bluuebunny/crossref_metadata_2025 huggingface dataset:
2B4, the Natural Killer and T Cell Immunoglobulin Superfamily Surface Protein, Is a Ligand for CD48
Authors: Brown Marion H. , Boles Kent , Anton van der Merwe P. , Kumar Vinay , Mathew Porunelloor A. , Neil Barclay A.
Published: July 2002

2B4 is a cell surface glycoprotein related to CD2 and implicated in the regulation of natural killer and T lymphocyte function. A recombinant protein containing the extracellular region of mouse (m)2B4 attached to avidin-coated fluorescent beads bound to rodent cells, and binding was completely blocked by CD48 monoclonal antibodies (mAbs). Using surface plasmon resonance, we showed that purified soluble mCD48 bound m2B4 with a six- to ninefold higher affinity (Kd ≈ 16 μM at 37°C) than its other ligand, CD2. Human CD48 bound human 2B4 with a similar affinity (Kd ≈ 8 μM). The finding of an additional ligand for CD48 provides an explanation for distinct functional effects observed on perturbing CD2 and CD48 with mAbs or by genetic manipulation.
T Cell Receptor (TCR) Interacting Molecule (TRIM), A Novel Disulfide-linked Dimer Associated with the TCR–CD3–ζ Complex, Recruits Intracellular Signaling Proteins to the Plasma Membrane
Authors: Bruyns Eddy , Marie-Cardine Anne , Kirchgessner Henning , Sagolla Karin , Shevchenko Andrej , Mann Matthias , Autschbach Frank , Bensussan Armand , Meuer Stefan , Schraven Burkhart
Published: July 2002

The molecular mechanisms regulating recruitment of intracellular signaling proteins like growth factor receptor–bound protein 2 (Grb2), phospholipase Cγ1, or phosphatidylinositol 3-kinase (PI3-kinase) to the plasma membrane after stimulation of the T cell receptor (TCR)– CD3–ζ complex are not very well understood. We describe here purification, tandem mass spectrometry sequencing, molecular cloning, and biochemical characterization of a novel transmembrane adaptor protein which associates and comodulates with the TCR–CD3–ζ complex in human T lymphocytes and T cell lines. This protein was termed T cell receptor interacting molecule (TRIM). TRIM is a disulfide-linked homodimer which is comprised of a short extracellular domain of 8 amino acids, a 19–amino acid transmembrane region, and a 159–amino acid cytoplasmic tail. In its intracellular domain, TRIM contains several tyrosine-based signaling motifs that could be involved in SH2 domain–mediated protein–protein interactions. Indeed, after T cell activation, TRIM becomes rapidly phosphorylated on tyrosine residues and then associates with the 85-kD regulatory subunit of PI3-kinase via an YxxM motif. Thus, TRIM represents a TCR-associated transmembrane adaptor protein which is likely involved in targeting of intracellular signaling proteins to the plasma membrane after triggering of the TCR.
Dual Signaling of the Fas Receptor: Initiation of Both Apoptotic and Necrotic Cell Death Pathways
Authors: Vercammen Dominique , Brouckaert Greet , Denecker Geertrui , Van de Craen Marc , Declercq Wim , Fiers Walter , Vandenabeele Peter
Published: July 2002

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, tetrapeptide inhibitors of caspase-1– and caspase-3–like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas–induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor κB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.
A Human Immunoglobulin (Ig)A Cα3 Domain Motif Directs Polymeric Ig Receptor–mediated Secretion
Authors: Hexham J. Mark , White Kendra D. , Carayannopoulos Leonidas N. , Mandecki Wlodeck , Brisette Renee , Yang Yih-Sheng , Capra J. Donald
Published: July 2002

Polymeric immunoglobulins provide immunological protection at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR). Using a panel of human IgA1/IgG1 constant region “domain swap” mutants, the binding site for the pIgR on dimeric IgA (dIgA) was localized to the Cα3 domain. Selection of random peptides for pIgR binding and comparison with the IgA sequence suggested amino acids 402–410 (QEPSQGTTT), in a predicted exposed loop of the Cα3 domain, as a potential binding site. Alanine substitution of two groups of amino acids in this area abrogated the binding of dIgA to pIgR, whereas adjacent substitutions in a β-strand immediately NH2-terminal to this loop had no effect. All pIgR binding IgA sequences contain a conserved three amino acid insertion, not present in IgG, at this position. These data localize the pIgR binding site on dimeric human IgA to this loop structure in the Cα3 domain, which directs mucosal secretion of polymeric antibodies. We propose that it may be possible to use a pIgR binding motif to deliver antigen-specific dIgA and small-molecule drugs to mucosal epithelia for therapy.
ZAP-70 Protein Promotes Tyrosine Phosphorylation of T Cell Receptor Signaling Motifs (ITAMs) in Immature CD4+8+ Thymocytes with Limiting p56lck
Authors: Ashe Jennifer M. , Wiest David L. , Abe Ryo , Singer Alfred
Published: July 2002

