bluuebunny/crossref_metadata_2025 huggingface dataset:
Electromechanical Coupling between Skeletal and Cardiac Muscle
Authors: Reinecke Hans , MacDonald Glen H. , Hauschka Stephen D. , Murry Charles E.
Published: July 2002

Skeletal myoblasts form grafts of mature muscle in injured hearts, and these grafts contract when exogenously stimulated. It is not known, however, whether cardiac muscle can form electromechanical junctions with skeletal muscle and induce its synchronous contraction. Here, we report that undifferentiated rat skeletal myoblasts expressed N-cadherin and connexin43, major adhesion and gap junction proteins of the intercalated disk, yet both proteins were markedly downregulated after differentiation into myo-tubes. Similarly, differentiated skeletal muscle grafts in injured hearts had no detectable N-cadherin or connexin43; hence, electromechanical coupling did not occur after in vivo grafting. In contrast, when neonatal or adult cardiomyocytes were cocultured with skeletal muscle, ∼10% of the skeletal myotubes contracted in synchrony with adjacent cardiomyocytes. Isoproterenol increased myotube contraction rates by 25% in coculture without affecting myotubes in monoculture, indicating the cardiomyocytes were the pacemakers. The gap junction inhibitor heptanol aborted myotube contractions but left spontaneous contractions of individual cardiomyocytes intact, suggesting myotubes were activated via gap junctions. Confocal microscopy revealed the expression of cadherin and connexin43 at junctions between myotubes and neonatal or adult cardiomyocytes in vitro. After microinjection, myotubes transferred dye to neonatal cardiomyocytes via gap junctions. Calcium imaging revealed synchronous calcium transients in cardiomyocytes and myotubes. Thus, cardiomyocytes can form electromechanical junctions with some skeletal myotubes in coculture and induce their synchronous contraction via gap junctions. Although the mechanism remains to be determined, if similar junctions could be induced in vivo, they might be sufficient to make skeletal muscle grafts beat synchronously with host myocardium.
Distinct Roles of Rock (Rho-Kinase) and Mlck in Spatial Regulation of Mlc Phosphorylation for Assembly of Stress Fibers and Focal Adhesions in 3t3 Fibroblasts
Authors: Totsukawa Go , Yamakita Yoshihiko , Yamashiro Shigeko , Hartshorne David J. , Sasaki Yasuharu , Matsumura Fumio
Published: July 2002

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions.

We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.
Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit
Authors: Ho Jennifer Hei-Ngam , Kallstrom George , Johnson Arlen W.
Published: July 2002

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.
The Role of Macrophages in Demyelinating Peripheral Nervous System of Mice Heterozygously Deficient in P0
Authors: Carenini Stefano , Mäurer Mathias , Werner Alexander , Blazyca Heinrich , Toyka Klaus V. , Schmid Christoph D. , Raivich Gennadij , Martini Rudolf
Published: July 2002

Mice heterozygously deficient in the p0 gene (P0+/−) are animal models for some forms of inherited neuropathies. They display a progressive demyelinating phenotype in motor nerves, accompanied by mild infiltration of lymphocytes and increase in macrophages. We have shown previously that the T lymphocytes are instrumental in the demyelination process. This study addresses the functional role of the macrophage in this monogenic myelin disorder.

In motor nerves of P0+/− mice, the number of macrophages in demyelinated peripheral nerves was increased by a factor of five when compared with motor nerves of wild-type mice. Immunoelectron microscopy, using a specific marker for mouse macrophages, displayed macrophages not only in the endoneurium of the myelin mutants, but also within endoneurial tubes, suggesting an active role in demyelination. To elucidate the roles of the macrophages, we crossbred the myelin mutants with a spontaneous mouse mutant deficient in macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the P0-deficient double mutants also deficient in M-CSF, the numbers of macrophages were not elevated in the demyelinating motor nerves and demyelination was less severe. These findings demonstrate an active role of macrophages during pathogenesis of inherited demyelination with putative impact on future treatment strategies.
Integrin-Mediated Adhesion Regulates ERK Nuclear Translocation and Phosphorylation of Elk-1
Authors: Aplin Andrew E. , Stewart Sheryl A. , Assoian Richard K. , Juliano R.L.
Published: July 2002

Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal–regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1–mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.
Toxoplasma gondii myosins B/C
Authors: Delbac Frédéric , Sänger Astrid , Neuhaus Eva M. , Stratmann Rolf , Ajioka James W. , Toursel Catherine , Herm-Götz Angelika , Tomavo Stanislas , Soldati Thierry , Soldati Dominique
Published: July 2002

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.
Expression of α-catenin in α-catenin–deficient cells increases resistance to sphingosine-induced apoptosis
Authors: Matsubara Shyuichiro , Ozawa Masayuki
Published: July 2002

α-Catenin, an intracellular protein, associates with the COOH-terminal region of cadherin cell adhesion molecules through interactions with either β-catenin or γ-catenin (plakoglobin). The full activity of cadherins requires a linkage to the actin cytoskeleton mediated by catenins. We transfected α-catenin–deficient colon carcinoma cells with a series of α-catenin constructs to determine that α-catenin expression increases the resistance to apoptosis induced by sphingosine. Two groups of constructs, containing deletions in either the middle segment of the molecule or the COOH terminus, induced morphological changes, cell compaction, and decreases in cell death. In α-catenin–expressing cells, inhibition of cadherin cell adhesion by treatment with anti–E-cadherin antibodies did not decrease the cells viability. α-Catenin expression partially suppressed the downregulation of Bcl-xL and the activation of caspase 3. Expression of p27kip1 protein, an inhibitor of cyclin-dependent kinases, was increased by α-catenin expression in low density cell cultures. The increased levels of p27kip1 correlated with both increased resistance to cell death and morphological changes in transfectants containing deletion mutants. Transfection-mediated upregulation of p27kip1 decreases sphingosine-induced cell death in α-catenin–deficient cells. We postulate that α-catenin mediates transduction of signals from the cadherin–catenin complex to regulate the apoptotic cascade via p27kip1.
E-cadherin regulates cell growth by modulating proliferation-dependent β-catenin transcriptional activity
Authors: Stockinger Andreas , Eger Andreas , Wolf Julia , Beug Hartmut , Foisner Roland
Published: July 2002

β-Catenin is essential for E-cadherin–mediated cell adhesion in epithelial cells, but it also forms nuclear complexes with high mobility group transcription factors. Using a mouse mammary epithelial cell system, we have shown previously that conversion of epithelial cells to a fibroblastoid phenotype (epithelial-mesenchymal transition) involves downregulation of E-cadherin and upregulation of β-catenin transcriptional activity. Here, we demonstrate that transient expression of exogenous E-cadherin in both epithelial and fibroblastoid cells arrested cell growth or caused apoptosis, depending on the cellular E-cadherin levels. By expressing E-cadherin subdomains, we show that the growth-suppressive effect of E-cadherin required the presence of its cytoplasmic β-catenin interaction domain and/or correlated strictly with the ability to negatively interfere with β-catenin transcriptional activity. Furthermore, coexpression of β-catenin or lymphoid enhancer binding factor-1 or T cell factor 3 with E-cadherin rescued β-catenin transcriptional activity and counteracted E-cadherin–mediated cell cycle arrest. Stable expression of E-cadherin in fibroblastoid cells decreased β-catenin activity and reduced cell growth. Since proliferating cells had a higher β-catenin activity than G1 phase–arrested or contact-inhibited cells, we conclude that β-catenin transcriptional activity is essential for cell proliferation and can be controlled by E-cadherin in a cell adhesion-independent manner.
Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)–related protein Sli15 during chromosome segregation
Authors: Kang Jung-seog , Cheeseman Iain M. , Kallstrom George , Velmurugan Soundarapandian , Barnes Georjana , Chan Clarence S.M.
Published: July 2002

We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1–Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome–spindle interactions or in the movement of kinetochores along microtubules.
Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole
Authors: van Eeden Fredericus J.M. , Palacios Isabel M. , Petronczki Mark , Weston Matthew J.D. , St Johnston Daniel
Published: July 2002

