bluuebunny/crossref_metadata_2025 huggingface dataset:
Fzf1p Regulates an Inducible Response to Nitrosative Stress in Saccharomyces cerevisiae
Authors: Sarver Aaron , DeRisi Joseph
Published: July 2005

The mechanisms by which microorganisms sense and detoxify nitric oxide (.NO) are of particular interest due to the central role this molecule plays in innate immunity. We investigated the genetic basis of inducible nitric oxide (.NO) detoxification in Saccharomyces cerevisiae by characterizing the genome-wide transcriptional response to exogenously supplied .NO. Exposure to the .NO-generating compound dipropylenetriamine NONOate resulted in both a general stress response as well as a specific response characterized by the induction of a small set of genes, including the yeast flavohemoglobin YHB1, SSU1, and three additional uncharacterized open reading frames. Transcriptional induction of SSU1, which encodes a putative sulfite transporter, has previously been shown to require the zinc finger transcription factor Fzf1p. Deletion of Fzf1p eliminated the nitrosative stress-specific transcriptional response, whereas overexpression of Fzf1p recapitulated this response in the absence of exogenously supplied .NO. A cis-acting sequence unique to the promoter regions of Fzf1p-dependent genes was found to be sufficient to activate reporter gene activity in an .NO- and Fzf1p-dependent manner. Our results suggest that the presence of .NO or .NO derivatives activates Fzf1p leading to transcriptional induction of a discrete set of target genes that function to protect the cell from .NO-mediated stress.
The Ferrier Lecture 1995 Behind the Seen: The functional specialization of the brain in space and time
Authors: Zeki Semir
Published: July 2005

The visual brain consists of many different visual areas, which are functionally specialized to process and perceive different attributes of the visual scene. However, the time taken to process different attributes varies; consequently, we see some attributes before others. It follows that there is a perceptual asynchrony and hierarchy in visual perception. Because perceiving an attribute is tantamount to becoming conscious of it, it follows that we become conscious of different attributes at different times. Visual consciousness is therefore distributed in time. Given that we become conscious of different visual attributes because of activity at different, functionally specialized, areas of the visual brain, it follows that visual consciousness is also distributed in space. Therefore, visual consciousness is not a single unified entity, but consists of many microconsciousnesses.
Drosophilabristles and the nature of quantitative genetic variation
Authors: Mackay Trudy F.C , Lyman Richard F
Published: July 2005

Numbers ofDrosophilasensory bristles present an ideal model system to elucidate the genetic basis of variation for quantitative traits. Here, we review recent evidence that the genetic architecture of variation for bristle numbers is surprisingly complex. A substantial fraction of theDrosophilagenome affects bristle number, indicating pervasive pleiotropy of genes that affect quantitative traits. Further, a large number of loci, often with sex- and environment-specific effects that are also conditional on background genotype, affect natural variation in bristle number. Despite this complexity, an understanding of the molecular basis of natural variation in bristle number is emerging from linkage disequilibrium mapping studies of individual candidate genes that affect the development of sensory bristles. We show that there is naturally segregating genetic variance for environmental plasticity of abdominal and sternopleural bristle number. For abdominal bristle number this variance can be attributed in part to anabnormal abdomen-like phenotype that resembles the phenotype of mutants defective in catecholamine biosynthesis.Dopa decarboxylase(Ddc) encodes the enzyme that catalyses the final step in the synthesis of dopamine, a majorDrosophilacatecholamine and neurotransmitter. We found that molecular polymorphisms atDdcare indeed associated with variation in environmental plasticity of abdominal bristle number.
Maternal experience of pain during external cephalic version at term
Authors: Fok Wing Yee , Chan Lin Wai , Leung Tak Yeung , Lau Tze Kin
Published: July 2005

Background.  This study was designed to investigate maternal pain perception during external cephalic version (ECV).

Methods.  This prospective study included 97 pregnant women with singleton breech presentation at or above 36 weeks of gestation undergoing ECV. No analgesia or anesthesia were used during ECV. The women were asked to rate the degree of pain on a 10‐cm visual analog scale after ECV.

Results.  Ninety‐eight ECVs were performed and the overall success rate was 66%. The median pain score was 5.7 (interquartile range 2.7–6.8). The visual analog pain score was significantly lower among those with a successful ECV than those with a failed procedure (median score 4.6 vs. 6.8, p < 0.001). Similarly, those procedures rated as easy by the operator were associated with a significantly lower pain score (3.4 vs. 6.4, p < 0.001). However, the pain score was not found to be related to the operator, parity or placental site. Among the five subjects with a pain score of 8.5 and beyond, the ECV procedure was still successful in four (80%).

Conclusion.  ECV performed without analgesia was associated with a moderate degree of pain that was well tolerated by the majority of patients because of its short duration.
Vaginal fluid β‐human chorionic gonadotropin level in the diagnosis of premature rupture of membranes
Authors: Kim Young‐Han , Park Yong‐Won , Kwon Han‐Sung , Kwon Ja‐Young , Kim Bok‐Ja
Published: July 2005

Background.  To determine whether the measurement of β‐human chorionic gonadotropin (β‐HCG) level in the vaginal washing fluid could be useful for the diagnosis of premature rupture of membranes.

