bluuebunny/crossref_metadata_2025 huggingface dataset:
Epithelial Cell Adhesion Molecule (Ep-CAM) Modulates Cell–Cell Interactions Mediated by Classic Cadherins
Authors: Litvinov Sergey V. , Balzar Maarten , Winter Manon J. , Bakker Hellen A.M. , Bruijn Inge H. Briaire-de , Prins Frans , Fleuren Gert Jan , Warnaar Sven O.
Published: July 2002

The contribution of noncadherin-type, Ca2+-independent cell–cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM–positive transfectants behave like cells with a decreased strength of cell–cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM–cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of α- and β-catenins decreased in cells overexpressing Ep-CAM. While the total β-catenin content remains unchanged, a reduction in total cellular α-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell–cell adhesions diminish, Ep-CAM–mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell–cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell–cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.
Integrin αvβ3 Requirement for Sustained Mitogen-activated Protein Kinase Activity during Angiogenesis
Authors: Eliceiri Brian P. , Klemke Richard , Strömblad Staffan , Cheresh David A.
Published: July 2002

Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5–120 min) was refractory to integrin antagonists, whereas the sustained activity (4–20 h) depended on integrin αvβ3, but not β1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained αvβ3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin αvβ3.
Collagen II Is Essential for the Removal of the Notochord and the Formation of Intervertebral Discs
Authors: Aszódi Attila , Chan Danny , Hunziker Ernst , Bateman John F. , Fässler Reinhard
Published: July 2002

Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II–deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice.

Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that α1(XI) and α2 (XI) chains form unstable collagen XI molecules, demonstrating that the α3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.
The Ligand for Osteoprotegerin (OPGL) Directly Activates Mature Osteoclasts
Authors: Burgess Teresa L. , Qian Yi-xin , Kaufman Stephen , Ring Brian D. , Van Gwyneth , Capparelli Charles , Kelley Michael , Hsu Hailing , Boyle William J. , Dunstan Colin R. , Hu Sylvia , Lacey David L.
Published: July 2002

Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309–319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165–176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.
The Zinc Finger Protein A20 Inhibits TNF-induced NF-κB–dependent Gene Expression by Interfering with an RIP- or TRAF2-mediated Transactivation Signal and Directly Binds to a Novel NF-κB–inhibiting Protein ABIN
Authors: Heyninck Karen , De Valck Dirk , Berghe Wim Vanden , Van Criekinge Wim , Contreras Roland , Fiers Walter , Haegeman Guy , Beyaert Rudi
Published: July 2002

The zinc finger protein A20 is a tumor necrosis factor (TNF)– and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-κB)–dependent gene expression. However, the molecular mechanism by which A20 exerts this effect is still unclear. We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-κB, although it completely prevents the TNF- induced activation of an NF-κB–dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage–colony stimulating factor gene expression. Moreover, NF-κB activation induced by overexpression of the TNF receptor–associated proteins TNF receptor–associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor–associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-κB activation induced by overexpression of NF-κB–inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected. These results demonstrate that A20 inhibits NF-κB–dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK–IκB kinase pathway and that is specifically involved in the transactivation of NF-κB. Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-κB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-κB inhibiting protein, ABIN.
A Novel Muscle-Specific β1 Integrin Binding Protein (Mibp) That Modulates Myogenic Differentiation
Authors: Li Ji , Mayne Richard , Wu Chuanyue
Published: July 2002

Myogenesis is regulated by cell adhesion receptors, including integrins of the β1 family. We report the identification of a novel muscle-specific β1 integrin binding protein (MIBP). MIBP binds to the membrane-proximal cytoplasmic region shared by β1A and β1D integrins, and the binding occurs in vivo as well as in vitro. Furthermore, we show that MIBP is abundantly expressed by C2C12 myogenic cells before fusion, and the expression of MIBP is dramatically downregulated during subsequent differentiation. Finally, we show that overexpression of MIBP in C2C12 cells resulted in a suppression of fusion and terminal differentiation, suggesting that MIBP may play a key role in controlling the progression of muscle differentiation.
Agrin Isoforms with Distinct Amino Termini
Authors: Burgess Robert W. , Skarnes William C. , Sanes Joshua R.
Published: July 2002

