bluuebunny/crossref_metadata_2025 huggingface dataset:
Jembrana Disease Virus Tat Can Regulate Human Immunodeficiency Virus (HIV) Long Terminal Repeat-Directed Gene Expression and Can Substitute for HIV Tat in Viral Replication
Authors: Chen Hexin , He Jun , Fong Steven , Wilcox Graham , Wood Charles
Published: July 2002

Jembrana disease virus (JDV) is a bovine lentivirus genetically similar to bovine immunodeficiency virus; it causes an acute and sometimes fatal disease in infected animals. This virus carries a very potent Tat that can strongly activate not only its own long terminal repeat (LTR) but also the human immunodeficiency virus (HIV) LTR. In contrast, HIV Tat cannot reciprocally activate the JDV LTR (H. Chen, G. E. Wilcox, G. Kertayadnya, and C. Wood, J. Virol. 73:658–666, 1999). This indicates that in transactivation JDV Tat may utilize a mechanism similar to but not the same as that of the HIV Tat. To further study the similarity of JDV and HIVtatin transactivation, we first tested the responses of a series of HIV LTR mutants to the JDV Tat. Cross-transactivation of HIV LTR by JDV Tat was impaired by mutations that disrupted the HIV type 1 transactivation response element (TAR) RNA stem-loop structure. Our results demonstrated that JDV Tat, like HIV Tat, transactivated the HIV LTR at least partially in a TAR-dependent manner. However, the sequence in the loop region of TAR was not as critical for the function of JDV Tat as it was for HIV Tat. The competitive inhibition of Tat-induced transactivation by the truncated JDV or HIV Tat, which consisted only of the activation domain, suggested that similar cellular factors were involved in both JDV and HIV Tat-induced transactivation. Based on the one-round transfection assay with HIVtatmutant proviruses, the cotransfected JDVtatplasmid can functionally complement the HIVtatdefect. To further characterize the effect of JDV Tat on HIV, a stable chimeric HIV carrying the JDVtatgene was generated. This chimeric HIV replicated in a T-cell line, C8166, and in peripheral blood mononuclear cells, which suggested that JDV Tat can functionally substitute for HIV Tat. Further characterization of this chimeric virus will help to elucidate how JDV Tat functions and to explain the differences between HIV and JDV Tat transactivation.
In Vivo T-Lymphocyte Activation and Transient Reduction of Viral Replication in Macaques Infected with Simian Immunodeficiency Virus
Authors: Chen Zheng W. , Shen Yun , Zhou Dejiang , Simon Meredith , Kou ZhongChen , Lee-Parritz David , Shen Ling , Sehgal Prabhat , Letvin Norman L.
Published: July 2002

While it is well established that cellular activation can increase human immunodeficiency virus (HIV) replication in T lymphocytes, it is also clear that both activated CD8+and CD4+T lymphocytes mediate anti-HIV activity. To assess the relative importance of these contrary effects on HIV replication in vivo, we evaluated the consequences ofMycobacterium bovisBCG and staphylococcal enterotoxin B (SEB) inoculation in vivo in rhesus monkeys chronically infected with simian immunodeficiency virus of macaques (SIVmac). BCG inoculation induced as much as a 2.5-log reduction of plasma and intracellular SIV RNA in SIVmac-infected monkeys. This down-regulation of virus replication persisted as long as 4 weeks after BCG inoculation. Similarly, SEB injection resulted in up to a 3-log decrease in plasma and intracellular SIV RNA in SIVmac-infected macaques. Interestingly, the short-term reduction of viremia in these monkeys correlated with the peak in vivo production of SEB- and BCG-induced cytokine responses. However, no long-term clinical benefit was observed in the SIVmac-infected macaques. These studies provide in vivo evidence that potent T-cell stimulation driven by antigens other than the virus itself can, under some circumstances, mediate short-term reduction of viremia in AIDS virus-infected individuals.
The Reovirus S4 Gene 3′ Nontranslated Region Contains a Translational Operator Sequence
Authors: Mochow-Grundy Michelle , Dermody Terence S.
Published: July 2002

