bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of Dipyridamole's Action in Inhibition of Venous and Arterial Smooth Muscle Cell Proliferation
Authors: Zhuplatov Sergey B. , Masaki Takahisa , Blumenthal Donald K. , Cheung Alfred K.
Published: November 2006

Abstract: Dipyridamole is a potential pharmacological agent to prevent vascular stenosis because of its antiproliferative properties. The mechanisms by which dipyridamole inhibits the growth of vascular smooth muscle cells, especially venous smooth muscle cells, are unclear. In the present study, dipyridamole transiently but significantly increased cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels in human venous and arterial smooth muscle cells in a time‐ and dose‐dependent manner. Peak concentrations of both cyclic nucleotides were achieved at 15–30 min. and correlated with inhibition of proliferation in both cell types. The antiproliferative effects of dipyridamole observed at 48 hr were similar whether drug exposure was only 15 min. or sustained for 48 hr. Specific competitive inhibitors of protein kinases A and G attenuated the antiproliferative effects of subsaturating concentrations of dipyridamole, with the effects of protein kinase inhibition being particularly pronounced in venous smooth muscle cells. Flow cytometry analysis showed that dipyridamole caused an enrichment of cells in G0/G1 and a corresponding reduction of cells in S phase. These data indicate that a transient increase in cGMP and cAMP is sufficient to induce downstream kinase activation and subsequent cell cycle arrest, and that protein kinase G may be more important than protein kinase A in mediating the growth inhibitory effect of dipyridamole in venous protein kinase.
Cardioprotective Effects of Subcutaneous Ebselen against Daunorubicin‐Induced Cardiomyopathy in Rats
Authors: Saad Sherif Y. , Najjar Tawfeeg A. , Arafah Maha M.
Published: November 2006

Abstract: Daunorubicin is an anthracycline antitumour agent that can cause severe cardiomyopathy leading to a frequently fatal congestive heart failure. Although the exact molecular mechanisms of cardiotoxicity are not well established, oxidative mechanisms involving daunorubicin‐induced superoxide anion production have been proposed. In the present study, we showed that ebselen a seleno‐organic compound exhibiting glutathione peroxidase‐like and antioxidant activities, significantly ameliorated daunorubicin‐induced cardiomyopathy. Subcutaneous administration of ebselen to daunorubicin‐treated rats showed significant improvement in serum cardiac indices including creatine kinase isoenzyme and lactate dehydrogenase as well as serum glutathione (GSH) peroxidase. Moreover, myocardium of daunorubicin/ebselen‐treated rats showed significant improvement in daunorubicin‐induced depletion of GSH peroxidase activity and reduced glutathione content, in addition to attenuation of daunorubicin‐induced increase in cardiac malondialdehyde production and total nitrate/nitrite concentration levels. These results were confirmed by histopathological examination of ventricles of daunorubicin/ebselen‐treated rats that revealed significant improvement of the characteristic cardiomyopathic changes induced by daunorubicin treatment. Interestingly, control rats treated with ebselen showed significant elevation in serum lactate dehydrogenase activity, cardiac malondialdehyde production and total nitrate/nitrite concentration levels compared with the untreated control animals. In conclusion, ebselen treatment significantly alleviates daunorubicin‐induced cardiomyopathy.
Increased Wall Tension in Response to Vasoconstrictors in Isolated Mesenteric Arterial Rings from Patients with High Blood Pressure
Authors: Tahvanainen Anna , Taurio Jyrki , Mäki‐Jouppi Jenni , Kööbi Peeter , Mustonen Jukka , Kähönen Mika , Sand Juhani , Nordback Isto , Pörsti Ilkka
Published: November 2006