Methods.  Totally, 120 pregnant women were enrolled in this study. Subjects were divided into four groups [group I, no preterm labor and term delivery (n = 38); group II, preterm labor and term delivery (n = 12); group III, preterm labor and consequent premature delivery (n = 24); group IV, preterm labor with premature rupture of membranes (PROM) and consequent premature delivery (n = 46)]. After irrigating the posterior vaginal fornix with 3 ml of sterile saline and the obtained vaginal washing fluid, we measured β‐HCG levels.

Results.  The median and range of vaginal fluid β‐HCG levels were 3.60 (0.09–30.52), 4.42 (0.33–10.02), 15.50 (0.25–378.62), and 512.53 (26.95–3507.20) mIU/ml in group I, group II, group III, and group IV, respectively. Vaginal β‐HCG level was significantly higher in patients with PROM followed by premature delivery (group IV) than patients in other groups (P < 0.01). From the receiver operating characteristic curve, 39.8mIU/ml was set as a cutoff value. Sensitivity, specificity, positive predictive value, and negative predictive value were 95.5, 94.7, 91.3, and 97.3%, respectively.

Conclusion.  Our study demonstrated that the measurement of vaginal fluid β‐HCG may be reliable, simple, and rapid test in diagnosing PROM and used as a adjunctive test in equivocal cases.
Serum acute phase protein concentrations after hysterectomy with and without low‐molecular‐weight heparin thrombosis prophylaxis
Authors: Maciejczyk‐Pencuła Monika , Polak Grzegorz , Kotarski Jan
Published: July 2005

Background.  Low‐molecular‐weight heparin (LMWH) is recommended for venous thrombosis prophylaxis. There are several lines of evidence that heparins exhibit anti‐inflammatory properties in addition to their anticoagulant activity. Therefore, we decided to estimate the concentrations of C‐reactive protein (CRP) and haptoglobin, as acute‐phase proteins, in the blood serum of patients who underwent total abdominal hysterectomy with and without the LMWH administration.

Method.  Forty‐two women were studied, including 26 patients treated with enoxaparin and a group of 16 women without thrombosis prophylaxis. Haptoglobin and CRP concentrations were measured in serum samples obtained before the operation and also 8, 24, 48, 72, 96, and 192 hr after surgery.

Results.  Serum CRP level measured 8 hr after the operation was significantly higher in the group without thrombosis prophylaxis, however, no statistically significant differences in the concentration of CRP between studied groups in the other time periods were found. There were no statistically significant differences in serum haptoglobin levels between the groups of patients in all time periods.

Conclusion.  Administration of low‐molecular‐weight heparin may affect CRP production, however, further studies are still needed in order to clarify the exact immunosuppressive action of heparins.
Gonadotrophin‐releasing hormone‐antagonist luteolysis during the preceding mid‐luteal phase is a feasible protocol in ovarian hyperstimulation before in vitro fertilization
Authors: Fridén Barbro E. , Nilsson Lars
Published: July 2005

Background.  The aims of this study were to investigate whether a high dose of gonadotrophin‐releasing hormone antagonist during the preceding luteal phase yields satisfactory luteolysis and downregulation prior to ovarian hyperstimulation in an in vitro fertilization program.

Methods.  The treatment protocol was designed as a prospective pilot study in IVF units at two university hospitals. Fifty‐one patients with documented poor or normal response to ovarian stimulation underwent 57 treatment cycles. Treatment consisted of 3 mg gonadotrophin‐releasing hormone antagonist (cetrorelix) given during the preceding mid‐luteal phase as gonadotrophin‐releasing hormone‐receptor inhibition before ovarian stimulation with follicle‐stimulating hormone.

Results.  All women experienced menstrual bleeding within 2–3 days after the injection of 3 mg cetrorelix. Follicle hormone stimulation could subsequently commence in all cycles. All but three patients developed growing follicles and underwent oocyte retrieval. Seventy‐five percent of started cycles reached transfer of one or two good‐quality embryos.

Conclusions.  A single high‐dose gonadotrophin‐releasing hormone antagonist given during the preceding luteal phase presents a new and feasible protocol to initiate controlled ovarian hyperstimulation in an in vitro fertilization program.
Follicle‐stimulating hormone receptor polymorphisms in a population of infertile women
Authors: Falconer Henrik , Andersson Eva , Aanesen Arthur , Fried Gabriel
Published: July 2005

Background.  There are two known polymorphisms of clinical relevance in the follicle‐stimulating hormone (FSH) receptor exon 10, alanine or threonine at position 307, and asparagine or serine at position 680, giving rise to two discrete allelic variants: Thr307/Asn680 and Ala307/Ser680. At position 680, three FSH receptor variants are possible: Asn/Asn, Asn/Ser, and Ser/Ser. We hypothesized an association between FSH receptor polymorphisms and ovarian reserve.

Methods.  FSH receptor polymorphisms at position 680 were studied in a population of 68 infertile women. We used serum FSH level at cycle day 3 as a screening for ovarian reserve. DNA was extracted from peripheral leukocytes in whole blood by using PCR and DNA sequencing in order to determine the type of FSH receptor.

