bluuebunny/crossref_metadata_2025 huggingface dataset:
Hormonal and metabolic relation to restraint and human handling in growing‐fattening steer
Authors: UETAKE Katsuji , ISHIWATA Toshie , ABE Naoshige , EGUCHI Yusuke , TANAKA Toshio
Published: May 2006

Endogenous relationship to restraint and human handling were studied with growing–fattening steers. Thirty‐five crossbred (Japanese Black × Holstein) steers aged 6–10 months were randomly assigned to three pens. They had free access to an Italian ryegrass hay and a restricted amount of high‐concentrate diets (total digestible nutrients (TDN) 70.5%, digestible crude protein (DCP) 10.0%) for the first 6 months of trial. Then they had free access to an oat hay and another vitamin A‐restricted diet (TDN 72.0%, DCP 10.0%) until slaughter. The steers were individually driven into a restraint stall, and bodyweight was recorded. Blood samples were then collected under haltered conditions. These serial handling procedures started at 2 h after the morning feeding were conducted in months 1, 3, 5 (growing stage, GS) and in months 7, 9, 11 (fattening stage, FS) of the trial. Mean peripheral blood concentrations of epinephrine (A; GS, 117.4 ± 76.4 pg/mL; FS, 64.1 ± 34.2 pg/mL), norepinephrine (NA; GS, 257.7 ± 95.0 pg/mL; FS, 125.9 ± 44.1 pg/mL), cortisol (GS, 1.6 ± 0.8 µg/dL; FS, 1.2 ± 0.4 µg/dL), glucose (GS, 83.1 ± 7.5 mg/dL; FS, 71.9 ± 6.9 mg/dL), non‐esterified fatty acid (NEFA; GS, 0.13 ± 0.06 mEQ/L; FS, 0.10 ± 0.06 mEQ/L) and vitamin A (GS, 90.5 ± 24.6 IU/dL; FS, 37.2 ± 21.3 IU/dL) were higher (allP < 0.01) in the GS than in the FS, whereas those of insulin (GS, 1.06 ± 0.82 µU/mL; FS, 1.36 ± 0.61 µU/mL) and leptin (GS, 4.5 ± 1.8 ng/mL human equivalent (HE); FS, 6.8 ± 2.4 ng/mL HE) were lower (bothP < 0.01). The metabolite that correlated with A and NA was glucose (A:r = 0.61,P < 0.001; NA:r = 0.53,P < 0.01) in the GS, and the metabolites correlating with A, NA and cortisol were NEFA (A:r = 0.31,P < 0.10; NA:r = 0.32,P < 0.10; cortisol:r = 0.41,P < 0.05) and triglyceride (A:r = −0.37,P < 0.05; NA:r = −0.39,P < 0.05) in the FS. Vitamin A was a mediator between A (r = −0.38,P < 0.05) and NA (r = −0.42,P < 0.05) and insulin (r = 0.31,P < 0.10) in the GS, and between NA (r = −0.33,P < 0.10) and leptin (r = 0.38,P < 0.05) in the FS. In conclusion, when changing from the growing to the fattening stages, the stress of handling and restraint had caused the pathways to shift from carbohydrate metabolism to lipid metabolism. In addition, vitamin A seemed to be an important mediator in the endogenous pathways in both stages.
Molecular approaches to developmental malformations using analogous forms of valproic acid
Authors: Okada Akinobu , Fujiwara Michio
Published: May 2006

ABSTRACT  The teratogenic potential of valproic acid has been well established both in experimental models and in human clinical studies. Evidence from many previous studies has shown that VPA is an appropriate drug model for studying chemical structure‐teratogenicity relationships. Using molecular techniques of DNA microarray (GeneChip system) or quantitative real‐time polymerase chain reaction with low teratogenic VPA analogs as comparative control drugs, we attempted to identify the genes involved with the molecular mechanisms of VPA teratogenicity in the neural tube and the axial skeleton of the mouse embryo. The recent development of DNA microarray enables a genome‐wide approach to the identification of genes correlated with the teratogenicity of chemicals (teratogenomics). The VPA‐induced changes in gene expression seen during mouse embryogenesis provides information for understanding how VPA disrupts normal embryonic development, and also provides leads for the development of safer medicines.
Clock mutant mice with Jcl/ICR background shows an impaired learning ability in water maze, but not in passive avoidance, at the beginning of dark phase
Authors: Sei Hiroyoshi , Oishi Katsutaka , Sano Atsuko , Seno Hiromasa , Ohmori Tetsuro , Morita Yusuke , Ishida Norio
Published: May 2006

ABSTRACT  We observed the learning ability in Clock mutant mice with Jcl/ICR background (Clockj), a mice model of evening‐type individuals, in the early part of dark phase. In order to estimate the learning ability, Morris water maze (WM) and passive avoidance (PA) test were performed. Release of acetylcholine, 5 hydroxytryptophan (5‐HT) and dopamine (DA) in hippocampus was measured by in vivo microdialysis method. Clockj showed the impaired learning ability in the WM, but not in PA test. Hippocampal acetylcholine release was significantly attenuated in the Clockj in comparison to the wild‐type mice. Neither 5‐HT nor DA in the hippocampus was affected by the Clock mutation. Clock, an essential gene controlling circadian rhythm, may have an important role on the spatial learning and hippocampal cholinergic function, at least, at the beginning of the dark phase.
Acute respiratory infections during pregnancy and congenital abnormalities: a population‐based case‐control study
Authors: Ács Nándor , Bánhidy Ferenc , Puhó Erzsébet H. , Czeizel Andrew E.
Published: May 2006

