bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Chronic Inflammatory Arthropathy Resembling Rheumatoid Arthritis in Interleukin 1 Receptor Antagonist–Deficient Mice
Authors: Horai Reiko , Saijo Shinobu , Tanioka Hidetoshi , Nakae Susumu , Sudo Katsuko , Okahara Akihiko , Ikuse Toshimi , Asano Masahide , Iwakura Yoichiro
Published: July 2002

Interleukin (IL)-1 is a proinflammatory cytokine that plays important roles in inflammation, host defense, and the neuro-immuno-endocrine network. IL-1 receptor antagonist (ra) is an endogenous inhibitor of IL-1 and is supposed to regulate IL-1 activity. However, its pathophysiological roles in a body remain largely unknown. To elucidate the roles of IL-1ra, IL-1ra–deficient mice were produced by gene targeting, and pathology was analyzed on different genetic backgrounds. We found that all of the mice on a BALB/cA background, but not those on a C57BL/6J background, spontaneously developed chronic inflammatory polyarthropathy. Histopathology showed marked synovial and periarticular inflammation, with articular erosion caused by invasion of granulation tissues closely resembling that of rheumatoid arthritis in humans. Moreover, elevated levels of antibodies against immunoglobulins, type II collagen, and double-stranded DNA were detected in these mice, suggesting development of autoimmunity. Proinflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor α were overexpressed in the joints, indicating regulatory roles of IL-1ra in the cytokine network. We thus show that IL-1ra gene deficiency causes autoimmunity and joint-specific inflammation and suggest that IL-1ra is important in maintaining homeostasis of the immune system. Possible involvement of IL-1ra gene deficiency in RA will be discussed.
The Gtpase Rho Controls a P53-Dependent Survival Checkpoint during Thymopoiesis
Authors: Costello Patrick S. , Cleverley Steve C. , Galandrini Ricciarda , Henning Stefan W. , Cantrell Doreen A.
Published: July 2002

During the early stages of thymopoiesis, cell survival is controlled by cytokines that regulate the expression of antiapoptotic proteins such as Bcl-2. At the pre-T cell stage, a critical checkpoint for β chain selection is monitored by the tumor suppressor p53: pre-T cells can survive and differentiate when p53 is removed genetically or when its proapoptotic function is inactivated physiologically as a consequence of signaling through the pre-T cell receptor complex. Previous work has shown that the guanine nucleotide binding protein Rho controls cell survival in T cell progenitors. Here we define the survival pathways controlled by Rho in pre-T cells and show that this GTPase is a pivotal regulator of the p53-mediated checkpoint operating at the time of β selection: loss of Rho function results in apoptosis in pre-T cells, but this cell death is prevented by loss of p53. The prevention of cell death by loss of p53 restored numbers of early T cell progenitors but did not fully restore thymic cellularity. Further analysis revealed that loss of Rho function caused survival defects in CD4/8 double-positive thymocytes that is independent of p53 but can be prevented by ectopic expression of Bcl-2. These studies highlight that the GTPase Rho is a crucial component of survival signaling pathways in at least two different thymocyte subpopulations: Rho controls the p53 survival checkpoint in pre-T cells and is also crucial for a p53 independent survival signaling pathway in CD4/8 double positives.
Candidate Microbicides Block HIV-1 Infection of Human Immature Langerhans Cells within Epithelial Tissue Explants
Authors: Kawamura Tatsuyoshi , Cohen Sandra S. , Borris Debra L. , Aquilino Elisabeth A. , Glushakova Svetlana , Margolis Leonid B. , Orenstein Jan M. , Offord Robin E. , Neurath A. Robert , Blauvelt Andrew
Published: July 2002

Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1Ba-L infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08–4.77%). HIV-1–infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1–1 μg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1Ba-L (an R5 HIV-1 strain) more efficiently infected LC–T cell cocultures when compared with HIV-1IIIB (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1.
Macrophage-Tropic HIV Induces and Exploits Dendritic Cell Chemotaxis
Authors: Lin Chen-Lung , Sewell Andrew K. , Gao George F. , Whelan Kathryn T. , Phillips Rodney E. , Austyn Jonathan M.
Published: July 2002

