bluuebunny/crossref_metadata_2025 huggingface dataset:
Transplantation of Quail Collagen-tailed Acetylcholinesterase Molecules Onto the Frog Neuromuscular Synapse
Authors: Rotundo Richard L. , Rossi Susana G. , Anglister Lili
Published: July 2002

The highly organized pattern of acetylcholinesterase (AChE) molecules attached to the basal lamina of the neuromuscular junction (NMJ) suggests the existence of specific binding sites for their precise localization. To test this hypothesis we immunoaffinity purified quail globular and collagen-tailed AChE forms and determined their ability to attach to frog NMJs which had been pretreated with high-salt detergent buffers. The NMJs were visualized by labeling acetylcholine receptors (AChRs) with TRITC-α-bungarotoxin and AChE by indirect immunofluorescence; there was excellent correspondence (>97%) between the distribution of frog AChRs and AChE. Binding of the exogenous quail AChE was determined using a speciesspecific monoclonal antibody. When frog neuromuscular junctions were incubated with the globular G4/G2 quail AChE forms, there was no detectable binding above background levels, whereas when similar preparations were incubated with the collagen-tailed A12 AChE form >80% of the frog synaptic sites were also immunolabeled for quail AChE attached. Binding of the A12 quail AChE was blocked by heparin, yet could not be removed with high salt buffer containing detergent once attached. Similar results were obtained using empty myofiber basal lamina sheaths produced by mechanical or freeze-thaw damage. These experiments show that specific binding sites exist for collagen-tailed AChE molecules on the synaptic basal lamina of the vertebrate NMJ and suggest that these binding sites comprise a “molecular parking lot” in which the AChE molecules can be released, retained, and turned over.
Saccharomyces cerevisiae Duo1p and Dam1p, Novel Proteins Involved in Mitotic Spindle Function
Authors: Hofmann Christian , Cheeseman Iain M. , Goode Bruce L. , McDonald Kent L. , Barnes Georjana , Drubin David G.
Published: July 2002

In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP–Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.
Slow Axonal Transport of Neurofilament Protein in Cultured Neurons
Authors: Koehnle Thomas J. , Brown Anthony
Published: July 2002

We have investigated the axonal transport of neurofilament protein in cultured neurons by constricting single axons with fine glass fibers. We observed a rapid accumulation of anterogradely and retrogradely transported membranous organelles on both sides of the constrictions and a more gradual accumulation of neurofilament protein proximal to the constrictions. Neurofilament protein accumulation was dependent on the presence of metabolic substrates and was blocked by iodoacetate, which is an inhibitor of glycolysis. These data indicate that neurofilament protein moves anterogradely in these axons by a mechanism that is directly or indirectly dependent on nucleoside triphosphates. The average transport rate was estimated to be at least 130 μm/h (3.1 mm/d), and ∼90% of the accumulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form. Electron microscopy demonstrated that there were an abnormally large number of neurofilament polymers proximal to the constrictions. These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation. This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.
Identification of a Suppressor of the Dictyostelium Profilin-minus Phenotype as a CD36/LIMP-II Homologue
Authors: Karakesisoglou Iakowos , Janssen Klaus-Peter , Eichinger Ludwig , Noegel Angelika A. , Schleicher Michael
Published: July 2002

Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme–mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type–like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin.
Microtubule-associated protein 1B
Authors: Franzen Rachelle , Tanner Sandra L. , Dashiell Suzanne M. , Rottkamp Catherine A. , Hammer Jeffrey A. , Quarles Richard H.
Published: July 2002

Myelin-associated glycoprotein (MAG) is expressed in periaxonal membranes of myelinating glia where it is believed to function in glia–axon interactions by binding to a component of the axolemma. Experiments involving Western blot overlay and coimmunoprecipitation demonstrated that MAG binds to a phosphorylated neuronal isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. The expression of some MAP1B as a neuronal plasma membrane glycoprotein (Tanner, S.L., R. Franzen, H. Jaffe, and R.H. Quarles. 2000. J. Neurochem. 75:553–562.), further documented here by its immunostaining without cell permeabilization, is consistent with it being a binding partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs colocalized with MAP1B on neuronal varicosities, and MAG and MAP1B also colocalized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was increased significantly when DRGNs were cocultured with MAG-transfected COS cells. The interaction of MAG with MAP1B is relevant to the known role of MAG in affecting the cytoskeletal structure and stability of myelinated axons.
Searching for a spindle matrix
Authors: Wells William A.
Published: July 2002

New evidence supports the idea of a nonmicrotubule spindle matrix, but the debate about the reality of this structure continues.
Lymphotoxin-α (LTα) Supports Development of Splenic Follicular Structure That Is Required for IgG Responses
Authors: Fu Yang-Xin , Molina Hector , Matsumoto Mitsuru , Huang Guangming , Min Jingjuan , Chaplin David D.
Published: July 2002

