Nonhematopoietic Cells for B Cell Localization within the Splenic White Pulp
Authors: Tkachuk Maria , Bolliger Stephan , Ryffel Bernhard , Pluschke Gerd , Banks Theresa A. , Herren Suzanne , Gisler Roland H. , Kosco-Vilbois Marie H.
Published: July 2002

During immune responses the initial activation of B cells takes place in T cell zones of periarteriolar lymphoid sheaths (PALS) of the splenic white pulp. After initial activation, B cells migrate into the primary follicles and, in association with follicular dendritic cells (FDCs), undergo clonal expansion and differentiation giving rise to germinal centers (GCs). Peanut agglutinin binding (PNA+) cells of the GC differentiate further into memory or plasma cells. Here we report that in tumor necrosis factor receptor 1–deficient mice (TNFR1−/−), the location of B cells was altered and that plasma cells were abnormally distributed in the splenic PALS. In contrast to lymphotoxin α–deficient mice (LTα−/−), bone marrow or fetal liver transplantation did not correct the abnormal organization of the spleen, location of B cells, the lack of an FDC network, nor the antibody response in TNFR1−/− mice. These results argue for a crucial role of TNFR1 expression on nonhematopoietic cells for the maintenance of the splenic architecture and proper B cell location. In addition, the lack in development of an FDC network after adoptive transfer suggests that either FDCs are not of bone marrow origin or that they depend on signals from nonhematopoietic cells for maturation.
α/β–T Cell Receptor (TCR)+CD4−CD8− (NKT) Thymocytes Prevent Insulin-dependent Diabetes Mellitus in Nonobese Diabetic (NOD)/Lt Mice by the Influence of Interleukin (IL)-4 and/or IL-10
Authors: Hammond Kirsten J.L. , Poulton Lynn D. , Palmisano Linda J. , Silveira Pablo A. , Godfrey Dale I. , Baxter Alan G.
Published: July 2002

We have previously shown that nonobese diabetic (NOD) mice are selectively deficient in α/β-T cell receptor (TCR)+CD4−CD8− NKT cells, a defect that may contribute to their susceptibility to the spontaneous development of insulin-dependent diabetes mellitus (IDDM). The role of NKT cells in protection from IDDM in NOD mice was studied by the infusion of thymocyte subsets into young female NOD mice. A single intravenous injection of 106 CD4−/lowCD8− or CD4−CD8− thymocytes from female (BALB/c × NOD)F1 donors protected intact NOD mice from the spontaneous onset of clinical IDDM. Insulitis was still present in some recipient mice, although the cell infiltrates were principally periductal and periislet, rather than the intraislet pattern characteristic of insulitis in unmanipulated NOD mice. Protection was not associated with the induction of “allogenic tolerance” or systemic autoimmunity. Accelerated IDDM occurs after injection of splenocytes from NOD donors into irradiated adult NOD recipients. When α/β-TCR+ and α/β-TCR− subsets of CD4−CD8− thymocytes were transferred with diabetogenic splenocytes and compared for their ability to prevent the development of IDDM in irradiated adult recipients, only the α/β-TCR+ population was protective, confirming that NKT cells were responsible for this activity. The protective effect in the induced model of IDDM was neutralized by anti–IL-4 and anti–IL-10 monoclonal antibodies in vivo, indicating a role for at least one of these cytokines in NKT cell-mediated protection. These results have significant implications for the pathogenesis and potential prevention of IDDM in humans.
Requirement of p21-activated Kinase (PAK) for Salmonella typhimurium–induced Nuclear Responses
Authors: Chen Li-Mei , Bagrodia Shubha , Cerione Richard A. , Galán Jorge E.
Published: July 2002

