bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the diets of sympatric dugongs and green turtles on the Orman Reefs, Torres Strait, Australia
Authors: André Jessica , Gyuris Emma , Lawler Ivan R.
Published: February 2005

This study investigated the diet of dugongs (Dugong dugon, Dugongidae) and green turtles (Chelonia mydas, Cheloniidae) on the Orman Reefs in Torres Strait, between Australia and Papua New Guinea, where large numbers of these animals live sympatrically. The stomach contents of dugongs and green turtles caught in an indigenous fishery were examined. Dugongs fed exclusively on seagrasses (mainly Thalassia hemprichii, Cymodocea spp. and Syringodium isoetifolium) whereas turtles consumed both seagrasses (especially T. hemprichii and Enhalus acoroides) and algae (mainly Hypnea spp., Laurencia spp. and Caulerpa spp.). The two herbivores showed no overlap in resource use except for the seagrass T. hemprichii, which was abundant in the feeding area. Both species appeared to feed selectively and did not just consume the most available food items. These results are suggestive of partitioning of food resources between dugongs and green turtles but a full explanation requires more detailed, and concurrent, study of the food resources and the animals’ movements.
Does the introduced bumblebee, Bombus terrestris (Apidae), prefer flowers of introduced or native plants in Australia?
Authors: Hingston Andrew B.
Published: February 2005

Proponents of importation of the European bumblebee, Bombus terrestris (L.), into Australia for pollination of commercial greenhouse crops argue that this species will have little impact on Australian native ecosystems because it prefers to forage on flowers of introduced species of plants rather than Australian native plants. However, data presented as evidence of preference for introduced plants have been equivocal. This study compared the attractiveness of introduced and Australian native plants to free-foraging B. terrestris in a garden at the interface between an urban area and native vegetation in the Australian island of Tasmania, where a feral population of B. terrestris had been established for over 10 years. No evidence was found to support the proposal that B. terrestris forages on flowers of introduced plants in preference to those of Australian native plants. The numbers of B. terrestris seen foraging per 1000 flowers did not differ significantly between introduced plants and Australian native plants, and the preferred food sources of B. terrestris included flowers of both introduced and Australian native species. Because B. terrestris forages frequently on many species of both introduced and native plants, assessments of its ecological impacts must include the effects of altered pollination on recruitment rates in both introduced weeds and native plants, and reduced quantities of nectar and pollen of native plants on recruitment rates of dependent fauna.
Structural polymorphism of the cytoskeleton: A model of linker-assisted filament aggregation
Authors: Borukhov Itamar , Bruinsma Robijn F. , Gelbart William M. , Liu Andrea J.
Published: February 2005

The phase behavior of charged rods in the presence of interrod linkers is studied theoretically as a model for the equilibrium behavior underlying the organization of actin filaments by linker proteins in the cytoskeleton. The presence of linkers in the solution modifies the effective interrod interaction and can lead to interfilament attraction. Depending on the composition and physical properties of the system, such as linker-binding energies, filaments will orient either perpendicular or parallel to each other, leading to network-like or bundled structures. We show that such a system can have one of three generic phase diagrams, one dominated by bundles, another by networks, and the third containing both bundle and network-like phases. The first two diagrams can be found over a wide range of interaction energies, whereas the third diagram occurs only for a narrow range. These results provide theoretical understanding of the classification of linker proteins as bundling proteins or crosslinking proteins. In addition, they suggest possible mechanisms by which the cell may control cytoskeletal morphology.
Dynamics of confined water molecules
Authors: Gilijamse J. J. , Lock A. J. , Bakker H. J.
Published: February 2005

We present femtosecond midinfrared pump–probe measurements of the molecular motion and energy-transfer dynamics of a water molecule that is enclosed by acetone molecules. These confined water molecules show hydrogen-bond and orientational dynamics that are much slower than in bulk liquid water. This behavior is surprising because the hydrogen bonds to the C=O groups of the acetone molecules are weaker than the hydrogen bonds in bulk water. The energy transfer between the O—H groups of the confined water molecules has a time constant of 1.3 ± 0.2 ps, which is >20 times slower than in bulk water. We find that this energy transfer is governed completely by the rate at which hydrogen bonds are broken and reformed, and we identify the short-lived molecular complex that forms the transition state of this process.
Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis
Authors: Dalton Timothy P. , He Lei , Wang Bin , Miller Marian L. , Jin Li , Stringer Keith F. , Chang Xiaoqing , Baxter C. Stuart , Nebert Daniel W.
Published: February 2005

