bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteolytic Activity of YibP Protein in
Escherichia coli
Authors: Ichimura Toshiharu , Yamazoe Mitsuyoshi , Maeda Maki , Wada Chieko , Hiraga Sota
Published: July 2002

Escherichia coli
YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family. A
yibP
disrupted mutant grows in rich medium at 37°C but not at 42°C. In the
yibP
null mutant, cell division and FtsZ ring formation are inhibited at 42°C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis. Five percent betaine suppresses the temperature sensitivity of the
yibP
disrupted mutation. The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain. YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction. Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space. Purified YibP has a protease activity that split the substrate β-casein.
Expression System for High Levels of GAG Lyase Gene Expression and Study of the
hepA
Upstream Region in
Flavobacterium heparinum
Authors: Blain Françoise , Tkalec A. Lydia , Shao Zhongqi , Poulin Catherine , Pedneault Marc , Gu Kangfu , Eggimann Bernhard , Zimmermann Joe , Su Hongsheng
Published: July 2002

A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in
Flavobacterium heparinum
is described.
hepA
, along with its regulatory region, as well as
hepB
,
hepC
,
cslA
, and
cslB
, cloned downstream of the
hepA
regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type
F. heparinum
grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type
F. heparinum
grown in chondroitin sulfate A-only medium. The
hepA
upstream region was also studied using
cslA
as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for
hepA
in both the wild-type
F. heparinum
and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.
Type 4 Pilus Biogenesis and Type II-Mediated Protein Secretion byVibrio choleraeOccur Independently of the TonB-Facilitated Proton Motive Force
Authors: Bose Niranjan , Payne Shelley M. , Taylor Ronald K.
Published: July 2002

InVibrio cholerae, elaboration of toxin-coregulated pilus and protein secretion by the extracellular protein secretion apparatus occurred in the absence of both TonB systems. In contrast, the cognate putative ATPases were required for each process and could not substitute for each other.
Comparison of Roche MONITOR and Organon Teknika NucliSens Assays To Quantify Human Immunodeficiency Virus Type 1 RNA in Cerebrospinal Fluid
Authors: Spearman Paul , Fiscus Susan A. , Smith Rita M. , Shepard Robin , Johnson Benjamin , Nicotera Janet , Harris Victoria L. , Clough Lisa A. , McKinsey Joel , Haas David W.
Published: July 2002

We compared Roche MONITOR and Organon Teknika NucliSens assays for human immunodeficiency virus type 1 (HIV-1) RNA in cerebrospinal fluid (CSF). Results of 282 assays were highly correlated (
r
= 0.826), with MONITOR values being 0.29 ± 0.4 log
10
copies/ml (mean ± standard deviation) values. Both assays can reliably quantify HIV-1 RNA in CSF.
Tomato Yellow Leaf Curl Geminivirus (TYLCV-Is) Is Transmitted among Whiteflies (
Bemisia tabaci
) in a Sex-Related Manner
Authors: Ghanim Murad , Czosnek Henryk
Published: July 2002

Tomato yellow leaf curl virus (TYLCV) is the name given to a complex of geminiviruses infecting tomato cultures worldwide. TYLCV is transmitted by a single insect species, the whitefly
Bemisia tabaci
. Herein we show that a TYLCV isolate from Israel (TYLCV-Is) can be transmitted among whiteflies in a sex-dependent manner, in the absence of any other source of virus. TYLCV was transmitted from viruliferous males to females and from viruliferous females to males but not among insects of the same sex. Transmission took place when insects were caged in groups or in couples, in a feeding chamber or on cotton plants, a TYLCV nonhost. The recipient insects were able to efficiently inoculate tomato test plants. Insect-to-insect virus transmission was instrumental in increasing the number of whiteflies capable of infecting tomato test plants in a whitefly population. TYLCV was present in the hemolymph of whiteflies caged with viruliferous insects of the other sex; therefore, the virus follows, at least in part, the circulative pathway associated with acquisition from infected plants. Taken as a whole, these results imply that a plant virus can be sexually transmitted from insect to insect.
Replacement of the Ectodomains of the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Recombinant Parainfluenza Virus Type 3 (PIV3) with Their Counterparts from PIV2 Yields Attenuated PIV2 Vaccine Candidates
Authors: Tao Tao , Skiadopoulos Mario H. , Davoodi Fatemeh , Riggs Jeffrey M. , Collins Peter L. , Murphy Brian R.
Published: July 2002

We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated
cp
45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955–2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503–510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the
cp
45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.
Murine Gammaherpesvirus 68 Cyclin D Homologue Is Required for Efficient Reactivation from Latency
Authors: Hoge Ann T. , Hendrickson Sara B. , Burns William H.
Published: July 2002