Reovirus mRNAs are efficiently translated within host cells despite the absence of 3′ polyadenylated tails. The 3′ nontranslated regions (3′NTRs) of reovirus mRNAs contain sequences that exhibit a high degree of gene-segment-specific conservation. To determine whether the 3′NTRs of reovirus mRNAs serve to facilitate efficient translation of viral transcripts, we used T7 RNA polymerase to express constructs engineered with full-length S4 gene cDNA or truncation mutants lacking sequences in the 3′NTR. Full-length and truncated s4 mRNAs were translated using rabbit reticulocyte lysates, and translation product ς3 was quantitated by phosphorimager analysis. In comparison to full-length s4 mRNA, translation of the s4 mRNA lacking the 3′NTR resulted in a 20 to 50% decrease in ς3 produced. Addition to translation reactions of an RNA oligonucleotide corresponding to the S4 3′NTR significantly enhanced translation of full-length s4 mRNA but had no effect on s4 mRNA lacking 3′NTR sequences. Translation of s4 mRNAs with smaller deletions within the 3′NTR identified a discrete region capable of translational enhancement and a second region capable of translational repression. Differences in translational efficiency of full-length and deletion-mutant mRNAs were independent of RNA stability. Protein complexes in reticulocyte lysates that specifically interact with the S4 3′NTR were identified by RNA mobility shift assays. RNA oligonucleotides lacking either enhancer or repressor sequences did not efficiently compete the binding of these complexes to full-length 3′NTR. These results indicate that the reovirus S4 gene 3′NTR contains a translational operator sequence that serves to regulate translational efficiency of the s4 mRNA. Moreover, these findings suggest that cellular proteins interact with reovirus 3′NTR sequences to regulate translation of the nonpolyadenylated reovirus mRNAs.
Gag-Pol Supplied in
trans
Is Efficiently Packaged and Supports Viral Function in Human Immunodeficiency Virus Type 1
Authors: Hill M. K. , Hooker C. W. , Harrich D. , Crowe S. M. , Mak J.
Published: July 2002

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.
Construction, Safety, and Immunogenicity in Nonhuman Primates of a Chimeric Yellow Fever-Dengue Virus Tetravalent Vaccine
Authors: Guirakhoo F. , Arroyo J. , Pugachev K. V. , Miller C. , Zhang Z.-X. , Weltzin R. , Georgakopoulos K. , Catalan J. , Ocran S. , Soike K. , Ratterree M. , Monath T. P.
Published: July 2002

We previously reported construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys (F. Guirakhoo et al., J. Virol. 74:5477–5485, 2000). In this paper, we describe construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ∼7.5 log10PFU/ml in Vero cells, were not neurovirulent in 3- to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose. When monkeys were boosted with a tetravalent YF/DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates.
Serum and Mucosal Immune Responses to an Inactivated Influenza Virus Vaccine Induced by Epidermal Powder Immunization
Authors: Chen Dexiang , Periwal Sangeeta B. , Larrivee Katherine , Zuleger Cindy , Erickson Cherie A. , Endres Ryan L. , Payne Lendon G.
Published: July 2002

Both circulating and mucosal antibodies are considered important for protection against infection by influenza virus in humans and animals. However, current inactivated vaccines administered by intramuscular injection using a syringe and needle elicit primarily circulating antibodies. In this study, we report that epidermal powder immunization (EPI) via a unique powder delivery system elicits both serum and mucosal antibodies to an inactivated influenza virus vaccine. Serum antibody responses to influenza vaccine following EPI were enhanced by codelivery of cholera toxin (CT), a synthetic oligodeoxynucleotide containing immunostimulatory CpG motifs (CpG DNA), or the combination of these two adjuvants. In addition, secretory immunoglobulin A (sIgA) antibodies were detected in the saliva and mucosal lavages of the small intestine, trachea, and vaginal tract, although the titers were much lower than the IgG titers. The local origin of the sIgA antibodies was further shown by measuring antibodies released from cultured tracheal and small intestinal fragments and by detecting antigen-specific IgA-secreting cells in the lamina propria using ELISPOT assays. EPI with a single dose of influenza vaccine containing CT or CT and CpG DNA conferred complete protection against lethal challenges with an influenza virus isolated 30 years ago, whereas a prime and boost immunizations were required for protection in the absence of an adjuvant. The ability to elicit augmented circulating antibody and mucosal antibody responses makes EPI a promising alternative to needle injection for administering vaccines against influenza and other diseases.
In Vitro Activation of Feline Immunodeficiency Virus in Ramified Microglial Cells from Asymptomatically Infected Cats
Authors: Hein Andreas , Martin Jean-Pierre , Dörries Rüdiger
Published: July 2002

Intravenous infection of cats with feline immunodeficiency virus was used as a model system to study activation of virus replication in brain-resident microglial cells in vitro. Virus release by ramified microglial cells isolated from subclinically infected animals was detectable in cell-free tissue culture supernatant only by reverse transcription and nested PCR ofgag-specific RNA sequences and not by virion-associated reverse transcriptase activity. In contrast, cocultivation of in vivo-infected microglial cells with mitogen-activated peripheral blood mononuclear cells (PBMC) regularly allows detection of high virus yields in cell-free tissue culture fluid. Besides uptake and multiplication of microglia-derived virus in PBMC, release of virus from microglia is stimulated by cell contact with PBMC. The data suggest that T lymphocytes patrolling the central nervous system could reactivate the semilatent state of lentiviruses in microglial cells in the course of clinically silent central nervous system infection.
An Epstein-Barr Virus Isolated from a Lymphoblastoid Cell Line Has a 16-Kilobase-Pair Deletion Which Includes gp350 and the Epstein-Barr Virus Nuclear Antigen 3A
Authors: Lee Wonkeun , Hwang Yoon-Ha , Lee Suk-Kyeong , Subramanian Chitra , Robertson Erle S.
Published: July 2002

