bluuebunny/crossref_metadata_2025 huggingface dataset:
Aczonin, a 550-Kd Putative Scaffolding Protein of Presynaptic Active Zones, Shares Homology Regions with Rim and Bassoon and Binds Profilin
Authors: Wang Xiaolu , Kibschull Mark , Laue Michael M. , Lichte Beate , Petrasch-Parwez Elisabeth , Kilimann Manfred W.
Published: July 2002

Neurotransmitter exocytosis is restricted to the active zone, a specialized area of the presynaptic plasma membrane. We report the identification and initial characterization of aczonin, a neuron-specific 550-kD protein concentrated at the presynaptic active zone and associated with a detergent-resistant cytoskeletal subcellular fraction. Analysis of the amino acid sequences of chicken and mouse aczonin indicates an organization into multiple domains, including two pairs of Cys4 zinc fingers, a polyproline tract, and a PDZ domain and two C2 domains near the COOH terminus. The second C2 domain is subject to differential splicing. Aczonin binds profilin, an actin-binding protein implicated in actin cytoskeletal dynamics. Large parts of aczonin, including the zinc finger, PDZ, and C2 domains, are homologous to Rim or to Bassoon, two other proteins concentrated in presynaptic active zones. We propose that aczonin is a scaffolding protein involved in the organization of the molecular architecture of synaptic active zones and in the orchestration of neurotransmitter vesicle trafficking.
Rhoa Function in Lamellae Formation and Migration Is Regulated by the α6β4 Integrin and Camp Metabolism
Authors: O'Connor Kathleen L. , Nguyen Bao-Kim , Mercurio Arthur M.
Published: July 2002

Clone A colon carcinoma cells develop fan-shaped lamellae and exhibit random migration when plated on laminin, processes that depend on the ligation of the α6β4 integrin. Here, we report that expression of a dominant negative RhoA (N19RhoA) in clone A cells inhibited α6β4-dependent membrane ruffling, lamellae formation, and migration. In contrast, expression of a dominant negative Rac (N17Rac1) had no effect on these processes. Using the Rhotekin binding assay to assess RhoA activation, we observed that engagement of α6β4 by either antibody-mediated clustering or laminin attachment resulted in a two- to threefold increase in RhoA activation, compared with cells maintained in suspension or plated on collagen. Antibody-mediated clustering of β1 integrins, however, actually suppressed Rho A activation. The α6β4-mediated interaction of clone A cells with laminin promoted the translocation of RhoA from the cytosol to membrane ruffles at the edges of lamellae and promoted its colocalization with β1 integrins, as assessed by immunofluorescence microscopy. In addition, RhoA translocation was blocked by inhibiting phosphodiesterase activity and enhanced by inhibiting the activity of cAMP-dependent protein kinase. Together, these results establish a specific integrin-mediated pathway of RhoA activation that is regulated by cAMP and that functions in lamellae formation and migration.
Coordinated Spindle Assembly and Orientation Requires Clb5p-Dependent Kinase in Budding Yeast
Authors: Segal Marisa , Clarke Duncan J. , Maddox Paul , Salmon E.D. , Bloom Kerry , Reed Steven I.
Published: July 2002

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Δ cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Δ cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.
The Acute Myeloid Leukemia-Associated Protein, Dek, Forms a Splicing-Dependent Interaction with Exon-Product Complexes
Authors: McGarvey Tim , Rosonina Emanuel , McCracken Susan , Li Qiyu , Arnaout Ramy , Mientjes Edwin , Nickerson Jeffrey A. , Awrey Don , Greenblatt Jack , Grosveld Gerard , Blencowe Benjamin J.
Published: July 2002

DEK is an ∼45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.
The Caenorhabditis elegans unc-78 Gene Encodes a Homologue of Actin-Interacting Protein 1 Required for Organized Assembly of Muscle Actin Filaments
Authors: Ono Shoichiro
Published: July 2002

Assembly and maintenance of myofibrils require dynamic regulation of the actin cytoskeleton. In Caenorhabditis elegans, UNC-60B, a muscle-specific actin depolymerizing factor (ADF)/cofilin isoform, is required for proper actin filament assembly in body wall muscle (Ono, S., D.L. Baillie, and G.M. Benian. 1999. J. Cell Biol. 145:491–502). Here, I show that UNC-78 is a homologue of actin-interacting protein 1 (AIP1) and functions as a novel regulator of actin organization in myofibrils. In unc-78 mutants, the striated organization of actin filaments is disrupted, and large actin aggregates are formed in the body wall muscle cells, resulting in defects in their motility. Point mutations in unc-78 alleles change conserved residues within different WD repeats of the UNC-78 protein and cause less severe phenotypes than a deletion allele, suggesting that these mutations partially impair the function of UNC-78. UNC-60B is normally localized in the diffuse cytoplasm and to the myofibrils in wild type but mislocalized to the actin aggregates in unc-78 mutants. Similar Unc-78 phenotypes are observed in both embryonic and adult muscles. Thus, AIP1 is an important regulator of actin filament organization and localization of ADF/cofilin during development of myofibrils.
Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly
Authors: Young Paul , Ehler Elisabeth , Gautel Mathias
Published: July 2002

