bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring Resistance to Human Immunodeficiency Virus Type 1 Protease Inhibitors by Pyrosequencing
Authors: O'Meara Deirdre , Wilbe Karin , Leitner Thomas , Hejdeman Bo , Albert Jan , Lundeberg Joakim
Published: July 2002

The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HIV-1-infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.
Molecular Typing and Epidemiological Study of
Salmonella enterica
Serotype Typhimurium Isolates from Cattle by Fluorescent Amplified-Fragment Length Polymorphism Fingerprinting and Pulsed-Field Gel Electrophoresis
Authors: Tamada Yukihiro , Nakaoka Yuji , Nishimori Kei , Doi Akira , Kumaki Takahiro , Uemura Nobuko , Tanaka Kiyoshi , Makino Sou-Ichi , Sameshima Toshiya , Akiba Masato , Nakazawa Muneo , Uchida Ikuo
Published: July 2002

One hundred twenty
Salmonella enterica
serotype Typhimurium strains, including 103 isolates from cattle gathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzed by using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles that formed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasingly common since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved 25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLP cluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGE cluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken place since 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporary isolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and 132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C, respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that of the 132-bp fragment shows homology with a segment of
traG
, which is an F plasmid conjugation gene. FAFLP is apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP can provide a source of molecular markers useful for studies of genetic variation in natural populations of serotype Typhimurium.
Frequency of Acyclovir-Resistant Herpes Simplex Virus in Clinical Specimens and Laboratory Isolates
Authors: Shin Young Kyoo , Cai Guang-Yun , Weinberg Adriana , Leary Jeffry J. , Levin Myron J.
Published: July 2002

The proportion of acyclovir (ACV)-resistant herpes simplex virus (HSV) isolates in clinical specimens and laboratory isolates was determined. HSV isolates in clinical specimens and laboratory isolates were cultured in the absence or presence of 5 μg of ACV per ml. The frequency of ACV-resistant HSV was calculated as (average virus titer in the wells with ACV)/(average virus titer in the wells without ACV). The mutation frequency of HSV type 1 isolates in clinical samples (directly from patient lesions) was 7.5 × 10
−4
± 2.5 × 10
−4
(mean ± standard error), and that of HSV type 2 isolates was 15.0 × 10
−4
± 4.6 × 10
−4
. The mutation frequencies of isolates derived in the laboratory from these clinical samples were very similar. Similarly, the 50% inhibitory concentrations for isolates in clinical samples and laboratory isolates were identical. The frequencies of ACV-resistant HSV types 1 and 2 were in a narrow range of 7.5 × 10
−4
to 15.0 × 10
−4
among isolates in clinical specimens and did not change for laboratory-derived pools of viral isolates.
Genetic Diversity of
Neisseria lactamica
Strains from Epidemiologically Defined Carriers
Authors: Alber Dirk , Oberkötter Mark , Suerbaum Sebastian , Claus Heike , Frosch Matthias , Vogel Ulrich
Published: July 2002

We assessed the genetic diversity of 26
Neisseria lactamica
strains from epidemiologically related sources, i.e., groups of kindergartens and primary schools in three Bavarian towns, by the partial sequencing of the
argF, rho, recA
, and 16S ribosomal genes. We found a total of 17 genotypes, of which 12 were found only in one strain. The genotypes comprised 5 alleles of the
argF
gene, 9 of
rho
, 8 of
recA
, and 10 of the 16S ribosomal DNA. Sequence analysis by determination of homoplasy ratios and split decomposition analysis revealed abundant recombination within
N. lactamica
.
Simultaneous Detection of
Mycobacterium leprae
and Its Susceptibility to Dapsone Using DNA Heteroduplex Analysis
Authors: Williams Diana L. , Pittman Tana L. , Gillis Thomas P. , Matsuoka Masanori , Kashiwabara Yoshiko
Published: July 2002

Currently recommended control measures for treating leprosy with multidrug therapy should control the spread of drug-resistant strains; however, dapsone (DDS) resistance continues to be reported. Comprehensive estimates of drug-resistant leprosy are difficult to obtain due to the cumbersome nature of the conventional drug susceptibility testing method using mouse footpad inoculation, which requires at least 6 months to obtain results. Recently, it has been determined that DDS-resistant strains contain missense mutations in codon 53 or 55 of the
folP1
gene of
Mycobacterium leprae
, and definitive evidence linking these mutations with DDS resistance in
M. leprae
has been obtained. Based on these mutations, a heteroduplex DDS
M. leprae
(HD-DDS-ML) assay was developed for the simultaneous detection of
M. leprae
and of its susceptibility to DDS. The assay relies on the PCR amplification of an
M. leprae
-specific 231-bp fragment of
folP1
containing codons 53 and 55. The PCR products are allowed to anneal to a universal heteroduplex generator, and the separation of the resultant DNA duplexes is accomplished by polyacrylamide gel electrophoresis.
M. leprae
was detected in crude cell lysates of skin biopsy specimen homogenates from eight leprosy patients and from
M. leprae
-infected mouse or armadillo tissues infected with 14 separate strains using the HD-DDS-ML assay. The assay was specific for
M. leprae
in a comparison with results obtained from 14 species of mycobacteria other than
M. leprae
and four bacterial species known to colonize human skin. The HD-DDS-ML assay detected as few as 100
M. leprae
organisms present in homogenates of human skin and demonstrated a 93% correlation with DDS susceptibility as determined by both DNA sequencing of
folP1
and mouse footpad susceptibility testing. The HD-DDS-ML assay provides a new tool for the simultaneous detection of
M. leprae
and of its susceptibility to DDS from a single specimen. The assay should prove useful for drug resistance surveillance in leprosy control programs when combined with similar molecular tests developed for other drug resistance markers.
Unwinding of a Herpes Simplex Virus Type 1 Origin of Replication (Ori
S
) by a Complex of the Viral Origin Binding Protein and the Single-Stranded DNA Binding Protein
Authors: He Xiaodun , Lehman I. R.
Published: July 2002