As a result of interaction with epithelial cells in the thymic cortex, immature CD4+8+ (double positive, DP) thymocytes express relatively few T cell receptors (TCRs) and contain diminished numbers of coreceptor-associated p56lck (lck) PTK molecules. As a result, TCR signal transduction in DP thymocytes is significantly impaired, despite its importance for repertoire selection. We report here that, in DP thymocytes, tyrosine phosphorylation of TCR signaling motifs (ITAMs) by lck, an early event in TCR signal transduction, is dependent upon ZAP-70 protein independent of ZAP-70's kinase activity. Furthermore, the dependence on ZAP-70 protein for ITAM phosphorylation diminishes as available lck increases. Importantly, ZAP-70's role in ITAM phosphorylation in DP thymocytes is not limited to protecting phosphorylated ITAMs from dephosphorylation. Rather, this study indicates that ZAP-70 protein augments ITAM phosphorylation in DP thymocytes and so compensates in part for the relative deficiency of coreceptor-associated lck.
Itm2a Is Induced during Thymocyte Selection and T Cell Activation and Causes Downregulation of Cd8 When Overexpressed in Cd4+Cd8+ Double Positive Thymocytes
Authors: Kirchner Jacqueline , Bevan Michael J.
Published: July 2002

To identify novel genes that are involved in positive selection of thymocytes, we performed polymerase chain reaction (PCR)-based subtractive hybridization between selecting and nonselecting thymi. OT-1 T cell receptor (TCR) transgenic thymocytes on a recombination activating gene (RAG) null background are efficiently selected into the CD8 lineage in H-2b mice (RAG-2−/−OT-1, selecting thymi), but are not selected on a transporter associated with antigen processing (TAP) null background (RAG-2−/−TAP-1−/−OT-1, nonselecting thymi). We report here our studies of one gene, ITM2A, whose expression is dramatically higher in T cells in the selecting thymus. The expression pattern of ITM2A in thymocyte subsets correlates with upregulation during positive selection. In addition, ITM2A expression is higher in the thymus than in either the spleen or lymph nodes, but can be upregulated in peripheral T cells upon activation. ITM2A expression was also induced in RAG-2−/− thymocytes in vivo upon CD3 cross-linking. We demonstrate that ITM2A is a type II membrane glycoprotein that exists as two species with apparent Mr of 45 and 43 kD and appears to localize primarily to large cytoplasmic vesicles and the Golgi apparatus, but is also expressed on the cell surface. Expression on the surface of EL4 cells increases with activation by phorbol myristate acetate (PMA) and ionomycin. Finally, overexpression of ITM2A under control of the lck proximal promoter in mice results in partial downregulation of CD8 in CD4+CD8+ double positive (DP) thymocytes, and a corresponding increase in the number of CD4+CD8lo thymocytes. Possible roles for this novel activation marker in thymocyte development are discussed.
Fibroblasts as Host Cells in Latent Leishmaniosis
Authors: Bogdan Christian , Donhauser Norbert , Döring Reinhard , Röllinghoff Martin , Diefenbach Andreas , Rittig Michael G.
Published: July 2002

Intracellular parasites are known to persist lifelong in mammalian hosts after the clinical cure of the disease, but the mechanisms of persistence are poorly understood. Here, we show by confocal laser microscopy that in the draining lymph nodes of mice that had healed a cutaneous infection with Leishmania major, 40% of the persisting parasites were associated with fibroblasts forming the reticular meshwork of the lymph nodes. In vitro, both promastigotes and amastigotes of L. major infected primary skin or lymph node fibroblasts. Compared with macrophages, cytokine-activated fibroblasts had a reduced ability to express type 2 nitric oxide synthase and to kill intracellular L. major. These data identify fibroblasts as an important host cell for Leishmania during the chronic phase of infection and suggest that they might serve as safe targets for the parasites in clinically latent disease.
Negative Regulation of Phagocytosis in Murine Macrophages by the Src Kinase Family Member, Fgr
Authors: Gresham Hattie D. , Dale Benjamin M. , Potter Jeffrey W. , Chang Peter W. , Vines Charlotte M. , Lowell Clifford A. , Lagenaur Carl F. , Willman Cheryl L.
Published: July 2002