ABSTRACT  Diseases of respiratory system caused by acute infections are among the most common maternal diseases during pregnancy. The objective of the study was to estimate the association between congenital abnormalities and acute respiratory infections during the first trimester of pregnancy. The data set of the Hungarian Case‐Control Surveillance of Congenital Abnormalities including 22 843 cases with congenital abnormalities, 38 151 population controls without congenital abnormalities and 834 malformed controls with Down syndrome between 1980 and 1996 was evaluated. 2118 cases with congenital abnormalities (9.3%), 3455 population controls (9.1%) and 92 malformed controls with Down syndrome (11.0%) had mothers with acute respiratory infections. Of 25 different congenital abnormality groups, esophageal atresia/stenosis showed a high adjusted prevalence odds ratios (POR) with 95% confidence interval (CI) for acute respiratory infections during the first trimester of pregnancy in case mothers compared with population controls (3.6, 1.4–9.1) and malformed controls (1.9, 1.0–3.5), respectively. In addition there was an association between medically recorded acute respiratory infections during the first trimester of pregnancy and a higher risk for some other congenital abnormalities, such as posterior cleft palate and multiple congenital abnormalities. In conclusion a possible association between some congenital abnormalities, particularly esophageal atresia/stenosis and maternal acute respiratory infections cannot be excluded due to the interactions of the microbial agents, related drug treatments and last but not least the indirect effect of maternal diseases, such as fever‐hyperthermia, hypoxia and dietary deficiency. However, periconceptional multivitamin/folic acid supplementation during the early pregnancy was able to reduce the acute respiratory infection related risk for congenital abnormalities.
Two‐generation reproduction toxicity study in rats with 1,1,1‐trichloro‐2,2‐bis(4‐chlorophenyl)ethane (p,p′‐DDT)
Authors: Hojo Hitoshi , Aoyama Hiroaki , Takahashi Ken L. , Shimizu Naoko , Araki Masayuki , Takizawa Yoshio , Sakasai Koji , Kuwahara Maki , Saka Machiko , Teramoto Shoji
Published: May 2006

ABSTRACT  DDT, an organochlorine pesticide, has been cited as a representative chemical suspected of having endocrine disrupting effects. In this study, the potential endocrine disrupting activities of p,p′‐DDT, a major component of DDT, were investigated in rats in a 2‐generation reproduction toxicity study in accordance with the most current test guidelines of the Ministry of Agriculture, Forestry and Fisheries in Japan, Organization for Economic Cooperation and Development (OECD) and United States Environmental Protection Agency (USEPA) with some modifications and additions. p,p′‐DDT was given to parental rats at dietary levels of 0, 5, 50 or 350 ppm. Systemic toxicities in the parental animals consisted of tremors and subsequent deaths (females only) and/or pathological alterations of the liver (both sexes of animals) of the 2 higher dose groups. Reproductive and postnatal developmental toxicities were not evident up to the highest dose level except for the decreased pup viability index on postnatal day 21 in the 350 ppm group. Changes in serum estradiol and progesterone levels and/or a delay in male sexual maturation were noted in the 2 higher dose groups in a dose‐dependent fashion, suggesting alterations of endogenous endocrine functions. However, these changes never resulted in substantial reproductive disorders.
Primary prevention of neural tube defects with folate in Western Australia: the value of the Western Australian Birth Defects Registry
Authors: Bower Carol
Published: May 2006

ABSTRACT  This paper reviews the role of the Western Australian Birth Defects Registry in the primary prevention of neural tube defects. The Registry provides complete and up‐to‐date information on all neural tube defects (NTD), including terminations of pregnancy. These data have been used to determine a baseline rate of NTD and to monitor trends in NTD over time, when health promotion of folic acid supplement use and voluntary fortification of food with folate were introduced. The register has also been used to investigate NTD in special populations (Indigenous infants in Australia) and as a sampling frame for case control studies. The data derived from these studies have been used to assist in assessing whether mandatory food fortification in Australia is indicated to prevent NTD.
Novel γ‐carboxyglutamic acid‐containing peptides from the venom of Conus textile
Authors: Czerwiec Eva , Kalume Dario E. , Roepstorff Peter , Hambe Björn , Furie Bruce , Furie Barbara C. , Stenflo Johan
Published: May 2006

The cone snail is the only invertebrate system in which the vitamin K‐dependent carboxylase (or γ‐carboxylase) and its product γ‐carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate γ‐carboxylase substrates. Four novel Gla‐containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)–TxVI, Gla(2)–TxVI/A, Gla(2)–TxVI/B and Gla(3)–TxVI each have six Cys residues and belong to the O‐superfamily of conotoxins. All four conopeptides contain 4‐trans‐hydroxyproline and the unusual amino acid 6‐l‐bromotryptophan. Gla(2)–TxVI/A and Gla(2)–TxVI/B are isoforms with an amidated C‐terminus that differ at positions +1 and +13. Three isoforms of Gla(3)–TxVI were observed that differ at position +7: Gla(3)–TxVI, Glu7–Gla(3)–TxVI and Asp7‐Gla(3)–TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids −46 to −27) were nearly identical and highly hydrophobic. The predicted propeptide region (−20 to −1) that contains the γ‐carboxylation recognition site (γ‐CRS) is very similar in Gla(2)–TxVI/A, Gla(2)–TxVI/B and Gla(3)–TxVI, but is more divergent for Gla(1)–TxVI. Kinetic studies utilizing the Conusγ‐carboxylase and synthetic peptide substrates localized the γ‐CRS of Gla(1)–TxVI to the region −14 to −1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)–TxVI lacking a propeptide to 24 µm when a 14‐residue propeptide was attached to the substrate. Similarly, addition of an 18‐residue propeptide to Gla(2)–TxVI/B reduced the Km value tenfold.
Exercise is the primary factor associated with Hsp70 induction in muscle of treadmill running rats
Authors: Noble E. G. , Ho R. , Dzialoszynski T.
Published: May 2006

