bluuebunny/crossref_metadata_2025 huggingface dataset:
Prediction of Gene Function in Methylthioadenosine Recycling from Regulatory Signals
Authors: Murphy Brooke A. , Grundy Frank J. , Henkin Tina M.
Published: July 2002

The S-box transcription termination control system, first identified in
Bacillus subtilis
, is used for regulation of gene expression in response to methionine availability. The presence of the S-box motif provided the first indication that the
ykrTS
and
ykrWXYZ
genes could play a role in recycling of 5′-methylthioadenosine, a by-product of polyamine biosynthesis that can be converted to methionine. In this study we demonstrate a role for the
ykrTS
and
ykrWXYZ
gene products in this pathway.
Detection of Ciprofloxacin-Resistant
Yersinia pestis
by Fluorogenic PCR Using the LightCycler
Authors: Lindler Luther E. , Fan Wei , Jahan Nazma
Published: July 2002

We have developed a fluorescence resonance energy transfer (FRET)-based assay to detect ciprofloxacin resistant (Cp
r
) mutants of the biothreat agent
Yersinia pestis
. We selected spontaneous mutants of the attenuated
Y. pestis
KIM 5 strain that were resistant to a ciprofloxacin (CIP) concentration of at least 1 μg/ml. DNA sequencing of
gyrA
encoded by 65 of these mutants revealed that all isolates contained one of four different point mutations within the quinolone resistance-determining region of
gyrA.
We developed a FRET-based assay that detected all of these mutations by using a single pair of fluorescent probes with sequences complementary to the wild-type
Y. pestis gyrA
sequence. Melting peak analysis revealed that the probe-PCR product hybrid was less stable when amplification occurred from any of the four mutant templates. This instability resulted in the PCR product obtained from the Cp
r
Y. pestis
strains displaying a 4 to 11°C shift in probe melting temperature. Following optimization of the reaction conditions, we were able to detect approximately 10 pg of purified wild-type template DNA or the presence of approximately 4 CFU of wild-type
Y. pestis
KIM 5 or Cp
r
mutants in crude lysates. Taken together, our results demonstrate the utility of FRET-based assays for detection of Cp
r
mutants of
Y. pestis
. This method is both sensitive and rapid.
Isolation of a Lysogenic Bacteriophage Carrying the
stx

1
OX3

Gene, Which Is Closely Associated with Shiga Toxin-Producing
Escherichia coli
Strains from Sheep and Humans
Authors: Koch Claudia , Hertwig Stefan , Lurz Rudi , Appel Bernd , Beutin Lothar
Published: July 2002

A specific PCR for the detection of a variant of the gene encoding Shiga toxin 1 (
stx
1
) called
stx

1
OX3

(GenBank accession no.
Z36901
) was developed. The PCR was used to investigate 148 Stx
1
-producing
Escherichia coli
strains from human patients (
n
= 72), cattle (
n
= 27), sheep (
n
= 48), and a goat (
n
= 1) for the presence of the
stx

1
OX3

gene. The
stx

1
OX3

gene was present in 38 Shiga toxin-producing
E. coli
(STEC) strains from sheep belonging to serogroups O5, O125, O128, O146, and OX3 but was absent from Stx
1
-positive ovine STEC O91 strains. The
stx

1
OX3

gene was also detected in 22 STEC strains from humans with nonbloody diarrhea and from asymptomatic excreters. Serotypes O146:H21 and O128:H2 were most frequently associated with
stx

1
OX3

-carrying STEC from sheep and humans. In contrast, Stx
1
-producing STEC strains from cattle and goats and 50 STEC strains from humans were all negative for the
stx

1
OX3

gene. The
stx

1
OX3

-negative strains belonged to 13 serotypes which were different from those of the
stx

1
OX3

-positive STEC strains. Moreover, the
stx

1
OX3

gene was not associated with STEC belonging to enterohemorrhagic
E. coli
(EHEC) serogroups O26, O103, O111, O118, O145, and O157. A bacteriophage carrying the
stx

1
OX3

gene (phage 6220) was isolated from a human STEC O146:H21 strain. The phage was able to lysogenize laboratory
E. coli
K-12 strain C600. Phage 6220 shared a similar morphology and a high degree of DNA homology with Stx
2
-encoding phage 933W, which originates from EHEC O157. In contrast, few similarities were found between phage 6220 and Stx
1
-encoding bacteriophage H-19B from EHEC O26.
Improved Antimicrobial Interventions Have Benefits
Authors: Barenfanger Joan , Short Michael A. , Groesch Alisa A.
Published: July 2002

