bluuebunny/crossref_metadata_2025 huggingface dataset:
Why are rural Indian women so thin? Findings from a village in Maharashtra
Authors: Chorghade GP , Barker M , Kanade S , Fall CHD
Published: February 2006

Objective

To identify social, behavioural and cultural factors that explain the thinness of young women relative to their men in rural Maharashtra, India.

Design

Twelve focus group discussions were conducted to explore the villagers' understanding of why women in their area might be thinner than men.

Setting

Pabal village and surrounding hamlets, in the Pune district of Maharashtra, India.

Subjects

Samples of young mothers and fathers, grandmothers and grandfathers were selected from families in the village with children below 10 years of age.

Results

Four factors were identified that the villagers felt contributed to the disparity in thinness. First, marriage isolated girls from their own families and villages, and brought the expectation of early motherhood. Young brides were often unable to relax and eat adequately. Second, marriage increased the workload of young women. They were expected to do the heaviest household chores as well as farm work in this predominantly agricultural community. Third, women had no financial autonomy or freedom of movement, and were therefore denied access to supplementary food sources available to men. Fourth, young women felt responsible for their household's health and success. They were encouraged to fast regularly to ensure this. Despite feeling responsible, young women had no control over factors that might affect the household's well being. This made them anxious and worried a great deal of the time.

Conclusions

Interventions to improve the nutritional status of young women in this region need to recognise the roles and responsibilities taken up by young brides.
Validity and reliability of the Block98 food-frequency questionnaire in a sample of Canadian women
Authors: Boucher Beatrice , Cotterchio Michelle , Kreiger Nancy , Nadalin Victoria , Block Torin , Block Gladys
Published: February 2006

Objective

To assess the validity and reliability of the most recent adaptation of Block's full-diet food-frequency questionnaire (FFQ) among a sample of Canadian women.

Design

Participants completed a self-administered FFQ (FFQ1), two unannounced 24-hour recalls (weekday and weekend) and a second FFQ (FFQ2) between October 2003 and February 2004. FFQs and recalls were analysed for 32 nutrients using Block Dietary Data Systems and the University of Minnesota's Nutrient Data System. Mean and median intakes were computed, along with crude and deattenuated Pearson correlation coefficients between FFQ1 and the average of two recalls (validity) and between FFQ1 and FFQ2 (reliability).

Setting

Ontario, Canada.

Subjects

A random population-based sample (n= 166) of women aged 25 to 74 years.

Results

One hundred and fifteen (69%) women completed FFQ1, 96 completed FFQ1 and both recalls, and 93 completed both FFQs, about 56 days apart. Mean intakes were similar for most nutrients. FFQ reliability was high, with Pearson correlation coefficients having a median of 0.75, ranging from 0.57 to 0.90 (macronutrients) and from 0.65 to 0.88 (micronutrients from supplements and food). FFQ validity was moderate to high, with deattenuated Pearson correlation coefficients having a median of 0.59, ranging from 0.11 to 0.73 (macronutrients) and from 0.50 to 0.76 (micronutrients from supplements and food). Our micronutrient correlations were similar to or higher than those of other studies that included supplements. Two correlations <0.40 were associated with fats.

Conclusions

The validity and reliability of this full-diet version of the Block FFQ were moderate to high, supporting its use in future studies among Canadian women.
Food insecurity among homeless and runaway adolescents
Authors: Whitbeck Les B , Chen Xiaojin , Johnson Kurt D
Published: February 2006

Objective

The purpose of this study was to investigate the prevalence of food insecurity and factors related to it among homeless and runaway adolescents.

Design

Computer-assisted personal interviews were conducted with homeless and runaway adolescents directly on the streets and in shelters.

Setting

Interviews were conducted in eight Midwest cities: Des Moines, Cedar Rapids, Iowa City, Kansas City, Lincoln, Omaha, St. Louis and Wichita.

Subjects

The subjects were 428 (187 males; 241 females) homeless and runaway adolescents aged 16–19 years. Average age of the adolescents was 17.4 (standard deviation 1.05) years.

Results

About one-third of the adolescents had experienced food insecurity in the past 30 days. Factors associated with food insecurity were age of adolescent, a history of caretaker neglect and abuse, having ever spent time directly on the street, a small post-runaway social network, and engaging in deviant and non-deviant street food-acquisition strategies.

Conclusions

Based on these findings, our conservative estimate is that nationally more than 165 000 homeless and runaway adolescents experienced food insecurity in the past 30 days. These adolescents are largely hidden from public notice and they are usually missed in studies that address national hunger.
Excess dietary iodine intake in long-term African refugees
Authors: Seal Andrew J , Creeke Paul I , Gnat Daniella , Abdalla Fathia , Mirghani Zahra
Published: February 2006

Objective

To assess the iodine status of long-term refugees dependent on international food aid and humanitarian assistance.

