bluuebunny/crossref_metadata_2025 huggingface dataset:
Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p
Authors: VanRheenen Susan M. , Cao Xiaochun , Sapperstein Stephanie K. , Chiang Elbert C. , Lupashin Vladimir V. , Barlowe Charles , Waters M. Gerard
Published: July 2002

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.
Cell Spreading and Lamellipodial Extension Rate Is Regulated by Membrane Tension
Authors: Raucher Drazen , Sheetz Michael P.
Published: July 2002

Cell spreading and motility require the extension of the plasma membrane in association with the assembly of actin. In vitro, extension must overcome resistance from tension within the plasma membrane. We report here that the addition of either amphiphilic compounds or fluorescent lipids that expanded the plasma membrane increased the rate of cell spreading and lamellipodial extension, stimulated new lamellipodial extensions, and caused a decrease in the apparent membrane tension. Further, in PDGF-stimulated motility, the increase in the lamellipodial extension rate was associated with a decrease in the apparent membrane tension and decreased membrane–cytoskeleton adhesion through phosphatidylinositol diphosphate hydrolysis. Conversely, when membrane tension was increased by osmotically swelling cells, the extension rate decreased. Therefore, we suggest that the lamellipodial extension process can be activated by a physical signal (perhaps secondarily), and the rate of extension is directly dependent upon the tension in the plasma membrane. Quantitative analysis shows that the lamellipodial extension rate is inversely correlated with the apparent membrane tension. These studies describe a physical chemical mechanism involving changes in membrane–cytoskeleton adhesion through phosphatidylinositol 4,5-biphosphate–protein interactions for modulating and stimulating the biochemical processes that power lamellipodial extension.
The Small Gtpase, Rap1, Mediates Cd31-Induced Integrin Adhesion
Authors: Reedquist Kris A. , Ross Ewan , Koop Elianne A. , Wolthuis Rob M.F. , Zwartkruis Fried J.T. , van Kooyk Yvette , Salmon Mike , Buckley Christopher D. , Bos Johannes L.
Published: July 2002

Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood. Here, we show that the cytoplasmic tail of CD31, an important integrin adhesion amplifier, propagates signals that induce T cell adhesion via β1 (VLA-4) and β2 (LFA-1) integrins. We identify the small GTPase, Rap1, as a critical mediator of this effect. Importantly, CD31 selectively activated the small Ras-related GTPase, Rap1, but not Ras, R-Ras, or Rap2. An activated Rap1 mutant stimulated T lymphocyte adhesion to intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as did the Rap1 guanine nucleotide exchange factor C3G and a catalytically inactive mutant of RapGAP. Conversely, negative regulators of Rap1 signaling blocked CD31-dependent adhesion. These findings identify a novel important role for Rap1 in regulating ligand-induced cell adhesion and suggest that Rap1 may play a more general role in coordinating adhesion-dependent signals during leukocyte migration and extravasation. Our findings also suggest an alternative mechanism, distinct from interference with Ras-proximal signaling, by which Rap1 might mediate transformation reversion.
Role of the Cytoplasmic Segments of Sec61α in the Ribosome-Binding and Translocation-Promoting Activities of the Sec61 Complex
Authors: Raden David , Song Weiqun , Gilmore Reid
Published: July 2002

The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH2 and COOH terminus of Sec61α was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61α. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.
Glycogen Synthase Kinase-3β Is a Negative Regulator of Cardiomyocyte Hypertrophy
Authors: Haq Syed , Choukroun Gabriel , Kang Zhao Bin , Ranu Hardeep , Matsui Takashi , Rosenzweig Anthony , Molkentin Jeffrey D. , Alessandrini Alessandro , Woodgett James , Hajjar Roger , Michael Ashour , Force Thomas
Published: July 2002

Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3β (GSK-3β), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase–dependent protein kinase that phosphorylates GSK-3β on ser 9. Using adenovirus-mediated gene transfer of GSK-3β containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3β is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3β regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3β as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway.
Focal Activation of a Mutant Allele Defines the Role of Stem Cells in Mosaic Skin Disorders
Authors: Arin Meral J. , Longley Mary Ann , Wang Xiao-Jing , Roop Dennis R.
Published: July 2002