Results.  The distribution of FSH receptor variants was Asn/Asn (AA) 35%, Asn/Ser (AS) 24%, and Ser/Ser (SS) 41%. In women with normal ovarian reserve, FSH levels at cycle day 3 were 5.6 ± 1.9 (AA group), 6.7 ± 1.3 (AS group), and 5.7 ± 1.7 (SS group) (non‐significant). Corresponding FSH levels at cycle day 10 were 6.9 ± 1.9, 6.3 ± 1.7, and 8.3 ± 2.8 (P < 0.01, AA and AS vs. SS group). In the SS group, FSH levels at cycle day 10 were significantly higher than they were at cycle day 3 (P < 0.05).

Conclusions.  The results show that Ser/Ser‐680 predominates in the studied infertile population. Furthermore, women with normal ovarian reserve and the Ser/Ser FSH receptor variant had significantly higher FSH levels, compared to women with Asn/Asn and Asn/Ser variants. FSH receptor genotyping may, thus, be interesting as an adjunct indicator of ovarian reserve for infertile women undergoing assisted reproduction, and may be helpful in the determination of the starting dosage of FSH in in vitro fertilization.
Magnetic resonance imaging in 300 cases of placenta accreta: surgical correlation of new findings
Authors: Palacios Jaraquemada José M. , Bruno Claudio H.
Published: July 2005

Background.  To establish the usefulness of placental magnetic resonance in patients with a diagnosis of placenta accreta through the correlation of diagnostic images and surgical findings.

Methods.  Three hundred patients with ultrasound signs of placenta accreta were studied. In 252 patients, magnetic resonance imaging (MRI) was performed in a closed 1.5 T‐resonator, and in 48 patients, open 0.23 T‐set was used. T1 and T2 slices in the three planes were performed, and placental invasion was classified in depth levels and topographic areas in relation to the posterior vesical wall. The final degree of invasion was established during surgery according to clinical and anatomical criteria. The information obtained with MRI was analyzed, thus establishing its relevance to the change in surgical technique.

Results.  In 286 cases, MRI provided topographic information of placental invasion, and in 90 patients, it modified invasion levels. Undiagnosed parametrium extent was determined in 11 cases, and 11 other cases were reclassified as placenta previa. Changes in conduct following MRI study included: recommendation to modify surgery date at week 35, recommendation for prophylactic ureteral catheterization, recommendation for the use of intraoperative blood salvage, possibility of approach through Pfannenstiel incision, probability of segmental myometrial approach, probability of aortic clamping, need to investigate subclinical disseminated intravascular coagulation, need of posterior pelvic dissection, and the possibility of uterine conservation.

Conclusions.  Magnetic resonance imaging turned out to be essential to define the topography and area of placental invasion. New findings modified surgical tactic and technique, allowing a reduction in historical morbidity and a significant increase in conservative surgeries.
Sample preparation procedures for biological atomic force microscopy
Authors: EL KIRAT K. , BURTON I. , DUPRES V. , DUFRENE Y. F.
Published: July 2005

Since the late 1980s, atomic force microscopy (AFM) has been increasingly used in biological sciences and it is now established as a versatile tool to address the structure, properties and functions of biological specimens. AFM is unique in that it provides three‐dimensional images of biological structures, including biomolecules, lipid films, 2D protein crystals and cells, under physiological conditions and with unprecedented resolution. A crucial prerequisite for successful, reliable biological AFM is that the samples need to be well attached to a solid substrate using appropriate, nondestructive methods. In this review, we discuss common techniques for immobilizing biological specimens for AFM studies.
Analysis of Expressed Sequence Tags (ESTs) in the Ciliated Protozoan Tetrahymena thermophila
Authors: FILLINGHAM JEFFREY S. , CHILCOAT N. DOANE , TURKEWITZ AARON P. , ORIAS EDUARDO , REITH MICHAEL , PEARLMAN RONALD E.
Published: July 2005

ABSTRACT. To assess the utility of expressed sequence tag (EST) sequencing as a method of gene discovery in the ciliated protozoan Tetrahymena thermophila, we have sequenced either the 5’or 3’ends of 157 clones chosen at random from two cDNA libraries constructed from the mRNA of vegetatively growing cultures. Of 116 total non‐redundant clones, 8.6% represented genes previously cloned in Tetrahymena. Fifty‐two percent had significant identity to genes from other organisms represented in GenBank, of which 92% matched human proteins. Intriguing matches include an opioid‐regulated protein, a glutamate‐binding protein for an NMDA‐receptor, and a stem‐cell maintenance protein. Eleven‐percent of the non‐Tetrahymena specific matches were to genes present in humans and other mammals but not found in other model unicellular eukaryotes, including the completely sequenced Saccharomyces cerevisiae. Our data reinforce the fact that Tetrahymena is an excellent unicellular model system for studying many aspects of animal biology and is poised to become an important model system for genome‐scale gene discovery and functional analysis.
Molecular Taxonomy of the Suborder Bodonina (Order Kinetoplastida), Including the Important Fish Parasite, Ichthyobodo necator
Authors: CALLAHAN HEATHER A. , LITAKER R. WAYNE , NOGA EDWARD J.
Published: July 2005

