bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Palmitoylation of Replicase Protein nsP1 on Alphavirus Infection
Authors: Ahola Tero , Kujala Pekka , Tuittila Minna , Blom Titta , Laakkonen Pirjo , Hinkkanen Ari , Auvinen Petri
Published: July 2002

The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.
Synergistic Upregulation of Interleukin-8 Secretion from Pulmonary Epithelial Cells by Direct and Monocyte-Dependent Effects of Respiratory Syncytial Virus Infection
Authors: Thomas Lynette H. , Wickremasinghe Melissa I. Y. , Sharland Mike , Friedland Jon S.
Published: July 2002

Respiratory syncytial virus (RSV) infection is the major cause of severe bronchiolitis in infants. Pathology of this infection is partly due to excessive proinflammatory leukocyte influx mediated by chemokines. Although direct infection of the respiratory epithelium by RSV may induce chemokine secretion, little is known about the role of cytokine networks. We investigated the effects of conditioned medium (CM) from RSV-infected monocytes (RSV-CM) on respiratory epithelial (A549) cell chemokine release. RSV-CM, but not control CM (both at a 1:5 dilution), stimulated interleukin-8 (IL-8) secretion from A549 cells within 2 h, and secretion increased over 72 h to 11,360 ± 1,090 pg/ml without affecting cell viability. In contrast, RSV-CM had only a small effect on RANTES secretion. RSV-CM interacted with direct RSV infection to synergistically amplify IL-8 secretion from respiratory epithelial cells (levels of secretion at 48 h were as follows: RSV-CM alone, 8,140 ± 2,160 pg/ml; RSV alone, 12,170 ± 300 pg/ml; RSV-CM plus RSV, 27,040 ± 5,260 pg/ml;P< 0.05). RSV-CM induced degradation of IκBα within 5 min but did not affect IκBβ. RSV-CM activated transient nuclear binding of NF-κB within 1 h, while activation of NF-IL6 was delayed until 8 h and was still detectable at 24 h. Promoter-reporter analysis demonstrated that NF-κB binding was essential and that NF-IL6 was important for IL-8 promoter activity in RSV-CM-activated cells. Blocking experiments revealed that the effects of RSV-CM depended on monocyte-derived IL-1 but that tumor necrosis factor alpha was not involved in this network. In summary, RSV infection of monocytes results in and amplifies direct RSV-mediated IL-8 secretion from respiratory epithelial cells by an NF-κB-dependent, NF-IL6-requiring mechanism.
The Valine Anticodon and Valylatability of
Peanut Clump Virus
RNAs Are Not Essential but Provide a Modest Competitive Advantage in Plants
Authors: Matsuda Daiki , Dunoyer Patrice , Hemmer Odile , Fritsch Christiane , Dreher Theo W.
Published: July 2002

The role of valine aminoacylation of the two genomic RNAs of
Peanut clump virus
(PCV) was studied by comparing the amplification in vivo of RNAs with GAC, GΔC, or CCA anticodons in the tRNA-like structure (TLS) present at the 3′ end of each viral RNA. The PCV RNA1 TLS of isolate PCV2 possesses a GAC anticodon and is capable of highly efficient valylation, whereas the RNA2 TLS has a GΔC anticodon that does not support valylation. The presence in RNA1 of GΔC or CCA anticodons that conferred nonvalylatability resulted in about 2- to 4-fold and a 14- to 24-fold reduction, respectively, in RNA accumulations in tobacco BY-2 protoplasts inoculated with the RNA1 variants together with wild-type RNA2(GΔC). No differences in RNA levels were observed among protoplasts inoculated with the three variant RNA2s in the presence of wild-type RNA1(GAC). All combinations of valylatable and nonvalylatable RNAs 1 and 2 were similarly infectious in
Nicotiana benthamiana
plants, and viral RNAs accumulated to similar levels; all input TLS sequences were present unchanged in apical leaves. In direct competition experiments in
N. benthamiana
plants, however, both RNA1 and RNA2 with GAC valylatable anticodons outcompeted the nonvalylatable variants. We conclude that valylation provides a small but significant replicational advantage to both PCV RNAs. Sequence analysis of the TLS from RNA2 of a second PCV isolate, PO2A, revealed the presence of an intact GAC valine anticodon, suggesting that the differential valylation of the genomic RNAs of isolate PCV2 is not a general characteristic of PCV.
Genetic Determinants of Altered Virulence of Taiwanese Foot-and-Mouth Disease Virus
Authors: Beard Clayton W. , Mason Peter W.
Published: July 2002

In 1997, a devastating outbreak of foot-and-mouth disease (FMD) in Taiwan was caused by a serotype O virus (referred to here as OTai) with atypical virulence. It produced high morbidity and mortality in swine but did not affect cattle. We have defined the genetic basis of the species specificity of OTai by evaluating the properties of genetically engineered chimeric viruses created from OTai and a bovine-virulent FMD virus. These studies have shown that an altered nonstructural protein, 3A, is a primary determinant of restricted growth on bovine cells in vitro and significantly contributes to bovine attenuation of OTai in vivo.
Loss of Heterozygosity at theInk4a/ArfLocus Facilitates Abelson Virus Transformation of Pre-B Cells
Authors: Mostecki Justin , Halgren Anne , Radfar Arash , Sachs Zohar , Ravitz James , Thome Kelly C. , Rosenberg Naomi
Published: July 2002