Testicular necrosis is a sensitive endpoint for cadmium (Cd
2+
, Cd) toxicity across all species tested. Resistance to Cd-induced testicular damage is a recessive trait assigned to the
Cdm
locus on mouse chromosome 3. We first narrowed the
Cdm
-gene-containing region to 880 kb. SNP analysis of this region from two sensitive and two resistant inbred strains demonstrated a 400-kb haplotype block consistent with the Cd-induced toxicity phenotype; in this region is the
Slc39a8
gene encoding a member of the solute-carrier superfamily.
Slc39a8
encodes SLC39A8 (ZIP8), whose homologs in plant and yeast are putative zinc transporters. We show here that ZRT-, IRT-like protein (ZIP)8 expression in cultured mouse fetal fibroblasts leads to a >10-fold increase in the rate of intracellular Cd influx and accumulation and 30-fold increase in sensitivity to Cd-induced cell death. The complete ZIP8 mRNA and intron-exon splice junctions have no nucleotide differences between two sensitive and two resistant strains of mice; by using situ hybridization, we found that ZIP8 mRNA is prominent in the vascular endothelial cells of the testis of the sensitive strains of mice but absent in these cells of resistant strains.
Slc39a8
is therefore the
Cdm
gene, defining sensitivity to Cd toxicity specifically in vascular endothelial cells of the testis.
Modulation of CD1d-restricted NKT cell responses by usingN-acyl variants of α-galactosylceramides
Authors: Yu Karl O. A. , Im Jin S. , Molano Alberto , Dutronc Yves , Illarionov Petr A. , Forestier Claire , Fujiwara Nagatoshi , Arias Isa , Miyake Sachiko , Yamamura Takashi , Chang Young-Tae , Besra Gurdyal S. , Porcelli Steven A.
Published: February 2005

A form of α-galactosylceramide, KRN7000, activates CD1d-restricted Vα14-invariant (Vα14i) natural killer (NK) T cells and initiates multiple downstream immune reactions. We report that substituting the C26:0N-acyl chain of KRN7000 with shorter, unsaturated fatty acids modifies the outcome of Vα14i NKT cell activation. One analogue containing a diunsaturated C20 fatty acid (C20:2) potently induced a T helper type 2-biased cytokine response, with diminished IFN-γ production and reduced Vα14i NKT cell expansion. C20:2 also exhibited less stringent requirements for loading onto CD1d than KRN7000, suggesting a mechanism for the immunomodulatory properties of this lipid. The differential cellular response elicited by this class of Vα14i NKT cell agonists may prove to be useful in immunotherapeutic applications.
The initial substrate-binding site of γ-secretase is located on presenilin near the active site
Authors: Kornilova Anna Y. , Bihel Frédéric , Das Chittaranjan , Wolfe Michael S.
Published: February 2005

γ-Secretase is a structurally enigmatic multiprotein complex that catalyzes intramembrane proteolysis of a variety of substrates, including the amyloid β-protein precursor of Alzheimer's disease and the Notch receptor essential to cell differentiation. The active site of this transmembrane aspartyl protease apparently lies at the interface between two subunits of presenilin-1 (PS1); however, evidence suggests the existence of an initial substrate-binding site that is distinct from the active site. Here, we report that photoaffinity probes based on potent helical peptide inhibitors and designed to mimic the amyloid β-protein precursor substrate bind specifically to the PS subunit interface, at a site close to the active site. The location of the helical peptide-binding site suggests that substrate passes between the two PS1 subunits to access the active site. An aggressive Alzheimer-causing mutation in PS1 strongly reduced photolabeling by a transition-state analogue but not by helical peptides, providing biochemical evidence that the pathological effect of this PS mutation is due to alteration of the active-site topography.
Chlamydia pneumoniae
infection promotes a proliferative phenotype in the vasculature through Egr-1 activation
in vitro
and
in vivo
Authors: Rupp Jan , Hellwig-Burgel Thomas , Wobbe Viola , Seitzer Ulrike , Brandt Ernst , Maass Matthias
Published: February 2005

Atherosclerosis is characterized by inflammation and proliferation of vascular cells. The intracellular bacterium
Chlamydia (Chlamydophila
)
pneumoniae
uses blood monocytes [peripheral blood mononuclear cells (PBMCs)] for dissemination, has been found to persist in atherosclerotic lesions, and has been implicated in atherogenesis by small GTPase activation and T lymphocyte recruitment. Infection of human coronary artery smooth muscle cells with
C. pneumoniae
significantly induced mRNA and protein for the angiogenic transcription factor Egr-1, resulting in enhanced coronary artery smooth muscle cell proliferation, which was reduced by transfection with small interfering RNA duplexes targeted at Egr-1 mRNA. These effects required viable chlamydiae and depended on p44/42 mitogen-activated protein kinase activity but not on the p38 mitogen-activated protein kinase pathway. Postinfectious Egr-1 mRNA up-regulation in arterial vessels was confirmed
ex vivo
in a rat aortic ring model of focal vascular chlamydial infection. An
in vivo
model based on the injection of
C. pneumoniae
-infected PBMCs into mice confirmed Egr-1 mRNA up-regulation within 24 h of endovascular infection. Arterial injury from repeated direct chlamydial infections and cell-to-cell contact with
C. pneumoniae
-infected PBMCs might represent a chronic focus of proliferative activity linked to the media proliferation seen in advanced atherosclerosis. Overall, chlamydial infection induces a proliferative phenotype in vascular cells via transcription factor Egr-1 activation
in vitro, ex vivo
, and
in vivo
.
The time course and specificity of perceptual deterioration
Authors: Mednick Sara C. , Arman A. Cyrus , Boynton Geoffrey M.
Published: February 2005