The localization of Oskar at the posterior pole of the Drosophila oocyte induces the assembly of the pole plasm and therefore defines where the abdomen and germ cells form in the embryo. This localization is achieved by the targeting of oskar mRNA to the posterior and the localized activation of its translation. oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus end–directed motor kinesin I, but nothing is known about how the RNA is coupled to the motor. Here, we describe barentsz, a novel gene required for the localization of oskar mRNA. In contrast to all other mutations that disrupt this process, barentsz-null mutants completely block the posterior localization of oskar mRNA without affecting bicoid and gurken mRNA localization, the organization of the microtubules, or subsequent steps in pole plasm assembly. Surprisingly, most mutant embryos still form an abdomen, indicating that oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with oskar mRNA, and this localization is oskar mRNA dependent. Thus, Barentsz is essential for the posterior localization of oskar mRNA and behaves as a specific component of the oskar RNA transport complex.
Treatment of Experimental Autoimmune Encephalomyelitis with Genetically Modified Memory T Cells
Authors: Mathisen Peter M. , Yu Min , Johnson Justin M. , Drazba Judith A. , Tuohy Vincent K.
Published: July 2002

The migratory properties of memory T cells provide a model vector system for site-specific delivery of therapeutic transgene factors to autoimmune inflammatory lesions. Lymph node cells from (SWR×SJL)F1 mice immunized with the p139–151 determinant of myelin proteolipid protein (PLP) were transfected with a DNA construct that placed the anti-inflammatory cytokine interleukin-10 (IL-10) cDNA under control of an antigen-inducible IL-2 promoter region. Isolated T cell clones demonstrated antigen-inducible expression of transgene IL-10 and expressed cell surface markers consistent with the phenotype of normal memory T cells. Upon adoptive transfer, transfected T cell clones were able to inhibit onset of experimental autoimmune encephalomyelitis (EAE) and to treat EAE animals therapeutically after onset of neurologic signs. Semiquantitative immunocytochemistry showed a significant correlation between decreased demyelination and treatment with the transfected T cells. Taken together, these data indicate the autoreactive T cells can be genetically designed to produce therapeutic factors in an antigen-inducible manner resulting in a decreased severity of clinical and histological autoimmune demyelinating disease.
Enhancer Complexes Located Downstream of Both Human Immunoglobulin Cα Genes
Authors: Mills Frederick C. , Harindranath Nagaradona , Mitchell Mary , Max Edward E.
Published: July 2002

To investigate regulation of human immunoglobulin heavy chain expression, we have cloned DNA downstream from the two human Cα genes, corresponding to the position in the mouse IgH cluster of a locus control region (LCR) that includes an enhancer which regulates isotype switching. Within 25 kb downstream of both the human immunoglobulin Cα1 and Cα2 genes we identified several segments of DNA which display B lymphoid–specific DNase I hypersensitivity as well as enhancer activity in transient transfections. The corresponding sequences downstream from each of the two human Cα genes are nearly identical to each other. These enhancers are also homologous to three regions which lie in similar positions downstream from the murine Cα gene and form the murine LCR. The strongest enhancers in both mouse and human have been designated HS12. Within a 135-bp core homology region, the human HS12 enhancers are ∼90% identical to the murine homolog and include several motifs previously demonstrated to be important for function of the murine enhancer; additional segments of high sequence conservation suggest the possibility of previously unrecognized functional motifs. On the other hand, certain functional elements in the murine enhancer, including a B cell–specific activator protein site, do not appear to be conserved in human HS12. The human homologs of the murine enhancers designated HS3 and HS4 show lower overall sequence conservation, but for at least two of the functional motifs in the murine HS4 (a κB site and an octamer motif  ) the human HS4 homologs are exactly conserved. An additional hypersensitivity site between human HS3 and HS12 in each human locus displays no enhancer activity on its own, but includes a region of high sequence conservation with mouse, suggesting the possibility of another novel functional element.
Eosinophil Lipid Bodies: Specific, Inducible Intracellular Sites for Enhanced Eicosanoid Formation
Authors: Bozza Patricia T. , Yu Wengui , Penrose John F. , Morgan Ellen S. , Dvorak Ann M. , Weller Peter F.
Published: July 2002

The specific intracellular sites at which enzymes act to generate arachidonate-derived eicosanoid mediators of inflammation are uncertain. We evaluated the formation and function of cytoplasmic lipid bodies. Lipid body formation in eosinophils was a rapidly (<1 h) inducible response which was platelet-activating factor (PAF) receptor–mediated, involved signaling through protein kinase C, and required new protein synthesis. In intact and enucleated eosinophils, the PAF-induced increases in lipid body numbers correlated with enhanced production of both lipoxygenase- and cyclooxygenase-derived eicosanoids. All principal eosinophil eicosanoid-forming enzymes, 5-lipoxygenase, leukotriene C4 synthase, and cyclooxygenase, were immunolocalized to native as well as newly induced lipid bodies in intact and enucleated eosinophils. Thus, lipid bodies are structurally distinct, inducible, nonnuclear sites for enhanced synthesis of paracrine eicosanoid mediators of inflammation.
Contribution of  Virus-specific CD8+ Cytotoxic T Cells to Virus Clearance or Pathologic Manifestations of Influenza Virus Infection in a T Cell Receptor Transgenic Mouse Model
Authors: Moskophidis Demetrius , Kioussis Dimitris
Published: July 2002