Studies have shown benefits to patients from improved interventions involving antimicrobial therapy. The purpose of the present study was to evaluate prospectively the impact of improved interventions by (i) the use of TheraTrac 2, a computer software program which electronically links susceptibility testing results immediately to the pharmacy and alerts pharmacists of potential interventions, and (ii) the education of pharmacists involving microbiologic topics. The study group had the new intervention program. The control group had interventions performed the way that they had previously been done by manually reviewing hard copies of susceptibility testing data. In a 5-month period, all inpatients whose last names began with A to K were the study group; inpatients whose last names began with L to Z were controls. Three analyses were done; one analysis (analysis A) involved only patients with interventions, one analysis (analysis B) involved all patients for whom antimicrobial testing was done and who were matched for diagnosis-related groups (DRGs), regardless of whether an intervention occurred, and one analysis (analysis C) involved these DRG-matched patients by using severity-adjusted data. In analysis A, the study group had a 4.8% decreased rate of mortality, an average of a 16.5-day decreased length of stay per patient, and $20,886 decreased variable direct costs per patient. None of these differences was statistically significant. In analysis B, the study patients had a 1.2% higher mortality rate (
P
= 0.741), an average of a 2.7-day decreased length of stay per patient (
P
= 0.035), and $2,626 decreased variable direct costs per patient (
P
= 0.008). In analysis C, the study patients had a 1.4% lower mortality rate, a 1.2-day decreased length of stay per patient, and $1,466 decreased variable direct costs per patient. In conclusion, the institution of this program caused substantial cost savings.
Direct Identification of Mycobacteria from MB/BacT Alert 3D Bottles: Comparative Evaluation of Two Commercial Probe Assays
Authors: Scarparo Claudio , Piccoli Paola , Rigon Alessandra , Ruggiero Giuliana , Nista Domenico , Piersimoni Claudio
Published: July 2002

The new INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium), a reverse-hybridization-based line probe assay, and the AccuProbe assay (Gen-Probe Inc., San Diego, Calif.) were applied to MB/BacT Alert 3D (MB/BacT) system (Organon Teknika, Boxtel, The Netherlands) culture bottles and evaluated for mycobacterial identification. From 2,532 respiratory and extrapulmonary specimens submitted for culture, 168 were flagged positive by the MB/BacT system and promptly evaluated for identification (within 24 h). Each of 163 vials grew one mycobacterial isolate, including
Mycobacterium tuberculosis
complex (
n
= 73),
M. avium
complex (
n
= 3),
M. avium
(
n
= 8),
M. intracellulare
(
n
= 5),
M. kansasii
(
n
= 15),
M. gordonae
(
n
= 8),
M. malmoense
(
n
= 3),
M. chelonae
(
n
= 13),
M. abscessus
(
n
= 2),
M. xenopi
(
n
= 11),
M. scrofulaceum
(
n
= 2),
M. fortuitum
(
n
= 7),
M. terrae
(
n
= 3),
M. simiae
(
n
= 2),
M. celatum
(
n
= 3),
M. flavescens
(
n
= 1),
M. interjectum
(
n
= 1),
M. bohemicum
(
n
= 1), and
M. pulveris
(
n
= 2). Five cultures yielded mixed growth of two mycobacterial species:
M. tuberculosis
complex plus
M. gordonae
(
n
= 2),
M. tuberculosis
complex plus
M. chelonae
(
n
= 1),
M. tuberculosis
complex plus
M. xenopi
(
n
= 1), and
M. avium
plus
M. chelonae
(
n
= 1). In testing of one-isolate vials, both systems showed excellent sensitivity and specificity for all species and complexes for which they are licensed (nine for INNO-LiPA Mycobacteria versus six for AccuProbe). There were minor discrepancies in results for two isolates identified by INNO-LiPA Mycobacteria as
M. avium - M. intracellulare - M. scrofulaceum
(MAIS) complex and by AccuProbe as
M. intracellulare
. In testing of two-isolate vials, INNO-LiPA Mycobacteria correctly identified all isolates, while the AccuProbe assay failed to identify three
M. tuberculosis
complex isolates and one
M. avium
isolate. The AccuProbe assay was completed within 2 h, while INNO-LiPA Mycobacteria required a 6-h period. In our opinion, INNO-LiPA Mycobacteria offers the following advantages: (i) it contains a genus-specific probe that, in addition to being used in genus identification, may be used as an internal control for both the amplification and hybridization steps; (ii) it simultaneously identifies
M. tuberculosis
complex, MAIS complex, and seven other mycobacterial species, even from mixed cultures; (iii) its mycobacterial DNA amplification ensures reliable results independent from the concentration of viable microorganisms; and (iv) it genotypically identifies
M. kansasii
and
M. chelonae
. In conclusion, even though INNO-LiPA Mycobacteria is considerably less easy to use than AccuProbe, requiring personnel skilled in molecular biology techniques, it represents an excellent approach for routine identification of frequently encountered mycobacteria.
Aspergillus
Galactomannan Antigen in the Cerebrospinal Fluid of Bone Marrow Transplant Recipients with Probable Cerebral Aspergillosis
Authors: Viscoli Claudio , Machetti Marco , Gazzola Paola , De Maria Andrea , Paola Dimitri , Van Lint Maria Teresa , Gualandi Francesca , Truini Mauro , Bacigalupo Andrea
Published: July 2002