LTα-deficient (LTα−/−) mice show altered splenic microarchitecture. This includes loss of normal B cell–T cell compartmentalization, of follicular dendritic cell (FDC) clusters, and of ability to form germinal centers (GC). LTα−/− mice immunized with sheep red blood cells (SRBC) produced high levels of antigen-specific IgM but no IgG in either primary or secondary responses, demonstrating failure of Ig class switching. This inability to switch to IgG could have been due to the altered splenic microarchitecture in these mice. Alternatively, it could have been due directly to a requirement for LTα expression by lymphocytes cooperating in the antibody response. To investigate this, we performed reciprocal spleen cell transfers. When irradiated LTα−/− mice were reconstituted with wild-type splenocytes and immunized immediately with SRBC, splenic microarchitecture remained disturbed and there was no IgG response. In contrast, when irradiated wild-type animals received splenocytes from LTα−/− mice, follicle structure and a strong IgG response were retained. These data indicate that LTα-deficient B cells and T cells have no intrinsic defect in ability to generate an IgG response. Rather, the altered microenvironment characteristic of LTα−/− mice appears to result in impaired ability to switch to a productive IgG response. To investigate whether prolonged expression of LTα could alter the structure and function of spleen follicles, reciprocal bone marrow (BM) transplantation was performed. Six weeks after reconstitution of LTα−/− mice with wild-type BM, spleen follicle structure was partially restored, with return of FDC clusters and GC. B cell/T cell compartmentalization remained abnormal and white pulp zones were small. This was accompanied by restoration of IgG response to SRBC. Reconstitution of wild-type mice with LTα−/− BM resulted in loss of FDC clusters and GC, and loss of the IgG response, although compartmentalized B cell and T cell zones were largely retained. Thus, defective IgG production is not absolutely associated with abnormal B cell and T cell compartmentalization. Rather, expression of LTα supports the maturation of spleen follicle structure, including the development and maintenance of FDC clusters, which supports Ig class switching and an effective IgG response.
Crucial Role of Jak3 in Negative Selection of Self-reactive T Cells
Authors: Saijo Kaoru , Park Seung Yong , Ishida Yasuo , Arase Hisashi , Saito Takashi
Published: July 2002

Jak3 mediates growth signals through cytokine receptors such as interleukin-2 (IL-2), IL-4, and IL-7, and its deficiency results in autosomal recessive SCID in mice and humans. In spite of the severely reduced number of lymphocytes in Jak3-deficient mice, the differentiation profile of thymocytes was normal and mature T cells accumulated in the periphery with age. However, we found that self-reactive T cells were not deleted in the thymus and the peripheral tissues in Jak3-deficient mice. All peripheral T cells were in the activation state and thus were unable to be activated further, as demonstrated by the failure of eliciting Ca2+ response upon T cell receptor (TCR) stimulation. From the analysis of TCR-transgenic Jak3-deficient mice, only self-reactive T cells appeared to be in the activated state and anergic. These findings demonstrate a crucial function of Jak3 in the negative selection of autoreactive T cells and the maintenance of functional peripheral T cells.
Conditions That Induce Tolerance in Mature CD4+ T Cells
Authors: Lanoue Astrid , Bona Constantin , von Boehmer Harald , Sarukhan Adelaida
Published: July 2002

Establishment of antigen-specific tolerance among mature T cells has been a long debated, yet poorly understood issue. In this study we have used transgenic mice bearing a class II–restricted TCR specific for the hemmagglutinin of the influenza virus in order to test the behavior of CD4+ T cells upon exposure to antigen in different forms and doses. We first studied the fate of T cells expressing the transgenic TCR (6.5) in double transgenic mice where HA was expressed as a self antigen by hemapoietic cells. In these mice, we found some mature T cells in periphery that had escaped thymic deletion and that showed signs of activation but which were anergic. Mature CD4+6.5+ cells that were transferred into antigen-containing recipients went through an initial phase of expansion after which most cells were deleted and those remaining became unresponsive, as previously described for CD8+ cells. Inducing tolerance in CD4+6.5+ cells in situ in single transgenic mice proved a difficult task: classical protocols using single doses of soluble or deaggregated antigen as well as feeding antigen all failed to induce antigen-specific unresponsiveness. It was only after decreasing cell numbers by CD4 antibody treatment and by repeatedly reintroducing antigen thereafter that unresponsiveness of 6.5+ cells was achieved and maintained. In no case could we observe the appearance of antigen-specific T cells with a Th2 cytokine profile among the remaining cells and therefore conclude that deletion and anergy represent the major mechanisms of tolerance in our studies.
Induction of Apoptosis and T Helper 2 (Th2) Responses Correlates with Peptide Affinity for the Major Histocompatibility Complex in Self-reactive T Cell Receptor Transgenic Mice
Authors: Pearson C.I. , van Ewijk W. , McDevitt H.O.
Published: July 2002