Design

A series of cross-sectional two-stage cluster or systematic random sample surveys which assessed urinary iodine excretion and the prevalence of visible goitre. Salt samples were also collected and tested for iodine content by titration.

Setting

Six refugee camps in East, North and Southern Africa.

Subjects

Male and female adolescents aged 10–19 years.

Main results

The median urinary iodine concentration (UIC) ranged from 254 to 1200 μg l−1 and in five of the camps exceeded the recommended maximum limit of 300 μg l−1, indicating excessive iodine intake. Visible goitre was assessed in four surveys where it ranged from 0.0 to 7.1%. The camp with the highest UIC also had the highest prevalence of visible goitre. The iodine concentrations in 11 salt samples from three camps were measured by titration and six of these exceeded the production-level concentration of 20 to 40 ppm recommended by the World Health Organization (WHO), but were all less than 100 ppm.

Conclusions

Excessive consumption of iodine is occurring in most of the surveyed populations. Urgent revision of the level of salt iodisation is required to meet current WHO recommendations. However, the full cause of excessive iodine excretion remains unknown and further investigation is required urgently to identify the cause, assess any health impact and identify remedial action.
Nucleoplasmic β-actin exists in a dynamic equilibrium between low-mobility polymeric species and rapidly diffusing populations
Authors: McDonald Darin , Carrero Gustavo , Andrin Christi , de Vries Gerda , Hendzel Michael J.
Published: February 2006

β-Actin, once thought to be an exclusively cytoplasmic protein, is now known to have important functions within the nucleus. Nuclear β-actin associates with and functions in chromatin remodeling complexes, ribonucleic acid polymerase complexes, and at least some ribonucleoproteins. Proteins involved in regulating actin polymerization are also found in the interphase nucleus. We define the dynamic properties of nuclear actin molecules using fluorescence recovery after photobleaching. Our results indicate that actin and actin-containing complexes are reduced in their mobility through the nucleoplasm diffusing at ∼0.5 μm2 s−1. We also observed that ∼20% of the total nuclear actin pool has properties of polymeric actin that turns over rapidly. This pool could be detected in endogenous nuclear actin by using fluorescent polymeric actin binding proteins and was sensitive to drugs that alter actin polymerization. Our results validate previous reports of polymeric forms of nuclear actin observed in fixed specimens and reveal that these polymeric forms are very dynamic.
Drosophila melanogaster γ-TuRC is dispensable for targeting γ-tubulin to the centrosome and microtubule nucleation
Authors: Vérollet Christel , Colombié Nathalie , Daubon Thomas , Bourbon Henri-Marc , Wright Michel , Raynaud-Messina Brigitte
Published: February 2006

In metazoans, γ-tubulin acts within two main complexes, γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs). In higher eukaryotes, it is assumed that microtubule nucleation at the centrosome depends on γ-TuRCs, but the role of γ-TuRC components remains undefined.

For the first time, we analyzed the function of all four γ-TuRC–specific subunits in Drosophila melanogaster: Dgrip75, Dgrip128, Dgrip163, and Dgp71WD. Grip-motif proteins, but not Dgp71WD, appear to be required for γ-TuRC assembly. Individual depletion of γ-TuRC components, in cultured cells and in vivo, induces mitotic delay and abnormal spindles. Surprisingly, γ-TuSCs are recruited to the centrosomes. These defects are less severe than those resulting from the inhibition of γ-TuSC components and do not appear critical for viability. Simultaneous cosilencing of all γ-TuRC proteins leads to stronger phenotypes and partial recruitment of γ-TuSC. In conclusion, γ-TuRCs are required for assembly of fully functional spindles, but we suggest that γ-TuSC could be targeted to the centrosomes, which is where basic microtubule assembly activities are maintained.
Nuclear actin: to polymerize or not to polymerize
Authors: Hofmann Wilma A. , de Lanerolle Primal
Published: February 2006

The form and function of actin in the nucleus have been enigmatic for over 30 years. Recently actin has been assigned numerous functional roles in the nucleus, but its form remains a mystery. The intricate relationship between actin form and function in the cytoplasm implies that understanding the structural properties of nuclear actin is elementary to fully understanding its function. In this issue, McDonald et al. (p. 541) use fluorescence recovery after photobleaching (FRAP) to tackle the question of whether nuclear actin exists as monomers or polymers.
Crystal structures arising from representations of 𝐺𝐿(𝑚|𝑛)
Authors: Kujawa Jonathan
Published: February 2006

This paper provides results on the modular representation theory of the supergroup

G
L
(
m

|

n
)
.

GL(m|n).