In many tumor systems, analysis of cells for loss of heterozygosity (LOH) has helped to clarify the role of tumor suppressor genes in oncogenesis. Two important tumor suppressor genes,p53and theInk4a/Arflocus, play central roles in the multistep process of Abelson murine leukemia virus (Ab-MLV) transformation. p53 and the p53 regulatory protein, p19Arf, are required for the apoptotic crisis that characterizes the progression of primary transformed pre-B cells to fully malignant cell lines. To search for other tumor suppressor genes which may be involved in the Ab-MLV transformation process, we used endogenous proviral markers and simple-sequence length polymorphism analysis to screen Abelson virus-transformed pre-B cells for evidence of LOH. Our survey reinforces the role of the p53-p19 regulatory pathway in transformation; 6 of 58 cell lines tested had lost sequences on mouse chromosome 4, including theInk4a/Arflocus. Consistent with this pattern, a high frequency of primary pre-B-cell transformants derived fromInk4a/Arf+/− mice became established cell lines. In addition, half of them retained the single copy of the locus when the transformation process was complete. These data demonstrate that a single copy of theInk4a/Arflocus is not sufficient to fully mediate the effects of these genes on transformation.
Human Receptor for Measles Virus (CD46) Enhances Nitric Oxide Production and Restricts Virus Replication in Mouse Macrophages by Modulating Production of Alpha/Beta Interferon
Authors: Katayama Yuko , Hirano Akiko , Wong Timothy C.
Published: July 2002

Complement regulatory protein CD46 is a human cell receptor for measles virus (MV). In this study, we investigated why mouse macrophages expressing human CD46 restricted MV replication and produced higher levels of nitric oxide (NO) in response to MV and gamma interferon (IFN-γ). Treatment of MV-infected CD46-expressing mouse macrophages with antibodies against IFN-α/β blocked NO production. Antibodies against IFN-α/β also inhibited the augmenting effect of MV on IFN-γ-induced NO production in CD46-expressing mouse macrophages. These antibodies did not affect NO production induced by IFN-γ alone. These data suggest that MV enhances NO production in CD46-expressing mouse macrophages through action of IFN-α/β. Mouse macrophages expressing a human CD46 mutant lacking the cytoplasmic domains were highly susceptible to MV. These cells produced much lower levels of NO and IFN-α/β upon infection by MV, suggesting the CD46 cytoplasmic domains enhanced IFN-α/β production. When mouse macrophages expressing tailless human CD46 were exposed to culture medium from MV-infected mouse macrophages expressing intact human CD46, viral protein synthesis and development of cytopathic effects were suppressed. Pretreating the added culture medium with antibodies against IFN-α/β abrogated these antiviral effects. Taken together, these findings suggest that expression of human CD46 in mouse macrophages enhances production of IFN-α/β in response to MV infection, and IFN-α/β synergizes with IFN-γ to enhance NO production and restrict viral protein synthesis and virus replication. This novel function of human CD46 in mouse macrophages requires the CD46 cytoplasmic domains.
The SU and TM Envelope Protein Subunits of Bovine Leukemia Virus Are Linked by Disulfide Bonds, both in Cells and in Virions
Authors: Johnston Elizabeth R. , Radke Kathryn
Published: July 2002

After the polyprotein precursor of retroviral envelope proteins is proteolytically cleaved, the surface (SU) and transmembrane (TM) subunits remain associated with each other by noncovalent interactions or by disulfide bonds. Disulfide linkages confer a relatively stable association between the SU and TM envelope protein subunits of Rous sarcoma virus and murine leukemia virus. In contrast, the noncovalent association between SU and TM of human immunodeficiency virus leads to significant shedding of SU from the surface of infected cells. The SU and TM proteins of bovine leukemia virus (BLV) initially were reported to be disulfide linked but later were concluded not to be, since TM is often lost during purification of SU protein. Here, we show that SU and TM of BLV do, indeed, associate through disulfide bonds, whether the envelope proteins are overexpressed in transfected cells, are produced in virus-infected cells, or are present in newly produced virions.
Binding of Recombinant Feline Immunodeficiency Virus Surface Glycoprotein to Feline Cells: Role of CXCR4, Cell-Surface Heparans, and an Unidentified Non-CXCR4 Receptor
Authors: de Parseval Aymeric , Elder John H.
Published: July 2002

To address the role of CXCR4 in the cell-surface attachment of the feline immunodeficency virus (FIV), a soluble fusion protein, gp95-Fc, consisting of the surface glycoprotein (SU, gp95) of either a primary (PPR) or cell line-adapted (34TF10) FIV strain was fused in frame with the Fc domain of human immunoglobulin G1. The recombinant SU-immunoadhesins were used as probes to investigate the cellular binding of FIV SU. In agreement with the host cell range properties of both viruses, binding of 34TF10 gp95-Fc was observed for all cell lines tested, whereas PPR gp95-Fc bound only to primary feline T cells. 34TF10 gp95-Fc also bound to Jurkat and HeLa cells, consistent with the ability of FIV to use human CXCR4 as a fusion receptor. As expected, 34TF10 gp95-Fc binding to Jurkat cells was blocked by addition of stromal cell-derived factor 1α (SDF-1α), as was binding to the 3201 feline lymphoma cell line. However, SDF-1α, RANTES, macrophage inflammatory protein 1β, and heparin all failed to inhibit the binding of either gp95-Fc to primary T cells, suggesting that a non-CXCR4 receptor is involved in the binding of FIV SU. In this regard, an unidentified 40-kDa protein species from the surface of primary T cells but not Jurkat and 3201 cells specifically coprecipitated with both gp95-Fc. Yet another type of binding of 34TF10 gp95-Fc to adherent kidney cells was noted. SDF-1α failed to block the binding of 34TF10 gp95-Fc to either HeLa, Crandel feline leukemia, or G355-5 cells. However, binding was severely impaired in the presence of soluble heparin, as well as after enzymatic removal of surface heparans or on cells deficient in heparan expression. These overall findings suggest that in addition to CXCR4, a non-CXCR4 receptor and cell-surface heparans also play an important role in FIV gp95 cell surface interactions on specific target cells.
Activation of TRAF5 and TRAF6 Signal Cascades Negatively Regulates the Latent Replication Origin of Epstein-Barr Virus through p38 Mitogen-Activated Protein Kinase
Authors: Shirakata Masaki , Imadome Ken-Ichi , Okazaki Kenji , Hirai Kanji
Published: July 2002

