bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a Novel Simian Immunodeficiency Virus (SIVmonNG1) Genome Sequence from a Mona Monkey (Cercopithecus mona)
Authors: Barlow Katrina L. , Ajao Adebowale Oluwafemi , Clewley Jonathan P. 
Published: May 2003

ABSTRACTA novel simian immunodeficiency virus (SIV) sequence has been recovered from RNA extracted from the serum of a mona monkey (Cercopithecus mona) wild born in Nigeria. The sequence was obtained by using novel generic (degenerate) PCR primers and spans from two-thirds into thegaggene to the 3′ poly(A) tail of the SIVmonNG1 RNA genome. Analysis of the open reading frames revealed that the SIVmonNG1 genome codes for a Vpu protein, in addition to Gag, Pol, Vif, Vpr, Tat, Rev, Env, and Nef proteins. Previously, only lentiviruses infecting humans (human immunodeficiency virus type 1 [HIV-1]) and chimpanzees (SIVcpz) were known to have avpugene; more recently, this has also been found in SIVgsn fromCercopithecus nictitans. Overall, SIVmonNG1 most closely resembles SIVgsn: theenvgene sequence groups with HIV-1/SIVcpzenvsequences, whereas thepolgene sequence clusters closely with thepolsequence of SIVsyk fromCercopithecus albogaris. By bootscanning and similarity plotting, the first half ofpolresembles SIVsyk, whereas the latter part is closer to SIVcol fromColobus guereza. The similarities between the complex mosaic genomes of SIVmonNG1 and SIVgsn are consistent with a shared or common lineage. These data further highlight the intricate nature of the relationships between the SIVs from different primate species and will be helpful for unraveling these associations.
EBNA1 Partitions Epstein-Barr Virus Plasmids in Yeast Cells by Attaching to Human EBNA1-Binding Protein 2 on Mitotic Chromosomes
Authors: Kapoor Priya , Frappier Lori 
Published: May 2003

ABSTRACT

Epstein-Barr virus (EBV) episomal genomes are stably maintained in human cells and are partitioned during cell division by mitotic chromosome attachment. Partitioning is mediated by the viral EBNA1 protein, which binds both the EBV segregation element (FR) and a mitotic chromosomal component. We previously showed that the segregation of EBV-based plasmids can be reconstituted in
Saccharomyces cerevisiae
and is absolutely dependent on EBNA1, the EBV FR sequence, and the human EBNA1-binding protein 2 (EBP2). We have now used this yeast system to elucidate the functional contribution of human EBP2 to EBNA1-mediated plasmid partitioning. Human EBP2 was found to attach to yeast mitotic chromosomes in a cell cycle-dependent manner and cause EBNA1 to associate with the mitotic chromosomes. The domain of human EBP2 that binds both yeast and human chromosomes was mapped and shown to be functionally distinct from the EBNA1-binding domain. The functionality and localization of human EBP2 mutants and fusion proteins indicated that the attachment of EBNA1 to mitotic chromosomes is crucial for EBV plasmid segregation in
S. cerevisiae
, as it is in humans, and that this is the contribution of human EBP2. The results also indicate that plasmid segregation in
S. cerevisiae
can occur through chromosome attachment.
A Novel Human Antibody against Human Immunodeficiency Virus Type 1 gp120 Is V1, V2, and V3 Loop Dependent and Helps Delimit the Epitope of the Broadly Neutralizing Antibody Immunoglobulin G1 b12
Authors: Zwick Michael B. , Kelleher Robert , Jensen Richard , Labrijn Aran F. , Wang Meng , Quinnan Gerald V. , Parren Paul W. H. I. , Burton Dennis R. 
Published: May 2003

