bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes
Authors: Vickery Ross G. , von Zastrow Mark
Published: July 2002

D1 and D2 dopamine receptors are structurally homologous G protein–coupled receptors that serve distinct physiological functions both in neurons and nonneural cell types. We have observed that these receptors are selectively endocytosed in HEK293 cells by distinct dynamin-dependent and -independent mechanisms. Although these endocytic mechanisms operate with similarly rapid kinetics, they differ in their regulation by agonist and deliver D1 and D2 receptors specifically to different primary endocytic vesicles. After this segregation into different endocytic membranes, both D1 and D2 receptors recycle to the plasma membrane. Similar results are observed in Neuro2A neuroblastoma cells coexpressing both receptors at high levels. These findings establish that “classical” dynamin-dependent and “alternative” dynamin-independent endocytic mechanisms differ in their physiological regulation, sort structurally homologous signaling receptors in the plasma membrane, and mediate distinct early endocytic pathways leading to recycling endosomes. Our results also refute the previous hypothesis that dynamin-independent endocytosis targets G protein–coupled receptors selectively to lysosomes, and they suggest a new role of endocytic sorting mechanisms in physically segregating structurally homologous signaling receptors at the cell surface.
Proteins Connecting the Nuclear Pore Complex with the Nuclear Interior
Authors: Strambio-de-Castillia Caterina , Blobel Günter , Rout Michael P.
Published: July 2002

While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC.
Expression of the 180-kD Ribosome Receptor Induces Membrane Proliferation and Increased Secretory Activity in Yeast
Authors: Becker Frank , Block-Alper Laura , Nakamura Gerald , Harada Josephine , Wittrup K. Dane , Meyer David I.
Published: July 2002

Expression of the canine 180-kD ribosome receptor (p180) in yeast cells resulted in a marked proliferation of intracellular membranes. The type of membranes observed varied with the expression of specific portions of p180. Rough membranes predominated when the ribosome binding domain of p180 was present, whereas expression constructs lacking this region resulted in smooth membranes. Northern analysis indicated that expression of the NH2-terminal 767 amino acids (ΔCT), which include the ribosome binding domain, upregulated the transcription and translation of genes involved in exocytosis. The membranes that were proliferated were functional as these cells overcame a temperature-sensitive translocation defect. Most significantly, cells that overexpressed ΔCT and proliferated rough endoplasmic reticulum exhibited severalfold higher levels of secretion of an ectopically expressed secretory protein. We conclude that p180 expression triggers a cascade of events leading to an increase in secretory potential akin to the terminal differentiation of mammalian secretory cells and tissues.
Presenilin 1 Suppresses the Function of C-Jun Homodimers via Interaction with Qm/Jif-1
Authors: Imafuku I. , Masaki T. , Waragai M. , Takeuchi S. , Kawabata M. , Hirai S.-i. , Ohno S. , Nee L.E. , Lippa C.F. , Kanazawa I. , Imagawa M. , Okazawa H.
Published: July 2002

Presenilin 1 (PS1) is the causative gene for an autosomal dominant familial Alzheimer's disease (AD) mapped to chromosome 14. Here we show that QM/Jun-interacting factor (Jif)-1, a negative regulator of c-Jun, is a candidate to mediate the function of PS1 in the cell. We screened for proteins that bind to PS1 from a human embryonic brain cDNA library using the two-hybrid method and isolated one clone encoding the QM/Jif-1 gene. The binding of QM/Jif-1 to full-length PS1 was confirmed in vitro by pull-down assay, and in vivo by immunoprecipitation assays with human samples, including AD brains. Immunoelectronmicroscopic analysis showed that QM/Jif-1 and PS1 are colocalized at the endoplasmic reticulum, and the nuclear matrix in human brain neurons. Chloramphenicol acetyltransferase assays in F9 cells showed that PS1 suppresses transactivation by c-Jun/c-Jun but not by c-Jun/c-Fos heterodimers, consistent with the reported function of QM/Jif-1. By monitoring fluorescent recombinant protein and by gel mobility shift assays, PS1 was shown to accelerate the translocation of QM from the cytoplasm to the nucleus and to thereby suppress the binding of c-Jun homodimer to 12-O-tetradecanoylphorbol-13- acetate (TPA)-responsive element (TRE). PS1 suppressed c-jun–associated apoptosis by retinoic acid in F9 embryonic carcinoma cells, whereas this suppression of apoptosis is attenuated by mutation in PS1. Collectively, the novel function of PS1 via QM/Jif-1 influences c-jun–mediated transcription and apoptosis.
Human 76p
Authors: Fava Fabienne , Raynaud-Messina Brigitte , Leung-Tack Jeanne , Mazzolini Laurent , Li Min , Guillemot Jean Claude , Cachot Didier , Tollon Yvette , Ferrara Pascual , Wright Michel
Published: July 2002

