bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatitis C Virus Envelope Protein E2 Does Not Inhibit PKR by Simple Competition with Autophosphorylation Sites in the RNA-Binding Domain
Authors: Taylor Deborah R. , Tian Bin , Romano Patrick R. , Hinnebusch Alan G. , Lai Michael M. C. , Mathews Michael B.
Published: July 2002

Double-stranded-RNA (dsRNA)-dependent protein kinase PKR is induced by interferon and activated upon autophosphorylation. We previously identified four autophosphorylated amino acids and elucidated their participation in PKR activation. Three of these sites are in the central region of the protein, and one is in the kinase domain. Here we describe the identification of four additional autophosphorylated amino acids in the spacer region that separates the two dsRNA-binding motifs in the RNA-binding domain. Eight amino acids, including these autophosphorylation sites, are duplicated in hepatitis C virus (HCV) envelope protein E2. This region of E2 is required for its inhibition of PKR although the mechanism of inhibition is not known. Replacement of all four of these residues in PKR with alanines did not dramatically affect kinase activity in vitro or in yeast
Saccharomyces cerevisiae
. However, when coupled with mutations of serine 242 and threonines 255 and 258 in the central region, these mutations increased PKR protein expression in mammalian cells, consistent with diminished kinase activity. A synthetic peptide corresponding to this region of PKR was phosphorylated in vitro by PKR, but phosphorylation was strongly inhibited after PKR was preincubated with HCV E2. Another synthetic peptide, corresponding to the central region of PKR and containing serine 242, was also phosphorylated by active PKR, but E2 did not inhibit this peptide as efficiently. Neither of the PKR peptides was able to disrupt the HCV E2-PKR interaction. Taken together, these results show that PKR is autophosphorylated on serine 83 and threonines 88, 89, and 90, that this autophosphorylation may enhance kinase activation, and that the inhibition of PKR by HCV E2 is not solely due to duplication of and competition with these autophosphorylation sites.
Interaction of JC Virus Agno Protein with T Antigen Modulates Transcription and Replication of the Viral Genome in Glial Cells
Authors: Safak Mahmut , Barrucco Robert , Darbinyan Armine , Okada Yuki , Nagashima Kazuo , Khalili Kamel
Published: July 2002

In addition to encoding the structural and regulatory proteins, many viruses encode auxiliary proteins, some of which have been shown to play important roles in lytic and latent states of the viruses. The human neurotropic JC virus (JCV) genome encodes an auxiliary protein called Agno whose function remains unknown. Here, we investigated the functional role of JCV Agno protein on transcription and replication of the viral genome in glial cells. Results from transfection of human glial cells showed that Agno protein suppresses both T-antigen-mediated transcription of the viral late gene promoter and T-antigen-induced replication of viral DNA. Affinity chromatography and coimmunoprecipitation assays demonstrated that the Agno protein and T antigen physically interact with each other. Through the use of a series of deletion mutants, we demonstrated that the T-antigen-interacting region of Agno protein is localized to its amino-terminal half and the Agno-interacting domain of T antigen maps to its central portion. Furthermore, utilizing various Agno deletion mutants in functional studies, we confirmed the importance of the Agno-T antigen interaction in the observed down-modulation of T antigen function upon viral gene transcription and DNA replication by Agno protein. Taken together these data suggest that the Agno protein of JCV, which is produced late during the late phase of the lytic cycle, can physically and functionally interact with the viral early protein, T antigen, and downregulate viral gene expression and DNA replication. The importance of these observations in the lytic cycle of JCV is discussed.
Effect of Intragenic Rearrangement and Changes in the 3′ Consensus Sequence on NSP1 Expression and Rotavirus Replication
Authors: Patton John T. , Taraporewala Zenobia , Chen Dayue , Chizhikov Vladimir , Jones Melinda , Elhelu Azza , Collins Megan , Kearney Karen , Wagner Mariam , Hoshino Yasutaka , Gouvea Vera
Published: July 2002

The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3′ termini ended with 5′-UGAACC-3′ instead of the 3′ consensus sequence 5′-UGACC-3′, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3′ consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3′ consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3′ end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3′ consensus sequence.
Interactions of Herpes Simplex Virus Type 1 with ND10 and Recruitment of PML to Replication Compartments
Authors: Burkham Jennifer , Coen Donald M. , Hwang Charles B. C. , Weller Sandra K.
Published: July 2002

Many of the events required for productive herpes simplex virus type 1 (HSV-1) infection occur within globular nuclear domains called replication compartments, whose formation appears to depend on interactions with cellular nuclear domains 10 (ND10). We have previously demonstrated that the formation of HSV-1 replication compartments involves progression through several stages, including the disruption of intact ND10 (stage I to stage II) and the formation of PML-associated prereplicative sites (stage III) and replication compartments (stage IV) (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100–10107, 1998). In this paper, we show that some, but not all, PML isoforms are recruited to stage III foci and replication compartments. Genetic experiments showed that the recruitment of PML isoforms to stage III prereplicative sites and replication compartments requires the localization of the HSV-1 polymerase protein (UL30) to these foci but does not require polymerase catalytic activity. We also examined the stages of viral infection under conditions affecting ND10 integrity. Treatment with factors that increase the stability of ND10, arsenic trioxide and the proteasome inhibitor MG132, inhibited viral disruption of ND10, formation of replication compartments, and production of progeny virus. These results strengthen the previously described correlation between ND10 disruption and productive viral infection.
Route of Simian Immunodeficiency Virus Inoculation Determines the Complexity but Not the Identity of Viral Variant Populations That Infect Rhesus Macaques
Authors: Greenier Jennifer L. , Miller Christopher J. , Lu Ding , Dailey Peter J. , Lü Fabien X. , Kunstman Kevin J. , Wolinsky Steven M. , Marthas Marta L.
Published: July 2002