ABSTRACTThe V1/V2 and V3 loops are proximal to the CD4 binding site (CD4bs) of human immunodeficiency virus type 1 (HIV-1) gp120 and undergo conformational change upon CD4 receptor engagement by the HIV-1 envelope spike. Nearly all of the reported monoclonal antibodies (MAbs) against the CD4bs exhibit a very limited capacity to neutralize HIV-1. However, one such human MAb, immunoglobulin G1 (IgG1) b12, is uniquely able to neutralize primary isolates across subtypes with considerable potency. The molecular basis for the anti-HIV-1 activity of b12 is not fully understood but is relevant to vaccine design. Here we describe a novel human MAb, 4KG5, whose binding to monomeric gp120 is moderately enhanced by IgG1 b12. In sharp contrast, 4KG5 binding to gp120 is inhibited by soluble CD4 (sCD4) and by all other (n= 14) anti-CD4bs MAbs tested. 4KG5 is unable to recognize gp120 in which either V1, V2, or V3 has been deleted, and MAbs against the V2 or V3 loops inhibit the binding of 4KG5 to gp120. Moreover, 4KG5 is able to inhibit the binding of the CD4-induced MAbs 17b and X5 in the absence of sCD4, whereas 17b and X5 only weakly inhibit the binding of 4KG5 to gp120. Mutagenesis of gp120 provides further evidence of a discontinuous epitope of 4KG5 that is formed by the V1/V2 loop, the V3 loop, and a portion of the bridging sheet (C4). 4KG5 was isolated as a single-chain Fv from a phage display library constructed from the bone marrow of an HIV-1-seropositive subject (FDA2) whose serum neutralizes HIV-1 across subtypes. Despite its source, we observed no significant neutralization with 4KG5 against the autologous (R2) virus and several other strains of HIV-1. The results suggest a model in which antibody access to the CD4bs on the envelope spike of HIV-1 is restricted by the orientation and/or dynamics of the V1/V2 and V3 loops, and b12 avoids these restrictions.
Identification of Amino Acid Residues in the Capsid Proteins of Adeno-Associated Virus Type 2 That Contribute to Heparan Sulfate Proteoglycan Binding
Authors: Opie Shaun R. , Warrington, Kenneth H. , Agbandje-McKenna Mavis , Zolotukhin Sergei , Muzyczka Nicholas 
Published: May 2003

ABSTRACT
The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.
Prime-Boost Immunization Schedules Based on Influenza Virus and Vaccinia Virus Vectors Potentiate Cellular Immune Responses against Human Immunodeficiency Virus Env Protein Systemically and in the Genitorectal Draining Lymph Nodes
Authors: Gherardi M. Magdalena , Nájera José Luis , Pérez-Jiménez Eva , Guerra Susana , García-Sastre Adolfo , Esteban Mariano 
Published: May 2003

ABSTRACTVaccines that elicit systemic and mucosal immune responses should be the choice to control human immunodeficiency virus (HIV) infections. We have previously shown that prime-boost immunizations with influenza virus Env and vaccinia virus (VV) WR Env recombinants induced an enhanced systemic CD8+T-cell response against HIV-1 Env antigen. In this report, we analyzed in BALB/c mice after priming with influenza virus Env the ability of two VV recombinants expressing HIV-1 Env B (VV WR Env and the highly attenuated modified VV Ankara [MVA] Env) to boost cellular immune responses in the spleen and in the lymph nodes draining the genital and rectal tracts. Groups of mice were primed by the intranasal route with 104PFU of influenza virus Env and boosted 14 days later by the intraperitoneal or intranasal route with 107PFU of MVA Env or VV WR Env, while the control group received two immunizations with influenza virus Env. We found that the combined immunization (Flu/VV) increased more than 60 times the number of gamma interferon-specific CD8+T cells compared to the Flu/Flu scheme. Significantly, boosting with MVA Env by the intraperitoneal route induced a response 1.25 or 2.5 times (spleen or genital lymph nodes) higher with respect to that found after the boost with VV WR Env. Mice with an enhanced CD8+T-cell response also had an increased Th1/Th2 ratio, evaluated by the cytokine pattern secreted following in vitro restimulation with gp160 protein and by the specific immunoglobulin G2a (IgG2a)/IgG1 ratio in serum. By the intranasal route recombinant WR Env booster gave a more efficient immune response (10 and 1.3 times in spleen and genital lymph nodes, respectively) than recombinant MVA Env. However, the scheme influenza virus Env/MVA Env increased four times the response in the spleen, giving a low but significant response in the genital lymph nodes compared with a single intranasal immunization with MVA Env. These results demonstrate that the combination Flu/MVA in prime-booster immunization regimens is an effective vaccination approach to generate cellular immune responses to HIV antigens at sites critical for protective responses.
Progressive Truncations C Terminal to the Membrane-Spanning Domain of Simian Immunodeficiency Virus Env Reduce Fusogenicity and Increase Concentration Dependence of Env for Fusion
Authors: Lin Xiaoxu , Derdeyn Cynthia A. , Blumenthal Robert , West John , Hunter Eric 
Published: May 2003