Salmonella typhimurium has sustained a long-standing association with its host and therefore has evolved sophisticated strategies to multiply and survive within this environment. Central to Salmonella pathogenesis is the function of a dedicated type III secretion system that delivers bacterial effector proteins into the host cell cytoplasm. These effectors stimulate nuclear responses and actin cytoskeleton reorganization leading to the production of proinflammatory cytokines and bacterial internalization. The stimulation of these responses requires the function of Cdc42, a member of the Rho family of small molecular weight GTPases, and SopE, a bacterial effector protein that stimulates guanine nucleotide exchange on Rho GTPases. However, nothing is known about the role of Cdc42 effector proteins in S. typhimurium–induced responses. We showed here that S. typhimurium infection of cultured epithelial cells results in the activation of p21-activated kinase (PAK), a serine/threonine kinase that is an effector of Cdc42-dependent responses. Transient expression of a kinase-defective PAK blocked both S. typhimurium– and SopE-induced c-Jun NH2-terminal kinase (JNK) activation but did not interfere with bacteria-induced actin cytoskeleton rearrangements. Similarly, expression of SH3-binding mutants of PAK did not block actin-mediated S. typhimurium entry into cultured cells. However, expression of an effector loop mutant of Cdc42Hs (Cdc42HsC40) unable to bind PAK and other CRIB (for Cdc42/Rac interacting binding)-containing target proteins resulted in abrogation of both S. typhimurium–induced nuclear and cytoskeletal responses. These results show that PAK kinase activity is required for bacteria-induced nuclear responses but it is not required for cytoskeletal rearrangements, indicating that S. typhimurium stimulates cellular responses through different Cdc42 downstream effector activities. In addition, these results demonstrate that the effector loop of Cdc42 implicated in the binding of PAK and other CRIB-containing target proteins is required for both responses.
Autoantigen-Specific B Cell Activation in FAS-Deficient Rheumatoid Factor Immunoglobulin Transgenic Mice
Authors: Wang Haowei , Shlomchik Mark J.
Published: July 2002

In systemic autoimmune disease, self-tolerance fails, leading to autoantibody production. A central issue in immunology is to understand the origins of activated self-reactive B cells. We have used immunoglobulin (Ig) transgenic mice to investigate the regulation of autoreactive B cells with specificity for self-IgG2a (the rheumatoid factor [RF] specificity) to understand how normal mice regulate RF autoantibodies and how this fails in autoimmune mice. We previously showed that normal mice do not tolerize the AM14 RF clone, nor do they appear to activate it. Here we show that in Fas-deficient autoimmune mice, the picture is quite different. RF B cells are activated to divide and secrete, but only when the autoantigen is present. Thus, B cells that are ignored rather than anergized in normal mice can be stimulated to produce autoantibody in Fas-deficient mice. This demonstrates a novel developmental step at which intact Fas–Fas ligand signaling is required to regulate B cells in order to prevent autoimmunity. These data also establish the relevance of ignorant self-specific B cells to autoantibody production in disease and prove that in the case of the RF specificity, the nominal autoantigen IgG2a is the driving autoantigen in vivo.
Rel Induces Interferon Regulatory Factor 4 (IRF-4) Expression in Lymphocytes
Authors: Grumont Raelene J. , Gerondakis Steve
Published: July 2002

In lymphocytes, the Rel transcription factor is essential in establishing a pattern of gene expression that promotes cell proliferation, survival, and differentiation. Here we show that mitogen-induced expression of interferon (IFN) regulatory factor 4 (IRF-4), a lymphoid-specific member of the IFN family of transcription factors, is Rel dependent. Consistent with IRF-4 functioning as a repressor of IFN-induced gene expression, the absence of IRF-4 expression in c-rel−/− B cells coincided with a greater sensitivity of these cells to the antiproliferative activity of IFNs. In turn, enforced expression of an IRF-4 transgene restored IFN modulated c-rel−/− B cell proliferation to that of wild-type cells. This cross-regulation between two different signaling pathways represents a novel mechanism that Rel/nuclear factor κB can repress the transcription of IFN-regulated genes in a cell type–specific manner.
Essential Role of Nuclear Factor (NF)-κB–Inducing Kinase and Inhibitor of κb (Iκb) Kinase α in Nf-κb Activation through Lymphotoxin β Receptor, but Not through Tumor Necrosis Factor Receptor I
Authors: Matsushima Akemi , Kaisho Tsuneyasu , Rennert Paul D. , Nakano Hiroyasu , Kurosawa Kyoko , Uchida Daisuke , Takeda Kiyoshi , Akira Shizuo , Matsumoto Mitsuru
Published: July 2002

Both nuclear factor (NF)-κB–inducing kinase (NIK) and inhibitor of κB (IκB) kinase (IKK) have been implicated as essential components for NF-κB activation in response to many external stimuli. However, the exact roles of NIK and IKKα in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKα in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin β receptor (LTβR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IκBα in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTβR stimulation induced NF-κB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKα-deficient mice. Consistent with the essential role of IKKα in LTβR signaling, we found that development of Peyer's patches was defective in IKKα-deficient mice. These results demonstrate that both NIK and IKKα are essential for the induction of NF-κB through LTβR, whereas the NIK–IKKα pathway is dispensable in TNFR-I signaling.
Differential Effects of Notch Ligands Delta-1 and Jagged-1 in Human Lymphoid Differentiation
Authors: Jaleco Ana C. , Neves Hélia , Hooijberg Erik , Gameiro Paula , Clode Nuno , Haury Matthias , Henrique Domingos , Parreira Leonor
Published: July 2002

