bluuebunny/crossref_metadata_2025 huggingface dataset:
Ascaris suum-Derived Products Induce Human Neutrophil Activation via a G Protein-Coupled Receptor That Interacts with the Interleukin-8 Receptor Pathway
Authors: Falcone Franco H. , Rossi Adriano G. , Sharkey Rose , Brown Alan P. , Pritchard David I. , Maizels Rick M.
Published: July 2002

Infection with tissue-migrating helminths is frequently associated with intense granulocyte infiltrations. Several host-derived factors are known to mediate granulocyte recruitment to the tissues, but less attention has been paid to how parasite-derived products trigger this process. Parasite-derived chemotactic factors which selectively recruit granulocytes have been described, but nothing is known about which cellular receptors respond to these agents. The effect of products from the nematodesAscaris suum, Toxocara canis, andAnisakis simplexon human neutrophils were studied. We monitored four parameters of activation: chemotaxis, cell polarization, intracellular Ca2+transients, and priming of superoxide anion production. Body fluids ofA. suum(ABF) andT. canis(TcBF) induced strong directional migration, shape change, and intracellular Ca2+transients. ABF also primed neutrophils for production of superoxide anions. Calcium mobilization in response toA. suum-derived products was completely abrogated by pretreatment with pertussis toxin, implicating a classical G protein-coupled receptor mechanism in the response to ABF. Moreover, pretreatment with interleukin-8 (IL-8) completely abrogated the response to ABF, demonstrating desensitization of a common pathway. However, ABF was unable to fully desensitize the response to IL-8, and binding to CXCR1 or CXCR2 was excluded in experiments using RBL-2H3 cells transfected with the two human IL-8 receptors. Our results provide the first evidence for a direct interaction between a parasite-derived chemotactic factor and the host's chemotactic network, via a novel G protein-coupled receptor which interacts with the IL-8 receptor pathway.
Identification and Cloning of a Cryptococcal Deacetylase That Produces Protective Immune Responses
Authors: Biondo Carmelo , Beninati Concetta , Delfino Demetrio , Oggioni Marco , Mancuso Giuseppe , Midiri Angelina , Bombaci Mauro , Tomaselli Giuseppe , Teti Giuseppe
Published: July 2002

Cell-mediated immunity plays a crucial role in host defenses againstCryptococcus(Filobasidiella)neoformans. Therefore, the identification of cryptococcal antigens capable of producing T-cell-mediated responses, such as delayed-type hypersensitivity (DTH) reactions, may be useful in the development of immune-based strategies to control cryptococcosis. In order to characterize DTH-producing antigens, culture supernatants from the unencapsulated Cap-67 strain were separated by anion-exchange chromatography. After further fractionation by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a purified protein with an apparent molecular mass of 25 kDa was found to produce DTH, as evidenced by increased footpad swelling in mice immunized with culture supernatants, relative to unimmunized mice. The 20-amino-acid N-terminal sequence of the 25-kDa protein was used to search data of theC. neoformansGenome Project. Based on the genomic DNA sequence, a DNA probe was used to screen a λ cDNA library prepared from strain B3501. Clones were isolated containing the full-length gene (d25), which showed homology with a number of polysaccharide deacetylases from fungi and bacteria. The recombinant d25 protein expressed inEscherichia coliwas similar to the natural one in DTH-producing activity. Moreover, immunization with either the natural or the recombinant protein prolonged survival and decreased fungal burden in mice challenged with the highly virulentC. neoformansstrain H99. In conclusion, we have described the first cryptococcal gene whose product, a 25-kDa extracellular polysaccharide deacetylase, has been shown to induce protective immunity responses.
Persistence of Protective Immunity to Malaria Induced by DNA Priming and Poxvirus Boosting: Characterization of Effector and Memory CD8+-T-Cell Populations
Authors: Sedegah Martha , Brice Gary T. , Rogers William O. , Doolan Denise L. , Charoenvit Yupin , Jones Trevor R. , Majam Victoria F. , Belmonte Arnel , Lu Minh , Belmonte Maria , Carucci Daniel J. , Hoffman Stephen L.
Published: July 2002

The persistence of immunity to malaria induced in mice by a heterologous DNA priming and poxvirus boosting regimen was characterized. Mice were immunized by priming with DNA vaccine plasmids encoding thePlasmodium yoeliicircumsporozoite protein (PyCSP) and murine granulocyte-macrophage colony-stimulating factor and boosting with recombinant vaccinia encoding PyCSP. BALB/c mice immunized with either high-dose (100 μg of pPyCSP plus 30 μg of pGM-CSF) or low-dose (1 μg of pPyCSP plus 1 μg of pGM-CSF DNA) priming were protected against challenge with 50P. yoeliisporozoites. Protection 2 weeks after immunization was 70 to 100%, persisted at this level for at least 20 weeks, and declined to 30 to 40% by 28 weeks. Eight of eight mice protected at 20 weeks were still protected when rechallenged at 40 weeks. The antigen (Ag)-specific effector CD8+-T-cell population present 2 weeks after boosting had ex vivo Ag-specific cytolytic activity, expressed both gamma interferon (IFN-γ) and tumor necrosis factor alpha, and constituted 12 to 20% of splenic CD8+T cells. In contrast, the memory CD8+-Ag-specific-cell population at 28 weeks lacked cytolytic activity and constituted only 6% of splenic CD8+T cells, but at the single-cell level it produced significantly higher levels of IFN-γ than the effectors. High levels of Ag- or parasite-specific antibodies present 2 weeks after boosting had declined three- to sevenfold by 28 weeks. Low-dose priming was similarly immunogenic and as protective as high-dose priming against a 50-, but not a 250-, sporozoite challenge. These results demonstrate that a heterologous priming and boosting vaccination can provide lasting protection against malaria in this model system.
Use of Heme Compounds as Iron Sources by Pathogenic Neisseriae Requires the Product of the
hemO
Gene
Authors: Zhu Wenming , Hunt Desiree J. , Richardson Anthony R. , Stojiljkovic Igor
Published: July 2002