ABSTRACT. Ichthyobodo necator is an important fish ectoparasite with a broad host and ecological range. A novel method, involving the use of an anesthetic, allowed the collection of large numbers of parasites from the skin and gills of hybrid striped bass (Morone saxatilis male ×M. chrysops female). Genomic DNA from these samples was used to amplify and clone the 18S rRNA gene. The 18S rRNA gene was similarly cloned from Bodo caudatus, Bodo edax, Bodo saltans, an unidentified Bodo species, and Dimastigella trypaniformis. The resulting sequences were aligned with other representative kinetoplastid species using pileup and similarities in secondary structure. Phylogenetic relationships within the suborder Bodonina and representatives of the suborder Trypanosomatina were determined using maximum‐likelihood statistics. The phylogenetic analyses strongly supported the order Kinetoplastida as a monophyletic assemblage consisting of at least two major lineages. One lineage consisted exclusively of 1. necator, indicating that it may represent a new suborder. The second lineage consisted of all other kinetoplastid species. This second lineage appeared to contain at least 8 bodonine sublineages, none of which correlated with currently recognized families. For three sublineages, there was a close correspondence between the 18S phylogeny and the classical taxonomy of Dimastigella, Rhynchobodo, and Rhynchomonas. In contrast, Bodo and Cryptobia were polyphyletic, containing species in two or more sublineages that may represent separate genera.
Spironucleus vortens (Diplomonadida) in the Ide, Leuciscus idus (L.) (Cyprinidae): a Warm Water Hexamitid Flagellate Found in Northern Europe
Authors: STERUD ERIK , POYNTON SARAH L.
Published: July 2005

ABSTRACT. The hexamitid flagellate Spironucleus vortens, previously reported from Pterophyllum scalare from Florida, was found in the intestine of Leuciscus idus in Norway. The flagellate was cultivated and studied by scanning and transmission electron microscopy. Identification was based on a suite of ultrastructural features unique for S. vortens: compound lateral ridges, a swirled posterior end, and a distinctive microtubular cytoskeleton. Microfibrillar structures with a periodicity of 0.13 μm in the right peripheral part of the compound lateral ridges were shown to be responsible for the distinctive rope‐like appearance of the peripheral ridge seen in scanning electron micrographs, and not previously reported for S. vortens. The present results show a wide geographic distribution and a wide temperature tolerance for S. vortens. The flagellate was successfully cultivated at 5°C and 15°C, having previously been cultivated between 2–34°C. Spironucleus vortens is believed to be endemic in Norwegian waters, but an introduction hypothesis is also discussed. The similarity is striking between S. vortens and S. elegans, previously described from amphibians and fish in Europe, and the possibility of conspecificity is believed to be high.
Brachiola algerae Spore Membrane Systems, their Activity During Extrusion, and a New Structural Entity, the Multilayered Interlaced Network, Associated with the Polar Tube and the Sporoplasm
Authors: CALI ANN , WEISS LOUIS M. , TAKVORIAN PETER M.
Published: July 2005

ABSTRACT. The microsporidial genus, Brachiola, contains three species: the type species Brachiola vesicularum (identified from an AIDS patient) and two species transferred from the genus Nosema, becoming Brachiola connori and Brachiola algerae. A developmental feature of the genus Brachiola is the “thickened” plasmalemma from sporoplasm through sporoblast stage. The sporoplasm has been reported to have a thick plasmalemma at 1‐h postextrusion. The purpose of this investigation was to observe B. algerae spores before, during and after germination to determine if the plasmalemma is thick at the point of extrusion and if not, when and how it forms. New understandings regarding the polar filament position inside the spore, places it outside the sporoplasm proper with the sporoplasm limiting membrane imaginations surrounding it. These invaginations, present a possible location for aquaporins. The multilayered interlaced network (MIN), a new organelle (possibly of Golgi origin from the sporoblast), was observed inside the spore and sporoplasm; it formed an attachment to the end of the extruded polar tube and contributed to the thickening of the sporoplasm plasmalemma. A thin “unit limiting membrane”, present on the sporoplasm at the time of extrusion, is connected to the MIN by many cross‐connections forming the “thick blistered” surface by 30 min‐postextrusion.
Trends in Clinical Toxicology: Advances That May Change Your Practice
Authors: Wiener Sage W. , Hoffman Robert S.
Published: July 2005

Abstract: Recent studies have had a significant impact on the practice of Medical Toxicology. We review selected articles that have advanced our thinking about consequential issues such as gastrointestinal decontamination, paracetamol poisoning, ethanol withdrawal, cocaine‐associated chest pain, carbon monoxide poisoning and over‐anticoagulation.
Essential Amino Acids of the Hantaan Virus N Protein in Its Interaction with RNA
Authors: Severson William , Xu Xiaolin , Kuhn Michaela , Senutovitch Nina , Thokala Mercy , Ferron François , Longhi Sonia , Canard Bruno , Jonsson Colleen B.
Published: July 2005

The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.
Impaired Virus Control and Severe CD8+T-Cell-Mediated Immunopathology in Chimeric Mice Deficient in Gamma Interferon Receptor Expression on both Parenchymal and Hematopoietic Cells
Authors: Henrichsen Pernille , Bartholdy Christina , Christensen Jan Pravsgaard , Thomsen Allan Randrup
Published: July 2005