Immature dendritic cells (iDCs) express the CC chemokine receptor (CCR)5, which promotes chemotaxis toward the CC chemokines regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β. By contrast, mature DCs downregulate CCR5 but upregulate CXC chemokine receptor (CXCR)4, and as a result exhibit enhanced chemotaxis toward stromal cell–derived factor (SDF)-1α. CCR5 and CXCR4 also function as coreceptors for macrophage-tropic (M-tropic) and T cell–tropic (T-tropic) human immunodeficiency virus (HIV)-1, respectively. Here, we demonstrate chemotaxis of iDCs toward M-tropic (R5) but not T-tropic (X4) HIV-1. Furthermore, preexposure to M-tropic HIV-1 or its recombinant envelope protein prevents migration toward CCR5 ligands. The migration of iDCs toward M-tropic HIV-1 may enhance formation of DC–T cell syncytia, thus promoting viral production and destruction of both DC and T helper lymphocytes. Therefore, disturbance of DC chemotaxis by HIV-1 is likely to contribute to immunosuppression in primary infection and AIDS. In addition, migration of iDCs toward HIV-1 may aid the capture of R5 HIV-1 virions by the abundant DC cell surface protein DC-specific intercellular adhesion molecule (ICAM)3-grabbing nonintegrin (DC-SIGN). HIV-1 bound to DC cell–specific DC-SIGN retains the ability to infect replication-permissive T cells in trans for several days. Consequently, recruitment of DC by HIV-1 could combine with the ability of DC-SIGN to capture and transmit the virus to T cells, and so facilitate dissemination of virus within an infected individual.
The Transmembrane Adaptor Protein Trim Regulates T Cell Receptor (Tcr) Expression and Tcr-Mediated Signaling via an Association with the Tcr ζ Chain
Authors: Kirchgessner Henning , Dietrich Jes , Scherer Jeanette , Isomäki Pia , Korinek Vladimir , Hilgert Ivan , Bruyns Eddy , Leo Albrecht , Cope Andrew P. , Schraven Burkhart
Published: July 2002

T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-ζ chains and to a lesser extent via the CD3-ε/γ heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca2+ mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.
Cellular Immune Responses and Viral Diversity in Individuals Treated during Acute and Early HIV-1 Infection
Authors: Altfeld Marcus , Rosenberg Eric S. , Shankarappa Raj , Mukherjee Joia S. , Hecht Frederick M. , Eldridge Robert L. , Addo Marylyn M. , Poon Samuel H. , Phillips Mary N. , Robbins Gregory K. , Sax Paul E. , Boswell Steve , Kahn James O. , Brander Christian , Goulder Philip J.R. , Levy Jay A. , Mullins James I. , Walker Bruce D.
Published: July 2002

Immune responses induced during the early stages of chronic viral infections are thought to influence disease outcome. Using HIV as a model, we examined virus-specific cytotoxic T lymphocytes (CTLs), T helper cells, and viral genetic diversity in relation to duration of infection and subsequent response to antiviral therapy. Individuals with acute HIV-1 infection treated before seroconversion had weaker CTL responses directed at fewer epitopes than persons who were treated after seroconversion. However, treatment-induced control of viremia was associated with the development of strong T helper cell responses in both groups. After 1 yr of antiviral treatment initiated in acute or early infection, all epitope-specific CTL responses persisted despite undetectable viral loads. The breadth and magnitude of CTL responses remained significantly less in treated acute infection than in treated chronic infection, but viral diversity was also significantly less with immediate therapy. We conclude that early treatment of acute HIV infection leads to a more narrowly directed CTL response, stronger T helper cell responses, and a less diverse virus population. Given the need for T helper cells to maintain effective CTL responses and the ability of virus diversification to accommodate immune escape, we hypothesize that early therapy of primary infection may be beneficial despite induction of less robust CTL responses. These data also provide rationale for therapeutic immunization aimed at broadening CTL responses in treated primary HIV infection.
Substantial Differences in Specificity of HIV-Specific Cytotoxic T Cells in Acute and Chronic HIV Infection
Authors: Goulder Philip J.R. , Altfeld Marcus A. , Rosenberg Eric S. , Nguyen Thi , Tang Yanhua , Eldridge Robert L. , Addo Marylyn M. , He Suqin , Muckerjee Joia S. , Phillips Mary N. , Bunce Michael , Kalams Spyros A. , Sekaly Rafick P. , Walker Bruce D. , Brander Christian
Published: July 2002

Cytotoxic T lymphocytes (CTLs) play a vital part in controlling viral replication during human viral infections. Most studies in human infections have focused on CTL specificities in chronic infection and few data exist regarding the specificity of the initial CTL response induced in acute infection. In this study, HIV-1 infection in persons expressing human histocompatibility leukocyte antigen (HLA)-A*0201 was used as a means of addressing this issue. In chronic infection, the dominant HLA-A*0201–restricted CTL response is directed towards the epitope SLYNTVATL (“SL9”) in p17 Gag (residues 77–85). This epitope is targeted by 75% of HLA-A*0201–positive adults, and the magnitude of this A*0201-SL9 response shows a strong negative association with viral load in progressive infection. Despite using the highly sensitive peptide–major histocompatibility complex tetramer and intracellular cytokine assays, responses to the SL9 epitope were not detectable in any of 11 HLA-A*0201–positive subjects with acute HIV-1 infection (P = 2 × 10−6), even when assays were repeated using the SL9 peptide variant that was encoded by their autologous virus. In contrast, multiple responses (median 3) to other epitopes were evident in 7 of the 11 A*0201–positive subjects. Longitudinal study of two subjects confirmed that the A*0201-SL9 response emerged later than other CTL responses, and after viral set point had been reached. Together, these data show that the CTL responses that are present and that even may dominate in chronic infection may differ substantially from those that constitute the initial antiviral CTL response. This finding is an important consideration in vaccine design and in the evaluation of vaccine candidates.
Critical Contribution of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (Trail) to Apoptosis of Human Cd4+T Cells in HIV-1–Infected Hu-Pbl-Nod-Scid Mice
Authors: Miura Yoshiharu , Misawa Naoko , Maeda Naoyoshi , Inagaki Yoshio , Tanaka Yuetsu , Ito Mamoru , Kayagaki Nobuhiko , Yamamoto Naoki , Yagita Hideo , Mizusawa Hidehiro , Koyanagi Yoshio
Published: July 2002