Stem cells are crucial for the formation and maintenance of tissues and organs. The role of stem cells in the pathogenesis of mosaic skin disorders remains unclear. To study the molecular and cellular basis of mosaicism, we established a mouse model for the autosomal-dominant skin blistering disorder, epidermolytic hyperkeratosis (MIM 113800), which is caused by mutations in either keratin K1 or K10. This genetic model allows activation of a somatic K10 mutation in epidermal stem cells in a spatially and temporally controlled manner using an inducible Cre recombinase. Our results indicate that lack of selective pressure against certain mutations in epidermal stem cells leads to mosaic phenotypes. This finding has important implications for the development of new strategies for somatic gene therapy of dominant genodermatoses.
Regulation of Cdc42 Gtpase by Proline-Rich Tyrosine Kinase 2 Interacting with Psgap, a Novel Pleckstrin Homology and Src Homology 3 Domain Containing Rhogap Protein
Authors: Ren Xiu-Rong , Du Quan-Sheng , Huang Yang-Zhong , Ao Shi-Zhou , Mei Lin , Xiong Wen-Cheng
Published: July 2002

Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain–dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.
A Link between the Synthesis of Nucleoporins and the Biogenesis of the Nuclear Envelope
Authors: Marelli Marcello , Lusk C. Patrick , Chan Honey , Aitchison John D. , Wozniak Richard W.
Published: July 2002

The nuclear pore complex (NPC) is a multicomponent structure containing a subset of proteins that bind nuclear transport factors or karyopherins and mediate their movement across the nuclear envelope. By altering the expression of a single nucleoporin gene, NUP53, we showed that the overproduction of Nup53p altered nuclear transport and had a profound effect on the structure of the nuclear membrane. Strikingly, conventional and immunoelectron microscopy analysis revealed that excess Nup53p entered the nucleus and associated with the nuclear membrane. Here, Nup53p induced the formation of intranuclear, tubular membranes that later formed flattened, double membrane lamellae structurally similar to the nuclear envelope. Like the nuclear envelope, the intranuclear double membrane lamellae enclosed a defined cisterna that was interrupted by pores but, unlike the nuclear envelope pores, they lacked NPCs. Consistent with this observation, we detected only two NPC proteins, the pore membrane proteins Pom152p and Ndc1p, in association with these membrane structures. Thus, these pores likely represent an intermediate in NPC assembly. We also demonstrated that the targeting of excess Nup53p to the NPC and its specific association with intranuclear membranes were dependent on the karyopherin Kap121p and the nucleoporin Nup170p. At the nuclear envelope, the abilities of Nup53p to associate with the membrane and drive membrane proliferation were dependent on a COOH-terminal segment containing a potential amphipathic α-helix. The implications of these results with regards to the biogenesis of the nuclear envelope are discussed.
Presence of Cyclophilin A in Synovial Fluids of Patients with Rheumatoid Arthritis
Authors: Billich Andreas , Winkler Gottfried , Aschauer Heinrich , Rot Antal , Peichl Peter
Published: July 2002

Cyclophilins have been suggested to act as leukocyte chemotactic factors produced in the course of inflammation. Therefore we looked for the presence of cyclophilins in the synovial fluids (SF) from patients with rheumatoid arthritis (RA). Peptidyl prolyl cis–trans isomerase activity (PPIase) was measured in SF from knee punctures of 26 patients with RA and five patients with knee osteoarthritis (OA). PPIase was detected in SF from RA patients, but not in samples from OA patients. Enzyme activity was sensitive to inhibition by cyclosporin A (IC50 = 28–50 nM). Estimated concentrations of the SF-derived cyclophilin based on the enzyme activity were in the range of 11 to 705 nM. The presence of cyclophilin in the SF showed disease correlation; its concentration correlated with the number of cells in the SF (r   = 0.91, P <0.0001) and with the percentage of neutrophils in the cellular infiltrate and was higher in more acute cases of joint swelling. In immunoblots of partially purified preparations of SF from RA patients, an ∼18-kD protein band reacted with polyclonal antibodies that recognize cyclophilin A and B, but not with antibodies specific for cyclophilin B. Sequencing of this protein revealed identity of the NH2-terminal amino acids with those of human cyclophilin A. The finding is unexpected since cyclophilin B rather than A is generally regarded as the secreted isoform, the presence of cyclophilin A being confined to the cytoplasm. Our data support the hypothesis that cyclophilins may contribute to the pathogenesis of inflammatory diseases, possibly by acting as cytokines. This may offer a possible explanation of the effectiveness of cyclosporin A in RA, in addition to the known immunosuppressive effects of the drug.
Highly Conserved Neisseria meningitidis Surface Protein Confers Protection against Experimental Infection
Authors: Martin Denis , Cadieux Nathalie , Hamel Josée , Brodeur Bernard R.
Published: July 2002