Notch signaling is known to differentially affect the development of lymphoid B and T cell lineages, but it remains unclear whether such effects are specifically dependent on distinct Notch ligands. Using a cell coculture assay we observed that the Notch ligand Delta-1 completely inhibits the differentiation of human hematopoietic progenitors into the B cell lineage while promoting the emergence of cells with a phenotype of T cell/natural killer (NK) precursors. In contrast, Jagged-1 did not disturb either B or T cell/NK development. Furthermore, cells cultured in the presence of either Delta-1 or Jagged-1 can acquire a phenotype of NK cells, and Delta-1, but not Jagged-1, permits the emergence of a de novo cell population coexpressing CD4 and CD8. Our results thus indicate that distinct Notch ligands can mediate differential effects of Notch signaling and provide a useful system to further address cell-fate decision processes in lymphopoiesis.
Semiclassical limit of the nonlinear Schrödinger equation in small time
Authors: Grenier E.
Published: July 2002

We study the semi-classical limit of the nonlinear Schrödinger equation for initial data with Sobolev regularity, before shocks appear in the limit system, and in particular the validity of the WKB method.
On the continuity of the evaluation mapping associated with a group and its character group
Authors: Turnwald Gerhard
Published: July 2002

For an abelian Hausdorff group

G
G

, let

G

∗

G^{\ast }

denote the character group endowed with the compact-open topology and let

α

G

:
G
→

G

∗
∗

\alpha _{G}:G\rightarrow G^{\ast \ast }

denote the canonical homomorphism. We show that the evaluation mapping from

G

∗

×
G

G^{\ast }\times G

into the torus is continuous if and only if

G

∗

G^{\ast }

is locally compact and

α

G

\alpha _{G}

is continuous. If

α

G

\alpha _{G}

is injective and open, then the evaluation mapping is continuous if and only if

G
G

is locally compact. Several examples and counterexamples are given.
Two-level additive Schwarz preconditioners for nonconforming finite element methods
Authors: Brenner Susanne
Published: July 2002

Two-level additive Schwarz preconditioners are developed for the nonconforming P1 finite element approximation of scalar second-order symmetric positive definite elliptic boundary value problems, the Morley finite element approximation of the biharmonic equation, and the divergence-free nonconforming P1 finite element approximation of the stationary Stokes equations. The condition numbers of the preconditioned systems are shown to be bounded independent of mesh sizes and the number of subdomains in the case of generous overlap.
Macromolecular Uptake Is a Spontaneous Event during Mitosis in Cultured Fibroblasts: Implications for Vector-dependent Plasmid Transfection
Authors: Pellegrin Pierre , Fernandez Anne , Lamb Ned J. C. , Bennes René
Published: July 2002

The process through which macromolecules penetrate the plasma membrane of mammalian cells remains poorly defined. We have examined whether natural cellular events modulate the capacity of cells to take up agents applied extraneously. Herein, we report that during mitosis and in a cell type-independent manner, cells exhibit a natural ability to absorb agents present in the extracellular environment up to 150 kDa as assessed using fluorescein isothiocyanate-dextrans. This event is exclusive to the mitotic period and not observed during G0, G1, S, or G2 phase. During mitosis, starting in advanced prophase, oligonucleotides, active enzymes, and polypeptides are efficiently taken into mitotic cells. This uptake of macromolecules during mitosis still takes place in the presence of cytochalasin D or nocodazole, showing no requirement for intact microtubules or actin filaments in this process. However, cell rounding up, which still takes place in the presence of either of these drugs in mitotic cells, appears to be a key event in this process. Indeed, limited trypsinization of adherent cells mimics both the cell retraction and macromolecule uptake observed as cells enter mitosis. A plasmid DNA encoding green fluorescent protein (3.3Mda) coated with an 18 amino acid peptide is efficiently expressed when applied onto synchronized G2/M fibroblasts, whereas little or no expression is observed when the coated plasmid is applied onto asynchronous cell cultures. This shows that such coating peptides are only efficient for their encapsulating and protective effect on the plasmid DNA to be “vectorized” rather than acting as true vectors.
Dynamin-dependent Transferrin Receptor Recycling by Endosome-derived Clathrin-coated Vesicles
Authors: van Dam Ellen M. , Stoorvogel Willem
Published: July 2002

Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a temperature-sensitive mutant of dynamin-1 (dynamin-1G273D) or, as a control, dynamin-1 wild type. In dynamin-1G273D–expressing cells, 29–36% of endocytosed transferrin failed to recycle at the nonpermissive temperature and remained associated with tubular recycling endosomes. Sorting of endocytosed transferrin from fluid-phase endocytosed markers in early endosome antigen 1-labeled sorting endosomes was not inhibited. Dynamin-1G273Dassociated with accumulated clathrin-coated buds on extended tubular recycling endosomes. Brefeldin A interfered with the assembly of clathrin coats on endosomes and reduced the extent of transferrin recycling in control cells but did not further affect recycling by dynamin-1G273D–expressing cells. Together, these data indicate that the pathway from recycling endosomes to the plasma membrane is mediated, at least in part, by endosome-derived clathrin-coated vesicles in a dynamin-dependent manner.
Pairwise Assembly Determines the Intrinsic Potential for Self-Organization and Mechanical Properties of Keratin Filaments
Authors: Yamada Soichiro , Wirtz Denis , Coulombe Pierre A.
Published: July 2002

Most type I and II keratin genes are spatially and temporally regulated in a pairwise manner in epithelial tissues, where they represent the major structural proteins. Epithelia can be partitioned into simple (single-layered) and complex (multilayered) types. We compared the structural and mechanical properties of natural keratin polymers occurring in complex (K5-K14) and simple (K8-K18) epithelia. The intrinsic properties of these distantly related keratin filaments, whether dispersed or bundled in vitro, were surprisingly similar in all respects when at high polymer concentration. When type I and II assembly partners were switched to give rise to mismatched polymers (K5-K18; K8-K14), the interfilament interactions, which determine the structural and mechanical properties of keratin polymers, were significantly altered. We also show that a K5-K16 polymer exhibits lesser elasticity than K5- K14, which may help explain the inability of K16 to fully rescue the skin blistering characteristic of K14 null mice. The property of self-interaction exhibited by keratin filaments is likely to assist their function in vivo and may account for the relative paucity of cytoplasmic and keratin-specific cross-linkers. Our findings underscore the fundamental importance of pairwise polymerization and have implications for the functional significance of keratin sequence diversity.
AMADA: analysis of microarray data
Authors: Xia Xuhua , Xie Zheng
Published: July 2002

Summary: AMADA is a Windows program for identifying co-expressed genes from microarray data. It performs data transformation, principal component analysis, a variety of cluster analyses and extensive graphic functions for visualizing expression profiles.

Availability: http://web.hku.hk/~xxia/software/AMADA.htm, free.

Contact: xxia@hkusua.hku.hk
ENDscript: a workflow to display sequence and structure information
Authors: Gouet Patrice , Courcelle Emmanuel
Published: July 2002

Summary: ENDscript is a web server grouping popular programs such as BLAST, Multalin and DSSP. It uses as query the co-ordinates file of a protein in Protein Data Bank format and generates PostScript and png figures showing: residues conserved after a multiple alignment against homologous sequences, secondary structure elements, accessibility, hydropathy and intermolecular contacts. Thus, the user can relate quickly 1D, 2D and 3D information of a protein of known structure.

Availability: http://genopole.toulouse.inra.fr/ENDscript

Contact: p.gouet@ibcp.fr

* To whom correspondence should be addressed.
The TGF‐β family member derrière is involved in regulation of the establishment of left–right asymmetry
Authors: Hanafusa Hiroshi , Masuyama Norihisa , Kusakabe Morioh , Shibuya Hiroshi , Nishida Eisuke
Published: July 2002

Although a number of genes that are involved in the establishment of left–right asymmetry have been identified, earlier events in the molecular pathway developing left–right asymmetry remain to be elucidated. Here we present evidence suggesting that the transforming growth factor‐β family member derrière is involved in the development of left–right asymmetry in Xenopus embryos. Ectopic expression of derrière on the right side can fully invert cardiac and visceral left–right orientation and nodal expression, and expression of a dominant‐negative form of derrière on the left side can partially randomize the left–right orientation and nodal expression. Moreover, while expression of the dominant‐negative derrière does not inhibit the activity of Vg1 directly, it can rescue the altered left–right orientation induced by Vg1. Vg1 can induce derrière in animal cap explants. These results suggest that derrière is involved in earlier molecular pathways developing the left–right asymmetry.
Peptidyl‐prolyl isomerases: a new twist to transcription
Authors: Shaw Peter E
Published: July 2002