The
Aspergillus
galactomannan test was performed on cerebrospinal fluid and serum samples from 5 patients with probable cerebral aspergillosis and from 16 control patients. Cerebrospinal fluid galactomannan levels were significantly higher in aspergillosis patients, and most galactomannan was produced intrathecally. Comparison of serum galactomannan values in pulmonary and cerebral aspergillosis patients showed significant overlapping. Detection of
Aspergillus
galactomannan in cerebrospinal fluid may be diagnostic of cerebral aspergillosis.
Reassessment of a Midwestern Lyme Disease Focus for
Borrelia burgdorferi
and the Human Granulocytic Ehrlichiosis Agent
Authors: Jackson Craig A. , Lovrich Steven D. , Agger William A. , Callister Steven M.
Published: July 2002

Previous studies from the late 1980s defined the risk of human Lyme disease by determining the prevalence of
Borrelia burgdorferi
infection in
Ixodes scapularis
ticks and
Peromyscus
sp. mice captured from areas around La Crosse, Wis. High percentages of
B. burgdorferi
-infected
I. scapularis
ticks and
P. leucopus
mice were common in areas located north of Interstate 90 but were not detected in areas south of this major east-west thoroughfare. In this study, we reevaluated the extent of
B. burgdorferi
infection. High percentages of mice captured from sites north of the interstate were still infected with
B. burgdorferi
. In addition,
B. burgdorferi
was recovered from 12 (67%) of 18 mice captured from a site well south of the highway. However, none of 104 mice or 713
I. scapularis
ticks captured from the study sites were infected with
Ehrlichia
spp. The results confirmed the continued high risk for humans to contract infection with
B. burgdorferi
and the significant southward expansion of the area in which Lyme disease is endemic. In contrast, the risk of acquiring human granulocytic ehrlichiosis remains minimal despite the abundance of appropriate vector ticks and reservoir rodents.
Genotyping Study of
Scedosporium apiospermum
Isolates from Patients with Cystic Fibrosis
Authors: Defontaine Alain , Zouhair Rachid , Cimon Bernard , Carrère Jacqueline , Bailly Eric , Symoens Françoise , Diouri Mohammed , Hallet Jean-Noel , Bouchara Jean-Philippe
Published: July 2002

Usually a saprophyte,
Scedosporium apiospermum
often colonizes the respiratory tracts of patients with cystic fibrosis (CF). In order to improve our understanding of the molecular epidemiology of the airway colonization, 129 sequential and multiple isolates collected from January 1998 to March 1999 from nine CF patients monitored in three hospitals in France were typed by random amplification of polymorphic DNA with primers GC70, UBC-701, and UBC-703. Among these primers, UBC-703 was the most discriminating, allowing the differentiation of 14 genotypes. Combining the results obtained with this three-primer set resulted in the differentiation of 16 genotypes. No common genotype was found among the different patients, and no clustering according to geographic origin of the isolates was seen. In addition, five of the patients were colonized by a single genotype. The others usually exhibited a predominant genotype accompanied by one or two others, which were found occasionally and were genetically close to the predominant genotype. Thus, our study demonstrates the persistence of the fungus despite antifungal treatments and therefore reinforces the need for the development of new antifungals that are more efficient against this species.
Lymphoid Organs as a Major Reservoir for Human T-Cell Leukemia Virus Type 1 in Experimentally Infected Squirrel Monkeys (Saimiri sciureus): Provirus Expression, Persistence, and Humoral and Cellular Immune Responses
Authors: Kazanji Mirdad , Ureta-Vidal Abel , Ozden Simona , Tangy Frédéric , de Thoisy Benoit , Fiette Laurence , Talarmin Antoine , Gessain Antoine , de Thé Guy
Published: July 2002

The aim of this study was to investigate the distribution of human T-cell leukemia virus type 1 (HTLV-1) in various organs of serially sacrificed squirrel monkeys (Saimiri sciureus) in order to localize the reservoir of the virus and to evaluate the relationship between viral expression and the humoral or cellular immune response during infection. Six squirrel monkeys infected with HTLV-1 were sacrificed 6, 12, and 35 days and 3, 6, and 26 months after inoculation, and 20 organs and tissues were collected from each animal. PCR and reverse transcription-PCR (RT-PCR) were performed withgagandtaxprimers. Proviral DNA was detected by PCR in peripheral blood mononuclear cells (PBMCs) of monkeys sacrificed 6 days after inoculation and in PBMCs, spleens, and lymph nodes of monkeys sacrificed 12 and 35 days and 3, 6, and 26 months after inoculation. Furthermore,tax/rexmRNA was detected by RT-PCR in the PBMCs of two monkeys 8 to 12 days after inoculation and in the spleens and lymph nodes of the monkey sacrificed on day 12. In this animal, scattered HTLV-1tax/rexmRNA-positive lymphocytes were detected by in situ hybridization in frozen sections of the spleen, around the germinal centers and close to the arterial capillaries. Anti-HTLV-1 cell-mediated immunity was evaluated at various times after inoculation. Anti-p40Taxand anti-Env cytolytic T-cell responses were detected 2 months after infection and remained detectable thereafter. When Tax peptides were used, this response appeared to be directed against various Tax epitopes. Our results indicate that squirrel monkeys represent a promising animal model for studying the early events of HTLV-1 infection and for evaluating candidate vaccines against HTLV-1.
Isolation and Characterization of a New Subtype of Borna Disease Virus
Authors: Nowotny Norbert , Kolodziejek Jolanta , Jehle Christian O. , Suchy Angelika , Staeheli Peter , Schwemmle Martin
Published: July 2002