A new surface protein, named NspA, which is distinct from the previously described Neisseria meningitidis outer membrane proteins was identified. An NspA-specific mAb, named Me-1, reacted with 99% of the meningococcal strains tested indicating that the epitope recognized by this particular mAb is widely distributed and highly conserved. Western immunoblotting experiments indicated that mAb Me-1 is directed against a protein band with an approximate molecular mass of 22,000, but also recognized a minor protein band with an approximate molecular mass of 18,000. This mAb exhibited bactericidal activity against four meningococcal strains, two isolates of serogroup B, and one isolate from each serogroup A and C, and passively protected mice against an experimental infection. To further characterize the NspA protein and to evaluate the protective potential of recombinant NspA protein, the nspA gene was identified and cloned into a low copy expression vector. Nucleotide sequencing of the meningococcal insert revealed an ORF of 525 nucleotides coding for a polypeptide of 174 amino acid residues, with a predicted molecular weight of 18,404 and a isoelectric point of 9.93. Three injections of either 10 or 20 μg of the affinity-purified recombinant NspA protein efficiently protected 80% of the mice against a meningococcal deadly challenge comparatively to the 20% observed in the control groups. The fact that the NspA protein can elicit the production of bactericidal and protective antibodies emphasize its potential as a vaccine candidate.
Infected Cell Protein (ICP)47 Enhances Herpes Simplex Virus Neurovirulence by Blocking the CD8+ T Cell Response
Authors: Goldsmith Kim , Chen Wei , Johnson David C. , Hendricks Robert L.
Published: July 2002

The herpes simplex virus (HSV) infected cell protein (ICP)47 blocks CD8+ T cell recognition of infected cells by inhibiting the transporter associated with antigen presentation (TAP). In vivo, HSV-1 replicates in two distinct tissues: in epithelial mucosa or epidermis, where the virus enters sensory neurons; and in the peripheral and central nervous system, where acute and subsequently latent infections occur. Here, we show that an HSV-1 ICP47− mutant is less neurovirulent than wild-type HSV-1 in mice, but replicates normally in epithelial tissues. The reduced neurovirulence of the ICP47− mutant was due to a protective CD8+ T cell response. When compared with wild-type virus, the ICP47− mutant expressed reduced neurovirulence in immunologically normal mice, and T cell–deficient nude mice after reconstitution with CD8+ T cells. However, the ICP47− mutant exhibited normal neurovirulence in mice that were acutely depleted of CD8+ T cells, and in nude mice that were not reconstituted, or were reconstituted with CD4+ T cells. In contrast, CD8+ T cell depletion did not increase the neurovirulence of an unrelated, attenuated HSV-1 glycoprotein (g)E− mutant. ICP47 is the first viral protein shown to influence neurovirulence by inhibiting CD8+ T cell protection.
Induction and Exhaustion of Lymphocytic Choriomeningitis Virus–specific Cytotoxic T Lymphocytes Visualized Using Soluble Tetrameric Major Histocompatibility Complex Class I–Peptide Complexes
Authors: Gallimore Awen , Glithero Ann , Godkin Andrew , Tissot Alain C. , Plückthun Andreas , Elliott Tim , Hengartner Hans , Zinkernagel Rolf
Published: July 2002

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated β2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33–41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33–41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8+ T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.
Spontaneous Skin Ulceration and Defective T Cell Function in CD18 Null Mice
Authors: Scharffetter-Kochanek Karin , Lu Huifang , Norman Keith , van Nood Nicole , Munoz Flor , Grabbe Stephan , McArthur Mark , Lorenzo Isabel , Kaplan Sheldon , Ley Klaus , Wayne Smith C. , Montgomery Charles A. , Rich Susan , Beaudet Arthur L.
Published: July 2002

A null mutation was prepared in the mouse for CD18, the β2 subunit of leukocyte integrins. Homozygous CD18 null mice develop chronic dermatitis with extensive facial and submandibular erosions. The phenotype includes elevated neutrophil counts, increased immunoglobulin levels, lymphadenopathy, splenomegaly, and abundant plasma cells in skin, lymph nodes, gut, and kidney. Very few neutrophils were found in spontaneously occurring skin lesions or with an induced toxic dermatitis. Intravital microscopy in CD18 null mice revealed a lack of firm neutrophil attachment to venules in the cremaster muscle in response to N-formyl- methionyl-leucyl-phenylalanine. A severe defect in T cell proliferation was found in the CD18 null mice when T cell receptors were stimulated either by staphylococcal enterotoxin A or by major histocompatibility complex alloantigens demonstrating a greater role of CD11/CD18 integrins in T cell responses than previously documented. The null mice are useful for delineating the functions of CD18 in vivo.
Major Histocompatibility Complex Class II Expression by Intrinsic Renal Cells Is Required for Crescentic Glomerulonephritis
Authors: Li Shuo , Kurts Christian , Köntgen Frank , Holdsworth Stephen R. , Tipping Peter G.
Published: July 2002

