bluuebunny/crossref_metadata_2025 huggingface dataset:
Large-scale Chromosomal Movements During Interphase Progression in Drosophila
Authors: Csink Amy K. , Henikoff Steven
Published: July 2002

We examined the effect of cell cycle progression on various levels of chromosome organization in Drosophila. Using bromodeoxyuridine incorporation and DNA quantitation in combination with fluorescence in situ hybridization, we detected gross chromosomal movements in diploid interphase nuclei of larvae. At the onset of S-phase, an increased separation was seen between proximal and distal positions of a long chromsome arm. Progression through S-phase disrupted heterochromatic associations that have been correlated with gene silencing. Additionally, we have found that large-scale G1 nuclear architecture is continually dynamic. Nuclei display a Rabl configuration for only ∼2 h after mitosis, and with further progression of G1-phase can establish heterochromatic interactions between distal and proximal parts of the chromosome arm. We also find evidence that somatic pairing of homologous chromosomes is disrupted during S-phase more rapidly for a euchromatic than for a heterochromatic region. Such interphase chromosome movements suggest a possible mechanism that links gene regulation via nuclear positioning to the cell cycle: delayed maturation of heterochromatin during G1-phase delays establishment of a silent chromatin state.
Fab1p Is Essential for PtdIns(3)P 5-Kinase Activity and the Maintenance of Vacuolar Size and Membrane Homeostasis
Authors: Gary Jonathan D. , Wurmser Andrew E. , Bonangelino Cecilia J. , Weisman Lois S. , Emr Scott D.
Published: July 2002

The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH2-terminus and a kinase domain at its COOH terminus. Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (Yamamoto et al., 1995). Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synthesize PtdIns(4,5)P2. Consistent with this, we find that unlike wild-type cells, fab1Δ, fab1tsf, and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P2, a phosphoinositide recently identified both in yeast and mammalian cells. PtdIns(4,5)P2 synthesis, on the other hand, is only moderately affected even in fab1Δ mutants. The presence of PtdIns(3)P in fab1 mutants, combined with previous data, indicate that PtdIns(3,5)P2 synthesis is a two step process, requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p- dependent phosphorylation of PtdIns(3)P yielding PtdIns(3,5)P2. Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P2 by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P2 has a distinct function. The major phenotypes resulting from Fab1p kinase inactivation include temperature-sensitive growth, vacuolar acidification defects, and dramatic increases in vacuolar size. Based on our studies, we hypothesize that whereas Vps34p is essential for anterograde trafficking of membrane and protein cargoes to the vacuole, Fab1p may play an important compensatory role in the recycling/turnover of membranes deposited at the vacuole. Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns(3,5)P2, suggesting that Vac7p functions as an upstream regulator, perhaps in a complex with Fab1p. We propose that Fab1p and Vac7p are components of a signal transduction pathway which functions to regulate the efflux or turnover of vacuolar membranes through the regulated production of PtdIns(3,5)P2.
Spatial and Temporal Dynamics of DNA Replication Sites in Mammalian Cells
Authors: Ma Hong , Samarabandu Jagath , Devdhar Rekandu S. , Acharya Raj , Cheng Ping-chin , Meng Chunling , Berezney Ronald
Published: July 2002

Fluorescence microscopic analysis of newly replicated DNA has revealed discrete granular sites of replication (RS). The average size and number of replication sites from early to mid S-phase suggest that each RS contains numerous replicons clustered together. We are using fluorescence laser scanning confocal microscopy in conjunction with multidimensional image analysis to gain more precise information about RS and their spatial-temporal dynamics. Using a newly improved imaging segmentation program, we report an average of ∼1,100 RS after a 5-min pulse labeling of 3T3 mouse fibroblast cells in early S-phase. Pulse-chase-pulse double labeling experiments reveal that RS take ∼45 min to complete replication. Appropriate calculations suggest that each RS contains an average of 1 mbp of DNA or ∼6 average-sized replicons. Double pulse–double chase experiments demonstrate that the DNA sequences replicated at individual RS are precisely maintained temporally and spatially as the cell progresses through the cell cycle and into subsequent generations. By labeling replicated DNA at the G1/S borders for two consecutive cell generations, we show that the DNA synthesized at early S-phase is replicated at the same time and sites in the next round of replication.
Induction of Cell Scattering by Expression of β1 Integrins in β1-Deficient Epithelial Cells Requires Activation of Members of the Rho Family of Gtpases and Downregulation of Cadherin and Catenin Function
Authors: Gimond Clotilde , van der Flier Arjan , van Delft Sanne , Brakebusch Cord , Kuikman Ingrid , Collard John G. , Fässler Reinhard , Sonnenberg Arnoud
Published: July 2002

Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and β1 integrins influence each other using two different β1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of β1A or the cytoplasmic splice variant β1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of β1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of β1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-β1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM–cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length β1A. This indicates that the disruption of cell–cell adhesion is not simply the consequence of the stimulated cell migration. Expression of β1 integrins in GE11 cells resulted in a decrease in cadherin and α-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of α-catenin protein levels by β1 integrins is likely to play a role in the morphological transition, since overexpression of α-catenin in GE11 cells before β1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of β1A, β1D, or IL2R-β1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell–cell adhesions when expressed before β1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-β1A cells. Our results indicate that β1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of α-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.
Exocytotic Insertion of Calcium Channels Constrains Compensatory Endocytosis to Sites of Exocytosis
Authors: Smith Robert M. , Baibakov Boris , Ikebuchi Yoshihide , White Benjamin H. , Lambert Nevin A. , Kaczmarek Leonard K. , Vogel Steven S.
Published: July 2002

Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.
DNA Dendrimers Localize Myod mRNA in Presomitic Tissues of the Chick Embryo
Authors: Gerhart Jacquelyn , Baytion Michael , DeLuca Steven , Getts Robert , Lopez Christian , Niewenhuis Robert , Nilsen Thor , Olex Scott , Weintraub Harold , George-Weinstein Mindy
Published: July 2002

MyoD expression is thought to be induced in somites in response to factors released by surrounding tissues; however, reverse transcription-PCR and cell culture analyses indicate that myogenic cells are present in the embryo before somite formation. Fluorescently labeled DNA dendrimers were used to identify MyoD expressing cells in presomitic tissues in vivo. Subpopulations of MyoD positive cells were found in the segmental plate, epiblast, mesoderm, and hypoblast. Directly after laying, the epiblast of the two layered embryo contained ∼20 MyoD positive cells. These results demonstrate that dendrimers are precise and sensitive reagents for localizing low levels of mRNA in tissue sections and whole embryos, and that cells with myogenic potential are present in the embryo before the initiation of gastrulation.
Region-Specific Microtubule Transport in Motile Cells
Authors: Yvon Anne-Marie C. , Wadsworth Patricia
Published: July 2002

Photoactivation and photobleaching of fluorescence were used to determine the mechanism by which microtubules (MTs) are remodeled in PtK2 cells during fibroblast-like motility in response to hepatocyte growth factor (HGF). The data show that MTs are transported during cell motility in an actomyosin-dependent manner, and that the direction of transport depends on the dominant force in the region examined. MTs in the leading lamella move rearward relative to the substrate, as has been reported in newt cells (Waterman-Storer, C.M., and E.D. Salmon. 1997. J. Cell Biol. 139:417–434), whereas MTs in the cell body and in the retraction tail move forward, in the direction of cell locomotion. In the transition zone between the peripheral lamella and the cell body, a subset of MTs remains stationary with respect to the substrate, whereas neighboring MTs are transported either forward, with the cell body, or rearward, with actomyosin retrograde flow. In addition to transport, the photoactivated region frequently broadens, indicating that individual marked MTs are moved either at different rates or in different directions. Mark broadening is also observed in nonmotile cells, indicating that this aspect of transport is independent of cell locomotion. Quantitative measurements of the dissipation of photoactivated fluorescence show that, compared with MTs in control nonmotile cells, MT turnover is increased twofold in the lamella of HGF-treated cells but unchanged in the retraction tail, demonstrating that microtubule turnover is regionally regulated.
Internal Modification of U2 Small Nuclear (Snrna) Occurs in Nucleoli of Xenopus Oocytes
Authors: Yu Yi-Tao , Shu Mei-Di , Narayanan Aarthi , Terns Rebecca M. , Terns Michael P. , Steitz Joan A.
Published: July 2002

U2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5′ trimethylated guanosine cap, 13 pseudouridines, and 10 2′-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.
The Orphan Nuclear Estrogen Receptor–Related Receptor α (Errα) Is Expressed Throughout Osteoblast Differentiation and Regulates Bone Formation in Vitro
Authors: Bonnelye E. , Merdad L. , Kung V. , Aubin J.E.
Published: July 2002