Multiple sclerosis is an autoimmune disease thought to be mediated by CD4+ T helper cells (Th). Experimental autoimmune encephalomyelitis is a rodent model of multiple sclerosis and has been used extensively to explore a variety of immunotherapies using soluble protein or peptide antigens. The underlying mechanisms of such therapy have been attributed to induction of T cell anergy, a switch in Th1 to Th2 responses, or peripheral deletion of autoreactive T cells. In this study, we have developed transgenic mice expressing a T cell receptor (TCR) specific for the NH2-terminal peptide Ac1-11 of the autoantigen myelin basic protein to explore the mechanism of soluble peptide therapy. T cells from these mice are highly skewed toward the CD4 population and have an abnormal thymic architecture, a phenomenon found in other TCR transgenic mice that exhibit a highly skewed CD4/CD8 ratio. Soluble Ac1-11 or the analogues Ac1-11[4A] or Ac1-11[4Y] (which bind to the major histocompatibility complex [MHC] class II molecule I-Au with increasing affinities) given intravenously activates T cells, rendering cells hyperresponsive in vitro for at least two days after injection. Concomitantly, T cells apoptose in the periphery, the degree of which correlates with the affinity of the peptide for the MHC. In addition, a shift in the T helper phenotype of the surviving T cells occurs such that the low affinity peptide, Ac1-11, induces primarily a Th1 response, whereas the highest affinity peptide, Ac1-11[4Y], induces primarily a Th2 type response. These data show that both the nature and the presumed number of the peptide–MHC complexes formed during specific peptide therapy affect both the degree of peripheral programmed cell death as well as the outcome of the T helper subset response in vivo, leading to amelioration of disease.
Rat Spleen Dendritic Cells Express Natural Killer Cell Receptor Protein 1 (NKR-P1) and Have Cytotoxic Activity to Select Targets via a Ca2+-dependent Mechanism
Authors: Josien Régis , Heslan Michèle , Soulillou Jean-Paul , Cuturi Maria-Cristina
Published: July 2002

Dendritic cells (DC) are a subset of leukocytes whose major function is antigen presentation. We investigated the phenotype and function of enriched (95–98.5%) rat DC. We show that both spleen and thymus DC express the natural killer cell receptor protein 1 (NKR-P1) as a disulfide linked homodimer of 60 kD. Freshly isolated DC express a low level of NKR-P1, which is strongly upregulated after overnight culture. Spleen, but not thymus DC, were able to kill the NK-sensitive YAC-1 cell line in vitro, and since this killing was Ca2+ dependent, a Fas ligand–Fas interaction was probably not involved. Besides their potent antigen-presenting function, DC can thus be cytotoxic for some tumor targets.
Two Distinct Steps during Thymocyte Maturation from CD4−CD8− to CD4+CD8+ Distinguished in the Early Growth Response (Egr)-1 Transgenic Mice with a Recombinase-activating Gene–Deficient Background
Authors: Miyazaki Toru
Published: July 2002

The early growth response (Egr)-1 is a zinc finger–containing transcription factor belonging to the immediate–early genes. Its expression in CD4/CD8 double negative (DN) immature thymocytes suggests that Egr-1 expression may be involved in early thymocyte development. In transgenic mice overexpressing Egr-1 in a recombinase-activating gene–deficient background, thymocytes bypassed the block at the CD25+CD44− DN stage and matured to the immature CD8 single-positive (ISP) cell stage, but not further to the CD4/CD8 double-positive (DP) cell stage. When these mice were irradiated, thymocytes did develop to the DP stage, suggesting transcriptional induction of additional genes by irradiation that are required to promote thymocyte development from the ISP to the DP stage. These results provide genetic evidence for two distinct steps during early thymocyte development from the CD25+CD44− DN to the DP stage. The first step, from the CD25+CD44− DN to the ISP stage, can be entirely promoted by overexpression of Egr-1.
Long-Term Persistence of Activated Cytotoxic T Lymphocytes after Viral Infection of the Central Nervous System
Authors: Hawke Simon , Stevenson Philip G. , Freeman Samantha , Bangham Charles R.M.
Published: July 2002