Repeated within-day testing on a texture discrimination task leads to retinotopically specific decreases in performance. Although perceptual learning has been shown to be highly specific to the retinotopic location and characteristics of the trained stimulus, the specificity of perceptual deterioration has not been studied. We investigated the similarities between learning and deterioration by examining whether deterioration transfers to new distractor or target orientations or to the untrained eye. Participants performed a texture discrimination task in three one-hour sessions. We tested the specificity of deterioration in the final session by switching either the orientation of the background or the target elements by 90°. We found that performance deteriorated steadily both within and across the first two sessions and was specific to the target but not the distractor orientation. In a separate experiment, we found that deterioration transferred to the untrained eye. Changes in performance were independent of reported sleepiness and awareness of stimulus changes, arguing against the possibility that perceptual deterioration is due to general fatigue. Rather, we hypothesize that perceptual deterioration may be caused by changes in the ability for attention to selectively enhance the responses of relatively low-level orientation-selective sensory neurons, possibly within the primary visual cortex. Further, the differences in specificity profiles between learning and deterioration suggest separate underlying mechanisms that occur within the same cortical area.
Mutations in
Serac1
or
Synj2
cause proximal
t
haplotype-mediated male mouse sterility but not transmission ratio distortion
Authors: Schimenti John C. , Reynolds Jennifer L. , Planchart Antonio
Published: February 2005

Transmission ratio distortion (TRD) and sterility are male-specific quantitative trait phenomena associated with the mouse
t
haplotype. TRD occurs in
t
haplotype-heterozygous males and is caused by the deleterious action of distorter products on sperm bearing a wild-type responder locus. It has been proposed that
t
-mediated male sterility is a severe manifestation of TRD caused by homozygosity for distorter loci; thus, the distorter and sterility loci would be identical. In this, study a transgenic approach was used to identify the proximal sterility locus,
tcs1
(S1), and test its role in TRD. Mice transgenic for a wild-type bacterial artificial chromosome (BAC) derived from the S1-critical region were bred onto
t
haplotype backgrounds. Mating results conclusively showed that the BAC is sufficient to restore fertility in otherwise sterile males. Multiple mutations were identified in the
t
alleles of
Synj2
and
Serac1
, two genes in the BAC; thus, they are candidates for S1. In addition, whereas the BAC transgene rescued sterility, it had no effect on TRD. These results uncouple the proximal
t
haplotype sterility locus, S1, from TRD, demonstrating that S1 and the proximal distorter locus, D1, are not the same gene.
Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes
Authors: Bacia Kirsten , Schwille Petra , Kurzchalia Teymuras
Published: February 2005

The existence of lipid rafts in biological membranes
in vivo
is still debated. In contrast, the formation of domains in model systems has been well documented. In giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol, a clear separation of liquid-disordered and sphingomyelin-enriched, liquid-ordered phases could be observed. This phase separation can lead to the fission of the liquid-ordered phase from the vesicle. Here we show that in cholesterol-containing GUVs, the phase separation can involve dynamic redistribution of lipids from one phase into another as a result of a cross-linking perturbation. We found that the molecular structure of a sterol used for the preparation of GUVs determines (
i
) its ability to induce phase separation and (
ii
) the curvature (positive or negative) of the formed liquid-ordered phase. As a consequence, the latter can pinch off to the outside or inside of the vesicle. Remarkably, some mixtures of sterols induce liquid-ordered domains exhibiting both positive and negative curvature, which can lead to a new type of budding behavior in GUVs. Our findings could have implications for the role of sterols in various cell-biological processes such as budding of secretory vesicles, endocytosis, or formation of multivesicular bodies.
Seeing the light: Illumination as a contextual cue to color choice behavior in bumblebees
Authors: Lotto R. Beau , Chittka Lars
Published: February 2005

The principal challenge faced by any color vision system is to contend with the inherent ambiguity of stimulus information, which represents the interaction between multiple attributes of the world (e.g., object reflectance and illumination). How natural systems deal with this problem is not known, although traditional hypotheses are predicated on the idea that vision represents object reflectance accurately by discounting early in processing the conflating effects of illumination. Here, we test the merits of this general supposition by confronting bumblebees (Bombus terrestris) with a color discrimination task that can be solved only if information about the illuminant is not discounted but maintained in processing and thus available to higher-order learned behavior. We show that bees correctly use the intensity and chromaticity of illumination as a contextual cue to guide them to different target colors. In fact, we trained bees to choose opposite, rather than most similar, target colors after an illumination change. This performance cannot be explained with a simple color-constancy mechanism that discounts illumination. Further tests show that bees do not use a simple assessment of the overhead illumination, but that they assess the spectral relationships between a floral target and its background. These results demonstrate that bees can be color-constant without discounting the illuminant; that, in fact, they can use information about the illuminant itself as a salient source of information.
Mutation of chicken anemia virus VP2 differentially affects serine/threonine and tyrosine protein phosphatase activities
Authors: Peters Michelle A. , Jackson David C. , Crabb Brendan S. , Browning Glenn F.
Published: February 2005