Ingestion of opsonized pathogens by professional phagocytes results in the generation and release of microbicidal products that are essential for normal host defense. Because these products can result in significant tissue injury, phagocytosis must be regulated to limit damage to the host while allowing for optimal clearance and destruction of opsonized pathogens. To pursue negative regulation of phagocytosis, we assessed the effect of the Src kinase family member, Fgr, on opsonin-dependent phagocytosis by mouse macrophages. We chose Fgr because it is present in high concentrations in circulating phagocytes but is not essential for Fcγ receptor–mediated ingestion by mouse macrophages. Although expression of Fgr both in a macrophage cell line and in primary macrophages significantly attenuates ingestion mediated by Fcγ receptors and CR3, it does not affect macropinocytosis or receptor-mediated endocytosis. This selective effect of Fgr is independent of its tyrosine kinase function. After Fcγ receptor cross-linking, Fgr becomes associated with the immunoreceptor tyrosine-based inhibition motif (ITIM)–containing receptor, SIRPα (a member of the signal-regulatory protein family, also known as Src homology 2 domain–containing protein tyrosine phosphatase [SHP] substrate 1 [SHPS-1], brain immunoglobulin-like molecule with tyrosine-based activation motifs [BIT], and P84) and potentiates the association of the phosphatase SHP-1 with SIRPα. This association is responsible, at least in part, for decreasing positive signaling essential for optimal phagocytosis. These data demonstrate an important negative regulatory role for this Src kinase family member and suggest that this homeostatic function must be overcome for optimal uptake and clearance of opsonized pathogens.
P-Selectin Glycoprotein Ligand 1 (Psgl-1) Is Expressed on Platelets and Can Mediate Platelet–Endothelial Interactions in Vivo
Authors: Frenette Paul S. , Denis Cécile V. , Weiss Linnea , Jurk Kerstin , Subbarao Sangeetha , Kehrel Beate , Hartwig John H. , Vestweber Dietmar , Wagner Denisa D.
Published: July 2002

The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin–immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin–bound ligands were eluted with ethylenediaminetetraacetic acid. An ∼210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti–human PSGL-1 antibody and could be blotted with P-selectin–IgG. Under reducing conditions, both the predicted PSGL-1 ∼210-kD dimer and the ∼120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25–100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti–PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.
Inflammatory Responses Induced by the Filarial Nematode Brugia malayi Are Mediated by Lipopolysaccharide-like Activity from Endosymbiotic Wolbachia Bacteria
Authors: Taylor Mark J. , Cross Helen F. , Bilo Katja
Published: July 2002

The pathogenesis of filarial disease is characterized by acute and chronic inflammation. Inflammatory responses are thought to be generated by either the parasite, the immune response, or opportunistic infection. We show that soluble extracts of the human filarial parasite Brugia malayi can induce potent inflammatory responses, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and nitric oxide (NO) from macrophages. The active component is heat stable, reacts positively in the Limulus amebocyte lysate assay, and can be inhibited by polymyxin B. TNF-α, IL-1β, and NO responses were not induced in macrophages from lipopolysaccharide (LPS)-nonresponsive C3H/HeJ mice. The production of TNF-α after chemotherapy of microfilariae was also only detected in LPS-responsive C3H/HeN mice, suggesting that signaling through the Toll-like receptor 4 (TLR4) is necessary for these responses. We also show that CD14 is required for optimal TNF-α responses at low concentrations. Together, these results suggest that extracts of B. malayi contain bacterial LPS. Extracts from the rodent filaria, Acanthocheilonema viteae, which is not infected with the endosymbiotic Wolbachia bacteria found in the majority of filarial parasites, failed to induce any inflammatory responses from macrophages, suggesting that the source of bacterial LPS in extracts of B. malayi is the Wolbachia endosymbiont. Wolbachia extracts derived from a mosquito cell line induced similar LPS-dependent TNF-α and NO responses from C3H/HeN macrophages, which were eliminated after tetracycline treatment of the bacteria. Thus, Wolbachia LPS may be one of the major mediators of inflammatory pathogenesis in filarial nematode disease.
High Mobility Group 1 Protein (Hmg-1) Stimulates Proinflammatory Cytokine Synthesis in Human Monocytes
Authors: Andersson Ulf , Wang Haichao , Palmblad Karin , Aveberger Ann-Charlotte , Bloom Ona , Erlandsson-Harris Helena , Janson Alfred , Kokkola Riikka , Zhang Minghuang , Yang Huan , Tracey Kevin J.
Published: July 2002

Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a “late” mediator of endotoxin lethality. Anti–HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1α, IL-1β, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, and MIP-1β; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1–induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.
Lymphocyte Cc Chemokine Receptor 9 and Epithelial Thymus-Expressed Chemokine (Teck) Expression Distinguish the Small Intestinal Immune Compartment
Authors: Kunkel Eric J. , Campbell James J. , Haraldsen Guttorm , Pan Junliang , Boisvert Judie , Roberts Arthur I. , Ebert Ellen C. , Vierra Mark A. , Goodman Stuart B. , Genovese Mark C. , Wardlaw Andy J. , Greenberg Harry B. , Parker Christina M. , Butcher Eugene C. , Andrew David P. , Agace William W.
Published: July 2002

