bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of CALNUC (Nucleobindin) Increases Agonist and Thapsigargin Releasable Ca2+ Storage in the Golgi
Authors: Lin Ping , Yao Yong , Hofmeister Robert , Tsien Roger Y. , Farquhar Marilyn Gist
Published: July 2002

We previously demonstrated that CALNUC, a Ca2+-binding protein with two EF-hands, is the major Ca2+-binding protein in the Golgi by 45Ca2+ overlay (Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries, and M. Farquhar. 1998. J. Cell Biol. 141:1515–1527). In this study we investigated CALNUC's properties and the Golgi Ca2+ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 μg CALNUC/mg Golgi protein, 2.5 × 105 CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca2+-binding site (Kd = 6.6 μM, binding capacity = 1.1 μmol Ca2+/μmol CALNUC). 45Ca2+ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand α helix from CALNUC completely abolished its Ca2+-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, α-mannosidase II (Man II). Approximately 70% of the 45Ca2+ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca2+ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP3 alone (permeabilized cells) also resulted in a significant increase in Ca2+ release from Golgi stores. By immunofluorescence, the IP3 receptor type 1 (IP3R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca2+ in vivo and together with SERCA and IP3R is involved in establishment of the agonist-mobilizable Golgi Ca2+ store.
A Role for the Vesicle Tethering Protein, P115, in the Post-Mitotic Stacking of Reassembling Golgi Cisternae in a Cell-Free System
Authors: Shorter James , Warren Graham
Published: July 2002

During telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)–sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.
Biogenesis of Tom40, Core Component of the Tom Complex of Mitochondria
Authors: Rapaport Doron , Neupert Walter
Published: July 2002

Tom40 is an essential component of the preprotein translocase of the mitochondrial outer membrane (TOM complex) in which it constitutes the core element of the protein conducting pore. We have investigated the biogenesis of Tom40. Tom40 is inserted into the outer membrane by the TOM complex. Initially, Tom40 is bound as a monomer at the mitochondrial surface. The import receptor Tom20 is involved in this initial step; it stimulates both binding and efficient insertion of the Tom40 precursor. This step is followed by the formation of a further intermediate at which the Tom40 precursor is partially inserted into the outer membrane. Finally, Tom40 is integrated into preexisting TOM complexes. Efficient import appears to require the Tom40 precursor to be in a partially folded conformation. Neither the NH2 nor the COOH termini are necessary to target Tom40 to the outer membrane. However, the NH2-terminal segment is required for Tom40 to become assembled into the TOM complex. A model for the biogenesis of Tom40 is presented.
Morphological Control of Inositol-1,4,5-Trisphosphate–Dependent Signals
Authors: Fink Charles C. , Slepchenko Boris , Moraru Ion I. , Schaff James , Watras James , Loew Leslie M.
Published: July 2002

Inositol-1,4,5-trisphosphate (InsP3)-mediated calcium signals represent an important mechanism for transmitting external stimuli to the cell. However, information about intracellular spatial patterns of InsP3 itself is not generally available. In particular, it has not been determined how the interplay of InsP3 generation, diffusion, and degradation within complex cellular geometries can control the patterns of InsP3 signaling. Here, we explore the spatial and temporal characteristics of [InsP3]cyt during a bradykinin-induced calcium wave in a neuroblastoma cell. This is achieved by using a unique image-based computer modeling system, Virtual Cell, to integrate experimental data on the rates and spatial distributions of the key molecular components of the process. We conclude that the characteristic calcium dynamics requires rapid, high-amplitude production of [InsP3]cyt in the neurite. This requisite InsP3 spatiotemporal profile is provided, in turn, as an intrinsic consequence of the cell's morphology, demonstrating how geometry can locally and dramatically intensify cytosolic signals that originate at the plasma membrane. In addition, the model predicts, and experiments confirm, that stimulation of just the neurite, but not the soma or growth cone, is sufficient to generate a calcium response throughout the cell.
Molecular Dissection of Zyxin Function Reveals Its Involvement in Cell Motility
Authors: Drees Beth E. , Andrews Katy M. , Beckerle Mary C.
Published: July 2002