Aim:  The cytoprotective, inducible stress protein, Hsp70, increases in muscles of rodents subjected to strenuous treadmill running. Most treadmill running protocols employ negative reinforcement to encourage animals to exercise. As these stimuli may themselves activate stress responses, the present investigation was conducted to determine their contribution to the exercise‐induced expression of Hsp70.

Methods:  Twenty‐one male Sprague–Dawley rats were randomly divided into three equal groups including an exercise group (EX), which ran on a treadmill at 30 m min−1 for 60 min; a stimulation group (STIM), which was not allowed to run, but was stimulated with compressed air and mild electric shock concurrently with their exercising cohort; and a control group (CON), which was housed in the treadmill room during the exercise period. Animals were killed 24 h post‐experiment and hearts (H), soleii (SOL) and white gastrocnemii (WG) were harvested and analysed for Hsp70 content (mean% ± SEM of standard).

Results:  Significant increases in Hsp70 (as a % of standard) were noted in H and WG (H = 77.4 ± 8.5; WG = 93.9 ± 18.4) of EX but not in STIM (H = 32.5 ± 4.6; WG = 32.0 ± 3.4) or CON (H = 20.5 ± 3.7; WG = 32.4 ± 7.4). In SOL, Hsp70 expression in EX (126.7 ± 6.2) was different from STIM (98.3 ± 10.9) only. This occurred, despite the fact that all groups were exposed to a stressful environment and exhibited elevated (P < 0.001) temperatures (EX −41.2 ± 0.1 °C > STIM −40.5 ± 0.2 °C > CON −39.0 ± 0.1 °C) indicative of a general stress response.

Conclusions:  These data suggest that exercise per se, rather than environmental conditions or noxious stimuli, are responsible for the induction of Hsp70 in rat muscle during treadmill running.
Hereditary Bone and Joint Disease in the Dog
Authors: Burton Neil
Published: May 2006

Hereditary Bone and Joint Disease in the Dog
 Joe Morgan, Alinda Wind & Autumn Davidson Published by Manson Publishing, 2003. Hardback, 328 pages Price £61.00. ISBN 3‐87706‐548‐1
The Arg16Gly polymorphism of the β2‐adrenergic receptor and the natriuretic response to rapid saline infusion in humans
Authors: Snyder Eric M. , Turner Stephen T. , Joyner Michael J. , Eisenach John H. , Johnson Bruce D.
Published: May 2006

The β2‐adrenergic receptors (β2ARs) play a role in renal Na+ regulation. Subjects homozygous for glycine at amino acid 16 (Gly16) of the β2AR have been shown to have enhanced β2‐mediated vascular relaxation when compared to subjects homozygous for arginine (Arg16). However, Gly16 subjects have been shown to have higher blood pressure than Arg16 subjects. Given the dominant role of the kidneys in long‐term blood pressure regulation, we sought to determine whether there were differences in renal Na+ handling between Gly16 (n= 17) and Arg16 (n= 14) subjects (Gly16: age, 30 ± 2 years; body mass index (BMI), 25 ± 11 kg m−2; Arg16: age, 30 ± 2 years; BMI, 25 ± 1 kg m−2). We measured urinary Na+ content before and for 3 h following rapid intravenous saline infusion (30 ml kg−1 in ∼16 min). Prior to the infusion, there were no differences in 24‐h Na+ excretion between Gly16 and Arg16 subjects (Gly16, 183 ± 21 mmol; Arg16, 184 ± 20 mmol); however, systolic blood pressure (SBP) was significantly higher in Gly16 than Arg16 subjects with no differences observed in diastolic blood pressure (DBP) or mean arterial pressure (MAP) (SBP: Gly16, 117 ± 3 mmHg; Arg16, 109 ± 2 mmHg; DBP: Gly16, 78 ± 2 mmHg; Arg16, 77 ± 2 mmHg; MAP: Gly16, 90 ± 2 mmHg; Arg16, 89 ± 2 mmHg). With rapid saline infusion, MAP increased in both genotype groups (Gly16, 6.7%; Arg16, 3.4%; P > 0.05). In the 3 h following Na+ infusion, Na+ excretion was less in Gly16 when compared to Arg16 subjects, with a trend towards significance when expressed as total Na+ excreted (Gly16, 66 ± 7 mmol; Arg16, 85 ± 9 mmol; P= 0.07), and a significant difference when expressed as a fraction of the administered load (Gly16, 0.18 ± 0.02; Arg16, 0.28 ± 0.03; P < 0.01). These results suggest that the Arg16Gly polymorphism of the β2AR is associated with differences in natriuretic response to rapid saline infusion, which may influence long‐term regulation of blood pressure.
The involvement of nitric oxide in the cutaneous vasoconstrictor response to local cooling in humans
Authors: Hodges Gary J. , Zhao Kun , Kosiba Wojciech A. , Johnson John M.
Published: May 2006