Heme compounds are an important source of iron for neisseriae. We have identified a neisserial gene,
hemO
, that is essential for heme, hemoglobin (Hb), and haptoglobin-Hb utilization. The
hemO
gene is located 178 bp upstream of the
hmbR
Hb receptor gene in
Neisseria meningitidis
isolates. The product of the
hemO
gene is homologous to enzymes that degrade heme; 21% of its amino acid residues are identical, and 44% are similar, to those of the human heme oxygenase-1. DNA sequences homologous to
hemO
were ubiquitous in commensal and pathogenic neisseriae. HemO genetic knockout strains of
Neisseria gonorrhoeae
and
N. meningitidis
were unable to use any heme source, while the assimilation of transferrin-iron and iron-citrate complexes was unaffected. A phenotypic characterization of a conditional
hemO
mutant, constructed by inserting an isopropyl-β-
d
-thiogalactopyranoside (IPTG)-regulated promoter upstream of the ribosomal binding site of
hemO
, confirmed the indispensability of the HemO protein in heme utilization. The expression of HemO also protected
N. meningitidis
cells against heme toxicity.
hemO
mutants were still able to transport heme into the cell, since both heme and Hb could complement an
N. meningitidis hemA hemO
double mutant for growth. The expression of the HmbR receptor was reduced significantly by the inactivation of the
hemO
gene, suggesting that
hemO
and
hmbR
are transcriptionally linked. The expression of the unlinked Hb receptor, HpuAB, was not altered. Comparison of the polypeptide patterns of the wild type and the
hemO
mutant led to detection of six protein spots with an altered expression pattern, suggesting a more general role of HemO in the regulation of gene expression in
Neisseriae.
Characterization of
tpp1
+
as Encoding a Main Trehalose-6P Phosphatase in the Fission Yeast
Schizosaccharomyces pombe
Authors: Franco Alejandro , Soto Teresa , Vicente-Soler Jero , Guillen Pedro Valero , Cansado Jose , Gacto Mariano
Published: July 2002

We have characterized an open reading frame of 2,454 bp on chromosome I of
Schizosaccharomyces pombe
as the gene encoding trehalose-6P phosphatase (
tpp1
+
). Disruption of
tpp1
+
caused in vivo accumulation of trehalose-6P upon heat shock and prevented cell growth at 37 to 40°C. Accumulation of trehalose-6P in cells bearing a chromosomal disruption of the
tpp1
+
gene and containing a plasmid with
tpp1
+
under the control of the thiamine-repressible promotor correlated with
tpp1
+
repression. The level of
tpp1
+
mRNA rose upon heat shock, osmostress, or oxidative stress and was negatively controlled by cyclic AMP-dependent protein kinase activity. Expression of
tpp1
+
during oxidative or osmotic stress, but not during heat shock, was under positive control by the
wis1-sty1
(equivalent to
phh1
and
spc1
) mitogen-activated protein kinase pathway. Analysis of Tpp1 protein levels suggests that the synthesis of trehalose-6P phosphatase may also be subjected to translational or posttranslational control.
Sinorhizobium meliloti putA
Gene Regulation: a New Model within the Family
Rhizobiaceae
Authors: Soto María José , Jiménez-Zurdo José Ignacio , van Dillewijn Pieter , Toro Nicolás
Published: July 2002

Proline dehydrogenase (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. In
Sinorhizobium meliloti
, as in other microorganisms, the
putA
gene is transcriptionally activated in response to proline. In
Rhodobacter capsulatus
,
Agrobacterium
, and most probably in
Bradyrhizobium
, this activation is dependent on an Lrp-like protein encoded by the
putR
gene, located immediately upstream of
putA
. Interestingly, sequence and genetic analysis of the region upstream of the
S. meliloti putA
gene did not reveal such a
putR
locus or any other encoded transcriptional activator of
putA
. Furthermore, results obtained with an
S. meliloti putA
null mutation indicate the absence of any proline-responsive transcriptional activator and that PutA serves as an autogenous repressor. Therefore, the model of
S. meliloti putA
regulation completely diverges from that of its
Rhizobiaceae
relatives and resembles more that of enteric bacteria. However, some differences have been found with the latter model: (i)
S. meliloti putA
gene is not catabolite repressed, and (ii) the gene encoding for the major proline permease (
putP
) does not form part of an operon with the
putA
gene.
Characterization of the
dnaK
Multigene Family in the Cyanobacterium
Synechococcus
sp. Strain PCC7942
Authors: Nimura Kaori , Takahashi Hideo , Yoshikawa Hirofumi
Published: July 2002