A better understanding of the host and viral factors associated with human immunodeficiency virus (HIV) transmission is essential to developing effective strategies to curb the global HIV epidemic. Here we used the rhesus macaque-simian immunodeficiency virus (SIV) animal model of HIV infection to study the range of viral genotypes that are transmitted by different routes of inoculation and by different types of viral inocula. Analysis of transmitted variants was undertaken in outbred rhesus macaques inoculated intravenously (IV) or intravaginally (IVAG) with a genetically heterogeneous SIVmac251 stock derived from a well-characterized rhesus macaque viral isolate. In addition, we performed serial IV and IVAG passage experiments using plasma from SIV-infected macaques as the inoculum. We analyzed the V1-V2 region of the SIV envelope gene from virion-associated RNA in plasma from infected animals by the heteroduplex mobility assay (HMA) and by DNA sequence analysis. We found that a more diverse population of SIV genetic variants was present in the earliest virus-positive plasma samples from all five IV SIVmac251-inoculated monkeys and from two of five IVAG SIVmac251-inoculated monkeys. In contrast, we found a relatively homogeneous population of SIV envelope variants in three of five monkeys inoculated IVAG with SIVmac251 stock and in two monkeys infected after IVAG inoculation with plasma from an SIV-infected animal. In some IVAG-inoculated animals, the transmitted SIV variant was the most common variant in the inoculum. However, a specific viral variant in the SIVmac251 stock was not consistently transmitted by IVAG inoculation. Thus, it is likely that host factors or stochastic processes determine the specific viral variants that infect an animal after IVAG SIV exposure. In addition, our results clearly demonstrate that the route of inoculation is associated with the extent and breadth of the genetic complexity of the viral variant population in the earliest stages of systemic infection.
A Single Amino Acid Substitution in Nonstructural Protein 3A Can Mediate Adaptation of Foot-and-Mouth Disease Virus to the Guinea Pig
Authors: Núñez Jose Ignacio , Baranowski Eric , Molina Nicolas , Ruiz-Jarabo Carmen M. , Sánchez Carmen , Domingo Esteban , Sobrino Francisco
Published: July 2002

The genetic changes selected during the adaptation of a clonal population of foot-and-mouth disease virus (FMDV) to the guinea pig have been analyzed. FMDV clone C-S8c1 was adapted to the guinea pig by serial passage in the animals until secondary lesions were observed. Analysis of the virus directly recovered from the lesions developed by the animals revealed the selection of variants with two amino acid substitutions in nonstructural proteins, I
248
→T in 2C and Q
44
→R in 3A. On further passages, an additional mutation, L
147
→P, was selected in an important antigenic site located in the G-H loop of capsid protein VP1. The amino acid substitution Q
44
→R in 3A, either alone or in combination with the replacement I
248
→T in 2C, was sufficient to give FMDV the ability to produce lesions. This was shown by using infectious transcripts which generated chimeric viruses with the relevant amino acid substitutions. Clinical symptoms produced by the artificial chimeras were similar to those produced by the naturally adapted virus. These results obtained with FMDV imply that one or very few replacements in nonstructural viral proteins, which should be within reach of the mutant spectra of replicating viral quasispecies, may result in adaptation of a virus to a new animal host.
Kaposi's Sarcoma-Associated Herpesvirus OX2 Glycoprotein Activates Myeloid-Lineage Cells To Induce Inflammatory Cytokine Production
Authors: Chung Young-Hwa , Means Robert E. , Choi Joong-Kook , Lee Bok-Soo , Jung Jae U.
Published: July 2002

Kaposi's sarcoma is an inflammatory cytokine-mediated angioproliferative disease which is triggered by infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV contains an open reading frame, K14, that has significant homology with cellular OX2, designated viral OX2 (vOX2). In this report, we demonstrate that vOX2 encodes a glycosylated cell surface protein with an apparent molecular mass of 55 kDa. Purified glycosylated vOX2 protein dramatically stimulated primary monocytes, macrophages, and dendritic cells to produce the inflammatory cytokines interleukin 1β (IL-1β), IL-6, monocyte chemoattractant protein 1, and TNF-α. Furthermore, expression of vOX2 on B lymphocytes stimulated monocytes to produce inflammatory cytokines in mixed culture. These results demonstrate that like its cellular counterpart, vOX2 targets myeloid-lineage cells, but unlike cellular OX2, which delivers a restrictive signal, KSHV vOX2 provides an activating signal, resulting in the production of inflammatory cytokines. Thus, this is a novel viral strategy where KSHV has acquired the cellular OX2 gene to induce inflammatory cytokine production, which potentially promotes the cytokine-mediated angiogenic proliferation of KSHV-infected cells.
Immunogenicity and Protective Efficacy of Replication-Incompetent Influenza Virus-Like Particles
Authors: Watanabe Tokiko , Watanabe Shinji , Neumann Gabriele , Kida Hiroshi , Kawaoka Yoshihiro
Published: July 2002