Cutaneous vascular conductance (CVC) declines in response to local cooling (LC). Previous work indicates that at least part of the vasoconstrictor response to LC may be through an inhibitory effect on nitric oxide synthase (NOS) activity. In this study we further tested that notion. A total of eight (6 male, 2 female) subjects participated (Part 1 n= 7; Part 2 n= 5, 4 of whom participated in Part 1). Skin blood flow was monitored by laser‐Doppler flowmetry. Control of local skin and body temperatures was achieved with Peltier cooler/heater probe holders and water perfused suits, respectively. Microdialysis fibres were inserted aseptically. Saline, l‐NAME (20 mm; to inhibit NOS activity) and sodium nitroprusside (SNP 10 μm) were infused by microdialysis. Bretylium tosylate (BT), to block adrenergic function, was administered by iontophoresis. CVC was calculated from blood flow and blood pressure. Part 1 was designed to determine the relative roles of the NO and the adrenergic systems. The infusion of l‐NAME elicited a 35 ± 4% decrease in CVC at the l‐NAME and BT +l‐NAME sites (P < 0.05); subsequent slow LC (34–24°C) for 35 min caused a significant (P < 0.05) decrease in CVC at control sites (68 ± 4%) and at the BT treated sites (39 ± 5%). LC caused a further 23 ± 5% of initial baseline decrease in CVC at the l‐NAME treated sites (P < 0.05). Importantly, CVC at the BT +l‐NAME sites was unaffected by LC (P > 0.05). Part 2 was designed to test whether LC influences were specific to the NOS enzymes. Two sites were pretreated with both BT and l‐NAME. After 50 min, SNP was added as an NO donor to restore baseline CVC at one site. The same LC process as in Part 1 was applied. There was a 24 ± 10% decrease (P < 0.05) in CVC at sites with baseline CVC restored, while, as in Part 1, there was no change (P > 0.05) at sites treated with BT +l‐NAME only. These data suggest that the vasoconstriction with slow LC is due to a combination of increased noradrenaline release and decreased activity of both NOS per se and of process(es) downstream of NOS.
A Methodology for Product Family Ontology Development Using Formal Concept Analysis and Web Ontology Language
Authors: Nanda Jyotirmaya , Simpson Timothy W. , Kumara Soundar R. T. , Shooter Steven B.
Published: May 2006

The use of ontologies for information sharing is well documented in the literature, but the lack of a comprehensive and systematic methodology for constructing product ontologies has limited the process of developing ontologies for design artifacts. In this paper we introduce the Product Family Ontology Development Methodology (PFODM), a novel methodology to develop formal product ontologies using the Semantic Web paradigm. Within PFODM, Formal Concept Analysis (FCA) is used first to identify similarities among a finite set of design artifacts based on their properties and then to develop and refine a product family ontology using Web Ontology Language (OWL). A family of seven one-time-use cameras is used to demonstrate the steps of the PFODM to construct such an ontology. The benefit of PFODM lies in providing a systematic and consistent methodology for constructing ontologies to support product family design. The resulting ontologies provide a hierarchical conceptual clustering of related design artifacts, which is particularly advantageous for product family design where parts, processes, and most important, information is intentionally shared and reused to reduce complexity, lead-time, and development costs. Potential uses of the resulting ontologies and FCA representations within product family design are also discussed.
Optical Conformal Mapping
Authors: Leonhardt Ulf
Published: May 2006

An invisibility device should guide light around an object as if nothing were there, regardless of where the light comes from. Ideal invisibility devices are impossible, owing to the wave nature of light. This study develops a general recipe for the design of media that create perfect invisibility within the accuracy of geometrical optics. The imperfections of invisibility can be made arbitrarily small to hide objects that are much larger than the wavelength. With the use of modern metamaterials, practical demonstrations of such devices may be possible. The method developed here can also be applied to escape detection by other electromagnetic waves or sound.
p53 Regulates Mitochondrial Respiration
Authors: Matoba Satoaki , Kang Ju-Gyeong , Patino Willmar D. , Wragg Andrew , Boehm Manfred , Gavrilova Oksana , Hurley Paula J. , Bunz Fred , Hwang Paul M.
Published: May 2006

The energy that sustains cancer cells is derived preferentially from glycolysis. This metabolic change, the Warburg effect, was one of the first alterations in cancer cells recognized as conferring a survival advantage. Here, we show that p53, one of the most frequently mutated genes in cancers, modulates the balance between the utilization of respiratory and glycolytic pathways. We identify Synthesis of Cytochrome c Oxidase 2 (SCO2) as the downstream mediator of this effect in mice and human cancer cell lines. SCO2 is critical for regulating the cytochrome c oxidase (COX) complex, the major site of oxygen utilization in the eukaryotic cell. Disruption of the
SCO2
gene in human cancer cells with wild-type p53 recapitulated the metabolic switch toward glycolysis that is exhibited by p53-deficient cells. That SCO2 couples p53 to mitochondrial respiration provides a possible explanation for the Warburg effect and offers new clues as to how p53 might affect aging and metabolism.
Regulation of Epstein-Barr Virus Latency Type by the Chromatin Boundary Factor CTCF
Authors: Chau Charles M. , Zhang Xiao-Yong , McMahon Steven B. , Lieberman Paul M.
Published: May 2006