Mice intranasally inoculated with influenza A/X-31 are protected against a subsequent intracerebral challenge with the neurovirulent influenza A/WSN and this heterotypic protection is mediated by CD8+ cytotoxic T lymphocytes. We have studied the kinetics of this secondary immune response and found that despite the elimination of replication-competent virus by day 10, we were able to recover activated influenza-specific cytotoxic T lymphocytes (CTLs) that killed freshly ex vivo from the brains of mice for at least 320 d after the intracerebral inoculation. The activated antiviral CTLs expressed high levels of the early activation marker CD69, suggesting continuing TCR signaling despite a lack of viral protein and major histocompatibility complex staining by immunohistochemistry in the brain parenchyma and barely detectable levels of viral nucleic acid by single and two-step reverse transcription PCR. Local persistence of activated lymphocytes may be important for efficient long-term responses to viruses prone to recrudesce in sites of relative immune privilege.
Mouse Ly-49D Recognizes H-2Dd and Activates Natural Killer Cell Cytotoxicity
Authors: Nakamura Mary C. , Linnemeyer Paul A. , Niemi Eréne C. , Mason Llewellyn H. , Ortaldo John R. , Ryan James C. , Seaman William E.
Published: July 2002

Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D–ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti– Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor– bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab′)2 anti–Ly-49D (12A8) and F(ab′)2 anti–H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2d mice (BALB/c, B10.D2, and DBA/2) but not from H-2b or H-2k mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.
A Critical Role for Cd40–Cd40 Ligand Interactions in Amplification of the Mucosal Cd8 T Cell Response
Authors: Lefrançois Leo , Olson Sara , Masopust David
Published: July 2002

The role of CD40 ligand (CD40L) in CD8 T cell activation was assessed by tracking antigen-specific T cells in vivo using both adoptive transfer of T cell receptor transgenic T cells and major histocompatibility complex (MHC) class I tetramers. Soluble antigen immunization induced entry of CD8 cells into the intestinal mucosa and cytotoxic T lymphocyte (CTL) differentiation, whereas CD8 cells in secondary lymphoid tissue proliferated but were not cytolytic. Immunization concurrent with CD40L blockade or in the absence of CD40 demonstrated that accumulation of CD8 T cells in the mucosa was CD40L dependent. Furthermore, activation was mediated through CD40L expressed by the CD8 cells, since inhibition by anti-CD40L monoclonal antibodies occurred after adoptive transfer to CD40L-deficient mice. However, mucosal CD8 T cells in normal and CD40−/− mice were equivalent killers, indicating that CD40L was not required for CTL differentiation. Appearance of virus-specific mucosal, but not splenic, CD8 cells also relied heavily on CD40–CD40L interactions. The mucosal CTL response of transferred CD8 T cells was MHC class II and interleukin 12 independent. The results established a novel pathway of direct CD40L-mediated CD8 T cell activation.
Id2 and Id3 Inhibit Development of Cd34+ Stem Cells into Predendritic Cell (Pre-Dc)2 but Not into Pre-Dc1
Authors: Spits Hergen , Couwenberg Franka , Bakker Arjen Q. , Weijer Kees , Uittenbogaart Christel H.
Published: July 2002

We found previously that Id3, which inhibits transcriptional activities of many basic helix-loop-helix transcription factors, blocked T and B cell development but stimulated natural killer (NK) cell development. Here we report that ectopic expression of Id3 and another Id protein, Id2, strongly inhibited the development of primitive CD34+CD38− progenitor cells into CD123high dendritic cell (DC)2 precursors. In contrast, development of CD34+CD38− cells into CD4+CD14+ DC1 precursors and mature DC1 was not affected by ectopic Id2 or Id3 expression. These observations support the notion of a common origin of DC2 precursors, T and B cells. As Id proteins did not block development of NK cells, a model presents itself in which these proteins drive common lymphoid precursors to develop into NK cells by inhibiting their options to develop into T cells, B cells, and pre-DC2.
An Instructive Component in T Helper Cell Type 2 (Th2) Development Mediated by Gata-3
Authors: Farrar J. David , Ouyang Wenjun , Löhning Max , Assenmacher Mario , Radbruch Andreas , Kanagawa Osami , Murphy Kenneth M.
Published: July 2002

Although interleukin (IL)-12 and IL-4 polarize naive CD4+ T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4–dependent redirection of phenotype in cells that have already committed to a non–IL-4–producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.
The Mouse Cd1d-Restricted Repertoire Is Dominated by a Few Autoreactive T Cell Receptor Families
Authors: Park Se-Ho , Weiss Angela , Benlagha Kamel , Kyin Tim , Teyton Luc , Bendelac Albert
Published: July 2002