Spatially controlled actin filament assembly is critical for numerous processes, including the vectorial cell migration required for wound healing, cell- mediated immunity, and embryogenesis. One protein implicated in the regulation of actin assembly is zyxin, a protein concentrated at sites where the fast growing ends of actin filaments are enriched. To evaluate the role of zyxin in vivo, we developed a specific peptide inhibitor of zyxin function that blocks its interaction with α-actinin and displaces it from its normal subcellular location. Mislocalization of zyxin perturbs cell migration and spreading, and affects the behavior of the cell edge, a structure maintained by assembly of actin at sites proximal to the plasma membrane. These results support a role for zyxin in cell motility, and demonstrate that the correct positioning of zyxin within the cell is critical for its physiological function. Interestingly, the mislocalization of zyxin in the peptide-injected cells is accompanied by disturbances in the distribution of Ena/VASP family members, proteins that have a well-established role in promoting actin assembly. In concert with previous work, our findings suggest that zyxin promotes the spatially restricted assembly of protein complexes necessary for cell motility.
Toxoplasma gondiiExploits Host Low-Density Lipoprotein Receptor-Mediated Endocytosis for Cholesterol Acquisition
Authors: Coppens Isabelle , Sinai Anthony P. , Joiner Keith A.
Published: July 2002

The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.
Inhibitors of Copi and Copii Do Not Block PEX3-Mediated Peroxisome Synthesis
Authors: South Sarah T. , Sacksteder Katherine A. , Li Xiaoling , Liu Yifei , Gould Stephen J.
Published: July 2002

In humans, defects in peroxisome biogenesis are the cause of lethal diseases typified by Zellweger syndrome. Here, we show that inactivating mutations in human PEX3 cause Zellweger syndrome, abrogate peroxisome membrane synthesis, and result in reduced abundance of peroxisomal membrane proteins (PMPs) and/or mislocalization of PMPs to the mitochondria. Previous studies have suggested that PEX3 may traffic through the ER en route to the peroxisome, that the COPI inhibitor, brefeldin A, leads to accumulation of PEX3 in the ER, and that PEX3 overexpression alters the morphology of the ER. However, we were unable to detect PEX3 in the ER at early times after expression. Furthermore, we find that inhibition of COPI function by brefeldin A has no effect on trafficking of PEX3 to peroxisomes and does not inhibit PEX3-mediated peroxisome biogenesis. We also find that inhibition of COPII-dependent membrane traffic by a dominant negative SAR1 mutant fails to block PEX3 transport to peroxisomes and PEX3-mediated peroxisome synthesis. Based on these results, we propose that PEX3 targeting to peroxisomes and PEX3-mediated peroxisome membrane synthesis may occur independently of COPI- and COPII-dependent membrane traffic.
Rac Mediates Cytoskeletal Rearrangements and Increased Cell Motility Induced by Urokinase-Type Plasminogen Activator Receptor Binding to Vitronectin
Authors: Kjøller Lars , Hall Alan
Published: July 2002

The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell motility in a variety of cell types. We show here that expression of human uPAR in growing murine fibroblasts leads to a dramatic reorganization of the actin cytoskeleton. uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA and activation of the RGD-binding activity of integrins but require uPAR binding to vitronectin (VN). The actin reorganization is blocked by coexpression of dominant negative versions of either Rac (N17Rac) or p130Cas, but not by inhibitors of Cdc42 or Rho, and is accompanied by a Rac-dependent increase in cell motility. In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression. We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses. This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.
The nucleoporin Nup60p functions as a Gsp1p–GTP-sensitive tether for Nup2p at the nuclear pore complex
Authors: Denning Daniel , Mykytka Brook , Allen Nadia P.C. , Huang Lan , Burlingame Al , Rexach Michael
Published: July 2002

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p–Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p–GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p–Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p–GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p–Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p–GTP, between Nup60p and Nup2p at the NPC.
Overlapping functions of the cell adhesion molecules Nr-CAM and L1 in cerebellar granule cell development
Authors: Sakurai Takeshi , Lustig Marc , Babiarz Joanne , Furley Andrew J.W. , Tait Steven , Brophy Peter J. , Brown Stephen A. , Brown Lucia Y. , Mason Carol A. , Grumet Martin
Published: July 2002