The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4+ and CD8+ T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9+, and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9−. TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses.
Reactive Oxygen Species (Ros-Induced) Ros Release
Authors: Zorov Dmitry B. , Filburn Charles R. , Klotz Lars-Oliver , Zweier Jay L. , Sollott Steven J.
Published: July 2002

We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, ΔΨ. This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein (∼620 daltons) concurrently with depolarization. These photodynamically produced “triggering” ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial “ROS-induced ROS release” (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.
Apoptosis Control in Syncytia Induced by the HIV Type 1–Envelope Glycoprotein Complex
Authors: Ferri Karine F. , Jacotot Etienne , Blanco Julià , Esté José A. , Zamzami Naoufal , Susin Santos A. , Xie Zhihua , Brothers Greg , Reed John C. , Penninger Josef M. , Kroemer Guido
Published: July 2002

Syncytia arising from the fusion of cells expressing a lymphotropic HIV type 1–encoded envelope glycoprotein complex (Env) with cells expressing the CD4/CXC chemokine receptor 4 complex spontaneously undergo cell death. Here we show that this process is accompanied by caspase activation and signs of mitochondrial membrane permeabilization (MMP), including the release of intermembrane proteins such as cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF) from mitochondria. In Env-induced syncytia, caspase inhibition did not suppress AIF- and Cyt-c translocation, yet it prevented all signs of nuclear apoptosis. Translocation of Bax to mitochondria led to MMP, which was inhibited by microinjected Bcl-2 protein or bcl-2 transfection. Bcl-2 also prevented the subsequent nuclear chromatin condensation and DNA fragmentation. The release of AIF occurred before that of Cyt-c and before caspase activation. Microinjection of AIF into syncytia sufficed to trigger rapid, caspase-independent Cyt-c release. Neutralization of endogenous AIF by injection of an antibody prevented all signs of spontaneous apoptosis occurring in syncytia, including the Cyt-c release and nuclear apoptosis. In contrast, Cyt-c neutralization only prevented nuclear apoptosis, and did not affect AIF release. Our results establish that the following molecular sequence governs apoptosis of Env-induced syncytia: Bax-mediated/Bcl-2–inhibited MMP → AIF release → Cyt-c release → caspase activation → nuclear apoptosis.
Human Placental Cytotrophoblasts Attract Monocytes and Cd56bright Natural Killer Cells via the Actions of Monocyte Inflammatory Protein 1α
Authors: Drake Penelope M. , Gunn Michael D. , Charo Israel F. , Tsou Chia-Lin , Zhou Yan , Huang Ling , Fisher Susan J.
Published: July 2002

During human pregnancy, the specialized epithelial cells of the placenta (cytotrophoblasts) come into direct contact with immune cells in several locations. In the fetal compartment of the placenta, cytotrophoblast stem cells lie adjacent to macrophages (Hofbauer cells) that reside within the chorionic villus stroma. At sites of placental attachment to the mother, invasive cytotrophoblasts encounter specialized maternal natural killer (NK) cells (CD56bright), macrophages, and T cells that accumulate within the uterine wall during pregnancy. Here we tested the hypothesis that fetal cytotrophoblasts can direct the migration of these maternal immune cells. First, we assayed the chemotactic activity of cytotrophoblast conditioned medium samples, using human peripheral blood mononuclear cells as targets. The placental samples preferentially attracted NK cells (both CD56dim and CD56bright), monocytes, and T cells, suggesting that our hypothesis was correct. A screen to identify chemokine activity through the induction of a Ca2+ flux in cells transfected with individual chemokine receptors suggested that cytotrophoblasts secreted monocyte inflammatory protein (MIP)-1α. This was confirmed by localizing the corresponding mRNA and protein, both in vitro and in vivo. MIP-1α protein in conditioned medium was further characterized by immunoblotting and enzyme-linked immunosorbent assay. Immunodepletion of MIP-1α from cytotrophoblast conditioned medium showed that this chemokine was responsible for a significant portion of the induced monocyte and CD56bright NK cell chemotax-is. These data suggest the specific conclusion that cytotrophoblasts can attract monocytes and CD56bright NK cells by producing MIP-1α and the more general hypothesis that these cells may organize and act on leukocytes at the maternal–fetal interface.
Secreted Lymphotoxin-α Is Essential for the Control of an Intracellular Bacterial Infection
Authors: Roach Daniel R. , Briscoe Helen , Saunders Bernardette , France Malcolm P. , Riminton Sean , Britton Warwick J.
Published: July 2002