Epstein-Barr virus
(EBV) is associated with human cancers, including nasopharyngeal carcinoma, Burkitt's lymphoma, gastric carcinoma and, somewhat controversially, breast carcinoma. EBV infects and efficiently transforms human primary B lymphocytes in vitro. A number of EBV-encoded genes are critical for EBV-mediated transformation of human B lymphocytes. In this study we show that an EBV-infected lymphoblastoid cell line obtained from the spontaneous outgrowth of B cells from a leukemia patient contains a deletion, which involves a region of approximately 16 kbp. This deletion encodes major EBV genes involved in both infection and transformation of human primary B lymphocytes and includes the glycoprotein gp350, the entire open reading frame of EBNA3A, and the amino-terminal region of EBNA3B. A fusion protein created by this deletion, which lies between the BMRF1 early antigen and the EBNA3B latent antigen, is truncated immediately downstream of the junction 21 amino acids into the region of the EBNA3B sequence, which is out of frame with respect to the EBNA3B protein sequence, and indicates that EBNA3B is not expressed. The fusion is from EBV coordinate 80299 within the BMRF1 sequence to coordinate 90998 in the EBNA3B sequence. Additionally, we have shown that there is no detectable induction in viral replication observed when SNU-265 is treated with phorbol esters, and no transformants were detected when supernatant is used to infect primary B lymphocytes after 8 weeks in culture. Therefore, we have identified an EBV genome with a major deletion in critical genes involved in mediating EBV infection and the transformation of human primary B lymphocytes that is incompetent for replication of this naturally occurring EBV isolate.
Caspase Activation by Adenovirus E4orf4 Protein Is Cell Line Specific and Is Mediated by the Death Receptor Pathway
Authors: Livne Adi , Shtrichman Ronit , Kleinberger Tamar
Published: July 2002

Adenovirus E4orf4 protein has been shown to induce transformed cell-specific, protein phosphatase 2A-dependent, and p53-independent apoptosis. It has been further reported that the E4orf4 apoptotic pathway is caspase-independent in CHO cells. Here, we show that E4orf4 induces caspase activation in the human cell lines H1299 and 293T. Caspase activation is required for apoptosis in 293T cells, but not in H1299 cells. Dominant negative mutants of caspase-8 and the death receptor adapter protein FADD/MORT1 inhibit E4orf4-induced apoptosis in 293T cells, suggesting that E4orf4 activates the death receptor pathway. Cytochromecis released into the cytosol in E4orf4-expressing cells, but caspase-9 is not required for induction of apoptosis. Furthermore, E4orf4 induces accumulation of reactive oxygen species (ROS) in a caspase-8- and FADD/MORT1-dependent manner, and inhibition of ROS generation by 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) inhibits E4orf4-induced apoptosis. Thus, our results demonstrate that E4orf4 engages the death receptor pathway to generate at least part of the molecular events required for E4orf4-induced apoptosis.
Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus A20 Gene: Temperature-Sensitive Mutants Have a DNA-Minus Phenotype and Are Defective in the Production of Processive DNA Polymerase Activity
Authors: Punjabi Almira , Boyle Kathleen , DeMasi Joseph , Grubisha Olivera , Unger Beth , Khanna Marilyn , Traktman Paula
Published: July 2002

Although the vaccinia virus DNA polymerase is inherently distributive, a highly processive form of the enzyme exists within the cytoplasm of infected cells (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168–175, 1997). In the accompanying report we outline the purification of the 49-kDa A20 protein as a stoichiometric component of the processive polymerase complex (N. Klemperer, W. McDonald, K. Boyle, B. Unger, and P. Traktman, J. Virol. 75:12298–12307, 2001). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these,
ts
A20-6 and
ts
A20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the
ts
A20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA
−
phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.
NSP4 Enterotoxin of Rotavirus Induces Paracellular Leakage in Polarized Epithelial Cells
Authors: Tafazoli Farideh , Zeng Carl Q. , Estes Mary K. , Magnusson Karl-Erik , Svensson Lennart
Published: July 2002

The nonstructural NSP4 protein of rotavirus has been described as the first viral enterotoxin. In this study we have examined the effect of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable filters. Apical but not basolateral administration of NSP4 was found to cause a reduction in the transepithelial electrical resistance, redistribution of filamentous actin, and an increase in paracellular passage of fluorescein isothiocyanate-dextran. Significant effects on transepithelial electrical resistance were noted after a 20- to 30-h incubation with 1 nmol of NSP4. Most surprisingly, the epithelium recovered its original integrity and electrical resistance upon removal of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented not only development of a permeability barrier but also lateral targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1) protein. Taken together, these data indicate new and specific effects of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on polarized epithelia.
L1 Interaction Domains of Papillomavirus L2 Necessary for Viral Genome Encapsidation
Authors: Okun Martin M. , Day Patricia M. , Greenstone Heather L. , Booy Frank P. , Lowy Douglas R. , Schiller John T. , Roden Richard B. S.
Published: July 2002

BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.
Calcineurin-Dependent Mitochondrial Disturbances in Calcium-Induced Apoptosis of Human Immunodeficiency Virus gp160-Expressing CD4+Cells
Authors: Sasaki Masafumi , Miyazaki Kozo , Koga Yasuhiro , Kimura Genki , Nomoto Kikuo , Yoshida Hiroki
Published: July 2002