The ability of influenza virus to evade immune surveillance by neutralizing antibodies (Abs) directed against its variable surface antigens provides a challenge to the development of effective vaccines. CD8+ cytotoxic T lymphocytes (CTLs) restricted by class I major histocompatibility complex molecules are important in establishing immunity to influenza virus because they recognize internal viral proteins which are conserved between multiple viral strains. In contrast, protective Abs are strain-specific. However, the precise role of effector CD8+ CTLs in protection from influenza virus infection, critical for understanding disease pathogenesis, has not been well defined. In transgenic mice with a very high frequency of antiinfluenza CTL precursors, but without protective Abs, CD8+ CTLs conferred protection against low dose viral challenge, but exacerbated viral pathology and caused mortality at high viral dose. The data suggest a dual role for CD8+ CTLs against influenza, which may present a challenge to the development of effective CTL vaccines. Effector mechanisms used by CD8+ CTLs in orchestrating clearance of virus and recovery from experimental influenza infection, or potentiation of lethal pathology, are discussed.
Structure and Expression of an Adhesive Protein–like Molecule of Mosquito Invasive-stage Malarial Parasite
Authors: Yuda Masao , Sawai Toshiki , Chinzei Yasuo
Published: July 2002

Invasion of the malarial parasite into a vector mosquito begins when the motile ookinete transverses the gut epithelium. Adhesive proteins that may mediate this invasive process have not been identified to date. We found that a molecule with an adhesive protein–like structure was expressed in the ookinete of Plasmodium berghei. This protein is structurally homologous to circumsporozoite protein and thrombospondin-related adhesive protein (TRAP)-related protein, CTRP, of Plasmodium falciparum. We named it P. berghei CTRP (PbCTRP) and report here its structure and manner of expression. PbCTRP has six integrin I region–like domains and seven thrombospondin-like domains in its putative extracellular region. This structure is similar to that of CTRP and TRAPs of malaria sporozoite. The putative transmembrane and cytoplasmic regions of PbCTRP, CTRP, and TRAP also have conserved amino acid sequences. PbCTRP is produced at least 10 h after fertilization when zygotes begin transformation to ookinetes. In the mature ookinete, PbCTRP is located mainly in the anterior cytoplasm. The staining pattern was also similar to TRAP in the sporozoite. We suggest that PbCTRP may play a role in ookinete invasive motility and belongs to a protein family together with TRAP and other structurally related proteins of apicomplexan parasites.
The Glycoprotein Ib-IX-V Complex Is a Platelet Counterreceptor for P-Selectin
Authors: Romo Gabriel M. , Dong Jing-Fei , Schade Alicia J. , Gardiner Elizabeth E. , Kansas Geoffrey S. , Li Chester Q. , McIntire Larry V. , Berndt Michael C. , López José A.
Published: July 2002

We have identified platelet glycoprotein (GP) Ibα as a counterreceptor for P-selectin. GP Ibα is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibα, and GP Ibα–expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin–dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibα. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibα required neither calcium nor carbohydrate core-2 branching or α(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibα, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.
Trance, a Tumor Necrosis Factor Family Member, Enhances the Longevity and Adjuvant Properties of Dendritic Cells in Vivo
Authors: Josien Régis , Li Hong-Li , Ingulli Elizabeth , Sarma Supria , R.Wong Brian , Vologodskaia Maria , Steinman Ralph M. , Choi Yongwon
Published: July 2002