ABSTRACT
The simian immunodeficiency virus (SIV) transmembrane (TM) protein, gp41, has multiple functions, which include anchoring the glycoprotein complex in the lipid envelope of the virus and mediating fusion of the virus and host cell membranes. Recently, a series of mutants of the SIVmac239 TM protein that have truncations at the carboxyl terminus of the membrane-spanning domain (MSD) have been characterized (J. T. West, P. Johnston, S. R. Dubay, and E. Hunter, J. Virol. 75:9601-9612, 2001). These mutants retained membrane anchorage but demonstrated reduced fusogenicity and infectivity as the MSD length was shortened. We have established a novel three-color fluorescence assay, which allows qualitative confocal and quantitative flow cytometric analyses, to further characterize the nature of the fusion defect in five of the MSD mutants: TM185, TM186, TM187, TM188, and TM189. Our analysis showed that each mutant could mediate complete lipid and aqueous dye transfer at early time points after effector and target cell mixing. No hemifusion with only lipid dye flux was detected. However, another intermediate fusion stage, which appears to involve small-fusion-pore formation that allowed small aqueous dye transfer but prevented the exchange of large cytoplasmic components, was identified infrequently in mutant-Env-expressing cell and target cell mixtures. Quantitative flow cytometric analysis of these mutants demonstrated that the TM187, TM188, and TM189 mutants were significantly more fusogenic than TM185 and TM186 but remained significantly impaired compared to the wild type. Moreover, fusion efficiency showed an increased dependence on the expression level of glycoproteins, suggesting that, for these mutants, formation of an active fusion complex was an increasingly stochastic event.
Cross-Reactivity of HLA-B
*
1801-Restricted T-Lymphocyte Clones with Target Cells Expressing Variants of the Human Cytomegalovirus 72kDa-IE1 Protein
Authors: Prod'homme Virginie , Retière Christelle , Valtcheva Ralitza , Bonneville Marc , Hallet Marie-Martine 
Published: May 2003

ABSTRACT

The impact of natural polymorphism in a cytomegalovirus-dominant HLA-B
*
1801-restricted epitope, IE1
199-206
, on the specific responses of T-cell clones was assessed by measuring their cytolytic activity against target cells expressing mutated recombinant IE1 proteins. Our results suggest an in vivo selection of T lymphocytes that cross-react with multiple IE1 variants.
Inhibition of NF-κB by a TAT-NEMO–binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis
Authors: Choi Mira , Rolle Susanne , Wellner Maren , Cardoso M. Cristina , Scheidereit Claus , Luft Friedrich C. , Kettritz Ralph 
Published: May 2003

AbstractDelivery of biologically active peptides into human polymorphonuclear neutrophils (PMNs) has implications for studying cellular functions and may be therapeutically relevant. The transcription factor nuclear factor-κB (NF-κB) regulates the expression of multiple genes controlling inflammation, proliferation, and cell survival. PMNs play a crucial role in first-line defense. Targeting NF-κB in these cells may promote apoptosis and therefore facilitate resolution of inflammation. We used an 11-amino acid sequence NEMO-binding domain (NBD) that selectively inhibits the IKKγ (NEMO)/IKKβ interaction, preventing NF-κB activation. An HIV-TAT sequence served as a highly effective transducing shuttle. We show that lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), and dexamethasone (DEX) significantly reduced apoptosis after 20 hours. LPS, but not GM-CSF or DEX, activated NF-κB as shown by IκBα degradation, NF-κB DNA binding, and transcriptional activity. The TAT-NBD blocked LPS-induced NF-κB activation and NF-κB–dependent gene expression. TAT-NBD accelerated constitutive PMN apoptosis dose dependently and abrogated LPS-delayed apoptosis. These results provide a proof of principle for peptide delivery by TAT-derived protein transduction domains to specifically inhibit NF-κB activity in PMNs. This strategy may help in controlling various cellular functions even in short-lived, transfection-resistant primary human cells.
Selective depletion of donor alloreactive T cells without loss of antiviral or antileukemic responses
Authors: Amrolia Persis J. , Muccioli-Casadei Giada , Yvon Eric , Huls Helen , Sili Uluhan , Wieder Eric D. , Bollard Catherine , Michalek Jaroslav , Ghetie Victor , Heslop Helen E. , Molldrem Jeffrey J. , Rooney Cliona M. , Schlinder John , Vitetta Ellen , Brenner Malcolm K. 
Published: May 2003