The requirement for major histocompatibility complex class II (MHC II) to initiate immune renal injury was studied in a murine model of CD4+ T cell–dependent crescentic glomerulonephritis (GN). C57BL/6 (MHC II+/+) mice developed crescentic GN with glomerular CD4+ T cell infiltration and renal injury, in response to a nephritogenic antigen (sheep globulin) planted on their glomerular basement membrane. MHC II–deficient C57BL/6 mice (MHC II−/−) did not develop crescentic GN, CD4+ T cell infiltration, or injury, indicating that this form of immune glomerular injury is MHC II dependent. The requirement for MHC II expression by intrinsic renal cells was studied in chimeric mice, which expressed MHC II on bone marrow–derived cells and in the thymus, but not in the kidneys. These chimeric mice had normal T and B cell populations and MHC II expression in their spleens and lymph nodes and developed an immune response to systemically and cutaneously administered sheep globulin. However, they did not develop crescentic GN, CD4+ T cell infiltration, or renal injury in response to the sheep globulin planted in their glomeruli. These studies demonstrate that interaction of CD4+ T cells with intrinsic renal cells expressing MHC II is required for development of cell-mediated immune renal injury.
Transgenic Mice Overexpressing the Complement Inhibitor Crry as a Soluble Protein Are Protected from Antibody-induced Glomerular Injury
Authors: Quigg Richard J. , He Chun , Lim Alice , Berthiaume Dawn , Alexander Jessy J. , Kraus Damian , Michael Holers V.
Published: July 2002

Complement receptor 1–related gene/protein y (Crry) is a potent murine membrane complement regulator that inhibits classical and alternative pathway C3 convertases. In nephrotoxic serum (NTS) nephritis, injected antibodies (Abs) bind to glomeruli, leading to complement activation and subsequent glomerular injury and albuminuria. To study the phenotypic effects of continuous complement pathway blockade, transgenic mice were created that express recombinant soluble (rs) Crry directed by the broadly active and heavy metal-inducible metallothionein-I promoter. One transgenic line expressing high levels of rsCrry was propagated. Serum rsCrry levels were 18.7 ± 2.7 μg/ml (n = 5) at basal level and increased to 118.1 ± 20.6 μg/ml 4 d after addition of zinc to the drinking water. By reverse transcription polymerase chain reaction (RT-PCR), transgene messenger (m)RNA was present in liver, kidney, brain, lung, and spleen, but not in heart. By in situ RT-PCR analysis of kidneys, transgene mRNA was widely expressed both in renal glomeruli and tubules. Urinary excretion of rsCrry was 113.4 ± 22.4 μg/ml with a fractional excretion relative to creatinine of 13.2 ± 2.7%, consistent with local renal production of rsCrry and secretion into urine. The founder and all transgene positive adult animals have remained healthy with no mortality or apparent phenotypic abnormalities, including infection or immune complex disease. To determine whether rsCrry blocked complement-mediated injury, NTS nephritis was induced by injection of NTS immunoglobulin (Ig)G, followed by an 18-h urine collection to quantitate the excretion of albumin as a measure of glomerular injury. In transgene-negative littermates (n = 15), transgene-positive animals (n = 10), and transgene-positive animals fed zinc (n = 10), albuminuria was 4,393 ± 948, 1,783 ± 454, and 1,057 ± 277 μg/mg creatinine, respectively (P < 0.01 by ANOVA). Glomerular C3 was evident by immunofluorescence staining in 12/15 transgene-negative animals, but in none of the transgene-positive animals fed zinc. Thus, we have produced the first transgenic animals that overexpress a soluble C3 convertase inhibitor. rsCrry expression markedly ameliorates an Ab-induced disease model in vivo. These results support the hypothesis that continuous complement inhibition at the C3 convertase step is feasible and effective in complement-mediated injury states.
Human Cd4+ T Lymphocytes Consistently Respond to the Latent Epstein-Barr Virus Nuclear Antigen Ebna1
Authors: Münz Christian , Bickham Kara L. , Subklewe Marion , Tsang Ming L. , Chahroudi Ann , Kurilla Michael G. , Zhang Dan , O'Donnell Michael , Steinman Ralph M.
Published: July 2002