Murine gammaherpesvirus 68 (MHV68) is a gammaherpesvirus that was first isolated from murid rodents. MHV68 establishes a latent infection in the spleen and other lymphoid organs. Several gammaherpesviruses, including herpesvirus saimiri, human herpesvirus 8, and MHV68, encode proteins with extensive homology to the D-type cyclins. To study the function of the cyclin homologue, a recombinant MHV68 has been constructed that lacks the cyclin homologue and expresses β-galactosidase as a marker (MHV68cy−). MHV68cy−grows in vitro with kinetics and to titers similar to those of the wild type. BALB/c mice infected with mixtures of equivalent amounts of the wild type and MHV68cy−show deficient growth of the MHV68cy−in an acute infection. Infection of SCID mice with virus mixtures also showed decreased MHV68cy−virus growth, indicating that the deficiency is not mediated by T or B cells. Although mice infected with mixtures containing 100 times as much MHV68cy−had greater splenic titers of the mutant virus than wild-type virus in acute infection, at 28 days postinfection splenocytes from these mice reactivated primarily wild-type virus. Quantitative PCR data indicate that equivalent genomes were present in the latent state. Reinsertion of the cyclin homologue into the cyclin-deleted virus restored the wild-type phenotype. These results indicate that the MHV68 cyclin D homologue mediates important functions in the acute infection and is required for efficient reactivation from latency.
Dynamics of CCR5 Expression by CD4+T Cells in Lymphoid Tissues during Simian Immunodeficiency Virus Infection
Authors: Veazey Ronald S. , Mansfield Keith G. , Tham Irene C. , Carville Angela C. , Shvetz Daniel E. , Forand Amy E. , Lackner Andrew A.
Published: July 2002

Early viral replication and profound CD4+T-cell depletion occur preferentially in intestinal tissues of macaques infected with simian immunodeficiency virus (SIV). Here we show that a much higher percentage of CD4+T cells in the intestine express CCR5 compared with those found in the peripheral blood, spleen, or lymph nodes. In addition, the selectivity and extent of the CD4+T-cell loss in SIV infection may depend upon these cells coexpressing CCR5 and having a “memory” phenotype (CD45RA−). Following intravenous infection with SIVmac251, memory CD4+CCR5+T cells were selectively eliminated within 14 days in all major lymphoid tissues (intestine, spleen, and lymph nodes). However, the effect on CD4+T-cell numbers was most profound in the intestine, where cells of this phenotype predominate. The CD4+T cells that remain after 14 days of infection lacked CCR5 and/or were naive (CD45RA+). Furthermore, when animals in the terminal stages of SIV infection (with AIDS) were examined, virtually no CCR5-expressing CD4+T cells were found in lymphoid tissues, and all of the remaining CD4+T cells were naive and coexpressed CXCR4. These findings suggest that chemokine receptor usage determines which cells are targeted for SIV infection and elimination in vivo.
Antigenic Variation within the CD4 Binding Site of Human Immunodeficiency Virus Type 1 gp120: Effects on Chemokine Receptor Utilization
Authors: Hammond Anthea L. , Lewis Julie , May Jackie , Albert Jan , Balfe Peter , McKeating Jane A.
Published: July 2002

To assess the antigenicity of envelope glycoproteins derived from primary human immunodeficiency virus type 1 populations, their interactions with the receptor CD4, and their coreceptor usage, we have cloned and expressed multiple gp120 proteins from a number of primary virus isolates. Characterization of these proteins showed a high degree of antigenic polymorphism both within the CD4 binding site and in defined neutralization epitopes, which may partially account for the general resistance of primary isolates to neutralizing agents. Furthermore, chimeric viruses expressing gp120 proteins with reduced CD4 binding abilities are viable, suggesting that primary viruses may require a less avid interaction with the receptor CD4 to initiate infection than do their laboratory-adapted counterparts. The coreceptor usage of chimeric viruses was related to the ability of the virus to bind CD4, with reduced CD4 binding correlating with preferential usage of CXCR4. Changes in coreceptor usage mapped to sequence changes in the C2 and V4 regions, with no changes seen in the V3 region.
RNA Recombination between Persisting Pestivirus and a Vaccine Strain: Generation of Cytopathogenic Virus and Induction of Lethal Disease
Authors: Becher Paul , Orlich Michaela , Thiel Heinz-Jürgen
Published: July 2002

Molecular analysis of a cytopathogenic (cp) bovine viral diarrhea virus (BVDV) isolate (1741) obtained from a case of mucosal disease (MD) led to the identification of five different viral subgenomic RNAs in addition to a noncytopathogenic (noncp) strain (NCP 1741). For each of the subgenomes, a large internal deletion was found together with an inserted sequence encoding part of ribosomal protein S27a fused to an N-terminally truncated ubiquitin monomer. Surprisingly, the two cellular insertions together with flanking viral sequences encoding parts of NS3 and NS4B are >99% identical to the previously described sequence of BVDV vaccine strain RIT (P. Becher, M. Orlich, and H.-J. Thiel, J. Virol. 72:8697–8704, 1998), while the remainder of the subgenomes is derived from the genome of NCP 1741. Further analyses including molecular cloning and nucleotide sequencing of the recombination partners revealed that both homologous and nonhomologous RNA recombination contributed to the generation of the viral subgenomes. Interestingly, for another cp BVDV isolate (CP 4584) from an independent case of MD, again an insertion of a RIT-derived sequence element was detected. In contrast to CP 1741, for CP 4584 a duplication of the genomic region encoding NS3 and parts of NS4A and NS4B was found. Transfection of bovine cells with RNA transcribed from a chimeric cDNA construct showed that the RIT-derived insertion together with the CP 4584-specific duplication of viral sequences represents the genetic basis of cytopathogenicity of CP 4584. Remarkably, passages of the recovered cp virus in cell culture led to emergence of noncp BVDV and a number of viral subgenomes whose genome organization was similar to that in BVDV 1741.
Induction and Characterization of Neutralizing Antibodies against a Human Immunodeficiency Virus Type 1 Primary Isolate
Authors: Chen Chin-Ho , Jin Lei , Zhu Chongbin , Holz-Smith Sonia , Matthews Thomas J.
Published: July 2002