The structurally related cell adhesion molecules L1 and Nr-CAM have overlapping expression patterns in cerebellar granule cells. Here we analyzed their involvement in granule cell development using mutant mice. Nr-CAM–deficient cerebellar granule cells failed to extend neurites in vitro on contactin, a known ligand for Nr-CAM expressed in the cerebellum, confirming that these mice are functionally null for Nr-CAM. In vivo, Nr-CAM–null cerebella did not exhibit obvious histological defects, although a mild size reduction of several lobes was observed, most notably lobes IV and V in the vermis. Mice deficient for both L1 and Nr-CAM exhibited severe cerebellar folial defects and a reduction in the thickness of the inner granule cell layer. Additionally, anti-L1 antibodies specifically disrupted survival and maintenance of Nr-CAM–deficient granule cells in cerebellar cultures treated with antibodies. The combined results indicate that Nr-CAM and L1 play a role in cerebellar granule cell development, and suggest that closely related molecules in the L1 family have overlapping functions.
Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction
Authors: Hatch Victoria , Zhi Gang , Smith Lula , Stull James T. , Craig Roger , Lehman William
Published: July 2002

Ca2+–calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK–actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228–232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.
Spatial organization of active and inactive genes and noncoding DNA within chromosome territories
Authors: Mahy Nicola L. , Perry Paul E. , Gilchrist Susan , Baldock Richard A. , Bickmore Wendy A.
Published: July 2002

The position of genes within the nucleus has been correlated with their transcriptional activity. The interchromosome domain model of nuclear organization suggests that genes preferentially locate at the surface of chromosome territories. Conversely, high resolution analysis of chromatin fibers suggests that chromosome territories do not present accessibility barriers to transcription machinery.

To clarify the relationship between the organization of chromosome territories and gene expression, we have used fluorescence in situ hybridization to analyze the spatial organization of a contiguous ∼1 Mb stretch of the Wilms' tumor, aniridia, genitourinary anomalies, mental retardation syndrome region of the human genome and the syntenic region in the mouse. These regions contain constitutively expressed genes, genes with tissue-restricted patterns of expression, and substantial regions of intergenic DNA. We find that there is a spatial organization within territories that is conserved between mouse and humans: certain sequences do preferentially locate at the periphery of the chromosome territories in both species. However, we do not detect genes necessarily at the periphery of chromosome territories or at the surface of subchromosomal domains. Intraterritory organization is not different among cell types that express different combinations of the genes under study.

Our data demonstrate that transcription of both ubiquitous and tissue-restricted genes is not confined to the periphery of chromosome territories, suggesting that the basal transcription machinery and transcription factors can readily gain access to the chromosome interior.
A Human Immunoglobulin λ Locus Is Similarly Well Expressed in Mice and Humans
Authors: Popov Andrei V. , Zou Xiangang , Xian Jian , Nicholson Ian C. , Brüggemann Marianne
Published: July 2002

Transgenic mice carrying a 380-kb region of the human immunoglobulin (Ig) λ light (L) chain locus in germline configuration were created. The introduced translocus on a yeast artificial chromosome (YAC) accommodates the most proximal Igλ variable region (V) gene cluster, including 15 Vλ genes that contribute to >60% of λ L chains in humans, all Jλ-Cλ segments, and the 3′ enhancer. HuIgλYAC mice were bred with animals in which mouse Igκ production was silenced by gene targeting. In the κ−/− background, human Igλ was expressed by ∼84% of splenic B cells. A striking result was that human Igλ was also produced at high levels in mice with normal κ locus. Analysis of bone marrow cells showed that human Igλ and mouse Igκ were expressed at similar levels throughout B cell development, suggesting that the Igλ translocus and the endogenous κ locus rearrange independently and with equal efficiency at the same developmental stage. This is further supported by the finding that in hybridomas expressing human Igλ the endogenous L chain loci were in germline configuration. The presence of somatic hypermutation in the human Vλ genes indicated that the Igλ-expressing cells function normally. The finding that human λ genes can be utilized with similar efficiency in mice and humans implies that L chain expression is critically dependent on the configuration of the locus.
Augmentation of Antigen Receptor–mediated Responses by Histamine H1 Receptor Signaling
Authors: Banu Yasmin , Watanabe Takeshi
Published: July 2002