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. γ-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and γ-tubulin are associated in the γ-tubulin complexes. (3) γ-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human γ-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.
Dynamics of DNA Replication Factories in Living Cells
Authors: Leonhardt Heinrich , Rahn Hans-Peter , Weinzierl Peter , Sporbert Anje , Cremer Thomas , Zink Daniele , Cardoso M. Cristina
Published: July 2002

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.
Regulation of the Growth of Multinucleated Muscle Cells by an Nfatc2-Dependent Pathway
Authors: Horsley Valerie , Friday Bret B. , Matteson Sarah , Kegley Kristy Miller , Gephart Jonathan , Pavlath Grace K.
Published: July 2002

The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the development and differentiation of several tissue types. Here, we examine the role of NFATC2 in skeletal muscle by analyzing adult NFATC2−/− mice. These mice exhibit reduced muscle size due to a decrease in myofiber cross-sectional area, suggesting that growth is blunted. Muscle growth was examined during regeneration after injury, wherein NFATC2-null myofibers form normally but display impaired growth. The growth defect is intrinsic to muscle cells, since the lack of NFATC2 in primary muscle cultures results in reduced cell size and myonuclear number in myotubes. Retroviral-mediated expression of NFATC2 in the mutant cells rescues this cellular phenotype. Myonuclear number is similarly decreased in NFATC2−/− mice. Taken together, these results implicate a novel role for NFATC2 in skeletal muscle growth. We demonstrate that during growth of multinucleated muscle cells, myoblasts initially fuse to form myotubes with a limited number of nuclei and that subsequent nuclear addition and increases in myotube size are controlled by a molecular pathway regulated by NFATC2.
Mammalian Abp1, a Signal-Responsive F-Actin–Binding Protein, Links the Actin Cytoskeleton to Endocytosis via the Gtpase Dynamin
Authors: Kessels Michael M. , Engqvist-Goldstein Åsa E.Y. , Drubin David G. , Qualmann Britta
Published: July 2002

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin–binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.
Merotelic Kinetochore Orientation Is a Major Mechanism of Aneuploidy in Mitotic Mammalian Tissue Cells
Authors: Cimini Daniela , Howell Bonnie , Maddox Paul , Khodjakov Alexey , Degrassi Francesca , Salmon E.D.
Published: July 2002

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 μM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2–5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.
Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export
Authors: Frosst Phyllis , Guan Tinglu , Subauste Cecilia , Hahn Klaus , Gerace Larry
Published: July 2002

Tpr is a coiled-coil protein found near the nucleoplasmic side of the pore complex. Since neither the precise localization of Tpr nor its functions are well defined, we generated antibodies to three regions of Tpr to clarify these issues. Using light and EM immunolocalization, we determined that mammalian Tpr is concentrated within the nuclear basket of the pore complex in a distribution similar to Nup153 and Nup98. Antibody localization together with imaging of GFP-Tpr in living cells revealed that Tpr is in discrete foci inside the nucleus similar to several other nucleoporins but is not present in intranuclear filamentous networks (Zimowska et al., 1997) or in long filaments extending from the pore complex (Cordes et al., 1997) as proposed. Injection of anti-Tpr antibodies into mitotic cells resulted in depletion of Tpr from the nuclear envelope without loss of other pore complex basket proteins. Whereas nuclear import mediated by a basic amino acid signal was unaffected, nuclear export mediated by a leucine-rich signal was retarded significantly. Nuclear injection of anti-Tpr antibodies in interphase cells similarly yielded inhibition of protein export but not import. These results indicate that Tpr is a nucleoporin of the nuclear basket with a role in nuclear protein export.
The protein storage vacuole
Authors: Jiang Liwen , Phillips Thomas E. , Hamm Christopher A. , Drozdowicz Yolanda M. , Rea Philip A. , Maeshima Masayoshi , Rogers Sally W. , Rogers John C.
Published: July 2002