Epstein Barr virus (EBV) can establish distinct latency types with different growth-transforming properties. Type I latency and type III latency can be distinguished by the expression of EBNA2, which has been shown to be regulated, in part, by the EBNA1-dependent enhancer activity of the origin of replication (OriP). Here, we report that CTCF, a chromatin boundary factor with well-established enhancer-blocking activity, binds to EBV sequences between the OriP and the RBP-Jκ response elements of the C promoter (Cp) and regulates transcription levels of EBNA2 mRNA. Using DNA affinity, electrophoretic mobility shift assay, DNase I footprinting, and chromatin immunoprecipitation (ChIP), we found that CTCF binds both in vitro and in vivo to the EBV genome between OriP and Cp, with an ∼50-bp footprint at EBV coordinates 10515 to 10560. Deletion of this CTCF binding site in a recombinant EBV bacterial artificial chromosome (BAC) increased EBNA2 transcription by 3.5-fold compared to a wild-type EBV BAC. DNA affinity and ChIP showed more CTCF binding at this site in type I latency cell lines (MutuI and KemI) than in type III latency cell lines (LCL3456 and Raji). CTCF protein and mRNA expression levels were higher in type I than type III cell lines. Short interfering RNA depletion of CTCF in type I MutuI cells stimulated EBNA2 mRNA levels, while overexpression of CTCF in type III Raji cells inhibited EBNA2 mRNA levels. These results indicate that increased CTCF can repress EBNA2 transcription. We also show that c-MYC, as well as EBNA2, can stimulate CTCF mRNA levels, suggesting that CTCF levels may contribute to B-cell differentiation as well as EBV latency type determination.
Properties and Dissemination of H5N1 Viruses Isolated during an Influenza Outbreak in Migratory Waterfowl in Western China
Authors: Chen Hualan , Li Yanbing , Li Zejun , Shi Jianzhong , Shinya Kyoko , Deng Guohua , Qi Qiaoling , Tian Guobin , Fan Shufang , Zhao Haidan , Sun Yingxiang , Kawaoka Yoshihiro
Published: May 2006

H5N1 influenza A viruses are widely distributed among poultry in Asia, but until recently, only a limited number of wild birds were affected. During late April through June 2005, an outbreak of H5N1 virus infection occurred among wild birds at Qinghai Lake in China. Here, we describe the features of this outbreak. First identified in bar-headed geese, the disease soon spread to other avian species populating the lake. Sequence analysis of 15 viruses representing six avian species and collected at different times during the outbreak revealed four different H5N1 genotypes. Most of the isolates possessed lysine at position 627 in the PB2 protein, a residue known to be associated with virulence in mice and adaptation to humans. However, neither of the two index viruses possessed this residue. All of the viruses tested were pathogenic in mice, with the exception of one index virus. We also tested the replication of two viruses isolated during the Qinghai Lake outbreak and one unrelated duck H5N1 virus in rhesus macaques. The Qinghai Lake viruses did not replicate efficiently in these animals, producing no evidence of disease other than transient fever, while the duck virus replicated in multiple organs and caused symptoms of respiratory illness. Importantly, H5N1 viruses isolated in Mongolia, Russia, Inner Mongolia, and the Liaoning Province of China after August 2005 were genetically closely related to one of the genotypes isolated during the Qinghai outbreak, suggesting the dominant nature of this genotype and underscoring the need for worldwide intensive surveillance to minimize its devastating consequences.
The Putative Terminase Subunit of Herpes Simplex Virus 1 Encoded by U
L
28 Is Necessary and Sufficient To Mediate Interaction between pU
L
15 and pU
L
33
Authors: Yang Kui , Baines Joel D.
Published: May 2006

Viral terminases play essential roles as components of molecular motors that package viral DNA into capsids. Previous results indicated that the putative terminase subunits of herpes simplex virus 1 (HSV-1) encoded by U
L
15 and U
L
28 (designated pU
L
15 and pU
L
28, respectively) coimmunoprecipitate with the U
L
33 protein from lysates of infected cells. All three proteins are among six required for HSV-1 DNA packaging but dispensable for assembly of immature capsids. The current results show that in both infected- and uninfected-cell lysates, pU
L
28 coimmunoprecipitates with either pU
L
33 or pU
L
15, whereas pU
L
15 and pU
L
33 do not coimmunoprecipitate unless pU
L
28 is present. The U
L
28 protein was sufficient to stabilize pU
L
33 from proteasomal degradation in an engineered cell line and was necessary to stabilize pU
L
33 in infected cells, whereas pU
L
15 had no such effects. The presence of pU
L
33 was dispensable for the pU
L
15/pU
L
28 interaction in lysates of both infected and uninfected cells but augmented the tendency for pU
L
15 and pU
L
28 to coimmunoprecipitate. These data suggest that pU
L
28 and pU
L
33 interact directly and that pU
L
15 interacts directly with pU
L
28 but only indirectly with pU
L
33. It is logical to propose that the indirect interaction of pU
L
15 and pU
L
33 is mediated through the interaction of both proteins with pU
L
28. The data also suggest that one function of pU
L
33 is to optimize the pU
L
15/pU
L
28 interaction.
Vaccine-Induced Cellular Immune Responses Reduce Plasma Viral Concentrations after Repeated Low-Dose Challenge with Pathogenic Simian Immunodeficiency Virus SIVmac239
Authors: Wilson Nancy A. , Reed Jason , Napoe Gnankang S. , Piaskowski Shari , Szymanski Andy , Furlott Jessica , Gonzalez Edna J. , Yant Levi J. , Maness Nicholas J. , May Gemma E. , Soma Taeko , Reynolds Matthew R. , Rakasz Eva , Rudersdorf Richard , McDermott Adrian B. , O'Connor David H. , Friedrich Thomas C. , Allison David B. , Patki Amit , Picker Louis J. , Burton Dennis R. , Lin Jing , Huang Lingyi , Patel Deepa , Heindecker Gwendolyn , Fan Jiang , Citron Michael , Horton Melanie , Wang Fubao , Liang Xiaoping , Shiver John W. , Casimiro Danilo R. , Watkins David I.
Published: May 2006