Latent Epstein-Barr virus (EBV) is maintained by the virus replication originoriPthat initiates DNA replication with the viraloriP-binding factor EBNA1. However, it is not known whetheroriP's replicator activity is regulated by virus proteins or extracellular signals. By using a transient replication assay, we found that a low level of expression of viral signal transduction activator latent membrane protein 1 (LMP1) suppressedoriPactivity. The binding site of the tumor necrosis factor receptor-associated factor (TRAF) of LMP1 was essential for this suppressive effect. Activation of the TRAF signal cascade by overexpression of TRAF5 and/or TRAF6 also suppressedoriPactivity. Conversely, blocking of TRAF signaling with dominant negative mutants of TRAF5 and TRAF6, as well as inhibition of a downstream signal mediator p38 MAPK, released the LMP1-inducedoriPsuppression. Furthermore, activation of TRAF6 signal cascade by lipopolysaccharides (LPS) resulted in loss of EBV from Burkitt's lymphoma cell line Akata, and inhibition of p38 MAPK abolished the suppressive effect of LPS. These results suggested that the level oforiPactivity is regulated by LMP1 and extracellular signals through TRAF5- and TRAF6-mediated signal cascades.
Octamer-Binding Sequence Is a Key Element for the Autoregulation of Kaposi's Sarcoma-Associated Herpesvirus ORF50/Lyta Gene Expression
Authors: Sakakibara Shuhei , Ueda Keiji , Chen Jiguo , Okuno Toshiomi , Yamanishi Koichi
Published: July 2002

The expression of the Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) protein, Lyta (lytic transactivator), marks the switch from latent KSHV infection to the lytic phase. ORF50/Lyta upregulates several target KSHV genes, such as K8 (K-bZip), K9 (vIRF1), and ORF57, finally leading to the production of mature viruses. The auto-upregulation of ORF50/Lyta is thought to be an important mechanism for efficient lytic viral replication. In this study, we focused on this autoregulation and identified the promoter element required for it. An electrophoretic mobility shift assay indicated that the octamer-binding protein 1 (Oct-1) bound to this element. Mutations in the octamer-binding motif resulted in refractoriness of the ORF50/Lyta promoter to transactivation by ORF50/Lyta, and Oct-1 expression enhanced this transactivation. These results suggest that the autoregulation of ORF50/Lyta is mediated by Oct-1.
Copper Binding to the PrP Isoforms: a Putative Marker of Their Conformation and Function
Authors: Shaked Yuval , Rosenmann Hana , Hijazi Nuha , Halimi Michele , Gabizon Ruth
Published: July 2002

We show here that PrPC, the normal isoform of the prion protein (PrPSc), could be retained by a Cu2+-loaded resin through two different binding sites. Contrarily, PrPScwas not retained at all by such resin. This constitutes a new prion-specific property of PrPSc, which in addition to protease resistance and β-sheet content, may result from its aberrant conformation.
Adenovirus DNA Binding Protein Interacts with the SNF2-Related CBP Activator Protein (SrCap) and Inhibits SrCap-Mediated Transcription
Authors: Xu Xiequn , Chackalaparampil Isaac , Monroy M. Alexandra , Cannella Maria T. , Pesek Elizabeth , Chrivia John , Yaciuk Peter
Published: July 2002

The SNF2-related CBP activator protein, SrCap (pronounced “sir cap”), shares homology with the SNF2/SWI2 protein family. SrCap was cloned through its ability to bind CBP. SrCap can function as a CBP coactivator and can activate transcription in a reporter assay when expressed as a Gal-SrCap fusion protein. A monoclonal antibody raised against the carboxyl terminus of SrCap coimmunoprecipitates CBP/p300, supporting the model that SrCap is a CBP binding protein and that these proteins can be found together in a cellular protein complex. In addition, several cellular proteins are coimmunoprecipitated by the SrCap-specific antibody. Since adenovirus E1A proteins interact with CBP/p300 proteins, we examined what proteins could be copurified in a SrCap-specific coimmunoprecipitation assay from lysates of adenovirus-infected cells. While E1A proteins were not detected in this complex, to our surprise, we observed the presence of an infected-cell-specific band of 72 kDa, which we suspected might be the adenovirus DNA binding protein, DBP. The adenovirus DBP is a multifunctional protein involved in several aspects of the adenovirus life cycle, including an ability to modulate transcription. The identity of DBP was confirmed by DBP-specific Western blot analysis and by reimmunoprecipitating DBP from denatured SrCap-specific protein complexes. Using in vitro-translated DBP and SrCap proteins, we demonstrated that these proteins interact. To determine whether this interaction could affect SrCap-mediated transcription, we tested whether increasing amounts of DBP could modulate the Gal-SrCap transcription activity. We observed that DBP inhibited Gal-SrCap transcription activity in a dose-dependent manner. These data suggest a novel mechanism of adenovirus host cell control by which DBP binds to and inactivates SrCap, a member of the SNF2 chromatin-remodeling protein family.
Epstein-Barr Virus and the Somatic Hypermutation of Immunoglobulin Genes in Burkitt's Lymphoma Cells
Authors: Harris Reuben S. , Croom-Carter Debbie S. G. , Rickinson Alan B. , Neuberger Michael S.
Published: July 2002