Despite the success of influenza virus vaccines in reducing severe illness, their efficacy is suboptimal. We describe here the immunogenicity and protective capacity of replication-incompetent influenza virus-like particles (VLPs) which were generated entirely from cDNAs and lacked either the entire NS gene (encoding both the NS1 and NS2 protein) or only the NS2 gene. In mammalian cells infected with NS gene-deficient VLPs, the nucleoprotein, but not other viral proteins including hemagglutinin (HA) and neuraminidase (NA), was detected. In contrast, cells infected with VLPs expressing NS1 but not NS2 (NS2 knockout) expressed multiple viral proteins, including HA and NA. When challenged with lethal doses of an antigenically homologous mouse-adapted influenza virus, 94% of mice vaccinated with the NS2-knockout VLPs survived, compared with less than 10% of those given the NS-deficient VLPs. These results demonstrate the potential of replication-incompetent NS2-knockout VLPs as novel influenza vaccines and perhaps also as vectors to express genes from entirely unrelated pathogens.
Genetically Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for Treatment of Malignant Disease
Authors: Fernandez Marilyn , Porosnicu Mercedes , Markovic Dubravka , Barber Glen N.
Published: July 2002

We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.
Processing of Nonstructural Protein 1a of Human Astrovirus
Authors: Geigenmüller Ute , Chew Teri , Ginzton Nancy , Matsui Suzanne M.
Published: July 2002

Astrovirus contains three open reading frames (ORF) on its genomic RNA, ORF1a, ORF1b, and ORF2. ORF1a encodes a 920-amino-acid (aa) nonstructural protein, nsP1a, which displays a 3C-like serine protease motif. Little is known about the processing of nsP1a or whether the protease it contains is active and involved in autocatalytic processing. Here we address both of these matters. Intact and N-terminally deleted forms of ORF1a from human astrovirus serotype 1 were expressed in BHK cells, and nsP1a-derived processing products were immunoprecipitated with an nsP1a-specific antibody or an antibody specific for an N-terminally linked epitope tag. The mapping of the main processing products, p20 and p27, suggests cleavage sites near aa 170, 410, and 655 of nsP1a. Cleavages at around aa 410 and 655, but not aa 170, were abolished when a 9-aa substitution was introduced into the protease motif in nsP1a. The p27 processing product was also found in Caco-2 cells that had been infected with human astrovirus serotype 1, confirming the presence of the cleavage sites at approximately aa 410 and 655.
A Human Immunodeficiency Virus Type 1 Isolate from an Infected Person Homozygous for CCR5Δ32 Exhibits Dual Tropism by Infecting Macrophages and MT2 Cells via CXCR4
Authors: Naif Hassan M. , Cunningham Anthony L. , Alali Mohammed , Li Shan , Nasr Najla , Buhler Marc M. , Schols Dominique , de Clercq Erik , Stewart Graeme
Published: July 2002

The mechanisms of human immunodeficiency virus (HIV) infection of a man (VH) homozygous for theCCR5Δ32mutation were investigated, and coreceptors other than CCR5 used by HIV type 1 (HIV-1) isolated from this individual were identified. In contrast to previous reports, this individual's rate of disease progression was not accelerated. Homozygosity forCCR5Δ32mutation was demonstrated by PCR and DNA sequencing (R. Biti et al., Nat. Med. 3:252-253, 1997). CCR5 surface expression was absent on T lymphocytes and macrophages. HIV was isolated by coculture with peripheral blood mononuclear cells (PBMCs) from siblings who were homozygous (VM) or wild type (WT) for theCCR5Δ32mutation. The virus demonstrated dual tropism for infection of MT2 cell line and primary macrophages. Sequencing of the full HIV genome directly from the patient's PBMCs revealed 21 nucleotide insertions in the V1 region of gp120. The VH envelope sequence segregated apart from both the T-cell-line-adapted tropic strains NL4-3 and SF2 and M-tropic strain JRFL or YU2 by phylogenetic tree analysis. VH was shown to utilize predominantly CXCR4 for entry into T lymphocytes and macrophages by HOS.CD4 cell infection assay, direct envelope protein fusion, and inhibition by anti-CXCR4 monoclonal antibody (12G5), SDF-1, and AMD3100. Microsatellite mapping demonstrated the separate inheritance of CXCR4 by both homozygote brothers (VH and VM). Our study demonstrates the ability of certain strains of HIV to readily use CXCR4 for infection or entry into macrophages, which is highly relevant to the pathogenesis of late-stage disease and presumably also HIV transmission.
Cre Levels Limit Packaging Signal Excision Efficiency in the Cre/
loxP
Helper-Dependent Adenoviral Vector System
Authors: Ng Philip , Evelegh Carole , Cummings Derek , Graham Frank L.
Published: July 2002