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is ∼800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3–like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH2-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca2+/calmodulin, as well as G protein–coupled signal transduction in the sarcomere.
Inducible dimerization of FGFR1
Authors: Welm Bryan E. , Freeman Kevin W. , Chen Mercy , Contreras Alejandro , Spencer David M. , Rosen Jeffrey M.
Published: July 2002

To develop an inducible and progressive model of mammary gland tumorigenesis, transgenic mice were generated with a mouse mammary tumor virus–long terminal repeat–driven, conditional, fibroblast growth factor (FGF)–independent FGF receptor (FGFR)1 (iFGFR1) that can be induced to dimerize with the drug AP20187. Treatment of transgenic mice with AP20187 resulted in iFGFR1 tyrosine phosphorylation, increased proliferation, activation of mitogen-activated protein kinase and Akt, and lateral budding. Lateral buds appeared as early as 3 d after AP20187 treatment and initially consisted of bilayered epithelial cells and displayed apical and basolateral polarity appeared after 13 d of AP20187 treatment. Invasive lesions characterized by multicell-layered lateral buds, decreased myoepithelium, increased vascular branching, and loss of cell polarity were observed after 2–4 wk of treatment. These data indicate that acute iFGFR1 signaling results in increased lateral budding of the mammary ductal epithelium, and that sustained activation induces alveolar hyperplasia and invasive lesions.
Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae
Authors: Mironov Alexander A. , Beznoussenko Galina V. , Nicoziani Paolo , Martella Oliviano , Trucco Alvar , Kweon Hee-Seok , Di Giandomenico Daniele , Polishchuk Roman S. , Fusella Aurora , Lupetti Pietro , Berger Eric G. , Geerts Willie J.C. , Koster Abraham J. , Burger Koert N.J. , Luini Alberto
Published: July 2002

Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.
Structural requirements for localization and activation of protein kinase C μ (PKCμ) at the Golgi compartment
Authors: Hausser Angelika , Link Gisela , Bamberg Linda , Burzlaff Annett , Lutz Sylke , Pfizenmaier Klaus , Johannes Franz-Josef
Published: July 2002

We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC)μ involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKCμ–green fluorescent protein fusion proteins costained with the Golgi compartment–specific markers p24 and p230. Deletions of either the NH2-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKCμ completely abrogated Golgi localization of PKCμ. As an NH2-terminal PKCμ fragment was colocalized with p24, this region of PKCμ is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKCμ to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinase-dead PKCμ found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKCμ, in which Golgi compartment recruitment precedes and is essential for activation loop phoshorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of NH2-terminal serine(s) in the regulatory domain. PKCμ activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKCμ function at the Golgi compartment.
Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection
Authors: Shao Hui , Kono Dwight H. , Chen Ling-Yu , Rubin Elyssa M. , Kaye Jonathan
Published: July 2002

There is little known about the regulation of gene expression during TCR-mediated differentiation of immature CD4+8+ (double positive) thymocytes into mature T cells. Using the DPK CD4+8+thymocyte precursor cell line, we demonstrate that the early growth response-1 gene (Erg-1), encoding a zinc finger transcription factor, is rapidly upregulated after TCR stimulation. We also report that Egr-1 is expressed by a subset of normal double positive thymocytes in the thymic cortex, as well by a majority of medullary single positive thymocytes. Expression of Egr-1 is dramatically reduced in the thymus of major histocompatibility complex knockout mice, but can be induced by anti-CD3 antibody stimulation of isolated thymocytes from these animals. These and other data suggest that high level expression of Egr-1 in the thymus is a consequence of selection. A similar pattern of expression is found for family members Egr-2 and Egr-3. Using the DPK cell line, we also demonstrate that expression of Egr-1, 2, and 3 is dependent upon ras activation, as is the initiation of differentiation to a single positive cell. In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1. The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.
Interleukin-10 Is a Natural Suppressor of Cytokine Production and Inflammation in a Murine Model of Allergic Bronchopulmonary Aspergillosis
Authors: Grünig Gabriele , Corry David B. , Leach Michael W. , Seymour Brian W.P. , Kurup Viswanath P. , Rennick Donna M.
Published: July 2002