In CD4+UE160 cells with inducible expression of gp160, mechanisms of apoptosis induced by human immunodeficiency virus (HIV) Env protein were analyzed. Induction of gp160 caused intracellular calcium increase followed by the release of cytochromecfrom mitochondria, which was inhibited by calcineurin inhibitors. Association of BAD with Bcl-xL was observed, and a portion of BAD was dephosphorylated after induction of gp160. These data suggested that calcineurin plays a role in the HIV Env-induced apoptosis in a mitochondrion-dependent way.
Substitution in the Murine Nectin1 Receptor of a Single Conserved Amino Acid at a Position Distal from the Herpes Simplex Virus gD Binding Site Confers High-Affinity Binding to gD
Authors: Menotti Laura , Casadio Rita , Bertucci Carlo , Lopez Marc , Campadelli-Fiume Gabriella
Published: July 2002

By analogy with its human nectin1 counterpart, murine nectin1 serves as a cellular receptor for the entry of herpes simplex virus (HSV) into murine cells. HSV entry mediated by either receptor is dependent on the viral glycoprotein D (gD). Whereas human nectin1 binds gD at high affinity and in a saturable manner, murine nectin1 binds gD in a barely detectable fashion, depending on the sensitivity of the assay. The immunoglobulin type V domain of murine nectin differs from its human counterpart in 11 amino acids. To identify the key residues responsible for the high-affinity binding of gD to human nectin1, we replaced each of the 11 divergent amino acids with the human counterparts singly or in groups in an incremental manner. Replacement in murine nectin1 of six amino acids that lie within the gD binding region of human nectin1 (previously mapped to residues 64 to 94, likely the CC′C″ surface) increased the gD binding activity to a limited extent. In contrast, the single P138L substitution, which lies distal from the gD binding site, markedly increased gD binding. This substitution, when coupled with downstream substitutions, exerted the greatest effect. Three-dimensional modeling of the nectin1 V domain suggested that P138 in murine nectin1 might decrease the stability of the V domain by reducing the size of β-strand G. The results support the notion that the overall structure of V nectin1 plays a pivotal role in its ability to bind HSV gD.
Vector Infection Determinants of Venezuelan Equine Encephalitis Virus Reside within the E2 Envelope Glycoprotein
Authors: Brault Aaron C. , Powers Ann M. , Weaver Scott C.
Published: July 2002

Epizootic subtype IAB and IC Venezuelan equine encephalitis viruses (VEEV) readily infect the epizootic mosquito vector
Aedes taeniorhynchus
. The inability of enzootic subtype IE viruses to infect this mosquito species provides a model system for the identification of natural viral determinants of vector infectivity. To map mosquito infection determinants, reciprocal chimeric viruses generated from epizootic subtype IAB and enzootic IE VEEV were tested for mosquito infectivity. Chimeras containing the IAB epizootic structural gene region and, more specifically, the IAB PE2 envelope glycoprotein E2 precursor gene demonstrated an efficient infection phenotype. Introduction of the PE2 gene from an enzootic subtype ID virus into an epizootic IAB or IC genetic backbone resulted in lower infection rates than those of the epizootic parent. The finding that the E2 envelope glycoprotein, the site of epitopes that define the enzootic and epizootic subtypes, also encodes mosquito infection determinants suggests that selection for efficient infection of epizootic mosquito vectors may mediate VEE emergence.
Parvovirus Initiator Protein NS1 and RPA Coordinate Replication Fork Progression in a Reconstituted DNA Replication System
Authors: Christensen Jesper , Tattersall Peter
Published: July 2002

We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5′ end and displacing the other strand while progressing in a 3′-to-5′ direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5′ end of the nick in the process and providing a 3′ OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO
4
oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of
Escherichia coli
single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase δ, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with
E. coli
SSB or other proteins included as controls.
Tumor Control in a Model of Bone Marrow Transplantation and Acute Liver-Infiltrating B-Cell Lymphoma: an Unpredicted Novel Function of Cytomegalovirus
Authors: Erlach Katja C. , Podlech Jürgen , Rojan Aysel , Reddehase Matthias J.
Published: July 2002