The Epstein-Barr virus (EBV)-encoded nuclear antigen EBNA1 is critical for the persistence of the viral episome in replicating EBV-transformed human B cells. Therefore, all EBV-induced tumors express this foreign antigen. However, EBNA1 is invisible to CD8+ cytotoxic T lymphocytes because its Gly/Ala repeat domain prevents proteasome-dependent processing for presentation on major histocompatibility complex (MHC) class I. We now describe that CD4+ T cells from healthy adults are primed to EBNA1. In fact, among latent EBV antigens that stimulate CD4+ T cells, EBNA1 is preferentially recognized. We present evidence that the CD4+ response may provide a protective role, including interferon γ secretion and direct cytolysis after encounter of transformed B lymphocyte cell lines (B-LCLs). Dendritic cells (DCs) process EBNA1 from purified protein and from MHC class II–mismatched, EBNA1-expressing cells including B-LCLs. In contrast, B-LCLs and Burkitt's lymphoma lines likely present EBNA1 after endogenous processing, as their capacity to cross-present from exogenous sources is weak or undetectable. By limiting dilution, there is a tight correlation between the capacity of CD4+ T cell lines to recognize autologous B-LCL–expressing EBNA1 and DCs that have captured EBNA1. Therefore, CD4+ T cells can respond to the EBNA1 protein that is crucial for EBV persistence. We suggest that this immune response is initiated in vivo by DCs that present EBV-infected B cells, and that EBNA1-specific CD4+ T cell immunity be enhanced to prevent and treat EBV-associated malignancies.
The Role of Aquaporins in Dendritic Cell Macropinocytosis
Authors: de Baey Annegret , Lanzavecchia Antonio
Published: July 2002

Immature dendritic cells (DCs) constitutively take up large volumes of fluid by macropinocytosis and concentrate the macrosolutes in the endocytic compartment. This concentration mechanism that is the basis of their high capacity to present soluble antigens requires that DCs be capable of rapidly exchanging water across their membranes. We report that two members of the aquaporin family, AQP3 and AQP7, are expressed in immature DCs and are downregulated after maturation. Treatment of DCs with p-chloromercuribenzenesulphonate (pCMBS), a mercuric drug that blocks aquaporins, inhibited uptake and concentration of macrosolutes taken up by fluid phase endocytosis and led to dramatic cell swelling. In contrast, pCMBS did not affect receptor-mediated endocytosis via the mannose receptor. These findings indicate that aquaporins represent essential elements of a volume control mechanism that allows DCs to concentrate macrosolutes taken up via macropinocytosis.
Fcγ Receptor Iib–Deficient Mice Develop Goodpasture's Syndrome upon Immunization with Type IV Collagen
Authors: Nakamura Akira , Yuasa Takae , Ujike Azusa , Ono Masao , Nukiwa Toshihiro , Ravetch Jeffrey V. , Takai Toshiyuki
Published: July 2002

The combination of hemorrhagic pneumonitis and rapidly progressive glomerulonephritis is a characteristic feature of Goodpasture's syndrome (GPS), an autoimmune disease resulting from the interaction of pathogenic anti–collagen type IV (C-IV) antibodies with alveolar and glomerular basement membranes. Lack of a suitable animal model for this fatal disease has hampered both a basic understanding of its etiology and the development of therapeutic strategies. We now report a novel model for GPS using mice deficient in a central regulatory receptor for immunoglobulin (Ig)G antibody expression and function, the type IIB Fc receptor for IgG (FcγRIIB). Mutant mice immunized with bovine C-IV reproducibly develop massive pulmonary hemorrhage with neutrophil and macrophage infiltration and crescentic glomerulonephritis. The distinctive linear, ribbon-like deposition of IgG immune complex seen in GPS was observed along the glomerular and tubulointerstitial membranes of diseased animals. These results highlight the role of FcγRIIB in maintaining tolerance and suggest that it may play a role in the pathogenesis of human GPS.
Perivascular Macrophages Are the Primary Cell Type Productively Infected by Simian Immunodeficiency Virus in the Brains of Macaques
Authors: Williams Kenneth C. , Corey Sarah , Westmoreland Susan V. , Pauley Doug , Knight Heather , deBakker Colin , Alvarez Xavier , Lackner Andrew A.
Published: July 2002

The macrophage is well established as a target of HIV and simian immunodeficiency virus (SIV) infection and a major contributor to the neuropathogenesis of AIDS. However, the identification of distinct subpopulations of monocyte/macrophages that carry virus to the brain and that sustain infection within the central nervous system (CNS) has not been examined. We demonstrate that the perivascular macrophage and not the parenchymal microglia is the primary cell productively infected by SIV. We further demonstrate that although productive viral infection of the CNS occurs early, thereafter it is not easily detectable until terminal AIDS. The biology of perivascular macrophages, including their rate of turnover and replacement by peripheral blood monocytes, may explain the timing of neuroinvasion, disappearance, and reappearance of virus in the CNS, and questions the ability of the brain to function as a reservoir for productive infection by HIV/SIV.
Determinants of Viral Clearance and Persistence during Acute Hepatitis C Virus Infection
Authors: Thimme Robert , Oldach David , Chang Kyong-Mi , Steiger Carola , Ray Stuart C. , Chisari Francis V.
Published: July 2002