Although the essential role of tumor necrosis factor (TNF) in the control of intracellular bac-terial infection is well established, it is uncertain whether the related cytokines lymphotoxin-α (LTα3) and lymphotoxin-β (LTβ) have independent roles in this process. Using C57Bl/6 mice in which the genes for these cytokines have been disrupted, we have examined the relative contribution of secreted LTα3 and membrane-bound LTβ in the host response to aerosol Mycobacterium tuberculosis infection. To overcome the lack of peripheral lymph nodes in LTα−/− and LTβ−/− mice, bone marrow chimeric mice were constructed. LTα−/− chimeras, which lack both secreted LTα3 and membrane-bound LTβ (LTα1β2 and LTα2β1), were highly susceptible and succumbed 5 wk after infection. LTβ−/− chimeras, which lack only the membrane-bound LTβ, controlled the infection in a comparable manner to wild-type (WT) chimeric mice. T cell responses to mycobacterial antigens and macrophage responses in LTα−/− chimeras were equivalent to those of WT chimeras, but in LTα−/− chimeras, granuloma formation was abnormal. LTα−/− chimeras recruited normal numbers of T cells into their lungs, but the lymphocytes were restricted to perivascular and peribronchial areas and were not colocated with macrophages in granulomas. Therefore, LTα3 is essential for the control of pulmonary tuberculosis, and its critical role lies not in the activation of T cells and macrophages per se but in the local organization of the granulomatous response.
Control of Mitochondrial Membrane Permeabilization by Adenine Nucleotide Translocator Interacting with HIV-1 Viral Protein R and Bcl-2
Authors: Jacotot Etienne , Ferri Karine F. , El Hamel Chahrazed , Brenner Catherine , Druillennec Sabine , Hoebeke Johan , Rustin Pierre , Métivier Didier , Lenoir Christine , Geuskens Maurice , Vieira Helena L.A. , Loeffler Markus , Belzacq Anne-Sophie , Briand Jean-Paul , Zamzami Naoufal , Edelman Lena , Xie Zhi Hua , Reed John C. , Roques Bernard P. , Kroemer Guido
Published: July 2002

Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein–protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96–induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT–Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT–Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.
HIV-1 Vpr Enhances Viral Burden by Facilitating Infection of Tissue Macrophages but Not Nondividing CD4+ T Cells
Authors: Eckstein Daniel A. , Sherman Michael P. , Penn Michael L. , Chin Peggy S. , De Noronha Carlos M.C. , Greene Warner C. , Goldsmith Mark A.
Published: July 2002

Prior experiments in explants of human lymphoid tissue have demonstrated that human immunodeficiency virus type 1 (HIV-1) productively infects diverse cellular targets including T cells and tissue macrophages. We sought to determine the specific contribution of macrophages and T cells to the overall viral burden within lymphoid tissue. To block infection of macrophages selectively while preserving infection of T cells, we used viruses deficient for viral protein R (Vpr) that exhibit profound replication defects in nondividing cells in vitro. We inoculated tonsil histocultures with matched pairs of congenic viruses that differed only by the presence of a wild-type or truncated vpr gene. Although these viruses exhibited no reduction in the infection or depletion of T cells, the ability of the Vpr-deficient R5 virus to infect tissue macrophages was severely impaired compared with matched wild-type R5 virus. Interestingly, the Vpr-deficient R5 virus also exhibited a 50% reduction in overall virus replication compared with its wild-type counterpart despite the fact that macrophages represent a small fraction of the potential targets of HIV-1 infection in these tissues. Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1.
Cd47 (Integrin-Associated Protein) Engagement of Dendritic Cell and Macrophage Counterreceptors Is Required to Prevent the Clearance of Donor Lymphohematopoietic Cells
Authors: Blazar Bruce R. , Lindberg Frederik P. , Ingulli Elizabeth , Panoskaltsis-Mortari Angela , Oldenborg Per-Arne , Iizuka Koho , Yokoyama Wayne M. , Taylor Patricia A.
Published: July 2002