Tumor relapse and cytomegalovirus (CMV) infection are major concerns in the therapy of hematopoietic malignancies by bone marrow transplantation (BMT). Little attention so far has been given to a possible pathogenetic interplay between CMV and lymphomas. CMV inhibits stem cell engraftment and hematopoietic reconstitution. Thus, by causing maintenance of bone marrow aplasia and immunodeficiency, CMV could promote tumor relapse. Alternatively, CMV could aid tumor remission. One might think of cytopathogenic infection of tumor cells, induction of apoptosis or inhibitory cytokines, interference with tumor cell extravasation or tumor vascularization, or bystander stimulation of an antitumoral immune response. To approach these questions, the established model of experimental BMT and murine CMV infection was extended by the introduction of liver-infiltrating, highly tumorigenic variant clone E12E of BALB/c-derived B-cell lymphoma A20. We document a remarkable retardation of lymphoma progression. First-guess explanations were ruled out: (i) lymphoma cells were not infected; (ii) lymphoma cells located next to infected hepatocytes did not express executioner caspase 3 but were viable and proliferated; (iii) an inhibitory effect of virus on the formation of tumor nodules in the liver became apparent by day 7 after BMT, long before the reconstitution of immune cells; and (iv) recombinant tumor necrosis factor alpha (TNF-α) did not substitute for virus; accordingly anti-TNF-α did not prevent the inhibition. Notably, while the antitumoral effect required replicative virus, prevention of cytopathogenic infection of the liver by antiviral CD8 T cells did not abolish lymphoma control. These findings are paradigmatic for a novel virus-associated antitumoral mechanism distinct from oncolysis.
Induction of Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD8+and CD4+T-Cell Reactivity by Dendritic Cells Loaded with HIV-1 X4-Infected Apoptotic Cells
Authors: Zhao Xiao-Qing , Huang Xiao-Li , Gupta Phalguni , Borowski Luann , Fan Zheng , Watkins Simon C. , Thomas Elaine K. , Rinaldo Charles R.
Published: July 2002

T-cell responses to X4 strains of human immunodeficiency virus type 1 (HIV-1) are considered important in controlling progression of HIV-1 infection. We investigated the ability of dendritic cells (DC) and various forms of HIV-1 X4 antigen to induce anti-HIV-1 T-cell responses in autologous peripheral blood mononuclear cells from HIV-1-infected persons. Immature DC loaded with HIV-1 IIIB-infected, autologous, apoptotic CD8−cells and matured with CD40 ligand induced gamma interferon production in autologous CD8+and CD4+T cells. In contrast, mature DC loaded with HIV-1 IIIB-infected, necrotic cells or directly infected with cell-free HIV-1 IIIB were poorly immunogenic. Thus, HIV-1-infected cells undergoing apoptosis serve as a rich source of X4 antigen for CD8+and CD4+T cells by DC. This may be an important mechanism of HIV-1 immunogenicity and provides a strategy for immunotherapy of HIV-1-infected patients on combination antiretroviral therapy.
Regions and Activities of Simian Virus 40 T Antigen That Cooperate with an Activated
ras
Oncogene in Transforming Primary Rat Embryo Fibroblasts
Authors: Beachy Tina M. , Cole Sara L. , Cavender Jane F. , Tevethia Mary J.
Published: July 2002

Prolonged expression of a
ras
oncogene in primary cells accelerates the natural process of senescence. This
ras
-induced permanent growth arrest is bypassed in cells expressing the simian virus 40 large T antigen. Previously we showed that two regions of T antigen, a region consisting of the N-terminal 147 amino acids and a region consisting of amino acids 251 to 708 (T251-708), independently overcome
ras
-induced senescence. Coexpression of either T-antigen fragment and Ras results in the appearance of dense foci of transformed cells. Using a series of mutants that produce shorter T-antigen fragments, we show that the C-terminal limit of the N-terminal T-antigen fragment that cooperates with Ras lies between amino acids 83 and 121. The N-terminal limit of the C-terminal T-antigen fragment lies between amino acids 252 and 271. In addition, we present evidence that cooperation between the N-terminal fragment and Ras depends upon an intact T-antigen J domain and the ability of the T antigen to bind and inactivate the growth-suppressive effect of the tumor suppressor Rb. Introduction of specific amino acid substitutions surrounding residue 400 into T251-708 prevented the fragment from cooperating with Ras. T251-708 proteins with these same substitutions inhibited the transcriptional transactivating potential of p53 as effectively as did the wild-type protein. Thus, at least one activity contained within T251-708, other than inactivating p53 as a transcriptional transactivator, is likely to be required to bypass Ras-induced senescence.
The ITAM in Nef Influences Acute Pathogenesis of AIDS-Inducing Simian Immunodeficiency Viruses SIVsm and SIVagm without Altering Kinetics or Extent of Viremia
Authors: Dehghani Houman , Brown Charles R. , Plishka Ronald , Buckler-White Alicia , Hirsch Vanessa M.
Published: July 2002