Storage proteins are deposited into protein storage vacuoles (PSVs) during plant seed development and maturation and stably accumulate to high levels; subsequently, during germination the storage proteins are rapidly degraded to provide nutrients for use by the embryo. Here, we show that a PSV has within it a membrane-bound compartment containing crystals of phytic acid and proteins that are characteristic of a lytic vacuole. This compound organization, a vacuole within a vacuole whereby storage functions are separated from lytic functions, has not been described previously for organelles within the secretory pathway of eukaryotic cells. The partitioning of storage and lytic functions within the same vacuole may reflect the need to keep the functions separate during seed development and maturation and yet provide a ready source of digestive enzymes to initiate degradative processes early in germination.
Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex
Authors: Gleizes Pierre-Emmanuel , Noaillac-Depeyre Jacqueline , Léger-Silvestre Isabelle , Teulières Frédéric , Dauxois Jean-Yves , Pommet Denys , Azum-Gelade Marie-Claude , Gas Nicole
Published: July 2002

To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus. This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus. Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p–Nup159p–Nsp1p complex. In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome. Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export. Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport. Thus, the Nup82p–Nup159p–Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.
The Peripheral Deletion of Autoreactive CD8+ T Cells Induced by Cross-presentation of Self-antigens Involves Signaling through CD95 (Fas, Apo-1)
Authors: Kurts Christian , Heath William R. , Kosaka Hiroshi , Miller Jacques F.A.P. , Carbone Francis R.
Published: July 2002

Recently, we demonstrated that major histocompatibility complex class I–restricted cross-presentation of exogenous self-antigens can induce peripheral T cell tolerance by deletion of autoreactive CD8+ T cells. In these studies, naive ovalbumin (OVA)-specific CD8+ T cells from the transgenic line OT-I were injected into transgenic mice expressing membrane-bound OVA (mOVA) under the control of the rat insulin promoter (RIP) in pancreatic islets, kidney proximal tubules, and the thymus. Cross-presentation of tissue-derived OVA in the renal and pancreatic lymph nodes resulted in activation, proliferation, and then the deletion of OT-I cells. In this report, we investigated the molecular mechanisms underlying this form of T cell deletion. OT-I mice were crossed to tumor necrosis factor receptor 2 (TNFR2) knockout mice and to CD95 (Fas, Apo-1) deficient mutant lpr mice. Wild-type and TNFR2-deficient OT-I cells were activated and then deleted when transferred into RIP-mOVA mice, whereas CD95-deficient OT-I cells were not susceptible to deletion by cross-presentation. Furthermore, cross-presentation led to upregulation of the CD95 molecule on the surface of wild-type OT-I cells in vivo, consistent with the idea that this is linked to rendering autoreactive T cells susceptible to CD95-mediated signaling. This study represents the first evidence that CD95 is involved in the deletion of autoreactive CD8+ T cells in the whole animal.
Repression of B7.2 on Self-reactive B Cells Is Essential to Prevent Proliferation and Allow Fas-mediated Deletion by CD4+ T Cells
Authors: Rathmell Jeffrey C. , Fournier Sylvie , Weintraub Bennett C. , Allison James P. , Goodnow Christopher C.
Published: July 2002

Peripheral tolerance mechanisms normally prevent delivery of T cell help to anergic self-reactive B cells that accumulate in the T zones of spleen and lymph nodes. Chronic exposure to self-antigens desensitizes B cell antigen receptor (BCR) signaling on anergic B cells so that they are not stimulated into clonal expansion by CD4+ T cells but instead are eliminated by Fas (CD95)-induced apoptosis. Because a range of BCR-induced signals and responses are repressed in anergic B cells, it is not known which of these are critical to regulate for Fas-mediated peripheral tolerance. Display of the costimulatory molecule, B7.2 (CD86), represents a potentially important early response to acute BCR engagement that is poorly induced by antigen on anergic B cells. We show here that restoring B7.2 expression on tolerant B cells using a constitutively expressed B7.2 transgene is sufficient to prevent Fas-mediated deletion and to trigger extensive T cell–dependent clonal expansion and autoantibody secretion in the presence of specific T cells. Dysregulated expression of B7.2 on tolerant B cells caused a more extreme reversal of peripheral tolerance than that caused by defects in Fas or Fas ligand, and resulted in T cell–dependent clonal expansion and antibody secretion comparable in magnitude to that made by foreign antigen-specific B cells. These findings demonstrate that repression of B7.2 is critical to eliminate autoreactive B cells by Fas in B cell–T cell interactions. The possible role of B7.2 dysregulation in systemic autoimmune diseases is discussed.
Identification of Functional Human Splenic Memory B Cells by Expression of CD148 and CD27
Authors: Tangye Stuart G. , Liu Yong-Jun , Aversa Gregorio , Phillips Joseph H. , de Vries Jan E.
Published: July 2002