Borna disease virus (BDV), the causative agent of severe meningoencephalitis in a wide variety of animal species, has been considered to be genetically invariable and to form a single type within the genus
Bornavirus
of the family
Bornaviridae
. BDV infections are of particular interest, because for the first time a virus infection appears to be linked to human psychiatric disorders. We now describe a new subtype of BDV isolated from a horse which was euthanatized due to severe, incurable neurological disease. The nucleotide sequence of this new strain, named No/98, differs from the reference strains by more than 15%, and the subtype is difficult to detect by standard reverse transcriptase PCR protocols. The nucleotide exchanges of the novel BDV isolate have surprisingly little effect on the primary structures of most viral proteins, with the notable exception of the X protein (p10), which is only 81% identical to its counterpart in reference strains. Our data indicate that the genome of BDV is far more variable than previously assumed and that naturally occurring subtypes may escape detection by currently used diagnostic assays.
Selective Membrane Permeabilization by the Rotavirus VP5* Protein Is Abrogated by Mutations in an Internal Hydrophobic Domain
Authors: Dowling William , Denisova Evgeniya , LaMonica Rachel , Mackow Erich R.
Published: July 2002

Rotavirus infectivity is dependent on the proteolytic cleavage of the VP4 spike protein into VP8* and VP5* proteins. Proteolytically activated virus, as well as expressed VP5*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of VP5* which effects rapid viral entry. The VP5* protein contains a single long hydrophobic domain (VP5*-HD, residues 385 to 404) at an internal site. In order to address the role of the VP5*-HD in permeabilizing cellular membranes, we analyzed the entry of
o
-nitrophenyl-β-
d
-galactopyranoside (ONPG) into cells induced to express VP5* or mutated VP5* polypeptides. Following IPTG (isopropyl-β-
d
-thiogalactopyranoside) induction, VP5* and VP5* truncations containing the VP5*-HD permeabilized cells to the entry and cleavage of ONPG, while VP8* and control proteins had no effect on cellular permeability. Expression of VP5* deletions containing residues 265 to 474 or 265 to 404 permeabilized cells; however, C-terminal truncations which remove the conserved GGA (residues 399 to 401) within the HD abolished membrane permeability. Site-directed mutagenesis of the VP5-HD further demonstrated a requirement for residues within the HD for VP5*-induced membrane permeability. Functional analysis of mutant VP5*s indicate that conserved glycines within the HD are required and suggest that a random coiled structure rather than the strictly hydrophobic character of the domain is required for permeability. Expressed VP5* did not alter bacterial growth kinetics or lyse bacteria following induction. Instead, VP5*-mediated size-selective membrane permeability, releasing 376-Da carboxyfluorescein but not 4-kDa fluorescein isothiocyanate-dextran from preloaded liposomes. These findings suggest that the fundamental role for VP5* in the rotavirus entry process may be to expose triple-layered particles to low [Ca]
i
, which uncoats the virus, rather than to effect the detergent-like lysis of early endosomal membranes.
Functional Dissection of the Major Structural Protein of Bluetongue Virus: Identification of Key Residues within VP7 Essential for Capsid Assembly
Authors: Limn Chang-Kwang , Staeuber Norbert , Monastyrskaya Katherine , Gouet Patrice , Roy Polly
Published: July 2002

A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.
A Putative G Protein-Coupled Receptor, RDC1, Is a Novel Coreceptor for Human and Simian Immunodeficiency Viruses
Authors: Shimizu Nobuaki , Soda Yasushi , Kanbe Katsuaki , Liu Hui-yu , Mukai Ryozaburo , Kitamura Toshio , Hoshino Hiroo
Published: July 2002

More than 10 G protein-coupled receptors (GPCRs) have been shown to act as coreceptors for infection of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We have isolated HIV-1 variants infectious to primary brain-derived CD4-positive cells (BT-3 and BT-20/N) and U87/CD4 glioma cells that are resistant to T-cell line-tropic (T-tropic), macrophage-tropic (M-tropic), and T- and M-tropic (dualtropic) (X4, R5, and R5X4) HIV-1 strains. These primary brain-derived cells were also highly susceptible to HIV-2
ROD
, HIV-2
SBL6669
, and SIV
mndGB-1
. A factor or coreceptor that determines the susceptibility of these brain-derived cells to these HIV and SIV strains has not been fully identified. To identify this coreceptor, we examined amino acid sequences of all known HIV and SIV coreceptors and noticed that tyrosine residues are well conserved in their extracellular amino-terminal domains. By this criterion, we selected 18 GPCRs as candidates of coreceptors for HIV and SIV strains infectious to these brain-derived cells. mRNA expression of an orphan GPCR, RDC1, was detected in the brain-derived cells, the C8166 T-cell line, and peripheral blood lymphocytes, all of which are susceptible to HIV-1 variants, but not in macrophages, which are resistant to them. When a CD4-expressing cell line, NP-2/CD4, which shows strict resistance to infection not only with HIV-1 but also with HIV-2 or SIV, was transduced with the RDC1 gene, the cells became highly susceptible to HIV-2 and SIV
mnd
strains but to neither M- nor T-tropic HIV-1 strains. The cells also acquired a low susceptibility to the HIV-1 variants. These findings indicate that RDC1 is a novel coreceptor for several HIV-1, HIV-2, and SIV strains which infect brain-derived cells.
Hepatitis A Virus-Specific Immunoglobulin A Mediates Infection of Hepatocytes with Hepatitis A Virus via the Asialoglycoprotein Receptor
Authors: Dotzauer Andreas , Gebhardt Ulrike , Bieback Karen , Göttke Ulrich , Kracke Anja , Mages Jörg , Lemon Stanley M. , Vallbracht Angelika
Published: July 2002