Histamine is considered one of the important mediators of immediate hypersensitivity and inflammation, and acts via G protein–coupled receptors. Here, we report that histamine may affect antigen receptor–mediated immune responses of T and B cells via a signal(s) from histamine H1 receptors (H1Rs). Histamine exhibited enhancing effects on the in vitro proliferative responses of anti-CD3ε– or anti-IgM–stimulated spleen T and B cells, respectively, at the culture condition that the fetal calf serum was dialyzed before culture and c-kit–positive cells were depleted from the spleen cells. In studies of histamine H1R knockout mice, H1R-deficient T cells had low proliferative responses to anti-CD3ε cross-linking or antigen stimulation in vitro. B cells from H1R-deficient mice were also affected, demonstrating low proliferative responses to B cell receptor cross-linking. Antibody production against trinitrophenyl-Ficoll was reduced in H1R-deficient mice. Other aspects of T and B cell function were normal in the H1R knockout mice. H1R-deficient T and B cells showed normal responses upon stimulation with interleukin (IL)-2, IL-4, CD40 ligand, CD40 ligand plus IL-4, and lipopolysaccharide. Collectively, these results imply that the signal generated by histamine through H1R augments antigen receptor–mediated immune responses, suggesting cross-talk between G protein–coupled receptors and antigen receptor–mediated signaling.
Expression of the c-myc Proto-oncogene Is Essential for HIV-1 Infection in Activated T Cells
Authors: Sun Yu , Clark Edward A.
Published: July 2002

We previously found that activation of primary CD4+ T cells via both the T cell antigen receptor (TCR) and CD28 is required for HIV-1 DNA to be translocated from the cytoplasm to the nucleus. Here we report that expression of c-Myc protein in CD4+ T cells is induced only after such costimulation. In addition, cyclosporin A not only inhibits nuclear import of HIV-1 DNA but also inhibits expression of c-Myc protein. Because of these correlations, we tested whether c-Myc is necessary for nuclear import of HIV-1 DNA. Specific c-myc antisense, but not sense or non-sense, phosphorothioate oligodeoxynucleotides selectively induced the accumulation of two NH2-terminally truncated c-Myc proteins and abolished HIV-1 genome entry into host nuclei. Consequently, both virus replication and HIV-1–induced apoptotic cell death were inhibited. Synthesis of viral full-length DNA was not affected. Specific c-myc antisense oligonucleotide inhibited HIV-1 infection under conditions that did not affect cell cycle entry or proliferation. Thus, c-Myc appears to regulate HIV-1 DNA nuclear import via a mechanism distinct from those controlling entry into the cell cycle.
Molecular Mimicry of Human Cytochrome P450 by Hepatitis C Virus at the Level of Cytotoxic T Cell Recognition
Authors: Kammer Andreas R. , van der Burg Sjoerd H. , Grabscheid Benno , Hunziker Isabelle P. , Kwappenberg Kitty M.C. , Reichen Jürg , Melief Cornelis J.M. , Cerny Andreas
Published: July 2002