The orphan nuclear estrogen receptor–related receptor α (ERRα), is expressed by many cell types, but is very highly expressed by osteoblastic cells in which it transactivates at least one osteoblast-associated gene, osteopontin. To study the putative involvement of ERRα in bone, we first assessed its expression in rat calvaria (RC) in vivo and in RC cells in vitro. ERRα mRNA and protein were expressed at all developmental stages from early osteoprogenitors to bone-forming osteoblasts, but protein was most abundant in mature cuboidal osteoblasts. To assess a functional role for ERRα in osteoblast differentiation and bone formation, we blocked its expression by antisense oligonucleotides in either proliferating or differentiating RC cell cultures and found inhibition of cell growth and a proliferation-independent inhibition of differentiation. On the other hand, ERRα overexpression in RC cells increased differentiation and maturation of progenitors to mature bone-forming cells. Our findings show that ERRα is highly expressed throughout the osteoblast developmental sequence and plays a physiological role in differentiation and bone formation at both proliferation and differentiation stages. In addition, we found that manipulation of receptor levels in the absence of known ligand is a fruitful approach for functional analysis of this orphan receptor and identification of potential target genes.
Vav2 is required for cell spreading
Authors: Marignani Paola A. , Carpenter Christopher L.
Published: July 2002

Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor– or epidermal growth factor–dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor–dependent activation of Rac leading to lamellipodia.
Distinct Roles of Lymphotoxin α and the Type I Tumor Necrosis Factor (TNF) Receptor in the Establishment of Follicular Dendritic Cells from Non–Bone Marrow–derived Cells
Authors: Matsumoto Mitsuru , Fu Yang-Xin , Molina Hector , Huang Guangming , Kim Jinho , Thomas Dori A. , Nahm Moon H. , Chaplin David D.
Published: July 2002

In mice deficient in either lymphotoxin α (LT-α) or type I tumor necrosis factor receptor (TNFR-I), organized clusters of follicular dendritic cells (FDC) and germinal centers (GC) are absent from the spleen. We investigated the role of LT-α and TNFR-I in the establishment of spleen FDC and GC structure by using reciprocal bone marrow (BM) transfer. When LT-α–deficient mice were reconstituted with wild-type BM, FDC organization and the ability to form GC were restored, indicating that the LT-α–expressing cells required to establish organized FDC are derived from BM. The role of LT-α in establishing organized FDC structure was further investigated by the transfer of complement receptor 1 and 2 (CR1/2)–deficient BM cells into LT-α–deficient mice. Organized FDC were identified with both the FDC-M1 and anti-CR1 monoclonal antibodies in these BM-chimeric mice, indicating that these cells were derived from the LT-α–deficient recipient. Thus, expression of LT-α in the BM-derived cells, but not in the non–BM-derived cells, is required for the maturation of FDC from non-BM precursor cells. In contrast, when TNFR-I–deficient mice were reconstituted with wild-type BM, they showed no detectable FDC clusters or GC formation. This indicates that TNFR-I expression on non–BM-derived cellular components is necessary for the establishment of these lymphoid structures. TNFR-I–deficient BM was able to restore FDC organization and GC formation in LT-α–deficient mice, indicating that formation of these structures does not require TNFR-I expression on BM-derived cells. The data in this study demonstrate that FDC organization and GC formation are controlled by both LT-α–expressing BM-derived cells and by TNFR-I-expressing non–BM-derived cells.
Dendritic Cells Genetically Modified with an Adenovirus Vector Encoding the cDNA for a Model Antigen Induce Protective and Therapeutic Antitumor Immunity
Authors: Song Wenru , Kong Hwai-Loong , Carpenter Heather , Torii Hideshi , Granstein Richard , Rafii Shahin , Moore Malcolm A.S. , Crystal Ronald G.
Published: July 2002

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the initiation of antitumor immune responses. In this study, we show that genetic modifications of a murine epidermis-derived DC line and primary bone marrow–derived DCs to express a model antigen β-galactosidase (βgal) can be achieved through the use of a replication-deficient, recombinant adenovirus vector, and that the modified DCs are capable of eliciting antigen-specific, MHC-restricted CTL responses. Importantly, using a murine metastatic lung tumor model with syngeneic colon carcinoma cells expressing βgal, we show that immunization of mice with the genetically modified DC line or bone marrow DCs confers potent protection against a lethal tumor challenge, as well as suppression of preestablished tumors, resulting in a significant survival advantage. We conclude that genetic modification of DCs to express antigens that are also expressed in tumors can lead to antigen-specific, antitumor killer cells, with a concomitant resistance to tumor challenge and a decrease in the size of existing tumors.
Acidic Sphingomyelinase (ASM) Is Necessary for Fas-induced GD3 Ganglioside Accumulation and Efficient Apoptosis of Lymphoid Cells
Authors: De Maria Ruggero , Rippo Maria Rita , Schuchman Edward H. , Testi Roberto
Published: July 2002