The mechanisms underlying the hepatotropism of hepatitis A virus (HAV) and the relapsing courses of HAV infections are unknown. In this report, we show for a mouse hepatocyte model that HAV-specific immunoglobulin A (IgA) mediates infection of hepatocytes with HAV via the asialoglycoprotein receptor, which binds and internalizes IgA molecules. Proof of HAV infection was obtained by detection of HAV minus-strand RNA, which is indicative for virus replication, and quantification of infectious virions. We demonstrate that human hepatocytes also ingest HAV–anti-HAV IgA complexes by the same mechanism, resulting in infection of the cells, by using the HepG2 cell line and primary hepatocytes. The relevance of this surrogate receptor mechanism in HAV pathogenesis lies in the fact that HAV, IgA, and antigen-IgA complexes use the same pathway within the organism, leading from the gastrointestinal tract to the liver via blood and back to the gastrointestinal tract via bile fluid. Therefore, HAV-specific IgA antibodies produced by gastrointestinal mucosa-associated lymphoid tissue may serve as carrier and targeting molecules, enabling and supporting HAV infection of IgA receptor-positive hepatocytes and, in the case of relapsing courses, allowing reinfection of the liver in the presence of otherwise neutralizing antibodies, resulting in exacerbation of liver disease.
Establishment and Characterization of Molecular Clones of Porcine Endogenous Retroviruses Replicating on Human Cells
Authors: Czauderna Frank , Fischer Nicole , Boller Klaus , Kurth Reinhard , Tönjes Ralf R.
Published: July 2002

The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of theenvgene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restoredenvstart codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p.t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomicenvtranscript of 3.1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.
Targeted Disruption of theCeacam1(MHVR) Gene Leads to Reduced Susceptibility of Mice to Mouse Hepatitis Virus Infection
Authors: Blau Dianna M. , Turbide Claire , Tremblay Michel , Olson Melanie , Létourneau Stéphanie , Michaliszyn Eva , Jothy Serge , Holmes Kathryn V. , Beauchemin Nicole
Published: July 2002

The CEACAM1 glycoproteins (formerly called biliary glycoproteins; BGP, C-CAM, CD66a, or MHVR) are members of the carcinoembryonic antigen family of cell adhesion molecules. In the mouse, splice variants of CEACAM1 have either two or four immunoglobulin (Ig) domains linked through a transmembrane domain to either a short or a long cytoplasmic tail. CEACAM1 has cell adhesion activity and acts as a signaling molecule, and long-tail isoforms inhibit the growth of colon and prostate tumor cells in rodents. CEACAM1 isoforms serve as receptors for several viral and bacterial pathogens, including the murine coronavirus mouse hepatitis virus (MHV) andHaemophilus influenzae,Neisseria gonorrhoeae,andNeisseria meningitidisin humans. To elucidate the mechanisms responsible for the many biological activities of CEACAM1, we modified the expression of the mouseCeacam1gene in vivo. Manipulation of theCeacam1gene in mouse embryonic stem cells that contained theCeacam1aallele yielded a partial knockout. We obtained one line of mice in which the insert in theCeacam1agene had sustained a recombination event. This resulted in the markedly reduced expression of the two CEACAM1a isoforms with four Ig domains, whereas the expression of the two isoforms with two Ig domains was doubled relative to that in wild-type BALB/c (+/+) mice. Homozygous (p/p)Ceacam1a-targeted mice (Ceacam1aΔ4D) had no gross tissue abnormalities and were viable and fertile; however, they were more resistant to MHV A59 infection and death than normal (+/+) mice. Following intranasal inoculation with MHV A59, p/p mice developed markedly fewer and smaller lesions in the liver than +/+ or heterozygous (+/p) mice. The titers of virus produced in the livers were 50- to 100-fold lower in p/p mice than in +/p or +/+ mice. p/p mice survived a dose 100-fold higher than the lethal dose of virus for +/+ mice. +/p mice were intermediate between +/+ and p/p mice in susceptibility to liver damage, virus growth in liver, and susceptibility to killing by MHV.Ceacam1a-targeted mice provide a new model to study the effects of modulation of receptor expression on susceptibility to MHV infection in vivo.
Mutational Analysis of the Repeated Open Reading Frames, ORFs 63 and 70 and ORFs 64 and 69, of Varicella-Zoster Virus
Authors: Sommer Marvin H. , Zagha Edward , Serrano Oscar K. , Ku Chia Chi , Zerboni Leigh , Baiker Armin , Santos Richard , Spengler Mary , Lynch Jennifer , Grose Charles , Ruyechan William , Hay John , Arvin Ann M.
Published: July 2002