Novel dual-specificity protein phosphatases (DSPs), which catalyse the removal of phosphate from both phosphotyrosine and phosphoserine/phosphothreonine substrates, have recently been identified in two viruses within the familyCircoviridae. Viral protein 2 (VP2) of chicken anemia virus (CAV) and ORF2 of TT virus have been shown to possess DSP activityin vitro. CAV VP2 is unusual in possessing two vicinal cysteines within the protein phosphatase signature motif. The first cysteine residue (C95) within the motif has been identified by mutagenesis as the essential catalytic cysteine. In this study, it was shown that virus mutated at this residue displayed a marked inhibition of growth, with titres reduced 104-fold, and reduced cytopathogenic effect in cell culture, indicating that viral DSP activity may be significant during infection. As with virus mutated at the first cysteine residue, mutation of the second cysteine (C97) within the motif resulted in a marked reduction in viral growth and attenuation of cytopathogenicity in infected cell cultures. However, mutagenesis of this second cysteine only reduced phosphotyrosine phosphatase activity to 70 % of that of wild-type VP2, but increased phosphoserine/phosphothreonine phosphatase activity by as much as 700 %. The differential effect of the C97S mutation on VP2 activity does not appear to have parallels in other DSPs and suggests a unique role for the second cysteine in the function of these viral proteins, particularlyin vivo.
Foot-and-mouth disease virus replication sites form next to the nucleus and close to the Golgi apparatus, but exclude marker proteins associated with host membrane compartments
Authors: Knox Caroline , Moffat Katy , Ali Shireen , Ryan Martin , Wileman Thomas
Published: February 2005

Picornavirus infection of cells generally results in the production of membranous vesicles containing the viral proteins necessary for viral RNA synthesis. To determine whether foot-and-mouth disease virus (FMDV) infection induced similar structures, and which cellular components were involved, the subcellular distribution of FMDV proteins was compared with protein markers of cellular membrane compartments. Using immunofluorescence analysis and digital deconvolution, it was shown that FMDV structural and non-structural proteins co-localize to punctate structures in juxtanuclear virus assembly sites close to the Golgi complex. Significantly, viral protein 2C did not co-localize with marker proteins of thecis- ormedial-Golgi compartments ortrans-Golgi network. Furthermore, incubation of infected cells with brefeldin A caused a redistribution of Golgi proteins to the endoplasmic reticulum, but did not affect the distribution of 2C and, by inference, the integrity of the virus assembly site. Taken with the observation that 2C was membrane-associated, but failed to fractionate with Golgi markers on density gradients, it was possible to conclude that Golgi membranes were not a source of structures containing 2C. Further immunofluorescence analysis showed that 2C was also separate from marker proteins of the endoplasmic reticulum, endoplasmic reticulum intermediate compartment, endosomes and lysosomes. The results suggest that the membranes generated at FMDV assembly sites are able to exclude organelle-specific marker proteins, or that FMDV uses an alternative source of membranes as a platform for assembly and replication.
Phenotype of disease-associated PrP accumulation in the brain of bovine spongiform encephalopathy experimentally infected sheep
Authors: González Lorenzo , Martin Stuart , Houston Fiona E. , Hunter Nora , Reid Hugh W. , Bellworthy Sue J. , Jeffrey Martin
Published: February 2005

In view of the established link between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt–Jakob disease and of the susceptibility of sheep to experimental BSE, the detection of potential cases of naturally occurring BSE in sheep has become of great importance. In this study, the immunohistochemical (IHC) phenotype of disease-associated prion protein (PrPd) accumulation has been determined in the brain of 64 sheep, of various breeds and PrP genotypes, that had developed neurological disease after experimental BSE challenge with different inocula by a range of routes. Sheep BSE was characterized by neuron-associated intra- and extracellular PrPd aggregates and by conspicuous and consistent deposits in the cytoplasm of microglia-like cells. The stellate PrPd type was also prominent in most brain areas and marked linear deposits in the striatum and midbrain were distinctive. Sheep of the ARR/ARR and ARQ/AHQ genotypes displayed lower levels of PrPd than other sheep, and intracerebral BSE challenge resulted in higher levels of PrPd accumulating in the brain compared with other routes. The PrP genotype and the route of challenge also appeared to affect the incubation period of the disease, giving rise to complex combinations of magnitude of PrPd accumulation and incubation period. Despite these differences, the phenotype of PrPd accumulation was found to be very consistent across the different factors tested (notably after subpassage of BSE in sheep), thus highlighting the importance of detailed IHC examination of the brain of clinically affected sheep for the identification of potential naturally occurring ovine BSE.
Ets-2 repressor factor recruits histone deacetylase to silence human cytomegalovirus immediate-early gene expression in non-permissive cells
Authors: Wright Edward , Bain Mark , Teague Linda , Murphy Jane , Sinclair John
Published: February 2005

Previous work from this laboratory has shown that expression of human cytomegalovirus (HCMV) immediate-early (IE) genes from the major immediate-early promoter (MIEP) is likely to be regulated by chromatin remodelling around the promoter affecting the acetylation state of core histone tails. The HCMV MIEP contains sequences that bind cellular transcription factors responsible for its negative regulation in undifferentiated, non-permissive cells. Ets-2 repressor factor (ERF) is one such factor that binds to such sequences and represses IE gene expression. Although it is not known how cellular transcription factors such as ERF mediate transcriptional repression of the MIEP, it is likely to involve differentiation-specific co-factors. In this study, the mechanism by which ERF represses HCMV IE gene expression was analysed. ERF physically interacts with the histone deacetylase, HDAC1, bothin vitroandin vivoand this physical interaction between ERF and HDAC1 mediates repression of the MIEP. This suggests that silencing of viral IE gene expression, associated with histone deacetylation events around the MIEP, is mediated by differentiation-dependent cellular factors such as ERF, which specifically recruit chromatin remodellers to the MIEP in non-permissive cells.
Immunohistochemical characteristics of disease-associated PrP are not altered by host genotype or route of inoculation following infection of sheep with bovine spongiform encephalopathy
Authors: Martin Stuart , González Lorenzo , Chong Angela , Houston Fiona E. , Hunter Nora , Jeffrey Martin
Published: February 2005