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.
Microtubule-Independent Motility and Nuclear Targeting of Adenoviruses with Fluorescently Labeled Genomes
Authors: Glotzer Jolanta B. , Michou Anne-Isabelle , Baker Adam , Saltik Mediyha , Cotten Matt
Published: July 2002

A novel adenovirus system for analyzing the adenovirus entry pathway has been developed that contains green fluorescent protein bound to the encapsidated viral DNA (AdLite viruses). AdLite viruses enter host cells and accumulate around the nuclei and near the microtubule organizing centers (MTOC). In live cells, individual AdLite particles were observed trafficking both toward and away from the nucleus. Depolymerization of microtubules during infection prevented AdLite accumulation around the MTOC; however, it did not abolish perinuclear localization of AdLite particles. Furthermore, depolymerization of microtubules did not affect AdLite motility and did not affect gene expression from wild-type adenovirus and adenovirus-derived vectors. These data revealed that adenovirus intracellular motility and nuclear targeting can be supported by a mechanism that does not rely on the microtubule network.
Systemic Immunity and Mucosal Immunity Are Induced against Human Immunodeficiency Virus Gag Protein in Mice by a New Hyperattenuated Strain ofListeria monocytogenes
Authors: Rayevskaya Marina V. , Frankel Fred R.
Published: July 2002

Vaccines designed to control chronic infections by intracellular agents such as human immunodeficiency virus type 1 (HIV-1) require the induction of cell-mediated immune responses to rid the host of pathogen-infected cells.Listeria monocytogeneshas characteristics that make it an attractive vaccine vector for use against such infections. Here we show that parenteral immunization with a new highly attenuated strain of this organism provided complete protection against systemic and mucosal challenges with a recombinant vaccinia virus expressing HIV-1gag. Immunization also generated a strong, long-term memory cytotoxic-T-lymphocyte (CTL) response in spleen, mesenteric lymph nodes, and Peyer's patches directed against thegagprotein. Oral immunization with this attenuated strain also produced complete, long-lasting protection against the recombinant virus but only against mucosal virus challenge. Curiously, oral immunization was associated with a transient CTL response in the three lymphoid tissues examined.
Role of Natural Killer Cells in Resistance against Friend Retrovirus-Induced Leukemia
Authors: Iwanami Norimasa , Niwa Atsuko , Yasutomi Yasuhiro , Tabata Nobutada , Miyazawa Masaaki
Published: July 2002

We have previously shown that immunization with a synthetic peptide that contains a single CD4+T-cell epitope protects mice against immunosuppressive Friend retrovirus infection. Cells producing infectious Friend virus were rapidly eliminated from the spleens of mice that had been immunized with the single-epitope peptide. However, actual effector mechanisms induced through T-helper-cell responses after Friend virus inoculation were unknown. When cytotoxic effector cells detected in the early phase of Friend retrovirus infection were separated based on their expression of cell surface markers, those lacking CD4 and CD8 but expressing natural killer cell markers were found to constitute the majority of effector cells that lysed Friend virus-induced leukemia cells. Depletion of natural killer cells by injecting anti-asialo-ganglio-N-tetraosylceramide antibody did not affect the number of CD4+or CD8+T cells in the spleen, virus antigen-specific proliferative responses of CD4+T cells, or cytotoxic activity against Friend virus-induced leukemia cells exerted by CD8+effector cells. However, the same treatment markedly reduced the killing activity of CD4−CD8−effector cells and completely abolished the effect of peptide immunization. Although the above enhancement of natural killer cell activity in the early stage of Friend virus infection was also observed in mice given no peptide, these results have demonstrated the importance and requirement of natural killer cells in vaccine-induced resistance against the retroviral infection.
Viral Regulation of RANTES Expression during Human Cytomegalovirus Infection of Endothelial Cells
Authors: Billstrom Schroeder Marcella , Worthen G. Scott
Published: July 2002

Human cytomegalovirus (HCMV) evades healthy immune responses during infection, and this evasion may allow HCMV to establish latency in the host. The human vasculature has been recognized as a site of HCMV infection and may also be a site of latent HCMV infection. As the interface between circulating cells and underlying parenchymal cells, the vascular endothelium provides signals for local reaction of inflammatory cells. We propose that HCMV down-regulates expression of the proinflammatory chemokine RANTES from the infected endothelium, which may result in reduced recruitment of mononuclear cells to the site of infection. Abortive HCMV infection of primary endothelial cells with the clinical isolate HCMV 4010, under conditions in which viral gene expression could not occur, induced high levels of RANTES expression. Replicative HCMV infection, however, induced cells in parallel cultures to express significantly lower levels of RANTES. Expression of the chemokines interleukin 8 and MCP-1 by endothelial cells was found to be unaffected by replicative HCMV infection and thus may not play an important role during early HCMV infection of the endothelium. HCMV may regulate RANTES expression from endothelial cells as a mechanism to evade the local immune response to infection.
Transcriptional Activation of the Telomerase hTERT Gene by Human Papillomavirus Type 16 E6 Oncoprotein
Authors: Veldman Tim , Horikawa Izumi , Barrett J. Carl , Schlegel Richard
Published: July 2002