Helper-dependent (HD) adenovirus vectors devoid of all viral coding sequences have a large cloning capacity and provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of producing HD vectors involves coinfecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by
loxP
sites. Cre-mediated packaging signal excision renders the helper virus genome unpackageable but still able to replicate and provide helper functions for HD vector propagation. Typically, helper virus contamination is ≤1% pre- and ≤0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. However, this objective has not been realized since the Cre/
loxP
system was first developed. This lack of progress is due, at least in part, to our lack of understanding of the origins of the contaminating helper virus, thus rendering its reduction or elimination difficult to achieve. This study was designed to investigate the possible sources of contaminating helper virus persisting during HD vector amplification. The results revealed that Cre is limiting in helper virus-infected Cre-expressing 293 cells, thereby permitting helper viruses to escape packaging signal excision and propagate. The results of this study should provide a foundation for developing rational strategies to further reduce or possibly eliminate the contaminating helper virus.
Rafts Promote Assembly and Atypical Targeting of a Nonenveloped Virus, Rotavirus, in Caco-2 Cells
Authors: Sapin Catherine , Colard Odile , Delmas Olivier , Tessier Cedric , Breton Michelyne , Enouf Vincent , Chwetzoff Serge , Ouanich Jocelyne , Cohen Jean , Wolf Claude , Trugnan Germain
Published: July 2002

Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.
Homogeneous Nucleation in Supersaturated Vapors of n-Nonane
Authors: Ždímal Vladimír , Smolík Jiří , Meijer Ivo G. N.
Published: July 2002

The upward thermal diffusion cloud chamber was used to measure the supersaturations of n-nonane vapors required to cause an observed rate of homogeneous nucleation of 1 droplet/cm3 s. New temperature-dependent correlations for the surface tension and the density of n-nonane have been applied in theoretical prediction of the corresponding supersaturation. The measurements are found to be in reasonable agreement with the predictions of the classical (Becker-Doering) theory whereas both the Dillmann-Meier and the Girshick-Chiu theories lead to a significant underestimation of supersaturation in the whole range of temperatures measured.
Alkaline Hydrolysis of 4-Nitrophenyl Acetate and Diphenyl (4-Nitrophenyl) Phosphate Catalyzed by Iodosoarenesulfonic Acids
Authors: Kulič Jiří , Ptáček Aleš
Published: July 2002

Alkaline hydrolysis of 4-nitrophenyl acetate and of diphenyl (4-nitrophenyl) phosphate catalyzed by 2-iodosobenzenesulfonic and 2-iodoso-1-naphthalenesulfonic acids was studied in the presence of hexadecyltrimethylammonium bromide as the micellar agent. It was found that 2-iodosobenzenesulfonic acid is the better catalyst for the hydrolysis of phenyl acetate while 2-iodoso-1-naphthalenesulfonic acid is more efficient for the hydrolysis of the phosphate.
Crystal Structures of Ergot Alkaloid Derivatives. Ergometrine Maleate and Methylergometrine Maleate
Authors: Čejka Jan , Hušák Michal , Kratochvíl Bohumil , Jegorov Alexandr , Cvak Ladislav
Published: July 2002

Two ergot alkaloid derivatives were examined by X-ray diffraction techniques. The crystal and molecular structure of ergometrine maleate (1) was evaluated from single-crystal data and the molecular structure of related methylergometrine maleate (2) was obtained by molecular modelling. The measured and calculated X-ray powder patterns of 1 and 2 were compared. Derivative 1 crystallizes in the orthorhombic space group P212121 with a = 5.7112(7) Å, b = 12.337(2) Å, c = 33.276(6) Å, Z = 4, V = 2 344.5(6) Å3. Derivative 2 is nearly isostructural with 1, it has slightly expanded unit cell, a = 5.724(2) Å, b = 12.762(6) Å, c = 33.25(2) Å, Z = 4, V = 2 428(1) Å3. No effect of an additional methyl group on the molecular packing of 2 was found.
Electrochemical Investigations of the Nickel(II)-Penicillamine System. Inert and Labile Complexes Detected by Polarography
Authors: Ion Ana , Banica Florinel G. , Luca Constantin
Published: July 2002

The effect of penicillamine on nickel diffusion current demonstrates that Ni22- is a kinetically inert complex whereas NiL is labile (L2- = (CH3)2C(S-)CH(NH2)COO-)). The difference in kinetic stability between the analogous bis-ligand complexes of Ni(II) with either penicillamine or cysteine is tentatively explained taking into account their structures. It is suggested that nickel detoxication by chelate ligands depends on the inert character rather than on the thermodynamic stability of the relevant nickel complex.
Electrochemical Investigations of the Nickel(II)-Penicillamine System. 2. Direct Identification of Complex Species Involved in Catalytic Nickel Reduction on the Dropping Mercury Electrode
Authors: Banica Florinel G. , Ion Ana
Published: July 2002