The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Here we demonstrate that the murine agrin gene generates two proteins with different NH2 termini, and present evidence that these isoforms differ in subcellular localization, tissue distribution, and function. The two isoforms share ∼1,900 amino acids (aa) of common sequence following unique NH2 termini of 49 or 150 aa; we therefore call them short NH2-terminal (SN) and long NH2-terminal (LN) isoforms. In the mouse genome, LN-specific exons are upstream of an SN-specific exon, which is in turn upstream of common exons. LN-agrin is expressed in both neural and nonneural tissues. In spinal cord it is expressed in discrete subsets of cells, including motoneurons. In contrast, SN-agrin is selectively expressed in the nervous system but is widely distributed in many neuronal cell types. Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated. Differential expression of the two isoforms appears to be transcriptionally regulated, whereas the unique SN and LN sequences direct their distinct subcellular localizations. Insertion of a “gene trap” construct into the mouse genome between the LN and SN exons abolished expression of LN-agrin with no detectable effect on expression levels of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absent from all basal laminae in mice lacking LN-agrin transcripts. The formation of the neuromuscular junctions was as drastically impaired in these mutants as in mice lacking all forms of agrin. Thus, basal lamina–associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.
Dissection of Autophagosome Biogenesis into Distinct Nucleation and Expansion Steps
Authors: Abeliovich Hagai , Dunn William A. , Kim John , Klionsky Daniel J.
Published: July 2002

Rapamycin, an antifungal macrolide antibiotic, mimics starvation conditions in Saccharomyces cerevisiae through activation of a general G0 program that includes widespread effects on translation and transcription. Macroautophagy, a catabolic membrane trafficking phenomenon, is a prominent part of this response. Two views of the induction of autophagy may be considered. In one, up-regulation of proteins involved in autophagy causes its induction, implying that autophagy is the result of a signal transduction mechanism leading from Tor to the transcriptional and translational machinery. An alternative hypothesis postulates the existence of a dedicated signal transduction mechanism that induces autophagy directly. We tested these possibilities by assaying the effects of cycloheximide and specific mutations on the induction of autophagy. We find that induction of autophagy takes place in the absence of de novo protein synthesis, including that of specific autophagy-related proteins that are up-regulated in response to rapamycin. We also find that dephosphorylation of Apg13p, a signal transduction event that correlates with the onset of autophagy, is also independent of new protein synthesis. Finally, our data indicate that autophagosomes that form in the absence of protein synthesis are significantly smaller than normal, indicating a role for de novo protein synthesis in the regulation of autophagosome expansion. Our results define the existence of a signal transduction-dependent nucleation step and a separate autophagosome expansion step that together coordinate autophagosome biogenesis.
Expression Profiling in the Muscular Dystrophies
Authors: Chen Yi-Wen , Zhao Po , Borup Rehannah , Hoffman Eric P.
Published: July 2002

We used expression profiling to define the pathophysiological cascades involved in the progression of two muscular dystrophies with known primary biochemical defects, dystrophin deficiency (Duchenne muscular dystrophy) and α-sarcoglycan deficiency (a dystrophin-associated protein). We employed a novel protocol for expression profiling in human tissues using mixed samples of multiple patients and iterative comparisons of duplicate datasets. We found evidence for both incomplete differentiation of patient muscle, and for dedifferentiation of myofibers to alternative lineages with advancing age. One developmentally regulated gene characterized in detail, α-cardiac actin, showed abnormal persistent expression after birth in 60% of Duchenne dystrophy myofibers. The majority of myofibers (∼80%) remained strongly positive for this protein throughout the course of the disease. Other developmentally regulated genes that showed widespread overexpression in these muscular dystrophies included embryonic myosin heavy chain, versican, acetylcholine receptor α-1, secreted protein, acidic and rich in cysteine/osteonectin, and thrombospondin 4. We hypothesize that the abnormal Ca2+ influx in dystrophin- and α-sarcoglycan–deficient myofibers leads to altered developmental programming of developing and regenerating myofibers. The finding of upregulation of HLA-DR and factor XIIIa led to the novel identification of activated dendritic cell infiltration in dystrophic muscle; these cells mediate immune responses and likely induce microenvironmental changes in muscle. We also document a general metabolic crisis in dystrophic muscle, with large scale downregulation of nuclear-encoded mitochondrial gene expression. Finally, our expression profiling results show that primary genetic defects can be identified by a reduction in the corresponding RNA.
Cofactor Requirements for Nuclear Export of Rev Response Element (Rre)–And Constitutive Transport Element (Cte)–Containing Retroviral Rnas
Authors: Hofmann Wilma , Reichart Beate , Ewald Andrea , Müller Eleonora , Schmitt Iris , Stauber Roland H. , Lottspeich Friedrich , Jockusch Brigitte M. , Scheer Ulrich , Hauber Joachim , Dabauvalle Marie-Christine
Published: July 2002