Mature dendritic cells (DCs) are powerful antigen presenting cells that have the unique capacity to migrate to the T cell zone of draining lymph nodes after subcutaneous injection. Here we report that treatment of antigen-pulsed mature DCs with tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a TNF family member, before immunization enhances their adjuvant capacity and elicits improved T cell priming in vivo, such that both primary and memory T cell immune responses are enhanced. By enumerating migratory DCs in the draining lymph nodes and by studying their function in stimulating naive T cells, we show that one of the underlying mechanisms for enhanced T cell responses is an increase in the number of ex vivo antigen-pulsed DCs that are found in the T cell areas of lymph nodes. These results suggest that the longevity and abundance of mature DCs at the site of T cell priming influence the strength of the DC-initiated T cell immunity in situ. Our findings have the potential to improve DC-based immunotherapy; i.e., the active immunization of humans with autologous DCs that have been pulsed with clinically significant antigens ex vivo.
Association between Kinin B1 Receptor Expression and Leukocyte Trafficking across Mouse Mesenteric Postcapillary Venules
Authors: McLean Peter G. , Ahluwalia Amrita , Perretti Mauro
Published: July 2002

Using intravital microscopy, we examined the role played by B1 receptors in leukocyte trafficking across mouse mesenteric postcapillary venules in vivo. B1 receptor blockade attenuated interleukin (IL)-1β–induced (5 ng intraperitoneally, 2 h) leukocyte–endothelial cell interactions and leukocyte emigration (∼50% reduction). The B1 receptor agonist des-Arg9bradykinin (DABK), although inactive in saline- or IL-8–treated mice, caused marked neutrophil rolling, adhesion, and emigration 24 h after challenge with IL-1β (when the cellular response to IL-1β had subsided). Reverse transcriptase polymerase chain reaction and Western blot revealed a temporal association between the DABK-induced response and upregulation of mesenteric B1 receptor mRNA and de novo protein expression after IL-1β treatment. DABK-induced leukocyte trafficking was antagonized by the B1 receptor antagonist des-arg10HOE 140 but not by the B2 receptor antagonist HOE 140. Similarly, DABK effects were maintained in B2 receptor knockout mice. The DABK-induced responses involved the release of neuropeptides from C fibers, as capsaicin treatment inhibited the responses. Treatment with the neurokinin (NK)1 and NK3 receptor antagonists attenuated the responses, whereas NK2, calcitonin gene-related peptide, or platelet-activating factor receptor antagonists had no effect. Substance P caused leukocyte recruitment that, similar to DABK, was inhibited by NK1 and NK3 receptor blockade. Mast cell depletion using compound 48/80 reduced DABK-induced leukocyte trafficking, and DABK treatment was shown histologically to induce mast cell degranulation. DABK-induced trafficking was inhibited by histamine H1 receptor blockade. Our findings provide clear evidence that B1 receptors play an important role in the mediation of leukocyte–endothelial cell interactions in postcapillary venules, leading to leukocyte recruitment during an inflammatory response. This involves activation of C fibers and mast cells, release of substance P and histamine, and stimulation of NK1, NK3, and H1 receptors.
Human Cd25+Cd4+ T Regulatory Cells Suppress Naive and Memory T Cell Proliferation and Can Be Expanded in Vitro without Loss of Function
Authors: Levings Megan K. , Sangregorio Romina , Roncarolo Maria-Grazia
Published: July 2002

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4+ Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25+CD4+ Tr cells in humans has not been reported. Here we show that human CD25+CD4+ Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25+CD4+ Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25+CD4+ Tr cells strongly inhibited the proliferative responses of both naive and memory CD4+ T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25+CD4+ Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25+CD4+ Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.
Signal Transducer and Activator of Transcription 6 Controls Chemokine Production and T Helper Cell Type 2 Cell Trafficking in Allergic Pulmonary Inflammation
Authors: Mathew Anuja , MacLean James A. , DeHaan Elliot , Tager Andrew M. , Green Francis H.Y. , Luster Andrew D.
Published: July 2002

Antigen-specific CD4 T helper type 2 (Th2) cells play a pivotal role in the induction of allergic asthma, but the mechanisms regulating their recruitment into the airways are unknown. Signal transducer and activator of transcription factor (Stat)6 is a transcription factor essential for Th2 cell differentiation. Here we show that Stat6 also controls Th2 cell recruitment and effector function in allergic inflammation in vivo. To isolate the role of Stat6 in regulating Th2 cell trafficking and effector function from its role in Th2 cell differentiation, we used a murine model of asthma in which in vitro–differentiated Stat6+/+ antigen-specific Th2 cells were adoptively transferred into naive Stat6−/− and Stat6+/+ mice followed by aerosol antigen challenge. We found that all of the features of asthma, including Th2 cell accumulation, Th2 and eosinophil-active chemokine production, and airway eosinophilia, mucus production, and hyperresponsiveness seen in Stat6+/+ mice, were dramatically absent in Stat6−/− mice that received Stat6+/+ antigen-specific Th2 cells. Our findings establish Stat6 as essential for Th2 cell trafficking and effector function and suggest that interruption of Stat6 signaling in resident cells of the lung is a novel approach to asthma therapy.
Discrete Generation of Superoxide and Hydrogen Peroxide by T Cell Receptor Stimulation
Authors: Devadas Satish , Zaritskaya Luba , Rhee Sue Goo , Oberley Larry , Williams Mark S.
Published: July 2002