Memory B cells isolated from human tonsils are characterized by an activated cell surface phenotype, localization to mucosal epithelium, expression of somatically mutated immunoglobulin (Ig) variable (V) region genes, and a preferential differentiation into plasma cells in vitro. In spleens of both humans and rodents, a subset of memory B cells is believed to reside in the marginal zone of the white pulp. Similar to tonsil-derived memory B cells, splenic marginal zone B cells can be distinguished from naive follicular B cells by a distinct cell surface phenotype and by the presence of somatic mutations in their Ig V region genes. Although differences exist between human naive and memory B cells, no cell surface molecules have been identified that positively identify all memory B cells. In this study, we have examined the expression of the receptor-type protein tyrosine phosphatase CD148 on human B cells. CD148+ B cells present in human spleen exhibited characteristics typical of memory B cells. These included an activated phenotype, localization to the marginal zone, the expression of somatically mutated Ig V region genes, and the preferential differentiation into plasma cells. In contrast, CD148− B cells appeared to be naive B cells due to localization to the mantle zone, the expression of surface antigens typical of unstimulated B cells, and the expression of unmutated Ig V region genes. Interestingly, CD148+ B cells also coexpressed CD27, whereas CD148− B cells were CD27−. These results identify CD148 and CD27 as markers which positively identify memory B cells present in human spleen. Thus, assessing expression of these molecules may be a convenient way to monitor the development of memory B cell responses in immunocompromised individuals or in vaccine trials.
Crystal Structure of the Cysteine-Rich Domain of Mannose Receptor Complexed with a Sulfated Carbohydrate Ligand
Authors: Liu Yang , Chirino Arthur J. , Misulovin Ziva , Leteux Christine , Feizi Ten , Nussenzweig Michel C. , Bjorkman Pamela J.
Published: July 2002

The macrophage and epithelial cell mannose receptor (MR) binds carbohydrates on foreign and host molecules. Two portions of MR recognize carbohydrates: tandemly arranged C-type lectin domains facilitate carbohydrate-dependent macrophage uptake of infectious organisms, and the NH2-terminal cysteine-rich domain (Cys-MR) binds to sulfated glycoproteins including pituitary hormones. To elucidate the mechanism of sulfated carbohydrate recognition, we determined crystal structures of Cys-MR alone and complexed with 4-sulfated-N-acetylgalactosamine at 1.7 and 2.2 Å resolution, respectively. Cys-MR folds into an approximately three-fold symmetric β-trefoil shape resembling fibroblast growth factor. The sulfate portions of 4-sulfated-N-acetylgalactosamine and an unidentified ligand found in the native crystals bind in a neutral pocket in the third lobe. We use the structures to rationalize the carbohydrate binding specificities of Cys-MR and compare the recognition properties of Cys-MR with other β-trefoil proteins.
A Hierarchical Role for Classical Pathway Complement Proteins in the Clearance of Apoptotic Cells in Vivo
Authors: Taylor Philip R. , Carugati Anna , Fadok Valerie A. , Cook H. Terence , Andrews Mark , Carroll Michael C. , Savill John S. , Henson Peter M. , Botto Marina , Walport Mark J.
Published: July 2002

The strongest susceptibility genes for the development of systemic lupus erythematosus (SLE) in humans are null mutants of classical pathway complement proteins. There is a hierarchy of disease susceptibility and severity according to the position of the missing protein in the activation pathway, with the severest disease associated with C1q deficiency. Here we demonstrate, using novel in vivo models of apoptotic cell clearance during sterile peritonitis, a similar hierarchical role for classical pathway complement proteins in vivo in the clearance of apoptotic cells by macrophages. Our results constitute the first demonstration of an impairment in the phagocytosis of apoptotic cells by macrophages in vivo in a mammalian system. Apoptotic cells are thought to be a major source of the autoantigens of SLE, and impairment of their removal by complement may explain the link between hereditary complement deficiency and the development of SLE.
Cis3/Socs3/Ssi3 Plays a Negative Regulatory Role in Stat3 Activation and Intestinal Inflammation
Authors: Suzuki Asuka , Hanada Toshikatsu , Mitsuyama Keiichi , Yoshida Takafumi , Kamizono Shintaro , Hoshino Tomoaki , Kubo Masato , Yamashita Atsuko , Okabe Masaru , Takeda Kiyoshi , Akira Shizuo , Matsumoto Satoshi , Toyonaga Atsushi , Sata Michio , Yoshimura Akihiko
Published: July 2002