Ceramides deriving from sphingomyelin hydrolysis are important mediators of apoptotic signals originating from Fas (APO-1/CD95). However, definitive evidence for the role played by individual sphingomyelinases is still lacking. We have analyzed lymphoblastoid cell lines derived from patients affected by Niemann Pick disease (NPD), an autosomal recessive disorder caused by loss-of-function mutations within the acidic sphingomyelinase (ASM) gene. NPD lymphoblasts, which display normal neutral sphingomyelinase activity, fail to activate ASM in response to Fas cross-linking, unlike normal lymphoblasts. NPD lymphoblasts also fail to accumulate GD3 ganglioside, a downstream mediator of ceramide-induced cell death (De Maria, R., L. Lenti, F. Malisan, F. D'Agostino, B. Tomassini, A. Zeuner, M.R. Rippo, R. Testi. 1997. Science. 277:1652–1655), and display a substantially inefficient apoptosis after Fas cross-linking. Inefficient apoptosis is due to lack of ASM activity, because proximal signaling from Fas in NPD lymphoblasts is not impaired and apoptosis can be efficiently triggered by passing the ASM defect with exogenous ceramides. Moreover, mannose receptor–mediated transfer of ASM into NPD lymphoblasts rescues their ability to transiently activate ASM, accumulate GD3, and rapidly undergo apoptosis after Fas cross-linking. These results provide definitive genetic evidence for the role of ASM in the progression of apoptotic signals originating from Fas.
Vasostatin, a Calreticulin Fragment, Inhibits Angiogenesis and Suppresses Tumor Growth
Authors: Pike Sandra E. , Yao Lei , Jones Karen D. , Cherney Barry , Appella Ettore , Sakaguchi Kazuyasu , Nakhasi Hira , Teruya-Feldstein Julie , Wirth Peter , Gupta Ghanshyam , Tosato Giovanna
Published: July 2002

An endothelial cell inhibitor was purified from supernatant of an Epstein-Barr virus–immortalized cell line and identified as fragments of calreticulin. The purified recombinant NH2-terminal domain of calreticulin (amino acids 1–180) inhibited the proliferation of endothelial cells, but not cells of other lineages, and suppressed angiogenesis in vivo. We have named this NH2-terminal domain of calreticulin vasostatin. When inoculated into athymic mice, vasostatin significantly reduced growth of human Burkitt lymphoma and human colon carcinoma. Compared with other inhibitors of angiogenesis, vasostatin is a small, soluble, and stable molecule that is easy to produce and deliver. As an angiogenesis inhibitor that specifically targets proliferating endothelial cells, vasostatin has a unique potential for cancer treatment.
Activated Murine B Lymphocytes and Dendritic Cells Produce a Novel CC Chemokine which Acts Selectively on Activated T Cells
Authors: Schaniel Christoph , Pardali Evangelia , Sallusto Federica , Speletas Mattheos , Ruedl Christiane , Shimizu Takeyuki , Seidl Thomas , Andersson Jan , Melchers Fritz , Rolink Antonius G. , Sideras Paschalis
Published: July 2002

Genes were isolated using the suppression subtractive hybridization method by stimulation of pro/pre B cells with anti-CD40 and interleukin (IL)-4 to mature Sμ-Sε–switched cells. One of the strongly upregulated genes encodes a novel murine CC chemokine we have named ABCD-1. The ABCD-1 gene has three exons separated by 1.2- and 2.7-kb introns. It gives rise to a 2.2-kb transcript containing an open reading frame of 276 nucleotides. Two polyadenylation sites are used, giving rise to cDNAs with either 1550 or 1850 bp of 3′ untranslated regions. The open reading frame encodes a 24 amino acid–long leader peptide and a 68 amino acid–long mature protein with a predicted molecular mass of 7.8 kD. ABCD-1 mRNA is found in highest quantities in activated splenic B lymphocytes and dendritic cells. Little chemokine mRNA is present in lung, in unstimulated splenic cells, in thymocytes, and in lymph node cells. No ABCD-1 mRNA is detected in bone marrow, liver, kidney, or brain, in peritoneal exudate cells as well as in the majority of all unstimulated B lineage cells tested. It is also undetectable in Concanavalin A–activated/IL-2–restimulated splenic T cells, and in bone marrow–derived IL-2–induced natural killer cells and IL-3–activated macrophages. Recombinant ABCD-1 revealed a concentration-dependent and specific migration of activated splenic T lymphoblasts in chemotaxis assays. FACS® analyses of migrated cells showed no preferential difference in migration of CD4+ versus CD8+ T cell blasts. Murine as well as human T cells responded to ABCD-1. Freshly isolated cells from bone marrow, thymus, spleen, and lymph node, IL-2–activated NK cells, and LPS-stimulated splenic cells, all did not show any chemotactic response. Thus, ABCD-1 is the first chemokine produced in large amounts by activated B cells and acting selectively on activated T lymphocytes. Therefore, ABCD-1 is expected to play an important role in the collaboration of dendritic cells and B lymphocytes with T cells in immune responses.
HIV-specific T Cell Cytotoxicity Mediated by RANTES Via the Chemokine Receptor CCR3
Authors: Hadida Fabienne , Vieillard Vincent , Autran Brigitte , Clark-Lewis Ian , Baggiolini Marco , Debré Patrice
Published: July 2002

