bluuebunny/crossref_metadata_2025_split huggingface dataset:
Translocation-Specific Conformation of Adenylate Cyclase Toxin from
Bordetella pertussis
Inhibits Toxin-Mediated Hemolysis
Authors: Gray Mary C. , Lee Sang-Jin , Gray Lloyd S. , Zaretzky Franca R. , Otero Angela S. , Szabo Gabor , Hewlett Erik L. 
Published: July 2002

ABSTRACT

Bordetella pertussis
adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three- to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, ΔAC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to ΔAC enhances its hemolytic activity three- to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, ΔN489 (missing amino acids 6 to 489) and ΔN518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while ΔN549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.
Characterization of Intragenic Recombination in a Hyperthermophilic Archaeon via Conjugational DNA Exchange
Authors: Reilly Michelle S. , Grogan Dennis W. 
Published: July 2002

ABSTRACT

Sulfolobus acidocaldarius
is so far the only hyperthermophilic archaeon in which genetic recombination can be assayed by conjugation and simple selections. Crosses among spontanteous
pyr
mutants were able to resolve closely spaced chromosomal mutations, identify deletions and rearrangements, and map mutations to a given deletion interval. Frameshift mutations in
pyrE
exerted polar effects that depressed orotidine-5′-monophosphate decarboxylase activity (encoded by
pyrF
), whereas base pair substitutions and an 18-bp deletion had no effect.
Dynamics of Genome Architecture in
Rhizobium
sp. Strain NGR234
Authors: Mavingui Patrick , Flores Margarita , Guo Xianwu , Dávila Guillermo , Perret Xavier , Broughton William J. , Palacios Rafael 
Published: July 2002

ABSTRACT

Bacterial genomes are usually partitioned in several replicons, which are dynamic structures prone to mutation and genomic rearrangements, thus contributing to genome evolution. Nevertheless, much remains to be learned about the origins and dynamics of the formation of bacterial alternative genomic states and their possible biological consequences. To address these issues, we have studied the dynamics of the genome architecture in
Rhizobium
sp. strain NGR234 and analyzed its biological significance. NGR234 genome consists of three replicons: the symbiotic plasmid pNGR234
a
(536,165 bp), the megaplasmid pNGR234
b
(>2,000 kb), and the chromosome (>3,700 kb). Here we report that genome analyses of cell siblings showed the occurrence of large-scale DNA rearrangements consisting of cointegrations and excisions between the three replicons. As a result, four new genomic architectures have emerged. Three consisted of the cointegrates between two replicons: chromosome-pNGR234
a
, chromosome-pNGR234
b
, and pNGR234
a
-pNGR234
b
. The other consisted of a cointegrate of the three replicons (chromosome-pNGR234
a
-pNGR234
b
). Cointegration and excision of pNGR234
a
with either the chromosome or pNGR234
b
were studied and found to proceed via a Campbell-type mechanism, mediated by insertion sequence elements. We provide evidence showing that changes in the genome architecture did not alter the growth and symbiotic proficiency of
Rhizobium
derivatives.
Cyclospora cayetanensis
in Three Populations at Risk in Guatemala
Authors: Pratdesaba Rafael A. , González Mario , Piedrasanta Evelyn , Mérida Claudia , Contreras Karen , Vela Carlos , Culajay Francisco , Flores Luis , Torres Olga 
Published: July 2002

ABSTRACT

In 1996 and 1997, outbreaks of
Cyclospora cayetanensis
in North America were linked to Guatemalan raspberries. From April 1999 to April 2000, we undertook a survey for
C. cayetanensis
in raspberry farm workers, malnourished children, and human immunodeficiency virus and AIDS patients in Guatemala. Stool samples were analyzed using ethylacetate-formalin concentration, wet preparation, modified acid-fast staining method, and epifluorescence. Oocysts were found in 1.5% of the subjects, none of whom were raspberry farm workers.
E-Test Method for Testing Susceptibilities of
Aspergillus
spp. to the New Triazoles Voriconazole and Posaconazole and to Established Antifungal Agents: Comparison with NCCLS Broth Microdilution Method
Authors: Espinel-Ingroff Ana , Rezusta A. 
Published: July 2002