The virological and immunological features of hepatitis C virus (HCV) infection were studied weekly for 6 months after accidental needlestick exposure in five health care workers, four of whom developed acute hepatitis that progressed to chronicity while one subject cleared the virus. In all subjects, viremia was first detectable within 1–2 weeks of inoculation, 1 month or more before the appearance of virus-specific T cells. The subject who cleared the virus experienced a prolonged episode of acute hepatitis that coincided with a CD38+ IFN-γ− CD8+ T cell response to HCV and a small reduction in viremia. Subsequently, a strong CD4+ T cell response emerged and the CD8+ T cells became CD38− and started producing IFN-γ in response to HCV, coinciding with a rapid 100,000-fold decrease in viremia that occurred without a corresponding surge of disease activity. Chronic infection developed in two subjects who failed to produce a significant T cell response and in two other subjects who initially mounted strong CD4+ T cell responses that ultimately waned. In all subjects, viremia was higher at the peak of acute hepatitis than it was when the disease began, and the disease improved during the viremia. These results provide the first insight into the host–virus relationship in humans during the incubation phase of acute HCV infection, and they provide the only insight to date into the virological and immunological characteristics of clinically asymptomatic acute HCV infection, the commonest manifestation of this disease. In addition, the results suggest that the vigor and quality of the antiviral T cell response determines the outcome of acute HCV infection, that the ability of HCV to outpace the T cell response may contribute to its tendency to persist; that the onset of hepatitis coincides with the onset of the CD8+T cell response, that disease pathogenesis and viral clearance are mediated by different CD8+ T cell populations that control HCV by both cytolytic and noncytolytic mechanisms, and that there are different pathways to viral persistence in asymptomatic and symptomatic acute HCV infection.
Processing of Mycobacterium tuberculosis Antigen 85B Involves Intraphagosomal Formation of Peptide–Major Histocompatibility Complex II Complexes and Is Inhibited by Live Bacilli that Decrease Phagosome Maturation
Authors: Ramachandra Lakshmi , Noss Erika , Boom W. Henry , Harding Clifford V.
Published: July 2002

Mycobacterium tuberculosis (MTB) inhibits phagosomal maturation to promote its survival inside macrophages. Control of MTB infection requires CD4 T cell responses and major histocompatibility complex (MHC) class II (MHC-II) processing of MTB antigens (Ags). To investigate phagosomal processing of MTB Ags, phagosomes containing heat-killed (HK) or live MTB were purified from interferon-γ (IFN-γ)–activated macrophages by differential centrifugation and Percoll density gradient subcellular fractionation. Flow organellometry and Western blot analysis showed that MTB phagosomes acquired lysosome-associated membrane protein-1 (LAMP-1), MHC-II, and H2-DM. T hybridoma cells were used to detect MTB Ag 85B(241–256)–I-Ab complexes in isolated phagosomes and other subcellular fractions. These complexes appeared initially (within 20 min) in phagosomes and subsequently (>20 min) on the plasma membrane, but never within late endocytic compartments. Macrophages processed HK MTB more rapidly and efficiently than live MTB; phagosomes containing live MTB expressed fewer Ag 85B(241–256)–I-Ab complexes than phagosomes containing HK MTB. This is the first study of bacterial Ag processing to directly show that peptide–MHC-II complexes are formed within phagosomes and not after export of bacterial Ags from phagosomes to endocytic Ag processing compartments. Live MTB can alter phagosome maturation and decrease MHC-II Ag processing, providing a mechanism for MTB to evade immune surveillance and enhance its survival within the host.
Positive Selection of Cd4+ T Cells Is Induced in Vivo by Agonist and Inhibited by Antagonist Peptides
Authors: Kraj Piotr , Pacholczyk Rafal , Ignatowicz Hanna , Kisielow Pawel , Jensen Peter , Ignatowicz Leszek
Published: July 2002

The nature of peptides that positively select T cells in the thymus remains poorly defined. Here we report an in vivo model to study the mechanisms of positive selection of CD4+ T cells. We have restored positive selection of TCR transgenic CD4+ thymocytes, arrested at the CD4+CD8+ stage, due to the lack of the endogenously selecting peptide(s), in mice deficient for H2-M and invariant chain. A single injection of soluble agonist peptide(s) initiated positive selection of CD4+ transgenic T cells that lasted for up to 14 days. Positively selected CD4+ T cells repopulated peripheral lymphoid organs and could respond to the antigenic peptide. Furthermore, coinjection of the antagonist peptide significantly inhibited agonist-driven positive selection. Hence, contrary to the prevailing view, positive selection of CD4+ thymocytes can be induced in vivo by agonist peptides and may be a result of accumulation of signals from TCR engaged by different peptides bound to major histocompatibility complex class II molecules. We have also identified a candidate natural agonist peptide that induces positive selection of CD4+ TCR transgenic thymocytes.
Inflammatory Chemokine Transport and Presentation in HEV
Authors: Palframan Roger T. , Jung Steffen , Cheng Guiying , Weninger Wolfgang , Luo Yi , Dorf Martin , Littman Dan R. , Rollins Barrett J. , Zweerink Hans , Rot Antal , von Andrian Ulrich H.
Published: July 2002