The goal of an AIDS vaccine regimen designed to induce cellular immune responses should be to reduce the viral set point and preserve memory CD4 lymphocytes. Here we investigated whether vaccine-induced cellular immunity in the absence of any Env-specific antibodies can control viral replication following multiple low-dose challenges with the highly pathogenic SIVmac239 isolate. EightMamu-A*01-positive Indian rhesus macaques were vaccinated with simian immunodeficiency virus (SIV)gag,tat,rev, andnefusing a DNA prime-adenovirus boost strategy. Peak viremia (P= 0.007) and the chronic phase set point (P= 0.0192) were significantly decreased in the vaccinated cohort, out to 1 year postinfection. Loss of CD4+memory populations was also ameliorated in vaccinated animals. Interestingly, only one of the eight vaccinees developed Env-specific neutralizing antibodies after infection. The control observed was significantly improved over that observed in animals vaccinated with SIVgagonly. Vaccine-induced cellular immune responses can, therefore, exert a measure of control over replication of the AIDS virus in the complete absence of neutralizing antibody and give us hope that a vaccine designed to induce cellular immune responses might control viral replication.
Crystal Structures of Human Immunodeficiency Virus Type 1 (HIV-1) Neutralizing Antibody 2219 in Complex with Three Different V3 Peptides Reveal a New Binding Mode for HIV-1 Cross-Reactivity
Authors: Stanfield Robyn L. , Gorny Miroslaw K. , Zolla-Pazner Susan , Wilson Ian A.
Published: May 2006

Human monoclonal antibody 2219 is a neutralizing antibody isolated from a human immunodeficiency virus type 1-infected individual. 2219 was originally selected for binding to a V3 fusion protein and can neutralize primary isolates from subtypes B, A, and F. Thus, 2219 represents a cross-reactive, human anti-V3 antibody. Fab 2219 binds to one face of the variable V3 β-hairpin, primarily contacting conserved residues on the N-terminal β-strand of V3, leaving the V3 crown or tip largely accessible. Three V3/2219 complexes reveal the antibody-bound conformations for both the N- and C-terminal regions that flank the V3 crown and illustrate how twisting of the V3 loop alters the relative dispositions and pairing of the amino acids in the adjacent V3 β-strands and how the antibody can accommodate V3 loops with different sequences.
Disruption of Microtubule Organization and Centrosome Function by Expression ofTobacco Mosaic VirusMovement Protein
Authors: Ferralli Jacqueline , Ashby Jamie , Fasler Monika , Boyko Vitaly , Heinlein Manfred
Published: May 2006

The movement protein (MP) ofTobacco mosaic virusmediates the cell-to-cell transport of viral RNA through plasmodesmata, cytoplasmic cell wall channels for direct cell-to-cell communication between adjacent cells. Previous in vivo studies demonstrated that the RNA transport function of the protein correlates with its association with microtubules, although the exact role of microtubules in the movement process remains unknown. Since the binding of MP to microtubules is conserved in transfected mammalian cells, we took advantage of available mammalian cell biology reagents and tools to further address the interaction in flat-growing and transparent COS-7 cells. We demonstrate that neither actin, nor endoplasmic reticulum (ER), nor dynein motor complexes are involved in the apparent alignment of MP with microtubules. Together with results of in vitro coprecipitation experiments, these findings indicate that MP binds microtubules directly. Unlike microtubules associated with neuronal MAP2c, MP-associated microtubules are resistant to disruption by microtubule-disrupting agents or cold, suggesting that MP is a specialized microtubule binding protein that forms unusually stable complexes with microtubules. MP-associated microtubules accumulate ER membranes, which is consistent with a proposed role for MP in the recruitment of membranes in infected plant cells and may suggest that microtubules are involved in this process. The ability of MP to interfere with centrosomal γ-tubulin is independent of microtubule association with MP, does not involve the removal of other tested centrosomal markers, and correlates with inhibition of centrosomal microtubule nucleation activity. These observations suggest that the function of MP in viral movement may involve interaction with the microtubule-nucleating machinery.
High-Molecular-Weight Protein (pUL48) of Human Cytomegalovirus Is a Competent Deubiquitinating Protease: Mutant Viruses Altered in Its Active-Site Cysteine or Histidine Are Viable
Authors: Wang Jianlei , Loveland Amy N. , Kattenhorn Lisa M. , Ploegh Hidde L. , Gibson Wade
Published: May 2006

We show here that the high-molecular-weight protein (HMWP or pUL48; 253 kDa) of human cytomegalovirus (HCMV) is a functionally competent deubiquitinating protease (DUB). By using a suicide substrate probe specific for ubiquitin-binding cysteine proteases (DUB probe) to screen lysates of HCMV-infected cells, we found just one infected-cell-specific DUB. Characteristics of this protein, including its large size, expression at late times of infection, presence in extracellular virus particles, and reactivity with an antiserum to the HMWP, identified it as the HMWP. This was confirmed by constructing mutant viruses with substitutions in two of the putative active-site residues, Cys24Ile and His162Ala. HMWP with these mutations either failed to bind the DUB probe (C24I) or had significantly reduced reactivity with it (H162A). More compellingly, the deubiquitinating activity detected in wild-type virus particles was completely abolished in both the C24I and H162A mutants, thereby directly linking HMWP with deubiquitinating enzyme activity. Mutations in these active-site residues were not lethal to virus replication but slowed production of infectious virus relative to wild type and mutations of other conserved residues. Initial studies, by electron microscopy, of cells infected with the mutants revealed no obvious differences at late times of replication in the general appearance of the cells or in the distribution, relative numbers, or appearance of virus particles in the cytoplasm or nucleus.
Endosomal Proteolysis by Cathepsins Is Necessary for Murine Coronavirus Mouse Hepatitis Virus Type 2 Spike-Mediated Entry
Authors: Qiu Zhaozhu , Hingley Susan T. , Simmons Graham , Yu Christopher , Das Sarma Jayasri , Bates Paul , Weiss Susan R.
Published: May 2006