Chimpanzees infected with the primary isolate DH012 mount potent neutralizing antibodies. This DH012 neutralizing activity is highly strain specific. Immune sera from guinea pigs immunized with recombinant DH012 gp120 could also neutralize this primary isolate. The neutralizing activity in chimpanzee and guinea pig sera against wild-type DH012 appears to be independent of a linear epitope in the V3 region of gp120. Interestingly, the neutralization escape mutant derived from growing DH012 in the presence of the potent neutralizing chimpanzee serum is at least 50-fold more sensitive than wild-type DH012 to neutralization by guinea pig immune sera. The unusually potent neutralizing activity against the DH012 neutralization-resistant virus is due to the presence of anti-V3 antibodies in guinea pig sera. These results suggested that recombinant gp120 could induce neutralizing antibodies against primary isolate DH012. The V3 of wild-type DH012 is poorly immunogenic in infected chimpanzees and is not accessible to neutralizing V3 antibodies. It is likely that this cryptic V3 region became exposed when the virus escaped the neutralizing activity of the chimpanzee serum.
No Selection for CCR5 Coreceptor Usage during Parenteral Transmission of Macrophagetropic Syncytium-Inducing Human Immunodeficiency Virus Type 1
Authors: Koning Fransje A. , Schols Dominique , Schuitemaker Hanneke
Published: July 2002

In two cases of parenteral transmission of human immunodeficiency virus type 1 (HIV-1) syncitium-inducing (SI) variants, we previously observed selection for macrophagetropic variants. Although infection of macrophages is generally mediated via CCR5, we found no selection for SI variants that could use CCR5 as coreceptor in addition to CXCR4, suggesting that features other than coreceptor usage account for the macrophagetropism of these transmitted SI HIV-1 variants.
Evidence for Similar Recognition of the Conserved Neutralization Epitopes of Human Immunodeficiency Virus Type 1 Envelope gp120 in Humans and Macaques
Authors: Malenbaum Susan E. , Yang David , Cheng-Mayer Cecilia
Published: July 2002

We compared the immune responses to the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins in humans and macaques with the use of clade A and clade B isogenic V3 loop glycan-possessing and -deficient viruses. We found that the presence or absence of the V3 loop glycan affects to similar extents immune recognition by a panel of anti-HIV human and anti-simian/human immunodeficiency virus (anti-SHIV) macaque sera. All sera tested neutralized the glycan-deficient viruses, in which the conserved CD4BS and CD4i epitopes are more exposed, better than the glycan-containing viruses. The titer of broadly neutralizing antibodies appears to be higher in the sera of macaques infected with glycan-deficient viruses. Collectively, our data add legitimacy to the use of SHIV-macaque models for testing the efficacy of HIV-1 Env-based immunogens. Furthermore, they suggest that antibodies to the CD4BS and CD4i sites of gp120 are prevalent in human and macaque sera and that the use of immunogens in which these conserved neutralizing epitopes are more exposed is likely to increase their immunogenicity.
Cross-Linking of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Template-Primer
Authors: Peletskaya Elena N. , Boyer Paul L. , Kogon Alex A. , Clark Patrick , Kroth Heiko , Sayer Jane M. , Jerina Donald M. , Hughes Stephen H.
Published: July 2002

Cross-linking experiments were performed with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Two approaches were used—photoaffinity cross-linking and disulfide chemical cross-linking (using an oligonucleotide that contained an N
2
-modified dG with a reactive thiol group). In the former case, cross-linking can occur to any nucleotide in either DNA strand, and in the latter case, a specific cross-link is produced between the template and the enzyme. Neither the introduction of the unique cysteine residues into the fingers nor the modification of these residues with photocross-linking reagents caused a significant decrease in the enzymatic activities of RT. We were able to use this model system to investigate interactions between specific points on the fingers domain of RT and double-stranded DNA (dsDNA). Photoaffinity cross-linking of the template to the modified RTs with Cys residues in positions 65, 67, 70, and 74 of the fingers domain of the p66 subunit was relatively efficient. Azide-modified Cys residues produced 10 to 25% cross-linking, whereas diazirine modified residues produced 5 to 8% cross-linking. Disulfide cross-linking yields were up to 90%. All of the modified RTs preferentially photocross-linked to the 5′ extended template strand of the dsDNA template-primer substrate. The preferred sites of interactions were on the extended template, 5 to 7 bases beyond the polymerase active site. HIV-1 RT is quite flexible. There are conformational changes associated with substrate binding. Cross-linking was used to detect intramolecular movements associated with binding of the incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreases the efficiency of cross-linking, but causes only modest changes in the preferred positions of cross-linking. This suggests that the interactions between the fingers of p66 and the extended template involve the “open” configuration of the enzyme with the fingers away from the active site rather than the closed configuration with the fingers in direct contact with the incoming dNTP. This experimental approach can be used to measure distances between any site on the surface of the protein and an interacting molecule.
Vaccinia Virus Intracellular Movement Is Associated with Microtubules and Independent of Actin Tails
Authors: Ward Brian M. , Moss Bernard
Published: July 2002

Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens
Listeria
and
Shigella
, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr
112
and Tyr
132
of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 μm/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.
Interleukin-4 Up-Regulates Mouse Mammary Tumor Virus Expression yet Is Not Required for In Vivo Virus Spread
Authors: Czarneski Jennifer , Meyers Jennifer , Peng Tao , Abraham Valsamma , Mick Rosemarie , Ross Susan R.
Published: July 2002

The mouse mammary tumor virus (MMTV) superantigen induces T-cell production of cytokines, such as interleukin-4, which in turn increase MMTV transcription. However, interleukin-4 is not required for in vivo virus spread, because mice lacking interleukin-4 or the STAT6 transcription factor showed wild-type infection of lymphoid and mammary tissue. In spite of this, mammary tumor incidence was decreased in STAT6 null mice.
Signals for Bidirectional Nucleocytoplasmic Transport in the Duck Hepatitis B Virus Capsid Protein
Authors: Mabit Helene , Breiner Klaus M. , Knaust Andreas , Zachmann-Brand Beate , Schaller Heinz
Published: July 2002

Hepadnavirus genome replication involves cytoplasmic and nuclear stages, requiring balanced targeting of cytoplasmic nucleocapsids to the nuclear compartment. In this study, we analyze the signals determining capsid compartmentalization in the duck hepatitis B virus (DHBV) animal model, as this system also allows us to study hepadnavirus infection of cultured primary hepatocytes. Using fusions to the green fluorescent protein as a functional assay, we have identified a nuclear localization signal (NLS) that mediates nuclear pore association of the DHBV nucleocapsid and nuclear import of DHBV core protein (DHBc)-derived polypeptides. The DHBc NLS mapped is unique. It bears homology to repetitive NLS elements previously identified near the carboxy terminus of the capsid protein of hepatitis B virus, the human prototype of the hepadnavirus family, but it maps to a more internal position. In further contrast to the hepatitis B virus core protein NLS, the DHBc NLS is not positioned near phosphorylation target sites that are generally assumed to modulate nucleocytoplasmic transport. In functional assays with a knockout mutant, the DHBc NLS was found to be essential for nuclear pore association of the nucleocapsid. The NLS was found to be also essential for virus production from the full-length DHBV genome in transfected cells and from hepatocytes infected with transcomplemented mutant virus. Finally, the DHBc additionally displayed activity indicative of a nuclear export signal, presumably counterbalancing NLS function in the productive state of the infected cell and thereby preventing nucleoplasmic accumulation of nucleocapsids.
Molecular and Functional Dissection of the H-2Db-Restricted Subdominant Cytotoxic T-Cell Response to Lymphocytic Choriomeningitis Virus
Authors: Hudrisier Denis , Riond Joëlle , Gairin Jean Edouard
Published: July 2002

Infection ofH-2bmice with lymphocytic choriomeningitis virus (LCMV) generates an H-2Db-restricted cytotoxic T-lymphocyte (CTL) response whose subdominant component is directed against the GP92-101 (CSANNSHHYI) epitope. The aim of this study was to identify the functional parameters accounting for this subdominance. We found that the two naturally occurring (genetically encoded and posttranslationally modified) forms of LCMV GP92-101 were immunogenic, did not act as T-cell antagonists, and bound efficiently to but were unable to form stable complexes with H-2Db, a crucial factor for immunodominance. Thus, the H-2Db-restricted subdominant CTL response to LCMV resulted not from altered T-cell activation but from impaired major histocompatibility complex presentation properties.
Novel, Live Attenuated Simian Immunodeficiency Virus Constructs Containing Major Deletions in Leader RNA Sequences
Authors: Guan Yongjun , Whitney James B. , Liang Chen , Wainberg Mark A.
Published: July 2002

We have constructed a series of simian immunodeficiency virus (SIV) mutants containing deletions within a 97-nucleotide (nt) region of the leader sequence. Deletions in this region markedly decreased the replication capacity in tissue culture, i.e., in both the C8166 and CEMx174 cell lines, as well as in rhesus macaque peripheral blood mononuclear cells. In addition, these deletions adversely affected the packaging of viral genomic RNA into virions, the processing of Gag precursor proteins, and patterns of viral proteins in virions, as assessed by biochemical labeling and polyacrylamide gel electrophoresis. Different levels of attenuation were achieved by varying the size and position of deletions within this 97-nt region, and among a series of constructs that were generated, it was possible to rank in vitro virulence relative to that of wild-type virus. In all of these cases, the most severe impact on viral replication was observed when the deletions that were made were located at the 3′ rather than 5′ end of the leader region. The potential of viral reversion over protracted periods was investigated by repeated viral passage in CEMx174 cells. The results showed that several of these constructs showed no signs of reversion after more than 6 months in tissue culture. Thus, a series of novel, attenuated SIV constructs have been developed that are significantly impaired in replication capacity yet retain all viral genes. One of these viruses, termed SD4, may be appropriate for study with rhesus macaques, in order to determine whether reversions will occur in vivo and to further study this virus as a candidate for attenuated vaccination.
Coalescence of the Sites of Cowpea Mosaic Virus RNA Replication into a Cytopathic Structure
Authors: Carette Jan E. , Gühl Kerstin , Wellink Joan , Van Kammen Ab
Published: July 2002