A herpes simplex virus type 1 (HSV-1) Ori
S
analogue in which the A+T sequence linking the box I and II elements was replaced by two single-stranded oligo(dT)s is unwound by the UL9 protein-ICP8 complex. Unwinding of wild-type Ori
S
by the UL9 protein-ICP8 complex was also observed under conditions which destabilize the A+T sequence. These experiments support a model for the unwinding of Ori
S
in which destabilization of the A+T sequence can generate a single-stranded DNA binding site for ICP8, which then associates with the UL9 protein bound to boxes I and II to promote the bidirectional unwinding of Ori
S
.
Association of RNA Polymerase Complexes of the Parasitic Protozoan
Cryptosporidium parvum
with Virus-Like Particles: Heterogeneous System
Authors: Khramtsov Nikolai V. , Upton Steve J.
Published: July 2002

RNA polymerase complexes were purified from
Cryptosporidium parvum
, a parasitic protozoan known to infect many species of mammals including humans. Western blot analysis revealed the association of the complexes with two different proteins, encoded by large and small segments of viral double-stranded RNAs. Each complex was found to contain only double-stranded RNA, both double- and single-stranded RNA, or only single-stranded RNA. Maximum RNA-dependent RNA polymerase activity was observed within the complexes containing both double- and single-stranded RNAs. These complexes possessed both transcriptase and replicase polymerase activities. Virus-like particles with a diameter of 31 nm were copurified with RNA polymerase complexes, and buoyant density and polymerase studies suggest that
C. parvum
harbors a putative double-stranded RNA virus which separately encapsidates the large and small RNA segments. The mechanism of replication and other characteristics of this virus are similar to those of the viruses of the family
Partitiviridae
, previously identified only in fungi and plants.
Increased Neutralization Sensitivity and Reduced Replicative Capacity of Human Immunodeficiency Virus Type 1 after Short-Term In Vivo or In Vitro Passage through Chimpanzees
Authors: Beaumont Tim , Broersen Silvia , van Nuenen Ad , Huisman Han G. , de Roda Husman Ana-Maria , Heeney Jonathan L. , Schuitemaker Hanneke
Published: July 2002

Development of disease is extremely rare in chimpanzees when inoculated with either T-cell-line-adapted neutralization-sensitive or primary human immunodeficiency virus type 1 (HIV-1), at first excluding a role for HIV-1 neutralization sensitivity in the clinical course of infection. Interestingly, we observed that short-term in vivo and in vitro passage of primary HIV-1 isolates through chimpanzee peripheral blood mononuclear cells (PBMC) resulted in a neutralization-sensitive phenotype. Furthermore, an HIV-1 variant reisolated from a chimpanzee 10 years after experimental infection was still sensitive to neutralization by soluble CD4, the CD4 binding site recognizing antibody IgG1b12 and autologous chimpanzee serum samples, but had become relatively resistant to neutralization by polyclonal human sera and neutralizing monoclonal antibodies. The initial adaptation of HIV-1 to replicate in chimpanzee PBMC seemed to coincide with a selection for viruses with low replicative kinetics. Neither coreceptor usage nor the expression level of CD4, CCR5, or CXCR4 on chimpanzee PBMC compared to human cells could explain the phenotypic changes observed in these chimpanzee-passaged viruses. Our data suggest that the increased neutralization sensitivity of HIV-1 after replication in chimpanzee cells may in part contribute to the long-term asymptomatic HIV-1 infection in experimentally infected chimpanzees.
Functional Role of Residues Corresponding to Helical Domain II (Amino Acids 35 to 46) of Human Immunodeficiency Virus Type 1 Vpr
Authors: Singh Satya P. , Tomkowicz Brian , Lai Derhsing , Cartas Maria , Mahalingam Sundarasamy , Kalyanaraman Vaniambadi S. , Murali Ramachandran , Srinivasan Alagarsamy
Published: July 2002

Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G
2
, nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLΔVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprΔ35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization, unlike other mutants and control Vpr-FL. While wild-type Vpr registered cell cycle arrest at G
2
, mutant Vpr showed an intermediary effect with the exception of VprΔ35-50 and VprΔ35-50-H. These results suggest that residues in the HII domain are essential for Vpr functions.
CXC-Chemokine Receptor 4 Is Not a Coreceptor for Human Herpesvirus 7 Entry into CD4+T Cells
Authors: Zhang Ying , Hatse Sigrid , De Clercq Erik , Schols Dominique
Published: July 2002

Human herpesvirus 7 (HHV-7) is a T-lymphotropic virus which utilizes the CD4 receptor as its main receptor to enter the target cells. Hence, HHV-7 can interfere with human immunodeficiency virus type 1 (HIV-1) infection in CD4+T cells. It was recently suggested that the CXC chemokine receptor 4 (CXCR4), which was found to be a crucial coreceptor for T-tropic HIV-1 strains, may also play a role in the HHV-7 infection process. However, the results presented here demonstrate that CXCR4 is not involved in HHV-7 infection. The natural ligand of CXCR4, SDF-1α, was not able to inhibit HHV-7 infection in SupT1 cells or in CD8+T-cell-depleted peripheral blood mononuclear cells. Also, AMD3100, a specific CXCR4 antagonist with potent antiviral activity against T-tropic HIV strains (50% inhibitory concentration [IC50], 1 to 10 ng/ml), completely failed to inhibit HHV-7 infection (IC50, >250 μg/ml). Thus, two different agents known to specifically interact with CXCR4 were not able to inhibit HHV-7 infection. Other T-lymphoid cell lines, expressing both CD4 and CXCR4 (e.g., HUT-78 and MT-4) could not be infected by HHV-7. In addition, the CD4-transfected cell lines HOS.CD4 and U87.CD4 and the CD4/CXCR4 double-transfected cell lines HOS.CD4.CXCR4 and U87.CD4.CXCR4 were not infectable with HHV-7. Also, we found no down-regulation of surface-bound or intracellular CXCR4 in HHV-7-infected CD4+T cells. As compared to uninfected SupT1 cells, stromal cell-derived factor 1α (SDF-1α)/CXCR4-mediated intracellular calcium flux was unchanged in SupT1 cells that were acutely or persistently infected with HHV-7. All these data argue against CXCR4 as a receptor involved in the HHV-7 infection process.
Nuclear Accumulation of IE62, the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein, Is Inhibited by Phosphorylation Mediated by the VZV Open Reading Frame 66 Protein Kinase
Authors: Kinchington Paul R. , Fite Karen , Turse Stephanie E.
Published: July 2002

IE62, the major transcriptional activator protein encoded by varicella-zoster virus (VZV), locates to the nucleus when expressed in transfected cells. We show here that cytoplasmic forms of IE62 accumulate in transfected and VZV-infected cells as the result of the protein kinase activity associated with VZV open reading frame 66 (ORF66). Expression of the ORF66 protein kinase but not the VZV ORF47 protein kinase impaired the ability of coexpressed IE62 to transactivate promoter-reporter constructs. IE62 that was coexpressed with the ORF66 protein accumulated predominantly in the cytoplasm, whereas the normal nuclear localization of other proteins was not affected by the ORF66 protein. In cells infected with VZV, IE62 accumulated in the cytoplasm at late times of infection, whereas in cells infected with a VZV recombinant unable to express ORF66 protein (ROka66S), IE62 was completely nuclear. Point mutations introduced into the predicted serine/threonine catalytic domain and ATP binding domain of ORF66 abrogated its ability to influence IE62 nuclear localization, indicating that the protein kinase activity was required. The region of IE62 that was targeted by ORF66 was mapped to amino acids 602 to 733. IE62 peptides containing this region were specifically phosphorylated in cells coexpressing the ORF66 protein kinase and in cells infected with wild-type VZV but were not phosphorylated in cells infected with ROka66S. We conclude that the ORF66 protein kinase phosphorylates IE62 to induce its cytoplasmic accumulation, most likely by inhibiting IE62 nuclear import.
Naturally Occurring TAP-Dependent Specific T-Cell Tolerance for a Variant of an Immunodominant Retroviral Cytotoxic T-Lymphocyte Epitope
Authors: Kim Victor , Yewdell Jonathan W. , Green William R.
Published: July 2002