It has previously been reported that disease-associated prion protein (PrPd) derived from natural scrapie and from sheep infected experimentally with bovine spongiform encephalopathy (BSE) differed in respect of their immunohistochemical and immunoblotting properties. For BSE, however, these initial observations were restricted to orally challenged sheep of the ARQ/ARQ PrP genotype. Here, extended examinations were performed on 28 sheep that developed neurological signs after BSE experimental infection by one of three routes. Intracerebrally infected ARQ/ARQ sheep showed more widespread and abundant accumulations of PrPd in tissues of the lymphoreticular system (LRS) than VRQ/VRQ animals, whereas no peripheral PrPd was detected in ARR/ARR sheep. The intensity and dissemination of PrPd accumulation in LRS tissues were less than those found previously in orally dosed sheep. AHQ/AHQ sheep challenged orally and ARQ/AHQ and ARQ/ARQ animals infected intravenously showed similar LRS-tissue PrPd distributions and levels to those of ARQ/ARQ sheep infected intracerebrally. The patterns of intra- and extracellular immunoreactivity to different PrP antibodies in brain and LRS tissues and the immunoblotting characteristics of PrPres from brain samples remained constant, irrespective of the route of inoculation and the PrP genotype, and were the same as described previously for ARQ/ARQ sheep dosed orally with BSE. These results suggest that the intracellular truncation of BSE PrPd and the proteinase K cleavage site of BSE PrPres are not altered by PrP genotype or by route of inoculation and that, therefore, screening tests based on these properties can be applied to identify potential sheep BSE cases occurring naturally.
Polygenic variation and transmission factors involved in the resistance/susceptibility to scrapie in a Romanov flock
Authors: Díaz Clara , Vitezica Zulma G. , Rupp Rachel , Andréoletti Olivier , Elsen Jean Michel
Published: February 2005

Data from 4049 Romanov sheep belonging to a flock affected by natural scrapie were analysed by using survival-analysis techniques. Failure time was defined as the period of time between first exposure to infection and the date that animals left the flock with scrapie signs. Four hundred and forty-seven sheep were identified as ‘scrapie animals’. Several models, including level of exposure as a time-dependent effect, PrP genotype, sex, age at first exposure, litter size and factors related to vertical transmission, were tested. The best model was extended to a sire–dam frailty model, in order to estimate the polygenic variation in addition to that in the Prnp gene. A combined effect of rearing type and the dam's disease status was detected. Thus, only sheep with a low degree of exposure to infection as lambs (lambs reared artificially and born out of a healthy dam) showed less risk than others. Animals first exposed to infection at older ages seemed to be less susceptible to scrapie. In this Romanov population, new genotypes (AHQ/AHQ, AHQ/VRQ, ARR/VRQ and ARR/ARQ) were associated with risk, suggesting the effect of genotypes on the incubation period of animals. Polygenic variance was responsible for 21 % of the total genetic variability that was related to susceptibility to scrapie. Therefore, the genetic susceptibility to scrapie may be explained by the joint effect of point mutations at the Prnp major gene and a number of genes that modulate its effect.
Rhesus brain microvascular endothelial cells are permissive for rhesus cytomegalovirus infection
Authors: Carlson James R. , Chang W. L. William , Zhou Shan Shan , Tarantal Alice F. , Barry Peter A.
Published: February 2005

Endothelial cells (EC) are an important cell type for human cytomegalovirus (CMV) pathogenesis. To characterize better the role of EC in primate CMV natural history, rhesus macaque microvascular EC (MVEC) were purified from fetal brain and analysed for infectivity by rhesus cytomegalovirus (RhCMV). Rhesus brain MVEC (BrMVEC) in culture were positive for von Willebrand factor and CD105 expression, uptake of acetylated low-density lipoprotein, and formation of capillary-like tubules on Matrigel, all phenotypic hallmarks of EC. BrMVEC were fully permissive for infection by RhCMV strain 68-1, and detectable plaques formed within 5 days of infection. Infectivity of BrMVEC by RhCMV could be reduced, but not abolished, by treatment of cells either before or during infection with pro-inflammatory mediators tumour necrosis factor-α, interleukin-1βor phorbol 12-myristate 13-acetate. These results demonstrate thatin vitroinfection of rhesus BrMVEC is a dynamic process that is influenced by activation conditions.
Epstein–Barr virus latent membrane protein 2A mimics B-cell receptor-dependent virus reactivation
Authors: Schaadt Eveline , Baier Barbara , Mautner Josef , Bornkamm Georg W. , Adler Barbara
Published: February 2005