Working over a field of arbitrary characteristic, we prove that the explicit combinatorics of certain crystal graphs describe the representation theory of a modular analogue of the Bernstein-Gelfand-Gelfand category

O

\mathcal {O}

. In particular, we obtain a linkage principle and describe the effect of certain translation functors on irreducible supermodules. Furthermore, our approach accounts for the fact that

G
L
(
m

|

n
)

GL(m|n)

has non-conjugate Borel subgroups and we show how Serganova’s odd reflections give rise to canonical crystal isomorphisms.
Proteasome-mediated Degradation of Rac1-GTP during Epithelial Cell Scattering
Authors: Lynch Emma A. , Stall Jennifer , Schmidt Gudila , Chavrier Philippe , D'Souza-Schorey Crislyn
Published: February 2006

Epithelial cells disassemble their adherens junctions and “scatter” during processes such as tumor cell invasion as well as some stages of embryonic development. Control of actin polymerization is a powerful mechanism for regulating the strength of cell–cell adhesion. In this regard, studies have shown that sustained activation of Rac1, a well-known regulator of actin dynamics, results in the accumulation of polymerized actin at cell–cell contacts in epithelia and an increase in E-cadherin–mediated adhesion. Here we show that active Rac1 is ubiquitinated and subject to proteasome-mediated degradation during the early stages of epithelial cell scattering. These findings delineate a mechanism for the down-regulation of Rac1 in the disassembly of epithelial cell–cell contacts and support the emerging theme that UPS-mediated degradation of the Rho family GTPases may serve as an efficient mechanism for GTPase deactivation in the sustained presence of Dbl-exchange factors.
The Fission Yeast Transforming Acidic Coiled Coil–related Protein Mia1p/Alp7p Is Required for Formation and Maintenance of Persistent Microtubule-organizing Centers at the Nuclear Envelope
Authors: Zheng Liling , Schwartz Cindi , Wee Liangmeng , Oliferenko Snezhana
Published: February 2006

Microtubule-organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in spatial and temporal manner. How MTOCs occur remains an exciting question in cell biology. Here, we show that the transforming acidic coiled coil–related protein Mia1p/Alp7p functions in emergence of large MTOCs in interphase fission yeast cells. We found that Mia1p was a microtubule-binding protein that preferentially localized to the minus ends of microtubules and was associated with the sites of microtubule attachment to the nuclear envelope. Cells lacking Mia1p exhibited less microtubule bundles. Microtubules could be nucleated and bundled but were frequently released from the nucleation sites in mia1Δ cells. Mia1p was required for stability of microtubule bundles and persistent use of nucleation sites both in interphase and postanaphase array dynamics. The γ-tubulin–rich material was not organized in large perinuclear or microtubule-associated structures in mia1Δ cells. Interestingly, absence of microtubules in dividing wild-type cells prevented appearance of large γ-tubulin–rich MTOC structures in daughters. When microtubule polymerization was allowed, MTOCs were efficiently assembled de novo. We propose a model where MTOC emergence is a self-organizing process requiring the continuous association of microtubules with nucleation sites.
The Ras-GRF1 Exchange Factor Coordinates Activation of H-Ras and Rac1 to Control Neuronal Morphology
Authors: Yang Huibin , Mattingly Raymond R.
Published: February 2006

The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRFΔN, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRFΔC, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRFΔC binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size.
Ran Is Required before Metaphase for Spindle Assembly and Chromosome Alignment and after Metaphase for Chromosome Segregation and Spindle Midbody Organization
Authors: Silverman-Gavrila Rosalind V. , Wilde Andrew
Published: February 2006

The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles.
Function of the Neuron-specific Alternatively Spliced Isoforms of Nonmuscle Myosin II-B during Mouse Brain Development
Authors: Ma Xuefei , Kawamoto Sachiyo , Uribe Jorge , Adelstein Robert S.
Published: February 2006

We report that the alternatively spliced isoforms of nonmuscle myosin heavy chain II-B (NHMC II-B) play distinct roles during mouse brain development. The B1-inserted isoform of NMHC II-B, which contains an insert of 10 amino acids near the ATP-binding region (loop 1) of the myosin heavy chain, is involved in normal migration of facial neurons. In contrast, the B2-inserted isoform, which contains an insert of 21 amino acids near the actin-binding region (loop 2), is important for postnatal development of cerebellar Purkinje cells. Deletion of the B1 alternative exon, together with reduced expression of myosin II-B, results in abnormal migration and consequent protrusion of facial neurons into the fourth ventricle. This protrusion is associated with the development of hydrocephalus. Restoring the amount of myosin II-B expression to wild-type levels prevents these defects, showing the importance of total myosin activity in facial neuron migration. In contrast, deletion of the B2 alternative exon results in abnormal development of cerebellar Purkinje cells. Cells lacking the B2-inserted isoform show reduced numbers of dendritic spines and branches. Some of the B2-ablated Purkinje cells are misplaced in the cerebellar molecular layer. All of the B2-ablated mice demonstrated impaired motor coordination.
Genome-wide Mapping of DNA Synthesis in Saccharomyces cerevisiae Reveals That Mechanisms Preventing Reinitiation of DNA Replication Are Not Redundant
Authors: Green Brian M. , Morreale Richard J. , Özaydin Bilge , DeRisi Joseph L. , Li Joachim J.
Published: February 2006