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative
Avr
II site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.
Human Immunodeficiency Virus Type 1 IIIB Selected for Replication In Vivo Exhibits Increased Envelope Glycoproteins in Virions without Alteration in Coreceptor Usage: Separation of In Vivo Replication from Macrophage Tropism
Authors: Miller Eric D. , Duus Karen M. , Townsend Michael , Yi Yanjie , Collman Ronald , Reitz Marvin , Su Lishan
Published: July 2002

Analysis of viral replication and pathogenicity after in vivo selection of human immunodeficiency virus type 1 (HIV-1) attenuated in vitro will help to define the functions involved in replication and pathogenesis in vivo. Using the SCID-hu Thy/Liv mouse and human fetal thymus organ culture as in vivo models, we previously defined HIV-1envdeterminants (HXB2/LW) which were reverted for replication in vivo (L. Su et al., Virology 227:46–52, 1997). In this study, we examined the replication of four highly related HIV-1 clones directly derived from Lai/IIIB or after selection in vivo to investigate the envelope gp120 determinants associated with replication in macrophages and in the thymus models in vivo. The LW/C clone derived from the IIIB-infected laboratory worker and HXB2/LW both efficiently infected monocyte-derived macrophages (MDM) and the human thymus. Although the laboratory worker (LW) isolates showed altered tropism from IIIB, they still predominantly used CXCR4 as coreceptors for infecting peripheral blood mononuclear cells, macrophages, and the thymus. Interestingly, a single amino acid mutation in the V3 loop associated with resistance to neutralizing antibodies was also essential for the replication activity of the LW virus in the thymus models but not for its activity in infecting MDM. The LW virions were equally sensitive to a CXCR4 antagonist. We further demonstrated that the LW HIV-1 isolate selected in vivo produced more infectious viral particles that contained higher levels of the Env protein gp120. Thus, selection of the laboratory-attenuated Lai/IIIB isolate in vivo leads to altered tropism but not coreceptor usage of the virus. The acquired replication activity in vivo is correlated with an early A-to-T mutation in the V3 loop and increased virion association of HIV-1 Env gp120, but it is genetically separable from the acquired replication activity in macrophages.
Folding of Hepatitis C Virus E1 Glycoprotein in a Cell-Free System
Authors: Merola Marcello , Brazzoli Michela , Cocchiarella Fabienne , Heile Jens M. , Helenius Ari , Weiner Amy J. , Houghton Michael , Abrignani Sergio
Published: July 2002

The hepatitis C virus (HCV) envelope proteins, E1 and E2, form noncovalent heterodimers and are leading candidate antigens for a vaccine against HCV. Studies in mammalian cell expression systems have focused primarily on E2 and its folding, whereas knowledge of E1 folding remains fragmentary. We used a cell-free in vitro translation system to study E1 folding and asked whether the flanking proteins, Core and E2, influence this process. We translated the polyprotein precursor, in which the Core is N-terminal to E1, and E2 is C-terminal, and found that when the core protein was present, oxidation of E1 was a slow, E2-independent process. The half-time for E1 oxidation was about 5 h in the presence or absence of E2. In contrast with previous reports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially folded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lower efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 could be detected. Release from calnexin and calreticulin was used to assess formation of mature E1. When E1 was expressed in the absence of Core and E2, its oxidation was impaired. We conclude that E1 folding is a process that is affected not only by E2, as previously shown, but also by the Core. The folding of viral proteins can thus depend on complex interactions between neighboring proteins within the polyprotein precursor.
Residual Viral Replication during Antiretroviral Therapy Boosts Human Immunodeficiency Virus Type 1-Specific CD8+T-Cell Responses in Subjects Treated Early after Infection
Authors: Ortiz Gabriel M. , Hu Jennifer , Goldwitz Joshua A. , Chandwani Rohit , Larsson Marie , Bhardwaj Nina , Bonhoeffer Sebastian , Ramratnam Bharat , Zhang Linqi , Markowitz Martin M. , Nixon Douglas F.
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1)-infected subjects treated early after infection have preserved HIV-1-specific CD4+T-cell function. We studied the effect of highly active antiretroviral therapy (HAART) on the frequency of HIV-1-specific CD8+T cells in patients treated during early (n= 31) or chronic (n= 23) infection. The degree of viral suppression and time of initiation of treatment influenced the magnitude of the CD8+T-cell response. HIV-1-specific CD8+T cells can increase in number after HAART in subjects treated early after infection who have episodes of transient viremia.
Identification of the Lymphocytic Choriomeningitis Virus (LCMV) Proteins Required To Rescue LCMV RNA Analogs into LCMV-Like Particles
Authors: Lee Ki Jeong , Perez Mar , Pinschewer Daniel D. , de la Torre Juan Carlos
Published: July 2002