Cowpea mosaic virus (CPMV) replication induces an extensive proliferation of endoplasmic reticulum (ER) membranes, leading to the formation of small membranous vesicles where viral RNA replication takes place. Using fluorescent in situ hybridization, we found that early in the infection of cowpea protoplasts, CPMV plus-strand RNA accumulates at numerous distinct subcellular sites distributed randomly throughout the cytoplasm which rapidly coalesce into a large body located in the center of the cell, often near the nucleus. The combined use of immunostaining and a green fluorescent protein ER marker revealed that during the course of an infection, CPMV RNA colocalizes with the 110-kDa viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, indicating that these sites correspond to the membranous site of viral replication. Experiments with the cytoskeleton inhibitors oryzalin and latrunculin B point to a role of actin and not tubulin in establishing the large central structure. The induction of ER membrane proliferations in CPMV-infected protoplasts did not coincide with increased levels of BiP mRNA, indicating that the unfolded-protein response is not involved in this process.
Prior Vaccination Increases the Epitopic Breadth of the Cytotoxic T-Lymphocyte Response That Evolves in Rhesus Monkeys following a Simian-Human Immunodeficiency Virus Infection
Authors: Santra Sampa , Barouch Dan H. , Kuroda Marcelo J. , Schmitz Jörn E. , Krivulka Georgia R. , Beaudry Kristin , Lord Carol I. , Lifton Michelle A. , Wyatt Linda S. , Moss Bernard , Hirsch Vanessa M. , Letvin Norman L.
Published: July 2002

Although recent evidence has confirmed the importance of cytotoxic T-lymphocyte (CTL) responses in controlling human immunodeficiency virus type 1 and simian immunodeficiency virus replication, the relevance of the epitopic breadth of those CTL responses remains unexplored. In the present study, we sought to determine whether vaccination can expand CTL populations which recognize a repertoire of viral epitopes that is greater than is typically generated in the course of a viral infection. We demonstrate that potent secondary CTL responses to subdominant epitopes are rapidly generated following a pathogenic simian-human immunodeficiency virus challenge of rhesus monkeys vaccinated with plasmid DNA or recombinant modified vaccinia virus Ankara vaccines. These data indicate that prior vaccination can increase the breadth of the CTL response that evolves after an AIDS virus infection.
trans-Complementation Rescue of Cyclophilin A-Deficient Viruses Reveals that the Requirement for Cyclophilin A in Human Immunodeficiency Virus Type 1 Replication Is Independent of Its Isomerase Activity
Authors: Saphire Andrew C. S. , Bobardt Michael D. , Gallay Philippe A.
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) requires the incorporation of cyclophilin A (CypA) for replication. CypA is packaged by binding to the capsid (CA) region of Gag. This interaction is disrupted by cyclosporine (CsA). Preventing CypA incorporation, either by mutations in the binding region of CA or by the presence of CsA, abrogates virus infectivity. Given that CypA possesses an isomerase activity, it has been proposed that CypA acts as an uncoating factor by destabilizing the shell of CA that surrounds the viral genome. However, because the same domain of CypA is responsible for both its isomerase activity and its capacity to be packaged, it has been challenging to determine if isomerase activity is required for HIV-1 replication. To address this issue, we fused CypA to viral protein R (Vpr), creating a Vpr-CypA chimera. Because Vpr is packaged via the p6 region of Gag, this approach bypasses the interaction with CA and allows CypA incorporation even in the presence of CsA. Using this system, we found that Vpr-CypA rescues the infectivity of viruses lacking CypA, either produced in the presence of CsA or mutated in the CypA packaging signal of CA. Furthermore, a Vpr-CypA mutant which has no isomerase activity and no capacity to bind to CA also rescues HIV-1 replication. Thus, this study demonstrates that the isomerase activity of CypA is not required for HIV-1 replication and suggests that the interaction of the catalytic site of CypA with CA serves no other function than to incorporate CypA into viruses.
Genomic but Not Antigenomic Hepatitis Delta Virus RNA Is Preferentially Exported from the Nucleus Immediately after Synthesis and Processing
Authors: Macnaughton Thomas B. , Lai Michael M. C.
Published: July 2002