To maintain genomic stability, reinitiation of eukaryotic DNA replication within a single cell cycle is blocked by multiple mechanisms that inactivate or remove replication proteins after G1 phase. Consistent with the prevailing notion that these mechanisms are redundant, we previously showed that simultaneous deregulation of three replication proteins, ORC, Cdc6, and Mcm2-7, was necessary to cause detectable bulk re-replication in G2/M phase in Saccharomyces cerevisiae. In this study, we used microarray comparative genomic hybridization (CGH) to provide a more comprehensive and detailed analysis of re-replication. This genome-wide analysis suggests that reinitiation in G2/M phase primarily occurs at a subset of both active and latent origins, but is independent of chromosomal determinants that specify the use and timing of these origins in S phase. We demonstrate that re-replication can be induced within S phase, but differs in amount and location from re-replication in G2/M phase, illustrating the dynamic nature of DNA replication controls. Finally, we show that very limited re-replication can be detected by microarray CGH when only two replication proteins are deregulated, suggesting that the mechanisms blocking re-replication are not redundant. Therefore we propose that eukaryotic re-replication at levels below current detection limits may be more prevalent and a greater source of genomic instability than previously appreciated.
The Transitional Junction: A New Functional Subcellular Domain at the Intercalated Disc
Authors: Bennett Pauline M. , Maggs Alison M. , Baines Anthony J. , Pinder Jennifer C.
Published: February 2006

We define here a previously unrecognized structural element close to the heart muscle plasma membrane at the intercalated disc where the myofibrils lead into the adherens junction. At this location, the plasma membrane is extensively folded. Immunofluorescence and immunogold electron microscopy reveal a spectrin-rich domain at the apex of the folds. These domains occur at the axial level of what would be the final Z-disc of the terminal sarcomere in the myofibril, although there is no Z-disc-like structure there. However, a sharp transitional boundary lies between the myofibrillar I-band and intercalated disc thin filaments, identifiable by the presence of Z-disc proteins, α-actinin, and N-terminal titin. This allows for the usual elastic positioning of the A-band in the final sarcomere, whereas the transduction of the contractile force normally associated with the Z-disc is transferred to the adherens junctions at the plasma membrane. The axial conjunction of the transitional junction with the spectrin-rich domains suggests a mechanism for direct communication between intercalated disc and contractile apparatus. In particular, it provides a means for sarcomeres to be added to the ends of the cells during growth. This is of particular relevance to understanding myocyte elongation in dilated cardiomyopathy.
A Second SNARE Role for Exocytic SNAP25 in Endosome Fusion
Authors: Aikawa Yoshikatsu , Lynch Kara L. , Boswell Kristin L. , Martin Thomas F.J.
Published: February 2006

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicle–plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.
The DIMA web resource—exploring the protein domain network
Authors: Pagel Philipp , Oesterheld Matthias , Stümpflen Volker , Frishman Dmitrij
Published: February 2006

Summary: Conserved domains represent essential building blocks of most known proteins. Owing to their role as modular components carrying out specific functions they form a network based both on functional relations and direct physical interactions. We have previously shown that domain interaction networks provide substantially novel information with respect to networks built on full-length protein chains. In this work we present a comprehensive web resource for exploring the Domain Interaction MAp (DIMA), interactively. The tool aims at integration of multiple data sources and prediction techniques, two of which have been implemented so far: domain phylogenetic profiling and experimentally demonstrated domain contacts from known three-dimensional structures. A powerful yet simple user interface enables the user to compute, visualize, navigate and download domain networks based on specific search criteria.

Availability:  

Contact:  p.pagel@gsf.de
An ensemble of K-local hyperplanes for predicting protein–protein interactions
Authors: Nanni Loris , Lumini Alessandra
Published: February 2006

Prediction of protein–protein interaction is a difficult and important problem in biology. In this paper, we propose a new method based on an ensemble of K-local hyperplane distance nearest neighbor (HKNN) classifiers, where each HKNN is trained using a different physicochemical property of the amino acids. Moreover, we propose a new encoding technique that combines the amino acid indices together with the 2-Grams amino acid composition. A fusion of HKNN classifiers combined with the ‘Sum rule’ enables us to obtain an improvement over other state-of-the-art methods. The approach is demonstrated by building a learning system based on experimentally validated protein–protein interactions in human gastric bacterium Helicobacter pylori and in Human dataset.