Peptidyl‐prolyl isomerases (PPIs) catalyse the cis‐trans isomerisation of peptide bonds N‐terminal to proline residues in polypeptide chains. They have roles in the folding of newly synthesised proteins and in the function of the immune system. In addition, members of the parvulin‐like family of PPIs have been implicated in cell cycle control. Their activity is directed by the prior phosphorylation of target proteins in both yeast and mammalian cells. More recent data have illustrated that they may also influence other nuclear events. This review examines PPI activity in the context of eukaryotic transcriptional regulation. The findings are consistent with a two‐step model of conformational control, in which the outcome depends on the transcription factor involved.
Non-respiratory blood vessels in Latimeria gill filaments
Authors: Vogel W. O. P. , Hughes G. M. , Mattheus U.
Published: July 2002

A study of the blood pathways within the gills ofLatimeriahas been carried out using light and transmission electron microscopy. Clear evidence has been found for the presence of a secondary non–respiratory circulation in addition to the well–established respiratory pathway through the gill lamellae. All essential components of this system have been observed and have the same relationships and basic structure as comparable secondary systems in actinopterygian and elasmobranch fishes. These include a central venous sinus (CVS), arterio–venous anastomoses (AVAs) and central filament arteries (CFAs). AVAs connect both arterial vessels of the primary circulation and CFAs of the secondary circulation to the CVS. The latter contained many red blood cells. The presence of this secondary circulation inLatimeriagills contrasts with the situation in the gills of the three living genera of lungfishes where a system possessing the essential features of the tetrapod lymphatic vessel system has been recognized. No suggestions of a true lymphatic vessel system were observed inLatimeria. Other features of gill and vascular anatomy inLatimeriashow its closer relationship to dipnoans than other groups of living fishes but evidence derived from this study of the secondary circulation clearly supports the view that the Dipnoi rather thanLatimeriarepresent the living fishes most closely related to the tetrapods.
The population genetics ofTrypanosoma bruceiand the origin of human infectivity
Authors: MacLeod Annette , Tait Andy , Turner C. Michel R.
Published: July 2002

The African trypanosome,Trypanosoma brucei, is a zoonotic parasite transmitted by tsetse flies. Two of the three subspecies,T. brucei gambienseandT.b. rhodesiense, cause sleeping sickness in humans whereas the third subspecies,T.b. brucei, is not infective to humans. We propose that the key to understanding genetic relationships within this species is the analysis of gene flow to determine the importance of genetic exchange within populations and the relatedness of populations.T.bruceiparasites undergo genetic exchange when present in infections of mixed genotypes in tsetse flies in the laboratory, although this is not an obligatory process. Infections of mixed genotype are surprisingly common in field isolates from tsetse flies such that there is opportunity for genetic exchange to occur. Population genetic analyses, taking into account geographical and host species of origin, show that genetic exchange occurs sufficiently frequently in the field to be an important determinant of genetic diversity, except where particular clones have acquired the ability to infect humans. Thus,T. bruceipopulations have an ‘epidemic’ genetic structure, but the better-characterized human-infective populations have a ‘clonal’ structure. Remarkably, the ability to infect humans appears to have arisen on multiple occasions in different geographical locations in sub-Saharan Africa. Our data indicate that the classical subspecies terminology forT. bruceiis genetically inappropriate. It is an implicit assumption in most infectious disease biology that when a zoonotic pathogen acquires the capability to infect humans, it does so once and then spreads through the human population from that single-source event. For at least one major pathogen in tropical medicine,T. brucei, this assumption is invalid.
LAF1, a MYB transcription activator for phytochrome A signaling
Authors: Ballesteros Marı́a L. , Bolle Cordelia , Lois Luisa M. , Moore James M. , Vielle-Calzada Jean-Philippe , Grossniklaus Ueli , Chua Nam-Hai
Published: July 2002

The photoreceptor phytochrome (phy) A has a well-defined role in regulating gene expression in response to specific light signals. Here, we describe a new Arabidopsis mutant, laf1(long after far-red light1) that has an elongated hypocotyl specifically under far-red light. Gene expression studies showed that laf1 has reduced responsiveness to continuous far-red light but retains wild-type responses to other light wavelengths. As far-red light is only perceived by phyA, our results suggest that LAF1 is specifically involved in phyA signal transduction. Further analyses revealed thatlaf1 is affected in a subset of phyA-dependent responses and the phenotype is more severe at low far-red fluence rates. LAF1encodes a nuclear protein with strong homology with the R2R3–MYB family of DNA-binding proteins. Experiments using yeast cells identified a transactivation domain in the C-terminal portion of the protein. LAF1 is constitutively targeted to the nucleus by signals in its N-terminal portion, and the full-length protein accumulates in distinct nuclear speckles. This accumulation in speckles is abolished by a point mutation in a lysine residue (K258R), which might serve as a modification site by a small ubiquitin-like protein (SUMO).