Apoptosis is a key for CD4+ T cell destruction in HIV-1–infected patients. In this study, human peripheral blood lymphocyte (PBL)-transplanted nonobese diabetic (NOD)-severe combined immunodeficient (SCID) (hu-PBL-NOD-SCID) mice were used to examine in vivo apoptosis after HIV-1 infection. As the hu-PBL-NOD-SCID mouse model allowed us to see extensive infection with HIV-1 and to analyze apoptosis in human cells in combination with immunohistological methods, we were able to quantify the number of apoptotic cells with HIV-1 infection. As demonstrated by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL), massive apoptosis was predominantly observed in virus-uninfected CD4+ T cells in the spleens of HIV-1–infected mice. A combination of TUNEL and immunostaining for death-inducing tumor necrosis factor (TNF) family molecules indicated that the apoptotic cells were frequently found in conjugation with TNF-related apoptosis-inducing ligand (TRAIL)-expressing CD3+CD4+ human T cells. Administration of a neutralizing anti-TRAIL mAb in HIV-1–infected mice markedly inhibited the development of CD4+ T cell apoptosis. These results suggest that a large number of HIV-1–uninfected CD4+ T cells undergo TRAIL-mediated apoptosis in HIV-infected lymphoid organs.
Point Mutation in Essential Genes with Loss or Mutation of the Second Allele
Authors: Beck-Engeser Gabriele B. , Monach Paul A. , Mumberg Dominik , Yang Farley , Wanderling Sherry , Schreiber Karin , Espinosa Rafael , Le Beau Michelle M. , Meredith Stephen C. , Schreiber Hans
Published: July 2002

Antigens that are tumor specific yet retained by tumor cells despite tumor progression offer stable and specific targets for immunologic and possibly other therapeutic interventions. Therefore, we have studied two CD4+ T cell–recognized tumor-specific antigens that were retained during evolution of two ultraviolet-light–induced murine cancers to more aggressive growth. The antigens are ribosomal proteins altered by somatic tumor-specific point mutations, and the progressor (PRO) variants lack the corresponding normal alleles. In the first tumor, 6132A-PRO, the antigen is encoded by a point-mutated L9 ribosomal protein gene. The tumor lacks the normal L9 allele because of an interstitial deletion from chromosome 5. In the second tumor, 6139B-PRO, both alleles of the L26 gene have point mutations, and each encodes a different tumor-specific CD4+ T cell–recognized antigen. Thus, for both L9 and L26 genes, we observe “two hit” kinetics commonly observed in genes suppressing tumor growth. Indeed, reintroduction of the lost wild-type L9 allele into the 6132A-PRO variant suppressed the growth of the tumor cells in vivo. Since both L9 and L26 encode proteins essential for ribosomal biogenesis, complete loss of the tumor-specific target antigens in the absence of a normal allele would abrogate tumor growth.
Elucidating the Autoimmune and Antitumor Effector Mechanisms of a Treatment Based on Cytotoxic T Lymphocyte Antigen-4 Blockade in Combination with a B16 Melanoma Vaccine
Authors: van Elsas Andrea , Sutmuller Roger P.M. , Hurwitz Arthur A. , Ziskin Jennifer , Villasenor Jennifer , Medema Jan-Paul , Overwijk Willem W. , Restifo Nicholas P. , Melief Cornelis J.M. , Offringa Rienk , Allison James P.
Published: July 2002