We have used interleukin-10 (IL-10) gene knockout mice (IL-10−/−) to examine the role of endogenous IL-10 in allergic lung responses to Aspergillus fumigatus Ag. In vitro restimulated lung cells from sensitized IL-10−/− mice produced exaggerated amounts of IL-4, IL-5, and interferon-γ (IFN-γ) compared with wild-type (WT) lung cells. In vivo, the significance of IL-10 in regulating responses to repeated A. fumigatus inhalation was strikingly revealed in IL-10−/− outbred mice that had a 50–60% mortality rate, while mortality was rare in similarly treated WT mice. Furthermore, IL-10−/− outbred mice exhibited exaggerated airway inflammation and heightened levels of IL-5 and IFN-γ in bronchoalveolar lavage (BAL) fluids. In contrast, the magnitude of the allergic lung response was similar in intranasally (i.n.) sensitized IL-10−/− and wild-type mice from a different strain (C57BL/6). Using a different route of priming (intraperitoneal) followed by one i.n. challenge we found that IL-10−/− C57BL/6 mice had heightened eosinophilic airway inflammation, BAL–IL-5 levels, and numbers of αβT cells in the lung tissues compared with WT mice. We conclude that IL-10 can suppress inflammatory Th2-like lung responses as well as Th1-like responses given the constraints of genetic background and route of priming.
Impairment of  T Cell Development in δ
EF1 Mutant Mice
Authors: Higashi Yujiro , Moribe Hiroki , Takagi Tsuyoshi , Sekido Ryohei , Kawakami Kiyoshi , Kikutani Hitoshi , Kondoh Hisato
Published: July 2002

Using the method of gene targeting in mouse embryonic stem cells, regulatory function of δEF1, a zinc finger and homeodomain-containing transcription factor, was investigated in vivo by generating the δEF1 mutant mice. The mutated allele of δEF1 produced a truncated form of the δEF1 protein lacking a zinc finger cluster proximal to COOH terminus. The homozygous δEF1 mutant mice had poorly developed thymi with no distinction of cortex and medulla. Analysis of the mutant thymocyte showed reduction of the total cell number by two orders of magnitude accompanying the impaired thymocyte development. The early stage intrathymic c-kit+ T precursor cells were largely depleted. The following thymocyte development also seemed to be affected as assessed by the distorted composition of CD4- or CD8-expressing cells. The mutant thymocyte showed elevated α4 integrin expression, which might be related to the T cell defect in the mutant mice. In the peripheral lymph node tissue of the mutant mice, the CD4−CD8+ single positive cells were significantly reduced relative to CD4+CD8− single positive cells. In contrast to T cells, other hematopoietic lineages appeared to be normal. The data indicated that δEF1 is involved in regulation of T cell development at multiple stages.
Multiple Hemopoietic Defects and Lymphoid Hyperplasia in Mice Lacking the Transcriptional Activation Domain of the c-Rel Protein
Authors: Carrasco Daniel , Cheng Janet , Lewin Anne , Warr Glenn , Yang Hyekyung , Rizzo Cheryl , Rosas Fabio , Snapper Clifford , Bravo Rodrigo
Published: July 2002

The c-rel protooncogene encodes a member of the Rel/nuclear factor (NF)-κB family of transcriptional factors. To assess the role of the transcriptional activation domain of c-Rel in vivo, we generated mice expressing a truncated c-Rel (Δc-Rel) that lacks the COOH-terminal region, but retains a functional Rel homology domain. Mice with an homozygous mutation in the c-rel region encoding the COOH terminus of c-Rel (c-relΔCT/ΔCT) display marked defects in proliferative and immune functions. c-relΔCT/ΔCT animals present histopathological alterations of hemopoietic tissues, such as an enlarged spleen due to lymphoid hyperplasia, extramedullary hematopoiesis, and bone marrow hypoplasia. In older c-relΔCT/ΔCT mice, lymphoid hyperplasia was also detected in lymph nodes, liver, lung, and stomach. These animals present a more severe phenotype than mice lacking the entire c-Rel protein. Thus, in c-relΔCT/ΔCT mice, the lack of c-Rel activity is less efficiently compensated by other NF-κB proteins.
Challenging Cytokine Redundancy: Inflammatory Cell Movement and Clinical Course of Experimental Autoimmune Encephalomyelitis Are Normal in Lymphotoxin-deficient, but Not Tumor Necrosis Factor–deficient, Mice
Authors: Sean Riminton D. , Körner Heinrich , Strickland Deborah H. , Lemckert Frances A. , Pollard John D. , Sedgwick Jonathon D.
Published: July 2002