Interstitial fluid is constantly drained into lymph nodes (LNs) via afferent lymph vessels. This conduit enables monocyte-derived macrophages and dendritic cells to access LNs from peripheral tissues. We show that during inflammation in the skin, a second recruitment pathway is evoked that recruits large numbers of blood-borne monocytes to LNs via high endothelial venules (HEVs). Inhibition of monocyte chemoattractant protein (MCP)-1 blocked this inflammation-induced monocyte homing to LNs. MCP-1 mRNA in inflamed skin was over 100-fold upregulated and paralleled MCP-1 protein levels, whereas in draining LNs MCP-1 mRNA induction was much weaker and occurred only after a pronounced rise in MCP-1 protein. Thus, MCP-1 in draining LNs was primarily derived from inflamed skin. In MCP-1−/− mice, intracutaneously injected MCP-1 accumulated rapidly in the draining LNs where it enhanced monocyte recruitment. Intravital microscopy showed that skin-derived MCP-1 was transported via the lymph to the luminal surface of HEVs where it triggered integrin-dependent arrest of rolling monocytes. These findings demonstrate that inflamed peripheral tissues project their local chemokine profile to HEVs in draining LNs and thereby exert “remote control” over the composition of leukocyte populations that home to these organs from the blood.
Estimating the Precursor Frequency of Naive Antigen-specific CD8 T Cells
Authors: Blattman Joseph N. , Antia Rustom , Sourdive David J.D. , Wang Xiaochi , Kaech Susan M. , Murali-Krishna Kaja , Altman John D. , Ahmed Rafi
Published: July 2002

The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner. The precursor frequency is equal to the number of transferred cells when the transgenic and endogenous responses are of equal magnitude. Using this method we have estimated the precursor frequency of naive CD8 T cells specific for the H-2Db–restricted GP33–41 epitope of LCMV to be 1 in 2 × 105. Thus, in an uninfected mouse containing ∼2-4 × 107 naive CD8 T cells we estimate there to be 100–200 epitope-specific cells. After LCMV infection these 100–200 GP33-specific naive CD8 T cells divide >14 times in 1 wk to reach a total of ∼107 cells. Approximately 5% of these activated GP33-specific effector CD8 T cells survive to generate a memory pool consisting of ∼5 × 105 cells. Thus, an acute LCMV infection results in a >1,000-fold increase in precursor frequency of DbGP33-specific CD8 T cells from 2 × 102 naive cells in uninfected mice to 5 × 105 memory cells in immunized mice.
Activation-induced Deaminase (AID)-directed Hypermutation in the Immunoglobulin Sμ Region
Authors: Nagaoka Hitoshi , Muramatsu Masamichi , Yamamura Namiko , Kinoshita Kazuo , Honjo Tasuku
Published: July 2002

Somatic hypermutation (SHM) and class switch recombination (CSR) cause distinct genetic alterations at different regions of immunoglobulin genes in B lymphocytes: point mutations in variable regions and large deletions in S regions, respectively. Yet both depend on activation-induced deaminase (AID), the function of which in the two reactions has been an enigma. Here we report that B cell stimulation which induces CSR but not SHM, leads to AID-dependent accumulation of SHM-like point mutations in the switch μ region, uncoupled with CSR. These findings strongly suggest that AID itself or a single molecule generated by RNA editing function of AID may mediate a common step of SHM and CSR, which is likely to be involved in DNA cleavage.
Submaximal Responses in Calcium-triggered Exocytosis Are Explained by Differences in the Calcium Sensitivity of Individual Secretory Vesicles
Authors: Blank Paul S. , Cho Myoung-Soon , Vogel Steven S. , Kaplan Doron , Kang Albert , Malley James , Zimmerberg Joshua
Published: July 2002

A graded response to calcium is the defining feature of calcium-regulated exocytosis. That is, there exist calcium concentrations that elicit submaximal exocytotic responses in which only a fraction of the available population of secretory vesicles fuse. The role of calcium-dependent inactivation in defining the calcium sensitivity of sea urchin egg secretory vesicle exocytosis in vitro was examined. The cessation of fusion in the continued presence of calcium was not due to calcium-dependent inactivation. Rather, the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous. Any specific calcium concentration above threshold triggered subpopulations of vesicles to fuse and the size of the subpopulations was dependent upon the magnitude of the calcium stimulus. The existence of multiple, stable subpopulations of vesicles is consistent with a fusion process that requires the action of an even greater number of calcium ions than the numbers suggested by models based on the assumption of a homogeneous vesicle population.
Topological centers of certain dual algebras
Authors: Lau Anthony , Ülger Ali
Published: July 2002