To define the phenotype and T cell receptor (TCR) repertoire of CD1d-dependent T cells, we compared the populations of T cells that persisted in major histocompatibility complex (MHC)-deficient mice, which lack mainstream T cells, with those from MHC/CD1d doubly deficient mice, which lack both mainstream and CD1d-dependent T cells. Surprisingly, up to 80% of the CD1d-dependent T cells were stained by tetramers of CD1d/α-galactosylceramide, which specifically identify the previously described CD1d autoreactive Vα14-Jα18/Vβ8 natural killer (NK) T cells. Furthermore, zooming in on the CD1d-dependent non-Vα14 T cells, we found that, like Vα14 NK T cells, they mainly expressed recurrent, CD1d autoreactive TCR families and had a natural memory phenotype. Thus, CD1d-restricted T cells differ profoundly from MHC-peptide–specific T cells by their predominant use of autoreactive and semiinvariant, rather than naive and diverse, TCRs. They more closely resemble other lineages of innate lymphocytes such as B-1 B cells, γδ T cells, and NK cells, which express invariant or semiinvariant autoreactive receptors. Finally, we demonstrate that the MHC-restricted TCR repertoire is essentially non–cross-reactive to CD1d. Altogether, these findings imply that lipid recognition by CD1d-restricted T cells may have largely evolved as an innate rather than an adaptive arm of the mouse immune system.
Friend of GATA-1 Represses GATA-3–dependent Activity in CD4+ T Cells
Authors: Zhou Meixia , Ouyang Wenjun , Gong Qian , Katz Samuel G. , White J. Michael , Orkin Stuart H. , Murphy Kenneth M.
Published: July 2002

The development of naive CD4+ T cells into a T helper (Th) 2 subset capable of producing interleukin (IL)-4, IL-5, and IL-13 involves a signal transducer and activator of transcription (Stat)6-dependent induction of GATA-3 expression, followed by Stat6-independent GATA-3 autoactivation. The friend of GATA (FOG)-1 protein regulates GATA transcription factor activity in several stages of hematopoietic development including erythrocyte and megakaryocyte differentiation, but whether FOG-1 regulates GATA-3 in T cells is uncertain. We show that FOG-1 can repress GATA-3–dependent activation of the IL-5 promoter in T cells. Also, FOG-1 overexpression during primary activation of naive T cells inhibited Th2 development in CD4+ T cells. FOG-1 fully repressed GATA-3–dependent Th2 development and GATA-3 autoactivation, but not Stat6-dependent induction of GATA-3. FOG-1 overexpression repressed development of Th2 cells from naive T cells, but did not reverse the phenotype of fully committed Th2 cells. Thus, FOG-1 may be one factor capable of regulating the Th2 development.
Clonality Analysis of Synchronous Lesions of Cervical Carcinoma Based on X Chromosome Inactivation Polymorphism, Human Papillomavirus Type 16 Genome Mutations, and Loss of Heterozygosity
Authors: Hu Xinrong , Pang Tianyun , Asplund Anna , Pontén Jan , Nistér Monica
Published: July 2002

One of the most common forms of carcinoma in women, cervical invasive squamous cell carcinoma (CIC), often coexists with multiple lesions of cervical intraepithelial neoplasia (CIN). CIC and CIN show heterogeneity with respect to both histopathology and biology. To understand the causes, origin, and model of progression of cervical carcinoma, we assessed the clonality of a case with multiple synchronous lesions by analyzing X chromosome inactivation polymorphism, human papillomavirus type 16 (HPV16) sequence variation/mutations, and loss of heterozygosity (LOH). Microdissection was performed on 24 samples from this case, representing the entire lesional situation. The combination of different X chromosome inactivation patterns, two HPV16 point mutations, and LOH at three genomic microsatellite loci, led to the identification of five different “monoclonal” lesions (CIN II, CIN III, and invasive carcinoma nests) and five different “polyclonal” areas (CIN II and normal squamous epithelium). This finding indicated that CIC can originate from multiple precursor cells, from which some clones might progress via multiple steps, namely via CIN II and CIN III, whereas others might develop independently and possibly directly from the carcinoma precursor cells. Our results also supported the view that HPV16 as a “field factor” causes cervical carcinoma, which is probably promoted by the loss of chromosomal material as indicated by the LOH.
Structural Regions of the Cardiac Ca Channel α1C Subunit Involved in Ca-dependent Inactivation
Authors: Adams Brett , Tanabe Tsutomu
Published: July 2002