Upon immunization and restimulation with tumors induced by the endogenous AKR/Gross murine leukemia virus (MuLV), C57BL/6 mice generate vigorousH-2Kb-restricted cytotoxic T-lymphocyte (CTL) responses to a determinant (KSPWFTTL) derived from the p15E transmembrane portion of the viral envelope glycoprotein. By contrast, the highly homologous determinant RSPWFTTL, expressed by tumor cells induced by Friend/Moloney/Rauscher (FMR) MuLV, is not immunogenic, even when presented to the immune system as vaccinia virus-encoded cytosolic or endoplasmic reticulum (ER)-targeted minigene products. Such minigene products are usually highly immunogenic since they bypass the need for cells to liberate the peptide or transport the peptide into the ER by the transporter associated with antigen processing (TAP). Using KSPWFTTL-specific CTLs that cross-react with RSPWFTTL, we previously demonstrated that presentation of RSPWFTTL from its natural viral gene product is TAP dependent. Here, we show first that C57BL/6 mice express mRNA encoding RSPWFTTL but not KSPWFTTL and second that the ER-targeted RSPWFTTL minigene product is highly immunogenic in C57BL/6 mice with a targeted deletion in TAP1. These findings provide the initial demonstration of TAP-dependent tolerance induction to a specific self peptide and demonstrate that this contributes to the differential recognition of RSPWFTTL and KSPWFTTL by C57BL/6 mice.
Active and Selective Transcytosis of Cell-Free Human Immunodeficiency Virus through a Tight Polarized Monolayer of Human Endometrial Cells
Authors: Hocini Hakim , Becquart Pierre , Bouhlal Hicham , Chomont Nicolas , Ancuta Petronela , Kazatchkine Michel D. , Bélec Laurent
Published: July 2002

We report that both primary and laboratory-adapted infectious human immunodeficiency virus type 1 (HIV-1) isolates in a cell-free form are capable of transcytosis through a tight and polarized monolayer of human endometrial cells. Trancytosis of cell-free HIV occurs in a strain-selective fashion and appears to be dependent on interactions between HIV envelope glycoproteins and lectins on the apical membrane of the epithelial cells. These findings provide new insights into the initial events occurring during heterosexual transmission of the virus.
Functional Interaction between Fluorodeoxyuridine-Induced Cellular Alterations and Replication of a Ribonucleotide Reductase-Negative Herpes Simplex Virus
Authors: Petrowsky Henrik , Roberts Gretchen D. , Kooby David A. , Burt Bryan M. , Bennett Joseph J. , Delman Keith A. , Stanziale Stephen F. , Delohery Thomas M. , Tong William P. , Federoff Howard J. , Fong Yuman
Published: July 2002

G207 is an oncolytic herpes simplex virus (HSV) which is attenuated by inactivation of viral ribonucleotide reductase (RR) and deletion of both
γ

1

34.5
genes. The cellular counterparts that can functionally substitute for viral RR and the carboxyl-terminal domain of ICP34.5 are cellular RR and the corresponding homologous domain of the growth arrest and DNA damage protein 34 (GADD34), respectively. Because the thymidylate synthetase (TS) inhibitor fluorodeoxyuridine (FUdR) can alter expression of cellular RR and GADD34, we examined the effect of FUdR on G207 bioactivity with the hypothesis that FUdR-induced cellular changes will alter viral proliferation and cytotoxicity. Replication of wild-type HSV-1 was impaired in the presence of 10 nM FUdR, whereas G207 demonstrated increased replication under the same conditions. Combined use of FUdR and G207 resulted in synergistic cytotoxicity. FUdR exposure caused elevation of RR activity at 10 and 100 nM, whereas GADD34 was induced only at 100 nM. The effect of enhanced viral replication by FUdR was suppressed by hydroxyurea, a known inhibitor of RR. These results demonstrate that the growth advantage of G207 in FUdR-treated cells is primarily based on an RR-dependent mechanism. Although our findings show that TS inhibition impairs viral replication, the FUdR-induced RR elevation may overcome this disadvantage, resulting in enhanced replication of G207. These data provide the cellular basis for the combined use of RR-negative HSV mutants and TS inhibitors in the treatment of cancer.
Simian Virus 40 Vp1 DNA-Binding Domain Is Functionally Separable from the Overlapping Nuclear Localization Signal and Is Required for Effective Virion Formation and Full Viability
Authors: Li Peggy P. , Nakanishi Akira , Shum Dorothy , Sun Peter C.-K. , Salazar Adler M. , Fernandez Cesar F. , Chan Sze-Wai , Kasamatsu Harumi
Published: July 2002

A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major capsid protein Vp1, and the domain's function in the SV40 life cycle was examined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1ΔC58 pentamer bound DNA with a

K
d

of 1.8 × 10
−9
M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1ΔC17 had comparable apparent

K
d

s of 5.3 × 10
−9
to 7.3 × 10
−9
M in terms of the protein monomers. Previously identified on Vp1 was a nuclear localization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4-KRK-6) and NLS2 (15-KKPK-18). Vp1ΔC58 pentamers harboring multiple-point mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1 · 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1ΔC58 level for NLSm1 · 2 Vp1. These data, along with those of N-terminally truncated proteins, placed the DBD in overlap with the bipartite NLS. The role of the Vp1 DBD during infection was investigated by taking advantage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209–8216, 1994), in which an NLS-defective Vp1 could localize to the nucleus in the presence of wild-type minor capsid proteins Vp2 and Vp3. This approach made it possible to dissect the role of the bifunctional Vp1 NLS-DBD in virion assembly in the nucleus. Mutants of the viable nonoverlaping SV40 (NO-SV40) DNA NLSm1, NLSm2, and NLSm1 · 2 replicated normally following transfection into host cells and produced capsid proteins at normal levels. All mutant Vp1s were able to interact with Vp3 in vitro. The mutants NLSm1 and NLSm1 · 2 were nonviable, and the mutant Vp1s unexpectedly failed to localize to the nucleus though Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant signal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the mutant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, displayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected cell lysate revealed a 10-fold reduction in the level of DNase I-protected viral DNA, and yet virion-like particles were found among the DNase I-resistant material. Collective results support a role for Vp1 NLS2-DBD2 in the assembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.
Effective Human Herpesvirus 8 Infection of Human Umbilical Vein Endothelial Cells by Cell-Mediated Transmission
Authors: Sakurada Shinsaku , Katano Harutaka , Sata Tetsutaro , Ohkuni Hisashi , Watanabe Toshiki , Mori Shigeo
Published: July 2002