Latent membrane protein 2A (LMP2A) of Epstein–Barr virus (EBV) shares protein motifs with the B-cell receptor that play a role in B-cell receptor signalling and has been shown to mimic an activated B-cell receptor by providing a survival signal for mature B cells in transgenic mice. Conversely, LMP2A has been reported not to support but to inhibit B-cell receptor signalling with respect to virus reactivation and to block lytic virus induction after anti-Ig treatment of EBV-infected B cells. To solve this apparent paradox, the role of LMP2A in lytic-cycle induction was re-examined in B cells conditionally immortalized by EBV. It was shown that, in the absence of other stimuli, LMP2A expression alone could lead to induction of the virus lytic cycle. Similarly to B-cell receptor stimulation by anti-Ig treatment, this LMP2A-mediated reactivation was dependent on the mitogen-activated protein kinase pathway and could be inhibited by the viral LMP1. Our data reinforce the notion that LMP2A is a functional homologue of the B-cell receptor, not only with respect to B-cell survival but also with respect to regulation of the lytic cycle.
Intracellular location and translocation of silent and active poliovirus replication complexes
Authors: Egger Denise , Bienz Kurt
Published: February 2005

Replication of poliovirus (PV) genomic RNA in HeLa cells has previously been found to start at distinct sites at the nuclear periphery. In the present study, the earliest steps in the virus replication cycle, i.e. the appearance and intracellular translocation of viral protein and negative-strand RNA prior to positive-strand RNA synthesis, were followed. During translation, positive-strand RNA and newly synthesized viral protein presented as a dispersed endoplasmic reticulum (ER)-like pattern. Concomitant with translation, individual PV vesicle clusters emerged at the ER and formed nascent replication complexes, which contained newly synthesized negative-strand RNA. The complexes rapidly moved centripetally, in a microtubule-dependent way, to the perinuclear area to engage in positive-strand viral RNA synthesis. Replication complexes made transcriptionally silent with guanidine/HCl followed the anterograde membrane pathway to the Golgi complex within the microtubule-organizing centre (MTOC), whereas replication complexes active in positive-strand RNA synthesis were retained at the nuclear periphery. If the silent replication complexes that had accumulated at the MTOC were released from the guanidine block, transcription was not readily resumed. Rather, positive-strand RNA was redistributed back to the ER to start, after a lag phase, translation, followed by negative- and positive-strand RNA synthesis in replication complexes migrating to the nuclear periphery. As some of the findings appear to be in contrast to events reported in cell-free guanidine-synchronized translation/transcription systems, implications for the comparison ofin vitrosystems with the living cell are discussed.
Phylogenetic analysis of human rhinovirus capsid protein VP1 and 2A protease coding sequences confirms shared genus-like relationships with human enteroviruses
Authors: Laine Pia , Savolainen Carita , Blomqvist Soile , Hovi Tapani
Published: February 2005

Phylogenetic analysis of the capsid protein VP1 coding sequences of all 101 human rhinovirus (HRV) prototype strains revealed two major genetic clusters, similar to that of the previously reported VP4/VP2 coding sequences, representing the established two species, Human rhinovirus A (HRV-A) and Human rhinovirus B (HRV-B). Pairwise nucleotide identities varied from 61 to 98 % within and from 46 to 55 % between the two HRV species. Interserotypic sequence identities in both HRV species were more variable than those within any Human enterovirus (HEV) species in the same family. This means that unequivocal serotype identification by VP1 sequence analysis used for HEV strains may not always be possible for HRV isolates. On the other hand, a comprehensive insight into the relationships between VP1 and partial 2A sequences of HRV and HEV revealed a genus-like situation. Distribution of pairwise nucleotide identity values between these genera varied from 41 to 54 % in the VP1 coding region, similar to those between heterologous members of the two HRV species. Alignment of the deduced amino acid sequences revealed more fully conserved amino acid residues between HRV-B and polioviruses than between the two HRV species. In phylogenetic trees, where all HRVs and representatives from all HEV species were included, the two HRV species did not cluster together but behaved like members of the same genus as the HEVs. In conclusion, from a phylogenetic point of view, there are no good reasons to keep these two human picornavirus genera taxonomically separated.
Regulation of human immunodeficiency virus 1 transcription by nef microRNA
Authors: Omoto Shinya , Fujii Yoichi R.
Published: February 2005

MicroRNAs (miRNAs) are ∼21–25 nt long and interact with mRNAs to lead to either translational repression or RNA cleavage through RNA interference. A previous study showed that human immunodeficiency virus 1 (HIV-1) nef dsRNA from AIDS patients who are long-term non-progressors inhibited HIV-1 transcription. In the study reported here, nef-derived miRNAs in HIV-1-infected and nef transduced cells were identified, and showed that HIV-1 transcription was suppressed by nef-expressing miRNA, miR-N367, in human T cells. The miR-N367 could reduce HIV-1 LTR promoter activity through the negative responsive element of the U3 region in the 5′-LTR. Therefore, nef miRNA produced in HIV-1-infected cells may downregulate HIV-1 transcription through both a post-transcriptional pathway and a transcriptional neo-pathway.
Downregulation of Bax mRNA expression and protein stability by the E6 protein of human papillomavirus 16
Authors: Magal Sharon Shnitman , Jackman Anna , Ish-Shalom Shahar , Botzer Liat Edri , Gonen Pinhas , Schlegel Richard , Sherman Levana
Published: February 2005