Abstract: Essential hypertension is associated with several alterations in arterial function. A wealth of information from animal models is available concerning hypertensive changes in the mesenteric circulation, while only few studies have examined human mesenteric arterial function. The tone of isolated mesenteric arterial segments (outer diameter 0.7–0.9 mm) was examined from individuals with high (n=17) or normal (n=22) blood pressure, grouped using the current definition of elevated blood pressure (140/90 mmHg). Since the majority of them were operated because of malignancies, we evaluated whether functional vascular properties provided information about patient prognosis. Wall tension development (mN/mm) in response to vasoconstrictors (noradrenaline, 5–hydroxy tryptamine, potassium chloride) was higher in mesenteric arterial rings from patients with high than normal blood pressure. There was no difference in vasoconstrictor sensitivity, or endothelium‐dependent and endothelium‐independent vasorelaxation. Arterial segment weight was higher in hypertensive subjects, suggesting vascular wall hypertrophy. The 10–year follow‐up showed no differences in the control of arterial tone between the surviving (n=14) or deceased (n=25) patients. In conclusion, isolated mesenteric arterial segments from hypertensive patients showed increased wall tension in response to vasoconstrictors. Since the mesenteric circulation is an important regulator of peripheral arterial resistance, possible functional alterations in this vascular bed should be further investigated in hypertensive patients.
Formation of Porous SiC Ceramics by Pyrolysis of Wood Impregnated with Silica
Authors: Vyshnyakova Kateryna , Yushin Gleb , Pereselentseva Ludmila , Gogotsi Yury
Published: November 2006

Biomorphous β‐SiC ceramics were produced at 1400°C from pine wood impregnated with silica. This one‐step carbothermal reduction process decreases the cost of manufacturing of SiC ceramics compared with siliconization of carbonized wood in silicon vapor. The synthesized sample exhibits a 14 m2/g surface area and has a hybrid pore structure with large 5–20 μm tubular macropores and small (<50 nm) slit‐shaped mesopores. SiC whiskers of 20–400 nm in diameter and 5–20 μm in length formed within the tubular pores. These whiskers are expected to improve the filtration by removing dust particles that could otherwise penetrate through large pores. After ultrasonic milling, the powdered sample showed an average particle size of ∼30 nm. The SiC nanopowder produced in this process may be used for manufacturing SiC ceramics for structural, tribological, and other applications.
Fabrication of a Multilayered Low‐Temperature Cofired Ceramic Micro‐Plasma‐Generating Device
Authors: Baker Amanda , Randall Clive , Stewart Randall , Fantazier Richard , Wise Fred
Published: November 2006

Plasma technology is currently being used in innumerable industrial applications. Some of the common uses of this technology include surface cleaning and treatment, sputtering and etching of semiconductor devices, excitation source for chemical analyses, cutting, environmental cleanup, sterilization, and phototherapy. The harsh conditions that these devices must endure require robust refractory materials systems for their fabrication and reliability. Low‐temperature cofired ceramic (LTCC) material systems provide a durable and cost‐effective platform for the manufacture of such devices, and allow for possible integration into meso‐scale microsystems. Our designs are based on RF microstriplines that capacitively couple and ionize small gas discharge sites over the top electrode. In this paper, we have built several iterations of this micro‐plasma generating device using LTCC material systems. The impact of electrode ink selection and processing, lamination methods, dielectric layer thickness, and electrode design has been investigated. Several micro‐plasma‐generating devices were then evaluated for power requirements, output stability, and long‐term reliability.
Properties of Lead Zirconate Titanate Thick‐Film Piezoelectric Actuators on Ceramic Substrates
Authors: Belavic Darko , Zarnik Marina Santo , Holc Janez , Hrovat Marko , Kosec Marija , Drnovsek Silvo , Cilensek Jena , Macek Srecko
Published: November 2006