We have previously shown that small B16 melanomas can be successfully treated using a combination of anti–cytotoxic T lymphocyte antigen (CTLA)-4 monoclonal antibody with a granulocyte/macrophage colony-stimulating factor (GM-CSF) producing irradiated tumor cell vaccine. Regression of tumors results in long-lasting immunity and is frequently accompanied by autoimmune depigmentation. Here we examine the cellular and molecular mechanisms of this combined treatment. Histological examination of depigmented lesions revealed infiltration of polymorphonuclear cells and deposition of antibody. The combination therapy also induced tumor rejection and skin depigmentation in B cell–deficient and in CD4+ T cell–depleted mice. Both effects of the treatment absolutely required CD8+ T cells. Analysis of the response in successfully treated mice revealed elevated levels of CD8+ T cells specific for a nonameric peptide consisting of residues 180–188 of the melanocyte differentiation antigen tyrosinase-related protein (TRP)2. There was no evidence of reactivity to the melanocyte antigens gp100, tyrosinase, Mart1/MelanA, or TRP1. Fas–FasL interactions and perforin played a role in mounting the effector response, whereas the tumor necrosis factor pathway was not required. The cellular requirements for tumor rejection in this therapeutic setting were strikingly different from those in a prophylactic setting. In particular, if mice received a prophylactic vaccine consisting of anti–CTLA-4 and B16–GM-CSF before tumor challenge, full protection was obtained even in the absence of CD8+ T cells. Our data demonstrate that therapeutic autoreactive CD8+ T cell responses can effectively be generated in tumor-bearing mice and stresses the value of studying tumor immunity in a therapeutic rather than a prophylactic setting.
Interleukin 15 Skews Monocyte Differentiation into Dendritic Cells with Features of Langerhans Cells
Authors: Mohamadzadeh Mansour , Berard Frederic , Essert Gregory , Chalouni Cecile , Pulendran Bali , Davoust Jean , Bridges George , Palucka A. Karolina , Banchereau Jacques
Published: July 2002

Langerhans cells (LCs) represent a subset of immature dendritic cells (DCs) specifically localized in the epidermis and other mucosal epithelia. As surrounding keratinocytes can produce interleukin (IL)-15, a cytokine that utilizes IL-2Rγ chain, we analyzed whether IL-15 could skew monocyte differentiation into LCs. Monocytes cultured for 6 d with granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-15 differentiate into CD1a+HLA-DR+CD14−DCs (IL15-DCs). Agents such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)α, and CD40L induce maturation of IL15-DCs to CD83+, DC-LAMP+ cells. IL15-DCs are potent antigen-presenting cells able to induce the primary (mixed lymphocyte reaction [MLR]) and secondary (recall responses to flu-matrix peptide) immune responses. As opposed to cultures made with GM-CSF/IL-4 (IL4-DCs), a proportion of IL15-DCs expresses LC markers: E-Cadherin, Langerin, and CC chemokine receptor (CCR)6. Accordingly, IL15-DCs, but not IL4-DCs, migrate in response to macrophage inflammatory protein (MIP)-3α/CCL20. However, IL15-DCs cannot be qualified as “genuine” Langerhans cells because, despite the presence of the 43-kD Langerin, they do not express bona fide Birbeck granules. Thus, our results demonstrate a novel pathway in monocyte differentiation into dendritic cells.
Osteopontin Is Involved in the Initiation of Cutaneous Contact Hypersensitivity by Inducing Langerhans and Dendritic Cell Migration to Lymph Nodes
Authors: Weiss J.M. , Renkl A.C. , Maier C.S. , Kimmig M. , Liaw L. , Ahrens T. , Kon S. , Maeda M. , Hotta H. , Uede T. , Simon J.C.
Published: July 2002

Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and αv integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.
Resistance to Granzyme B-mediated Cytochrome c Release in Bak-deficient Cells
Authors: Wang Gui-Qiang , Wieckowski Eva , Goldstein Leslie A. , Gastman Brian R. , Rabinovitz Asaf , Gambotto Andrea , Li Shuchen , Fang Bingliang , Yin Xiao-Ming , Rabinowich Hannah
Published: July 2002

Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.
Pacemaker Synchronization of Electrically Coupled Rabbit Sinoatrial Node Cells
Authors: Verheijck E. Etienne , Wilders Ronald , Joyner Ronald W. , Golod David A. , Kumar Rajiv , Jongsma Habo J. , Bouman Lennart N. , Ginneken Antoni C.G. van
Published: July 2002

The effects of intercellular coupling conductance on the activity of two electrically coupled isolated rabbit sinoatrial nodal cells were investigated. A computer-controlled version of the “coupling clamp” technique was used in which isolated sinoatrial nodal cells, not physically in contact with each other, were electrically coupled at various values of ohmic coupling conductance, mimicking the effects of mutual interaction by electrical coupling through gap junctional channels. We demonstrate the existence of four types of electrical behavior of coupled spontaneously active cells. As the coupling conductance is progressively increased, the cells exhibit: (a) independent pacemaking at low coupling conductances, (b) complex dynamics of activity with mutual interactions, (c) entrainment of action potential frequency at a 1:1 ratio with different action potential waveforms, and (d) entrainment of action potentials at the same frequency of activation and virtually identical action potential waveforms. The critical value of coupling conductance required for 1:1 frequency entrainment was <0.5 nS in each of the five cell pairs studied. The common interbeat interval at a relatively high coupling conductance (10 nS), which is sufficient to produce entrainment of frequency and also identical action potential waveforms, is determined most by the intrinsically faster pacemaker cell and it can be predicted from the diastolic depolarization times of both cells. Evidence is provided that, at low coupling conductances, mutual pacemaker synchronization results mainly from the phase-resetting effects of the action potential of one cell on the depolarization phase of the other. At high coupling conductances, the tonic, diastolic interactions become more important.
Structural Similarities between Glutamate Receptor Channels and K+ Channels Examined by Scanning Mutagenesis
Authors: Panchenko Victor A. , Glasser Carla R. , Mayer Mark L.
Published: July 2002