AbstractPoor immune reconstitution after haploidentical stem cell transplantation results in a high mortality from viral infections and relapse. One approach to overcome this problem is to selectively deplete the graft of alloreactive cells using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient peripheral blood mononuclear cells (PBMCs), and this can result in graft versus host disease (GVHD). We have refined this approach using recipient Epstein-Barr virus (EBV)–transformed lymphoblastoid cell lines (LCLs) as stimulators to activate donor alloreactive T cells. Our studies demonstrate that allodepletion with an anti-CD25 immunotoxin following stimulation with HLA-mismatched host LCLs more consistently depleted in vitro alloreactivity than stimulation with host PBMCs, as assessed in primary mixed lymphocyte reactions (MLRs). Allodepletion using this approach specifically abrogates cytotoxic T-cell responses against host LCLs. In interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assays, antiviral responses to adenovirus and cytomegalovirus (CMV) were preserved following allodepletion. Likewise, using HLA-A2–pp65 tetramers, we have shown that the frequency of CMV-specific T cells is unaffected by allodepletion. Moreover, the donor anti-EBV response is partially retained by recognition of EBV antigens through the nonshared haplotype. Finally, we studied whether allodepletion affects the response to candidate tumor antigens in myeloid malignancies. Using HLA-A2–PR1 tetramer analysis, we found that the frequency of T cells recognizing the PR1 epitope of proteinase 3 was not significantly different in allodepleted and unmanipulated PBMCs from patients with chronic myeloid leukemia (CML) undergoing transplantation. Based on these data, we have embarked on a phase 1 clinical trial of addback of allo-LCL–depleted donor T cells in the haplo-identical setting.
Adoptive transfer of costimulated T cells induces lymphocytosis in patients with relapsed/refractory non-Hodgkin lymphoma following CD34+-selected hematopoietic cell transplantation
Authors: Laport Ginna G. , Levine Bruce L. , Stadtmauer Edward A. , Schuster Stephen J. , Luger Selina M. , Grupp Stephan , Bunin Nancy , Strobl Frank J. , Cotte Julio , Zheng Zhaohui , Gregson Brian , Rivers Patricia , Vonderheide Robert H. , Liebowitz David N. , Porter David L. , June Carl H. 
Published: May 2003

AbstractWe explored the feasibility and toxicity of administering escalating doses of anti-CD3/CD28 ex vivo costimulated T cells as a therapeutic adjunct for patients with relapsed, refractory, or chemotherapy-resistant, aggressive non-Hodgkin lymphoma (NHL) following high-dose chemotherapy and CD34+-selected hematopoietic cell transplantation (HCT). Sixteen patients had infusions on day 14 after HCT of autologous T cells that had been stimulated using beads coated with anti-CD3 and anti-CD28 monoclonal antibodies. At baseline, the subjects had severe quantitative and functional T-cell impairments. The culture procedure partially reversed impaired cytokine responsiveness in T cells in vitro and in vivo. Transient dose-dependent infusion toxicities were observed. There was a rapid reconstitution of lymphocytes; however, there were persistent defects in CD4 T cells. Most interestingly, 5 patients had a delayed lymphocytosis between day 30 and day 120 after HCT. Maximal clinical responses included 5 patients with a complete response (CR), 7 patients with a partial response (PR), and 4 patients with stable disease. At a median follow-up of 33 months (range, 26-60 months), 5 patients are alive with stable or relapsed disease and 3 patients remain in CR. In conclusion, this phase 1 trial demonstrates that adoptive transfer of autologous costimulated T cells (1) is feasible in heavily pretreated patients with advanced NHL, (2) is associated with a rapid recovery of lymphocyte counts, (3) reverses cytokine activation deficits in vitro, and (4) is associated with delayed lymphocytosis in a subset of patients.
Imatinib mesylate inhibits autonomous erythropoiesis in patients with polycythemia vera in vitro
Authors: Oehler Leopold , Jaeger Eva , Eser Alexander , Sillaber Christian , Gisslinger Heinz , Geissler Klaus 
Published: May 2003