The catalytic polarographic nickel prewave was investigated by making appropriate correlations between prewave current and complex species concentrations as calculated by means of available formation constants. It was concluded that the active species is (D-penicillaminato-N,S)nickel(II) [NiL], whereas the bis-ligand complex, [NiL2]2-, is inert and does not play any role in the electrode process. The catalytic character of the electrode process originates from the regeneration of [NiL] by the reaction of adsorbed ligand molecules with free nickel ions available in the bulk of the solution. Conversely, all the complex species in the Ni2+-cysteine system are labile. Consequently, the reaction mechanism in this case may include the dissociation of the complex [NiL2]2- as an alternative path for the generation of the active species, [NiL]. The bell-shaped form of the prewave was interpreted in terms of potential-dependent catalyst adsorption.
Full Configuration Interaction Energies, Geometries, and Quartic Force Fields of the Nitrenium Ion
Authors: Van Huis Timothy J. , Leininger Matthew L. , Sherrill C. David , Schaefer Henry F.
Published: July 2002

Benchmark configuration interaction energies, geometries, dipole moments, and harmonic vibrational frequencies are obtained for four low-lying electronic states of NH2+ and for X2B1 NH2 by solving the electronic Schrodinger equation exactly within a double-ζ plus polarization (DZP) basis set and restricting the nitrogen 1s-like core orbital to remain doubly occupied. In addition, full quartic force fields have been determined, and sets of anharmonic spectroscopic constants and fundamental frequencies are reported for the ã 1A1 state of NH2+. Vertical and adiabatic ionization potentials of NH2 are also determined exactly within a DZP basis. The capability of less-complete electron correlation treatments to match these exact, full configuration interaction (full CI) results is assessed. The efficacy of obtaining anharmonic force fields at nonstationary geometries is also examined, and in agreement with previous work, it is found that the self-consistent field method can provide high quality cubic and quartic force constants when they are evaluated at the full CI equilibrium geometry. Both the CCSD(T) and CASSCF-SOCI methods provide geometric and spectroscopic data in excellent agreement with the Full CI results and are competitive in their reliability with more expensive procedures (e.g. CISDTQ). The second 1A1 state in C2v symmetry is predicted by the TZ2P(f,d) CASSCF-SOCI wavefunction to be linear (thus properly labeled as c1Σg+), even though it is found to be quasilinear at the DZP full CI level.
Role of Energy Dissipation and Surface Mobility in Heterogeneous Catalysis by Metals
Authors: Knor Zlatko , Plšek Jan
Published: July 2002

The fundamental role of excess energy dissipation and of surface species mobility, both in the preparation of model metallic catalysts (bimetallic catalysts, bimetallic oxide-supported catalysts), and in the activation of reactant molecules on the catalyst surfaces (single crystals, bimetallic and bimetallic oxide-supported catalysts) are discussed. A generalized model of surface interactions is proposed which satisfactorily explains the trapping of particles from the gas phase at the atomic steps and recovery of the trapping sites due to migration of the trapped species towards flat terraces. Higher probability of encounter of these species with other reactants on flat terraces and easier desorption of the reaction products from there in comparison with the bound state at the edges of atomic steps can be expected. Results of FIM and FEM studies of Pt, Dy-W, Pd-W and Pd-Mo systems are used to illustrate selected features of the proposed general model.
Selective Reduction of Peptidic Ergot Alkaloids
Authors: Cvak Ladislav , Stuchlík Josef , Schreiberová Magdalena , Sedmera Petr , Havlíček Vladimír , Flieger Miroslav , Čejka Jan , Kratochvíl Bohumil , Jegorov Alexandr
Published: July 2002

Five 6'-deoxoergopeptines were prepared in 51-68% yield by selective reduction of parent alkaloids with lithium aluminium hydride in tetrahydrofuran at low temperature. New compounds were characterized by mass spectrometry and NMR spectroscopy. The conformation of the peptide part in starting compounds and reduced derivatives is discussed on the basis of crystal structure determination of 6'-deoxo-9,10-dihydroergotamine dihydrate butan-2-one solvate as a representative member of the series.
Hydrogenation of 3-(Dimethylamino)propionitrile Over Palladium Catalysts
Authors: Krupka Jiří , Pašek Josef , Navrátilová Markéta
Published: July 2002

Hydrogenation of 3-(dimethylamino)propionitrile over palladium catalysts was studied. Besides the expected amines, N,N,N',N'-tetramethylpropane-1,3-diamine, N,N-dimethyl-N'-propylpropane-1,3-diamine, and N,N-bis[3-(dimethylamino)propyl]propylamine were found. Reaction pathways of their formation were discussed. Effects of reaction conditions, type of catalyst, and addition of ammonia or an amine into the charge on the hydrogenation selectivity were studied.
Synthesis of Carba Analogues of Deoxy-4-C-(hydroxymethyl)hexopyranoses, Intermediates in the Synthesis of Carbocyclic Nucleosides
Authors: Hřebabecký Hubert , Holý Antonín
Published: July 2002