The pores of glutamate receptors and K+ channels share sequence homology, suggesting a conserved secondary structure. Scanning mutagenesis with substitution of alanine and tryptophan in GluR6 channels was performed based on the structure of KcsA. Our assay used disruption of voltage-dependent polyamine block to test for changes in the packing of pore-forming regions. Alanine scanning from D567 to R603 revealed reduced rectification resulting from channel block in two regions. A periodic pattern from F575 to M589 aligned with the pore helix in KcsA, whereas a cluster of sensitive positions around Q590, a site regulated by RNA editing, mapped to the selectivity filter in KcsA. Tryptophan scanning from D567 to R603 revealed similar patterns, but with a complete disruption of spermine block for 7 out of the 37 positions and a pM dissociation constant for Q590W. Molecular modeling with KcsA coordinates showed that GluR6 pore helix mutants disrupting polyamine block pack against M1 and M2, and are not exposed in the ion channel pore. In the selectivity filter, tryptophan creates an aromatic cage consistent with the pM dissociation constant for Q590W. A scan with glutamate substitution was used to map the cytoplasmic entrance to the pore based on charge neutralization experiments, which established that E594 was uniquely required for high affinity polyamine block. In E594Q mutants, introduction of glutamate at positions S593–L600 restored polyamine block at positions corresponding to surface-exposed residues in KcsA. Our results reinforce proposals that the pore region of glutamate receptors contains a helix and pore loop analogous to that found in K+ channels. At the cytoplasmic entrance of the channel, a negatively charged amino acid, located in an extended loop with solvent-exposed side chains, is required for high affinity polyamine block and probably attracts cations via a through space electrostatic mechanism.
Different Fast-Gate Regulation by External Cl− and H+ of the Muscle-Type Clc Chloride Channels
Authors: Chen Mei-Fang , Chen Tsung-Yu
Published: July 2002

The fast gate of the muscle-type ClC channels (ClC-0 and ClC-1) opens in response to the change of membrane potential (V). This gating process is intimately associated with the binding of external Cl− to the channel pore in a way that the occupancy of Cl− on the binding site increases the channel's open probability (Po). External H+ also enhances the fast-gate opening in these channels, prompting a hypothesis that protonation of the binding site may increase the Cl− binding affinity, and this is possibly the underlying mechanism for the H+ modulation. However, Cl− and H+, modulate the fast-gate Po-V curve in different ways. Varying the external Cl− concentrations ([Cl−]o) shifts the Po-V curve in parallel along the voltage axis, whereas reducing external pH mainly increases the minimal Po of the curve. Furthermore, H+ modulations at saturating and nonsaturating [Cl−]o are similar. Thus, the H+ effect on the fast gating appears not to be a consequence of an increase in the Cl− binding affinity. We previously found that a hyperpolarization-favored opening process is important to determine the fast-gate Po of ClC-0 at very negative voltages. This [Cl−]o-independent mechanism attracted little attention, but it appears to be the opening process that is modulated by external H+.
Asymptotic properties of Banach spaces under renormings
Authors: Odell E. , Schlumprecht Th.
Published: July 2002

It is shown that a separable Banach space

X
X

can be given an equivalent norm

|

|

|

⋅

|

|

|

|||\, \cdot |||\,

with the following properties: If

(

x

n

)
⊆
X

(x_{n})\subseteq X

is relatively weakly compact and
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="limit Underscript m right-arrow normal infinity Endscripts limit Underscript n right-arrow normal infinity Endscripts StartAbsoluteValue EndAbsoluteValue StartAbsoluteValue x Subscript m Baseline plus x Subscript n Baseline EndAbsoluteValue StartAbsoluteValue EndAbsoluteValue equals 2 limit Underscript m right-arrow normal infinity Endscripts StartAbsoluteValue EndAbsoluteValue StartAbsoluteValue x Subscript m Baseline EndAbsoluteValue StartAbsoluteValue EndAbsoluteValue">

lim

m
→
∞

lim

n
→
∞

|

|

|

x

m

+

x

n

|

|

|

=
2

lim

m
→
∞

|

|

|

x

m

|

|

|

\lim _{m\to \infty } \lim _{n\to \infty } |||\, x_{m}+x_{n}|||\, = 2\lim _{m\to \infty } |||\, x_{m}|||\,

, then

(

x

n

)

(x_{n})

converges in norm. This yields a characterization of reflexivity once proposed by V.D. Milman. In addition it is shown that some spreading model of a sequence in

(
X
,

|

|

|

⋅

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)