Bone marrow chimeras were used to determine the cellular target(s) for the antiviral activity of gamma interferon (IFN-γ). By transfusing such mice with high numbers of naive virus-specific CD8+T cells, a system was created in which the majority of virus-specific CD8+T cells would be capable of responding to IFN-γ, but expression of the relevant receptor on non-T cells could be experimentally controlled. Only when the IFN-γ receptor is absent on both radioresistant parenchymal and bone marrow-derived cells will chimeric mice challenged with a highly invasive, noncytolytic virus completely lack the ability to control the infection and develop severe wasting disease. Further, the study shows that IFN-γ receptor expression on parenchymal cells in the viscera is more important for virus control than IFN-γ receptor expression on bone marrow-derived cells.
The Severe Acute Respiratory Syndrome Coronavirus 3a Protein Up-Regulates Expression of Fibrinogen in Lung Epithelial Cells
Authors: Tan Yee-Joo , Tham Puay-Yoke , Chan Daphne Z. L. , Chou Chih-Fong , Shen Shuo , Fielding Burtram C. , Tan Timothy H. P. , Lim Seng Gee , Hong Wanjin
Published: July 2005

Here we analyzed the gene expression profile of cells that stably express the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein to determine its effects on host functions. A lung epithelial cell-line, A549, was chosen for this study because the lung is the primary organ infected by SARS-CoV and fatalities resulted mainly from pulmonary complications. Our results showed that the expression of 3a up-regulates the mRNA levels of all three subunits, Aα, Bβ, and γ, of fibrinogen. Consequently, the intracellular levels as well as the secretion of fibrinogen were increased. We also observed increased fibrinogen levels in SARS-CoV-infected Vero E6 cells.
Inhibition of Transcription and Translation in Sindbis Virus-Infected Cells
Authors: Gorchakov Rodion , Frolova Elena , Frolov Ilya
Published: July 2005

Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. The natural transmission cycle of alphaviruses requires their presence at high concentrations in the blood of amplification hosts for efficient infection of mosquito vectors. The high-titer viremia development implies multiple rounds of infection that proceed in the background of the developing antiviral cell response aimed at blocking virus spread on an organismal level. Therefore, as for many viruses, if not most of them, alphaviruses have evolved mechanisms directed toward downregulating different components of the antiviral cell reaction and increasing viremia to a level sufficient for the next round of transmission. Using Sindbis virus (SIN) as a model, we demonstrated that (i) the replication of wild-type SIN strongly affects major cellular processes, e.g., transcription and translation of mRNAs; (ii) transcriptional and translational shutoffs are distinctly independent events, and their development can be differentially manipulated by creating different mutations in SIN nonstructural protein nsP2; and (iii) inhibition of transcription, but not translation, is a critical mechanism that SIN employs to suppress the expression of cellular viral stress-inducible genes in cells of vertebrate origin. Downregulation of transcription of all of the cellular mRNAs appears to be a very efficient means of reducing the development of an antiviral response. The ability to cause transcriptional shutoff may partially determine SIN host range and replication in particular tissues.
An Envelope-Determined, pH-Independent Endocytic Route of Viral Entry Determines the Susceptibility of Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2 to Lv2 Restriction
Authors: Marchant David , Neil Stuart J. D. , Aubin Keith , Schmitz Christian , McKnight Áine
Published: July 2005

We identified a postentry restriction, termed Lv2, which determines the cellular tropism of two related human immunodeficiency virus type 2 (HIV-2) isolates and is dependent on the sequence of the capsid (CA) and envelope (Env) proteins. To explain the reliance on both CA and Env, we proposed that restrictive Envs deliver susceptible capsids to a compartment where Lv2 is active whereas nonrestrictive Envs deliver capsids into a compartment where Lv2 is either absent or less active. To test this model, we used compounds that affect endocytic pathways (ammonium chloride, bafilomycin A1, hypertonic sucrose) or lipid rafts (methyl-β-cyclodextrin) to treat restrictive cells and show that restricted virus can be rescued from Lv2 if a lipid-raft-dependent, pH-independent endocytic pathway is inhibited. Furthermore, viral entry into HeLa/CD4 cells containing a tailless CD4 receptor, located outside lipid rafts, was fully permissive. Finally, we show that a variety of primary HIV-1 and HIV-2 viruses are susceptible to Lv2. Thus, we show that the route of entry, determined by the viral envelope, can influence cellular tropism by avoiding intracellular blocks to infection.
Role of B-Cell Proliferation in the Establishment of Gammaherpesvirus Latency
Authors: Moser Janice M. , Upton Jason W. , Allen Robert D. , Wilson Christopher B. , Speck Samuel H.
Published: July 2005