ABSTRACT

NCCLS document M38-P describes standard parameters for testing the fungistatic activities (MICs) of established agents against filamentous fungi (molds). This study evaluated the in vitro susceptibilities of 15
Aspergillus flavus
isolates, 62
A. fumigatus
isolates, and 10 isolates each of
A. niger
,
A. nidulans
, and
A. terreus
to voriconazole, posaconazole, itraconazole, and amphotericin B by the E-test and NCCLS M38-P microdilution methods. The agreement (within 3 dilutions) between methods for voriconazole was independent of the E-test incubation time (93.3 to 100% for four of five species at both incubation times). In contrast, with amphotericin B, itraconazole, and posaconazole, E-test results were more dependent on the incubation time for certain species. For
A. fumigatus,
posaconazole E-test MICs had better concordance with reference values after 48 h (95.2%) than after 24 h (90%), while the highest agreement for itraconazole MICs was after 24 h (90.3 versus 74.2%) of incubation. Better agreement between the methods was also obtained with 24-h E-test amphotericin B MICs for
A. flavus
(73.3 versus 26.7%) and
A. fumigatus
(96.7 versus 64.5%). E-test MICs of the four agents had the lowest percentages of agreement with reference values for
A. nidulans
(60 to 80%). For isolates for which high MICs were obtained for the four agents by the reference method, high MICs were also obtained by E-test at both 24 and 48 h. The utility of in vitro results of either the E-test or the NCCLS broth microdilution (M38-P) method for
Aspergillus
spp. needs to be established in clinical trials.
Polymorphism in the Interleukin-4 Promoter Affects Acquisition of Human Immunodeficiency Virus Type 1 Syncytium-Inducing Phenotype
Authors: Nakayama Emi E. , Hoshino Yoshihiko , Xin Xiaomi , Liu Huanliang , Goto Mieko , Watanabe Nobukazu , Taguchi Hitomi , Hitani Akihiro , Kawana-Tachikawa Ai , Fukushima Masao , Yamada Kaneo , Sugiura Wataru , Oka Shin-Ichi , Ajisawa Atsushi , Sato Hironori , Takebe Yutaka , Nakamura Tetsuya , Nagai Yoshiyuki , Iwamoto Aikichi , Shioda Tatsuo 
Published: July 2002

ABSTRACTThe emergence of syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) in infected individuals is an indicator of poor prognosis and is often correlated with faster CD4+cell depletion and rapid disease progression. Interleukin-4 (IL-4) is a pleiotropic cytokine with various immune-modulating functions including induction of immunoglobulin E (IgE) production in B cells, down-regulation of CCR5 (a coreceptor for HIV-1 non-SI [NSI] strains), and up-regulation of CXCR4 (a coreceptor for HIV-1 SI variants). Here we show that homozygosity of a polymorphism in the IL-4 promoter region, IL-4 −589T, is correlated with increased rates of SI variant acquisition in HIV-1-infected individuals in Japan. This mutation was also shown to be associated with elevated serum IgE levels in HIV-1-infected individuals, especially in those at advanced stages of disease. In contrast, neither a triallele polymorphism in IL-10, another Th2 cytokine, nor a biallele polymorphism in the RANTES promoter affected acquisition of the SI phenotype. This finding suggested that IL-4-589T increases IL-4 production in the human body and thus accelerates the phenotypic switch of HIV-1 from NSI to SI and possibly disease progression of AIDS.
The E1∧E4 Protein of Human Papillomavirus Type 16 Associates with a Putative RNA Helicase through Sequences in Its C Terminus
Authors: Doorbar John , Elston Robert C. , Napthine Sawsan , Raj Kenneth , Medcalf Elizabeth , Jackson Deborah , Coleman Nick , Griffin Heather M. , Masterson Philip , Stacey Simon , Mengistu Yohannes , Dunlop Julia 
Published: July 2002

ABSTRACTHuman papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1∧E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1∧E4.L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1∧E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1∧E4 (in vivo and in vitro) and could be immunoprecipitated as an E1∧E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1∧E4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1∧E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with threeEscherichia coliDEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein ofSaccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1∧E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1∧E4-associated protein indicates a possible role for E1∧E4 in virus synthesis.
Immune Responses following Neonatal DNA Vaccination Are Long-Lived, Abundant, and Qualitatively Similar to Those Induced by Conventional Immunization
Authors: Hassett Daniel E. , Zhang Jie , Slifka Mark , Whitton J. Lindsay 
Published: July 2002

ABSTRACTVirus infections are devastating to neonates, and the induction of active antiviral immunity in this age group is an important goal. Here, we show that a single neonatal DNA vaccination induces cellular and humoral immune responses which are maintained for a significant part of the animal's life span. We employ a sensitive technique which permits the first demonstration and quantitation, directly ex vivo, of virus-specific CD8+T cells induced by DNA immunization. One year postvaccination, antigen-specific CD8+T cells were readily detectable and constituted 0.5 to 1% of all CD8+T cells. By several criteria—including cytokine production, perforin content, development of lytic ability, and protective capacity—DNA vaccine-induced CD8+memory T cells were indistinguishable from memory cells induced by immunization with a conventional (live-virus) vaccine. Analyses of long-term humoral immune responses revealed that, in contrast to the strong immunoglobulin G2a (IgG2a) skewing of the humoral response seen after conventional vaccination, IgG1 and IgG2a levels were similar in DNA-vaccinated neonatal and adult animals, indicating a balanced T helper response. Collectively, these results show that a single DNA vaccination within hours or days of birth can induce long-lasting CD8+T- and B-cell responses; there is no need for secondary immunization (boosting). Furthermore, the observed immune responses induced in neonates and in adults are indistinguishable by several criteria, including protection against virus challenge.
Superantigen Expression Is Driven by Both Mouse Mammary Tumor Virus Long Terminal Repeat-Associated Promoters in Transgenic Mice
Authors: Salmons Brian , Miethke Thomas , Wintersperger Stefan , Müller Mathias , Brem Gottfried , Günzburg Walter H. 
Published: July 2002