Cell-free transmission of human herpesvirus 8 (HHV-8) to human cells in vitro has been reported to be difficult, if not impossible. The present experiments were conducted with the idea that cell-cell contact may produce much more effective transmission, so-called cell-mediated transmission. Primary human umbilical vein endothelial cells (HUVECs) were cocultured with an HHV-8-infected lymphoma cell line, BCBL-1 cells. When a ratio of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated BCBL-1 cells to HUVECs of 10:1 was used, more than 20% of HUVECs were found to express the HHV-8 latency-associated nuclear antigen (LANA) 48 h after the start of coculturing; this value increased to more than 30% after 72 h. HHV-8-encoded ORF26, K8, K8.1, K10, K11, ORF59, and ORF65 proteins were not detected in these HHV-8-infected HUVECs until 72 h. The HHV-8 antigens were not observed in HUVECs cocultured with TPA-treated BCBL-1 cells separated by a membrane. Thirty days after removal of the BCBL-1 cells from the cell-mediated transmission experiment, the HUVECs still expressed LANA and the HHV-8 genome was detected by PCR in these cells. Moreover, the ORF59 protein, a DNA replication-associated protein of HHV-8, was expressed in such HUVECs in the presence of TPA stimulation. These results indicated a far more effective transmission mechanism, cell-cell contact, suggesting the possibility that such a mechanism works in vivo.
Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins
Authors: Satoi Jujin , Murata Kazumoto , Lechmann Martin , Manickan Elanchezhiyan , Zhang Zhensheng , Wedemeyer Heiner , Rehermann Barbara , Liang T. Jake
Published: July 2002

To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.
Propagation of Rat Parvovirus in Thymic Lymphoma Cell Line C58(NT)D and Subsequent Appearance of a Resistant Cell Clone after Lytic Infection
Authors: Ueno Yutaka , Harada Tanenobu , Iseki Hiroyoshi , Ohshima Takayuki , Sugiyama Fumihiro , Yagami Ken-ichi
Published: July 2002

Rat parvovirus (RPV) is nonpathogenic in rats but causes persistent lymphocytotropic infection. We found that RPV was propagated in rat thymic lymphoma cell line C58(NT)D and induced apoptosis. Interestingly, a resistant subclone, C58(NT)D/R, from surviving cells after lytic infection had differentiated phenotypic modifications, such as increased cell adherence, resistance to apoptosis, and suppressed tumorigenicity.
Derivation and Characterization of a Dengue Type 1 Host Range-Restricted Mutant Virus That Is Attenuated and Highly Immunogenic in Monkeys
Authors: Markoff Lewis , Pang Xiaou , Houng Huo-shu , Falgout Barry , Olsen Raymond , Jones Estella , Polo Stephanie
Published: July 2002

We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3′ stem-loop structure (3′-SL) that is conserved among all flavivirus species. The 3′-SL constitutes the downstream terminal ∼95 nucleotides of the 3′ noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a human-virulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3′-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.
Mutations That Confer Resistance to Template-Analog Inhibitors of Human Immunodeficiency Virus (HIV) Type 1 Reverse Transcriptase Lead to Severe Defects in HIV Replication
Authors: Fisher Timothy S. , Joshi Pheroze , Prasad Vinayaka R.
Published: July 2002

We isolated two template analog reverse transcriptase (RT) inhibitor-resistant mutants of human immunodeficiency virus (HIV) type 1 RT by using the DNA aptamer, RT1t49. The mutations associated, N255D or N265D, displayed low-level resistance to RT1t49, while high-level resistance could be observed when both mutations were present (Dbl). Molecular clones of HIV that contained the mutations produced replication-defective virions. All three RT mutants displayed severe processivity defects. Thus, while biochemical resistance to the DNA aptamer RT1t49 can be generated in vitro via multiple mutations, the overlap between the aptamer- and template-primer-binding pockets favors mutations that also affect the RT-template-primer interaction. Therefore, viruses with such mutations are replication defective. Potent inhibition and a built-in mechanism to render aptamer-resistant viruses replication defective make this an attractive class of inhibitors.
Synthesis of Some 1,2,4-Triazolo[4,3-c]quinazolines Based on 4-Quinazolylthiosemicarbazides
Authors: Špirková Katarína , Stankovský Štefan , Dandárová Miloslava
Published: July 2002