(X,|||\, \cdot |||\, )

is 1-equivalent to the unit vector basis of

ℓ

1

\ell _{1}

(respectively,

c

0

c_{0}

) implies that

X
X

contains an isomorph of

ℓ

1

\ell _{1}

(respectively,

c

0

c_{0}

).
TBP Dynamics in Living Human Cells: Constitutive Association of TBP with Mitotic Chromosomes
Authors: Chen Danyang , Hinkley Craig S. , Henry R. William , Huang Sui
Published: July 2002

The recruitment of TATA binding protein (TBP) to gene promoters is a critical rate-limiting step in transcriptional regulation for all three eukaryotic RNA polymerases. However, little is known regarding the dynamics of TBP in live mammalian cells. In this report, we examined the distribution and dynamic behavior of green fluorescence protein (GFP)-tagged TBP in live HeLa cells using fluorescence recovery after photobleaching (FRAP) analyses. We observed that GFP-TBP associates with condensed chromosomes throughout mitosis without any FRAP. These results suggest that TBP stably associates with the condensed chromosomes during mitosis. In addition, endogenous TBP and TBP-associated factors (TAFs), specific for RNA polymerase II and III transcription, cofractionated with mitotic chromatin, suggesting that TBP is retained as a TBP-TAF complex on transcriptionally silent chromatin throughout mitosis. In interphase cells, GFP-TBP distributes throughout the nucleoplasm and shows a FRAP that is 100-fold slower than the general transcription factor GFP-TFIIB. This difference supports the idea that TBP and, most likely, TBP-TAF complexes, remain promoter- bound for multiple rounds of transcription. Altogether, our observations demonstrate that there are cell cycle specific characteristics in the dynamic behavior of TBP. We propose a novel model in which the association of TBP-TAF complexes with chromatin during mitosis marks genes for rapid transcriptional activation as cells emerge from mitosis.
Kinesin-II Is Required for Flagellar Sensory Transduction during Fertilization inChlamydomonas
Authors: Pan Junmin , Snell William J.
Published: July 2002

The assembly and maintenance of eucaryotic flagella and cilia depend on the microtubule motor, kinesin-II. This plus end-directed motor carries intraflagellar transport particles from the base to the tip of the organelle, where structural components of the axoneme are assembled. Here we test the idea that kinesin-II also is essential for signal transduction. When mating-type plus (mt+) and mating-type minus (mt−) gametes of the unicellular green algaChlamydomonas are mixed together, binding interactions between mt+ and mt− flagellar adhesion molecules, the agglutinins, initiate a signaling pathway that leads to increases in intracellular cAMP, gamete activation, and zygote formation. A critical question inChlamydomonas fertilization has been how agglutinin interactions are coupled to increases in intracellular cAMP. Recently,fla10 gametes with a temperature-sensitive defect in FLA10 kinesin-II were found to not form zygotes at the restrictive temperature (32°C). We found that, although the rates and extents of flagellar adhesion in fla10 gametes at 32°C are indistinguishable from wild-type gametes, the cells do not undergo gamete activation. On the other hand, fla10 gametes at 32°C regulated agglutinin location and underwent gamete fusion when the cells were incubated in dibutyryl cAMP, indicating that their capacity to respond to the cAMP signal was intact. We show that the cellular defect in the fla10 gametes at 32°C is a failure to undergo increases in cAMP during flagella adhesion. Thus, in addition to being essential for assembly and maintenance of the structural components of flagella, kinesin-II/intraflagellar transport plays a role in sensory transduction in these organelles.
Towards detection of orthologues in sequence databases.
Authors: Yuan Y P , Eulenstein O , Vingron M , Bork P
Published: July 2002

MOTIVATION: Numerous homologous sequences from diverse species can be retrieved from databases using programs such as BLAST. However, due to multigene families, evolutionary relationship often cannot be easily determined and proper functional assignment becomes difficult. Thus, discrimination between orthologues and paralogues within BLAST output lists of homologous sequences becomes more and more important. RESULT: We therefore developed a method that attempts to construct a reconciled tree from a gene tree of selected sequences and its corresponding phylogenetic tree of the species involved (species tree). An interface on the Web is developed to enable users to analyse the BLAST result. BLAST outputs are parsed and, for the selected sequences, multiple alignments are constructed either globally or for local regions. Bootstrapped trees are returned and compared with the expected species tree. In cases of discrepancies, gene duplications are assumed and a reconciled tree is computed. The reconciled tree shows probable orthologues and paralogues as predicted.
W2H: WWW interface to the GCG sequence analysis package.
Authors: Senger M , Flores T , Glatting K , Ernst P , Hotz-Wagenblatt A , Suhai S
Published: July 2002