Integrin-associated protein (CD47) is a broadly expressed protein that costimulates T cells, facilitates leukocyte migration, and inhibits macrophage scavenger function. To determine the role of CD47 in regulating alloresponses, CD47+/+ or CD47−/− T cells were infused into irradiated or nonconditioned major histocompatibility complex disparate recipients. Graft-versus-host disease lethality was markedly reduced with CD47−/− T cells. Donor CD47−/− T cells failed to engraft in immunodeficient allogeneic recipients. CD47−/− marrow was unable to reconstitute heavily irradiated allogeneic or congenic immune–deficient CD47+/+ recipients. These data suggested that CD47−/− T cells and marrow cells were cleared by the innate immune system. To address this hypothesis, dye-labeled CD47−/− and CD47+/+ lymphocytes or marrow cells were infused in vivo and clearance was followed. Dye-labeled CD47−/− cells were engulfed by splenic dendritic cells and macrophages resulting in the clearance of virtually all CD47−/− lymphohematopoietic cells within 1 day after infusion. Host phagocyte-depleted CD47+/+ recipients partially accepted allogeneic CD47−/− T cells. Thus, dendritic cells and macrophages clear lymphohematopoietic cells that have downregulated CD47 density. CD47 expression may be a critical indicator for determining whether lymphohematopoietic cells will survive or be cleared.
Isolation and Characterization of Dermal Lymphatic and Blood Endothelial Cells Reveal Stable and Functionally Specialized Cell Lineages
Authors: Kriehuber Ernst , Breiteneder-Geleff Silvana , Groeger Marion , Soleiman Afschin , Schoppmann Sebastian F. , Stingl Georg , Kerjaschki Dontscho , Maurer Dieter
Published: July 2002

A plexus of lymphatic vessels guides interstitial fluid, passenger leukocytes, and tumor cells toward regional lymph nodes. Microvascular endothelial cells (ECs) of lymph channels (LECs) are difficult to distinguish from those of blood vessels (BECs) because both express a similar set of markers, such as CD31, CD34, podocalyxin, von Willebrand factor (vWF), etc. Analysis of the specific properties of LECs was hampered so far by lack of tools to isolate LECs. Recently, the 38-kD mucoprotein podoplanin was found to be expressed by microvascular LECs but not BECs in vivo. Here we isolated for the first time podoplanin+ LECs and podoplanin− BECs from dermal cell suspensions by multicolor flow cytometry. Both EC types were propagated and stably expressed VE-cadherin, CD31, and vWF. Molecules selectively displayed by LECs in vivo, i.e., podoplanin, the hyaluronate receptor LYVE-1, and the vascular endothelial cell growth factor (VEGF)-C receptor, fms-like tyrosine kinase 4 (Flt-4)/VEGFR-3, were strongly expressed by expanded LECs, but not BECs. Conversely, BECs but not LECs expressed VEGF-C. LECs as well as BECs formed junctional contacts with similar molecular composition and ultrastructural features. Nevertheless, the two EC types assembled in vitro in vascular tubes in a strictly homotypic fashion. This EC specialization extends to the secretion of biologically relevant chemotactic factors: LECs, but not BECs, constitutively secrete the CC chemokine receptor (CCR)7 ligand secondary lymphoid tissue chemokine (SLC)/CCL21 at their basal side, while both subsets, upon activation, release macrophage inflammatory protein (MIP)-3α/CCL20 apically. These results demonstrate that LECs and BECs constitute stable and specialized EC lineages equipped with the potential to navigate leukocytes and, perhaps also, tumor cells into and out of the tissues.
Targeted Deletion of a High-Affinity GATA-binding Site in the GATA-1 Promoter Leads to Selective Loss of the Eosinophil Lineage In Vivo
Authors: Yu Channing , Cantor Alan B. , Yang Haidi , Browne Carol , Wells Richard A. , Fujiwara Yuko , Orkin Stuart H.
Published: July 2002

Transcription factor GATA-1 reprograms immature myeloid cells to three different hematopoietic lineages-erythroid cells, megakaryocytes, and eosinophils. GATA-1 is essential for maturation of erythroid and megakaryocytic precursors, as revealed by gene targeting in mice. Here we demonstrate that deletion of a high-affinity GATA-binding site in the GATA-1 promoter, an element presumed to mediate positive autoregulation of GATA-1 expression, leads to selective loss of the eosinophil lineage. These findings suggest that GATA-1 is required for specification of this lineage during hematopoietic development. Mice lacking the ability to produce eosinophils should prove useful in ascertaining the role of eosinophils in a variety of inflammatory or allergic disorders.
An Evaluation Cost Model of Consideration Sets
Authors: Hauser John R. , Wernerfelt Birger
Published: July 2002

If utility (net of price) varies by consumption occasion, the consideration set of a rational consumer will represent trade-offs between decision costs and the incremental benefits of choosing from a larger set of brands. If evaluating a brand decreases biases and uncertainty in perceived utility, the decision to evaluate a brand for inclusion in a consideration set is different from the decision to consider an evaluated brand. The decision to consume is, in turn, different from the decision to consider. This article provides analytical expressions for these decision criteria and presents four aggregate implications of the model: (1 ) distributions of consideration set sizes, (2) order-of-entry penalties, (3) dynamic advertising response, and (4) competitive promotion intensity.
The Hausdorff dimension of graphs of Weierstrass functions
Authors: Hunt Brian
Published: July 2002