Hepatitis C virus (HCV) is thought to be involved in the pathogenesis of autoimmune hepatitis (AIH) type 2, which is defined by the presence of type I antiliver kidney microsome autoantibodies directed mainly against cytochrome P450 (CYP)2D6 and by autoreactive liver infiltrating T cells. Virus-specific CD8+ cytotoxic T lymphocytes (CTLs) that recognize infected cells and contribute to viral clearance and tissue injury during HCV infection could be involved in the induction of AIH. To explore whether the antiviral cellular immunity may turn against self-antigens, we characterized the primary CTL response against an HLA-A*0201–restricted HCV-derived epitope, i.e., HCV core 178–187, which shows sequence homology with human CYP2A6 and CYP2A7 8–17. To determine the relevance of these homologies for the pathogenesis of HCV-associated AIH, we used synthetic peptides to induce primary CTL responses in peripheral blood mononuclear cells of healthy blood donors and patients with chronic HCV infection. We found that the naive CTL repertoire of both groups contains cross-reactive CTLs inducible by the HCV peptide recognizing both CYP2A6 and CYP2A7 peptides as well as endogenously processed CYP2A6 protein. Importantly, we failed to induce CTLs with the CYP-derived peptides that showed a lower capacity to form stable complexes with the HLA-A2 molecule. These findings demonstrate the potential of HCV to induce autoreactive CD8+ CTLs by molecular mimicry, possibly contributing to virus-associated autoimmunity.
Endothelial Cells Modify the Costimulatory Capacity of Transmigrating Leukocytes and Promote Cd28-Mediated Cd4+ T Cell Alloactivation
Authors: Denton Mark D. , Geehan Christopher S. , Alexander Steve I. , Sayegh Mohamed H. , Briscoe David M.
Published: July 2002

Activated vascular endothelial cells (ECs) express major histocompatibility complex (MHC) class II molecules in vitro and in vivo in acute and chronic allograft rejection. However, human ECs may be limited in their ability to effectively activate CD4+ T cells, because they do not express members of the B7 family (CD80 and CD86) of costimulatory molecules. In this study, we show that ECs promote the full activation of CD4+ T cells via trans-costimulatory interactions. By reverse transcriptase polymerase chain reaction, Western blot, and FACS® analysis, we could not detect the expression of CD80 and CD86 on activated ECs and found minimal expression on purified CD4+ T cells. In contrast, both CD80 and CD86 were expressed in allogeneic CD4+ T cell–EC cocultures. Expression of CD86 peaked at early times between 12 and 24 h after coculture, whereas CD80 was not expressed until 72 h. Addition of anti-CD86 but not anti-CD80 monoclonal antibodies to cocultures inhibited IL-2 production and the proliferation of CD4+ T cells to allogeneic donor human umbilical vein ECs (HUVECs), as well as to skin and lung microvascular ECs. Furthermore, we found that interferon γ–activated ECs but not untreated ECs induced mRNA and cell surface expression of CD80 and CD86 on CD4+ T cells, and these T cells were functional to provide a trans-costimulatory signal to autologous CD4+ T cells. Blockade of MHC class II and lymphocyte function–associated antigen 3 but not other EC cell surface molecules on IFN-γ–activated ECs inhibited the induction of CD86 on CD4+ T cells. Transmigration of purified populations of monocytes across EC monolayers similarly resulted in the induction of functional CD86, but also induced the de novo expression of the cytokines interleukin (IL)-1α and IL-12. In addition, EC-modified monocytes supported enhanced proliferation of allogeneic and autologous CD4+ T cells. Taken together, these data define the ability of the endothelium to modify CD4+ T cells and monocytes for trans-costimulatory events. This unique function of the endothelium in alloimmune T cell activation has functional consequences for the direct and the indirect pathways of allorecognition.
Txk, a Nonreceptor Tyrosine Kinase of the Tec Family, Is Expressed in T Helper Type 1 Cells and Regulates Interferon γ Production in Human T Lymphocytes
Authors: Kashiwakura Jun-ichi , Suzuki Noboru , Nagafuchi Hiroko , Takeno Mitsuhiro , Takeba Yuko , Shimoyama Yoshihiro , Sakane Tsuyoshi
Published: July 2002