Receptor-stimulated generation of reactive oxygen species (ROS) has been shown to regulate signal transduction, and previous studies have suggested that T cell receptor (TCR) signals may involve or be sensitive to ROS. In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes. Furthermore, the data suggest the novel observation that superoxide anion and hydrogen peroxide are produced separately by distinct TCR-stimulated pathways. Unexpectedly, TCR-stimulated activation of the Fas ligand (FasL) promoter and subsequent cell death was dependent upon superoxide anion, but independent of hydrogen peroxide, while nuclear factor of activated T cells (NFAT) activation or interleukin 2 transcription was independent of all ROS. Anti-CD3 induced phosphorylation of extracellular signal–regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion. Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).
Regulation of KATP Channel Activity by Diazoxide and MgADP
Authors: Shyng S.-L. , Ferrigni T. , Nichols C.G.
Published: July 2002

KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945–951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4−. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.
Approximation with interpolatory constraints
Authors: Mhaskar H. , Narcowich F. , Sivakumar N. , Ward J.
Published: July 2002

Given a triangular array of points on

[
−
1
,
1
]

[-1,1]

satisfying certain minimal separation conditions, a classical theorem of Szabados asserts the existence of polynomial operators that provide interpolation at these points as well as a near-optimal degree of approximation for arbitrary continuous functions on the interval. This paper provides a simple, functional-analytic proof of this fact. This abstract technique also leads to similar results in general situations where an analogue of the classical Jackson-type theorem holds. In particular, it allows one to obtain simultaneous interpolation and a near-optimal degree of approximation by neural networks on a cube, radial-basis functions on a torus, and Gaussian networks on Euclidean space. These ideas are illustrated by a discussion of simultaneous approximation and interpolation by polynomials and also by zonal-function networks on the unit sphere in Euclidean space.
On the distributions of boundary values of Cauchy integrals
Authors: Poltoratski Alexei
Published: July 2002

We use new methods to give short proofs to some known results on the distributions of boundary values of Cauchy integrals. We also indicate some further generalizations.
Asymptotic behaviour of ground states
Authors: Hulshof Josephus , van der Vorst Robertus
Published: July 2002

We derive the asymptotic behaviour of the ground states of a system of two coupled semilinear Poisson equations with a strongly indefinite variational structure in the critical Sobolev growth case.
Essentially subnormal operators
Authors: Feldman Nathan
Published: July 2002

An operator is essentially subnormal if its image in the Calkin algebra is subnormal. We shall characterize the essentially subnormal operators as those operators with an essentially normal extension. In fact, it is shown that an essentially subnormal operator has an extension of the form “normal plus compact”. The essential normal spectrum is defined and is used to characterize the essential isometries. It is shown that every essentially subnormal operator may be decomposed as the direct sum of a subnormal operator and some irreducible essentially subnormal operators. An essential version of Putnam’s Inequality is proven for these operators. Also, it is shown that essential normality is a similarity invariant within the class of essentially subnormal operators. The class of essentially hyponormal operators is also briefly discussed and several examples of essentially subnormal operators are given.
Commuting Toeplitz operators with pluriharmonic symbols
Authors: Zheng Dechao
Published: July 2002

By making use of

M

\mathcal M

-harmonic function theory, we characterize commuting Toeplitz operators with bounded pluriharmonic symbols on the Bergman space of the unit ball or on the Hardy space of the unit sphere in

n
n

-dimensional complex space.
Noncommutative curves and noncommutative surfaces
Authors: Stafford J. , Van den Bergh M.
Published: July 2002