CC chemokines produced by CD8+ T cells are known to act as HIV-suppressive factors. We studied the possible role of these chemokines in HIV-1–specific killing of target cells. We found that the activity of cytotoxic T lymphocytes (CTLs) in CTL lines or freshly isolated peripheral blood mononuclear cells from HIV-1–infected individuals is markedly enhanced by RANTES (regulated on activation, normal T cell expressed and secreted) and virtually abolished by an antibody neutralizing RANTES or the RANTES receptor antagonist RANTES(9-68). Lysis was mediated by CD8+ major histocompatibility complex class I–restricted T cells and was obtained with target cells expressing epitopes of the HIV-1LAI proteins Gag, Pol, Env, and Nef. The cytolytic activity observed in the presence or absence of added RANTES could be abolished by pretreatment of the CTLs with pertussis toxin, indicating that the effect is mediated by a G protein–coupled receptor. The chemokines monocyte chemotactic protein (MCP)-3, MCP-4, and eotaxin acted like RANTES, whereas macrophage inflammatory protein (MIP)-1α, MIP-1β, MCP-1, and stromal cell–derived factor 1 were inactive, suggesting a role for the eotaxin receptor, CCR3, and ruling out the involvement of CCR1, CCR2, CCR5, and CXCR4. CTL activity was abrogated by an antibody that blocks CCR3, further indicating that specific lysis is triggered via this chemokine receptor. These observations reveal a novel mechanism for the induction of HIV-1–specific cytotoxicity that depends on RANTES acting via CCR3.
Severe Attenuation of the B Cell Immune Response in Msh2-deficient Mice
Authors: Vora Kalpit A. , Tumas-Brundage Kathleen M. , Lentz Vicky M. , Cranston Aaron , Fishel Richard , Manser Tim
Published: July 2002

Recently, results obtained from mice with targeted inactivations of postreplication DNA mismatch repair (MMR) genes have been interpreted to demonstrate a direct role for MMR in antibody variable (V) gene hypermutation. Here we show that mice that do not express the MMR factor Msh2 have wide-ranging defects in antigen-driven B cell responses. These include lack of progression of the germinal center (GC) reaction associated with increased intra-GC apoptosis, severely diminished antigen-specific immunoglobulin G responses, and near absence of anamnestic responses. Mice heterozygous for the Msh2 deficiency display an “intermediate” phenotype in these regards, suggesting that normal levels of Msh2 expression are critical for the B cell response. Interpretation of the impact of an MMR deficiency on the mechanism of V gene somatic hypermutation could be easily confounded by these perturbations.
Reduced Isotype Switching in Splenic B Cells from Mice Deficient in Mismatch Repair Enzymes
Authors: Schrader Carol E. , Edelmann Winfried , Kucherlapati Raju , Stavnezer Janet
Published: July 2002

Mice deficient in various mismatch repair (MMR) enzymes were examined to determine whether this repair pathway is involved in antibody class switch recombination. Splenic B cells from mice deficient in Msh2, Mlh1, Pms2, or Mlh1 and Pms2 were stimulated in culture with lipopolysaccharide (LPS) to induce immunoglobulin (Ig)G2b and IgG3, LPS and interleukin (IL)-4 to induce IgG1, or LPS, anti–δ-dextran, IL-4, IL-5, and transforming growth factor (TGF)-β1 to induce IgA. After 4 d in culture, cells were surface stained for IgM and non-IgM isotypes and analyzed by FACS®. B cells from MMR-deficient mice show a 35–75% reduction in isotype switching, depending on the isotype and on the particular MMR enzyme missing. IgG2b is the most affected, reduced by 75% in Mlh1-deficient animals. The switching defect is not due to a lack of maturation of the B cells, as purified IgM+IgD+ B cells show the same reduction. MMR deficiency had no effect on cell proliferation, viability, or apoptosis, as detected by [3H]thymidine incorporation and by propidium iodide staining. The reduction in isotype switching was demonstrated to be at the level of DNA recombination by digestion-circularization polymerase chain reaction (DC-PCR). A model of the potential role for MMR enzymes in class switch recombination is presented.
Cd4+ T Cell Division in Irradiated Mice Requires Peptides Distinct from Those Responsible for Thymic Selection
Authors: Bender Jeremy , Mitchell Tom , Kappler John , Marrack Philippa
Published: July 2002