The paper describes the cyclization reactions of substituted 1-(4'-quinazolyl)-4-phenylthiosemicarbazides (Ia - Ie). The thermal intramolecular cyclization gives 2H-1,2,4-triazolo[4,3-c]quinazoline-3-thiones (IIa - IId). Heating of I with HgO gives 3-anilino-1,2,4-triazolo[4,3-c]quinazolines (IIIa - IIIe). The IR and 1H NMR spectra of the compounds synthesized are presented.
The Diffusion Flow to a Rotating Line Electrode
Authors: Kimla Arnošt , Míčka Jiří
Published: July 2002

A mathematical model for convective diffusion to the so-called line electrode was solved by combination of the analytical and numerical approaches. All terms of the diffusion equation were respected. A semiempirical formula was derived, extending appreciably its applicability with respect to the decisive physical and geometric parameters.
Dependence of Gas Chromatographic Retention Data of Hydrocarbons on the Film Thickness of the Polydimethylsiloxane Stationary Phase
Authors: Krupčík Ján , Skačáni Ivan , Benická Eva , Sandra Pat
Published: July 2002

Kovats indices of cyclic and aromatic hydrocarbons, separated by capillary gas liquid chromatography on polydimethylsiloxane capillary columns, were found to increase with increasing stationary phase film thickness. This effect is explained in terms of adsorption of the stationary phase on the active sites of the inner surface of the capillary column. Since the number of active sites is limited, the overall polarity of the polydimethylsiloxane stationary phase is better defined in columns with thick stationary phase films. Interlaboratory reproducibility of retention indices of cyclic and aromatic hydrocarbons is therefore also better when using capillary columns with thick films of the polydimethylsiloxane stationary phase. Retention data obtained on such thick-film columns are influenced by adsorption of the stationary phase on the column walls to a lesser extent than in columns with thinner films.
Some Aspects of Discrete Relaxation Time Spectra as Obtained from Dynamic Moduli Data
Authors: Jakeš Jaromír
Published: July 2002

The problem of finding a relaxation time spectrum best fitting dynamic moduli data in the least-squares sense is shown to be well-posed and to yield a discrete spectrum, provided the data cannot be fitted exactly, i.e., without any deviation of data and calculated values. Properties of the resulting spectrum are discussed. Examples of discrete spectra obtained from simulated literature data and experimental literature data on polymers are given. The problem of smoothing discrete spectra when continuous ones are expected is discussed. A detailed study of an integral transform inversion under the non-negativity constraint is given in Appendix.
The Poisoning Effect of Co3O4 on γ-Al2O3 and MgO Supports During the Decomposition of Isopropyl Alcohol
Authors: Halawy Samih A. , Mohamed Mohamed A. , Abd El-Hafez Suzan F.
Published: July 2002

Catalysts containing 20 wt.% Co3O4 supported on γ-Al2O3 and MgO were prepared. The catalyst precursors were analyzed by TG, DTG and DTA, and the calcined catalysts (400-700 °C) were characterized by XRD. The amount of excess surface oxygen for the catalysts was estimated iodometrically. The vapour-phase decomposition of isopropyl alcohol over these catalysts was studied. Co3O4/γ-Al2O3 catalysts are selective towards propene formation, whereas Co3O4/MgO catalysts are selective towards acetone formation. The poisoning effect of Co3O4 on both supports and the activity of the catalysts is discussed.
Pregnane-Type Brassinosteroids with a Four-Carbon Ester Functionality in Position 20
Authors: Kohout Ladislav , Kasal Alexander , Strnad Miroslav
Published: July 2002

Analogues of brassinolide and castasterone, containing an ester functionality with four carbon atoms in the position 20 of the pregnane skeleton, have been prepared. In the bean second internode assay the most effective compounds were (20R)-2α,3α,20-trihydroxy-5α-pregnan-6-one 20-isobutyrate (6d) and (20R)-2α,3α,20-trihydroxy-B-homo-7-oxa-5α-pregnan-6-one 20-isobutyrate (7d). On the other hand, (20R)-2α,3α,20-trihydroxy-B-homo-6-oxa-5α-pregnan-7-one 20-heptafluorobutyrate (8) was the first compound with the retarding effect in the same test.
N-1-Trimethylsilyl Derivatives of Ergot Alkaloids
Authors: Křen Vladimír , Sedmera Petr
Published: July 2002

N-1-Trimethylsilyl derivatives of five different suitably protected parent ergot (clavine) alkaloids (agroclavine 1a, elymoclavine 2a, lysergol 3a, lysergene 4a, and 9,10-dihydrolysergol 5a) were prepared in 47-94% yields by refluxing the (protected) parent compounds with N-methyl-N-(trimethylsilyl)trifluoroacetamide in acetonitrile under nitrogen atmosphere.
Dipole Moments of 2- and 4-Substituted 9-Isothiocyanatoacridines
Authors: Danihel Ivan , Suchár Gejza , Kristian Pavol , Böhm Stanislav
Published: July 2002