The cyanobacterium
Synechococcus
sp. strain PCC7942 has three
dnaK
homologues (
dnaK1, dnaK2
, and
dnaK3
), and a gene disruption experiment was carried out for each
dnaK
gene by inserting an antibiotic resistance marker. Our findings revealed that DnaK1 was not essential for normal growth, whereas DnaK2 and DnaK3 were essential. We also examined the effect of heat shock on the levels of these three DnaK and GroEL proteins and found a varied response to heat shock, with levels depending on each protein. The DnaK2 and GroEL proteins exhibited a typical heat shock response, that is, their synthesis increased upon temperature upshift. In contrast, the synthesis of DnaK1 and DnaK3 did not respond to heat shock; in fact, the level of DnaK1 protein decreased. We also analyzed the effect of overproduction of each DnaK protein in
Escherichia coli
cells using an inducible expression system. Overproduction of DnaK1 or DnaK2 resulted in defects in cell septation and formation of cell filaments. On the other hand, overproduction of DnaK3 did not result in filamentous cells; rather a swollen and twisted cell morphology was observed. When expressed in an
E. coli dnaK756
mutant,
dnaK2
could suppress the growth deficiency at the nonpermissive temperature, while
dnaK1
and
dnaK3
could not suppress this phenotype. On the contrary, overproduction of DnaK1 or DnaK3 resulted in growth inhibition at the permissive temperature. These results suggest that different types of Hsp70 in the same cellular compartment have specific functions in the cell.
Expression of the
Moraxella catarrhalis
UspA1 Protein Undergoes Phase Variation and Is Regulated at the Transcriptional Level
Authors: Lafontaine Eric R. , Wagner Nikki J. , Hansen Eric J.
Published: July 2002

The UspA1 protein of
Moraxella catarrhalis
has been shown to function as an adhesin that mediates adherence to human epithelial cell lines in vitro (E. R. Lafontaine, L. D. Cope, C. Aebi, J. L. Latimer, G. H. McCracken, Jr., and E. J. Hansen, J. Bacteriol. 182:1364–1373, 2000). In the present study, cell lysates prepared from individual colonies of several
M. catarrhalis
wild-type strains were analyzed by Western blot analysis using monoclonal antibodies (MAbs) specific for the UspA1 protein. Expression of UspA1 was shown to exhibit phase variation that was correlated with both adherence ability in vitro and the number of guanine (G) residues contained within a homopolymeric [poly(G)]tract located upstream of the
uspA1
open reading frame (ORF). Nucleotide sequence analysis revealed that isolates expressing relatively high levels of UspA1 had 10 G residues in their
uspA1
poly(G)tracts, whereas isolates that expressed much lower levels of UspA1 had 9 G residues. This poly(G) tract was located 30 nucleotides (nt) upstream of the
uspA1
ORF and 168 nt downstream of the
uspA1
transcriptional start site. Primer extension experiments, RNA slot blot analysis, and
cat
reporter constructs were used to demonstrate that
M. catarrhalis
isolates with 10 G residues in their
uspA1
poly(G) tracts expressed two-to threefold more
uspA1
mRNA than did isolates which had 9 G residues in their poly(G)tracts. Northern hybridization analysis revealed that an intact
uspA1
mRNA was readily detectable in RNA from
M. catarrhalis
isolates that had 10 G residues in their
uspA1
poly(G) tracts, whereas no full-length
uspA1
mRNA was observed in isolates whose poly(G)tracts contained 9 G residues.
M. catarrhalis
strain O35E
uspA1
genes that contained wild-type and mutated poly(G) tracts were expressed in
Haemophilus influenzae
to demonstrate that the length and composition of the poly(G)tract affected expression of UspA1.
Substrate Recognition Properties of Oligopeptidase B fromSalmonella entericaSerovar Typhimurium
Authors: Morty Rory E. , Fülöp Vilmos , Andrews Norma W.
Published: July 2002

Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of theopdBhomologue fromSalmonella entericaserovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P1. While similar to its eukaryotic homologues in terms of substrate specificity,SalmonellaOpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP).SalmonellaOpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu576and Glu578, that define P1specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp460and Asp462, that may be involved in defining P2specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.
Rhodospirillum centenum
Utilizes Separate Motor and Switch Components To Control Lateral and Polar Flagellum Rotation
Authors: McClain Jonathan , Rollo David R. , Rushing Brenda G. , Bauer Carl E.
Published: July 2002

Rhodospirillum centenum
is a purple photosynthetic bacterium that is capable of differentiating from vibrioid swimming cells that contain a single polar flagellum into rod-shaped swarming cells that have a polar flagellum plus numerous lateral flagella. Microscopic studies have demonstrated that the polar flagellum is constitutively present and that the lateral flagella are found only when the cells are grown on solidified or viscous medium. In this study, we demonstrated that
R. centenum
contains two sets of motor and switch genes, one set for the lateral flagella and the other for the polar flagellum. Electron microscopic analysis indicated that polar and lateral flagellum-specific FliG, FliM, and FliN switch proteins are necessary for assembly of the respective flagella. In contrast, separate polar and lateral MotA and MotB motor subunits are shown to be required for motility but are not needed for the synthesis of polar and lateral flagella. Phylogenetic analysis indicates that the polar and lateral FliG, FliM, and FliN switch proteins are closely related and most likely arose as a gene duplication event. However, phylogenetic analysis of the MotA and MotB motor subunits suggests that the polar flagellum may have obtained a set of motor genes through a lateral transfer event.
Cloning and Characterization of a Complex DNA Fingerprinting Probe for
Candida parapsilosis
Authors: Enger Lee , Joly Sophie , Pujol Claude , Simonson Patricia , Pfaller Michael , Soll David R.
Published: July 2002