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)–mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev–NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).
Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-α
Authors: Robertson Janice , Beaulieu Jean-Martin , Doroudchi Mohammad M. , Durham Heather D. , Julien Jean-Pierre , Mushynski Walter E.
Published: July 2002

Peripherin, a neuronal intermediate filament protein associated with axonal spheroids in amyotrophic lateral sclerosis (ALS), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures. Peripherin overexpression led to the formation of cytoplasmic protein aggregates and caused the death not only of motor neurons, but also of dorsal root ganglion (DRG) neurons that were cultured from dissociated spinal cords of peripherin transgenic embryos. Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-α. This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.
The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP–dependent activation of TGF-β1
Authors: Mu Dezhi , Cambier Stephanie , Fjellbirkeland Lars , Baron Jody L. , Munger John S. , Kawakatsu Hisaaki , Sheppard Dean , Broaddus V. Courtney , Nishimura Stephen L.
Published: July 2002

Întegrins, matrix metalloproteases (MMPs), and the cytokine TGF-β have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-β exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-β. Because the latent domain of TGF-β1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-β (SLC) to the cell surface where TGF-β activation could be locally controlled. Here, we show that SLC binds to αvβ8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP–dependent release of active TGF-β, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell–matrix interactions.
The glutamine commute
Authors: Chaudhry Farrukh A. , Reimer Richard J. , Edwards Robert H.
Published: July 2002

The transfer of glutamine between cells contributes to signaling as well as to metabolism. The recent identification and characterization of the system N and A family of transporters has begun to suggest mechanisms for the directional transfer of glutamine, and should provide ways to test its physiological significance in diverse processes from nitrogen to neurotransmitter release.
Spermine Inhibits Proinflammatory Cytokine Synthesis in Human Mononuclear Cells: A Counterregulatory Mechanism that Restrains the Immune Response
Authors: Zhang Minghuang , Caragine Theresa , Wang Haichao , Cohen Pamela S. , Botchkina Galina , Soda Kuniyasu , Bianchi Marina , Ulrich Peter , Cerami Anthony , Sherry Barbara , Tracey Kevin J.
Published: July 2002

The local production of proinflammatory cytokines mediates the host response to inflammation, infection, and injury, whereas an overexpression of these mediators can injure or kill the host. Recently, we identified a class of multivalent guanylhydrazone compounds that are effective inhibitors of proinflammatory cytokine synthesis in monocytes/macrophages. The structure of one such cationic molecule suggested a molecular mimicry with spermine, a ubiquitous endogenous biogenic amine that increases significantly at sites of inflammation and infection. Here, we addressed the hypothesis that spermine might counterregulate the innate immune response by downregulating the synthesis of potentially injurious cytokines. When spermine was added to cultures of human peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), it effectively inhibited the synthesis of the proinflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, MIP-1α, and MIP-1β. The inhibition of cytokine synthesis was specific and reversible, with significant inhibition of TNF synthesis occurring even when spermine was added after LPS. The mechanism of spermine-mediated cytokine suppression was posttranscriptional and independent of polyamine oxidase activity. Local administration of spermine in vivo protected mice against the development of acute footpad inflammation induced by carrageenan. These results identify a distinct molecular counterregulatory role for spermine in downregulating the monocyte proinflammatory cytokine response.
A Role for Mac-1 (CDIIb/CD18) in Immune Complex–stimulated Neutrophil Function In Vivo: Mac-1 Deficiency Abrogates Sustained Fcγ Receptor–dependent Neutrophil Adhesion and Complement-dependent Proteinuria in Acute Glomerulonephritis
Authors: Tang Tao , Rosenkranz Alexander , Assmann Karel J.M. , Goodman Michael J. , Gutierrez-Ramos Jose-Carlos , Carroll Michael C. , Cotran Ramzi S. , Mayadas Tanya N.
Published: July 2002