Contact:  lnanni@deis.unibo.it
maSigPro: a method to identify significantly differential expression profiles in time-course microarray experiments
Authors: Conesa Ana , Nueda María José , Ferrer Alberto , Talón Manuel
Published: February 2006

Motivation: Multi-series time-course microarray experiments are useful approaches for exploring biological processes. In this type of experiments, the researcher is frequently interested in studying gene expression changes along time and in evaluating trend differences between the various experimental groups. The large amount of data, multiplicity of experimental conditions and the dynamic nature of the experiments poses great challenges to data analysis.

Results: In this work, we propose a statistical procedure to identify genes that show different gene expression profiles across analytical groups in time-course experiments. The method is a two-regression step approach where the experimental groups are identified by dummy variables. The procedure first adjusts a global regression model with all the defined variables to identify differentially expressed genes, and in second a variable selection strategy is applied to study differences between groups and to find statistically significant different profiles. The methodology is illustrated on both a real and a simulated microarray dataset.

Availability: The method has been implemented in the statistical language R and is freely available from the Bioconductor contributed packages repository and from

Contact:  aconesa@ivia.es; mj.nueda@ua.es
Role of a conserved tripeptide in the endodomain of Sindbis virus glycoprotein E2 in virus assembly and function
Authors: West John , Brown Dennis T.
Published: February 2006

Envelopment of Sindbis virus (SV) at the plasma membrane begins with the interaction of the E2 glycoprotein endodomain with a hydrophobic cleft in the surface of the pre-assembled nucleocapsid. The driving force for this budding event is thought to reside in this virus type-specific association at the surface of the cell. The specific amino acids involved in this interaction have not been identified; however, it has been proposed that a conserved motif (TPY) at aa 398–400 in the E2 tail plays a critical role in this interaction. This interaction has been examined with virus containing mutations at two positions in this conserved domain, T398A and Y400N. The viruses produced have very low infectivity (as determined by particle : p.f.u. ratios); however, there appears to be no defect in assembly, as the virus has wild-type density and electron microscopy shows assembled particles with no obvious aberrant structural changes. The loss of infectivity in the double mutant is accompanied by the loss of the ability to fuse cells after brief exposure to acid pH. These data support the idea that these residues are vital for production of infectious/functional virus; however, they are dispensable for assembly. These results, combined with other published observations, expand our understanding of the interaction of the E2 endodomain with the capsid protein.
The glycosylation site in the envelope protein of West Nile virus (Sarafend) plays an important role in replication and maturation processes
Authors: Li J. , Bhuvanakantham R. , Howe J. , Ng M.-L.
Published: February 2006

The complete genome of West Nile (Sarafend) virus [WN(S)V] was sequenced. Phylogenetic trees utilizing the complete genomic sequence, capsid gene, envelope gene and NS5 gene/3′ untranslated region of WN(S)V classified WN(S)V as a lineage II virus. A full-length infectious clone of WN(S)V with a point mutation in the glycosylation site of the envelope protein (pWNS-S154A) was constructed. Both growth kinetics and the mode of maturation were affected by this mutation. The titre of the pWNS-S154A virus was lower than the wild-type virus. This defect was corrected by the expression of wild-type envelope protein in trans. The pWNS-S154A virus matured intracellularly instead of at the plasma membrane as shown for the parental WN(S)V.
Relaxed template specificity in fowl adenovirus 1 DNA replication initiation
Authors: Rademaker Hendrik J. , Fallaux Frits J. , Van den Wollenberg Diana J. M. , De Jong Rob N. , Van der Vliet Peter C. , Hoeben Rob C.
Published: February 2006

The fowl adenovirus 1 (FAdV-1) isolates PHELPS and OTE are highly similar, but have striking differences in the repeat region of the inverted terminal repeat (ITR). Whilst the repeat region in OTE conforms to the conventional human adenovirus repeat region (5′-CATCATC), that of PHELPS contains guanidine residues at positions 1, 4 and 7 (5′-GATGATG). This implies that the FAdV-1 isolates PHELPS and OTE have either distinct template specificity at replication initiation or, alternatively, a relaxed specificity for replication initiation. In this study, the distinct sequence variation at the origin of DNA replication in the ITRs of the FAdV-1 PHELPS and OTE isolates was confirmed. Sequence analyses of the pTP and Pol genes of both PHELPS and OTE did not reveal differences that could explain the distinct template specificity. Replication assays demonstrated that linear DNA fragments flanked by either 5′-CATCATC or 5′-GATGATG termini replicated in cells upon infection with FAdV-1 OTE and FAdV-1 PHELPS. This was evident from the appearance ofDpnI-resistant fragments in a minireplicon assay. From these data, it is concluded that FAdV-1 has relaxed, rather than changed, its template specificity at replication initiation.
Abnormal immune response of CCR5-deficient mice to ocular infection with herpes simplex virus type 1
Authors: Carr Daniel J. J. , Ash John , Lane Thomas E. , Kuziel William A.
Published: February 2006