Lymphotoxin (LT) is widely regarded as a proinflammatory cytokine with activities equivalent to tumor necrosis factor (TNF). The contribution of LT to experimental autoimmune encephalomyelitis (EAE) was examined using TNF/LTα−/− mice, TNF−/− mice, and a new LTα−/− line described here. All mice were generated directly in the C57BL/6 strain and used for the preparation of radiation bone marrow chimeras to reconstitute peripheral lymphoid organs and restore immunocompetence. This approach overcame the problems related to the lack of lymph nodes that results from LTα gene targeting. We show here that when LT is absent but TNF is present, EAE progresses normally. In contrast, when TNF is absent but LT is present, EAE is delayed in onset and inflammatory leukocytes fail to move normally into the central nervous system parenchyma, even at the peak of disease. In the absence of both cytokines, the clinical and histological picture is identical to that seen when TNF alone is deficient, including demyelination. Furthermore, the therapeutic inhibition of TNF and LTα with soluble TNF receptor in unmanipulated wild-type or TNF−/− mice exactly reproduces these outcomes. We conclude from these studies that TNF and LT are functionally distinct cytokines in vivo, and despite sharing common receptors, show no redundancy of function nor mutual compensation.
Anti–4-1bb Monoclonal Antibodies Abrogate T Cell–Dependent Humoral Immune Responses in Vivo through the Induction of Helper T Cell Anergy
Authors: Mittler Robert S. , Bailey Tina S. , Klussman Kerry , Trailsmith Mark D. , Hoffmann Michael K.
Published: July 2002

The 4-1BB receptor (CDw137), a member of the tumor necrosis factor receptor superfamily, has been shown to costimulate the activation of T cells. Here we show that anti–mouse 4-1BB monoclonal antibodies (mAbs) inhibit thymus-dependent antibody production by B cells. Injection of anti–4-1BB mAbs into mice being immunized with cellular or soluble protein antigens induced long-term anergy of antigen-specific T cells. The immune response to the type II T cell–independent antigen trinintrophenol-conjugated Ficoll, however, was not suppressed. Inhibition of humoral immunity occurred only when anti–4-1BB mAb was given within 1 wk after immunization. Anti–4-1BB inhibition was observed in mice lacking functional CD8+ T cells, indicating that CD8+ T cells were not required for the induction of anergy. Analysis of the requirements for the anti–4-1BB–mediated inhibition of humoral immunity revealed that suppression could not be adoptively transferred with T cells from anti–4-1BB–treated mice. Transfer of BALB/c splenic T cells from sheep red blood cell (SRBC)-immunized and anti–4-1BB–treated mice together with normal BALB/c B cells into C.B-17 severe combined immunodeficient mice failed to generate an anti-SRBC response. However, B cells from the SRBC-immunized, anti–4-1BB–treated BALB/c mice, together with normal naive T cells, exhibited a normal humoral immune response against SRBC after transfer, demonstrating that SRBC-specific B cells were left unaffected by anti–4-1BB mAbs.
Flice-Inhibitory Protein Expression during Macrophage Differentiation Confers Resistance to FAS-Mediated Apoptosis
Authors: Perlman Harris , Pagliari Lisa J. , Georganas Constantinos , Mano Toshiaki , Walsh Kenneth , Pope Richard M.
Published: July 2002

Macrophages differentiated from circulating peripheral blood monocytes are essential for host immune responses and have been implicated in the pathogenesis of rheumatoid arthritis and atherosclerosis. In contrast to monocytes, macrophages are resistant to Fas-induced cell death by an unknown mechanism. FLICE (Fas-associated death domain–like interleukin 1β–converting enzyme)–inhibitory protein (Flip), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis. Here, we show that monocyte differentiation into macrophages was associated with upregulation of Flip and a decrease in Fas-mediated apoptosis. Overexpression of Flip protected monocytes from Fas-mediated apoptosis, whereas acute Flip inhibition in macrophages induced apoptosis. Addition of an antagonistic Fas ligand antibody to Flip antisense–treated macrophages rescued cultures from apoptosis, demonstrating that endogenous Flip blocked Fas-induced cell death. Thus, the expression of Flip in macrophages conferred resistance to Fas-mediated apoptosis, which may contribute to the development of inflammatory disease.
Induction of B Cell Hyperplasia in Simian Immunodeficiency Virus–Infected Rhesus Macaques with the Simian Homologue of Kaposi's Sarcoma–Associated Herpesvirus
Authors: Wong Scott W. , Bergquam Eric P. , Swanson Ryan M. , Lee Felix W. , Shiigi Stanley M. , Avery Nancy A. , Fanton John W. , Axthelm Michael K.
Published: July 2002