The Weierstrass nowhere differentiable function, and functions constructed from similar infinite series, have been studied often as examples of functions whose graph is a fractal. Though there is a simple formula for the Hausdorff dimension of the graph which is widely accepted, it has not been rigorously proved to hold. We prove that if arbitrary phases are included in each term of the summation for the Weierstrass function, the Hausdorff dimension of the graph of the function has the conjectured value for almost every sequence of phases. The argument extends to a much wider class of Weierstrass-like functions.
The iteration formula of the Maslov-type index theory with applications to nonlinear Hamiltonian systems
Authors: Dong Di , Long Yiming
Published: July 2002

In this paper, the iteration formula of the Maslov-type index theory for linear Hamiltonian systems with continuous, periodic, and symmetric coefficients is established. This formula yields a new method to determine the minimality of the period for solutions of nonlinear autonomous Hamiltonian systems via their Maslov-type indices. Applications of this formula give new results on the existence of periodic solutions with prescribed minimal period for such systems, and unify known results under various convexity conditions.
Stability of multiple-pulse solutions
Authors: Sandstede Björn
Published: July 2002

In this article, stability of multiple-pulse solutions in semilinear parabolic equations on the real line is studied. A system of equations is derived which determines stability of

N
N

-pulses bifurcating from a stable primary pulse. The system depends only on the particular bifurcation leading to the existence of the

N
N

-pulses.

As an example, existence and stability of multiple pulses are investigated if the primary pulse converges to a saddle-focus. It turns out that under suitable assumptions infinitely many

N
N

-pulses bifurcate for any fixed

N
>
1

N>1

. Among them are infinitely many stable ones. In fact, any number of eigenvalues between 0 and

N
−
1

N-1

in the right half plane can be prescribed.
Why the characteristic polynomial factors
Authors: Sagan Bruce
Published: July 2002

We survey three methods for proving that the characteristic polynomial of a finite ranked lattice factors over the nonnegative integers and indicate how they have evolved recently. The first technique uses geometric ideas and is based on Zaslavsky’s theory of signed graphs. The second approach is algebraic and employs results of Saito and Terao about free hyperplane arrangements. Finally we consider a purely combinatorial theorem of Stanley about supersolvable lattices and its generalizations.
Wood dust exposure in the Danish furniture industry using conventional and passive monitors
Authors: Schlünssen Vivi , Vinzents Peter S. , Mikkelsen Anders B. , Schaumburg Inger
Published: July 2002

A study of wood dust exposure at furniture factories in one county in Denmark was performed as a cross sectional study. Dust exposure was measured with personal passive dust monitors and calibrated against active sampling on filters. Measurements of 1685 workers were included in the exposure assessment. The passive dust monitor conversion models for equivalent concentrations of inhalable dust and total dust based on data from the present study were not significantly different from the original models. Therefore models based on all available data were used. The parameters of the distribution of equivalent concentration of inhalable dust were 0.94 mg/m3 (geometric mean) and 2.10 (geometric standard deviation). Compared with a national cross sectional study from 1988 the exposure level (geometric mean) was reduced by a factor 2.0. Inhalable dust exposure was about 50% higher than exposure measured by the Danish ‘total’ dust method.
Xlandscape: the graphical display of word frequencies in sequences.
Authors: Levy S , Compagnoni L , Myers E W , Stormo G D
Published: July 2002

MOTIVATION: To provide a graphical interface for the generation, display and manipulation of a sequence landscape that will run on all X-windows-based Unix workstations. RESULTS: The sequence landscape approach enables the representation of the frequency of occurrence of all query sequence sub-words within a database. The landscape approach can detect tandem and other repeating word motifs, specific sub-words that are over-represented words in a particular database using Markov probability and the preference for sub-words belonging to either one of two databases. All these features aid in the classification of a query sequence. Given the open-text format for sequences and databases, the Xlandscape tool can be applied to a wide range of problems.
A new criterion to classify globular proteins based on their secondary structure contents.
Authors: Zhang C T , Zhang R
Published: July 2002