Differentiation of human T cells into T helper (Th)1 and Th2 cells is vital for the development of cell-mediated and humoral immunity, respectively. However, the precise mechanism responsible for the Th1 cell differentiation is not fully clarified. We have studied the expression and function of Txk, a member of the Tec family of nonreceptor tyrosine kinases. We found that Txk expression is restricted to Th1/Th0 cells with IFN-γ producing potential. Txk transfection of Jurkat T cells resulted in a several-fold increase of IFN-γ mRNA expression and protein production; interleukin (IL)-2 and IL-4 production were unaffected. Antisense oligodeoxynucleotide of Txk specifically inhibited IFN-γ production of normal peripheral blood lymphocytes, antigen-specific Th1 clones, and Th0 clones; IL-2 and IL-4 production by the T cells was unaffected. Txk cotransfection led to the enhanced luciferase activity of plasmid (p)IFN-γ promoter/enhancer (pIFN-γ[-538])-luciferase–transfected Jurkat cells upon mitogen activation. Txk transfection did not affect IL-2 and IL-4 promoter activities. Thus, Txk specifically upregulates IFN-γ gene transcription. In fact, Txk translocated from cytoplasm into nuclei upon activation and transfection with a mutant Txk expression plasmid that lacked a nuclear localization signal sequence did not enhance IFN-γ production by the cells, indicating that nuclear localization of Txk is obligatory for the enhanced IFN-γ production. In addition, IL-12 treatment of peripheral blood CD4+ T cells enhanced the Txk expression, whereas IL-4 treatment completely inhibited it. These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-γ production by the cells through Th1 cell–specific positive transcriptional regulation of the IFN-γ gene.
Ion-dependent Inactivation of Barium Current through L-type Calcium Channels
Authors: Ferreira Gonzalo , Yi Jianxun , Ríos Eduardo , Shirokov Roman
Published: July 2002

It is widely believed that Ba2+ currents carried through L-type Ca2+ channels inactivate by a voltage- dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca2+ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba2+ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, IBa decayed as the sum of two exponentials. The slow phase (τ ≈ 6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (τ ≈ 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na+ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba2+ current. The reduction of Ba2+ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba2+ entry during the pulse did not change the reversal potential for Ba2+. A simple model of Ca2+-dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005–1030) robustly accounts for fast Ba2+ current decay assuming the affinity of the inactivation site on the α1 subunit to be 100 times lower for Ba2+ than Ca2+.
Na+-dependent Ca2+ Extrusion Governs Response Recovery in Frog Olfactory Receptor Cells
Authors: Reisert Johannes , Matthews H.R.
Published: July 2002

To study the mechanism by which Ca2+, which enters during the odor response, is extruded during response recovery, recordings were made from isolated frog olfactory receptor cells using the suction pipette technique, while superfusing the olfactory cilia with solutions of modified ionic composition. When external Na+ was substituted with another cation, the response to odor was greatly prolonged. This prolongation of the response was similar irrespective of whether Na+ was replaced with Li+, which permeates the cyclic nucleotide-gated conductance, or choline, which does not. The prolonged current was greatly reduced by exposure to 300 μM niflumic acid, a blocker of the calcium-activated chloride channel, indicating that it is carried by this conductance, and abolished if Ca2+ was omitted from the external solution, demonstrating that Ca2+ influx is required for its generation. When the cilia were exposed to Na+-free solution after odor stimulation, the recovery of the response to a second stimulus from the adaptation induced by the first was greatly reduced. We conclude that a Na+-dependent Ca2+ extrusion mechanism is present in frog olfactory cilia and that it serves as the main mechanism that returns cytoplasmic Ca2+ concentration to basal levels after stimulation and mediates the normally rapid recovery of the odor response and the restoration of sensitivity after adaptation.
Sulfonylurea and K+-Channel Opener Sensitivity of KATP Channels
Authors: Koster J.C. , Sha Q. , Nichols C.G.
Published: July 2002

The sensitivity of KATP channels to high-affinity block by sulfonylureas and to stimulation by K+ channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4,5-bisphosphate (PIP2) profoundly antagonized ATP inhibition of KATP channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP2 drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant KATP channels (Kir6.2[ΔN30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an “uncoupled” phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6.2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP2 also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP2 application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP2. The net effect of increasing open state stability, either by PIP2 or mutagenesis, is an apparent “uncoupling” of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-l-lysine.
Localization and Molecular Determinants of the Hanatoxin Receptors on the Voltage-Sensing Domains of a K+ Channel
Authors: Li-Smerin Yingying , Swartz Kenton J.
Published: July 2002