Candida parapsilosis
accounts for a significant number of nosocomial fungemias, but in fact, no effective and verified genetic fingerprinting method has emerged for assessing the relatedness of independent isolates for epidemiological studies. A complex 15-kb DNA fingerprinting probe, Cp3-13, was therefore isolated from a library of
C. parapsilosis
genomic DNA fragments. The efficacy of Cp3-13 for DNA fingerprinting was verified by a comparison of its clustering capacity with those of randomly amplified polymorphic DNA analysis and internally transcribed spacer region sequencing, by testing species specificity, and by assessing its capacity to identify microevolutionary changes both in vitro and in vivo. Southern blot hybridization of
Eco
RI/
Sal
I-digested DNA with Cp3-13 provides a fingerprinting system that (i) identifies the same strain in independent isolates, (ii) discriminates between unrelated isolates, (iii) separates independent isolates into valid groups in a dendrogram, (iv) identifies microevolution in infecting populations, and (v) is amenable to automatic computer-assisted DNA fingerprint analysis. This probe is now available for epidemiological studies.
In Vitro Evolution of the Human Immunodeficiency Virus Type 1 Gag-Protease Region and Maintenance of Reverse Transcriptase Resistance following Prolonged Drug Exposure
Authors: La Seta Catamancio Simona , De Pasquale Maria Pia , Citterio Paola , Kurtagic Semir , Galli Massimo , Rusconi Stefano
Published: July 2002

We studied the human immunodeficiency virus type 1 phenotypic and genotypic profiles of a dual drug-resistant isolate (isolate 14aPost-DR) selected for zidovudine (ZDV) and lamivudine (3TC) resistance and then cultured in the presence of 3TC and a protease inhibitor: indinavir (IDV), ritonavir, or KNI-272. The IDV-treated virus was highly resistant to 3TC, ZDV, and IDV and accumulated protease mutations at positions M46I and V82F. A change from alanine to valine was observed in 4 of 10 clones in the P2 position of the p7-p1 Gag-protease cleavage site, linked to position M46I in the dominant viral quasispecies. Previous 3TC resistance did not impair the development of additional mutations in the protease and Gag-protease cleavage regions.
Comparison of DNA Hybridization and PCR Assays for Detection of Putative Pathogenic Enteroadherent
Escherichia coli
Authors: Scaletsky Isabel C. A. , Fabbricotti Sandra H. , Aranda Katia R. , Morais Mauro B. , Fagundes-Neto Ulysses
Published: July 2002

The correlation of the different adherence patterns with DNA probes and PCR primers for the identification of
Escherichia coli
was analyzed in isolates from children, less than 2 years of age with or without diarrhea, from different regions of Brazil. A total of 1,428 isolates obtained from 338 patients and 322 control children were studied. The enteropathogenic
E. coli
(EPEC) adherence factor (EAF) probe was shown to be as good as the HEp-2 adhesion assay for the detection of typical EPEC strains. The DNA probes used to detect diffusely adhering
E. coli
and enteroaggregative
E. coli
(EAEC) showed low sensitivities (64 and 50%, respectively), and the best method of identifying these organisms in clinical research remains the HEp-2 adherence assay. The “bundle-forming pilus” (BFP) and the EAEC PCR assays could be used instead of the DNA probes as a screening method for typical EPEC and EAEC carrying the EAEC probe sequence in the clinical laboratory. In our study, only typical EPEC strains that carried EAF and BFP were associated with acute diarrhea.
Genetic Identification of Rickettsiae Isolated from Ticks in Japan
Authors: Fournier Pierre-Edouard , Fujita Hiromi , Takada Nobuhiro , Raoult Didier
Published: July 2002