Ocular herpes simplex virus type 1 (HSV-1) infection elicits a strong inflammatory response that is associated with production of theβchemokines CCL3 and CCL5, which share a common receptor, CCR5. To gain insight into the role of these molecules in ocular immune responses, the corneas of wild-type (WT) and CCR5-deficient (CCR5−/−) mice were infected with HSV-1 and inflammatory parameters were measured. In the absence of CCR5, the early infiltration of neutrophils into the cornea was diminished. Associated with this aberrant leukocyte recruitment, neutrophils in CCR5−/− mice were restricted to the stroma, whereas in WT mice, these cells trafficked to the stroma and epithelial layers of the infected cornea. Virus titres and cytokine/chemokine levels in the infected tissue of these mice were similar for the first 5 days after infection. However, by day 7 post-infection, the CCR5−/− mice showed a significant elevation in the chemokines CCL2, CCL5, CXCL9 and CXCL10 in the trigeminal ganglion and brainstem, as well as a significant increase in virus burden. The increase in chemokine expression was associated with an increase in the infiltration of CD4 and/or CD8 T cells into the trigeminal ganglion and brainstem of CCR5−/− mice. Surprisingly, even though infected CCR5−/− mice were less efficient at controlling the progression of virus replication, there was no difference in mortality. These results suggest that, although CCR5 plays a role in regulating leukocyte trafficking and control of virus burden, compensatory mechanisms are involved in preventing mortality following HSV-1 infection.
Human herpesvirus 7 U47 gene products are glycoproteins expressed in virions and associate with glycoprotein H
Authors: Sadaoka Tomohiko , Yamanishi Koichi , Mori Yasuko
Published: February 2006

The function of the human herpesvirus 7 (HHV-7) U47 gene, which is a positional homologue of the genes encoding glycoprotein O (gO) in human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), was analysed. A monoclonal antibody (mAb) against the U47 gene product reacted in immunoblots with proteins migrating at 49 and 51 kDa in lysates of HHV-7-infected cells and with 49 and 51 kDa proteins in partially purified virions. Digestion of the 49 and 51 kDa proteins with endoglycosidase H and peptide N-glycosidase F indicated that the U47-encoded proteins were modified with N-linked oligosaccharides. Therefore, the U47 gene and its product were named gO, as in HCMV and HHV-6. In addition, the anti-gO mAb co-immunoprecipitated glycoprotein H (gH) in HHV-7-infected cells, indicating an association between HHV-7 gO and gH. The results suggest that the HHV-7 gO–gH complex might have a similar function to that in HCMV or HHV-6, such as cell–cell fusion in virus infection.
Genome sequence of an enhancin gene-rich nucleopolyhedrovirus (NPV) from Agrotis segetum: collinearity with Spodoptera exigua multiple NPV
Authors: Jakubowska Agata K. , Peters Sander A. , Ziemnicka Jadwiga , Vlak Just M. , van Oers Monique M.
Published: February 2006

The genome sequence of a Polish isolate of Agrotis segetum nucleopolyhedrovirus (AgseNPV-A) was determined and analysed. The circular genome is composed of 147 544 bp and has a G+C content of 45·7 mol%. It contains 153 putative, non-overlapping open reading frames (ORFs) encoding predicted proteins of more than 50 aa, together making up 89·8 % of the genome. The remaining 10·2 % of the DNA constitutes non-coding regions and homologous-repeat regions. One hundred and forty-three AgseNPV-A ORFs are homologues of previously reported baculovirus gene sequences. There are ten unique ORFs and they account for 3 % of the genome in total. All 62 lepidopteran baculovirus genes, including the 29 core baculovirus genes, were found in the AgseNPV-A genome. The gene content and gene order of AgseNPV-A are most similar to those of Spodoptera exigua (Se) multiple NPV and their shared homologous genes are 100 % collinear. Three putative enhancin genes were identified in the AgseNPV-A genome. In phylogenetic analysis, the AgseNPV-A enhancins form a cluster separated from enhancins of the Mamestra species NPVs.
CCR5 use by human immunodeficiency virus type 1 is associated closely with the gp120 V3 loop N-linked glycosylation site
Authors: Clevestig Peter , Pramanik Lotta , Leitner Thomas , Ehrnst Anneka
Published: February 2006