Mac-1 (αmβ2), a leukocyte adhesion receptor, has been shown in vitro to functionally interact with Fcγ receptors to facilitate immune complex (IC)–stimulated polymorphonuclear neutrophil (PMN) functions. To investigate the relevance of Mac-1–FcγR interactions in IC-mediated injury in vivo, we induced a model of Fc-dependent anti–glomerular basement membrane (GBM) nephritis in wild-type and Mac-1–deficient mice by the intravenous injection of anti-GBM antibody. The initial glomerular PMN accumulation was equivalent in Mac-1 null and wild-type mice, but thereafter increased in wild-type and decreased in mutant mice. The absence of Mac-1 interactions with obvious ligands, intercellular adhesion molecule 1 (ICAM-1), and C3 complement, is not responsible for the decrease in neutrophil accumulation in Mac-1– deficient mice since glomerular PMN accumulation in mice deficient in these ligands was comparable to those in wild-type mice. In vitro studies showed that spreading of Mac-1–null PMNs to IC-coated dishes was equivalent to that of wild-type PMNs at 5–12 min but was markedly reduced thereafter, and was associated with an inability of mutant neutrophils to redistribute filamentous actin. This suggests that in vivo, Mac-1 is not required for the initiation of Fc-mediated PMN recruitment but that Mac-1–FcγR interactions are required for filamentous actin reorganization leading to sustained PMN adhesion, and this represents the first demonstration of the relevance of Mac-1–FcγR interactions in vivo. PMN-dependent proteinuria, maximal in wild-type mice at 8 h, was absent in Mac-1 mutant mice at all time points. Complement C3–deficient mice also had significantly decreased proteinuria compared to wild-type mice. Since Mac-1 on PMNs is the principal ligand for ic3b, an absence of Mac-1 interaction with C3 probably contributed to the abrogation of proteinuria in Mac-1–null mice.
Fcγ Receptor–Mediated Phagocytosis in Macrophages Lacking the Src Family Tyrosine Kinases Hck, Fgr, and Lyn
Authors: Fitzer-Attas Cheryl J. , Lowry Malcolm , Crowley Mary T. , Finn Alexander J. , Meng Fanying , DeFranco Anthony L. , Lowell Clifford A.
Published: July 2002

Macrophage Fcγ receptors (FcγRs) mediate the uptake and destruction of antibody-coated viruses, bacteria, and parasites. We examined FcγR signaling and phagocytic function in bone marrow–derived macrophages from mutant mice lacking the major Src family kinases expressed in these cells, Hck, Fgr, and Lyn. Many FcγR-induced functional responses and signaling events were diminished or delayed in these macrophages, including immunoglobulin (Ig)G-coated erythrocyte phagocytosis, respiratory burst, actin cup formation, and activation of Syk, phosphatidylinositol 3-kinase, and extracellular signal–regulated kinases 1 and 2. Significant reduction of IgG-dependent phagocytosis was not seen in hck−/−fgr−/− or lyn−/− cells, although the single mutant lyn−/− macrophages did manifest signaling defects. Thus, Src family kinases clearly have roles in two events leading to FcγR-mediated phagocytosis, one involving initiation of actin polymerization and the second involving activation of Syk and subsequent internalization. Since FcγR-mediated phagocytosis did occur at modest levels in a delayed fashion in triple mutant macrophages, these Src family kinases are not absolutely required for uptake of IgG-opsonized particles.
T Cell Recognition of the Dominant I-Ak–Restricted Hen Egg Lysozyme Epitope
Authors: McAdam Stephen N. , Fleckenstein Burkhard , Rasmussen Ingunn B. , Schmid Dietmar G. , Sandlie Inger , Bogen Bjarne , Viner Nicholas J. , Sollid Ludvig M.
Published: July 2002