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins (ILs) and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activator of transcription (STAT) transcription factors. We recently identified two intrinsic Janus kinase (JAK) inhibitors, JAK binding protein (JAB; also referred to as suppressor of cytokine signaling [SOCS1]/STAT-induced STAT inhibitor [SSI1]) and cytokine-inducible SH2 protein (CIS)3 (or SOCS3/SSI3), which play an essential role in the negative regulation of cytokine signaling. We have investigated the role of STATs and these JAK inhibitors in intestinal inflammation. Among STAT family members, STAT3 was most strongly tyrosine phosphorylated in human ulcerative colitis and Crohn's disease patients as well as in dextran sulfate sodium (DSS)-induced colitis in mice. Development of colitis as well as STAT3 activation was significantly reduced in IL-6–deficient mice treated with DSS, suggesting that STAT3 plays an important role in the perpetuation of colitis. CIS3, but not JAB, was highly expressed in the colon of DSS-treated mice as well as several T cell–dependent colitis models. To define the physiological role of CIS3 induction in colitis, we developed a JAB mutant (F59D-JAB) that overcame the inhibitory effect of both JAB and CIS3 and created transgenic mice. DSS induced stronger STAT3 activation and more severe colitis in F59D-JAB transgenic mice than in their wild-type littermates. These data suggest that hyperactivation of STAT3 results in severe colitis and that CIS3 plays a negative regulatory role in intestinal inflammation by downregulating STAT3 activity.
BDCA-2, a Novel Plasmacytoid Dendritic Cell–specific Type II C-type Lectin, Mediates Antigen Capture and Is a Potent Inhibitor of Interferon α/β Induction
Authors: Dzionek Andrzej , Sohma Yoshiaki , Nagafune Jun , Cella Marina , Colonna Marco , Facchetti Fabio , Günther Gritt , Johnston Ian , Lanzavecchia Antonio , Nagasaka Tomoko , Okada Tsutomu , Vermi William , Winkels Gregor , Yamamoto Terumi , Zysk Monika , Yamaguchi Yasunori , Schmitz Jürgen
Published: July 2002

Plasmacytoid dendritic cells are present in lymphoid and nonlymphoid tissue and contribute substantially to both innate and adaptive immunity. Recently, we have described several monoclonal antibodies that recognize a plasmacytoid dendritic cell-specific antigen, which we have termed BDCA-2. Molecular cloning of BDCA-2 revealed that BDCA-2 is a novel type II C-type lectin, which shows 50.7% sequence identity at the amino acid level to its putative murine ortholog, the murine dendritic cell–associated C-type lectin 2. Anti–BDCA-2 monoclonal antibodies are rapidly internalized and efficiently presented to T cells, indicating that BDCA-2 could play a role in ligand internalization and presentation. Furthermore, ligation of BDCA-2 potently suppresses induction of interferon α/β production in plasmacytoid dendritic cells, presumably by a mechanism dependent on calcium mobilization and protein-tyrosine phosphorylation by src-family protein-tyrosine kinases. Inasmuch as production of interferon α/β by plasmacytoid dendritic cells is considered to be a major pathophysiological factor in systemic lupus erythematosus, triggering of BDCA-2 should be evaluated as therapeutic strategy for blocking production of interferon α/β in systemic lupus erythematosus patients.
Helical Structure and Packing Orientation of the S2 Segment in the Shaker K+ Channel
Authors: Monks Stephen A. , Needleman Daniel J. , Miller Christopher
Published: July 2002

Six transmembrane segments, S1–S6, cluster around the central pore-forming region in voltage-gated K+ channels. To investigate the structural characteristics of the S2 segment in the Shaker K+ channel, we replaced each residue in S2 singly with tryptophan (or with alanine for the native tryptophan). All but one of the 23 Trp mutants expressed voltage-dependent K+ currents in Xenopus oocytes. The effects of the mutations were classified as being of low or high impact on channel gating properties. The periodicity evident in the effects of these mutations supports an α-helical structure for the S2 segment. The high- and low-impact residues cluster onto opposite faces of a helical wheel projection of the S2 segment. The low-impact face is also tolerant of single mutations to asparagine. All results are consistent with the idea that the low-impact face projects toward membrane lipids and that changes in S2 packing occur upon channel opening. We conclude that the S2 segment is a transmembrane α helix and that the high-impact face packs against other transmembrane segments in the functional channel.
Multiple Modes of Calcium-Induced Calcium Release in Sympathetic Neurons II
Authors: Hongpaisan Jarin , Pivovarova Natalia B. , Colegrove Stephen L. , Leapman Richard D. , Friel David D. , Andrews S. Brian
Published: July 2002