Following the description in Japan of Japanese spotted fever, caused by
Rickettsia japonica
, a search for the vector of this disease led to the isolation of several rickettsiae from various tick species. Sixty-three rickettsial isolates were obtained from six different tick species, and six type strains were described by PCR and monoclonal antibody testing. We identified these six strains by amplification and sequencing of the genes encoding 16S rRNA and citrate synthase. We confirmed that the isolates from
Dermacentor taiwanensis
and
Haemaphysalis flava
ticks were
R. japonica
isolates. In
Ixodes ovatus
,
Ixodes persulcatus
, and
Ixodes monospinosus
, we identified a
Rickettsia
identical or closely related to
Rickettsia helvetica
, a species that is pathogenic for humans and that to date has only been found in Europe. Finally, we identified a new genotype of unknown pathogenicity, genotype AT, that was isolated from
Amblyomma testudinarium
ticks and that is closely related to a Slovakian genotype obtained from
Ixodes ricinus
ticks.
Nef Enhances Human Immunodeficiency Virus Type 1 Infectivity Resulting from Intervirion Fusion: Evidence Supporting a Role for Nef at the Virion Envelope
Authors: Zhou Jing , Aiken Christopher
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef stimulates viral infectivity by facilitating an early event in the HIV-1 life cycle. Although no structural or biochemical defects in Nef-defective HIV-1 particles have been demonstrated, the Nef protein is incorporated into HIV-1 particles. To localize the function of Nef within the virus particle, we developed a novel technology involving fusion of enveloped donor HIV-1 particles bearing core defects with envelope-defective target virions bearing HIV-1 receptors. Although neither virus alone was capable of infecting CD4
+
target cells, the incubation of donor and target virions prior to addition to target cells resulted in infection. This effect, termed “virion transcomplementation,” required a functional Env protein on the donor virus and CD4 and an appropriate coreceptor on target virions. To provide evidence for intervirion fusion as the mechanism of complementation, experiments were performed using dual-enveloped HIV-1 particles bearing both HIV-1 and ecotropic murine leukemia virus (E-MLV) Env proteins as donor virions. Infection of CD4-negative target cells bearing E-MLV receptors was prevented by HIV-1 entry inhibitors when added before, but not after, incubation of donor and target virions prior to the addition to cells. When we used Nef
+
and Nef
−
donor and target virions, Nef enhanced infection when present in donor virions. In contrast, no effect of Nef was detected when present in the target virus. These results reveal a potential mechanism for enhancing HIV-1 diversity in vivo through the rescue of defective viral genomes and provide a novel genetic system for the functional analysis of virion-associated proteins in HIV-1 infection.
Induction of Rapid and Extensive β-Chemokine Synthesis in Macrophages by Human Immunodeficiency Virus Type 1 and gp120, Independently of Their Coreceptor Phenotype
Authors: Choe Wonkyu , Volsky David J. , Potash Mary Jane
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) interacts with its target cells through CD4 and a coreceptor, generally CCR5 or CXCR4. Macrophages display CD4, CCR5, and CXCR4 that are competent for binding and entry of virus. Virus binding also induces several responses by lymphocytes and macrophages that can be dissociated from productive infection. We investigated the responses of macrophages to exposure to a series of HIV-1 species, R5 species that productively infect and X4 species that do not infect macrophages. We chose to monitor production of several physiologically relevant factors within hours of treatment to resolve virally induced effects that may be unlinked to HIV-1 production. Our novel findings indicate that independently of their coreceptor phenotype and independently of virus replication, exposure to certain R5 and X4 HIV-1 species induced secretion of high levels of macrophage inflammatory protein 1α (MIP-1α), MIP-1β, RANTES, and tumor necrosis factor alpha. However two of the six R5 species tested, despite efficient infection, were unable to induce rapid chemokine production. The acute effects of virus on macrophages could be mimicked by exposure to purified R5 or the X4 HIV-1 envelope glycoprotein gp120. Depletion of intracellular Ca2+or inhibition of protein synthesis blocked the chemokine induction, implicating Ca2+-mediated signal transduction and new protein synthesis in the response. The group of viruses able to induce this chemokine response was not consistent with coreceptor usage. We conclude that human macrophages respond rapidly to R5 and X4 envelope binding by production of high levels of physiologically active proteins that are implicated in HIV-1 pathogenesis.
Palmitoylation of the Rous Sarcoma Virus Transmembrane Glycoprotein Is Required for Protein Stability and Virus Infectivity
Authors: Ochsenbauer-Jambor Christina , Miller David C. , Roberts Charles R. , Rhee Sung S. , Hunter Eric
Published: July 2002

The Rous sarcoma virus (RSV) transmembrane (TM) glycoprotein is modified by the addition of palmitic acid. To identify whether conserved cysteines within the hydrophobic anchor region are the site(s) of palmitoylation, and to determine the role of acylation in glycoprotein function, cysteines at residues 164 and 167 of the TM protein were mutated to glycine (C164G, C167G, and C164G/C167G). In CV-1 cells, palmitate was added to
env
gene products containing single mutations but was absent in the double-mutant Env. Although mutant Pr95 Env precursors were synthesized with wild-type kinetics, the phenotypes of the mutants differed markedly. Env-C164G had properties similar to those of the wild type, while Env-C167G was degraded faster, and Env containing the double mutant C164G/C167G was very rapidly degraded. Degradation occurred after transient plasma membrane expression. The decrease in steady-state surface expression and increased rate of internalization into endosomes and lysosomes paralleled the decrease in palmitoylation observed for the mutants. The phenotypes of mutant viruses were assessed in avian cells in the context of the pATV8R proviral genome. Virus containing the C164G mutation replicated with wild-type kinetics but exhibited reduced peak reverse transcriptase levels. In contrast, viruses containing either the C167G or the C164G/C167G mutation were poorly infectious or noninfectious, respectively. These phenotypes correlated with different degrees of glycoprotein incorporation into virions. Infectious revertants of the double mutant demonstrated the importance of cysteine-167 for efficient plasma membrane expression and Env incorporation. The observation that both cysteines within the membrane-spanning domain are accessible for acylation has implications for the topology of this region, and a model is proposed.
Construction and Molecular Analysis of Gene Transfer Systems Derived from Bovine Immunodeficiency Virus
Authors: Berkowitz Robert , Ilves Heini , Lin Wei Yu , Eckert Karl , Coward Andrea , Tamaki Stan , Veres Gabor , Plavec Ivan
Published: July 2002

Because lentiviruses are able to infect nondividing cells, these viruses might be utilized in gene therapy applications where the target cell does not divide. However, it has been suggested that the introduction of primate lentivirus sequences, particularly those of human immunodeficiency virus, into human cells may pose a health risk for the patient. To avoid this concern, we have constructed gene transfer systems based on a nonprimate lentivirus, bovine immunodeficiency virus. A panel of vectors and packaging constructs was generated and analyzed in a transient expression system for virion production and maturation, vector expression and encapsidation, and envelope protein pseudotyping. Virion preparations were also analyzed for transduction efficiency in a panel of human and nonhuman primary cells and immortalized cell lines. The virion preparations transduced most of the target cell types, with efficiencies up to 90% and with titers of unconcentrated virus up to 5 × 10
5
infectious doses/ml. In addition, infection of nondividing human cells, including unstimulated hematopoietic stem cells and irradiated endothelial cells, was observed.
Identification of a Motif in the C Terminus of Herpes Simplex Virus Regulatory Protein ICP4 That Contributes to Activation of Transcription
Authors: Bruce James W. , Wilcox Kent W.
Published: July 2002