3-O-Benzyl-1,2-O-isopropylidene-α-D-glucofuranose-5,6-O-sulfate (1) was treated with sodium salt of dimethyl malonate to obtain, after hydrolysis, methyl 5-(3-O-benzyl-1,2-O-isopropylidene-α-D-erythrofuranos-4-yl)-2-oxotetrahydrofuran-3-carboxylate (3) which was converted to the mixture of methyl (2S,3R,4R)- (7) and (2R,3R,4R)-2-(acetyloxy)-3-(benzyloxy)-4-(formyloxy)-7-oxo-6-oxabicyclo[3.2.1]octane-1-carboxylate (8). The compound 7 was reduced with lithium aluminium hydride to give (1R,2R,3R,4S)-3-(benzyloxy)-5,5-bis(hydroxymethyl)cyclohexane-1,2,4-triol (9) which was transformed to (1R,2S,4R)-5,5-bis(hydroxymethyl)cyclohexane-1,2,4-triol (14). Treatment of sodium salt of diethyl malonate with 3-O-benzyl-5,6-dideoxy-6-iodo-1,2-O-isopropylidene-α-D-xylo-hexofuranose (19) gave diethyl (3-O-benzyl-5,6-dideoxy-1,2-O-isopropylidene-α-D-xylo-hexofuranos-6-yl)malonate (20) which was converted to (1R,3R)-4,4-bis(hydroxymethyl)cyclohexane-1,3-diol (28) by a similar procedure to that used for 14.
Radical Oxidation of 17-Functionalized 14α-Hydroxy Steroids
Authors: Khripach Vladimir A. , Zhabinskii Vladimir N. , Kotyatkina Anna I. , Fando Galina P. , Zhiburtovich Yuliya Y. , Lyakhov Alexander S. , Govorova Alla A. , Groen Marinus B. , Louw Jaap van der , Groot Aede de
Published: July 2002

The radical oxidation of 14α-hydroxy steroids with various functional groups at C-17 was studied. Lead tetraacetate and ceric ammonium nitrate were used as oxidizing agents. It was shown that reactions of this type afforded complex mixtures of compounds. However, the radical oxidation of 14α-hydroxy-17-oxo steroid (lead tetraacetate version of the hypoiodite reaction) proceeded smoothly with formation of the 13,14-secosteroid in up to 85% yield. The structure and conformation of the formed 13α-iodo-3α,5-cyclo-13,14-seco-5α-androst-5-ene-14,17-dione was determined by X-ray analysis.
Stereochemistry of Cuprate-Mediated Conjugate Additions of a 17-Iodoandrost-16-ene to E- and Z-6-Methylhept-2-en-4-one
Authors: Andersen Denise L. , Back Thomas G.
Published: July 2002

(3S,15R)-15-(Benzyloxy)-3-[(tert-butyldimethylsilyl)oxy]-17-iodoandrosta-5,16-diene (3) was prepared from the corresponding 17-hydrazone9with iodine in the presence of tetramethylguanidine. The vinyl iodide3was converted into the corresponding organocuprate and conjugate additions of the latter were performed with both (E)- and (Z)-6-methylhept-2-en-4-one (4aand4b, respectively). In each case, the resulting adduct5awas obtained exclusively with the unnatural 20Sconfiguration. (3S,15R)-15-(Benzyloxy)-3-[(tert-butyldimethylsilyl)oxy]androsta-5,16-diene (13) and the corresponding 17,17' homocoupling product14were obtained as byproducts. When the addition to theE-enone was repeated in the presence of chloro(trimethyl)silane, the yield of the conjugate addition product improved, but the stereochemistry at C-20 remained the same.
Association of the tumour necrosis factor α −308 but not the interleukin 10 −627 promoter polymorphism with genetic susceptibility to primary sclerosing cholangitis
Authors: Mitchell S A , Grove J , Spurkland A , Boberg K M , Fleming K A , Day C P , Schrumpf E , Chapman R W ,   the European Study Group of Primary Sclerosing Cholangitis
Published: July 2002

BACKGROUND AND AIMS

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown aetiology. Abnormalities in immune regulation and genetic associations suggest that PSC is an immune mediated disease. Several polymorphisms within the tumour necrosis factor α (TNF-α) and interleukin 10 (IL-10) promoter genes have been described which influence expression of these cytokines. This study examines the possible association between polymorphisms at the −308 and −627 positions in the TNF-α and IL-10 promoter genes, respectively, and susceptibility to PSC.

METHODS

TNF-α −308 genotypes were studied by polymerase chain reaction (PCR) in 160 PSC patients from Norway and the UK compared with 145 ethnically matched controls. IL-10 −627 genotypes were studied by PCR in 90 PSC patients compared with 84 ethnically matched controls.

RESULTS

A total of 16% of Norwegian PSC patients and 12% of British PSC patients were homozygous for the TNF2 allele compared with 3% and 6% of respective controls. The TNF2 allele was present in 60% of PSC patients versus 30% of controls (ORcombined data=3.2 (95% confidence intervals (CI) 1.8–4.5); pcorr=10−5). The association between the TNF2 allele and susceptibility to PSC was independent of the presence of concurrent inflammatory bowel disease (IBD) in the PSC patients; 61% of PSC patients without IBD had TNF2 compared with 30% of controls (ORcombined data=3.2 (95% CI 1.2–9.0); pcorr=0.006 ). There was no difference in the −627 IL-10 polymorphism distributions between patients and controls in either population. The increase in TNF2 allele in PSC patients only occurs in the presence of DRB1*0301 (DR3) and B8. In the combined population data, DRB1*0301 showed a stronger association with susceptibility to PSC than both the TNF2 and B8 alleles (ORcombined data=3.8, pcorr=10−6v ORcombined data=3.2, pcorr=10−5vORcombined data =3.41, pcorr=10−4, respectively).