Most strains of murine coronavirus mouse hepatitis virus (MHV) express a cleavable spike glycoprotein that mediates viral entry and pH-independent cell-cell fusion. The MHV type 2 (MHV-2) strain of murine coronavirus differs from other strains in that it expresses an uncleaved spike and cannot induce cell-cell fusion at neutral pH values. We show here that while infection of the prototype MHV-A59 strain is not sensitive to pretreatment with lysosomotropic agents, MHV-2 replication is significantly inhibited by these agents. By use of an A59/MHV-2 chimeric virus, the susceptibility to lysosomotropic agents is mapped to the MHV-2 spike, suggesting a requirement of acidification of endosomes for MHV-2 spike-mediated entry. However, acidification is likely not a direct trigger for MHV-2 spike-mediated membrane fusion, as low-pH treatment is unable to overcome ammonium chloride inhibition, and it also cannot induce cell-cell fusion between MHV-2-infected cells. In contrast, trypsin treatment can both overcome ammonium chloride inhibition and promote cell-cell fusion. Inhibitors of the endosomal cysteine proteases cathepsin B and cathepsin L greatly reduce MHV-2 spike-mediated entry, while they have little effect on A59 entry, suggesting that there is a proteolytic step in MHV-2 entry. Finally, a recombinant virus expressing a cleaved MHV-2 spike has the ability to induce cell-cell fusion at neutral pH values and does not require low pH and endosomal cathepsins during infection. These studies demonstrate that endosomal proteolysis by cathepsins is necessary for MHV-2 spike-mediated entry; this is similar to the entry pathway recently described for severe acute respiratory syndrome coronavirus and indicates that coronaviruses may use multiple pathways for entry.
Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms
Authors: Berkova Z. , Crawford S. E. , Trugnan G. , Yoshimori T. , Morris A. P. , Estes M. K.
Published: May 2006

Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.
Phosphorylation and Function of the Kaposin B Direct Repeats of Kaposi's Sarcoma-Associated Herpesvirus
Authors: McCormick Craig , Ganem Don
Published: May 2006

Kaposi's sarcoma-associated herpesvirus encodes a protein, kaposin B, which is composed of multiple copies of 23-amino-acid direct repeats, termed DR2 and DR1. Kaposin B enhances the release of pathogenetically important proinflammatory cytokines by activating the p38 mitogen-activated protein kinase (MAPK)-MK2 kinase pathway and blocking cytokine mRNA decay. Here, we show that this mRNA stabilization function requires both the DR2 and DR1 elements of kaposin B; a monomeric form of the protein consisting of one copy of each repeat retains function. Furthermore, we show that p38 MAPK is capable of directly phosphorylating kaposin B in vitro and map the site of phosphorylation to a specific serine residue in DR1. Mutational ablation of this serine abolishes phosphorylation of the protein by p38 MAPK but does not affect kaposin B's ability to extend mRNA half-life.
Efficient In Vitro System of Homologous Recombination in Brome Mosaic Bromovirus
Authors: Wierzchoslawski Rafal , Bujarski Jozef J.
Published: May 2006

Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol.
78:
8552-8564, 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (−)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA recombination.
Identification of a Novel Human Immunodeficiency Virus Type 1 Integrase Interactor, Gemin2, That Facilitates Efficient Viral cDNA Synthesis In Vivo
Authors: Hamamoto Seiji , Nishitsuji Hironori , Amagasa Teruo , Kannagi Mari , Masuda Takao
Published: May 2006

Retroviral integrase (IN) catalyzes the integration of viral cDNA into a host chromosome. Additional roles have been suggested for IN, including uncoating, reverse transcription, and nuclear import of the human immunodeficiency virus type 1 (HIV-1) genome. However, the underlying mechanism is largely unknown. Here, using a yeast two-hybrid system, we identified a survival motor neuron (SMN)-interacting protein 1 (Gemin2) that binds to HIV-1 IN. Reduction of Gemin2 with small interfering RNA duplexes (siGemin2) dramatically reduced HIV-1 infection in human primary monocyte-derived macrophages and also reduced viral cDNA synthesis. In contrast, siGemin2 did not affect HIV-1 expression from the integrated proviral DNA. Although Gemin2 was undetectable in cell-free viral particles, coimmunoprecipitation experiments using FLAG-tagged Gemin2 strongly suggested that Gemin2 interacts with the incoming viral genome through IN. Further experiments reducing SMN or other SMN-interacting proteins suggested that Gemin2 might act on HIV-1 either alone or with unknown proteins to facilitate efficient viral cDNA synthesis soon after infection. Thus, we provide the evidence for a novel host protein that binds to HIV-1 IN and facilitates viral cDNA synthesis and subsequent steps that precede integration in vivo.
Evaluation of the Roles of Specific Regions of the
Cucumber Necrosis Virus
Coat Protein Arm in Particle Accumulation and Fungus Transmission
Authors: Hui Elizabeth , Rochon D'Ann
Published: May 2006