CICR from an intracellular store, here directly characterized as the ER, usually refers to net Ca2+ release that amplifies evoked elevations in cytosolic free calcium ([Ca2+]i). However, the companion paper (Albrecht, M.A., S.L. Colegrove, J. Hongpaisan, N.B. Pivovarova, S.B. Andrews, and D.D. Friel. 2001. J. Gen. Physiol. 118:83–100) shows that in sympathetic neurons, small [Ca2+]i elevations evoked by weak depolarization stimulate ER Ca accumulation, but at a rate attenuated by activation of a ryanodine-sensitive CICR pathway. Here, we have measured depolarization-evoked changes in total ER Ca concentration ([Ca]ER) as a function of [Ca2+]i, and found that progressively larger [Ca2+]i elevations cause a graded transition from ER Ca accumulation to net release, consistent with the expression of multiple modes of CICR. [Ca]ER is relatively high at rest (12.8 ± 0.9 mmol/kg dry weight, mean ± SEM) and is reduced by thapsigargin or ryanodine (5.5 ± 0.7 and 4.7 ± 1.1 mmol/kg, respectively). [Ca]ER rises during weak depolarization (to 17.0 ± 1.6 mmol/kg over 120s, [Ca2+]i less than ∼350 nM), changes little in response to stronger depolarization (12.1 ± 1.1 mmol/kg, [Ca2+]i ∼700 nM), and declines (to 6.5 ± 1.0 mmol/kg) with larger [Ca2+]i elevations (>1 μM) evoked by the same depolarization when mitochondrial Ca2+ uptake is inhibited (FCCP). Thus, net ER Ca2+ transport exhibits a biphasic dependence on [Ca2+]i. With mitochondrial Ca2+ uptake enabled, [Ca]ER rises after repolarization (to 16.6 ± 1.8 mmol/kg at 15 min) as [Ca2+]i falls within the permissive range for ER Ca accumulation over a period lengthened by mitochondrial Ca2+ release. Finally, although spatially averaged [Ca]ER is unchanged during strong depolarization, net ER Ca2+ release still occurs, but only in the outermost ∼5-μm cytoplasmic shell where [Ca2+]i should reach its highest levels. Since mitochondrial Ca accumulation occurs preferentially in peripheral cytoplasm, as demonstrated here by electron energy loss Ca maps, the Ca content of ER and mitochondria exhibit reciprocal dependencies on proximity to sites of Ca2+ entry, possibly reflecting indirect mitochondrial regulation of ER Ca2+ transport.
On the exactness of an S-shaped bifurcation curve
Authors: Korman Philip , Li Yi
Published: July 2002

For a class of two-point boundary value problems we prove exactness of an S-shaped bifurcation curve. Our result applies to a problem from combustion theory, which involves nonlinearities like

e

a
u

/

(
u
+
a
)

e^{au/(u+a)}

for

a
>
0

a>0

.
The Lyapunov spectrum of families of time-varying matrices
Authors: Colonius Fritz , Kliemann Wolfgang
Published: July 2002

For

L

∞

L^{\infty }

-families of time varying matrices centered at an unperturbed matrix, the Lyapunov spectrum contains the Floquet spectrum obtained by considering periodically varying piecewise constant matrices. On the other hand, it is contained in the Morse spectrum of an associated flow on a vector bundle. A closer analysis of the Floquet spectrum based on geometric control theory in projective space and, in particular, on control sets, is performed. Introducing a real parameter

ρ
≥
0

\rho \ge 0

, which indicates the size of the

L

∞

L^{\infty }

-perturbation, we study when the Floquet spectrum, the Morse spectrum, and hence the Lyapunov spectrum all coincide. This holds, if an inner pair condition is satisfied, for all up to at most countably many

ρ
\rho

-values.
Construction of arbitrary Kazhdan-Lusztig polynomials in symmetric groups
Authors: Polo Patrick
Published: July 2002

To each polynomial

P
P

with integral nonnegative coefficients and constant term equal to

1
1

, of degree

d
d

, we associate a certain pair of elements

(
y
,
w
)

(y,w)

in the symmetric group

S
n

S_n

, where

n
=
1
+
d
+
P
(
1
)

n = 1 + d + P(1)