The role of the immunoreceptor tyrosine-based activation motif (ITAM) that is unique to the Nef protein of the acutely pathogenic simian immunodeficiency virus SIVsmPBj was studied in the context of two AIDS-inducing simian immunodeficiency virus molecular clones. NefY+variants of SIVagm9063-2 and SIVsmE543-3 replicated in and induced proliferation of unstimulated pig-tailed macaque PBMC. The pathogenesis of the NefY+and NefY−clones of SIVagm9063-2, SIVsmE543-3, and PBj6.6 were evaluated by intravenous inoculation of pig-tailed macaques (Macaca nemestrina). Introduction of the ITAM did not increase plasma viral RNA levels nor alter the kinetics of viremia compared with the NefY−versions of each clone. Clinical symptoms were not observed in animals inoculated with the NefY−variants. In contrast, characteristic PBj symptoms were observed in animals inoculated with any of the three NefY+clones. Blunting and fusion of intestinal villi and multifocal infiltration of mononuclear cells were observed in the gastrointestinal tracts of macaques inoculated with the NefY+versions. Lesions were associated with active viral replication, as demonstrated by simian immunodeficiency virus-specific in situ hybridization. However, only the macaque inoculated with wild-type NefY+SIVsmPBj developed fatal disease; lesions were more widespread and severe in this animal. A switch to macrophages as a viral reservoir and the presence of interleukin-6 in plasma was unique to the macaque infected with PBj6.6. Overall, these data suggest that the ITAM in SIV Nef alters the pathogenesis of simian immunodeficiency virus regardless of the viral background. The change in pathogenesis occurs without enhancement of viral replication. However, NefY+variants of SIVagm and SIVsm did not fully recapitulate the virulence of SIVsmPBj, implicating additional viral factors in this unique virus pathogenesis.
Examination of the Conditions of Extraction of Toxic Organic Bases and of Methods of Their Determination. III. Extraction of Ion-Associates of Dibenz[b,f]-1,4-oxazepin
Authors: Souček Jan , Belický Ladislav , Havel Josef
Published: July 2002

The protonation and distribution constants of dibenz[b, f]-1,4-oxazepin (CR) were measured. This substance forms stable ion-associates with Acid Red 88, extractable into chloroform. Reextraction with an aqueous phase containing basic fuchsine or safranine T brings about exchange of the CR cation for the basic dye cation. The associates so formed exhibit higher conditional extraction constants and higher molar absorptivities than the initial associates.
Synthesis of 2-Aryl-3-hydroxyquinolin-4(1H)-ones
Authors: Hradil Pavel , Jirman Josef
Published: July 2002

Eight substituted phenacyl anthranilates have been prepared by reaction of sodium anthranilate with substituted phenacyl halides in dimethylformamide in the yields from 31 to 93%. The phenacyl esters were cyclized in polyphosphoric acid or by mere heating to give the respective substituted 2-aryl-3-hydroxyquinolin-4(1H)-ones in the yields from 77 to 98%. All the compounds prepared have been characterized by their 1H and 13C NMR spectra.
Partial Modifications of Hydroxy Groups in 5-Methyl-5β-estr-9-ene Derivatives
Authors: Kasal Alexander , Buděšínský Miloš
Published: July 2002

In compounds of the Westphalen type the substituent in position 6 has been removed: the 6β-hydroxyl in 3β,17β-dibenzoyloxy-5-methyl-5β-estr-9-en-6β-ol (1) by oxidation to ketone 2 and Huang Minlon reduction, the 6β-chlorine atom in 3β-acetoxy-6β-chloro-5-methyl-5β-estr-9-en-17-one (13) by reduction with tributyltin hydride. Androstenedione, testosterone and methyltestosterone analogues 4, 5, and 16, respectively, were prepared from 3β,17β-dihydroxy-5-methyl-5β-estr-9-ene (3) by partial oxidation or partial acylation followed by oxidation. Alternatively, these derivatives were prepared from compound 13 without the need of partial transformations.
Coupling of Steroid O-(Carboxymethyl)oxime Derivatives with Amino Alcohols
Authors: Pouzar Vladimír , Černý Ivan
Published: July 2002

New approach to the preparation of steroids with connecting bridge, based on an O-carboxymethyloxime (CMO) structure, and with terminal hydroxy group, is presented. 17-CMO derivatives of 3β-acetoxy- and 3β-methoxymethoxyandrost-5-en-17-one were condensed with α,ω-amino alcohols to give derivatives with a chain of seven to nine atoms. After THP-protection, these compounds were converted to 3-keto-4-ene derivatives. An alternative synthesis consisted in transformation of 17-CMO derivatives with bonded amino acids by reduction of the terminal carboxyl. The resulting compounds were designed as building blocks for the preparation of bis-haptens for sandwich immunoassays.
Synthesis of 3-(6-Azauracil-5-yl)anthranilic Acid and Its Application to the Preparation of Other 1,2,4-Triazine Derivatives
Authors: Hlaváč Jan , Slouka Jan
Published: July 2002

The title compound was synthesized by alkaline recyclization of isatin-7-carboxylic acid semicarbazone and used for the preparation of 3-oxo-2,3-dihydro-5H-1,2,4-triazino[5,6-b]indol-6-carboxylic acid (8) and 3-oxo-2,3,4,6-tetrahydro-1,2,4-triazino[5,6-c]cinnoline-7-carboxylic acid (9).
Sterically Crowded Heterocycles. VI. Relative Configuration of Diastereoisomeric (Z)-1,3-Diphenyl-3-(5-methyl-2-phenylimidazo[1,2-a]pyridin-3-yl)prop-2-en-1-ols
Authors: Čejka Jan , Kubík Richard , Kratochvíl Bohumil , Kuthan Josef
Published: July 2002