Lead zirconate titanate (PZT) is a piezoelectric material that can sense or respond to mechanical deformations and can be used in ceramic electro‐mechanical systems (C‐MEMS). The microstructural, electrical, and piezoelectric characteristics of thick PZT films on low‐temperature cofired ceramics (LTCC) and alumina substrates were studied. The PZT composition was prepared with low‐melting‐point additives in order to decrease the sintering temperature and to be compatible with thick‐film technology. The integration of the PZT thick‐film materials on ceramic substrates could lead to degradation of the PZT's characteristics due to the interactions between an active PZT layer and a substrate, particularly with glassy LTCC material. To minimize the interactions with LTCC substrates, an intermediate PZT barrier layer was integrated. The value of the piezoelectric coefficientd33was found to be up to 120 pC/N on an alumina substrate and approximately 50 on an LTCC substrate. Based on these results, a cantilever‐type actuator was designed and fabricated on alumina substrates. Under an applied voltage of 200 V, the maximum tip deflection was about 5 μm.
Multilayer Construction with Various Ceramic Films for Electronic Devices Fabricated by Aerosol Deposition
Authors: Hatono Hironori , Ito Tomokazu , Iwata Kengo , Akedo Jun
Published: November 2006

Aerosol deposition (AD) is applicable as a fabrication technology for microstructures comprising different materials. We used this method for electronic devices that consist of ceramic films and metal electrodes. Various ceramic thick films (5–50 μm thickness), for example, Al2O3, 2MgO·SiO2, and BaTiO3, were deposited on substrates using room‐temperature aerosol deposition. The dielectric constant of BaTiO3 was 78 at 1 MHz. Multilayer constructions with ceramic films and copper electrodes were obtained using aerosol deposition and sputtering. During deposition, photoresist film masks were applied to produce patterns of ceramic films and connections between upper and lower electrodes through the ceramic films.
Soft Ferrite Materials for Multilayer Inductors
Authors: Töpfer Jörg , Mürbe Julia , Angermann Andre , Kracunovska Silvia , Barth Stefan , Bechtold Franz
Published: November 2006

Various families of soft ferrites (Ni–Cu–Zn; Mg–Cu–Zn; Co2–Z; Mn–Zn) with a sintering temperature of T≤950°C are used for multilayer inductors. It is shown for Ni–Cu–Zn and Mg–Cu–Zn ferrites that composition, powder particle size, and sintering additive concentration strongly affect the shrinkage, sintering behavior, microstructure, and magnetic properties. A maximum permeability of μi=700–900 is observed with a Bi2O3 addition of about 0.5 wt%. The maximum shrinkage can be shifted down to 700°C using nanosize powders. Z‐type hexaferrites with a permeability of 10 are used for high‐frequency applications up to 2 GHz.
Viscosity of LTCC Determined by Discontinuous Sinter‐Forging
Authors: Ollagnier Jean‐Baptiste , Guillon Olivier , Rödel Jürgen
Published: November 2006

The sintering behavior of the commercial material Ceramtape GC, a glass‐ceramic composite, is characterized with the help of the continuum mechanical description. The sinter‐forging technique permits to determine the uniaxial viscosity of the porous ceramic body as a function of density. At an isothermal temperature of 840°C, the uniaxial viscosity varies from 0.2 to 6 GPa.s with increasing relative density from 76% to 97%. Continuous and discontinuous sinter‐forging allows the characterization of the effect induced by the load.
Improved Cryopreservation of Sperm of Paddlefish (Polyodon spathula)
Authors: Horváth ÁKos , Urbányi Béla , Mims Steven D. , Bean William B. , Gomelsky Boris , Tiersch Terrence R.
Published: November 2006