Dipole moments of a series of 2- and 4-substituted 9-isothiocyanatoacridines were determined and compared with the values calculated by vector addition using bond and group dipole moments. The results are discussed from the viewpoint of geometrical and conformational structures of the compounds investigated.
ansa-Vanadocene Complexes - Syntheses, Structures and Ligand Exchange Reactions
Authors: Dorer Birgit , Prosenc Marc-Heinrich , Rief Ursula , Brintzinger Hans-Herbert
Published: July 2002

Tetramethylethano-bridged vanadocene dichlorides (CH3)4C2(3-R-C5H3)2VCl2 with R = H, Me, t-Bu are obtained by reacting the corresponding ligand MgCl salts with vanadium(III) acetylacetonato compounds, followed by oxidation with PCl3 or AgCl. Smooth ligand exchange reactions of these ansa-vanadocene dichlorides afford acetylacetonato cations and binaphtholate complexes; from these, the dichlorides are regenerated by exposure to MgCl2 in THF solution. Separation of meso and rac isomers (as well as enantiomer separation) is achieved via corresponding binaphtholate complexes; conversion to the vanadium(III) diisonitrile cations (CH3)4C2(3-R-C5H3)2V(CNt-Bu)2+ allows an assessment of diastereoisomer ratios by 1H NMR. Lewis-acidic bistriflate derivatives of these ansa-vanadocene complexes are obtained from the dichlorides by reaction with silver triflate.
Determination of Butyltin Compounds in Sediments by GC/MS After Their Conversion to Methyl Derivatives
Authors: Milde David , Plzák Zbyněk , Suchánek Miloslav
Published: July 2002

A selective and rapid procedure was developed for the determination of trace amounts of butyltin compounds in sediments by capillary gas chromatography with mass spectrometric detection. Analytes were isolated from the sediment samples by extraction with a methanolic solution of HCl (0.5 mol l-1) followed by reextraction into a hexane solution of tropolone (0.1%). The organotin compounds were converted to the nonpolar methyl derivatives by methylation with a Grignard reagent. The recovery was determined for each step of the procedure. This efficient separation method combined with the ion trap based mass detection ensures a high selectivity even for concentrations approaching the detection limit (0.15 pg for Bu3SnMe) in real sediments. Chemical ionization using acetonitrile as the reaction gas, exhibiting a similar sensitivity of determination and reducing the background level, can serve as a convenient alternative of detection for sediments heavily contaminated by organics. The method was applied to a set of real samples and validated by analysis of a certified reference material.
Efficient Stereoselective Synthesis of All Geometrical Isomers of Heptadeca-11,13-dienes
Authors: Svatoš Aleš , Šaman David
Published: July 2002

All geometrical isomers of heptacosa-11,13-dienes, 1-4, previously identified in termite Prorhinotermes simplex cuticular hydrocarbons, were efficiently synthesized according Peterson-Hudrlik olefination procedure in high stereoisomeric purity using syn and anti elimination of erythro alkenyl-β-hydroxysilanes (15 and 17). These (Z)- and (E)-alkenyl-β-hydroxysilanes are available from regioselective opening of (1R*,2S*)-1,2-epoxy-1-trimethylsilylpentadecane 13 with corresponding (Z)- and (E)-dodec-1-enyl cuprates (14 and 16). Stereoisomeric purity of obtained dienes 1-4 was higher than 95% (13C NMR).
Glycation of Human Serum Albumin by DL-Glyceraldehyde: A Fluorescence Quenching Study
Authors: Štěpánek Miroslav , Pavlíček Zdeněk
Published: July 2002

Process of glycation of human serum albumin (HSA) by DL-glyceraldehyde was studied using steady-state and time-resolved fluorescence spectroscopy in the course of 100 h. During this period, measurements of steady-state tryptophan (Trp) and non-tryptophan (non-Trp) fluorescence of the glycated HSA were carried out, together with measurement of the non-Trp fluorescence decay and steady-state quenching using potassium iodide as a quencher. Observed changes in both Trp and non-Trp fluorescence intensity, as well as changes in non-Trp fluorescence lifetimes and quenching efficiency are explained with respect to a probable mechanism of glycation.
Crystal Structure of Cyclosporin E
Authors: Hušák Michal , Kratochvíl Bohumil , Buchta Martin , Cvak Ladislav , Jegorov Alexandr
Published: July 2002

The structure of cyclosporin E was determined by X-ray diffraction methods and compared with the structure of related cyclosporins. In contrast to cyclosporin A, which crystallizes from acetone as tetragonal dihydrate, cyclosporin E acetone solvate monohydrate (C61H109N11O12·C3H6O·H2O) crystallizes under the same conditions in the monoclinic space group P21 with a = 15.698(2) Å, b = 21.333(3) Å, c = 13.224(2) Å, β = 103.74(1)°, Z = 2, and V = 4 302(1) Å3.
Formation of a Double Salt and Mixed Crystals in the Ca(HCOO)2-Cd(HCOO)2-H2O System
Authors: Vassileva Violeta Z.
Published: July 2002