The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5′ promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (−211 to +40). Furthermore, there is a 35-bp region (+5 to +40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.
Ikaros, a Lymphoid-Cell-Specific Transcription Factor, Contributes to the Leukemogenic Phenotype of a Mink Cell Focus-Inducing Murine Leukemia Virus
Authors: DiFronzo Nancy L. , Leung Cheuk T. , Mammel Mark K. , Georgopoulos Katia , Taylor Barbara J. , Pham Quynh N.
Published: July 2002

Mink cell focus-inducing (MCF) viruses induce T-cell lymphomas in AKR/J strain mice. MCF 247, the prototype of this group of nonacute murine leukemia viruses, transforms thymocytes, in part, by insertional mutagenesis and enhancer-mediated dysregulation of cellular proto-oncogenes. The unique 3′ (U3) regions in the long terminal repeats of other murine leukemia viruses contain transcription factor binding sites known to be important for enhancer function and for the induction of T-cell lymphomas. Although transcription factor binding sites important for the biological properties of MCF 247 have not been identified, pathogenesis studies from our laboratory suggested to us that binding sites for Ikaros, a lymphoid-cell-restricted transcriptional regulator, affect the biological properties of MCF 247. In this report, we demonstrate that Ikaros binds to predicted sites in U3 sequences of MCF 247 and that site-directed mutations in these sites greatly diminish this binding in vitro. Consistent with these findings, ectopic expression of Ikaros in murine cells that do not normally express this protein significantly increases transcription from the viral promoter in transient gene expression assays. Moreover, site-directed mutations in specific Ikaros-binding sites reduce this activity in T-cell lines that express Ikaros endogenously. To determine whether the Ikaros-binding sites are functional in vivo, we inoculated newborn mice with a variant MCF virus containing a mutant Ikaros-binding site. The variant virus replicated in thymocytes less efficiently and induced lymphomas with a delayed onset compared to the wild-type virus. These data are consistent with the hypothesis that the Ikaros-binding sites in the U3 region of MCF 247 are functional and cooperate with other DNA elements for optimal enhancer function in vivo.
Ambisense Sendai Viruses Are Inherently Unstable but Are Useful To Study Viral RNA Synthesis
Authors: Le Mercier Philippe , Garcin Dominique , Hausmann Stéphane , Kolakofsky Daniel
Published: July 2002

Ambisense Sendai virus (SeV) was prepared in order to study the control of viral RNA synthesis. In these studies, we found that the relative ratios of genomes/antigenomes formed during infection are largely determined by the relative strengths of the replication promoters, independent of the presence of a functional mRNA start site. We also found that the ability of the viral polymerase (vRdRP) to respond to an mRNA editing site requires prior (re)initiation at an mRNA start site, similar to the acquisition of vRdRP processivity in the absence of nascent chain coassembly. During these studies, the inherent instability of ambisense SeV upon passage in embryonated chicken eggs was noted and was found to be associated with a point mutation in the ambisense mRNA (ambi-mRNA) start site that severely limited its expression. Since the interferon (IFN)-induced antiviral state is mediated in part via double-stranded RNA (dsRNA), the efficiency of the ambi-mRNA poly(A)/stop site was examined. This site was found to operate in a manner similar to that of other SeV mRNA poly(A)/stop sites, i.e., at ∼95% efficiency. This modest level of vRdRP read-through is apparently tolerable for natural SeV because the potential to form dsRNA during infection remains limited. However, when mRNAs are expressed from ambisense SeV antigenomes, vRdRP read-through of the ambi-mRNA poly(A)/stop site creates a capped transcript that can potentially extend the entire length of the antigenome, since there are no further poly(A)/stop sites here. In support of this hypothesis, loss of ambi-mRNA expression during passage of ambisense SeV stocks in eggs is also characterized by conversion of virus that grows poorly in IFN-sensitive cultures and is relatively IFN sensitive to virus that grows well even in IFN-pretreated cells that restrict vesicular stomatitis virus replication, i.e., the wild-type SeV phenotype. The selection of mutants unable to express ambi-mRNA on passage in chicken eggs is presumably due to increased levels of dsRNA during infection. How natural ambisense viruses may deal with this dilemma is discussed.
Neuroblastoma Cell-Adapted Yellow Fever 17D Virus: Characterization of a Viral Variant Associated with Persistent Infection and Decreased Virus Spread
Authors: Vlaycheva Leonsia A. , Chambers Thomas J.
Published: July 2002