Hanatoxin inhibits voltage-gated K+ channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K+ channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1–S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K+ channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K+ channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20–25 Å away from the central pore axis.
Neutralization of Gating Charges in Domain II of the Sodium Channel α Subunit Enhances Voltage-Sensor Trapping by a β-Scorpion Toxin
Authors: Cestèle Sandrine , Scheuer Todd , Mantegazza Massimo , Rochat Hervé , Catterall William A.
Published: July 2002

β-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3–S4 loop at the extracellular end of the S4 voltage sensor in domain II of the α subunit. Here, we probe the role of gating charges in the IIS4 segment in β-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances β-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the β-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from −80 to −140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor–trapping model in which the β-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.
Voltage-Controlled Gating at the Intracellular Entrance to a Hyperpolarization-Activated Cation Channel
Authors: Rothberg Brad S. , Shin Ki Soon , Phale Prashant S. , Yellen Gary
Published: July 2002

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Our previous work using the specific HCN channel blocker ZD7288 provided evidence for an intracellular activation gate for these channels because it appears that ZD7288, applied from the intracellular side, can enter and leave HCN channels only at voltages where the activation gate is opened (Shin, K.S., B.S. Rothberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91–101). However, the ZD7288 molecule is larger than the Na+ or K+ ions that flow through the open channel. In the present study, we sought to resolve whether the voltage gate at the intracellular entrance to the pore for ZD7288 also can be a gate for permeant ions in HCN channels. Single residues in the putative pore-lining S6 region of an HCN channel (cloned from sea urchin; spHCN) were substituted with cysteines, and the mutants were probed with Cd2+ applied to the intracellular side of the channel. One mutant, T464C, displayed rapid irreversible block when Cd2+ was applied to opened channels, with an apparent blocking rate of ∼3 × 105 M−1s−1. The blocking rate was decreased for channels held at more depolarized voltages that close the channels, which is consistent with the Cd2+ access to this residue being gated from the intracellular side of the channel. 464C channels could be recovered from Cd2+ inhibition in the presence of a dithiol applied to the intracellular side. The rate of this recovery also was reduced when channels were held at depolarized voltages. Finally, Cd2+ could be trapped inside channels that were composed of WT/464C tandem-linked subunits, which could otherwise recover spontaneously from Cd2+ inhibition. Thus, Cd2+ escape is also gated at the intracellular side of the channel. Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore.
Euler-Lagrange and Hamiltonian formalisms in dynamic optimization
Authors: Ioffe Alexander
Published: July 2002

We consider dynamic optimization problems for systems governed by differential inclusions. The main focus is on the structure of and interrelations between necessary optimality conditions stated in terms of Euler–Lagrange and Hamiltonian formalisms. The principal new results are: an extension of the recently discovered form of the Euler–Weierstrass condition to nonconvex valued differential inclusions, and a new Hamiltonian condition for convex valued inclusions. In both cases additional attention was given to weakening Lipschitz type requirements on the set–valued mapping. The central role of the Euler type condition is emphasized by showing that both the new Hamiltonian condition and the most general form of the Pontriagin maximum principle for equality constrained control systems are consequences of the Euler–Weierstrass condition. An example is given demonstrating that the new Hamiltonian condition is strictly stronger than the previously known one.
Characterizations of spectra with 𝒰-injective cohomology which satisfy the Brown-Gitler property
Authors: Hunter David , Kuhn Nicholas
Published: July 2002

We work in the stable homotopy category of

p
p

–complete connective spectra having mod

p
p

homology of finite type.