MOTIVATION: The user-friendly, graphical X-windows interface (WPI) to the GCG sequence analysis package can often not be used due to the lack of an X-server on PC or Macintosh computers. Because Web browsers like Netscape are much more common on those platforms, we decided to develop W2H, a WWW interface to the GCG Sequence Analysis Software Package with nearly the same functionality as the X-windows interface WPI. RESULTS: The new WWW interface (W2H) to the GCG Sequence Analysis Software Package (Wisconsin Package) supports modern Web technologies, like client-pull method, or embedded scripting language, and provides a reasonable platform independence. The interface is quite comprehensive with advanced features like sequence selector, search set builder, enzyme chooser, access to sequence databases, uploading client files to the GCG server or displaying and manipulating graphical outputs in addition to GCG analysis programs. W2H also manages secure access to both GCG server and user data. For special environments, like workshops, conferences and company intranets, there is a special mode (Intranet mode) with less security constraints. The behaviour of W2H is mostly controlled by meta-data files describing the applications and giving a base for dynamic creation of HTML documents. This paper presents mainly the development approaches used, and architectural design aspects of W2H. AVAILABILITY: W2H is available by ftp://ftp.ebi.ac. uk/pub/software/unix/w2h or ftp://genome.dkfz-heidelberg.de/pub/w2h CONTACT: m.senger@ebi.ac.uk
Graphical interface to the genetic network database GeNet.
Authors: Serov V N , Spirov A V , Samsonova M G
Published: July 2002

SUMMARY: We designed a Java applet which enables the visualization of genetic networks and can be used as a Web publishing tool by molecular biologists studying the mechanisms of gene interactions. AVAILABILITY: http://www. csa.ru/Inst/gorb_dep/inbios/ genet/Graph/Genes_Graph.html CONTACT: samson@fn.csa.ru
DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis.
Authors: Rozas J , Rozas R
Published: July 2002

SUMMARY: DnaSP is a Windows integrated software package for the analysis of the DNA polymorphism from nucleotide sequence data. DnaSP version 3 incorporates several methods for estimating the amount and pattern of DNA polymorphism and divergence, and for conducting neutrality tests. AVAILABILITY: For academic uses, DnaSP is available free of charge from: http://www.bio.ub.es/julio/DnaSP.html CONTACT: julio@porthos.bio.ub.es
REPuter: fast computation of maximal repeats in complete genomes.
Authors: Kurtz S , Schleiermacher C
Published: July 2002

SUMMARY: A software tool was implemented that computes exact repeats and palindromes in entire genomes very efficiently. AVAILABILITY: Via the Bielefeld Bioinformatics Server (http://bibiserv.techfak.uni-bielefeld.de/rep uter/).
Saturated BLAST: an automated multiple intermediate sequence

search used to detect distant homology
Authors: Li Weizhong , Pio Frederic , Pawłowski Krzysztof , Godzik Adam
Published: July 2002

Motivation: Two proteins can have a similar 3-dimensional structure and biological function, but have sequences sufficiently different that traditional protein sequence comparison algorithms do not identify their relationship. The desire to identify such relations has led to the development of more sensitive sequence alignment strategies. One such strategy is the Intermediate Sequence Search (ISS), which connects two proteins through one or more intermediate sequences. In its brute-force implementation, ISS is a strategy that repetitively uses the results of the previous query as new search seeds, making it time-consuming and difficult to analyze.

Results: Saturated BLAST is a package that performs ISS in an efficient and automated manner. It was developed using Perl and Perl/Tk and implemented on the LINUX operating system. Starting with a protein sequence, Saturated BLAST runs a BLAST search and identifies representative sequences for the next generation of searches. The procedure is run until convergence or until some predefined criteria are met. Saturated BLAST has a friendly graphic user interface, a built-in BLAST result parser, several multiple alignment tools, clustering algorithms and various filters for the elimination of false positives, thereby providing an easy way to edit, visualize, analyze, monitor and control the search. Besides detecting remote homologies, Saturated BLAST can be used to maintain protein family databases and to search for new genes in genomic databases.

Availability: Free from http://bioinformatics.burnham-inst.org/xblast

Contact: liwz@sdsc.eduor adam@burnham-inst.org

* To whom correspondence should be addressed.
JESAM: CORBA software components to create and publish EST
alignments and clusters
Authors: Parsons J. D. , Rodriguez-Tomé P.
Published: July 2002

Motivation: Expressed Sequence Tags (ESTs) are cheap, easy and quick to obtain relative to full genomic sequencing and currently sample more eukaryotic genes than any other data source. They are particularly useful for developing Sequence Tag Sites (STSs for mapping), polymorphism discovery, disease gene hunting, mass spectrometer proteomics, and most ironically for finding genes and predicting gene structure after the great effort of genomic sequencing. However, ESTs have many problems and the public EST databases contain all the errors and high redundancy intrinsic to the submitted data so it is often found that derived database views, which reduce both errors and redundancy, are more effective starting points for research than the original raw submissions. Existing derived views such as EST cluster databases and consensus databases have never published supporting evidence or intermediary results leading to difficulties trusting, correcting, and customizing the final published database. These difficulties have lead many groups to wastefully repeat the complex intermediary work of others in order to offer slightly different final views. A better approach might be to discover the most expensive common calculations used by all the approaches and then publish all intermediary results. Given a globally accessible database with a suitable component interface, like the JESAM software described in this paper, the creation of customized EST-derived databases could be achieved with minimum effort.