Relative configuration of the titled racemic compounds 1a and 1b has been elucidated on the basis of a complete X-ray structure determined for the higher melting diastereoisomer. The compound 1a crystallizes in the orthorhombic space group P21212, a = 15.610(3) Å, b = 18.634(4) Å, c = 7.5672(7) Å, V = 2201.1(6) Å3, Z = 4. Preparative separation of both diastereoisomers is reported.
Synthesis and Resolution of Racemic 2-Amino-2'-hydroxy-1,1'-binaphthyl
Authors: Smrčina Martin , Vyskočil Štěpán , Polívková Jana , Poláková Jana , Kočovský Pavel
Published: July 2002

The title compound 3 has been prepared via a highly selective, Cu(II)-mediated cross-coupling of 2-aminonaphthalene 1 and 2-naphthol 2 and resolved into enantiomers via crystallization of diastereoisomeric salts with (1S)-(+)-10-camphorsulfonic acid. The method has been optimized and the use of chromatography eliminated.
Convenient Preparation of 1,3-Bis(dimethylamino)trimethinium Perchlorate, Tetrafluoroborate and Hexafluorophosphate
Authors: Arnold Zdeněk , Dvořák Dalimil , Havránek Miroslav
Published: July 2002

Formylation of ethyl vinyl ether with (chloromethylene)dimethylammonium chloride prepared from dimethylformamide and oxalyl chloride or phosgene followed by successive treatment with dimethylamine and 70% perchloric acid leads to 1,3-bis(dimethylamino)trimethinium perchlorate 1a in high yield. Similarly, work-up of the reaction mixture after formylation with hexafluorophosphoric acid or dimethylamine tetrafluoroborate afforded 1,3-bis(dimethylamino)trimethinium hexafluorophosphate 1b or tetrafluoroborate 1c, respectively.
[OsH4(PPh3)3]: New Catalyst for the Selective Cyclopropanation of Activated Olefins
Authors: Demonceau Albert , Simal François , Lemoine Corine A. , Noels Alfred F. , Chizhevsky Igor T. , Sorokin Pavel V.
Published: July 2002

The title compound was found to be an efficient catalyst for the selective cyclopropanation of activated olefins by ethyl diazoacetate. The cyclopropane yields range from moderate to good (75 to 95%) for activated olefins such as styrene and styrene derivatives, but are rather low (20 to 30%) for non-activated olefins such as terminal and cyclic alkenes. In the intermolecular competition, styrene was 45 times more reactive than cyclooctene. In all cases, trans (exo) cyclopropane predominated over the cis (endo) isomer.
Synthesis of New 2-Hydrazono-4-oxo-5-thiazolidineacetic Acids and Their Alkylamides
Authors: Sigmundová Ivica , Mečiarová Mária
Published: July 2002

The direct two step synthesis of three new 2-hydrazono-4-oxo-5-thiazolidineacetic acids from thiosemicarbazide, the appropriate ketones (acetophenone, cyclopentanone, cyclohexanone) and maleic anhydride is described. The prepared acids were transformed, via their mixed anhydrides with ethyl carbonate, into their alkylamides in a one-pot procedure.
Synthesis of 24-(Piperidin-1-yl, Morpholin-4-yl and 4-Methylpiperazin-1-yl)-5β-cholan-3α-ols and Four Hydroxylated 23-(4,5-Dihydroimidazol-2-yl)-24-nor-5β-cholanes
Authors: Nguyen Thi Thu Huong , Protiva Jiří , Klinotová Eva , Urban Jiří , Protiva Miroslav
Published: July 2002

Lithocholic (1a), chenodeoxycholic (1b), deoxycholic (1c) and cholic acid (1d) were used for the synthesis of the title compouds. Reactions of O-acetyllithocholic acid chloride with piperidine, morpholine and 1-methylpiperazine gave the corresponding amides 2a-2c which were reduced with lithium aluminium hydride to 24-(piperidin-1-yl)-5β-cholan-3α-ol (3a) and analogues 3b and 3c. Heating of the acids 1a-1d with ethylenediamine monotosylate afforded 23-(4,5-dihydroimidazol-2-yl)-24-nor-5β-cholan-3α-ol (4a) and analogues 4b-4d. Compound 4a was similarly obtained from 3α-acetoxy-24-nor-5β-cholane-23-carbonitrile. Identity of the products was corroborated by spectral characterization. Some of the products (in the form of salts) were tested for cancerostatic and antimicrobial activities in vitro with partially promising results.
Synthesis of Novel 4-Amino-5-(disubstituted amino)-2-phenyl-2H-pyridazin-3-ones
Authors: Konečný Václav , Žúžiová Jozefína , Kováč Štefan , Liptaj Tibor
Published: July 2002