Murine gammaherpesvirus 68 (γHV68) provides a tractable small animal model with which to study the mechanisms involved in the establishment and maintenance of latency by gammaherpesviruses. Similar to the human gammaherpesvirus Epstein-Barr virus (EBV), γHV68 establishes and maintains latency in the memory B-cell compartment following intranasal infection. Here we have sought to determine whether, like EBV infection, γHV68 infection in vivo is associated with B-cell proliferation during the establishment of chronic infection. We show that γHV68 infection leads to significant splenic B-cell proliferation as late as day 42 postinfection. Notably, γHV68 latency was found predominantly in the proliferating B-cell population in the spleen on both days 16 and 42 postinfection. Furthermore, virus reactivation upon ex vivo culture was heavily biased toward the proliferating B-cell population. DNA methyltransferase 1 (Dnmt1) is a critical maintenance methyltransferase which, during DNA replication, maintains the DNA methylation patterns of the cellular genome, a process that is essential for the survival of proliferating cells. To assess whether the establishment of γHV68 latency requires B-cell proliferation, we characterized infections of conditional Dnmt1 knockout mice by utilizing a recombinant γHV68 that expresses Cre-recombinase (γHV68-Cre). In C57BL/6 mice, the γHV68-Cre virus exhibited normal acute virus replication in the lungs as well as normal establishment and reactivation from latency. Furthermore, the γHV68-Cre virus also replicated normally during the acute phase of infection in the lungs of Dnmt1 conditional mice. However, deletion of the
Dnmt1
alleles from γHV68-infected cells in vivo led to a severe ablation of viral latency, as assessed on both days 16 and 42 postinfection. Thus, the studies provide direct evidence that the proliferation of latently infected B cells is critical for the establishment of chronic γHV68 infection.
Frequent Coinfection of Cells Explains Functional In Vivo Complementation between Cytomegalovirus Variants in the Multiply Infected Host
Authors: Čičin-Šain Luka , Podlech Jürgen , Messerle Martin , Reddehase Matthias J. , Koszinowski Ulrich H.
Published: July 2005

In contrast to many other virus infections, primary cytomegalovirus (CMV) infection does not fully protect against reinfection. Accordingly, clinical data have revealed a coexistence of multiple human CMV variants/strains in individual patients. Notably, the phenomenon of multiple infection was found to correlate with increased virus load and severity of CMV disease. Although of obvious medical relevance, the mechanism underlying this correlation is unknown. A weak immune response in an individual could be responsible for a more severe disease and for multiple infections. Alternatively, synergistic contributions of variants that differ in their biological properties can lead to qualitative changes in viral fitness by direct interactions such as genetic recombination or functional complementation within coinfected host cells. We have addressed this important question paradigmatically with the murine model by differently designed combinations of two viruses employed for experimental coinfection of mice. Specifically, a murine cytomegalovirus (MCMV) mutant expressing Cre recombinase was combined for coinfection with a mutant carrying Cre-inducible green fluorescent protein gene, and attenuated mutants were combined for coinfection with wild-type virus followed by two-color in situ hybridization studies visualizing the replication of the two viruses in infected host organs. These different approaches concurred in the conclusion that coinfection of host cells is more frequent than statistically predicted and that this coinfection alters virus fitness by functional
trans
-complementation rather than by genetic recombination. The reported findings make a major contribution to our molecular understanding of enhanced CMV pathogenicity in the multiply infected host.
The Long Terminal Repeat-Containing Retrotransposon Tf1 Possesses Amino Acids in Gag That Regulate Nuclear Localization and Particle Formation
Authors: Kim Min-Kyung , Claiborn Kathryn C. , Levin Henry L.
Published: July 2005

Tf1 is a long terminal repeat-containing retrotransposon of
Schizosaccharomyces pombe
that is studied to further our understanding of retrovirus propagation. One important application is to examine Tf1 as a model for how human immunodeficiency virus type 1 proteins enter the nucleus. The accumulation of Tf1 Gag in the nucleus requires an N-terminal nuclear localization signal (NLS) and the nuclear pore factor Nup124p. Here, we report that NLS activity is regulated by adjacent residues. Five mutant transposons were made, each with sequential tracts of four amino acids in Gag replaced by alanines. All five versions of Tf1 transposed with frequencies that were significantly lower than that of the wild type. Although all five made normal amounts of Gag, two of the mutations did not make cDNA, indicating that Gag contributed to reverse transcription. The localization of the Gag in the nucleus was significantly reduced by mutations A1, A2, and A3. These results identified residues in Gag that contribute to the function of the NLS. The Gags of A4 and A5 localized within the nucleus but exhibited severe defects in the formation of virus-like particles. Of particular interest was that the mutations in Gag-A4 and Gag-A5 caused their nuclear localization to become independent of Nup124p. These results suggested that Nup124p was only required for import of Tf1 Gag because of its extensive multimerization.
Utilization of Homotypic and Heterotypic Proteins of Vesicular Stomatitis Virus by Defective Interfering Particle Genomes for RNA Replication and Virion Assembly: Implications for the Mechanism of Homologous Viral Interference
Authors: Kim Gyoung Nyoun , Kang C. Yong
Published: July 2005