The
Cucumber necrosis virus
(CNV) particle is a T=3 icosahedron composed of 180 identical coat protein (CP) subunits. Each CP subunit includes a 34-amino-acid (aa) arm which connects the RNA binding and shell domains. The arm is comprised of an 18-aa “
β
” region and a 16-aa “ε” region, with the former contributing to a
β
-annular structure involved in particle stability and the latter contributing to quasiequivalence and virion RNA binding. Previous work has shown that specific regions of the CNV capsid play important roles in transmission by zoospores of the fungal vector
Olpidium bornovanus
and that particle expansion is essential for this process. To assess the importance of the two arm regions in particle accumulation, stability, and virus transmission, five CP arm deletion mutants were constructed. Our findings indicate that
β
(−) mutants are capable of producing particles in plants; however, the arm(−) and ε(−) mutants are not. In addition,
β
(−) particles bind zoospores less efficiently than wild-type CNV and are not fungally transmissible.
β
(−) particles are also less thermally stable and disassemble under swelling conditions. Our finding that
β
(−) mutants can accumulate in plants suggests that other features of the virion, such as RNA/CP interactions, may also be important for particle stability.
Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKε Molecular Complex from the Mitochondrial Outer Membrane by Hepatitis C Virus NS3-4A Proteolytic Cleavage
Authors: Lin Rongtuan , Lacoste Judith , Nakhaei Peyman , Sun Qiang , Yang Long , Paz Suzanne , Wilkinson Peter , Julkunen Ilkka , Vitour Damien , Meurs Eliane , Hiscott John
Published: May 2006

Intracellular RNA virus infection is detected by the cytoplasmic RNA helicase RIG-I that plays an essential role in signaling to the host antiviral response. Recently, the adapter molecule that links RIG-I sensing of incoming viral RNA to downstream signaling and gene activation events was characterized by four different groups; MAVS/IPS-1-1/VISA/Cardif contains an amino-terminal CARD domain and a carboxyl-terminal mitochondrial transmembrane sequence that localizes to the mitochondrial membrane. Furthermore, the hepatitis C virus NS3-4A protease complex specifically targets MAVS/IPS-1/VISA/Cardif for cleavage as part of its immune evasion strategy. With a novel search program written in python, we also identified an uncharacterized protein, KIAA1271 (K1271), containing a single CARD-like domain at the N terminus and a Leu-Val-rich C terminus that is identical to that of MAVS/IPS-1/VISA/Cardif. Using a combination of biochemical analysis, subcellular fractionation, and confocal microscopy, we now demonstrate that NS3-4A cleavage of MAVS/IPS-1/VISA/Cardif/K1271 results in its dissociation from the mitochondrial membrane and disrupts signaling to the antiviral immune response. Furthermore, virus-induced IKKε kinase, but not TBK1, colocalized strongly with MAVS at the mitochondrial membrane, and the localization of both molecules was disrupted by NS3-4A expression. Mutation of the critical cysteine 508 to alanine was sufficient to maintain mitochondrial localization of MAVS/IPS-1/VISA/Cardif and IKKε in the presence of NS3-4A. These observations provide an outline of the mechanism by which hepatitis C virus evades the interferon antiviral response.
Erythroblast Transformation by the Friend Spleen Focus-Forming Virus Is Associated with a Block in Erythropoietin-Induced STAT1 Phosphorylation and DNA Binding and Correlates with High Expression of the Hematopoietic Phosphatase SHP-1
Authors: Nishigaki Kazuo , Hanson Charlotte , Ohashi Takashi , Spadaccini Angelo , Ruscetti Sandra
Published: May 2006

Infection of mice with Friend spleen focus-forming virus (SFFV) results in a multistage erythroleukemia. In the first stage, the SFFV envelope glycoprotein interacts with the erythropoietin receptor and a short form of the receptor tyrosine kinase sf-Stk, resulting in constitutive activation of signal transducing molecules and the development of erythropoietin (Epo)-independent erythroid hyperplasia and polycythemia. The second stage results from the outgrowth of a rare virus-infected erythroid cell that expresses nonphysiological levels of the myeloid transcription factor PU.1. These cells exhibit a differentiation block and can be grown as murine erythroleukemia (MEL) cell lines. In this study, we examined SFFV MEL cells to determine whether their transformed phenotype was associated with a block in the activation of any Epo signal-transducing molecules. Our studies indicate that Epo- or SFFV-induced activation of STAT1/3 DNA binding activity is blocked in SFFV MEL cells. The block is at the level of tyrosine phosphorylation of STAT1, although Jak2 phosphorylation is not blocked in these cells. In contrast to Epo, alpha interferon can induce STAT1 phosphorylation and DNA binding in SFFV MEL cells. The SFFV-transformed cells were shown to express elevated levels of the hematopoietic phosphatase SHP-1, and treatment of the cells with a phosphatase inhibitor restored STAT1 tyrosine phosphorylation. MEL cells derived from Friend murine leukemia virus (MuLV) or ME26 MuLV-infected mice, which do not express PU.1, express lower levels of SHP-1 and are not blocked in STAT1/3 DNA-binding activity. Our studies suggest that SFFV-infected erythroid cells become transformed when differentiation signals activated by STAT1/3 are blocked due to high SHP-1 levels induced by inappropriate expression of the PU.1 protein.