Substituted 4-amino-2-phenyl-2H-pyridazin-3-ones 5a-5j have been prepared from 4-amino-5-chloro-2-phenyl-2H-pyridazin-3-one 1 which on reactions with acetyl chloride or acetic anhydride gives 4-acetylamino derivative 2 or 4-diacetylamino derivative 3, respectively. Derivatives 2 and 3 with dialkylamines and cyclic amines yielded appropriate 4-acetylamino-5-(disubstituted amino)-2-phenyl-2H-pyridazin-3-ones 4a-4j. Subsequent alkaline hydrolysis of the acetylamino derivatives 4a-4j let to the title compounds 5a-5j, which were screened for pesticidal activity, but none of them reached activity of the used standards.
Stereospecific Synthesis of (-)-allo-Muscarine from D-Glucose: Novel Routes to the Key Chiral Synthon
Authors: Popsavin Velimir , Beric Ostoja , Popsavin Mirjana , Csanádi János , Lajšic Stevan , Miljković Dušan
Published: July 2002

The key chiral synthon in a novel synthesis of (-)-allo-muscarine from D-glucose has been prepared by three independent routes. The most efficient one includes a four-step conversion via the 4-O-benzoyl derivatives of starting 2,5-anhydro-3,5-di-O-methanesulfonyl-L-idose ethylene acetal (2a) into 2,5-anhydro-3,6-dideoxy-L-lyxo-hexose ethylene acetal (4b). The intermediate 4b was efficiently converted into the chiral synthon 2,5-anhydro-4-O-benzoyl-3,6-dideoxy-L-arabino-hexose (4c) by Mitsunobu reaction.
Cyclic Dipeptides with 1-Aminocyclopropane-1-carboxylic Acid
Authors: Kasafírek Evžen , Moural Jaroslav , Vinšová Jarmila , Šturc Antonín , Taimr Jan
Published: July 2002

Cyclic dipeptides of the formula cyclo(-Acc-X-), where Acc = 1-aminocyclopropane-1-carboxylic acid, X = Gly, Ala, Val, Leu, Phe or Tyr, were synthesized. The prepared 2,5-piperazinediones showed no proliferative or antiproliferative activity on normal human embryo cells as well as on HL60 leukemia cells. Results of trace memory assays in mice were negative.
Preparation of Derivatives of 2-Cyano-3-(5-N-arylamino- or 5-N-Alkyl-N-phenylamino-2-furyl)propenoic Acids
Authors: Šafář Peter , Považanec František , Čepec Pavel , Prónayová Naďa
Published: July 2002

(5-Bromo-2-furyl)methylidenemalonodinitrile (1) reacted with substituted aromatic amines under formation of (5-N-arylamino-2-furyl)methylidenemalonodinitriles 2a-2h whereas no reaction was observed with N-alkyl-N-phenylamines. Derivatives of 2-cyano-3-(5-N-alkyl-N-phenylamino-2-furyl)- propenoic acid 5a-5o were prepared by reaction of the corresponding N-alkylanilines with 5-bromo-2-furancarbaldehyde, followed by hydrolysis of the obtained Eiji salts and reaction with malonic acid derivatives.
Prostanoids: Synthesis of Enantiomers of 15-Deoxy-16-hydroxy-16-methylprostaglandin E1
Authors: Terinek Miroslav , Kozmík Václav , Paleček Jaroslav
Published: July 2002

Four optically pure isomers of methyl 11,16-dihydroxy-16-methyl-9-oxoprost-13-enoate (1a-1d) were synthesized using an approach reverse to the classical Corey procedure, the key intermediates being the easily accessible (-)- and (+)-enantiomers of the Corey lactone, 2a and 2b.
Oxidation of Alkylaromatic Hydrocarbons Over V2O5-Sb2O3/TiO2 Catalyst
Authors: Antol Marcel , Cvengrošová Zuzana , Vrábel Imrich , Leško Ján , Hronec Milan
Published: July 2002

Monoalkylbenzenes, polymethylbenzenes, para-substituted toluenes and monomethylnaphthalenes were oxidized in the vapor phase by oxygen-containing gas in the presence of water over a Sb2O3-promoted V2O5/TiO2 catalyst. This type of catalyst yields carboxylic acids with high selectivity. In the oxidation of substituted alkylbenzenes only alkyl groups were oxidized. No products of oxidative dimerization were detected. Only in the oxidation of methylnaphthalenes, also products of aromatic ring oxidation are formed. A correlation between experimental data and results of quantum-chemical calculations of bond dissociation energies is discussed.
Ab initio Hartree-Fock Investigation of π-Conjugated Compounds Presenting Large βv/βe Ratio: Merocyanines
Authors: Champagne Benoît , Legrand Thierry , Perpete Eric A. , Quinet Olivier , André Jean-Marie
Published: July 2002

CHF/6-311G* calculations of the first electronic and vibrational hyperpolarizabilities reveal that merocyanines present a substantial βv/βe ratio under their quinonoid nonpolar form. It originates from a large vibrational first hyperpolarizability whereas its electronic counterpart is small for this class of push-pull π-conjugated molecules. The transition from the quinonoid to the aromatic configuration is accompanied by an increase of βe and a decrease of the βv/βe ratio as well as by a ≈ 180° rotation in the plane of the molecule of βe and βv with respect to the molecular frame. Our results support the recent experimental discovery that antiparallel aggregation of aromatic and quinonoid forms of merocyanine is energetically favoured and that their first hyperpolarizabilities, which combine constructively, present both electronic and non purely electronic origins.