We investigated the molecular basis for Ca-dependent inactivation of the cardiac L-type Ca channel. Transfection of HEK293 cells with the wild-type α1C or its 3′ deletion mutant (α1C−3′del) produced channels that exhibited prominent Ca-dependent inactivation. To identify structural regions of α1C involved in this process, we analyzed chimeric α1 subunits in which one of the major intracellular domains of α1C was replaced by the corresponding region from the skeletal muscle α1S subunit (which lacks Ca-dependent inactivation). Replacing the NH2 terminus or the III–IV loop of α1C with its counterpart from α1S had no appreciable effect on Ca channel inactivation. In contrast, replacing the I–II loop of α1C with the corresponding region from α1S dramatically slowed the inactivation of Ba currents while preserving Ca-dependent inactivation. A similar but less pronounced result was obtained with a II–III loop chimera. These results suggest that the I–II and II–III loops of α1C may participate in the mechanism of Ca-dependent inactivation. Replacing the final 80% of the COOH terminus of α1C with the corresponding region from α1S completely eliminated Ca-dependent inactivation without affecting inactivation of Ba currents. Significantly, Ca-dependent inactivation was restored to this chimera by deleting a nonconserved, 211–amino acid segment from the end of the COOH terminus. These results suggest that the distal COOH terminus of α1S can block Ca-dependent inactivation, possibly by interacting with other proteins or other regions of the Ca channel. Our findings suggest that structural determinants of Ca-dependent inactivation are distributed among several major cytoplasmic domains of α1C.
Block of Brain Sodium Channels by Peptide Mimetics of the Isoleucine, Phenylalanine, and Methionine (IFM) Motif from the Inactivation Gate
Authors: Eaholtz Galen , Colvin Anita , Leonard Daniele , Taylor Charles , Catterall William A.
Published: July 2002

Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block by the peptide ac-KIFMK-NH2, measured on sodium channels whose inactivation was slowed by the α-scorpion toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH2 with smaller, less hydrophobic residues prevented fast block. The positively charged tripeptide IFM-NH2 did not cause appreciable fast block, but the divalent cation IFM-NH(CH2)2NH2 was as effective as the pentapeptide ac-KIFMK-NH2. The constrained peptide cyclic KIFMK containing two positive charges did not cause fast block. These results indicate that the position of the positive charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH2 showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.
Interactions of Cyclic Nucleotide-Gated Channel Subunits and Protein Tyrosine Kinase Probed with Genistein
Authors: Molokanova Elena , Savchenko Alexei , Kramer Richard H.
Published: July 2002

The cGMP sensitivity of cyclic nucleotide–gated (CNG) channels can be modulated by changes in phosphorylation catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. Previously, we used genistein, a PTK inhibitor, to probe the interaction between PTKs and homomeric channels comprised of α subunits (RETα) of rod photoreceptor CNG channels expressed in Xenopus oocytes. We showed that in addition to inhibiting phosphorylation, genistein triggers a noncatalytic interaction between PTKs and homomeric RETα channels that allosterically inhibits channel gating. Here, we show that native CNG channels from rods, cones, and olfactory receptor neurons also exhibit noncatalytic inhibition induced by genistein, suggesting that in each of these sensory cells, CNG channels are part of a regulatory complex that contains PTKs. Native CNG channels are heteromers, containing β as well as α subunits. To determine the contributions of α and β subunits to genistein inhibition, we compared the effect of genistein on native, homomeric (RETα and OLFα), and heteromeric (RETα+β, OLFα+β, and OLFα+RETβ) CNG channels. We found that genistein only inhibits channels that contain either the RETα or the OLFβ subunits. This finding, along with other observations about the maximal effect of genistein and the Hill coefficient of genistein inhibition, suggests that the RETα and OLFβ subunits contain binding sites for the PTK, whereas RETβ and OLFα subunits do not.
Fundamental Gating Mechanism of Nicotinic Receptor Channel Revealed by Mutation Causing a Congenital Myasthenic Syndrome
Authors: Wang Hai-Long , Ohno Kinji , Milone Margherita , Brengman Joan M. , Evoli Amelia , Batocchi Anna-Paola , Middleton Lefkos T. , Christodoulou Kyproula , Engel Andrew G. , Sine Steven M.
Published: July 2002

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation εA411P in the amphipathic helix of the acetylcholine receptor (AChR) ε subunit. Myasthenic patients from three unrelated families are either homozygous for εA411P or are heterozygous and harbor a null mutation in the second ε allele, indicating that εA411P is recessive. We expressed human AChRs containing wild-type or A411P ε subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by εA411P. Prolines engineered into positions flanking residue 411 of the ε subunit greatly increase the range of activation kinetics similar to εA411P, whereas prolines engineered into positions equivalent to εA411 in β and δ subunits are without effect. Thus, the amphipathic helix of the ε subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.
A dual-dual mixed formulation for nonlinear exterior transmission problems
Authors: Gatica Gabriel , Meddahi Salim
Published: July 2002