We investigated the mechanism by which α/β T cells expand upon transfer to T cell–deficient host mice by injecting carboxyfluorescein diacetate succinimidyl ester–labeled T cells into mice depleted of T cells by sublethal irradiation. We found that CD4+ T cells divided when transferred to irradiated hosts and that the division of more than half of these cells required class II expression. However, division of transferred CD4+ T cells did not occur in irradiated hosts that expressed class II molecules occupied solely by the peptide responsible for thymic selection, indicating that peptides distinct from those involved in thymic selection cause the division of CD4+ T cells in irradiated mice. These data establish that class II–bound peptides control the expansion of CD4+ T cells transferred to T cell–deficient hosts and suggest that the same peptides contribute to the maintenance of T cell numbers in normal mice.
Receptor-Mediated Uptake of Antigen/Heat Shock Protein Complexes Results in Major Histocompatibility Complex Class I Antigen Presentation via Two Distinct Processing Pathways
Authors: Castellino Flora , Boucher Philip E. , Eichelberg Katrin , Mayhew Mark , Rothman James E. , Houghton Alan N. , Germain Ronald N.
Published: July 2002

Heat shock proteins (HSPs) derived from tumors or virally infected cells can stimulate antigen-specific CD8+ T cell responses in vitro and in vivo. Although this antigenicity is known to arise from HSP-associated peptides presented to the immune system by major histocompatibility complex (MHC) class I molecules, the cell biology underlying this presentation process remains poorly understood. Here we show that HSP 70 binds to the surface of antigen presenting cells by a mechanism with the characteristics of a saturable receptor system. After this membrane interaction, processing and MHC class I presentation of the HSP-associated antigen can occur via either a cytosolic (transporter associated with antigen processing [TAP] and proteasome–dependent) or an endosomal (TAP and proteasome–independent) route, with the preferred pathway determined by the sequence context of the optimal antigenic peptide within the HSP-associated material. These findings not only characterize two highly efficient, specific pathways leading to the conversion of HSP-associated antigens into ligands for CD8+ T cells, they also imply the existence of a mechanism for receptor-facilitated transmembrane transport of HSP or HSP-associated ligands from the plasma membrane or lumen of endosomes into the cytosol.
Platelet-activating Factor, a Molecular Sensor for Cellular Damage, Activates Systemic Immune Suppression
Authors: Walterscheid Jeffrey P. , Ullrich Stephen E. , Nghiem Dat X.
Published: July 2002

Ultraviolet (UV) radiation plays a critical role in the induction of nonmelanoma skin cancer. UV radiation is also immune suppressive, and the immune suppression induced by UV irradiation has been identified as a major risk factor for skin cancer induction. Previously, we showed that UV exposure activates a cytokine cascade involving prostaglandin (PG)E2, interleukin (IL)-4, and IL-10 that induces immune suppression. However, the earliest molecular events that occur immediately after UV exposure, especially those upstream of PGE2, are not well defined. UV-irradiated keratinocytes secrete the inflammatory phospholipid mediator, platelet-activating factor (PAF). Because PAF upregulates the production of immunomodulatory compounds, including PGE2, we tested the hypothesis that UV-induced PAF activates cytokine production and initiates UV-induced immune suppression. Both UV and PAF activated cyclooxygenase (COX)-2 and IL-10 reporter gene construct transcription. PAF mimicked the effects of UV in vivo and suppressed delayed-type hypersensitivity (DTH). Furthermore, immune suppression was blocked when UV-irradiated mice were injected with PAF receptor antagonists. In addition to the well-known role of PAF as a proinflammatory lipid mediator, we propose that the PAF receptor senses cellular damage through the recognition of PAF and/or PAF-like molecules, such as oxidized phosphatidylcholine, which activates cytokine transcription and induces systemic immune suppression.
Monovalent Ion Selectivity Sequences of the Rat Connexin43 Gap Junction Channel
Authors: Wang Hong-Zhan , Veenstra Richard D.
Published: July 2002