Defective interfering (DI) particles of Indiana serotype of vesicular stomatitis virus (VSV
Ind
) are capable of interfering with the replication of both homotypic VSV
Ind
and heterotypic New Jersey serotype (VSV
NJ
) standard virus. In contrast, DI particles from VSV
NJ
do not interfere with the replication of VSV
Ind
standard virus but do interfere with VSV
NJ
replication. The differences in the interfering activities of VSV
Ind
DI particles and VSV
NJ
DI particles against heterotypic standard virus were investigated. We examined the utilization of homotypic and heterotypic VSV proteins by DI particle genomic RNAs for replication and maturation into infectious DI particles. Here we show that the RNA-nucleocapsid protein (N) complex of one serotype does not utilize the polymerase complex (P and L) of the other serotype for RNA synthesis, while DI particle genomic RNAs of both serotypes can utilize the N, P, and L proteins of either serotype without serotypic restriction but with differing efficiencies as long as all three proteins are derived from the same serotype. The genomic RNAs of VSV
Ind
DI particles assembled and matured into DI particles by using either homotypic or heterotypic viral proteins. In contrast, VSV
NJ
DI particles could assemble only with homotypic VSV
NJ
viral proteins, although the genomic RNAs of VSV
NJ
DI particles could be replicated by using heterotypic VSV
Ind
N, P, and L proteins. Thus, we concluded that both efficient RNA replication and assembly of DI particles are required for the heterotypic interference by VSV DI particles.
Two Sp1/Sp3 Binding Sites in the Major Immediate-Early Proximal Enhancer of Human Cytomegalovirus Have a Significant Role in Viral Replication
Authors: Isomura Hiroki , Stinski Mark F. , Kudoh Ayumi , Daikoku Tohru , Shirata Noriko , Tsurumi Tatsuya
Published: July 2005

We previously demonstrated that the major immediate early (MIE) proximal enhancer containing one GC box and the TATA box containing promoter are minimal elements required for transcription and viral replication in human fibroblast cells (H. Isomura, T. Tsurumi, M. F. Stinski, J. Virol.
78:
12788-12799, 2004). After infection, the level of Sp1 increased while Sp3 remained constant. Here we report that either Sp1 or Sp3 transcription factors bind to the GC boxes located at approximately positions −55 and −75 relative to the transcription start site (+1). Both the Sp1 and Sp3 binding sites have a positive and synergistic effect on the human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. There was little to no change in MIE transcription or viral replication for recombinant viruses with one or the other Sp1 or Sp3 binding site mutated. In contrast, mutation of both the Sp1 and Sp3 binding sites caused inefficient MIE transcription and viral replication. These data indicate that the Sp1 and Sp3 binding sites have a significant role in HCMV replication in human fibroblast cells.
The Promiscuous CC Chemokine Receptor D6 Is a Functional Coreceptor for Primary Isolates of Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2 on Astrocytes
Authors: Neil Stuart J. D. , Aasa-Chapman Marlen M. I. , Clapham Paul R. , Nibbs Robert J. , McKnight Áine , Weiss Robin A.
Published: July 2005

The role of coreceptors other than CCR5 and CXCR4 in the pathogenesis of human immunodeficiency virus (HIV) disease is controversial. Here we show that a promiscuous CC chemokine receptor, D6, can function as a coreceptor for various primary dual-tropic isolates of HIV type 1 (HIV-1) and HIV-2. Furthermore, D6 usage is common among chimeric HIV-1 constructs bearing the gp120 proteins of isolates from early seroconverting patients. D6 mRNA and immunoreactivity were demonstrated to be expressed in HIV-1 target cells such as macrophages, peripheral blood mononuclear cells, and primary astrocytes. In primary astrocytes, an RNA interference-mediated knockdown of D6 expression inhibited D6-tropic isolate infection. D6 usage may account for some previous observations of alternative receptor tropism for primary human cells. Thus, D6 may be an important receptor for HIV pathogenesis in the brain and for the early dissemination of virus in the host.
Inhibition, Escape, and Attenuated Growth of Severe Acute Respiratory Syndrome Coronavirus Treated with Antisense Morpholino Oligomers
Authors: Neuman Benjamin W. , Stein David A. , Kroeker Andrew D. , Churchill Michael J. , Kim Alice M. , Kuhn Peter , Dawson Philip , Moulton Hong M. , Bestwick Richard K. , Iversen Patrick L. , Buchmeier Michael J.
Published: July 2005

The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targeted P-PMO and a random-sequence control P-PMO showed low inhibitory activity against SARS coronavirus. Certain other virus-targeted P-PMO reduced virus-induced cytopathology and cell-to-cell spread as a consequence of decreasing viral amplification. Active P-PMO were effective when administered at any time prior to peak viral synthesis and exerted sustained antiviral effects while present in culture medium. P-PMO showed low nonspecific inhibitory activity against translation of nontargeted RNA or growth of the arenavirus lymphocytic choriomeningitis virus. Two P-PMO targeting the viral transcription-regulatory sequence (TRS) region in the 5′ untranslated region were the most effective inhibitors tested. After several viral passages in the presence of a TRS-targeted P-PMO, partially drug-resistant SARS-CoV mutants arose which contained three contiguous base point mutations at the binding site of a TRS-targeted P-PMO. Those partially resistant viruses grew more slowly and formed smaller plaques than wild-type SARS-CoV. These results suggest PMO compounds have powerful therapeutic and investigative potential toward coronavirus infection.
Rmcf2
, a Xenotropic Provirus in the Asian Mouse Species
Mus castaneus
, Blocks Infection by Polytropic Mouse Gammaretroviruses
Authors: Wu Tiyun , Yan Yuhe , Kozak Christine A.
Published: July 2005