Type-B T cells raised against the immunodominant peptide in hen egg lysozyme (HEL48–62) do not respond to whole lysozyme, and this has been thought to indicate that peptide can bind to l-Ak in different conformations. Here we demonstrate that such T cells recognize a deamidated form of the HEL peptide and not the native peptide. The sequence of the HEL epitope facilitates rapid and spontaneous deamidation when present as a free peptide or within a flexible domain. However, this deamidated epitope is not created within intact lysozyme, most likely because it resides in a highly structured part of the protein. These findings argue against the existence of multiple conformations of the same peptide–MHC complex and have important implications for the design of peptide-based vaccines. Furthermore, as the type-B T cells are known to selectively evade induction of tolerance when HEL is expressed as a transgene, these results suggest that recognition of posttranslationally modified self-antigen may play a role in autoimmunity.
Telomere Shortening Accompanies Increased Cell Cycle Activity during Serial Transplantation of Hematopoietic Stem Cells
Authors: Allsopp Richard C. , Cheshier Samuel , Weissman Irving L.
Published: July 2002

Reactivation of telomerase and maintenance of telomere length can lead to the prevention of replicative senescence in some human somatic cells grown in vitro. To investigate whether telomere shortening might also play a role in the limitation of hematopoietic stem cell (HSC) division capacity in vivo, we analyzed telomere length during serial transplantation of murine HSCs. Southern blot analysis of telomere length in donor bone marrow cells revealed extensive shortening (∼7 kb) after just two rounds of HSC transplantation. The number of cycling HSCs increased after transplantation and remained elevated for at least 4 mo, while the frequency of HSCs in the bone marrow was completely regenerated by 2 mo after transplantation. Direct analysis of telomeres in HSCs by fluorescent in situ hybridization during serial transplantation also revealed a reduction in telomere size. Together, these data show that telomeres shorten during division of HSCs in vivo, and are consistent with the hypothesis that telomere shortening may limit the replicative capacity of HSCs.
Role of p75 Neurotrophin Receptor in the Neurotoxicity by β-amyloid Peptides and Synergistic Effect of Inflammatory Cytokines
Authors: Perini Giovanni , Della-Bianca Vittorina , Politi Valeria , Della Valle Giuliano , Dal-Pra Ilaria , Rossi Filippo , Armato Ubaldo
Published: July 2002

The neurodegenerative changes in Alzheimer's disease (AD) are elicited by the accumulation of β-amyloid peptides (Aβ), which damage neurons either directly by interacting with components of the cell surface to trigger cell death signaling or indirectly by activating astrocytes and microglia to produce inflammatory mediators. It has been recently proposed that the p75 neurotrophin receptor (p75NTR) is responsible for neuronal damage by interacting with Aβ. By using neuroblastoma cell clones lacking the expression of all neurotrophin receptors or engineered to express full-length or various truncated forms of p75NTR, we could show that p75NTR is involved in the direct signaling of cell death by Aβ via the function of its death domain. This signaling leads to the activation of caspases-8 and -3, the production of reactive oxygen intermediates and the induction of an oxidative stress. We also found that the direct and indirect (inflammatory) mechanisms of neuronal damage by Aβ could act synergistically. In fact, TNF-α and IL-1β, cytokines produced by Aβ-activated microglia, could potentiate the neurotoxic action of Aβ mediated by p75NTR signaling. Together, our results indicate that neurons expressing p75NTR, mostly if expressing also proinflammatory cytokine receptors, might be preferential targets of the cytotoxic action of Aβ in AD.
The Transcription Factor T-bet Regulates Mucosal T Cell Activation in Experimental Colitis and Crohn's Disease
Authors: Neurath M.F. , Weigmann B. , Finotto S. , Glickman J. , Nieuwenhuis E. , Iijima H. , Mizoguchi A. , Mizoguchi E. , Mudter J. , Galle P.R. , Bhan A. , Autschbach F. , Sullivan B.M. , Szabo S.J. , Glimcher L.H. , Blumberg R.S.
Published: July 2002