The relative permeability sequences of the rat connexin 43 (rCx43) gap junction channel to seven cations and chloride were examined by double whole cell patch clamp recording of single gap junction channel currents in rCx43 transfected neuroblastoma 2A (N2A) cell pairs. The measured maximal single channel slope conductances (γj, in pS) of the junctional current-voltage relationships in 115 mM XCl were RbCl (103) ≥ CsCl (102) > KCl (97) > NaCl (79) ≥ LiCl (78) > TMACl (65) > TEACl (53) and for 115 mM KY were KBr (105) > KCl (97) > Kacetate (77) > Kglutamate (61). The single channel conductance-aqueous mobility relationships for the test cations and anions were linear. However, the predicted minimum anionic and cationic conductances of these plots did not accurately predict the rCx43 channel conductance in 115 mM KCl. Instead, the conductance of the rCx43 channel in 115 mM KCl was accurately predicted from cationic and anionic conductance-mobility plots by applying a mobility scaling factor Dx/Do, which depends upon the relative radii of the permeant ions to an estimated pore radius. Relative permeabilities were determined for all of the monovalent cations and anions tested from asymmetric salt reversal potential measurements and the Goldman-Hodgkin-Katz voltage equation. These experiments estimate the relative chloride to potassium permeability to be 0.13. The relationship between the relative cation permeability and hydrated radius was modeled using the hydrodynamic equation assuming a pore radius of 6.3 ± 0.4 Å. Our data quantitatively demonstrate that the rCx43 gap junction channel is permeable to monovalent atomic and organic cations and anions and the relative permeability sequences are consistent with an Eisenman sequence II or I, respectively. These predictions about the rCx43 channel pore provide a useful basis for future investigations into the structural determinants of the conductance and permeability properties of the connexin channel pore.
Distinct Transient Outward Potassium Current (Ito) Phenotypes and Distribution of Fast-inactivating Potassium Channel Alpha Subunits in Ferret Left Ventricular Myocytes
Authors: Brahmajothi Mulugu V. , Campbell Donald L. , Rasmusson Randall L. , Morales Michael J. , Trimmer James S. , Nerbonne Jeanne M. , Strauss Harold C.
Published: July 2002

The biophysical characteristics and α subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21–22°C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [τrec, −70 mV = 51 ± 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [τrec, −70 mV = 3,002 ± 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 α subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 α subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito α subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4.2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito α subunits not only exists from LV epicardium to endocardium but also from apex to base.
Normal service continues — the role of intermediaries in electronic publishing
Authors: Prior Albert
Published: July 2002

The paper outlines some of the conditions which bring about the need for intermediaries in the electronic world, the views of end users and librarians towards electronic journals and the range of intermediary services which exist for Internet based full texts. It also briefly considers archiving and digitisation activities.
On the range of the sum of monotone operators in general Banach spaces
Authors: Riahi Hassan
Published: July 2002

The purpose of this paper is to generalize the Brézis-Haraux theorem on the range of the sum of monotone operators from a Hilbert space to general Banach spaces. The result obtained provides that the range

R

(

A
+
B

¯

τ

)

\mathcal R(\overline {A+B}{}^\tau )

is topologically almost equal to the sum

R

(
A
)
+

R

(
B
)

\mathcal R(A)+\mathcal R(B)

where

τ
\tau

is a compatible topology in

X

∗
∗

×

X
∗

X^{**}\times X^*

as proposed by Gossez. To illustrate the main result we consider some basic properties of densely maximal monotone operators.
Iteration of compact holomorphic maps on a Hilbert ball
Authors: Chu Cho-Ho , Mellon Pauline
Published: July 2002

Given a compact holomorphic fixed-point-free self-map,

f
f

, of the open unit ball of a Hilbert space, we show that the sequence of iterates,

(

f
n

)

(f^n)

, converges locally uniformly to a constant map

ξ
\xi

with

‖
ξ
‖
=
1

\Vert \xi \Vert = 1

. This extends results of Denjoy (1926), Wolff (1926), Hervé (1963) and MacCluer (1983). The result is false without the compactness assumption, nor is it true for all open balls of

J

∗

J^{*}

-algebras.
The normal homogeneous space (𝖲𝖴(3)×𝖲𝖮(3))/𝖴^{∙}(2) has positive sectional curvature
Authors: Wilking Burkhard
Published: July 2002

We give a new description of the positively curved seven-dimensional Aloff-Wallach spaces

M

k
l

7

M^7_{kl}

. In particular, this description exhibits

M

11

7

M^7_{11}

as a normal homogeneous space, although it does not occur in Berger’s classification (1961).
Verifying the Goldbach conjecture up to 4⋅10¹⁴
Authors: Richstein Jörg
Published: July 2002

Using a carefully optimized segmented sieve and an efficient checking algorithm, the Goldbach conjecture has been verified and is now known to be true up to

4
⋅

10

14

4\cdot 10^{14}

. The program was distributed to various workstations. It kept track of maximal values of the smaller prime

p
p

in the minimal partition of the even numbers, where a minimal partition is a representation