Hepatitis delta virus (HDV) contains a viroid-like circular RNA that replicates via a double rolling circle replication mechanism. It is generally assumed that HDV RNA is synthesized and remains exclusively in the nucleus until being exported to the cytoplasm for virion assembly. Using a [
32
P]orthophosphate metabolic labeling procedure to study HDV RNA replication (T. B. Macnaughton, S. T. Shi, L. E. Modahl, and M. M. C. Lai. J. Virol. 76:3920-3927, 2002), we unexpectedly found that a significant amount of newly synthesized HDV RNA was detected in the cytoplasm. Surprisingly, Northern blot analysis revealed that the genomic-sense HDV RNA is present almost equally in both the nucleus and cytoplasm, whereas antigenomic HDV RNA was mostly retained in the nucleus, suggesting the specific and highly selective export of genomic HDV RNA. Kinetic studies showed that genomic HDV RNA was exported soon after synthesis. However, only the monomer and, to a lesser extent, the dimer HDV RNAs were exported to the cytoplasm; very little higher-molecular-weight HDV RNA species were detected in the cytoplasm. These results suggest that the cleavage and processing of HDV RNA may facilitate RNA export. The export of genomic HDV RNA was resistant to leptomycin B, indicating that a cell region maintenance 1 (Crm1)-independent pathway was involved. The large form of hepatitis delta antigen (L-HDAg), which is responsible for virus packaging, was not required for RNA export, as a mutant HDV RNA genome unable to synthesize L-HDAg was still exported. The proportions of genomic HDV RNA in the nucleus and cytoplasm remained relatively constant throughout replication, indicating that export of genomic HDV RNA occurred continuously. In contrast, while antigenomic HDV RNA was predominately in the nucleus, there was a proportionally large fraction of antigenomic HDV RNA in the cytoplasm at early time points of RNA replication. These findings uncover a previously unrecognized presence of HDV RNA in the cytoplasm, which may have implications for viral RNA synthesis and packaging.
Structure of Sesquiterpenic Lactones of Some Species of Subtribe Centaureinae DUMORT.
Authors: Buděšínský Miloš , Nowak Gerard , Rychłewska Urszula , Hodgson Derek J. , Šaman David , Daniewski Włodzimierz M. , Drożdż Bohdan , Holub Miroslav
Published: July 2002

The authors describe in full the elucidation of complete structures of guaianolide and germacranolide lactones, isolated from some species of genera Centaurea L., Chartolepis CASS. and others belonging to subtribe Centaureinae DUMORT. The structures were determined mainly on the basis of detailed analysis of 1H NMR and CD spectra. The structure of linichlorin B (XV) was determined by X-ray diffraction analysis.
Dehalogenation of Chlorobenzenes with Sodium Dihydridobis(2-methoxyethoxo)aluminate
Authors: Včelák Jaroslav , Friesová Anna , Řeřicha Roman , Hetflejš Jiří
Published: July 2002

The dehalogenation of a series of 9 mono- to pentachlorinated benzenes with the title hydride in toluene has been found to be first order in the substrate and half order in the hydride. The reactivities of the chlorobenzenes, expressed by rate constants for the first-step dehalogenation, increased with increasing number of chlorine atoms over three orders of magnitude. The rate data revealed the unexpected acceleration of benzene formation during exhaustive dehalogenation of the higher chlorinated benzenes. For comparison, dehalogenation of several isomeric dibromobenzenes and bromochlorobenzenes with the same hydride and the product distribution for the dehalogenation of some chlorobenzenes with LiAlH4 are also reported.
1H, 13C and 15N NMR Spectra of Ni(II) Complex of Schiff Base from (S)-2-(N-Benzylprolyl)amino-5-methylbenzophenone and Glycine
Authors: Jirman Josef , Popkov Alexander
Published: July 2002

Nickel(II) complex of Schiff base derived from (S)-2-(N-benzylprolyl)amino-5-methylbenzophenone and glycine (I) was prepared. It was characterized by mass, 1H, 13C and 15N NMR spectra. The NMR signals were assigned and the data thus obtained can serve for study of products in the asymmetric synthesis of amino acids, starting from this complex.
Tethered Chains in Concave Volumes. A Monte Carlo Study
Authors: Procházka Karel , Limpouchová Zuzana
Published: July 2002

Monte Carlo study of tethered chain conformations in spherical cavities was performed in a relatively broad range of average segment densities (i.e. numbers of tethered chains with increasing length in the sphere). Simulations were performed on a tetrahedral lattice using (i) an equilibrated self-avoiding walk for systems containing a single tethered chain with increasing length, and (ii) a simultaneous self-avoiding walk of many tethered chains in the spherical cavity together with equilibration of the system which was performed by a modified algorithm similar to that of Siepmann and Frenkel. Only a geometric excluded volume effect of segments was considered (i.e. the prohibition principle of a double occupancy of one lattice site by two different segments). Various distribution functions (e.g. distribution of the end-to-end and the end-to-gravity center distances and their orientations with respect either to the radial direction, or to the direction of the first-to-second segment connection, etc.) were calculated and the effect of increasing average segment density in the sphere on conformational characteristics of individual chains was studied. It was found that conformational and orientational properties of relatively short tethered chains are only little affected by increasing segment density (i.e. by the number of chains in the spherical cavity), whereas arrangements of long tethered chains are significantly influenced by the density of the system.
Donor Numbers for Binary Mixtures of Dimethyl Sulfoxide with Dipolar Aprotic Solvents
Authors: Rzeszotarska Jadwiga , Ranachowski Przemyslaw , Kalinowski Marek K.
Published: July 2002