We have used a reverse genetic approach to identify the viral proteins required for packaging and assembly of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). Plasmids encoding individual LCMV proteins under the control of an RNA polymerase II promoter were cotransfected with a plasmid containing an LCMV minigenome (MG). Intracellular synthesis of the LCMV MG was driven by T7 RNA polymerase whose expression was also mediated by a Pol II promoter. The supernatant from transfected cells was passaged onto fresh cells that were subsequently infected with LCMV to provide the minimal viral
trans
-acting factors, NP and L, that are required for LCMV MG RNA replication and expression. Reconstitution of LCMV-specific packaging and passage was detected by expression of the chloramphenicol acetyl transferase (CAT) reporter gene present in the MG. NP and L did not direct detectable levels of MG passage. Addition of Z and GP resulted in high levels of passage of CAT activity, which could be prevented by LCMV neutralizing antibodies. Passage of LCMV MG was inhibited by omission of either GP or Z.
Human Monocytic Cell Lines Transformed In Vitro by Epstein-Barr Virus Display a Type II Latency and LMP-1-Dependent Proliferation
Authors: Masy Eric , Adriaenssens Eric , Montpellier Claire , Crépieux Pascale , Mougel Alexandra , Quatannens Brigitte , Goormachtigh Gautier , Faumont Nathalie , Meggetto Fabienne , Auriault Claude , Groux Hervé , Coll Jean
Published: July 2002

Epstein-Barr virus (EBV) classically infects and transforms B lymphocytes in vitro, yielding lymphoblastoid cell lines (LCLs). In contrast to other herpesviruses, EBV is not described as an infectious agent for monocytes. However, recent papers described in vitro infection of monocytes leading to abortive or transient viral expression. In the present study, we report the characterization of E1, a monocytic cell line infected and transformed by EBV. This cell line was derived from an LCL by a drastic electroporation and selection of neomycin-resistant cells, unfavorable to B-cell outgrowth. E1 expressed surface molecules of monocytic lineage (CD14, major histocompatibility complex class II, and CD80) and the c-fmsgene, a highly specific marker for the monocytic lineage. This cell line is able to phagocytose and secrete proinflammatory monokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. E1 cells are tumorigenic after injection in nude mice, and a monocytic cell line obtained from one of these tumors (TE1) displayed immunophenotype and functional properties similar to those of E1. We detected the presence of the EBV genome in both cell lines, as well as expression of theEBNA-1andLMP-1, but notEBNA-2, viral genes, characteristic of a type II latency. LMP-1 influences the phenotype of these monocytic cell lines, as demonstrated by down-regulation of cell proliferation and membrane intercellular adhesion molecule 1 expression due to anLMP-1antisense strategy. This is the first description of a latently infected human monocytic cell line and the first direct demonstration of an instrumental role for LMP-1 in the proliferation of EBV-transformed cell lines expressing a type II latency.
The UL48 Tegument Protein of Pseudorabies Virus Is Critical for Intracytoplasmic Assembly of Infectious Virions
Authors: Fuchs Walter , Granzow Harald , Klupp Barbara G. , Kopp Martina , Mettenleiter Thomas C.
Published: July 2002

The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein. UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-ΔUL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-ΔUL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.
Synthetic Analogues of Prostaglandins F2α and E2
Authors: Kozmík Václav , Paleček Jaroslav
Published: July 2002

The synthesis of new derivatives of prostaglandins F2α and E2 XIIa,b - XVa,b and XXa,b - XXIIIa,b containing the furan or thiophene nucleus in the upper chain has been accomplished starting from [3aα,4α,5β,6aα]-(±)-hexahydro-5-hydroxy-4-((E)-(3α)-hydroxy-1-octenyl)-2H-cyclopenta[b]furan-2-one (Ia) and [3aα,4α,5β,6aα]-(±)-hexahydro-5-hydroxy-4-[4-(3-chlorophenoxy)-(3α)-hydroxy-1-butenyl]cyclopenta[b]furan-2-one (Ib). The diols Ia and Ib have been converted into the above-mentioned analogues of prostaglandins F2α and E2 by protecting the hydroxyl groups, subsequent reduction and Wittig reaction with the ylides prepared from the phosphonium salts IV - VII, and final deprotection (or oxidation plus deprotection).
A Convenient Differential Protection Strategy for Functional Groups of Serine. Application to Boc-Ser(Bzl)-OH Synthesis
Authors: Kundu Bijoy , Shukla Sushma
Published: July 2002

Synthesis of Boc-Ser(Bzl)-OH has been carried out in high yield using a novel differential protection scheme.
The Deamino-Carba Analogs of Vasotocin. Synthesis and Some Biological Properties
Authors: Procházka Zdenko , Slaninová Jiřina , Melin Per , Aurell Carl-Johan , Lebl Michal
Published: July 2002