ABSTRACTIn addition to the usual retroviral promoter, the mouse mammary tumor virus (MMTV) long terminal repeat carries a second promoter located in the U3 region. Here we show that both of these promoters are independently able to give rise to superantigen activity in transgenic mice. The ability of multiple MMTV promoters to drive superantigen expression underscores its importance in the virus life cycle.
Generation of Cytomegalovirus-Specific Human T-Lymphocyte Clones by Using Autologous B-Lymphoblastoid Cells with Stable Expression of pp65 or IE1 Proteins: a Tool To Study the Fine Specificity of the Antiviral Response
Authors: Retière Christelle , Prod'homme Virginie , Imbert-Marcille Berthe-Marie , Bonneville Marc , Vié Henri , Hallet Marie-Martine 
Published: July 2002

ABSTRACTCytotoxic T lymphocytes (CTLs) play a central role in the control of persistent human cytomegalovirus (HCMV) infection in healthy virus carriers. Previous analyses of the specificity of HCMV-reactive CD8+CTLs drawn from in vitro models in which antigen-presenting cells were autologous fibroblasts infected with laboratory HCMV strains have shown focusing of CTL responses against the major tegument protein, pp65. By contrast, the 72-kDa major immediate-early protein (IE1) was identified as a minor target for this response. Here we have studied the fine specificity and T-cell-receptor features of T-cell clones generated against autologous B lymphoblastoid cell lines stably transfected with HCMV cDNA coding for either pp65 or a natural variant of IE1. This strategy allowed efficient generation of T-cell clones against IE1 and pp65 and led to the identification of several new IE1 and pp65 epitopes, including some located in polymorphic regions of IE1. Such an approach may provide relevant information about the characteristics of the CTL response to IE1 and the effect of viral polymorphism on the immune response against HCMV.
Identification of Two Sequences in the Cytoplasmic Tail of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein That Inhibit Cell Surface Expression
Authors: Bültmann Andreas , Muranyi Walter , Seed Brian , Haas Jürgen 
Published: July 2002

ABSTRACTDuring synthesis and export of protein, the majority of the human immunodeficiency virus type 1 (HIV-1) Env glycoprotein gp160 is retained in the endoplasmic reticulum (ER) and subsequently ubiquitinated and degraded by proteasomes. Only a small fraction of gp160 appears to be correctly folded and processed and is transported to the cell surface, which makes it difficult to identify negative sequence elements regulating steady-state surface expression of Env at the post-ER level. Moreover, poorly localized mRNA retention sequences inhibiting the nucleocytoplasmic transport of viral transcripts interfere with the identification of these sequence elements. Using two heterologous systems with CD4 or immunoglobulin extracellular/transmembrane domains in combination with the gp160 cytoplasmic domain, we were able to identify two membrane-distal, neighboring motifs,is1(amino acids 750 to 763) andis2(amino acids 764 to 785), which inhibited surface expression and induced Golgi localization of the chimeric proteins. To prove that these two elements act similarly in the homologous context of the Env glycoprotein, we generated a synthetic gp160 gene with synonymous codons, the transcripts of which are not retained within the nucleus. In accordance with the results in heterologous systems, an internal deletion of both elements considerably increased surface expression of gp160.
Simian Virus 40 Large-T-Antigen-Specific Rejection of mKSA Tumor Cells in BALB/c Mice Is Critically Dependent on both Strictly Tumor-Associated, Tumor-Specific CD8+Cytotoxic T Lymphocytes and CD4+T Helper Cells
Authors: Utermöhlen Olaf , Schulze-Garg Christine , Warnecke Gabriele , Gugel Roland , Löhler Jürgen , Deppert Wolfgang 
Published: July 2002

ABSTRACTProtective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8+and CD4+T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4+and CD8+cells both were equally important in establishing and maintaining a protective immune response. CD4+cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4+lymphocytes provide help to CD8+cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4+cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8+lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8+cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8+T cells. Moreover, this helper effect of CD4+T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8+CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4+T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.
Sequence Requirements for Sindbis Virus Subgenomic mRNA Promoter Function in Cultured Cells
Authors: Wielgosz Matthew M. , Raju Ramaswamy , Huang Henry V. 
Published: July 2002