By solubility method in the Ca(HCOO)2-Cd(HCOO)2-H2O system at 25 and 50 °C, the formation of a double salt CaCd(HCOO)4 and mixed crystals Ca1-xCdx(HCOO)2 was established. The double salt formation is explained by the electronic configuration of the metal ions involved. Formation of the mixed crystals, possessing the α-Ca(HCOO)2 structure, is attributed to the ability of the Cd2+ ions to accept the coordination environment, characteristic of the Ca2+ ions in α-Ca(HCOO)2.
Steric Effects in the Base-Catalyzed Cyclization of 1-[2-(Methoxycarbonyl)phenyl]-3-(2-substituted Phenyl)triazenes
Authors: Ludwig Miroslav , Bauerová Ingrid
Published: July 2002

Eleven model 1-[2-(methoxycarbonyl)phenyl]-3-(2-substituted phenyl)triazenes were synthesized and their cyclization kinetics examined in aqueous-methanolic buffer solutions (51 wt.% methanol) at various pH values. 3(2-Substituted phenyl)benzo[d][1,2,3]triazin-4(3H)-ones were identified as the cyclization products. The log kobs vs pH plot was linear with a slope of unity. Investigation of the steric and electronic effects of substituents in the ortho position revealed that substituents at the ring which is bonded to the N(3) nitrogen affect the cyclization rate through their steric effect only, while their electronic effects are statistically insignificant. This fact was explained in terms of the ring being tilted from the plane in which the remaining part of the conjugate base anion of the model substrate lies. The assumed and confirmed BAc2 mechanism involving specific base catalysis begins by deprotonation of the triazene giving rise to the conjugate base, continues with formation of a tetrahedral intermediate, and ends with elimination of the methanolate ion. Other mechanisms, such as the elimination-addition mechanism via a ketene intermediate or the mechanism involving general base catalysis, are unlikely.
Synthesis and Antinociceptive Activity of Some 3-Chlorophenyl- and 6-Chloropyridin-2-yl Derivatives
Authors: Rádl Stanislav , Hafner Wieland , Hezký Petr , Krejčí Ivan , Proška Jan , Hájíček Josef
Published: July 2002

Derivatives of 2-chloro-6-(4-hydroxy-1-methylpiperidin-4-yl)pyridine (2b) were prepared and tested as analgesics. 2-Chloro-6-lithiopyridine treated with quinuclidin-3-one, 1-methylpyrrolidin-3-one, 2-(dimethylaminomethyl)cyclohexanone, and ethyl 1-methylpiperidin-4-ylcarboxylate provided the corresponding alcohols 5, 6, 13a, and 6-chloro-2-pyridyl 1-methylpiperidin-4-yl ketone (9). The ketone was reduced with sodium borohydride or treated with methylmagnesium chloride or phenyllithium to provide the corresponding alcohols 11, 12a and 12b, respectively. 1-[4-(6-Chloro-2-pyridyl)1-methylpiperidin-4-yl]-1-methylethanol (4b) was prepared from 2-chloro-6-(1-methyl-1,2,5,6-tetrahydropyridin-4-yl)pyridine (14b) by treatment with butyllithium and acetone followed by reduction of intermediate 15b with sodium cyanoborohydride.
Synthesis and Antimicrobial Activity of New 4-(Benzylsulfanyl)pyridine Derivatives
Authors: Klimešová Věra , Svoboda Martin , Waisser Karel , Pour Milan , Kaustová Jarmila
Published: July 2002

A series of 4-(benzylsulfanyl)pyridine-2-carbonitriles 2 and 4-(benzylsulfanyl)pyridine-2- carbothioamides 3 was synthesized and their antimycobacterial and antifungal activities were tested. The prepared compounds exhibit significant activity both against M. tuberculosis and nontuberculous mycobacteria. The most active compounds show the MICs of 4 μmol/l. The antifungal activities of 2 are very weak, compounds 3 are inactive against all the tested fungi.
Convenient Synthesis of More Complex 2-Substituted 4(3H)-Quinazolinones via Lithiation of 2-Alkyl-4(3H)-quinazolinones
Authors: Smith Keith , El-Hiti Gamal A. , Abdel-Megeed Mohamed F. , Abdo Mohamed A.
Published: July 2002

2-Methylquinazolin-4(3H)-one has been doubly lithiated, at nitrogen and in the 2-methyl group, with n-butylithium. The lithium reagent thus obtained reacts with a variety of electrophiles (iodomethane, D2O, phenyl isocyanate, benzaldehyde, benzophenone, cyclopentanone, 2-butanone, carvone) to give the corresponding 2-substituted derivatives in very good yields. Reaction of the dilithio reagent with acetonitrile gives an α,β-unsaturated imine by tautomerization of the initial addition product. Double lithiation of 2-ethyl- and 2-propyl-4(3H)-quinazolinones can be achieved using lithium diisopropylamide and the lithiated reagents thus obtained react with similar electrophiles to give the corresponding products in very good yields. In the reaction of the dianion of the 2-ethyl-4(3H)-quinazolinone with iodine, an oxidatively dimerised product was obtained. Lithiation of 2-unsubstituted 4(3H)-quinazolinone does not take place on C-2 under similar conditions.