Experiments were performed to improve protocols for sperm cryopreservation of paddlefish (Polyodon spathula), a species for which there has been limited study. The first experiment was conducted to investigate the effects of two extenders (modified Tsvetkova’s extender: mT and modified Hanks’ balanced salt solution: mHBSS) in combination with methanol (MeOH) and dimethyl sulfoxide in two concentrations (5 and 10%) on the postthaw motility and fertilization rates of cryopreserved sperm. The highest postthaw motility (85 ± 5%) was observed when sperm were frozen using mT extender with 10% MeOH as cryoprotectant. Extenders (P = 0.0018) and cryoprotectants (P = 0.0040) each had a significant effect on the postthaw motility of paddlefish sperm. The highest fertilization (80 ± 3%) was found when eggs were fertilized with sperm frozen with mT extender in combination with 10% MeOH. However, there was no significant difference among fertilization rates when MeOH was used as a cryoprotectant in either concentration or in combination with either mT or mHBSS extenders. In the second experiment, 4000 eggs were fertilized with the pooled contents of five straws of thawed sperm (total volume of 1.25 mL) using mT extender in combination with 5% MeOH, and hatch rates as high as 79 ± 5% were observed. A third experiment was also conducted to clarify the role of MeOH concentration; however, no significant difference was found among fertilization and hatch rates when either 5 or 10% MeOH was used as a cryoprotectant. These results suggest that MeOH is a safe and reliable cryoprotectant for freezing of paddlefish sperm and obtaining viable postthaw sperm for consistent fertilization and hatch rates. Further, this experimental protocol is relatively simple and applicable for commercial hatchery production of paddlefish.
Effect of Photoperiod on Feeding, Intraperitoneal Fat, and Insulin‐like Growth Factor‐I in Sunshine Bass*
Authors: Davis Kenneth B. , McEntire Matthew
Published: November 2006

The length of the photoperiod has been implicated as affecting growth of sunshine bass. This study was done to determine if photoperiod length might affect insulin‐like growth factor‐I (IGF‐I), which is a major hormonal regulator of growth in fish. Growth, feed conversion, peritoneal fat content, and plasma concentrations of IGF‐I of sunshine bass were compared on fish held on a short (8 h) or a long (16 h) photoperiod, or after the fish were switched from one photoperiod to the other. Fish were fed daily to apparent satiation for up to 5 wk and were not fed for the last 2 wk of the experiment. Body weight and intraperitoneal fat content increased for the first 2 wk of the experiment and then remained steady for the remainder of the experiment, and feed consumption decreased from 3.5% during the first 2 wk to about 1.5% for the second 2 wk and finally to only 1% during the last week of feeding. Plasma IGF‐I concentrations fell steadily during the entire experiment and was at the lowest level during the last 2 wk when the fish were not fed. There was no consistent effect of the photoperiod in any of the conditions; however, feeding and IGF‐I concentrations were affected by fish density.
Comparison of Pond Production Efficiency, Fatty Acid Profiles, and Contaminants in Litopenaeus vannamei Fed Organic Plant‐based and Fish‐meal‐based Diets
Authors: Browdy Craig , Seaborn Gloria , Atwood Heidi , Davis D. Allen , Bullis Robert A. , Samocha Tzachi M. , Wirth Ed , Leffler John W.
Published: November 2006

Reduction or elimination of fish meal and fish oil from aquaculture diets can help to reduce the potential for contamination and dependence of the industry on pelagic fisheries while improving economic competitiveness. However, fish oil provides important omega‐3 (n‐3) fatty acids (FAs) essential to shrimp health and beneficial to humans. This study evaluated an organic, plant‐based diet formulated to replace fish meal and fish oil with plant proteins and docosahexaenoic acid (DHA) produced by algal fermentation. Shrimp cultured in replicate outdoor ponds at 25/m2 were fed either a diet composed of organically produced plant ingredients or a conventional commercial fish‐meal‐based feed. No significant differences were found in production parameters between the conventional fish‐meal‐based diet and the plant‐based diet (production: 4594 and 4592 
kg/ha; harvest size: 18.7 and 19.2 
g; survival: 93 and 88%; and feed conversion ratio: 1.4 and 1.3, respectively). At harvest, shrimp were analyzed for 147 chemical contaminants and 71 FAs. Contaminant levels were negligible for shrimp raised on both diets. The fish meal and fish oil diet provided significantly higher quantities of eicosapentaenoic acid and DHA than the plant‐based diet, and the shrimp fed the conventional diet reflected this with higher levels of these beneficial FAs in edible tissues. Differences between feeds and shrimp tissues suggest that essential n‐3 FAs may accumulate in shrimp tissues over time or that natural pond productivity may play a role in providing supplemental nutrition. Shrimp raised on the two diets and wild‐caught shrimp are clearly distinguishable by their FA profiles. Compared to alternative protein sources like beef, pork, or chicken, differences in lipid profiles of shrimp raised on either diet may be insignificant because both offer increased human health benefits.
Effect of Multiple‐batch Channel Catfish, Ictalurus punctatus, Stocking Density and Feeding Rate on Water Quality, Production Characteristics, and Costs
Authors: Southworth Brent E. , Engle Carole R. , Stone Nathan
Published: November 2006