Solid phase methodology on benzhydrylamine resin with linkage agent and Fmoc technique was used for the synthesis of two analogs of deamino carba-1 and carba-6 oxytocin with phenylalanine in position 2 and with ornithine in position 8. The following analogs were prepared: [Phe2,Orn8]deamino-carba-1-oxytocin (I) and [Phe2,Orn8]deamino-carba-6-oxytocin (II). Moreover, analogs with δ-amino group of ornithine protected by benzyloxycarbonyl group and its corresponding sulfoxides were isolated and tested. Both deprotected analogs were found to have high uterotonic activity in vivo, and moderate pressor and antidiuretic agonistic activity. Surprisingly, in uterotonic test they are only partial agonists. The analogs with protected ornithine have preserved high uterotonic in vivo activity in contrary to the pressor activity that was strongly decreased.
Synthesis of Some Epitestosterone Analogues
Authors: Chodounská Hana , Slavíková Barbora , Kasal Alexander
Published: July 2002

11β-Hydroxyandrost-4-ene-3,17-dione (III) was converted into a potential metabolite of epitestosterone - 11β,17α-dihydroxyandrost-4-en-3-one (II) in 5 steps, including the inversion of configuration of a 17β-hydroxy group. This inversion was not feasible in the preparation of the analogues X, XIV, XX, and XXII, where the 17α-hydroxy group was introduced first and only then was the rest of the molecule modified.
Synthesis of New Spiropyrrolidines and Michael Addition Products Using Phase Transfer Catalyzed Addition of Schiff Bases to 9-Arylmethylenefluorenes
Authors: Popandova-Yambolieva Kostadinka
Published: July 2002

As a continuation of our previous studies concerned with the reactivity of benzylidene- and furfurylidenefluorenes in addition reactions, in the present work we have prepared a series of new spiropyrrolidines by reactions of 9-arylmethylenefluorenes I with aryl- (II) and furylazomethines (IV) and some Michael addition products by using diphenylmethyleneazomethines VI and VIII. The structure B was assigned to the furylspiropyrrolidines Va - Vf based on 1H NMR data.
7a-Keto-B-homotestosterone and 7a-Keto-B-homoepitestosterone
Authors: Kohout Ladislav , Kasal Alexander
Published: July 2002

Synthesis of 7α-keto-B-homotestosterone (IX) and its 17α-isomer XIV from 3β-acetoxy-17β(or α)-benzoyloxyandrost-5-en-7-one (I or X) is described. The key reaction is the enlargement of the ring B in reaction with diazomethane in the presence of aluminium chloride.
Preparation of Racemic cis- and trans-2-(2-Alkoxyphenylcarbamoyloxy)cycloheptylmethylpiperidinium Chlorides
Authors: Gregáň Fridrich , Kettmann Viktor , Novomeský Pavol , Sivý Július
Published: July 2002

The synthesis of two pairs of the title diastereomers, which represent conformationally constrained analogues of the phenylcarbamate local anesthetics, is described. The synthesis was accomplished by starting from cycloheptanone and 2-alkoxyanilines and the intermediate diastereomers of 2-aminomethylcycloalkanols (VI, VII) were separated as their 4-nitrobenzoyl derivatives (IV, V) by extraction and fractional crystallization. The prepared compounds (VIIIa, VIIIb, IXa, and IXb) are assumed to be of help in interpreting the structure activity relationships within this class of drugs.
Synthesis of 2-Methylheptadecane and 14-Methylpentadecan-3-one
Authors: Singh Jasvinder , Arora Ajay K. , Kaur Amandeep , Kad Goverdhan L.
Published: July 2002

2-Methylheptadecane (I) is the sex pheromone of the family Arctiidae (tiger moth) and was isolated by Roelof and Carde and assigned structure I on the basis of spectral data. Haque et al. isolated a granular white solid (m.p. 80 °C) from the ether extract of dried and powdered plant of Parthenium hysterophorous L. (Compositae) and proposed its structure, 14-methylpentadecan-3-one (II) on the basis of spectroscopic data. We report herein a short and facile synthesis of these compounds.
2,3-Dihydro-2-oxoergolene Derivatives
Authors: Cvak Ladislav , Stuchlík Josef , Schreiberová Magdalena , Sedmera Petr , Havlíček Vladimír , Flieger Miroslav
Published: July 2002

A general procedure for the preparation of 2-oxo derivatives of 8- or 9-ergolene is reported. Mass and NMR spectra of these compounds are discussed.
Crystallization of Pentaerythritol II. Model Experiments
Authors: Karel Miloslav , Nývlt Jaroslav , Chianese Angelo
Published: July 2002

Kinetics of crystallization of the pentaerythritol has been determined in laboratory experiments: MSMPR (Mixed Suspension Mixed Product Removal) crystallizer experiments served for determination of the kinetics of secondary nucleation and of the kinetics of crystal growth.
Reaction of Ethyl 5-Substituted-2-furoylmalonates with Secondary Amines
Authors: Kada Rudolf , Bruncková Jarmila , Bobál' Pavol
Published: July 2002

In situ prepared magnesium salts of ethyl malonate react with 5-X-2-furoyl chlorides (X = Br, NO2, C6H5S and C6H5SO2) to give the corresponding ethyl 5-X-2-furoylmalonates. On treatment of these compounds with secondary amines no nucleophilic substitution of the group X in position 5 of the furan nucleus took place but, instead, amides of 5-X-2-furancarboxylic acids were isolated.