In this survey article we describe some geometric results in the theory of noncommutative rings and, more generally, in the theory of abelian categories. Roughly speaking and by analogy with the commutative situation, the category of graded modules modulo torsion over a noncommutative graded ring of quadratic, respectively cubic, growth should be thought of as the noncommutative analogue of a projective curve, respectively surface. This intuition has led to a remarkable number of nontrivial insights and results in noncommutative algebra. Indeed, the problem of classifying noncommutative curves (and noncommutative graded rings of quadratic growth) can be regarded as settled. Despite the fact that no classification of noncommutative surfaces is in sight, a rich body of nontrivial examples and techniques, including blowing up and down, has been developed.
Moduli Spaces of Singular Yamabe Metrics
Authors: Mazzeo Rafe , Pollack Daniel , Uhlenbeck Karen
Published: July 2002

Complete, conformally flat metrics of constant positive scalar curvature on the complement of

k
k

points in the

n
n

-sphere,

k
≥
2

k \ge 2

,

n
≥
3

n \ge 3

, were constructed by R. Schoen in 1988. We consider the problem of determining the moduli space of all such metrics. All such metrics are asymptotically periodic, and we develop the linear analysis necessary to understand the nonlinear problem. This includes a Fredholm theory and asymptotic regularity theory for the Laplacian on asymptotically periodic manifolds, which is of independent interest. The main result is that the moduli space is a locally real analytic variety of dimension

k
k

. For a generic set of nearby conformal classes the moduli space is shown to be a

k
k

-dimensional real analytic manifold. The structure as a real analytic variety is obtained by writing the space as an intersection of a Fredholm pair of infinite dimensional real analytic manifolds.
Regularity of the free boundary for the porous medium equation
Authors: Daskalopoulos P. , Hamilton R.
Published: July 2002

We study the regularity of the free boundary for solutions of the porous medium equation

u

t

=
Δ

u

m

u_{t}=\Delta u^{m}

,

m
>
1

m >1

, on

R

2

×
[
0
,
T
]

{\mathcal {R}}^{2} \times [0,T]

, with initial data

u

0

=
u
(
x
,
0
)

u^{0}=u(x,0)

nonnegative and compactly supported. We show that, under certain assumptions on the initial data

u

0

u^{0}

, the pressure

f
=
m

u

m
−
1

f=m\, u^{m-1}

will be smooth up to the interface

Γ
=
∂
{
u
>
0
}

\Gamma = \partial \{ u >0 \}

, when

0
>
t
≤
T

0>t\leq T

, for some

T
>
0

T >0

. As a consequence, the free-boundary

Γ
\Gamma

is smooth.
Concerning Nikodym-type sets in 3-dimensional curved spaces
Authors: Sogge Christopher
Published: July 2002

We investigate maximal functions involving averages over geo- desics in three-dimensional Riemannian manifolds. We first show that one can easily extend the Euclidean results of Bourgain and Wolff if one assumes constant curvature. These results need not hold if this assumption is dropped. Nonetheless, we formulate a generic geometric condition which allows favorable estimates. Curiously, this condition ensures that one is in some sense as far as possible from the constant curvature case. Assuming this, one can prove dimensional estimates for Nikodym-type sets which are essentially optimal. Optimal estimates for the related maximal functions are still open though.
TheSchizosaccharomyces pombeAurora–related Kinase Ark1 Interacts with the Inner Centromere Protein Pic1 and Mediates Chromosome Segregation and Cytokinesis
Authors: Leverson Joel D. , Huang Han-kuei , Forsburg Susan L. , Hunter Tony
Published: July 2002

The chromosomal passenger proteins aurora-B, survivin, and inner centromere protein (INCENP) have been implicated in coordinating chromosome segregation with cell division. This work describes the interplay between aurora, survivin, and INCENP orthologs in the fission yeast Schizosaccharomyces pombe and defines their roles in regulating chromosome segregation and cytokinesis. We describe the cloning and characterization of the aurora-related kinase geneark1+, demonstrating that it is an essential gene required for sister chromatid segregation. Cells lacking Ark1p exhibit the cut phenotype, DNA fragmentation, and other defects in chromosome segregation. Overexpression of a kinase-defective version of Ark1, Ark1-K147R, inhibits cytokinesis, with cells exhibiting an elongated, multiseptate phenotype. Ark1p interacts physically and/or genetically with the survivin and INCENP orthologs Bir1p and Pic1p. We identified Pic1p in a two-hybrid screen for Ark1-K147R interacting partners and went on to map domains in both proteins that mediate their binding. Pic1p residues 925–972 are necessary and sufficient for Ark1p binding, which occurs through the kinase domain. As with Ark1-K147R, overexpression of Ark1p-binding fragments of Pic1p leads to multiseptate phenotypes. We also provide evidence that the dominant-negative effect of Ark1-K147R requires Pic1p binding, indicating that the formation of Ark1p-Pic1p complexes is required for the execution of cytokinesis.
t-SNARE Phosphorylation Regulates Endocytosis in Yeast
Authors: Gurunathan Sangiliyandi , Marash Michael , Weinberger Adina , Gerst Jeffrey E.
Published: July 2002