, such that the Kazhdan-Lusztig polynomial

P

y
,
w

P_{y,w}

equals

P
P

. This pair satisfies

ℓ
(
w
)
−
ℓ
(
y
)
=
2
d
+
P
(
1
)
−
1

\ell (w) - \ell (y) = 2d + P(1) - 1

, where

ℓ
(
w
)

\ell (w)

denotes the number of inversions of

w
w

.
Identification of Sec36p, Sec37p, and Sec38p: Components of Yeast Complex That Contains Sec34p and Sec35p
Authors: Ram Rachna J. , Li Baojie , Kaiser Chris A.
Published: July 2002

The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4,COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached.
An iterative method for faster sum-of-pairs multiple sequence

alignment
Authors: Reinert Knut , Stoye Jens , Will Torsten
Published: July 2002

Motivation: Multiple sequence alignment is an important tool in computational biology. In order to solve the task of computing multiple alignments in affordable time, the most commonly used multiple alignment methods have to use heuristics. Nevertheless, the computation of optimal multiple alignments is important in its own right, and it provides a means of evaluating heuristic approaches or serves as a subprocedure of heuristic alignment methods.

Results: We present an algorithm that uses the divide-and-conquer alignment approach together with recent results on search space reduction to speed up the computation of multiple sequence alignments. The method is adaptive in that depending on the time one wants to spend on the alignment, a better, up to optimal alignment can be obtained. To speed up the computation in the optimal alignment step, we apply the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathcal{A}^{*}\) \end{document}algorithm which leads to a procedure provably more efficient than previous exact algorithms. We also describe our implementation of the algorithm and present results showing the effectiveness and limitations of the procedure.

Availability: http://bibiserv.techfak.uni-bielefeld.de/oma/

Contact: j.stoye@dkfz.de

To whom correspondence should be addressed.

**** Present address: mediaWays GmbH, Hülshorstweg 30, 33415 Verl, Germany.
MAPS: a microarray project system for gene expression

experiment information and data validation
Authors: Bushel Pierre R. , Hamadeh Hisham , Bennett Lee , Sieber Stella , Martin Karla , Nuwaysir Emile F. , Johnson Kate , Reynolds Kelli , Paules Richard S. , Afshari Cynthia A.
Published: July 2002

Summary: MAPS is a MicroArray Project System for management and interpretation of microarray gene expression experiment information and data. Microarray project information is organized to track experiments and results that are: (1) validated by performing analysis on stored replicate gene expression data; and (2) queried according to the biological classifications of genes deposited on microarray chips.

Availability: MAPS is accessible at http://www.dir.niehs.nih.gov/microarray/software/maps/

Contact: bushel@niehs.nih.gov

* To whom correspondence should be addressed.

3 Present address: NimbleGen Systems, LLC, University Research Park, 505 South Rosa Road, Madison, WI 53719, USA.
Inference of a genetic network by a combined approach of
cluster analysis and graphical Gaussian modeling
Authors: Toh Hiroyuki , Horimoto Katsuhisa
Published: July 2002

Motivation: Recent advances in DNA microarray technologies have made it possible to measure the expression levels of thousands of genes simultaneously under different conditions. The data obtained by microarray analyses are called expression profile data. One type of important information underlying the expression profile data is the ‘genetic network,’ that is, the regulatory network among genes. Graphical Gaussian Modeling (GGM) is a widely utilized method to infer or test relationships among a plural of variables.

Results: In this study, we developed a method combining the cluster analysis with GGM for the inference of the genetic network from the expression profile data. The expression profile data of 2467 Saccharomyces cerevisiae genes measured under 79 different conditions (Eisen et al. , Proc. Natl Acad. Sci. USA , 95, 14683–14868, 1998) were used for this study. At first, the 2467 genes were classified into 34 clusters by a cluster analysis, as a preprocessing for GGM. Then, the expression levels of the genes in each cluster were averaged for each condition. The averaged expression profile data of 34 clusters were subjected to GGM, and a partial correlation coefficient matrix was obtained as a model of the genetic network of S. cerevisiae. The accuracy of the inferred network was examined by the agreement of our results with the cumulative results of experimental studies.

Availability: A set of programs will be electronically sent upon request.

Contact: toh@beri.co.jp; horimoto@ged.saga-med.ac.jp

* To whom correspondence should be addressed.