CONCLUSIONS

This study identified a significant association between possession of the TNF2 allele, a G→A substitution at position −308 in the TNF-α promoter, and susceptibility to PSC. This association was secondary to the association of PSC with the A1-B8-DRB1*0301-DQA1*0501-DQB1*0201 haplotype. No association was found between the IL-10 −627 promoter polymorphism and PSC.
Routine DNA cytometry of benign and malignant pleural effusions by means of the remote quantitation server Euroquant: a prospective study
Authors: Kayser K , Blum S , Beyer M , Haroske G , Kunze K D , Meyer W
Published: July 2002

Aim—To analyse the practicability and potential assistance of static DNA cytometry performed by means of the remote quantitation server Euroquant and the internet in routine diagnostic analysis of pleural effusions, and to outline the role of DNA cytometry on pleural effusions in distinguishing between benign and malignant (and herein primary versus metastatic) effusions.

Materials and methods—Cytological smears of 294 pleural effusions were stained with the Feulgen method. The DNA content of a minimum of 300 randomly chosen analysis nuclei and 30 reference nuclei (lymphocytes) was measured by internet connection to the remote quantitation server Euroquant. Cytometric features were derived from the histograms, and the time needed for case evaluation, the reliability of staining and measurement procedures, and the contribution to the final diagnosis were evaluated.

Results—Only 120 of 294 pleural effusions could be measured. The total measurement time for each specimen was 60 minutes. The guidelines of the consensus report on DNA measurements were fulfilled. Seventy eight malignant (18 mesotheliomas, 60 metastatic tumours) and 42 benign effusions were measured. Seven of 78 malignant effusions were euploid and none of 42 benign effusions were aneuploid. The sensitivity and specificity were 91% and 100%, respectively, for distinguishing benign from malignant effusions, and 95% and 100%, respectively, for discriminating between benign and malignant effusions caused by metastatic malignant tumours.

Conclusions—Static DNA cytometry using the remote quantitation server Euroquant can be performed reliably in the routine diagnosis of pleural effusions; however, only 40% of effusions meet the technical requirements for static DNA cytometry. Within the measurable cases, static DNA cytometry made an important contribution to the confirmation/exclusion of malignancy.
Indirect immunofluorescence (IIF) of normal washed peripheral blood cells to demonstrate antineutrophil cytoplasmic antibodies (ANCA)
Authors: Paspaliaris B , Pamio M , Savige J
Published: July 2002

Background—The “International consensus document on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA)” requires all sera to be examined by indirect immunofluorescence (IIF). However, commercial neutrophil slides are expensive, fluorescence patterns can be difficult to interpret, and coincidental antinuclear antibodies (ANA) cannot be demonstrated; in addition, in house cytospin neutrophil preparations are time consuming to prepare and deteriorate with time.

Aims—To compare the IIF demonstration of ANCA, using washed peripheral blood cell smears, with commercial neutrophil preparations and with ANCA positivity as demonstrated by enzyme linked immunosorbent assay (ELISA).

Methods—Serum fluorescence positivity, pattern, and intensity using washed peripheral blood cell smears were compared with the results obtained using commercial neutrophil slides (INOVA). Fluorescence positivity, pattern, and intensity of 500 sera from consecutive patients with suspected vasculitis tested with washed peripheral blood cells were compared with binding in ELISAs for proteinase 3 (PR3) and myeloperoxidase (MPO).

Results—IIF of washed peripheral blood cell smears detected seven of eight sera with cytoplasmic fluorescence (C-ANCA), and 11 of 12 sera with perinuclear fluorescence (P-ANCA) demonstrated using commercial slides. The two sera that were negative by IIF were also negative in the ELISAs for both PR3-ANCA and MPO-ANCA. Of the 500 sera examined, there were 35 (7%) with C-ANCA, 65 (13%) with P-ANCA, and eight (2%) IIF negative sera that were positive by either ELISA. There was a strong correlation between C-ANCA fluorescence and PR3-ANCA values (p < 0.0001), and a moderate to strong correlation between P-ANCA fluorescence and MPO-ANCA values (p < 0.001) when ANCA fluorescence was demonstrated with washed peripheral blood cell smears.

Conclusions—Washed peripheral blood cells are a convenient and useful low cost alternative to commercial or cytospin neutrophil preparations for the IIF demonstration of ANCA.
Coexistence of an endocrine tumour in a serous cystadenoma (microcystic adenoma) of the pancreas, an unusual association
Authors: Üstün M Ö , TuĞyan N , Tunakan M
Published: July 2002

A pancreatic endocrine tumour arising within a serous cystadenoma is reported. A 49 year old woman was admitted with a history of epigastric pain, nausea, vomiting, and weight loss of two months duration. She had been diabetic for 12 years. An epigastric mass was palpated in the physical examination, and computed tomography revealed a multiloculated cystic lesion in the pancreas. Pathological examination of the pancreatic tumour revealed the coexistence of a serous cystadenoma and an endocrine tumour. The endocrine tumour, which was located inside the serous cystadenoma, was 1 cm in diameter. The first case of a serous cystadenoma of the pancreas containing a pancreatic endocrine tumour was reported in the literature recently. This paper reports another incidentally found pancreatic endocrine tumour arising within a serous cystadenoma.
What modifies the relation between tumour size and lymph node metastases in T1 breast carcinomas?
Authors: Arisio R , Sapino A , Cassoni P , Accinelli G , Cuccorese M C , Mano M P , Bussolati G
Published: July 2002

Aims—To evaluate which pathological and clinical parameters modify the relation between tumour size and lymph node metastases in invasive breast carcinomas < 20 mm.