A simian homologue of Kaposi's sarcoma–associated herpesvirus (KSHV), the eighth human herpesvirus (HHV8), was isolated from a simian immunodeficiency virus (SIV)-infected rhesus macaque (Macaca mulatta) that developed a multicentric lymphoproliferative disorder (LPD). This simian rhadinovirus is genetically similar to a recently described rhesus rhadinovirus (RRV) (Desrosiers, R.C., V.G. Sasseville, S.C. Czajak, X. Zhang, K.G. Mansfield, A. Kaur, R.P. Johnson, A.A. Lackner, and J.U. Jung. 1997. J. Virol. 71:9764–9769) and is designated RRV 17577. RRV 17577 was experimentally inoculated into rhesus macaques with and without SIVmac239 infection to determine if RRV played a role in development of the LPD observed in the index case. In contrast to control animals inoculated with SIVmac239 or RRV alone, two animals coinfected with SIVmac239 and RRV 17577 developed hyperplastic LPD resembling the multicentric plasma cell variant of Castleman's disease, characterized by persistent angiofollicular lymphadenopathy, hepatomegaly, splenomegaly, and hypergammaglobulinemia. Hypergammaglobulinemia was associated with severe immune-mediated hemolytic anemia in one RRV/SIV-infected macaque. Both RRV/SIV-infected macaques exhibited persistent RRV viremia with little or no RRV-specific antibody response. The macaques inoculated with RRV alone displayed transient viremia followed by a vigorous anti-RRV antibody response and lacked evidence of LPD in peripheral blood and lymph nodes. Infectious RRV and RRV DNA were present in hyperplastic lymphoid tissues of the RRV/SIV-infected macaques, suggesting that lymphoid hyperplasia is associated with the high levels of replication. Thus, experimental RRV 17577 infection of SIV-infected rhesus macaques induces some of the hyperplastic B cell LPDs manifested in AIDS patients coinfected with KSHV.
Sulfated Tyrosines Contribute to the Formation of the C5a Docking Site of the Human C5a Anaphylatoxin Receptor
Authors: Farzan Michael , Schnitzler Christine E. , Vasilieva Natalya , Leung Doris , Kuhn Jens , Gerard Craig , Gerard Norma P. , Choe Hyeryun
Published: July 2002

The complement anaphylatoxin C5a and its seven-transmembrane segment (7TMS) receptor play an important role in host defense and in a number of inflammation-associated pathologies. The NH2-terminal domain of the C5a receptor (C5aR/CD88) contributes substantially to its ability to bind C5a. Here we show that the tyrosines at positions 11 and 14 of the C5aR are posttranslationally modified by the addition of sulfate groups. The sulfate moieties of each of these tyrosines are critical to the ability of the C5aR to bind C5a and to mobilize calcium. A C5aR variant lacking these sulfate moieties efficiently mobilized calcium in response to a small peptide agonist, but not to C5a, consistent with a two-site model of ligand association in which the tyrosine-sulfated region of the C5aR mediates the initial docking interaction. A peptide based on the NH2 terminus of the C5aR and sulfated at these two tyrosines, but not its unsulfated analogue or a doubly sulfated control peptide, partially inhibited C5a association with its receptor. These observations clarify structural and mutagenic studies of the C5a/C5aR association and suggest that related 7TMS receptors are also modified by functionally important sulfate groups on their NH2-terminal tyrosines.
Constitutively Activated Akt-1 Is Vital for the Survival of Human Monocyte-Differentiated Macrophages
Authors: Liu Hongtao , Perlman Harris , Pagliari Lisa J. , Pope Richard M.
Published: July 2002

Recent data from mice deficient for phosphatase and tensin homologue deleted from chromosome 10 or src homology 2 domain–containing 5′ inositol phosphatase, phosphatases that negatively regulate the phosphatidylinositol 3-kinase (PI3K) pathway, revealed an increased number of macrophages in these animals, suggesting an essential role for the PI3K pathway for macro-phage survival. Here, we focused on the role of the PI3K-regulated serine/threonine kinase Akt-1 in modulating macrophage survival. Akt-1 was constitutively activated in human macrophages and addition of the PI3K inhibitor, LY294002, suppressed the activation of Akt-1 and induced cell death. Furthermore, suppression of Akt-1 by inhibition of PI3K or a dominant negative (DN) Akt-1 resulted in loss of mitochondrial transmembrane potential, activation of caspases-9 and -3, and DNA fragmentation. The effects of PI3K inhibition were reversed by the ectopic expression of constitutively activated Akt-1 or Bcl-xL. Inhibition of PI3K/Akt-1 pathway either by LY294002 or DN Akt-1 had no effect on the constitutive or inducible activation of nuclear factor (NF)-κB in human macrophages. However, after inhibition of the PI3K/Akt-1 pathway, a marked decrease in the expression of the antiapoptotic molecule Mcl-1, but not other Bcl-2 family members was observed, and Mcl-1 rescued macrophages from LY294002-induced cell death. Further, inhibition of Mcl-1 by antisense oligonucleotides, also resulted in macrophage apoptosis. Thus, our findings demonstrate that the constitutive activation of Akt-1 regulates macrophage survival through Mcl-1, which is independent of caspases, NF-κB, or Bad.
Knock-in Mutation of the Distal Four Tyrosines of Linker for Activation of T Cells Blocks Murine T Cell Development
Authors: Sommers Connie L. , Menon Rashmi K. , Grinberg Alexander , Zhang Weiguo , Samelson Lawrence E. , Love Paul E.
Published: July 2002