Serial passage of yellow fever 17D virus (YF5.2iv, derived from an infectious molecular clone) on mouse neuroblastoma (NB41A3) cells established a persistent noncytopathic infection associated with a variant virus. This virus (NB15a) was dramatically reduced in plaque formation and exhibited impaired replication kinetics on all cell lines examined compared to the parental virus. Nucleotide sequence analysis of NB15a revealed a substitution in domain III of the envelope (E) protein at residue 360, where an aspartic acid residue was replaced by glycine. Single mutations were also found within the NS2A and NS3 proteins. Engineering of YF5.2iv virus to contain the E
360
substitution yielded a virus (G360 mutant) whose plaque size and growth efficiency in cell culture resembled those of NB15a. Compared with YF5.2iv, both NB15a and G360 were markedly restricted for spread through Vero cell monolayers and mildly restricted in C6/36 cells. On NB41A3 cells, spread of the viruses was similar, but all three were generally inefficient compared with spread in other cell lines. Compared to YF5.2iv virus, NB15a was uniformly impaired in its ability to penetrate different cell lines, but a difference in cell surface binding was detected only on NB41A3 cells, where NB15a appeared less efficient. Despite its small plaque size, impaired growth, and decreased penetration efficiency, NB15a did not differ from YF5.2iv in mouse neurovirulence testing, based on mortality rates and average survival times after intracerebral inoculation of young adult mice. The data indicate that persistence of yellow fever virus in NB41A3 cells is associated with a mutation in the receptor binding domain of the E protein that impairs the virus entry process in cell culture. However, the phenotypic changes which occur in the virus as a result of the persistent infection in vitro do not correlate with attenuation during pathogenesis in the mouse central nervous system.
Disruption of Hematopoiesis and Thymopoiesis in the Early Premalignant Stages of Infection with SL3-3 Murine Leukemia Virus
Authors: Rulli Karen , Lenz Jack , Levy Laura S.
Published: July 2002

A time course analysis of SL3-3 murine leukemia virus (SL3) infection in thymus and bone marrow of NIH/Swiss mice was performed to assess changes that occur during the early stages of progression to lymphoma. Virus was detectable in thymocytes, bone marrow, and spleen as early as 1 to 2 weeks postinoculation (p.i.). In bone marrow, virus infection was detected predominantly in immature myeloid or granulocytic cells. Flow cytometry revealed significant reductions of the Ter-119+and Mac-1+populations, and significant expansions of the Gr-1+and CD34+populations, between 2 and 4 weeks p.i. Analysis of colony-forming potential confirmed these findings. In the thymus, SL3 replication was associated with significant disruption in thymocyte subpopulation distribution between 4 and 7 weeks p.i. A significant thymic regression was observed just prior to the clonal outgrowth of tumor cells. Proviral long terminal repeats (LTRs) with increasing numbers of enhancer repeats were observed to accumulate exclusively in the thymus during the first 8 weeks p.i. Observations were compared to the early stages of infection with a virtually nonpathogenic SL3 mutant, termed SL3ΔMyb5, which was shown by real-time PCR to be replication competent. Comparison of SL3 with SL3ΔMyb5 implicated certain premalignant changes in tumorigenesis, including (i) increased proportions of Gr-1+and CD34+bone marrow progenitors, (ii) a significant increase in the proportion of CD4−CD8−thymocytes, (iii) thymic regression prior to tumor outgrowth, and (iv) accumulation of LTR enhancer variants. A model in which disrupted bone marrow hematopoiesis and thymopoiesis contribute to the development of lymphoma in the SL3-infected animal is discussed.
New Method of Preparation of Brassinosteroids
Authors: Kohout Ladislav
Published: July 2002

Brassinosteroids with lactone B-ring (II and IV) were prepared by oxidation of the corresponding 6-keto derivatives I and III without protection of the hydroxyl groups against the action of the oxidation reagent.
Synthesis of Deoxy, Dideoxy and Didehydrodideoxy Analogs of 9-(4-C-Hydroxymethyl-α-L-pentofuranosyl)adenine
Authors: Hřebabecký Hubert , Holý Antonín
Published: July 2002

Condensation of 1,2-di-O-acetyl-3,5-di-O-benzoyl-4-C-benzoyloxymethyl-L-arabinofuranose with N6-benzoyladenine, catalyzed with tin tetrachloride, afforded nucleoside I, which upon partial deacetylation and subsequent mesylation was converted into 9-(3,5-di-O-benzoyl-4-C-benzoyloxymethyl-2-O-methanesulfonyl-α-L-arabinofuranosyl)adenine (III). 9-(2,5,6-Tri-O-acetyl-4-C-acetoxymethyl-3-O-methanesulfonyl-α-L-arabinofuranosyl)-N6-benzoyladenine (V) was obtained by condensation of 1,2,5-tri-O-acetyl-4-C-acetoxymethyl-3-O-methanesulfonyl-L-arabinose with N6-benzoyladenine. Reaction of mesyl derivatives III and V with methanolic sodium methoxide afforded 2',3'-anhydro nucleosides VIa and VIIa, which were acetylated to give 9-(5-O-acetyl-4-C-acetoxymethyl-2,3-anhydro-α-L-ribofuranosyl)adenine (VIb) and 9-(5-O-acetyl-4-C-acetoxymethyl-2,3-anhydro-α-L-lyxofuranosyl)adenine (VIIb). Epoxy derivative VIb was cleaved with bromotrimethylsilane to 9-(5-O-acetyl-4-C-acetoxymethyl-2-bromo-2-deoxy-α-L-arabinofuranosyl)adenine (VIIIa); the same reaction with epoxy derivative VIIb afforded a mixture of 9-(5-O-acetyl-4-C-acetoxymethyl- 2-bromo-2-deoxy-α-L-xylofuranosyl)adenine (IXa) and 9-(5-O-acetyl-4-C-acetoxymethyl-3-bromo- 3-deoxy-α-L-arabinofuranosyl)adenine (Xa). Their dehalogenation with tributylstannane and subsequent deacetylation led to 9-(2-deoxy-4-C-hydroxymethyl-α-L-erythro-pentofuranosyl)adenine (VIIIc), 9-(2-deoxy-4-C-hydroxymethyl- α-L-threo-pentofuranosyl)adenine (IXc) and 9-(3-deoxy-4-C-hydroxymethyl-α-L-threo-pentofuranosyl)adenine (Xc). 9-(2,5-Di-O-acetyl-4-C-acetoxymethyl-2-bromo-2-deoxy-α-L-arabinofuranosyl)adenine (VIIId), prepared by acetylation of VIIIa, on reductive elimination with Cu/Zn couple and subsequent deacetylation afforded 9-(2,3-dideoxy-4-C-hydroxymethyl-α-L-glycero-pent-2-enofuranosyl)adenine (XIb). 9-(2,3-Dideoxy-4-C-hydroxymethyl-α-L-glycero-pentofuranosyl)adenine (XIIb) was obtained either by catalytic hydrogenation of bromo derivative VIIId, followed by deacetylation, or by catalytic hydrogenation of didehydro derivative XIb. The nucleosides synthesized were tested for antiviral activity.
Preparation of (17E)-3β-Hydroxyandrost-5-en-17-one (O-Carboxymethyl)oxime Derivatives with Short Peptide Chain
Authors: Pouzar Vladimír , Černý Ivan
Published: July 2002