Abstract
The overproduction of red blood cells in patients with polycythemia vera (PV) is reflected in vitro by the formation of erythroid burst-forming units (BFU-Es) in the absence of exogenous erythropoietin. In contrast to other myeloproliferative disorders, the molecular mechanism of PV is unknown and no specific chromosomal abnormality has been described. We speculated that imatinib mesylate may reverse the pathological overproduction of red cells by inhibition of autonomous erythropoiesis. In the present study, imatinib mesylate was found to either block or strongly inhibit autonomous BFU-E formation in vitro in all patients tested. Moreover, autonomous BFU-E growth was also markedly reduced by exposure of PV cells to imatinib mesylate prior to cultivation in semisolid medium. The profound effect of imatinib mesylate on autonomous erythropoiesis suggests the involvement of an as yet unidentified kinase in the pathogenesis of PV and should provide the rationale for a forthcoming clinical trial.
Visual resolution of gratings by the compound eye of the bee Apis mellifera
Authors: Horridge G. Adrian 
Published: May 2003

SUMMARY
Early measurements of the resolution of horizontal versus vertical gratings were confirmed, with a limit near a period of 2.5°, and the resolution is similar when vertical or horizontal gratings are tested separately against grey. Bees were next trained to discriminate from a distance between gratings at 45° versus 135°, with no green contrast, on targets presented in a vertical plane at a fixed distance. As expected, they fail to learn; however, with green contrast but no modulation difference the resolution limit is near 3.5°. With vertical and horizontal gratings with no green contrast they discriminate but do not learn an orientation cue. In order to eliminate the orientation cue altogether, new bees were then trained with alternating vertical and horizontal gratings versus grey, or with a black and white checkerboard versusgrey. Tests of these trained bees with horizontal or with vertical gratings separately against grey again show a resolution down to a period near 2.5°. These results, taken together, show that when edge orientation alone is the cue, the limit of resolution is near 3.5°, but when receptor modulation is the cue, the limit is near 2.5°.
Molecular characterisation of the smooth endoplasmic reticulum Ca2+-ATPase ofPorcellio scaberand its expression in sternal epithelia during the moult cycle
Authors: Hagedorn Monica , Weihrauch Dirk , Towle David W. , Ziegler Andreas 
Published: May 2003

SUMMARYThe anterior sternal epithelial cells of the terrestrial isopod Porcellio scaber transport large amounts of calcium during the formation and resorption of intermittent calcium carbonate deposits. Recent investigations on epithelia involved in mineralisation processes suggest a role of the smooth endoplasmic reticulum Ca2+-ATPase (SERCA) in transcellular calcium transport. We present the first molecular characterisation of a SERCA within a crustacean mineralising epithelium. We cloned the SERCA from a cDNA library of the anterior sternal epithelium and used in situ hybridisation to compare the expression of the SERCA mRNA between three different moulting stages. The full-length SERCA cDNA has an open reading frame of 3006 nucleotides. The deduced 1002 amino-acid polypeptide has a predicted molecular mass of 109.7 kDa and 87% identity to the SERCA of Procambarus clarkii axial muscle isoform. In situ hybridisation confirmed expression within the anterior sternal epithelium and revealed an increase in SERCA mRNA abundance from the non-transporting, early premoult stage to the calcium transporting, late premoult and intramoult stage. The results support previous indications of a contribution by the smooth endoplasmic reticulum to transcellular calcium transport and suggest a transcriptional regulation of SERCA activity.