MOTIVATION: With the enlargement of protein structure databases, it is hoped that a method to classify proteins automatically will be developed. Although the classification criterion proposed by Nakashima et al. ( J. Biochem., 1986, 99, 153-162) was widely used in the literature, it leads to some inconsistencies with the classification databases currently available in the class assignment of protein structures. To improve their work, a new classification criterion is proposed relying on statistical analysis of the secondary structure contents of more than 200 proteins with well-known structural classes. The Fisher linear discriminant algorithm is used to derive the new classification criterion. RESULTS: Three cross-validation tests are performed to evaluate the new criterion. In the jackknife test, of the 210 proteins used to derive the criterion, 206 are correctly classified with an accuracy of 98.10%. Of the 16 proteins of purely intermediate structure (i.e. structures lying near borderlines between two classes) in the first test set, 15 are correctly classified with an accuracy of 93.75%. For the second test set which consists of 200 proteins selected randomly from SCOP, a testing accuracy of 94.00% is obtained. For comparison, the criterion of Nakashima et al. is also used to classify the 210, 16 and 200 proteins, respectively. Consequently, accuracies of 94.76%, 62.50% and 91.50% are obtained, respectively. On average, the accuracy of the new classification criterion is 4% higher than that of Nakashima et al. AVAILABILITY: The program is available on request from the first author. CONTACT: ctzhang@tju.edu.cn
VHMPT: a graphical viewer and editor for helical membrane protein topologies.
Authors: Lin W J , Hwang M J
Published: July 2002

MOTIVATION: Lacking structures resolved at atomic resolution, the great majority of membrane proteins have typically been depicted in a schematic two-dimensional (2D) topology consisting of putative transmembrane domains predicted from hydropathy plots. As more and more sequences of membrane proteins become available from genome projects, there is a need to automate the process of generating the schematic topology while allowing important information, such as the individual amino acid and the extent to which it is conserved in evolution, to be conveniently inspected. We addressed this need by developing a program called VHMPT. RESULTS: VHMPT (a graphical V iewer and editor for H elical line M embrane P rotein T opologies) can automatically generate a schematic 2D topology for a protein with transmembrane helices. Through an interactive graphical interface, VHMPT allows users to modify the layout of the generated topology, label specific amino acid or amino acid groups, and annotate with arrows and texts. Given a multiple sequence alignment file, VHMPT can also color code a normalized conservation score for each amino acid on the generated topology, allowing ready visual recognition of highly conserved (or variable) topological regions. VHMPT is written in Tcl/Tk and can run on platforms that have installed the Tcl/Tk interpreter. AVAILABILITY: The source code and a user manual for VHMPT are available for download at http://www. ibms.sinica.edu.tw/mjhwang/vhmpt. CONTACT: mjhwang@mail.ibms.sinica.edu.tw
Mapmerge: merge genomic maps.
Authors: Nadkarni P
Published: July 2002

MOTIVATION: When two or more genomic maps of a chromosomal region are available, it is useful to be able to synthesize them to create a merged map. RESULTS: We show that map merging is an exploratory process because there are multiple ways to combine data based upon what the user wishes to focus on, and upon which particular data subset emphasis is desired. We describe Mapmerge, a program for merging two genomic maps, discuss its limitations, and illustrate an example of its use. AVAILABILITY: Freely available (ANSI C source code, a Make file, test data files, documentation) on request from the author. CONTACT: Prakash.Nadkarni@yale.edu
CloneIt: finding cloning strategies, in-frame deletions and frameshifts.
Authors: Lindenbaum P
Published: July 2002

MOTIVATION: The CloneIt program searches for sub-cloning strategies, in-frame deletions and frameshifts within a plasmid sequence. AVAILABILITY: The program, written in ANSI-C language, is available at http://locus.jouy.inra.fr/soft/cloneit/clonei t.html CONTACT: lindenb@biotec.jouy.inra.fr
EUCLID: automatic classification of proteins in functional classes by their database annotations.
Authors: Tamames J , Ouzounis C , Casari G , Sander C , Valencia A
Published: July 2002

SUMMARY: A tool is described for the automatic classification of sequences in functional classes using their database annotations. The Euclid system is based on a simple learning procedure from examples provided by human experts. AVAILABILITY: Euclid is freely available for academics at http://www.gredos.cnb.uam.es/EUCLID, with the corresponding dictionaries for the generation of three, eight and 14 functional classes. Contact: E-mail: valencia@cnb.uam.es SUPPLEMENTARY INFORMATION: The results of the EUCLID classification of different genomes are available at http://www.sander.ebi.ac. uk/genequiz/. A detailed description of the different applications mentioned in the text is available at http://www.gredos.cnb.uam. es/EUCLID/Full_Paper
New phylogenetic venues opened by a novel implementation of the DNAml algorithm.
Authors: Trelles O , Ceron C , Wang H C , Dopazo J , Carazo J M
Published: July 2002

SUMMARY: This note describes the parallelization of a key biological application, such as the construction of phylogenetic trees, using the DNAml program. A careful run-time analysis allowed us to propose an efficient parallelization that scaled very well to large problem sizes. AVAILABILITY: By anonymous ftp at ftp.ac.uma. es (/pub/ots/pDNAml) or from our web site http://www.cnb.uam. es/www/software/software_index.htmlCONTACT: ots@ac.uma.es