The integral membrane adapter protein linker for activation of T cells (LAT) performs a critical function in T cell antigen receptor (TCR) signal transduction by coupling the TCR to downstream signaling pathways. After TCR engagement, LAT is tyrosine phosphorylated by ZAP-70 creating docking sites for multiple src homology 2–containing effector proteins. In the Jurkat T cell line, the distal four tyrosines of LAT bind PLCγ-1, Grb2, and Gads. Mutation of these four tyrosine residues to phenylalanine (4YF) blocked TCR-mediated calcium mobilization, Erk activation, and nuclear factor (NF)-AT activation. In this study, we examined whether these four tyrosine residues were essential for T cell development by generating LAT “knock-in” mutant mice that express the 4YF mutant protein under the control of endogenous LAT regulatory sequences. Significantly, the phenotype of 4YF knock-in mice was identical to LAT−/− (null) mice; thymocyte development was arrested at the immature CD4−CD8− stage and no mature T cells were present. Knock-in mice expressing wild-type LAT protein, generated by a similar strategy, displayed a normal T cell developmental profile. These results demonstrate that the distal four tyrosine residues of LAT are essential for preTCR signaling and T cell development in vivo.
Somatic Hypermutation Shapes the Antibody Repertoire of Memory B Cells in Humans
Authors: Meffre Eric , Catalan Nadia , Seltz Françoise , Fischer Alain , Nussenzweig Michel C. , Durandy Anne
Published: July 2002

High-affinity antibodies produced by memory B cells differ from antibodies produced in naive B cells in two respects. First, many of these antibodies show somatic hypermutation, and second, the repertoire of antibodies expressed in memory responses is highly selected. To determine whether somatic hypermutation is responsible for the shift in the antibody repertoire during affinity maturation, we analyzed the immunoglobulin lambda light chain (Igλ) repertoire expressed by naive and antigen-selected memory B cells in humans. We found that the Igλ repertoire differs between naive and memory B cells and that this shift in the repertoire does not occur in the absence of somatic hypermutation in patients lacking activation-induced cytidine deaminase (AID). Our work suggests that somatic hypermutation makes a significant contribution to shaping the antigen-selected antibody repertoire in humans.
Identification of the Transferrin Receptor as a Novel Immunoglobulin (Ig)a1 Receptor and Its Enhanced Expression on Mesangial Cells in Iga Nephropathy
Authors: Moura Ivan C. , Centelles Miguel N. , Arcos-Fajardo Michelle , Malheiros Denise M. , Collawn James F. , Cooper Max D. , Monteiro Renato C.
Published: July 2002

The biological functions of immunoglobulin (Ig)A antibodies depend primarily on their interaction with cell surface receptors. Four IgA receptors are presently characterized. The FcαRI (CD89) expressed by myeloid cells selectively binds IgA1 and IgA2 antibodies, whereas the poly-IgR, Fcα/μR, and asialoglycoprotein receptors bind other ligands in addition to IgA. IgA binding by mesangial cells, epithelial cells, and proliferating lymphocytes is also well documented, but the nature of the IgA receptors on these cells remains elusive. A monoclonal antibody (A24) is described here that specifically blocks IgA binding to epithelial and B lymphocyte cell lines. Both the A24 antibody and IgA1 myelomas bind a cell surface protein that is identified as the transferrin receptor (CD71). The transferrin receptor selectively binds IgA1 antibodies, monomeric better than polymeric forms, and the IgA1 binding is inhibitable by transferrin. Transferrin receptor expression is upregulated on cultured mesangial cells as well as on glomerular mesangial cells in patients with IgA nephropathy. The characterization of transferrin receptor as a novel IgA1 receptor on renal mesangial cells suggests its potential involvement in the pathogenesis of IgA nephropathy.
The Stress Kinase Mitogen-Activated Protein Kinase Kinase (Mkk)7 Is a Negative Regulator of Antigen Receptor and Growth Factor Receptor–Induced Proliferation in Hematopoietic Cells
Authors: Sasaki Takehiko , Wada Teiji , Kishimoto Hiroyuki , Irie-Sasaki Junko , Matsumoto Goichi , Goto Takayuki , Yao Zhengbin , Wakeham Andrew , Mak Tak W. , Suzuki Akira , Cho Sarah K. , Zuniga-Pflucker Juan Carlos , Oliveira-dos-Santos Antonio J. , Katada Toshiaki , Nishina Hiroshi , Penninger Josef M.
Published: July 2002

The dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7−/− mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal–regulated kinase (ERK)1/2 or p38 MAPK. mkk7−/− mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7−/− mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor–driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.
Intrinsic Defect in T Cell Production of Interleukin (IL)-13 in the Absence of Both IL-5 and Eotaxin Precludes the Development of Eosinophilia and Airways Hyperreactivity in Experimental Asthma
Authors: Mattes Joerg , Yang Ming , Mahalingam Surendran , Kuehr Joachim , Webb Dianne C. , Simson Ljubov , Hogan Simon P. , Koskinen Aulikki , McKenzie Andrew N.J. , Dent Lindsay A. , Rothenberg Marc E. , Matthaei Klaus I. , Young Ian G. , Foster Paul S.
Published: July 2002

Interleukin (IL)-5 and IL-13 are thought to play key roles in the pathogenesis of asthma. Although both cytokines use eotaxin to regulate eosinophilia, IL-13 is thought to operate a separate pathway to IL-5 to induce airways hyperreactivity (AHR) in the allergic lung. However, identification of the key pathway(s) used by IL-5 and IL-13 in the disease process is confounded by the failure of anti–IL-5 or anti–IL-13 treatments to completely inhibit the accumulation of eosinophils in lung tissue. By using mice deficient in both IL-5 and eotaxin (IL-5/eotaxin−/−) we have abolished tissue eosinophilia and the induction of AHR in the allergic lung. Notably, in mice deficient in IL-5/eotaxin the ability of CD4+ T helper cell (Th)2 lymphocytes to produce IL-13, a critical regulator of airways smooth muscle constriction and obstruction, was significantly impaired. Moreover, the transfer of eosinophils to IL-5/eotaxin−/− mice overcame the intrinsic defect in T cell IL-13 production. Thus, factors produced by eosinophils may either directly or indirectly modulate the production of IL-13 during Th2 cell development. Our data show that IL-5 and eotaxin intrinsically modulate IL-13 production from Th2 cells and that these signaling systems are not necessarily independent effector pathways and may also be integrated to regulate aspects of allergic disease.
Different Mechanisms of Ca2+ Transport in NMDA and Ca2+-permeable AMPA Glutamate Receptor Channels
Authors: Wollmuth Lonnie P. , Sakmann Bert
Published: July 2002

The channel of the glutamate N-methyl-d-aspartate receptor (NMDAR) transports Ca2+ approximately four times more efficiently than that of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs+ efflux and Ca2+ influx in recombinant NMDAR and Ca2+-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca2+ using whole-cell currents and fluorometric techniques (dye overload) with Cs+ internally and Ca2+ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was ∼2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca2+ and Cs+ as the reference ions, Ca2+ permeability (PCa/PCs) was concentration dependent, being highest around physiological concentrations (1–1.8 mM; PCa/PCs ≈ 6.1) and reduced at both higher (110 mM; PCa/PCs ≈ 2.6) and lower (0.18 mM; PCa/PCs ≈ 2.2) concentrations. PCa/PCs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3–110 mM Ca2+. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca2+ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca2+ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca2+ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca2+, whereas that of AMPAR channels only a single site.
Activation of Ca2+-Dependent K+ Channels Contributes to Rhythmic Firing of Action Potentials in Mouse Pancreatic β Cells
Authors: Göpel Sven O. , Kanno Takahiro , Barg Sebastian , Eliasson Lena , Galvanovskis Juris , Renström Erik , Rorsman Patrik
Published: July 2002

We have applied the perforated patch whole-cell technique to β cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K+ conductance. The current was dependent on Ca2+ influx but unaffected by apamin and charybdotoxin, two blockers of Ca2+-activated K+ channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K+ channels) but partially (>60%) blocked by high (10–20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca2+-activated K+ current plays an important role in the generation of oscillatory electrical activity in the β cell.
The groups of order at most 2000
Authors: Besche Hans , Eick Bettina , O’Brien E.
Published: July 2002

We announce the construction up to isomorphism of the

49

910

529

484

49\,910\,529\,484

groups of order at most 2000.
A complete Vinogradov 3-primes theorem under the Riemann hypothesis
Authors: Deshouillers J.-M. , Effinger G. , te Riele H. , Zinoviev D.
Published: July 2002

We outline a proof that if the Generalized Riemann Hypothesis holds, then every odd number above