17-(O-Carboxymethyl)oxime (CMO) derivatives of 3β-acetoxy-, 3β-methoxymethoxy-, 3β-hydroxyandrost-5-en-17-one, and of -androst-4-ene-3,17-dione (IV - VI, XXIX) were prepared. Methods for the attachment of amino acids to the CMO group and for the peptide chain elongation were tested. The mixed anhydride method was used for linking the first amino acid unit (Gly, βAla, or Gly-Gly dipeptide), further units were added by the N-hydroxysuccinimide method. Compounds with one to four amino acids were prepared. This concept is suitable for the preparation of haptens with variable bridge length for the binding studies.
Microplate Assay for Hyaluronan Useful in Clinical Practice
Authors: Stančíková Mária , Kéry Vladimír , Orviský Eduard , Cibírová Lívia
Published: July 2002

A microplate assay for determination of hyaluronan (HA) has been developed. In principle, HA in the sample and biotinylated HA compete for binding to hyaluronan binding protein (HABP) coated to the plate. The amount of the microplate-bound biotinylated HA is quantified with avidin peroxidase complex. This assay is useful for 10 - 500 μg/l with < 2% relative error with 50% inhibition at 80 μg/l. The assay is simple, sensitive and particularly suitable for the determination of HA in serum and other biological fluids. The assay correlated with only commercially available radioisotope kit, Pharmacia HA-50, with correlation coefficient 0.92 at p < 0.001. Serum HA levels of patients with osteoarthritis, rheumatoid arthritis and healthy donors determined by this assay correspond to those previously determined by different methods. These results suggest that this assay is suitable for clinical use.
One-Step Synthesis of 2-Amino-3-furancarboxylic Acid Derivatives from 2-Furancarbaldehyde
Authors: Šafář Peter , Čepec Pavel , Považanec František , Koreňová Anna , Prónayová Naďa
Published: July 2002

Reaction of derivatives of 2-furancarbaldehyde or 2-acetylfuran with malononitrile, methyl cyanoacetate or cyanoacetamide in the presence of secondary amines afforded derivatives of 2-amino-3-furancarboxylic acid in 45 - 88% yields.
1,2,4,6-Substituted Pyridinium Derivatives-Synthesis and Properties
Authors: Urban Jiří , Volke Jiří
Published: July 2002

In relation to the investigation of their electrochemical properties we prepared 24 pyridinium derivatives (I, IV, V, VI, VII) with alkyl and aryl substituents in positions 1, 2, 4 and 6, and 19 derivatives with two pyridinium nuclei (II, III, VIII, IX, X) connected by an aliphatic or an aromatic chain. All the compounds were prepared from the corresponding pyrylium derivatives by reacting them with primary amines. The pyrylium derivatives were synthesized by aldolization and acylation followed by cyclization leading to a heteroaromatic pyrylium system. In nucleophilic reactions with amines, the pyrylium derivatives yield pyridinium derivatives. The reaction was performed by adding the corresponding amine to an ethanolic suspension of the starting pyrylium salt.
Stereoselectivity of Michael Addition to 3-Phenyl-1-(η6-o-tolyltricarbonylchromium)propenone
Authors: Šebo Ľubomír , Šraga Ján , Rihs Grety , Toma Štefan
Published: July 2002

The stereoselectivity of the Michael addition of several C-nucleophiles to 3-phenyl-1-(η6-o-tolyltricarbonylchromium)propenone has been studied. The ratio of diastereoisomers formed varied from 65 : 35 (malonodinitrile), 72 : 28 (methyl cyanoacetate) and 78 : 22 (nitromethane) and to 90 : 10 (dimethyl malonate). The ratio of diastereoisomers 63 : 37 was found even when addition of dimethyl malonate was carried out at 75 °C in methanol using piperidine as the catalyst. The (S,R) or (R,S) configuration of the main pair of isomers was proved by the X-ray analysis of the dimethyl malonate adduct.
Using in situ IR Spectroscopy to Understand Selective Alkylation of Aromatics over Zeolites
Authors: Eder-Mirth Gabriele
Published: July 2002