ABSTRACT
The Sindbis virus minimal subgenomic mRNA promoter (spanning positions −19 to +5 relative to the subgenomic mRNA start site) is approximately three- to sixfold less active than the fully active −98 to +14 promoter region. We identified two elements flanking the −19 to +5 region which increase its transcription to levels comparable to the −98 to +14 region. These elements span positions −40 to −20 and +6 to +14 and act synergistically to enhance transcription. Nine different virus libraries were constructed containing blocks of five randomized nucleotides at various positions in the −40 to +14 region. On passaging these libraries in mosquito cells, a small subset of the viruses came to dominate the population. Sequence analysis at the population level and for individual clones revealed that in general, wild-type bases were preferred for positions −15 to +5 of the minimal promoter. Base mutagenesis experiments indicated that the selection of wild-type bases in this region was primarily due to requirements for subgenomic mRNA transcription. Outside of the minimal promoter, the −35 to −29 region contained four positions which also preferred wildtype bases. However, the remaining positions generally preferred non-wild-type bases. On passaging of the virus libraries on hamster cells, the −15 to +5 region again preferred the wild-type base but most of the remaining positions exhibited almost no base preference. The promoter thus consists of an essential central region from −15 to +5 and discrete flanking sites that render it fully active, depending on the host environment.
Human Cytomegalovirus US7, US8, US9, and US10 Are Cytoplasmic Glycoproteins, Not Found at Cell Surfaces, and US9 Does Not Mediate Cell-to-Cell Spread
Authors: Huber Mary T. , Tomazin Roman , Wisner Todd , Boname Jessica , Johnson David C. 
Published: July 2002

ABSTRACT

Human cytomegalovirus (HCMV) expresses a large number of membrane proteins with unknown functions. One class of these membrane proteins apparently acts to allow HCMV to escape detection by the immune system. The best characterized of these are the glycoproteins encoded within the US2 to US11 region of the HCMV genome that mediate resistance to CD8
+
and CD4
+
T cells. US2, US3, US6, and US11 block various aspects of the major histocompatibility complex (MHC) class I and class II antigen presentation pathways, functioning in cytoplasmic membranes to cause retention, degradation, or mislocalization of MHC proteins. Distantly homologous genes in this region, US7, US8, US9, and US10, are not well characterized. Here, we report expression of the glycoproteins encoded by US7 to US10 by using replication-defective adenovirus (Ad) vectors. US7, US9, and US10 remained sensitive to endoglycosidase H and were exclusively or largely present in the endoplasmic reticulum (ER) as determined by confocal microscopy. US8 reached the Golgi apparatus and trans-Golgi network and was more quickly degraded. Previous studies suggested that US9 could localize to cell junctions and mediate cell-to-cell spread in ARPE-19 retinal epithelial cells. We found no evidence of US9 at cell junctions of HEC-1A epithelial cells. HCMV recombinants lacking US9 produced smaller plaques on ARPE-19 cell monolayers but also exhibited defects in virus replication compared with wild-type HCMV in these cells. Other HCMV recombinants constructed in a similar fashion that were able to express US9 also produced small plaques and some of these exhibited defects in production of infectious progeny in ARPE-19 cells. Thus, there was no correlation between defects in cell-to-cell spread (plaque size) and loss of expression of US9, and it is possible that US9
−
mutants produce smaller plaques because they produce fewer progeny. Together, our results do not support the hypothesis that US9 plays a direct role in HCMV cell-to-cell spread.
Antiviral Effects of Pyrrolidine Dithiocarbamate on Human Rhinoviruses
Authors: Gaudernak Elisabeth , Seipelt Joachim , Triendl Andrea , Grassauer Andreas , Kuechler Ernst 
Published: July 2002

ABSTRACTHuman rhinoviruses (HRVs) are the predominant cause of the common cold. The frequency of HRV infections in industrial countries and the lack of effective therapeutical treatment underline the importance of research for new antiviral substances. As viral infections are often accompanied by the generation of oxidative stress inside the infected cells, several redox-active substances were tested as potential antivirals. In the course of these studies it was discovered that pyrrolidine dithiocarbamate (PDTC) is an extremely potent compound against HRV and poliovirus infection in cell culture. Besides the ability to dramatically reduce HRV production by interfering with viral protein expression, PDTC promotes cell survival and abolishes cytopathic effects in infected cells. PDTC also protects cells against poliovirus infection. These effects were highly specific, as several other antioxidants (vitamin C, Trolox, 2-mercaptoethanol, andN-acetyl-l-cysteine) are inactive against HRV infection. Synthesis of HRV proteins and cleavage of eucaryotic initiation factor 4G responsible for host cell shutoff of cellular protein synthesis are severely inhibited in the presence of PDTC.