Let

A
A

be a Banach algebra with a bounded approximate identity. Let

Z
1

Z_1

and

Z
~

2

\widetilde Z_2

be, respectively, the topological centers of the algebras

A

∗
∗

A^{**}

and

(

A
∗

A

)
∗

(A^*A)^*

. In this paper, for weakly sequentially complete Banach algebras, in particular for the group and Fourier algebras

L
1

(
G
)

L^1(G)

and

A
(
G
)

A(G)

, we study the sets

Z
1

Z_1

,

Z
~

2

\widetilde Z_2

, the relations between them and with several other subspaces of

A

∗
∗

A^{**}

or

A
∗

A^*

.
The second variation of nonorientable minimal submanifolds
Authors: Ross Marty
Published: July 2002

Suppose

M
M

is a complete nonorientable minimal submanifold of a Riemannian manifold

N
N

. We derive a second variation formula for the area of

M
M

with respect to certain perturbations, giving a sufficient condition for the instability of

M
M

. Some simple applications are given: we show that the totally geodesic

R

P

2

\mathbb {R} \mathbb {P}^{2}

is the only stable surface in

R

P

3

\mathbb {R} \mathbb {P}^{3}

, and we show the non-existence of stable nonorientable cones in

R

4

\mathbb {R}^{4}

. We reproduce and marginally extend some known results in the truly non-compact setting.
Secretion and Assembly of Zona Pellucida Glycoproteins by Growing Mouse Oocytes Microinjected with Epitope-tagged cDNAs for mZP2 and mZP3
Authors: Qi Huayu , Williams Zev , Wassarman Paul M.
Published: July 2002

The zona pellucida (ZP) is a highly organized extracellular coat that surrounds all mammalian eggs. The mouse egg ZP is composed of three glycoproteins, called mZP1–3, that are synthesized, secreted, and assembled into a ZP exclusively by growing oocytes. Here, we microinjected epitope-tagged (Myc andFlag) cDNAs for mZP2 and mZP3 into the germinal vesicle (nucleus) of growing oocytes isolated from juvenile mice. Specific antibodies and laser scanning confocal microscopy were used to follow nascent, recombinant ZP glycoproteins in both permeabilized and nonpermeabilized oocytes. When such cDNAs were injected, epitope-tagged mZP2 (Myc-mZP2) and mZP3 (Flag-mZP3) were synthesized, packaged into large intracellular vesicles, and secreted by the vast majority of oocytes. Secreted glycoproteins were incorporated into only the innermost layer of the thickening ZP, and the amount of nascent glycoprotein in this region increased with increasing time of oocyte culture. Consistent with prior observations, the putative transmembrane domain at the C terminus of mZP2 and mZP3 was missing from nascent glycoprotein incorporated into the ZP. When the consensus furin cleavage site near the C terminus of mZP3 was mutated, such that it should not be cleaved by furin, secretion and assembly of mZP3 was reduced. On the other hand, mZP3 incorporated into the ZP lacked the transmembrane domain downstream of the mutated furin cleavage site, suggesting that some other protease(s) excised the domain. These results strongly suggest that nascent mZP2 and mZP3 are incorporated into only the innermost layer of the ZP and that excision of the C-terminal region of the glycoproteins is required for assembly into the oocyte ZP.
Fast evaluation of internal loops in RNA secondary structure prediction.
Authors: Lyngs√∏ R B , Zuker M , Pedersen C N
Published: July 2002

MOTIVATION: Though not as abundant in known biological processes as proteins, RNA molecules serve as more than mere intermediaries between DNA and proteins. Research in the last 15 years demonstrates that RNA molecules serve in many roles, including catalysis. Furthermore, RNA secondary structure prediction based on free energy rules for stacking and loop formation remains one of the few major breakthroughs in the field of structure prediction, as minimum free energy structures and related quantities can be computed with full mathematical rigor. However, with the current energy parameters, the algorithms used hitherto suffer the disadvantage of either employing heuristics that risk (though highly unlikely) missing the optimal structure or becoming prohibitively time consuming for moderate to large sequences. RESULTS: We present a new method to evaluate internal loops utilizing currently used energy rules. This method reduces the time complexity of this part of the structure prediction from O(n4) to O(n3), thus reducing the overall complexity to O(n3). Even when the size of evaluated internal loops is bounded by k (a commonly used heuristic), the method presented has a competitive edge by reducing the time complexity of internal loop evaluation from O(k2n2) to O(kn2). The method also applies to the calculation of the equilibrium partition function. AVAILABILITY: Source code for an RNA secondary structure prediction program implementing this method is available at ftp://www.ibc.wustl.edu/pub/zuker/zuker .tar.Z