Earlier we demonstrated that activation of a ceramide-activated protein phosphatase (CAPP) conferred normal growth and secretion to yeast lacking their complement of exocytic v-SNAREs (Snc1,2) or bearing a temperature-sensitive mutation in an exocytic t-SNARE (Sso2). CAPP activation led to Sso dephosphorylation and enhanced the assembly of t-SNAREs into functional complexes. Thus, exocytosis in yeast is modulated by t-SNARE phosphorylation. Here, we show that endocytic defects in cells lacking the v- and t-SNAREs involved in endocytosis are also rescued by CAPP activation. Yeast lacking the Tlg1 or Tlg2 t-SNAREs, the Snc v-SNAREs, or both, undergo endocytosis after phosphatase activation. CAPP activation correlated with restored uptake of FM4-64 to the vacuole, the uptake and degradation of the Ste2 receptor after mating factor treatment, and the dephosphorylation and assembly of Tlg1,2 into SNARE complexes. Activation of the phosphatase by treatment with C2-ceramide,VBM/ELO gene inactivation, or by the overexpression of SIT4 was sufficient to confer rescue. Finally, we found that mutation of single PKA sites in Tlg1 (Ser31 to Ala31) or Tlg2 (Ser90 to Ala90) was sufficient to restore endocytosis, but not exocytosis, to snc cells. These results suggest that endocytosis is also modulated by t-SNARE phosphorylation in vivo.
FIAT EXCHANGE IN FINITE ECONOMIES
Authors: Kovenock Dan , De Vries Casper G.
Published: July 2002

The state of the art of rendering fiat money valuable is either to impose a boundary condition or to make the boundary condition unimportant through an infinite sequence of markets so as to circumvent backward induction. We show fiat exchange may nevertheless arise in finite economies if agents have incomplete information about their relative position in the trade cycle or when the barter and autarky equilibria of the one‐shot trading round support a monetary equilibrium with repeated trades.
From the cradle to the grave: molecular chaperones that may choose between folding and degradation
Authors: Höhfeld Jörg , Cyr Douglas M , Patterson Cam
Published: July 2002

Molecular chaperones are known to facilitate cellular protein folding. They bind non‐native proteins and orchestrate the folding process in conjunction with regulatory cofactors that modulate the affinity of the chaperone for its substrate. However, not every attempt to fold a protein is successful and chaperones can direct misfolded proteins to the cellular degradation machinery for destruction. Protein quality control thus appears to involve close cooperation between molecular chaperones and energy‐dependent proteases. Molecular mechanisms underlying this interplay have been largely enigmatic so far. Here we present a novel concept for the regulation of the eukaryotic Hsp70 and Hsp90 chaperone systems during protein folding and protein degradation.
Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms
Authors: Stad Robert , Little Natalie A , Xirodimas Dimitris P , Frenk Ruth , van der Eb Alex J , Lane David P , Saville Mark K , Jochemsen Aart G
Published: July 2002

The p53 protein maintains genomic integrity through its ability to induce cell cycle arrest or apoptosis in response to various forms of stress. Substantial regulation of p53 activity occurs at the level of protein stability, largely determined by the activity of the Mdm2 protein. Mdm2 targets both p53 and itself for ubiquitylation and subsequent proteasomal degradation by acting as an ubiquitin ligase, a function that needs an intact Mdm2 RING finger. For efficient degradation of p53 nuclear export appears to be required. The Mdmx protein, structurally homologous to Mdm2, does not target p53 for degradation, but even stabilizes both p53 and Mdm2, an activity most likely mediated by heterodimerization of the RING fingers of Mdm2 and Mdmx. Here we show that Mdmx expression leads to accumulation of ubiquitylated, nuclear p53 but does not significantly affect the Mdm2‐mediated ubiquitylation of p53. In contrast, Mdmx stabilizes Mdm2 by inhibiting its self‐ubiquitylation.