The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-γ/interleukin (IL)-4 and transforming growth factor (TGF)-β activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L+ CD4+ T cells exacerbated colitis in reconstituted SCID mice, T-bet–deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet–deficient CD62L− CD4+ T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-β signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-γ/IL-4 and TGF-β responses and the development of chronic intestinal inflammation in T cell–mediated colitis. Furthermore, TGF-β was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-β and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell–mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.
How Does the W434F Mutation Block Current in Shaker Potassium Channels?
Authors: Yang Youshan , Yan Yangyang , Sigworth Fred J.
Published: July 2002

The mutation W434F produces an apparently complete block of potassium current in Shaker channels expressed in Xenopus oocytes. Tandem tetrameric constructs containing one or two subunits with this mutation showed rapid inactivation, although the NH2-terminal inactivation domain was absent from these constructs. The inactivation showed a selective dependence on external cations and was slowed by external TEA; these properties are characteristic of C-type inactivation. Inactivation was, however, incompletely relieved by hyperpolarization, suggesting the presence of a voltage-independent component. The hybrid channels had near-normal conductance and ion selectivity. Single-channel recordings from patches containing many W434F channels showed occasional channel openings, consistent with open probabilities of 10−5 or less. We conclude that the W434F mutation produces a channel that is predominantly found in an inactivated state.
Endomorphisms of expansive systems on compact metric spaces and the pseudo-orbit tracing property
Authors: Nasu Masakazu
Published: July 2002

We investigate the interrelationships between the dynamical properties of commuting continuous maps of a compact metric space. Let

X
X

be a compact metric space. First we show the following. If

τ
:
X
→
X

\tau : X \rightarrow X

is an expansive onto continuous map with the pseudo-orbit tracing property (POTP) and if there is a topologically mixing continuous map

φ
:
X
→
X

\varphi : X \rightarrow X

with

τ
φ
=
φ
τ

\tau \varphi = \varphi \tau

, then

τ
\tau

is topologically mixing. If

τ
:
X
→
X

\tau : X \rightarrow X

and

φ
:
X
→
X

\varphi : X \rightarrow X

are commuting expansive onto continuous maps with POTP and if

τ
\tau

is topologically transitive with period

p
p

, then for some

k
k

dividing

p
p

,

X
=

⋃

i
=
0

l
−
1

B
i

X = \bigcup _{i=0}^{l-1} B_i

, where the

B
i

B_i

,

0
≤
i
≤
l
−
1

0 \leq i \leq l-1

, are the basic sets of

φ
\varphi

with

l
=
p

/

k

l = p/k

such that all

φ

|

B
i

:

B
i

→

B
i

\varphi |B_i : B_i \rightarrow B_i

have period

k
k

, and the dynamical systems

(

B
i

,
φ

|

B
i

)

(B_i,\varphi |B_i)

are a factor of each other, and in particular they are conjugate if

τ
\tau

is a homeomorphism. Then we prove an extension of a basic result in symbolic dynamics. Using this and many techniques in symbolic dynamics, we prove the following. If

τ
:
X
→
X

\tau : X \rightarrow X

is a topologically transitive, positively expansive onto continuous map having POTP, and

φ
:
X
→
X

\varphi : X \rightarrow X

is a positively expansive onto continuous map with

φ
τ
=
τ
φ

\varphi \tau = \tau \varphi

, then

φ
\varphi

has POTP. If

τ
:
X
→
X

\tau :X \rightarrow X

is a topologically transitive, expansive homeomorphism having POTP, and

φ
:
X
→
X

\varphi : X \rightarrow X

is a positively expansive onto continuous map with

φ
τ
=
τ
φ

\varphi \tau = \tau \varphi

, then

φ
\varphi

has POTP and is constant-to-one. Further we define ‘essentially LR endomorphisms’ for systems of expansive onto continuous maps of compact metric spaces, and prove that if

τ
:
X
→
X

\tau : X \rightarrow X

is an expansive homeomorphism with canonical coordinates and

φ
\varphi

is an essentially LR automorphism of

(
X
,
τ
)

(X,\tau )

, then

φ
\varphi

has canonical coordinates. We add some discussions on basic properties of the essentially LR endomorphisms.
Recent developments on the Ricci flow
Authors: Cao Huai-Dong , Chow Bennett
Published: July 2002