Results: Databases of EST and full-length mRNA sequences for four model organisms have been self-compared by searching for overlaps consistent with contiguity. The sequence comparisons are performed in parallel using a PVM process farm and previous results are stored to allow incremental updates with minimal effort. The overlap databases have been published with CORBA interfaces to enable flexible global access as demonstrated by example Java applet browsers. Simple cDNA supercluster databases built as alignment database clients are themselves published via CORBA interfaces browsable with prototypical applets. A comparison with UniGene Mouse and Rat databases revealed undesirable features in both and the advantages of contrasting perspectives on complex data.

Availability: The software is packaged as two Jar files available from: URL: http://corba.ebi.ac.uk/EST/jesam/jesam.html. One jar contains all the Java source code, and the other contains all the C, C++ and IDL code. Links to working examples of the alignment and cluster viewers (if remote firewall permits) can be found at http://corba.ebi.ac.uk/EST. All the Washington University mouse EST traces are available for browsing at the same URL.

Contact: jparsons@ebi.ac.uk
Secondary structure alone is generally not statistically
significant for the detection of noncoding RNAs
Authors: Rivas Elena , Eddy Sean R.
Published: July 2002

Motivation: Several results in the literature suggest that biologically interesting RNAs have secondary structures that are more stable than expected by chance. Based on these observations, we developed a scanning algorithm for detecting noncoding RNA genes in genome sequences, using a fully probabilistic version of the Zuker minimum-energy folding algorithm.

Results: Preliminary results were encouraging, but certain anomalies led us to do a carefully controlled investigation of this class of methods. Ultimately, our results argue that for the probabilistic model there is indeed a statistical effect, but it comes mostly from local base-composition bias and not from RNA secondary structure. For the thermodynamic implementation (which evaluates statistical significance by doing Monte Carlo shuffling in fixed-length sequence windows, thus eliminating the base-composition effect) the signals for noncoding RNAs are still usually indistinguishable from noise, especially when certain statistical artifacts resulting from local base-composition inhomogeneity are taken into account. We conclude that although a distinct, stable secondary structure is undoubtedly important in most noncoding RNAs, the stability of most noncoding RNA secondary structures is not sufficiently different from the predicted stability of a random sequence to be useful as a general genefinding approach.

Contact: eddy@genetics.wustl.edu

* To whom correspondence should be addressed.
Generation of patterns from gene expression data by assigning

confidence to differentially expressed genes
Authors: Manduchi Elisabetta , Grant Gregory R. , McKenzie Steven E. , Overton G. Christian , Surrey Saul , Stoeckert Jr. Christian J.
Published: July 2002

Motivation: A protocol is described to attach expression patterns to genes represented in a collection of hybridization array experiments. Discrete values are used to provide an easily interpretable description of differential expression. Binning cutoffs for each sample type are chosen automatically, depending on the desired false-positive rate for the predictions of differential expression. Confidence levels are derived for the statement that changes in observed levels represent true changes in expression. We have a novel method for calculating this confidence, which gives better results than the standard methods. Our method reflects the broader change of focus in the field from studying a few genes with many replicates to studying many (possibly thousands) of genes simultaneously, but with relatively few replicates. Our approach differs from standard methods in that it exploits the fact that there are many genes on the arrays. These are used to estimate for each sample type an appropriate distribution that is employed to control the false-positive rate of the predictions made. Satisfactory results can be obtained using this method with as few as two replicates.

Results: The method is illustrated through applications to macroarray and microarray datasets. The first is an erythroid development dataset that we have generated using nylon filter arrays. Clones for genes whose expression is known in these cells were assigned expression patterns which are in accordance with what was expected and which are not picked up by the standards methods. Moreover, genes differentially expressed between normal and leukemic cells were identified. These included genes whose expression was altered upon induction of the leukemic cells to differentiate. The second application is to the microarray data by Alizadeh et al. (2000). Our results are in accordance with their major findings and offer confidence measures for the predictions made. They also provide new insights for further analysis.

Availability: Software is available on request from the authors.

Contact: manduchi@pcbi.upenn.edu

To whom correspondence should be addressed.
Pro-Frame: similarity-based gene recognition in eukaryotic DNA

sequences with errors
Authors: Mironov Andrey A. , Novichkov Pavel S. , Gelfand Mikhail S.
Published: July 2002

Summary: Performance of existing algorithms for similarity-based gene recognition in eukaryotes drops when the genomic DNA has been sequenced with errors. A modification of the spliced alignment algorithm allows for gene recognition in sequences with errors, in particular frameshifts. It tolerates up to 5% of sequencing errors without considerable drop of prediction reliability when a sufficiently close homologous protein is available (normalized evolutionary distance similarity score 50% or higher).

Availability: The program is free for academic users and available upon request at http://www.anchorgen.com

Contact: mgelfand@anchorgen.com