2
n
=
p
+
q

2n = p + q

with

2
n
−

p
′

2n - p’

being composite for all

p
′

>
p

p’ > p

. The maximal prime

p
p

needed in the considered interval was found to be 5569 and is needed for the partition 389965026819938 = 5569 + 389965026814369.
Distribution properties of multiply-with-c arry random number generators
Authors: Couture Raymond , L’Ecuyer Pierre
Published: July 2002

We study the multiply-with-carry family of generators proposed by Marsaglia as a generalization of previous add-with-carry families. We define for them an infinite state space and focus our attention on the (finite) subset of recurrent states. This subset will, in turn, split into possibly several subgenerators. We discuss the uniformity of the

d
d

-dimensional distribution of the output of these subgenerators over their full period. In order to improve this uniformity for higher dimensions, we propose a method for finding good parameters in terms of the spectral test. Our results are stated in a general context and are applied to a related complementary multiply-with-carry family of generators.
Association of a Nonmuscle Myosin II with Axoplasmic Organelles
Authors: DeGiorgis Joseph A. , Reese Thomas S. , Bearer Elaine L.
Published: July 2002

Association of motor proteins with organelles is required for the motors to mediate transport. Because axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a ∼220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this ∼220 kDa protein as a nonmuscle myosin II. This myosin is also present in axoplasm, as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using nonmuscle myosin II isotype-specific antibodies detected a single ∼220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment, which separates organelles from cytoskeletal components. Of the total nonmuscle myosin II in axoplasm, 43.2% copurified with organelles in the 15% sucrose fraction, while the remainder (56.8%) was soluble and found in the supernatant. This myosin decorates the cytoplasmic surface of 21% of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, nonmuscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. Therefore, in addition to myosin V, this myosin is likely to be an axoplasmic organelle motor.
The Three Mouse Actin-depolymerizing Factor/Cofilins Evolved to Fulfill Cell-Type–specific Requirements for Actin Dynamics
Authors: Vartiainen Maria K. , Mustonen Tuija , Mattila Pieta K. , Ojala Pauli J. , Thesleff Irma , Partanen Juha , Lappalainen Pekka
Published: July 2002

Actin-depolymerizing factor (ADF)/cofilins are essential regulators of actin filament turnover. Several ADF/cofilin isoforms are found in multicellular organisms, but their biological differences have remained unclear. Herein, we show that three ADF/cofilins exist in mouse and most likely in all other mammalian species. Northern blot and in situ hybridization analyses demonstrate that cofilin-1 is expressed in most cell types of embryos and adult mice. Cofilin-2 is expressed in muscle cells and ADF is restricted to epithelia and endothelia. Although the three mouse ADF/cofilins do not show actin isoform specificity, they all depolymerize platelet actin filaments more efficiently than muscle actin. Furthermore, these ADF/cofilins are biochemically different. The epithelial-specific ADF is the most efficient in turning over actin filaments and promotes a stronger pH-dependent actin filament disassembly than the two other isoforms. The muscle-specific cofilin-2 has a weaker actin filament depolymerization activity and displays a 5–10-fold higher affinity for ATP-actin monomers than cofilin-1 and ADF. In steady-state assays, cofilin-2 also promotes filament assembly rather than disassembly. Taken together, these data suggest that the three biochemically distinct mammalian ADF/cofilin isoforms evolved to fulfill specific requirements for actin filament dynamics in different cell types.
Morphology and Dynamics of the Endocytic Pathway inDictyostelium discoideum
Authors: Neuhaus Eva M. , Almers Wolfhard , Soldati Thierry
Published: July 2002

Dictyostelium discoideum is a genetically and biochemically tractable social amoeba belonging to the crown group of eukaryotes. It performs some of the tasks characteristic of a leukocyte such as chemotactic motility, macropinocytosis, and phagocytosis that are not performed by other model organisms or are difficult to study.D. discoideum is becoming a popular system to study molecular mechanisms of endocytosis, but the morphological characterization of the organelles along this pathway and the comparison with equivalent and/or different organelles in animal cells and yeasts were lagging. Herein, we used a combination of evanescent wave microscopy and electron microscopy of rapidly frozen samples to visualize primary endocytic vesicles, vesicular-tubular structures of the early and late endo-lysosomal system, such as multivesicular bodies, and the specialized secretory lysosomes. In addition, we present biochemical and morphological evidence for the existence of a micropinocytic pathway, which contributes to the uptake of membrane along side macropinocytosis, which is the major fluid phase uptake process. This complex endosomal compartment underwent continuous cycles of tubulation/vesiculation as well as homo- and heterotypic fusions, in a way reminiscent of mechanisms and structures documented in leukocytes. Finally, egestion of fluid phase from the secretory lysosomes was directly observed.