Expression of most viral genes during productive infection by herpes simplex virus is regulated by the viral protein ICP4 (also called IE175 or Vmw175). The N-terminal portion of ICP4 contains well-defined transactivation, DNA binding, and dimerization domains that contribute to promoter regulation. The C-terminal half of ICP4 contributes to the activity of ICP4, but the functional motifs have not been well mapped. To localize functional motifs in the C-terminal half of ICP4, we have compared the relative specific activities of ICP4 variants in transient-transfection assays. Deletion of the C-terminal 56 residues reduces the specific activity more than 10-fold. Mutational analysis identified three consecutive residues (1252 to 1254) that are conserved in ICP4 orthologs and are essential for full activity, especially in the context of ICP4 variants with a deletion in the N-terminal transactivation domain. Recombinant viruses that encode variants of ICP4 with mutations in the N-terminal transactivation domain and/or the extreme C terminus were constructed. The phenotypes of these recombinant viruses support the hypothesis that efficient promoter activation by ICP4 requires motifs at both the N and C termini. The data suggest that the C terminus of ICP4 functions not as an independent transactivation domain but as an enhancer of the ICP4 N-terminal transactivation domain. The data provide further support for the hypothesis that some ICP4 motifs required for promoter activation are not required for promoter repression and suggest that ICP4 utilizes different cellular factors for activation or repression of viral promoters.
Sequential Partially Overlapping Gene Arrangement in the Tricistronic S1 Genome Segments of Avian Reovirus and Nelson Bay Reovirus: Implications for Translation Initiation
Authors: Shmulevitz Maya , Yameen Zareen , Dawe Sandra , Shou Jingyun , O’Hara David , Holmes Ian , Duncan Roy
Published: July 2002

Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the ςC viral cell attachment protein initiates over 600 nucleotides distal from the 5′ end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3′-proximal ORF present on the NBV S1 genome segment also encodes a ςC homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the ςC coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and ςC gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream ςC ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal ςC start site.
Envelope Gene-Mediated Neurovirulence in Feline Immunodeficiency Virus Infection: Induction of Matrix Metalloproteinases and Neuronal Injury
Authors: Johnston J. B. , Silva C. , Power C.
Published: July 2002

The release of neurotoxins by activated brain macrophages or microglia is one mechanism proposed to contribute to the development of neurological disease following infection by lentiviruses, including feline immunodeficiency virus (FIV). Since molecular diversity in the lentiviral envelope gene influences the expression of host molecules implicated in neuronal injury, the role of the envelope sequence in FIV neuropathogenesis was investigated by using the neurovirulent FIV strain V1CSF, the nonneurovirulent strain Petaluma, and a chimera (FIVCh) containing the V1CSF envelope gene in a Petaluma background. All three viruses replicated in primary feline macrophages with equal efficiency, but conditioned medium from V1CSF- or FIVCh-infected cells was significantly more neurotoxic than medium from Petaluma-infected cultures (
P
< 0.001) and could be attenuated in a dose-dependent manner by treatment with either the matrix metalloproteinase (MMP) inhibitor prinomastat (PMT) or function-blocking antibodies to MMP-2. Although FIV sequences were detectable by PCR in brain tissue from neonatal cats infected with each of the viral strains, immunohistochemistry revealed increased astrogliosis and macrophage activation in the brains of V1CSF- and FIVCh-infected cats relative to the other groups, together with elevated markers of neuronal stress that included morphological changes and increased c-
fos
immunoreactivity. Similarly, MMP-2, but not MMP-9, mRNA and protein expression was increased in brain tissues of V1CSF- and FIVCh-infected cats relative to Petaluma-infected animals (
P
< 0.01). Infection with V1CSF or FIVCh was also associated with greater CD4
+
cell depletion (
P
< 0.001) and neurodevelopmental delays (
P
< 0.005), than in Petaluma-infected animals; these deficits improved following PMT therapy. These findings indicated that diversity in the envelope gene sequence influenced the neurovirulence exhibited by FIV both in vitro and in vivo, possibly through a mechanism involving the differential induction of MMP-2.
Weak Oxytocin Antagonists from Minor Modification of the Cyclic Portion of Agonists
Authors: Assimomytis N. , Manessi-Zoupa E. , Cordopatis P. , Theodoropoulos D.
Published: July 2002

[(OMe)Ser5]-oxytocin (I), [(OMe)Thr5]-oxytocin (II) and [Aib3]-oxytocin (III) were synthesized by solid phase techniques. The three analogs were found to be relatively weak antagonists with pA2 values 5.7 (for I), 5.8 (for II) and 5.8 (for III), respectively, in the rat uterotonic in vitro assay. They were all inactive in the pressor and galactogonic assays.
Kinetics and Mechanism of Decomposition of 1,3-Bis(4-methylphenyl)triazene Catalyzed with Substituted Benzoic Acids in 25% Aqueous Methanol-General or Specific Catalysis?
Authors: Pytela Oldřich , Nevěčná Taťjana
Published: July 2002