H
∗

(
X
)

H^*(X)

means cohomology with

Z

/

p

\mathbf {Z}/p

coefficients, and is a left module over the Steenrod algebra

A

\mathcal {A}

. A spectrum

Z
Z

is called spacelike if it is a wedge summand of a suspension spectrum, and a spectrum

X
X

satisfies the Brown–Gitler property if the natural map
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="left-bracket upper X comma upper Z right-bracket right-arrow upper H o m Subscript script upper A Baseline left-parenthesis upper H Superscript asterisk Baseline left-parenthesis upper Z right-parenthesis comma upper H Superscript asterisk Baseline left-parenthesis upper X right-parenthesis right-parenthesis">

[
X
,
Z
]
→

Hom

A

⁡
(

H
∗

(
Z
)
,

H
∗

(
X
)
)

[X,Z] \rightarrow \operatorname {Hom}_{\mathcal {A}}(H^*(Z),H^*(X))

is onto, for all spacelike

Z
Z

. It is known that there exist spectra

T
(
n
)

T(n)

satisfying the Brown–Gitler property, and with

H
∗

(
T
(
n
)
)

H^*(T(n))

isomorphic to the injective envelope of

H
∗

(

S
n

)

H^*(S^n)

in the category

U

\mathcal {U}

of unstable

A

\mathcal {A}

–modules. Call a spectrum

X
X

standard if it is a wedge of spectra of the form

L
∧
T
(
n
)

L \wedge T(n)

, where

L
L

is a stable wedge summand of the classifying space of some elementary abelian

p
p

–group. Such spectra have

U

\mathcal {U}

–injective cohomology, and all

U

\mathcal {U}

–injectives appear in this way. Working directly with the two properties of

T
(
n
)

T(n)

stated above, we clarify and extend earlier work by many people on Brown–Gitler spectra. Our main theorem is that, if

X
X

is a spectrum with

U

\mathcal {U}

–injective cohomology, the following conditions are equivalent: (A) there exist a spectrum

Y
Y

whose cohomology is a reduced

U

\mathcal {U}

–injective and a map

X
→
Y

X \rightarrow Y

that is epic in cohomology, (B) there exist a spacelike spectrum

Z
Z

and a map

X
→
Z

X \rightarrow Z

that is epic in cohomology, (C)

ϵ
:

Σ

∞

Ω

∞

X
→
X

\epsilon :\Sigma ^{\infty }\Omega ^{\infty }X \rightarrow X

is monic in cohomology, (D)

X
X

satisfies the Brown–Gitler property, (E)

X
X

is spacelike, (F)

X
X

is standard. (

M
∈

U

M \in \mathcal {U}

is reduced if it has no nontrivial submodule which is a suspension.) As an application, we prove that the Snaith summands of

Ω
2

S
3

\Omega ^2S^3

are Brown–Gitler spectra–a new result for the most interesting summands at odd primes. Another application combines the theorem with the second author’s work on the Whitehead conjecture. Of independent interest, enroute to proving that (B) implies (C), we prove that the homology suspension has the following property: if an

n
n

–connected space

X
X

admits a map to an

n
n

–fold suspension that is monic in mod

p
p

homology, then

ϵ
:

Σ
n

Ω
n

X
→
X

\epsilon : \Sigma ^n\Omega ^n X \rightarrow X

is onto in mod

p
p

homology.
Compactly supported tight affine spline frames in 𝐿₂(ℝ^{𝕕})
Authors: Ron Amos , Shen Zuowei
Published: July 2002

The theory of fiberization is applied to yield compactly supported tight affine frames (wavelets) in

L

2

(

R

d

)

L_{2}(\mathbb {R}^{d})

from box splines. The wavelets obtained are smooth piecewise-polynomials on a simple mesh; furthermore, they exhibit a wealth of symmetries, and have a relatively small support. The number of “mother wavelets”, however, increases with the increase of the required smoothness. Two bivariate constructions, of potential practical value, are highlighted. In both, the wavelets are derived from four-direction mesh box splines that are refinable with respect to the dilation matrix

(

1

a
m
p
;
1
 
1

a
m
p
;
−
1

)

\begin {pmatrix}1&1\ 1&-1\end {pmatrix}

.
Calmodulin Regulates Intracellular Trafficking of Epidermal Growth Factor Receptor and the MAPK Signaling Pathway
Authors: Tebar Francesc , Villalonga Priam , Sorkina Tatiana , Agell Neus , Sorkin Alexander , Enrich Carlos
Published: July 2002

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13–mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.