To quantitatively define relationships among stocking densities, feeding rates, water quality, and production costs for channel catfish, Ictalurus punctatus, grown in multiple‐batch systems, twelve 0.1‐ha earthen ponds were stocked at 8,600, 17,300, 26,000, or 34,600 fingerlings/ha along with 2,268 kg/ha of carryover fish. Fish in all ponds were fed daily to apparent satiation using 32% protein floating feed. Temperature and dissolved oxygen in each pond were monitored twice daily; pH weekly; nitrite‐N, total ammonia nitrogen, and Secchi disk visibility every 2 wk; nitrate‐N, chlorophyll a, total nitrogen, total phosphorus, and chemical oxygen demand monthly; and chloride every other month. The costs of producing channel catfish at different stocking densities were estimated. There were no significant differences (P > 0.05) as a result of stocking density among treatment means of (1) gross or net yields, (2) mean weights at harvest, and (3) growth or survival of fingerlings (24–36%) and carryover fish (77–94%). Mean and maximum daily feeding rates ranged from 40 to 53 kg/ha/d and 123 to 188 kg/ha/d, respectively, and feed conversion ratios averaged 1.75. There were no differences in any feed‐related parameter as a result of density. Water quality variables showed few differences among densities at samplings and no differences when averaged across the production season. Yield of fingerlings increased as stocking density increased with significant differences between the two highest and the two lowest stocking densities. Breakeven prices were lower at the higher stocking densities as a result of the higher yield of understocked fish and similar mean individual fish weights produced at these higher stocking densities. Overall, varying stocking densities of fingerlings in multiple‐batch systems had little effect on production efficiency and water quality. Additional research on managing the population structure of carryover fish in commercial catfish ponds may be warranted.
Protozoans and Meiofauna Inhabiting a Bead Filter: A Preliminary Investigation of Their Role as Potential Bioindicators of Shrimp Production System Health
Authors: Decamp Olivier E. , Otoshi Clete A. , Moss Shaun M.
Published: November 2006

Abstract.— Protozoans are used as bioindicators of system health in wastewater treatment plants. The objective of this study was to determine if protozoans or meiofauna could be useful bioindicators of shrimp production system health. Specifically, we noted the relative abundance of protozoans and meiofauna inhabiting a propeller‐washed bead filter (PBF) used in a recirculating aquaculture system for intensive shrimp production during a 12‐wk grow‐out trial. Based on microscopic analysis of PBF backwash, results indicate that (1) the presence of nematodes and rotifers may reflect the age and/or organic loading of the filter; (2) feed quantity affects protozoan biomass, whereas feed quality impacts the nutritional group of protozoans that survive; (3) free‐living amoeba typically are surface associated in their active feeding state and were present in most PBF samples; and (4) ciliates likely play an important ecological role in PBF used in intensive shrimp culture.
Rearing Male Bluegills Indoors May Be Advantageous for Producing Food‐size Sunfish
Authors: Hayward Robert S. , Wang Han‐Ping
Published: November 2006