The kinetics of decomposition of 1,3-bis(4-methylphenyl)triazene catalyzed with 13 substituted benzoic acids of various concentrations have been measured in 25 vol.% aqueous methanol at 25.0 °C. The rate constants observed (297 data) have be used as values of independent variable in a series of models of the catalyzed decomposition. For the catalytic particles were considered the undissociated acid, its conjugated base, and the proton in both the specific and general catalyses. Some models presumed formation of reactive or nonreactive complexes of the individual reactants. The substituent effect is described by the Hammett equation. The statistically best model in which the observed rate constant is a superposition of a term describing the dependence on proton concentration and a term describing the dependence on the product of concentrations of proton and conjugated base is valid with the presumption of complete proton transfer from the catalyst acid to substrate, which has been proved. The behaviour of 4-dimethylamino, 4-amino, and 3-amino derivatives is anomalous (lower catalytic activity as compared with benzoic acid). This supports the presumed participation of conjugated base in the title process.
Synthesis of Several Hydroxylated 23-(Benzimidazol-2-yl-, Benzoxazol-2-yl and Benzothiazol-2-yl)norcholanes and Some Related Compounds
Authors: Nguyen Thi Thu Huong , Urban Jiří , Klinotová Eva , Sejbal Jan , Protiva Jiří , Drašar Pavel , Protiva Miroslav
Published: July 2002

The bile acids Ia - Id (lithocholic, chenodeoxycholic, deoxycholic, cholic) and their derivatives (O-acetylated acids and O-acetylated acid chlorides) reacted under various conditions with 1,2-diaminobenzene, 2-aminophenol and 2-aminothiophenol and afforded the title benzimidazoles II and VII, benzoxazoles V and benzothiazoles VI. Alkylation of the benzimidazole derivative IIa with 2-dimethylaminoethyl chloride resulted in 3α-hydroxy-23-[1-(2-dimethylaminoethyl)ben zimidazol-2-yl]- norcholane (IVa). The use of 1,2-diamino-4-methylbenzene enabled the preparation of 3α-acetoxy-23-[5(6)-methylbenzimidazol-2-y l]norcholane (VIII). Reactions of the 3α-hydroxy compounds IVa, Va and VIa with succinic anhydride resulted in the hemisuccinates IVi - VIi. The boric acid mediated condensation of O-acetyllithocholic acid (Ie) with 3,4-diaminopyridine gave compound X which was transformed to 3α-acetoxy-23-[1H-imidazo(4,5- c)pyridin-2-yl]norcholane (IX). The structure of the products was corroborated by the mass, IR, 1H NMR and 13C NMR spectra. Some of the compounds were tested for antileukemic and for the anti-HIV activity in vitro.
Oxidation of Butane to Maleic Anhydride on Unmodified and Modified Vanadium-Phosphorus Catalysts
Authors: Kladeková Daniela , Hanudeľ Alexander , Brutovský Milan , Novák Ján
Published: July 2002

Properties of modified vanadium-phosphorus catalysts were studied and compared with the unmodified catalyst in the oxidation of butane. The highest promoting effect has been achieved by introducing cobalt to the catalyst lattice. At 90% conversion, the yield of maleic anhydride amounted to 59 mole per cent. The catalyst modified by cobalt increased the specific rate of butane oxidation and maleic anhydride formation three times compared to the unmodified catalyst. The specific activity of the catalysts decreased in the following order: Co > U ~ Ce > V-P-O > K ~ Mo.
Solvent Effect on the Structure and Infrared Spectra of N-(3-Chloro-2-benzo[b]thienocarbonyl)-N'-ethylthiourea
Authors: Hritzová Oľga , Suchár Gejza , Danihel Ivan , Kutschy Peter
Published: July 2002

The solvent effect on the title compound was studied by examining changes in the positions of IR bands belonging to the NH and CO bonds and in the dipole moments in benzene and dioxane solutions. The results are discussed in terms of intramolecular hydrogen bonding and spatial arrangement of the CONHCSNH grouping.
Synthesis and Characterization of Palladium Complexes Containing 3,3'-Annelated 2,2'-Bipyridines and 3,3'-Annelated 2,2'-Biquinolines
Authors: Klein René A. , Belzen Ruud van , Vrieze Kees , Elsevier Cornelis J. , Thummel Randolph P. , Fraanje Jan , Goubitz Kees
Published: July 2002

The synthesis and characterization of a number of PdCl2(N⁀N) and Pd(N⁀N)(fn) complexes (N⁀N = 3,3'-annelated 2,2'-bipyridines and 3,3'-annelated 2,2'-biquinolines, fn = fumaronitrile) have been described. Despite the variation in dihedral angle and distance between the nitrogen lone pairs in these series of ligands, they are all capable of forming stable chelates. An investigation was made as to whether the 3,3'-trimethylene-2,2'-biquinoline (3-bq) and 3,3'-tetramethylene-2,2'-biquinoline (4-bq) ligands could serve as C2-chiral auxiliaries. The ligand 3-bq in Pd(3-bq)(fn) turned out to be conformationally rigid on the NMR time scale, but the diastereomers appeared to be indistinguishable, as could be concluded from the 1H NMR spectra. However, Pd(4-bq)(fn) exists in two diastereomeric forms and their ratio (57 : 43) could be determined from 1H NMR spectra. Assignment of the major and minor isomers was done with a 2D NOESY experiment. Epimerization of the diastereomers occurred on the laboratory time scale which was deduced from a spin saturation transfer experiment and also from the observation that diastereomerically enriched crystalline material of Pd(4-bq)(fn) at low temperature showed a ratio of 80 : 20 which deteriorated to a ratio of 57 : 43. From the spin saturation transfer experiment, the thermodynamic parameters for the inversion could be determined (∆H≠ = 29.1 kJ mol-1 and ∆S≠ = -136.3 J K-1 mol-1). This large negative entropy of activation favours an epimerization process via inversion of the coordinated 4-bq ligand over a mechanism via alkene dissociation/recoordination. The structures of the complexes have been studied in solution and in the solid state and the following X-ray crystal structures have been determined: (i) [dichloropalladium(II)](3,3'-trimethylene-2,2'-bipyridine-κ2N), monoclinic crystals, space group P21/n, with a = 8.4230(7) Å, b = 16.425(2) Å, c = 9.5836(7) Å, β = 106.316(7)°, Z = 4, final R = 0.033 for 2 309 reflections, (ii) [3,3'-bis(methoxycarbonyl)-2,2'-bipyridine-κ2N][dichloropalladium(II)], triclinic crystals, space group P1, with a = 7.340(4) Å, b= 10.012(3) Å, c= 11.954(3) Å, α = 98.10(2)°, β = 104.03(3)°, γ = 107.33(5)°, Z = 2, final R = 0.039 for 4 273 reflections, (iii) [chloromethylpalladium(II)](4,5-diazafluorene-κ2N), orthorhombic crystals, space group Pmcn, with a = 6.713(1) Å, b = 10.120(1) Å, c = 17.187(3) Å, Z = 4, final R = 0.048 for 949 reflections.
Zeolite Silylation for the Enhancement of para-Selectivity in Toluene Alkylation with Ethylene
Authors: Krtil Jan , Čejka Jiří , Wichterlová Blanka
Published: July 2002