Methods—In a retrospective study, 1075 patients with pT1 invasive breast carcinoma and with known nodal status were analysed. The size of the infiltrating tumour was microscopically evaluated, and the in situ component was not considered. The additional pathological parameters considered were: tumour grade, peritumoral vascular invasion, multicentricity, and angiogenesis. The immunophenotype of the tumour was determined as: the expression of oestrogen (ER) and progesterone (PR) receptors, p53, and c-erbB2. The patients were grouped by age as follows: < 50, 51–70, and > 70 years old.

Results—Three hundred and seventy four patients (34.8%) were node positive. Univariate analysis showed that nodal positivity was significantly correlated with large tumour size (> 10 mm), vascular invasion, grade 2–3, multicentricity, and high angiogenesis (> 100 microvessels/×20 high power frame). No significant correlation was found between nodal positivity and ER, PR, p53, or c-erbB2 status. Interestingly, the association with in situ carcinoma was correlated with lower nodal positivity in tumours presenting equally sized infiltrating components. Age was an independent variable and significantly modified the risk of nodal positivity in tumours < 1 cm. In fact, in patients under 51 years of age, the proportion of nodal positivity in pT1a tumours was sevenfold higher than in older patients. In patients from 51 to 70 years old, nodal positivity correlated with tumour size, and multicentricity was an additional risk factor.

Conclusions—These data suggest that, together with tumour size, the presence of in situ carcinoma, and vascular invasion, age is one of the most important predictors of metastatic diffusion in breast carcinomas.
Squamous cell carcinoma of skin with a rhabdoid phenotype: a case report
Authors: Mathers M E , O'Donnell M
Published: July 2002

A 67 year old man presented with a polypoid lesion on the temple that had all the light microscopic, immunohistochemical, and ultrastructural features of a rhabdoid tumour. There was an area of intraepidermal carcinoma and invasive squamous carcinoma at the base of the polyp. The tumour progressed aggressively and the patient died five months after primary excision. Cutaneous tumours with a rhabdoid morphology have been described previously and tend to have a very poor prognosis. No previously published report describes a clear squamous histogenesis.
Basal cell adenocarcinoma of the palate with squamous metaplasia
Authors: de Sousa Suzana Orsini Machado , Schwarzschild Monica , de Araújo Ney Soares , de Araújo Vera Cavalcanti
Published: July 2002

Basal cell adenocarcinoma is a rare salivary gland tumour, especially in minor glands. The clinical, histological, and immunohistochemical features of a case involving the palate are described. Formalin fixed, paraffin embedded sections of the tumour were examined in haematoxylin and eosin (H&E) sections and also using immunostaining for cytokeratins 7, 8, 13, 14, 18, 19, vimentin, muscle specific actin (HHF35), and laminin. H&E sections showed that the tumour was composed mainly of basaloid cells and a striking feature was the presence of squamous metaplasia. Neural invasion was also conspicuous. Immunohistochemical reactions indicated that cytokeratin 14 was expressed by all tumour cells and vimentin by all cells except those in the areas of squamous metaplasia. The remaining cytokeratins and actin were present in some of the tumour cells, while laminin showed discreet positivity around cell arrangements. The foci of squamous metaplasia and the immunohistochemical findings are helpful in distinguishing basal cell adenocarcinoma from other salivary gland tumours which show basaloid cells.
Effect of transforming growth factor β2on oestrogen metabolism in the MCF-7 breast cancer cell line
Authors: Erbas Hakan , Lai Leslie C
Published: July 2002

Transforming growth factor β (TGF-β) is a multifunctional regulator of cellular growth and differentiation in many cell types and has a growth inhibitory effect on mammary epithelial cells. The TGF-β2isoform has been shown to be present in high concentrations in breast cyst fluid and might have a protective role in breast cancer. In addition, oestrogens play an important role in breast cancer development, and oestrone sulphate (E1S) might be the main source of active oestrogens in the breast. The aim of this study was to assess the effect of TGF-β2on oestrogen synthesis in an attempt to understand the mechanism by which TGF-β2may exert a protective effect in breast cancer. In this study, higher concentrations of TGF-β2significantly inhibited the conversion of E1S to oestrone (E1) and the conversion of E1to the potent oestrogen, oestradiol (E2). TGF-β2did not have any effect on MCF-7 cell growth or on E2to E1conversion. In conclusion, TGF-β2might exert a protective role in breast cancer by reducing the amount of active oestrogens present in the breast.