Solvent effect on the visible absorption spectrum of (acetylacetonato)(N,N,N',N'-tetramethylethylene-diamine)copper(II) was applied to estimate DN, the donor numbers of the binary mixtures of dimethyl sulfoxide with a series of other dipolar aprotic solvents. It was found that resulting DN values may be treated as explanatory parameters in the description of the mixed solvent effect on the stability constants of complexes formed by some macrocyclic ligands with univalent cations.
4-Aryl-3-substituted Furans
Authors: Štetinová Jarmila , Leško Ján , Dandárová Miloslava , Kada Rudolf , Koreň Roman
Published: July 2002

4-Aryl-3-substituted furans IIIa - IIId were prepared by Diels Alder reaction of 4-phenyloxazole (I) with compounds IIa - IId, containing an activated triple bond, and subsequent thermal decomposition of the cycloadduct.
Adsorption and Accumulation of Neutral Red at Mercury/Solution Interface
Authors: Ibrahim Mohamed S. , Ahmed Zahra A. , Kamal Moustafa M. , Temerk Yassein M.
Published: July 2002

The voltammetric behaviour of protonated and nonprotonated neutral red (NR) has been studied at a hanging mercury drop electrode (HMDE) by using alternating current (AC) and cyclic voltammetry (CV). The AC voltammetric behaviour and the morphology of the adsorption redox peaks indicate that interactions between adsorbed molecules are substantial and that the nature of these interactions changes from moderately repulsive below pH 4 to strongly attractive above pH 6. Controlled adsorptive accumulation of NR on the HMDE provides the basis for the direct stripping measurement of that compound in the nanomolar concentration level. Adsorptive phase selective AC stripping voltammetry has been proved to be advantageous over any other assay technique, allowing a detection limit of 1.0 . 10-9 M NR to be reached.
Novel Products in the CO2-Laser Induced Reaction of Trichloroethylene
Authors: Earl Boyd L. , Titus Richard L.
Published: July 2002

Previous reports on the thermal or CO2-laser induced decomposition of trichloroethylene have identified only one condensible product, hexachlorobenzene (in addition to HCl and mono- and dichloroacetylene). We have found that trichloroethylene vapor exposed to cw irradiation on the P(24) line of the (001 - 100) band of the CO2 laser at incident power levels from 8 - 17 W produces numerous products, of which the 13 major ones have been identified using IR, GC/MS, GC/FTIR, and NMR methods. All of these products have 4, 6, or 8 carbons, are highly unsaturated, and are completely chlorinated or contain a single hydrogen. C4HCl5 and C6Cl6 isomers (three of each) account for S 55% to 85% of total products (based on peak areas in the total ion chromatograms in GC/MS runs), depending on reaction conditions. In addition to characterizing the products, we discuss the dependence of the product distribution on laser power, irradiation time, and cell geometry, and we outline a possible mechanism.
Synthesis and Biological Activity of Four Vasopressin Analogs Modified in Position 3 with β-Thienylalanine
Authors: Slaninová Jiřina , Czaja Malgorzata , Lammek Bernard
Published: July 2002

Four new analogs of arginine-vasopressin substituted in position 3 with β-thienylalanine were synthesized on chloromethylated resin using Boc strategy and DCC or DCC-HOBt to form peptide bonds. The activity of the agonists is not much different in comparison to the unsubstituted compounds. However, the potency of one of the antagonists is strongly reduced.
Substituted 1-(2-Thienyl)cyclohexylamines and Related Compounds as Potential NMDA Antagonists
Authors: Šindelář Karel , Dobrovský Karel , Krejčí Ivan , Polívka Zdeněk
Published: July 2002

Reactions of bisulfite addition compounds prepared in situ from cyclohexanone or 3,4,5,6-tetrahydro-2H-thiopyran-3-one with potassium cyanide and corresponding amines resulted with high yields in amino nitriles type of V and VI. These compounds were subjected to reactions with 2-thienylmagnesium bromide and in the case of the amino nitriles Vc and Vf with 5-bromo-2-thienylmagnesium bromide. Only in the case of compound Vf, a by-product X was isolated in addition to the desired 1-[1-(5-bromo-2-thienyl)-1-cyclohexyl]piperidine (VIIf). Compound VIIf was used for the synthesis of the carboxylic acid XI. The compounds prepared were tested by some methods of biochemical and behavioural pharmacology.
Synthesis of Guanosine-3'-(5-bromo-4-chloroindol-3-yl)p-hosphate (G-3'-BCIP)
Authors: Haikal Abdelfattah
Published: July 2002

N-2-Benzoyl-5'-O-dimethoxytrityl-2'-O-tetrahydropyranylguanosine (5) and 1-acetyl-5-bromo-4-chloroindol-3-yl-3-phosphorodichloridate (7) were synthesized and coupled to give the title compound 9.
Sterically Crowded Heterocycles. VII. Reduction of Some (Z)-1,3-Diphenyl-3-(2-phenylimidazo[1,2-a]pyridin-3-yl)prop-2-en-1-ones as Their Axial Chirality Probe
Authors: Kubík Richard , Böhm Stanislav , Kuthan Josef
Published: July 2002