This article reports recent developments of the research on Hamilton’s Ricci flow and its applications.
A variational principle for domino tilings
Authors: Cohn Henry , Kenyon Richard , Propp James
Published: July 2002

We formulate and prove a variational principle (in the sense of thermodynamics) for random domino tilings, or equivalently for the dimer model on a square grid. This principle states that a typical tiling of an arbitrary finite region can be described by a function that maximizes an entropy integral. We associate an entropy to every sort of local behavior domino tilings can exhibit, and prove that almost all tilings lie within

ε
\varepsilon

(for an appropriate metric) of the unique entropy-maximizing solution. This gives a solution to the dimer problem with fully general boundary conditions, thereby resolving an issue first raised by Kasteleyn. Our methods also apply to dimer models on other grids and their associated tiling models, such as tilings of the plane by three orientations of unit lozenges.
Searching DNA databases for similarities to DNA sequences: when is a match significant?
Authors: Anderson I , Brass A
Published: July 2002

MOTIVATION: Searching DNA sequences against a DNA database is an essential element of sequence analysis. However, few systematic studies have been carried out to determine when a match between two DNA sequences has biological significance and this is limiting the use that can be made of DNA searching algorithms. RESULTS: A test set of DNA sequences has been constructed consisting of artificially evolved and real sequences. This set has been used to test various database searching algorithms (BLAST, BLAST2, FASTA and Smith-Waterman) on a subset of the EMBL database. The results of this analysis have been used to determine the sensitivity and coverage of all of the algorithms. Guidelines have been produced which can be used to assess the significance of DNA database search results. The Smith-Waterman algorithm was shown to have the best coverage, but the worst sensitivity, whereas the default BLASTN algorithm (word length set to 11) was shown to have good sensitivity, but poor coverage. A sensible compromise between speed, sensitivity and coverage can be obtained using either the FASTA or BLAST (word length set to 6) algorithms. However, analysis of the results also showed that no algorithm works well when the length of the probe sequence is <200 bases. In general, matches can accurately be identified between coding regions of DNA sequences when there is >35% sequence identity between the corresponding proteins. Searching a DNA sequence against a DNA sequence database can, therefore, be a useful tool in sequence analysis. AVAILABILITY: The test sets used are available via anonymous ftp from mbisg2.sbc.man.ac.uk in the directory /pub/cabios/testdata/ CONTACT: I.Anderson@stud.man.ac.uk; abrass@man.ac.uk
A neural network classifier capable of recognizing the patterns
of all major subcellular structures in fluorescence microscope
images of HeLa cells
Authors: Boland Michael V. , Murphy Robert F.
Published: July 2002

Motivation: Assessment of protein subcellular location is crucial to proteomics efforts since localization information provides a context for a protein’s sequence, structure, and function. The work described below is the first to address the subcellular localization of proteins in a quantitative, comprehensive manner.

Results: Images for ten different subcellular patterns (including all major organelles) were collected using fluorescence microscopy. The patterns were described using a variety of numeric features, including Zernike moments, Haralick texture features, and a set of new features developed specifically for this purpose. To test the usefulness of these features, they were used to train a neural network classifier. The classifier was able to correctly recognize an average of 83% of previously unseen cells showing one of the ten patterns. The same classifier was then used to recognize previously unseen sets of homogeneously prepared cells with 98% accuracy.

Availability: Algorithms were implemented using the commercial products Matlab, S-Plus, and SAS, as well as some functions written in C. The scripts and source code generated for this work are available at http://murphylab.web.cmu.edu/software.

Contact: murphy@cmu.edu

* To whom correspondence should be addressed.
Cleavage of non‐tRNA substrates by eukaryal tRNA splicing endonucleases
Authors: Fruscoloni Paolo , Baldi Maria I , Tocchini‐Valentini Glauco P
Published: July 2002

Eukaryal tRNA splicing endonucleases use the mature domains of pre‐tRNAs as their primary recognition elements. However, they can also cleave in a mode that is independent of the mature domain, when substrates are able to form the bulge–helix–bulge structure (BHB), which is cleaved by archaeal tRNA endonucleases. We present evidence that the eukaryal enzymes cleave their substrates after forming a structure that resembles the BHB. Consequently, these enzymes can cleave substrates that lack the mature domain altogether. That raises the possibility that these enzymes could also cleave non‐tRNA substrates that already have a BHB. As predicted, they can do so, both in vitro and in vivo.
Multitude of ion channels in regulation of transmitter release
Authors: Rahamimoff Rami , Butkevich Alexander , Duridanova Dessislava , Ahdut Ronit , Harari Emanuel , Kachalsky Sylvia G.
Published: July 2002