We combine a dual-mixed finite element method with a Dirichlet-to-Neumann mapping (derived by the boundary integral equation method) to study the solvability and Galerkin approximations of a class of exterior nonlinear transmission problems in the plane. As a model problem, we consider a nonlinear elliptic equation in divergence form coupled with the Laplace equation in an unbounded region of the plane. Our combined approach leads to what we call adual-dualmixed variational formulation since the main operator involved has itself a dual-type structure. We establish existence and uniqueness of solution for the continuous and discrete formulations, and provide the corresponding error analysis by using Raviart-Thomas elements. The main tool of our analysis is given by a generalization of the usual Babuska-Brezzi theory to a class of nonlinear variational problems with constraints.
Efficiency of a posteriori BEM–error estimates for first-kind integral equations on quasi–uniform meshes
Authors: Carstensen Carsten
Published: July 2002

In the numerical treatment of integral equations of the first kind using boundary element methods (BEM), the author and E. P. Stephan have derived a posteriori error estimates as tools for both reliable computation and self-adaptive mesh refinement. So far, efficiency of those a posteriori error estimates has been indicated by numerical examples in model situations only. This work affirms efficiency by proving the reverse inequality. Based on best approximation, on inverse inequalities and on stability of the discretization, and complementary to our previous work, an abstract approach yields a converse estimate. This estimate proves efficiency of an a posteriori error estimate in the BEM on quasi–uniform meshes for Symm’s integral equation, for a hypersingular equation, and for a transmission problem.
ℂ*-actions on ℂ³ are linearizable
Authors: Kaliman S. , Koras M. , Makar-Limanov L. , Russell P.
Published: July 2002

We give the outline of the proof of the linearization conjecture: every algebraic

C

∗

\mathbb {C}^*

-action on

C

3

\mathbb {C}^3

is linear in a suitable coordinate system.
IN02, A Positive Regulator of Lipid Biosynthesis, Is Essential for the Formation of Inducible Membranes in Yeast
Authors: Block-Alper Laura , Webster Paul , Zhou Xianghong , Supeková Lubica , Wong Wing Hung , Schultz Peter G. , Meyer David I.
Published: July 2002

Expression of the 180-kDa canine ribosome receptor inSaccharomyces cerevisiae leads to the accumulation of ER-like membranes. Gene expression patterns in strains expressing various forms of p180, each of which gives rise to unique membrane morphologies, were surveyed by microarray analysis. Several genes whose products regulate phospholipid biosynthesis were determined by Northern blotting to be differentially expressed in all strains that undergo membrane proliferation. Of these, the INO2 gene product was found to be essential for formation of p180-inducible membranes. Expression of p180 in ino2Δ cells failed to give rise to the p180-induced membrane proliferation seen in wild-type cells, whereas p180 expression in ino4Δ cells gave rise to membranes indistinguishable from wild type. Thus, Ino2p is required for the formation of p180-induced membranes and, in this case, appears to be functional in the absence of its putative binding partner, Ino4p.
Detecting periodic patterns in biological sequences.
Authors: Coward E , Drabl√∏s F
Published: July 2002

MOTIVATION: The search for repeated patterns in DNA and protein sequences is important in sequence analysis. The rapid increase in available sequences, in particular from large-scale genome sequencing projects, makes it relevant to develop sensitive automatic methods for the identification of repeats. RESULTS: A new method for finding periodic patterns in biological sequences is presented. The method is based on evolutionary distance and 'phase shifts' corresponding to insertions and deletions. A given sequence is aligned to itself in a certain sense, trying to minimize a distance to periodicity. Relationships between different such periodicity measures are discussed. An iterative algorithm is used, and the running time is nearly proportional to the sequence length. The alignment produces a periodic consensus pattern. A 'phase score' is used to indicate a statistical significance of the periodicity. Three examples using both DNA and protein sequences illustrate how the method can be used to find patterns. AVAILABILITY: On request from the authors. CONTACT: evindc@mat nu.no; finn.drablos@unimed.sintef.no
THE DEMAND FOR CIGARETTES IN JAPAN: IMPACT OF INFORMATION DISSEMINATION ON CIGARETTE CONSUMPTION
Authors: Yorozu Isao , Zhou Yimin
Published: July 2002

The authors present a theoretical derivation of cigarette demand and estimate the demand in Japan with prefecturE‐level data. By examining the impact of information dissemination regarding the health hazards of smoking, the authors argue that information dissemination is an effective instrument of public health policy, supplementary to cigarette taxation and antismoking ordinances.
PRODUCTION, APPROPRIATION, AND INCOME TRANSFER
Authors: Noh Suk Jae
Published: July 2002