Previous studies have shown that human papillomavirus (HPV) 16 E6 inhibits apoptosis induced during terminal differentiation of primary human keratinocytes (PHKs) triggered by serum and calcium. E6 inhibition of apoptosis was accompanied with prolonged expression of Bcl-2 and reduced elevation of Bax levels. In the present study, the effect of E6 on Bax mRNA expression and protein stability was investigated. These studies indicate that stable E6 expression in differentiating keratinocytes reduced the steady-state levels of Bax mRNA and shortened the half-life of Bax protein. These results were confirmed in transiently transfected 293T cells where E6 degraded Bax in a dose-dependent manner. Bax degradation was also exhibited in Saos-2 cells that lack p53, indicating its p53 independence. E6 did not form complexes with Bax and did not induce Bax degradation in vitro under experimental conditions where p53 was degraded. Finally, E6 aa 120–132 were shown to be necessary for Bax destabilization and, more importantly, for abrogating the ability of Bax to induce cellular apoptosis, highlighting the functional consequences of the E6-induced alterations in Bax expression.
Intersegmental recombination between the haemagglutinin and matrix genes was responsible for the emergence of a highly pathogenic H7N3 avian influenza virus in British Columbia
Authors: Pasick John , Handel Katherine , Robinson John , Copps John , Ridd Deidre , Hills Kevin , Kehler Helen , Cottam-Birt Colleen , Neufeld James , Berhane Yohannes , Czub Stefanie
Published: February 2005

In February 2004 a highly pathogenic avian influenza (HPAI) outbreak erupted in British Columbia. Investigations indicated that the responsible HPAI H7N3 virus emerged suddenly from a low pathogenic precursor. Analysis of the haemagglutinin (HA) genes of the low and high pathogenic viruses isolated from the index farm revealed the only difference to be a 21 nt insert at the HA cleavage site of the highly pathogenic avian influenza virus. It was deduced that this insert most probably arose as a result of non-homologous recombination between the HA and matrix genes of the same virus. Over the course of the outbreak, a total of 37 isolates with, and 3 isolates without inserts were characterized. The events described here appear very similar to those which occurred in Chile in 2002 where the virulence shift of another H7N3 virus was attributed to non-homologous recombination between the HA and nucleoprotein genes.
Genetic variation and dynamics of hepatitis C virus replicons in long-term cell culture
Authors: Kato Nobuyuki , Nakamura Takashi , Dansako Hiromichi , Namba Katsuyuki , Abe Ken-ichi , Nozaki Akito , Naka Kazuhito , Ikeda Masanori , Shimotohno Kunitada
Published: February 2005

Hepatitis C virus (HCV) genomic sequences are known to vary widely among HCV strains, but to date there have been few reports on the genetic variations and dynamics of HCV in an experimental system of HCV replication. In this study, a genetic analysis of HCV replicons obtained in long-term culture of two HCV replicon cells (50-1 and 1B-2R1), which were established from two HCV strains, 1B-1 and 1B-2, respectively, was performed. One person cultured 50-1 cells for 18 months, and two people independently cultured 50-1 cells for 12 months. 1B-2R1 cells were also cultured for 12 months. The whole nucleotide sequences of the three independent replicon RNA clones obtained at several time points were determined. It was observed that genetic mutations in both replicons accumulated in a time-dependent manner, and that the mutation rates of both replicons were approximately 3·0×10−3base substitutions/site/year. The genetic diversity of both replicons was also enlarged in a time-dependent manner. The colony formation assay by transfection of total RNAs isolated from both replicon cells at different time points into naïve HuH-7 cells revealed that the genetic mutations accumulating with time in both replicons apparently improved colony formation efficiency. Taken together, these results suggest that the HCV replicon system is useful for the analysis of evolutionary dynamics and variations of HCV. Using this replicon cell culture system, it was demonstrated further that neither ribavirin nor its derivative mizoribine accelerated the mutation rate or the increase in the genetic diversity of HCV replicon.
Differences in viral and host genetic risk factors for development of human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis between Iranian and Japanese HTLV-1-infected individuals
Authors: Sabouri Amir H. , Saito Mineki , Usuku Koichiro , Bajestan Sepideh Naghibzadeh , Mahmoudi Mahmoud , Forughipour Mohsen , Sabouri Zahra , Abbaspour Zahra , Goharjoo Mohammad E. , Khayami Esmaeil , Hasani Ali , Izumo Shuji , Arimura Kimiyoshi , Farid Reza , Osame Mitsuhiro
Published: February 2005

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease observed only in 1–2 % of infected individuals. HTLV-1 provirus load, certain HLA alleles and HTLV-1 tax subgroups are reported to be associated with different levels of risk for HAM/TSP in Kagoshima, Japan. Here, it was determined whether these risk factors were also valid for HTLV-1-infected individuals in Mashhad in northeastern Iran, another region of endemic HTLV-1 infection. In Iranian HTLV-1-infected individuals (n=132, 58 HAM/TSP patients and 74 seropositive asymptomatic carriers), although HLA-DRB1*0101 was associated with disease susceptibility in the absence of HLA-A*02 (P=0·038; odds ratio=2·71) as observed in Kagoshima, HLA-A*02 and HLA-Cw*08 had no effect on either the risk of developing HAM/TSP or HTLV-1 provirus load. All Iranian subjects possessed tax subgroup A sequences, and the protective effects of HLA-A*02 were observed only in Kagoshima subjects with tax subgroup B but not in those with tax subgroup A. Both the prevalence of HTLV-1 subgroups and the host genetic background may explain the different risks levels for HAM/TSP development in these two populations.
Formation of Bombyx mori nucleopolyhedrovirus IE2 nuclear foci is regulated by the functional domains for oligomerization and ubiquitin ligase activity
Authors: Imai Noriko , Matsumoto Shogo , Kang WonKyung
Published: February 2005