The effect of zeolite silylation using tetraethyl and tetramethyl orthosilicate on the enhancement of para-selectivity and of yields of p-ethyltoluene in toluene alkylation with ethylene was investigated for different Si loadings. It was found that zeolite silylation caused a substantial increase in the selectivity and yield of p-ethyltoluene. Data on changes in sorption capacity, in the number of surface and inner strong acid OH groups, in the rate of uptake of p-ethyltoluene together with the catalytic activity of zeolite and its deactivation indicate that the silylation is restricted to surface and subsurface zeolite layers. The enhancement of para-selectivity and yield of p-ethyltoluene are controlled predominantly by changes in geometry of pore openings and thus by transport rates of ethyltoluenes.
Nickel(0) and Palladium(0) Complexes with 1,3,5-Triaza-7-phosphaadamantane. Catalysis of Buta-1,3-diene Oligomerization or Telomerization in an AqueousBiphasic System
Authors: Čermák Jan , Kvíčalová Magdalena , Blechta Vratislav
Published: July 2002

New homoleptic nickel(0) and palladium(0) complexes with a water-soluble ligand, 1,3,5-triaza-7-phosphaadamantane, were prepared and characterized by 1H, 13C, and 31P NMR spectra. The complexes, together with the known analogous Ni(0) and Pd(0) complexes with tris(hydroxymethyl)phosphine, were found to be catalysts for buta-1,3-diene oligomerization or telomerization with water in an aqueous biphasic system without a cosolvent or a modifier. Tetrakis[tris(hydroxymethyl)phosphine]nickel (7) preferentially catalyzes oligomerization (both linear and cyclic) in the first example of a nickel-catalyzed buta-1,3-diene oligomerization in an aqueous biphasic system. Palladium complexes give telomers or linear oligomers in quantitative yields. In the case of the triazaphosphaadamantane complex 4, high selectivity to octadienyl ethers (87%) was observed. High values of metal leaching into the product phase in these reactions suggest an easy extraction of starting or intermediate metal complexes caused by the fact that both monomer and products are good ligands for the metal complexes in this particular case.
The Belousov-Zhabotinskii Oscillatory Reaction Involving Some Organic Chiral Substrates
Authors: Adamčíková Ľubica , Kučárová Katarína , Ševčík Peter
Published: July 2002

Oscillatory systems of the Belousov-Zhabotinskii (BZ) type were studied using the following substrates: D-gluconic and D-galactonic acids, their lactones, and D-glucose and D-galactose. The systems were either nitrogen purged or left undisturbed. It is suggested that during the induction period (IP), reactions occur giving rise to the active substrate in the BZ system which need not be identical with the organic substance with which the oscillatory system starts up. A special dependence of the IP on sulfuric acid concentration was measured, exhibiting a minimum and a maximum; the dependence on perchloric acid concentration displayed a minimum.
Coordination Chemistry of N-(2-Hydroxybenzyl)-(S)-amino Acids. Absolute Configuration of LAMBDA-mer-R,R-[Co(ohb-(S)-Ala)2]- Anion
Authors: Pakhomova Svetlana , Ondráček Jan , Jursík František
Published: July 2002

The crystal and molecular structure of the Ag[Co(ohb-(S)-Ala)2] . 5 H2O have been determined by X-ray diffraction method. The crystals are hexagonal with a = b = 9.039(0.002) Å, c = 24.896(0.006) Å, space group P3221, and Z = 3. Each Ag+ counterion coordinated by one water molecule which is a part of hydrogen bond network, is bonded to aromatic rings. The anion adopts LAMBDA-mer absolute configuration. Mer geometry is in accord with the molecular mechanics calculations. Since CD spectroscopy does not reflect configurational chirality, the absolute configuration has been determined by the X-ray structure analysis and assigned using chirality descriptors. The arrangements at the chiral centers are R for both N atoms. The two five-membered rings each assume envelope conformation with C1 and C2 atoms exceptionally deviated (0.378(7) and 1.210(7) Å) from the N-Co-O plane. Both six-membered rings have delta twist boat conformations. The complex described has been also characterized by the electronic and 13C NMR spectroscopies.