Human immunodeficiency virus type 1 (HIV-1) enters cells through the chemokine receptors CCR5 (R5 virus) and/or CXCR4 (X4 virus). Loss of N-linked glycans and increased net charge of the third variable loop (V3) of the gp120 envelope glycoprotein have been observed to be important steps towards CXCR4 use. All reported sequences using CCR5 or CXCR4 exclusively, or using both, were gathered from the Los Alamos HIV Database and analysed with regard to the V3 N-linked glycosylation motifs (sequons) and charge. The V3 loop glycan had a sensitivity of 0·98 and a 0·92 positive predictive value in the context of CCR5 use. The difference from X4 was remarkable (P<10−12). Especially, the sequon motif NNT within the V3 loop was conserved in 99·2 % of the major clades. The results suggest a close association between the V3 loop glycan and CCR5 use and may provide new insight into HIV-1 tropism and help to improve phenotype-prediction models.
Tagging of NS5A expressed from a functional hepatitis C virus replicon
Authors: McCormick Christopher J. , Maucourant Sophie , Griffin Stephen , Rowlands David J. , Harris Mark
Published: February 2006

Knowledge of how hepatitis C virus (HCV) proteins associate with components of the host cell to form a functional replication complex is still limited. To address this issue, HCV replicon constructs were generated where either green fluorescent protein (GFP) or the Propionibacterium shermanii transcarboxylase domain (PSTCD) was introduced into the NS5A coding region. Insertion of both GFP and PSTCD was tolerated well, allowing formation of stable replicon-containing cell lines that contained viral protein and transcript levels that were comparable to those of an unmodified parental replicon. Cell lines generated from the GFP-tagged NS5A replicon allowed live-cell visualization of the location of NS5A. Cell lines generated from the PSTCD-tagged replicons allowed rapid and efficient precipitation of the PSTCD-tagged NS5A, as well as other HCV non-structural proteins, using streptavidin-coated magnetic beads. Both replicons represent useful tools that offer different but complementary ways of examining replication-complex formation in cells.
Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus
Authors: See Raymond H. , Zakhartchouk Alexander N. , Petric Martin , Lawrence David J. , Mok Catherine P. Y. , Hogan Robert J. , Rowe Thomas , Zitzow Lois A. , Karunakaran Karuna P. , Hitt Mary M. , Graham Frank L. , Prevec Ludvik , Mahony James B. , Sharon Chetna , Auperin Thierry C. , Rini James M. , Tingle Aubrey J. , Scheifele David W. , Skowronski Danuta M. , Patrick David M. , Voss Thomas G. , Babiuk Lorne A. , Gauldie Jack , Roper Rachel L. , Brunham Robert C. , Finlay B. Brett
Published: February 2006

Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated byβ-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.
Identification of protease and ADP-ribose 1″-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3
Authors: Putics Ákos , Gorbalenya Alexander E. , Ziebuhr John
Published: February 2006

The replicase polyproteins, pp1a and pp1ab, of porcine Transmissible gastroenteritis virus (TGEV) have been predicted to be cleaved by viral proteases into 16 non-structural proteins (nsp). Here, enzymic activities residing in the amino-proximal region of nsp3, the largest TGEV replicase processing product, were characterized. It was shown, by in vitro translation experiments and protein sequencing, that the papain-like protease 1, PL1pro, but not a mutant derivative containing a substitution of the presumed active-site nucleophile, Cys1093, cleaves the nsp2|nsp3 site at 879Gly|Gly880. By using an antiserum raised against the pp1a/pp1ab residues 526–713, the upstream processing product, nsp2, was identified as an 85 kDa protein in TGEV-infected cells. Furthermore, PL1pro was confirmed to be flanked at its C terminus by a domain (called X) that mediates ADP-ribose 1″-phosphatase activity. Expression and characterization of a range of bacterially expressed forms of this enzyme suggest that the active X domain comprises pp1a/pp1ab residues Asp1320–Ser1486.
Jab1 is a specificity factor for E2F1-induced apoptosis
Authors: Hallstrom Timothy C. , Nevins Joseph R.
Published: February 2006

The members of the E2F family of transcription factors are key regulators of genes involved in cell cycle progression, cell fate determination, DNA damage repair, and apoptosis. Many cell-based experiments suggest that E2F1 is a stronger inducer of apoptosis than the other E2Fs. Our previous work identified the E2F1 marked box and flanking region as critical for the specificity in E2F1 apoptosis induction. We have now used a yeast two-hybrid screen to identify proteins that bind the E2F1 marked box and flanking regions, with a potential role in E2F1 apoptosis induction. We identified Jab1 as an E2F1-specific binding protein and showed that Jab1 and E2F1 coexpression synergistically induce apoptosis, coincident with an induction of p53 protein accumulation. In contrast, Jab1 does not synergize with E2F1 to promote cell cycle entry. Cells depleted of Jab1 are deficient for both E2F1-induced apoptosis and induction of p53 accumulation. We suggest that Jab1 is an essential cofactor for the apoptotic function of E2F1.
Human telomerase and Cajal body ribonucleoproteins share a unique specificity of Sm protein association
Authors: Fu Dragony , Collins Kathleen
Published: February 2006