Baculovirus IE2 functions as a transregulator and is also involved in viral DNA replication. However, the mechanism for these functions remains unknown. It has previously been reported that Bombyx mori nucleopolyhedrovirus (BmNPV) IE2 has a ubiquitin ligase activity that is dependent on the RING finger domain and that IE2 can oligomerize through its C-terminal coiled-coil region. Here, confocal microscopy analysis demonstrated that IE2 formed nuclear foci only during the early phase of infection (2–6 h post-infection). Therefore, it was determined whether the IE2 functional regions described above could affect this characteristic distribution. Transient expression of ie2 also showed focus formation, suggesting that IE2 does not require any other viral factors. IE2 mutants lacking the C-terminal coiled-coil region did not form foci, while a mutant of the RING finger domain showed nuclear foci that appeared larger and brighter than those formed by wild-type IE2. In addition, IE2 exhibited enlarged foci in infected cells following treatment with a proteasome inhibitor, suggesting that foci enlargement resulted from accumulation of IE2 due to inhibition of the ubiquitin-proteasome pathway. These results suggest that BmNPV IE2 oligomerization and ubiquitin ligase activity functional domains regulate nuclear foci formation.
Polymorphisms at codons 108 and 189 in murine PrP play distinct roles in the control of scrapie incubation time
Authors: Barron Rona M. , Baybutt Herbert , Tuzi Nadia L. , McCormack James , King Declan , Moore Richard C. , Melton David W. , Manson Jean C.
Published: February 2005

Susceptibility to transmissible spongiform encephalopathies (TSEs) is associated strongly with PrP polymorphisms in humans, sheep and rodents. In mice, scrapie incubation time is controlled by polymorphisms at PrP codons 108 (leucine or phenylalanine) and 189 (threonine or valine), but the precise role of each polymorphism in the control of disease is unknown. The L108F and T189V polymorphisms are present in distinct structural regions of PrP and thus provide an excellent model with which to investigate the role of PrP structure and gene variation in TSEs. Two unique lines of transgenic mice, in which 108F and 189V have been targeted separately into the endogenous murine Prnp
a gene, have been produced. TSE inoculation of inbred lines of mice expressing all allelic combinations at codons 108 and 189 has revealed a complex relationship between PrP allele and incubation time. It has been established that both codons 108 and 189 control TSE incubation time, and that each polymorphism plays a distinct role in the disease process. Comparison of ME7 incubation times in mouse lines that are heterozygous at both codons has also identified a previously unrecognized intramolecular interaction between PrP codons 108 and 189.
Expressing engineered thymidylate kinase variants in human cells to improve AZT phosphorylation and human immunodeficiency virus inhibition
Authors: Wöhrl Birgitta M. , Loubière Laurence , Brundiers Ralf , Goody Roger S. , Klatzmann David , Konrad Manfred
Published: February 2005

The triphosphorylated form of the nucleoside analogue AZT (AZTTP) acts as a chain terminator during reverse transcription of the human immunodeficiency virus (HIV) genome. The bottleneck in the conversion of AZT to AZTTP is the phosphorylation of AZT monophosphate (AZTMP) by cellular thymidylate kinase. Human thymidylate kinase was engineered to exhibit highly improved activity for AZTMP to AZTDP conversion. It was demonstrated here that genetically modified human cells transiently expressing these enzyme variants show more than 10-fold higher intracellular concentrations of AZTDP and AZTTP. Stable clones expressing these enzymes appear to phosphorylate AZTMP less efficiently, but first experiments indicate they are still more potent in HIV inhibition than the parental cells. It was proposed that the concept of introducing into human cells a catalytically improved human enzyme, rather than an enzyme of viral, bacterial or yeast origin, may serve as a paradigm for ameliorating the metabolic activation of an established drug.
Isolation and identification of a new tetravirus from Dendrolimus punctatus larvae collected from Yunnan Province, China
Authors: Yi Fuming , Zhang Jiamin , Yu Haiyang , Liu Chuanfeng , Wang Junping , Hu Yuanyang
Published: February 2005

In this study, Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis ω virus (NωV). DpTV particles are isometric, with a diameter of about 40 nm and a buoyant density of 1·281 g cm−3 in CsCl. The virus has two capsid proteins (of 62 500 and 6800 Da) and two single-stranded RNA molecules (RNA1 and RNA2), which are 5492 and 2490 nt long, respectively. RNA1 has a large open reading frame (ORF) encoding a polypeptide of 180 kDa; RNA2 contains two partially overlapping ORFs encoding polypeptides of 17 and 70 kDa. The 180 kDa protein, which contains consensus motifs of a putative methyltransferase, helicase and RNA-dependent RNA polymerase, shows significant similarity to those of other tetraviruses. The 17 kDa protein is a PEST (Pro/Glu/Ser/Thr) protein of unknown function. The 70 kDa protein is the coat protein precursor and is predicted to be cleaved at an Asn–Phe site located after residue 570. The 70 kDa protein shows 86 and 66 % identity to its homologues in NωV and Helicoverpa armigera stunt virus, respectively. Secondary-structure analysis revealed that the RNAs of DpTV have tRNA-like structures at their 3′ termini.