Borohydride reduction of titled ketones 1a-1g gave diastereoisomeric mixtures of (Z)-1,3-diphenyl-3-(2-phenylimidazo[1,2-a]pyridin-3-yl)prop-2-en-1-ols 2a-2g and 3a-3g in which the former ones prevailed. Only individual racemic products were obtained after borohydride reduction of (E)-1,3-diphenyl-3-(2-phenylimidazo[1,2-a]pyridin-3-yl)-prop-2-en-1-one 4 to corresponding 1-hydroxy derivative 5 and by conversion of (Z)-1-oxo derivative 1a to 1,3-diphenyl-3-(2-phenylimidazo[1,2-a]pyridin-3-yl)propan-1-one (6) with sodium hydrogenselenide. Diastereoselectivity of the borohydride reduction is discussed using the PM3 calculations of the molecules 1a, 2a, 2b, 3a, 3b, 4, 5, and 6.
Synthesis and Antituberculotic Activity of 5-Alkyl-6-chloro-2-pyrazinecarboxamides and Corresponding Thioamides
Authors: Hartl Jiří , Doležal Martin , Krinková Jana , Lyčka Antonín , Odlerová Želmíra
Published: July 2002

Homolytic alkylation of 6-chloro-2-pyrazinecarbonitrile by alkanoic acid and subsequent partial hydrolysis afforded 5-alkyl-6-chloro-2-pyrazinecarboxamides 1a-1e. Reaction of amides 1a-1e by Lawesson's reagent afforded corresponding thioamides 2a-2e. The structure of compounds was confirmed by elemental analysis, IR and 1H NMR spectra. The assessment of in vitro antimycobacterial activity of the compounds was carried out. The highest antituberculotic activity against Mycobacterium tuberculosis and other mycobacterial strains in this series was shown by 5-(1,1-dimethylethyl)-6-chloro-2-pyrazinecarbothioamide (2e).
The Effect of Acidity of Al and Fe Silicates with MFI Structure on Benzene and Toluene Alkylation with Isopropyl Alcohol
Authors: Čejka Jiří , Žilková Naděžda , Wichterlová Blanka
Published: July 2002

Kinetic study of toluene and benzene alkylation with isopropyl alcohol on alumo- and ferrisilicates of MFI structure has shown that the alkylation activity does not follow the acidity (both the number and strength of bridging OH groups) of these molecular sieves. The rate of the overall reaction is controlled by the desorption/transport rate of bulky, strongly adsorbed cymenes and cumene. A higher concentration of n-propyltoluenes compared to n-propylbenzene, both undesired reaction products, formed via a bimolecular isomerization of isopropyl aromate with benzene or toluene, was due to the higher reactivity of isopropyltoluene with toluene in comparison with that of cumene with benzene. It is concluded that ferrisilicates of MFI structure possessing low strength acid sites appear to be promising catalysts for achieving both a high isopropyl- and para-selectivity in toluene alkylation to p-cymene.
Phenyl-Induced 3,1,11-Rearrangement in the Synthesis of Mixed Pyrrolyl/Dicarbollide Cobaltadicarbaboranes
Authors: Bertran Josep , Viñas Clara , Gomez Sílvia , Lamrani Mouad , Teixidor Francesc , Sillanpää Reijo , Kivekäs Raikko
Published: July 2002

Mixed pyrrolide cobaltadicarbollides have been prepared from the reaction involving potassium pyrrolide, anhydrous CoCl2 and the [7,8-(C6H5)2-nido-7,8-C2B9H10]- anion in dimethoxyethane. An alternative and higher-yield route consists in the reaction between 1,2-(C6H5)2-closo-1,2-C2B10H10 and potassium pyrrolide in the presence of anhydrous CoCl2. As confirmed by the X-ray diffraction analysis of [3-(η5-NC4H4)-1,11-(C6H5)2-closo-3,1,11-CoC2B9H9], the phenyl rings were found to enhance the rearrangement of the metallacarborane cluster carbons to produce the 3,1,11-isomer instead of the expected 3,1,2-derivative. As suggested by 1H and 13C NMR spectroscopy, the electron-withdrawing properties and steric requirements of the phenyl rings facilitate the rearrangement.
Donnan Equilibria in Polymeric Micellar Systems with Weak Polyelectrolyte Shells: The Lowering of the pH Value
Authors: Munk Petr , Tuzar Zdeněk , Procházka Karel
Published: July 2002

When two electrolyte solutions are separated and only some of the ions can cross the boundary, the concentrations of these ions are different on both sides of the boundary. This is the well-known Donnan effect. When weak electrolytes are involved, the imbalance includes also hydrogen ions: there is a difference of pH across the boundary and the dissociation of nondiffusible weak electrolytes is suppressed. The effect is very pronounced when the concentration of the weak electrolyte is high and ionic strength is low. The significance of this phenomenon is discussed for polyelectrolyte solutions, and particularly for block copolymer micelles with weak polyelectrolyte shells. The effect is quite dramatic in the latter case.