† Both authors contributed equally to this work.
Costs of treating infections caused by methicillin-resistant staphylococci and vancomycin-resistant enterococci
Authors: Carbon Claude
Published: July 2002

Infection with methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococcus faecium (VREF) increases the risk of mortality and results in prolonged hospitalization and high utilization of costly treatment modalities. Measures to prevent the spread of MRSA (and possibly VREF) include patient isolation and decontamination, hygiene measures, ward closure, and screening of patients and staff for carriage. In seriously ill patients, the increased use of vancomycin for the treatment of MRSA can lead to the emergence of VREF colonization/infection. Quinupristin/dalfopristin is effective in the treatment of MRSA infections, including nosocomial pneumonia, skin and soft tissue infection, and septicaemia. In the treatment of nosocomial pneumonia, clinical success rates were equivalent between quinupristin/dalfopristin and vancomycin. In the context of a hospital policy which emphasizes effective hygiene measures and the prudent use of antibacterials, quinupristin/dalfopristin is an effective antimicrobial that can help to control the high costs associated with multiresistant MRSA and VREF infections.
An Easy and Accurate Regression Model for Multiparty Electoral Data
Authors: Tomz Michael , Tucker Joshua A. , Wittenberg Jason
Published: July 2002

Katz and King have previously proposed a statistical model for multiparty election data. They argue that ordinary least-squares (OLS) regression is inappropriate when the dependent variable measures the share of the vote going to each party, and they recommend a superior technique. Regrettably, the Katz-King model requires a high level of statistical expertise and is computationally demanding for more than three political parties. We offer a sophisticated yet convenient alternative that involves seemingly unrelated regression (SUR). SUR is nearly as easy to use as OLS yet performs as well as the Katz-King model in predicting the distribution of votes and the composition of parliament. Moreover, it scales easily to an arbitrarily large number of parties. The model has been incorporated intoClarify, a statistical suite that is available free on the Internet.
Huehuecuetzpalli mixtecus gen. et sp. nov: a basal squamate (Reptilia) from the Early Cretaceous of Tepexi de Rodríguez, Central México
Authors: Reynoso Víctor-Hugo
Published: July 2002

Huehuecuetzpalli mixtecusgen. et sp. nov. is characterized by a combination of characters unlike those of any of the previously described Late Jurassic or Early Cretaceous lizards. It has most of the synapomorphies common to modern squamates, but still retains primitive features rare in living taxa. Autapomorphic characters include an anteroposteriorly elongated premaxilla that results in the elongation of the snout and the apparent retraction of the external nares. A small rounded postfrontal and a parietal foramen on the frontoparietal suture suggest affinities with iguanians, but the retention of divided premaxillae, amphicoelous vertebrae, thoracolumbar intercentra, entepicondylar foramen, and a second distal tarsal supports the hypothesis thatHuehuecuetzpallihas a more basal position relative to the extant squamates. Although its appearance is late in the fossil record of lizards,Huehuecuetzpalliis the first report of a basal squamate. It provides important information on early transformation of characters in lizard evolution. Many primitive characters present in some modern squamates are usually explained by paedomorphosis; however, these characters are common in early lizards suggesting that derived states may have been fixed later in lizard evolution. IfHuehuecuetzpalliis an iguanian, then it would be the earliest known representative of this lineage and extends their fossil record into the Albian. This paper presents an extensive review of the characters and character states used in previously published cladistic analyses of the Squamata.
Hydrological investigations of forest disturbance and land cover impacts in South–East Asia: a review
Authors: Douglas Ian
Published: July 2002

Investigations of land management impacts on hydrology are well developed in South–East Asia, having been greatly extended by national organizations in the last two decades. Regional collaborative efforts, such as the ASEAN–US watershed programme, have helped develop skills and long–running monitoring programmes. Work in different countries is significant for particular aspects: the powerful effects of both cyclones and landsliding in Taiwan, the significance of lahars in Java, of small–scale agriculture in Thailand and plantation establishment in Malaysia. Different aid programmes have contributed specialist knowledge such as British work on reservoir sedimentation, Dutch, Swedish and British work on softwood plantations and US work in hill–tribe agriculture. Much has been achieved through individual university research projects, including PhD and MSc theses. The net result is that for most countries there is now good information on changes in the rainfall–run–off relationship due to forest disturbance or conversion, some information on the impacts on sediment delivery and erosion of hillslopes, but relatively little about the dynamics and magnitude of nutrient losses. Improvements have been made in the ability to model the consequences of forest conversion and of selective logging and exciting prospects exist for the development of better predictions of transfer of water from the hillslopes to the stream channels using techniques such as multilevel modelling. Understanding of the processes involved has advanced through the detailed monitoring made possible at permanent field stations such as that at Danum Valley, Sabah.