5
5

is a sum of three prime numbers. The proof involves an asymptotic theorem covering all but a finite number of cases, an intermediate lemma, and an extensive computation.
Rab27a Is an Essential Component of Melanosome Receptor for Myosin Va
Authors: Wu Xufeng , Wang Fei , Rao Kang , Sellers James R. , Hammer John A.
Published: July 2002

Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface depends on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a functions wholly or in part as the melanosome receptor for myosin Va (Myo5a). Herein, we show that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null (dilute) phenotype in wild-type melanocytes is absolutely dependent on the presence of exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue their distribution indilute melanocytes requires exon F but not exon D. These results predict that an interaction between myosin Va and Rab27a should be exon F dependent. Consistent with this, Rab27a present in detergent lysates of melanocytes binds to beads coated with purified, full-length melanocyte myosin Va and melanocyte myosin Va lacking exon D, but not to beads coated with melanocyte myosin Va lacking exon F or brain myosin Va. Moreover, the preparation of melanocyte lysates in the presence of GDP rather than guanosine-5′-O-(3-thio)triphosphate reduces the amount of Rab27a bound to melanocyte myosin Va-coated beads by approximately fourfold. Finally, pure Rab27a does not bind to myosin Va-coated beads, suggesting that these two proteins interact indirectly. Together, these results argue that Rab27a is an essential component of a protein complex that serves as the melanosome receptor for myosin Va, suggest that this complex contains at least one additional protein capable of bridging the indirect interaction between Rab27a and myosin Va, and imply that the recruitment of myosin Va to the melanosome surface in vivo should be regulated by factors controlling the nucleotide state of Rab27a.
Formal design and implementation of an improved DDBJ DNA database with a new schema and object-oriented library.
Authors: Okayama T , Tamura T , Gojobori T , Tateno Y , Ikeo K , Miyazaki S , Fukami-Kobayashi K , Sugawara H
Published: July 2002

MOTIVATION: The DNA Data Bank of Japan (DDBJ) has developed a new DNA database system with a new schema design to accommodate rapid change and growth of requirements on the system. RESULTS: The new schema and systems were created using an object-oriented design approach. The design was accomplished in accordance with ANSI/SPARC three-level schema architecture. First, the conceptual schema was designed using a functional model named AIS (associative information structure) and was visualized in extended diagram format. The model is a natural extension of an ER (entity relationship) model and describes real-world objects in binary associations between entities with the concept of order. Second, the schema was mapped on a relational database as a physical schema. All details are concentrated in this schema and the layer lying above enjoys physical independence. Finally, as another layer, external modeling was introduced for the database applications interface. It provides set-at-a-time basis operations and was implemented as a C++ object-oriented library. On this common framework of a new schema, a new annotator's workbench named Yamato II and a World Wide Web (WWW) submission system named Sakura have been successfully developed to improve drastically daily transactions in the DDBJ. AVAILABILITY: Sakura is available at the following address: http://sakura.ddbj.nig.ac.jp. CONTACT: hsugawar@genes.nig.ac.jp
LIGAND: chemical database for enzyme reactions.
Authors: Goto S , Nishioka T , Kanehisa M
Published: July 2002

MOTIVATION: The existing molecular biology databases focus on the sequence and structural aspects of biological macromolecules, i.e. DNAs, RNAs and proteins. However, in order to understand the functional aspects, it is essential to computerize the interaction of these molecules. Furthermore, living cells contain additional molecules, such as metabolic compounds and metal ions, that may also be considered as parts of the basic building blocks of life, but are not well organized in public databases. LIGAND chemical database is our attempt to solve these problems, at least for enzymatic reactions. RESULTS: LIGAND consists of two sections: ENZYME and COMPOUND. The ENZYME section is an extension of previous studies (Suyama et al. , Comput. Applic. Biosci., 9, 9-15, 1993), and it is a flat-file representation of 3303 enzymes and 2976 enzymatic reactions in the chemical equation format that can be parsed by machine. The COMPOUND section has been newly constructed for information on the nomenclature and chemical structures of compounds. It contains 5383 chemical compounds. Both ENZYME and COMPOUND entries contain rich cross-reference information, most of which is automatically generated by the DBGET/LinkDB system, thus providing the linkage between chemical and biological databases. LIGAND is updated daily, tightly coupled with the KEGG metabolic pathway database, and forms the basis for reconstruction and computation of pathways. AVAILABILITY: LIGAND can be accessed through the DBGET/LinkDB and KEGG systems in the Japanese GenomeNet database service via http://www.genome.ad.jp/. The flat-file format of the LIGAND database can be downloaded by anonymous FTP via ftp://kegg. genome.adjp/molecules/ligand/. CONTACT: goto@kuicr.kyoto-u.ac.jp; nishioka@scl.kyoto-u.ac.jp; kanehisa@kuicr.kyoto-u.ac.jp