The presynaptic nerve terminal is of key importance in the communication in the nervous system. Its primary role is to release transmitter quanta on the arrival of an appropriate stimulus. The structural basis of these transmitter quanta are the synaptic vesicles that fuse with the surface membrane of the nerve terminal, to release their content of neurotransmitter molecules and other vesicular components. We subdivide the control of quantal release into two major classes: the processes that take place before the fusion of the synaptic vesicle with the surface membrane (the pre–fusion control) and the processes that occur after the fusion of the vesicle (the post–fusion control). The pre–fusion control is the main determinant of transmitter release. It is achieved by a wide variety of cellular components, among them the ion channels. There are reports of several hundred different ion channel molecules at the surface membrane of the nerve terminal, that for convenience can be grouped into eight major categories. They are the voltage–dependent calcium channels, the potassium channels, the calcium–gated potassium channels, the sodium channels, the chloride channels, the non–selective channels, the ligand gated channels and the stretch–activated channels. There are several categories of intracellular channels in the mitochondria, endoplasmic reticulum and the synaptic vesicles. We speculate that the vesicle channels may be of an importance in the post–fusion control of transmitter release.
Probability‐based structural parameters from three‐dimensional nuclear magnetic resonance images as predictors of trabecular bone strength
Authors: Hwang Scott N. , Wehrli Felix W. , Williams John L.
Published: July 2002

The mechanical competence of trabecular bone is a function of its apparent density and three‐dimensional (3D) distribution. Three‐dimensional structure is typically inferred from histomorphometry and stereology on a limited number of two‐dimensional anatomic sections. In this work 3D nuclear magnetic resonance (NMR) images of anisotropic trabecular bone from the distal radius were analyzed in terms of a series of new structural parameters which are obtainable at relatively crude resolution, i.e., in the presence of substantial partial volume blurring. Unlike typical feature extraction techniques requiring image segmentation, the method relies on spatial autocorrelation analysis, which is based on the probability of finding bone at specified locations. The structural parameters were measured from high‐resolution images ( voxels) of 23 trabecular bone specimens from the distal radius. Maximum‐likelihood bone volume fractions (BVF) were calculated for each voxel and a resolution achievable in vivo voxels) was simulated by averaging BVF's from neighboring voxels. The parameters derived from the low‐resolution images were found to account for 91% of the variation in Young's modulus. The results suggest that noninvasive assessment of the mechanical competence of trabecular bone in osteoporotic patients may be feasible.
Fan beam and double crosshole Lamb wave tomography for mapping flaws in aging aircraft structures
Authors: Malyarenko Eugene V. , Hinders Mark K.
Published: July 2002

As the worldwide aviation fleet continues to age, methods for accurately predicting the presence of structural flaws—such as hidden corrosion and disbonds—that compromise airworthiness become increasingly necessary. Ultrasonic guided waves, Lamb waves, allow large sections of aircraft structures to be rapidly inspected. However, extracting quantitative information from Lamb wave data has always involved highly trained personnel with a detailed knowledge of mechanical waveguide physics. The work summarized here focuses on a variety of different tomographic reconstruction techniques to graphically represent the Lamb wave data in quantitative maps that can be easily interpreted by technicians. Because the velocity of Lamb waves depends on thickness, for example, the traveltimes of the fundamental Lamb modes can be converted into a thickness map of the inspection region. This article describes two potentially practical implementations of Lamb wave tomographic imaging techniques that can be optimized for in-the-field testing of large-area aircraft structures. Laboratory measurements discussed here demonstrate that Lamb wave tomography using either a ring of transducers with fan beam reconstructions, or a square array of transducers with algebraic reconstruction tomography, is appropriate for detecting flaws in multilayer aircraft materials. The speed and fidelity of the reconstruction algorithms as well as practical considerations for person-portable array-based systems are discussed in this article.