Cajal bodies are nuclear structures that host RNA modification and assembly reactions. Some RNAs transit Cajal bodies, while others must concentrate in Cajal bodies to function. Here we report that at least a subfraction of human telomerase RNA and individual resident Cajal body RNAs is associated with Sm proteins. Surprisingly, of seven Sm proteins assembled into a heteroheptameric ring, only a subset copurifies telomerase and Cajal body ribonucleoproteins. We show that a Cajal body RNA localization motif determines this specificity. These discoveries expand the cellular repertoire of Sm protein assemblies and their involvement in ribonucleoprotein localization and function.
Histone deacetylase-associating Atrophin proteins are nuclear receptor corepressors
Authors: Wang Lei , Rajan Harini , Pitman Jeffrey L. , McKeown Michael , Tsai Chih-Cheng
Published: February 2006

Drosophila Tailless (Tll) is an orphan nuclear receptor involved in embryonic segmentation and neurogenesis. Although Tll exerts potent transcriptional repressive effects, the underlying molecular mechanisms have not been determined. Using the established regulation of knirps by tll as a paradigm, we report that repression of knirps by Tll involves Atrophin, which is related to vertebrate Atrophin-1 and Atrophin-2. Atrophin interacts with Tll physically and genetically, and both proteins localize to the same knirps promoter region. Because Atrophin proteins interact with additional nuclear receptors and Atrophin-2 selectively binds histone deacetylase 1/2 (HDAC1/2) through its ELM2 (EGL-27 and MTA1 homology 2)/SANT (SWI3/ADA2/N-CoR/TFIII-B) domains, our study establishes that Atrophin proteins represent a novel class of nuclear receptor corepressors.
Distinct roles for cysteine cathepsin genes in multistage tumorigenesis
Authors: Gocheva Vasilena , Zeng Wei , Ke Danxia , Klimstra David , Reinheckel Thomas , Peters Christoph , Hanahan Douglas , Joyce Johanna A.
Published: February 2006

Multiple types of degradative enzymes, including cathepsins of the cysteine protease family, have been implicated in the regulation of angiogenesis and invasion during cancer progression. Several cysteine cathepsins are up-regulated in a mouse model of pancreatic islet cell carcinogenesis (RIP1-Tag2), and tumor progression is impaired following their collective pharmacologic inhibition. Using null mutations of four of the implicated cysteine cathepsins, we have now dissected their individual roles in cancer development. Mutants ofcathepsins BorSimpaired tumor formation and angiogenesis, whilecathepsin BorLknockouts retarded cell proliferation and tumor growth. Absence of any one of these three genes impaired tumor invasion. In contrast, removal ofcathepsin Chad no effect on either tumor formation or progression. We have identified E-cadherin as a target substrate of cathepsins B, L, and S, but not cathepsin C, potentially explaining their differential effects on tumor invasion. Furthermore, we detected analogous increases in cathepsin expression in human pancreatic endocrine neoplasms, and a significant association between increased levels of cathepsins B and L and tumor malignancy. Thus individual cysteine cathepsin genes make distinctive contributions to tumorigenesis.
Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays
Authors: Lin Baochuan , Wang Zheng , Vora Gary J. , Thornton Jennifer A. , Schnur Joel M. , Thach Dzung C. , Blaney Kate M. , Ligler Adam G. , Malanoski Anthony P. , Santiago Jose , Walter Elizabeth A. , Agan Brian K. , Metzgar David , Seto Donald , Daum Luke T. , Kruzelock Russell , Rowley Robb K. , Hanson Eric H. , Tibbetts Clark , Stenger David A.
Published: February 2006

The exponential growth of pathogen nucleic acid sequences available in public domain databases has invited their direct use in pathogen detection, identification, and surveillance strategies. DNA microarray technology has offered the potential for the direct DNA sequence analysis of a broad spectrum of pathogens of interest. However, to achieve the practical attainment of this potential, numerous technical issues, especially nucleic acid amplification, probe specificity, and interpretation strategies of sequence detection, need to be addressed. In this report, we demonstrate an approach that combines the use of a custom-designed Affymetrix resequencing Respiratory Pathogen Microarray (RPM v.1) with methods for microbial nucleic acid enrichment, random nucleic acid amplification, and automated sequence similarity searching for broad-spectrum respiratory pathogen surveillance. Successful proof-of-concept experiments, utilizing clinical samples obtained from patients presenting adenovirus or influenza virus-induced febrile respiratory illness (FRI), demonstrate the ability of this approach for correct species- and strain-level identification with unambiguous statistical interpretation at clinically relevant sensitivity levels. Our results underscore the feasibility of using this approach to expedite the early surveillance of diseases, and provide new information on the incidence of multiple pathogens.