In situ IR spectroscopy in combination with kinetic measurements was used to clarify the mechanism of the methylation of toluene over zeolitic catalysts. Over basic zeolites (i.e. Rb-X), the main surface species under reaction conditions is toluene strongly bound with the aromatic ring to the cation and with the methyl group to the lattice oxygens of the zeolite. The activated carbon atom of the methyl group reacts with the positively activated C-atom of methanol (formaldehyde) to yield styrene as the main product. Over acidic zeolites (i.e. HZSM-5), methanol is preferentially adsorbed over toluene, the rate of methylation is directly proportional to the surface concentration of the activated methanol species (methoxonium ions at low and methoxy groups at high reaction temperatures). The aromatic ring of toluene is attacked by the reactive alcohol methyl group, giving the xylene isomers as reaction products.
On the Structure of Lead(II) Complexes in Aqueous Solutions. I. Trinuclear Clusters
Authors: Breza Martin , Manová Alena
Published: July 2002

Using semiempirical MNDO method of quantum chemistry the optimal geometries and corresponding electronic structures of [Pb3(OH)n]6-n model systems as well as of their hydrated [Pb3(OH)n(H2O)8-n]6-n analogues (n = 4, 5) are investigated. The most stable trinuclear lead(II) complexes present in aqueous solutions correspond to cyclo-(μ3-OH)(μ2-OH)3Pb32+, Pb(μ-OH)2Pb(μ-OH)2Pb2+, cyclo-(μ3-OH)2(μ2-OH)3Pb3+, Pb(OH)(μ-OH)2Pb(μ-OH)Pb(OH)+ and Pb(OH)(μ-OH)2Pb(μ-OH)2Pb+ systems. The key role of OH bridges (by vanishing direct Pb-Pb bonds) on the stability of individual isomers is discussed.
Theoretical Study of the Conformational Behaviour of Poly(oxypropylene) Chains
Authors: Stepto Robert F. T. , Taylor David J. R.
Published: July 2002

The present paper describes the calculation of the conformational behaviour of isotactic, syndiotactic and atactic poly(oxypropylene) (POP) chains, using rotational-isomeric-state (R-I-S) theory in the form of BIOSYM's Polymer computer-software. It is found that the conformational behaviour of the POP chain, in the region of an R methyl-substituted skeletal carbon atom, differs from that in the region of the corresponding S carbon, due to the differences in the steric interactions between the methyl side-group and the methylene group two skeletal atoms away in the POP backbone. Thus, the isotactic and syndiotactic chains have markedly different values of Cn, the characteristic ratio. Accordingly, Cn was calculated for isotactic, syndiotactic and atactic POP chains of various numbers of skeletal bonds, n. The results showed that the isotactic chains occupy more extended conformations (with the highest values of Cn), compared with the highly-coiled syndiotactic chains (lower values of Cn). For the atactic POP chains, Cn was found to lie between the two extremes of the isotactic and syndiotactic forms. The value of C* for the infinite atactic POP chain was found to be consistent with values determined from experimental measurements.
Assignment of Nitrogen Stereochemistry of Agroclavine and Elymoclavine 6-N-Oxides
Authors: Křen Vladimír , Němeček Jan , Přikrylová Věra
Published: July 2002

Both possible 6-N-oxides of agroclavine (I) and elymoclavine (II) were prepared by hydrogen peroxide oxidation. Their 1H and 13C NMR spectra were assigned and the conformation of the D ring (half-chair) was determined. Absolute configuration at 6-N was established by NMR and molecular modelling.
Gould-Jacobs Reaction of 5- and 6-Amino-2-substituted Benzoxazoles. II. Reaction with 3-Ethoxymethylene-2,4-pentanedione and Ethyl 2-Ethoxymethylene-3-oxobutanoate
Authors: Heleyová Katarína , Ilavský Dušan , Bobošík Vladimír , Prónayová Naďa
Published: July 2002

Nucleophilic reaction of 5-amino- and 6-amino-2-substituted benzoxazoles 1 and 2 with 3-ethoxymethylene-2,4-pentanedione (3) gave compounds 5 and 6. Compounds 1 and 2 reacted with ethyl ethoxymethylene-3-oxobutanoate (4) under formation of compounds 7and 8. The substitution products 7 and 8 underwent thermal cyclization at high temperature (boiling mixture of diphenyl ether and biphenyl) to give angularly and linearly annelated derivatives of 5-acetyl-4-oxo-oxazolo[4,5-f]quinoline 9 and 7-acetyl-8-oxo-oxazolo[5,4-g]quinoline 10 (from 7), and derivatives of 8-acetyl-9-oxo-oxazolo[5,4-f]quinoline 11 and 6-acetyl-5-oxo-oxazolo[4,5-g]quinoline 12 (from 8). The structure of the substitution products is discussed on the basis of their spectral characteristics (1H and 13C NMR, IR, UV, MS).
Reaction of O-Alkyl-N-substituted Iminothiocarbonates with Bromoacetyl Bromide. A General Method for the Synthesis of 3-Substituted 1,3-Thiazolidine-2,4-diones
Authors: Imrich Ján , Bernát Juraj , Kristian Pavol , Bušová Tatiana , Hočová Slávka
Published: July 2002

Reaction of sodium salts of O-methyl-N-substituted iminothiocarbonates with bromoacetyl bromide represents a new general method for preparation of 3-substituted 1,3-thiazolidine-2,4-diones in good yields and high purity. The structure of the synthesized products has been confirmed by 1H NMR, 13C NMR, IR and mass spectroscopy as well as by independent synthesis.