In a conflict between the prey and predator, the availability of income transfer option to the prey, given that defense is already an option, results in equilibrium where both defense and income transfer are utilized and the use of income transfer increases production efficiency by inducing either the prey or predator to divert resources from appropriation to production. The equilibrium amount of defense can be smaller because the prey has an interest in minimizing the amount of transfer that can be achieved in part by provoking a largest possible amount of offense from the predator if actual fight broke out.
An octopaminergic system in the CNS of the snails, Lymnaea stagnalis and Helix pomatia
Authors: Hiripi L. , Vehovszky Á , Juhos S. , Elekes K.
Published: July 2002

Octopamine (OA) levels in each ganglion of the terrestrial snail,Helix pomatia, and the pond snail,Lymnaea stagnalis, were measured by using the HPLC technique. In both species an inhomogeneous distribution of OA was found in the central nervous system. The buccal ganglia contained a concentration of OA (12.6 pmol mg-1and 18.8 pmol mg-1) that was two to three times higher than the pedal (4.93 pmol mg-1and 9.2 pmol mg-1) or cerebral (4.46 pmol mg-1and 4.9 pmol mg-1) ganglia ofHelixandLymnaea, respectively, whereas no detectable amount of OA could be assayed in the visceroparietal complex. InLymnaeaganglia, the OA uptake into the synaptosomal fraction had a high (Km1= 4.07 ± 0.51 νM,Vmax1= 0.56 ±0.11 pmol mg-1per 20 min), and a low (Km2= 47.6 ± 5.2 νM,Vmax2= 4.2 ± 0.27 pmol mg201per 20 min), affinity component. A specific and dissociable3H–OA binding to the membrane pellet prepared from the CNS of bothHelixandLymnaeawas demonstrated. The Scatchard analysis of the ligand binding data showed a one'binding site, representing a single receptor site. TheKdandBmaxvalues were found to be 33.7 ± 5.95 nM and 1678 ± 179 fmol g-1tissue inHelixand 84.9 + 17.4 nM and 3803 ± 515 fmol g-1tissue inLymnaeapreparation. The pharmacological properties of the putative molluscan OA receptor were characterized in both species and it was demonstrated that the receptor resembled the insect OA2rather than to the clonedLymnaeaOA receptor. Immunocytochemical labelling demonstrated the presence of OA–immunoreactive neurons and fibres in the buccal, cerebral and pedal ganglia in the central nervous system of both species investigated. Electrophysiological experiments also suggested that theLymnaeabrain possessed specific receptors for OA. Local application of OA onto the identified buccal B2 neuron evoked a hyperpolarization which could selectively be inhibited by the OAergic agents phentolamine, demethylchlordimeform and 2–chloro–4–methyl–2–(phenylimino)–imidazolidine. Among the dopamine antagonists, ergotamine reversibly inhibited the OA response, whereas sulpiride had no effect. Based on our findings, a neurotransmitter–modulator role of OA is suggested in the gastropod CNS.
Population dynamics of scrapie in a sheep flock
Authors: Woolhouse M. E. J. , Matthews L. , Coen P. , Stringer S. M. , Foster J. D. , Hunter N.
Published: July 2002

A detailed analysis of an outbreak of natural scrapie in a flock of Cheviot sheep is described. A total of 137 cases was reported over 13 years among 1307 sheep born into the flock. The epidemiology of scrapie can only be understood with reference to sheep demography, the population genetics of susceptibility to scrapie, pathogenesis during a long incubation period, and the rate of transmission (by both vertical and horizontal routes), all of which interact in complex ways. A mathematical model incorporating these features is described, parameter values and model inputs are derived from available information from the flock and from independent sources, and model outputs are compared with the field data. The model is able to reproduce key features of the outbreak, including its long duration and the ages of cases. The analysis supports earlier work suggesting that many infected sheep do not survive to show clinical signs, that most cases arise through horizontal tranmission, and that there is strong selection against susceptible genotypes. However, important aspects of scrapie epidemiology remain poorly understood, including the possible role of carrier genotypes and of an environmental reservoir of infectivity, and the mechanisms maintaining alleles giving susceptibility to scrapie in the sheep population.
Measurement of small inter‐scan fluctuations in voxel dimensions in magnetic resonance images using registration
Authors: Lemieux Louis , Barker Gareth J.
Published: July 2002

We present a method to estimate fluctuations in voxel dimensions in MR images. In the proposed method images of a standard test object are acquired serially and registered to an initial dataset. We present the results of experiments where changes in voxel dimensions between 0.1% and 2% were prescribed and measured with an accuracy of 0.02%. The method can be integrated as part of a standard MRI Quality Assurance program and may also be useful to correct for inter‐scan variability introduced by fluctuations in gradient performance.