Cells from the Asian wild mouse species
Mus castaneus
are resistant to infection by the polytropic host range group of mouse gammaretroviruses. Two factors are responsible for this resistance: a defective XPR1 cell surface receptor for polytropic murine leukemia viruses (P-MLVs), and a resistance factor detectable only in interspecies hybrids between
M. castaneus
and mice with an XPR1 variant that permits infection by xenotropic MLVs (X-MLVs) as well as P-MLVs. This second novel virus resistance phenotype has been associated with expression of viral Env glycoprotein; Northern blotting with specific hybridization probes identified a spliced X-MLV
env
message unique to virus-resistant mice. These observations suggest that resistance is due to expression of one or more endogenous X-MLV envelope genes that interfere with infection by exogenous P-MLVs.
M. castaneus
contains multiple X-MLV proviruses, but serial backcrosses reduced this proviral content and permitted identification of a single proviral
env
sequence inherited with resistance. The resistance phenotype and the provirus were mapped to the same site on distal chromosome 18. The provirus was shown to be a full-length provirus highly homologous to previously described X-MLVs. Use of viral pseudotypes confirmed that this resistance gene, termed
Rmcf2
, prevents entry of P-MLVs.
Rmcf2
resembles the virus resistance genes
Fv4
and
Rmcf
in that it produces Env glycoprotein but fails to produce infectious virus; the proviruses associated with all three resistance genes have fatal defects. This type of provirus Env-mediated resistance represents an important defense mechanism in wild mouse populations exposed to endemic infections.
Shutoff of RNA Polymerase II Transcription by Poliovirus Involves 3C Protease-Mediated Cleavage of the TATA-Binding Protein at an Alternative Site: Incomplete Shutoff of Transcription Interferes with Efficient Viral Replication
Authors: Kundu Pallob , Raychaudhuri Santanu , Tsai Weimin , Dasgupta Asim
Published: July 2005

The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C
pro
to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C
pro
involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C
pro
involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C
pro
is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C
pro
-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.
Deletion of the Herpes Simplex Virus VP22-Encoding Gene (UL49) Alters the Expression, Localization, and Virion Incorporation of ICP0
Authors: Elliott Gillian , Hafezi Wali , Whiteley Alison , Bernard Emmanuelle
Published: July 2005

The role of the herpes simplex virus tegument protein VP22 is not yet known. Here we describe the characterization of a virus in which the entire VP22 open reading frame has been deleted. We show that VP22 is not essential for virus growth but that virus lacking VP22 (Δ22) displays a cell-specific replication defect in epithelial MDBK cells. Virus particles assembled in the absence of VP22 show few obvious differences to wild-type (WT) particles, except for a moderate reduction in glycoproteins gD and gB. In addition, the Δ22 virus exhibits a general delay in the initiation of virus protein synthesis, but this is not due to a glycoprotein-related defect in virus entry. Intriguingly, however, the absence of VP22 has an obvious effect on the intracellular level of the immediate-early (IE) protein ICP0. Moreover, following translocation from the nucleus to the cytoplasm, ICP0 is unable to localize to the characteristic cytoplasmic sites observed in a WT infection. We demonstrate that, in WT-infected cells, VP22 and ICP0 are concentrated in the same cytoplasmic sites. Furthermore, we show that, while ICP0 and ICP4 are components of WT extracellular virions, the altered localization of ICP0 in the cytoplasm of Δ22-infected cells correlates with an absence of both ICP0 and ICP4 from Δ22 virions. Hence, while a role has not yet been defined for virion IE proteins in virus infection, our results suggest that their incorporation is a specific event requiring the tegument protein VP22. This report provides the first direct evidence that VP22 influences virus assembly.
The Capsid Protein of Satellite Panicum Mosaic Virus Contributes to Systemic Invasion and Interacts with Its Helper Virus
Authors: Omarov Rustem T. , Qi Dong , Karen-Beth Scholthof G.
Published: July 2005

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus (PMV) for replication and spread in host plants. The SPMV RNA encodes a 17-kDa capsid protein (CP) that is essential for formation of its 16-nm virions. The results of this study indicate that in addition to the expression of the full-length SPMV CP from the 5′-proximal AUG start codon, SPMV RNA also expresses a 9.4-kDa C-terminal protein from the third in-frame start codon. Differences in solubility between the full-length protein and its C-terminal product were observed. Subcellular fractionation of infected plant tissues showed that SPMV CP accumulates in the cytosol, cell wall-, and membrane-enriched fractions. However, the 9.4-kDa protein exclusively cofractionated with cell wall- and membrane-enriched fractions. Earlier studies revealed that the 5′-untranslated region (5′-UTR) from nucleotides 63 to 104 was associated with systemic infection in a host-specific manner in millet plants. This study shows that nucleotide deletions and insertions in the 5′-UTR plus simultaneous truncation of the N-terminal part of the CP impaired SPMV spread in foxtail millet, but not in proso millet plants. In contrast, the expression of the full-length version of SPMV CP efficiently compensated the negative effect of the 5′-UTR deletions in foxtail millet. Finally, immunoprecipitation assays revealed the presence of a specific interaction between the capsid proteins of SPMV and its helper virus (PMV). Our findings show that the SPMV CP has several biological functions, including facilitating efficient satellite virus infection and movement in millet plants.