Abstract.— An indicated demand for sunfish (Lepomis spp.) as a food fish remains untested because of continuing inability to efficiently rear these fishes to required large sizes (≥227 g; 0.5 lbs). A recent study involving parallel, indoor rearing of two sunfishes under favorable conditions showed that bluegills Lepomis macrochirus possessed markedly higher growth capacity than B × G hybrids (F1: male bluegill × female green sunfish, Lepomis cyanellus). B × G hybrids had been thought to possess superior growth capacity and so have received more attention than bluegills as a food‐fish candidate in many areas of the USA. Reanalysis of data from the recent study with emphasis on gender differences revealed that the male bluegills possessed much higher growth capacity than male B × G hybrids, at least from May to March as Age 1 and then early Age 2 fish. Male bluegills reached 67% of food‐market weight within the 10‐mo period from a starting weight of 7 g; male B × G hybrids reached only 24% of this weight. Male bluegills’ more rapid growth versus male B × G hybrids’ apparently involved less growth energy allocation to gonad development. Female bluegills grew slower than male bluegills but also outgrew male and female B × G hybrids whose growth trajectories declined in midsummer (June) and remained largely flat through March. The previous view that B × G hybrids possess higher growth capacity than bluegills was fostered by studies in ponds where bluegill growth can be impeded by high reproduction rates and, as this study reveals, by high densities and exposure to suboptimal temperatures when their growth potential is high. Indoor rearing of male bluegills should ameliorate most pond‐related growth impediments and take fuller advantage of their rapid growth capacity.
Effects of Temperature on Production and Specificity of Antibodies in Rainbow Trout (Oncorhynchus mykiss)
Authors: Mikkelsen Heidi , Lindenstrøm Thomas , Nielsen Michael Engelbrecht
Published: November 2006

Abstract.— The effect of temperature on production and affinity of antibodies against antigens from the parasitic ciliate Ichthyophthirius multifiliis were studied in rainbow trout (Oncorhynchus mykiss). Fish were immunized with I. multifiliis antigens and reared at three different temperatures, 5, 12, and 20 C for 56 d. The production of specific antibodies was examined by enzyme‐linked immunosorbent assay (ELISA). The results showed that temperature has a pronounced effect on the assay result of the ELISA technique. Plasma samples analyzed at 5 C showed that the specific antibody response of fish reared at 5 C was similar to fish reared at 12 and 20 C. However, when samples were assayed at 12 and 20 C, the measured antibody response tended to be higher for the samples from trout reared at 12 and 20 C. Additionally, it was found that rainbow trout reared at 5 C showed a delayed but not hampered antibody response compared to fish reared at 12 and 20 C, indicating a correlation between low temperature and delayed antibody production.
Views of Jihad Throughout History
Authors: Afsaruddin Asma
Published: November 2006

The Arabic term jihad has primarily come to mean “armed struggle/combat” and is frequently translated into English as “holy war.” But a close scrutiny of the occurrence of this term in the Qur’an and in early hadith literature demonstrates that this exclusive understanding of the term cannot be supported for the earliest period of Islam (roughly mid‐seventh through the late eighth centuries). The essay traces the transformations in the meanings of jihad– and the related concepts of martyr and martyrdom – from the earliest period of Islam through the late medieval period and down to our present time.
Opening New Doors: The Jewish Women's Experience in the Early American West, 1848–1930
Authors: Abrams Jeanne
Published: November 2006

Opening New Doors examines the lives of Jewish women in the early American West to provide a deeper understanding of their diverse roles. Given the still‐forming social landscape, beginning with the 1848 Gold Rush, Jews were able to integrate more fully into local communities than they had in the East. Jewish women in the West took advantage of the unsettled nature of the region to “open new doors” for themselves in ways often not yet possible elsewhere in the country. As the stories of the Jewish women in this study demonstrate, they played an integral role in settling America's western frontier as well as promoting Jewish continuity as they built new lives in a region that offered them a host of expanded possibilities.
Analysis of the Cytosolic Domains of the Hepatitis B Virus Envelope Proteins for Their Function in Viral Particle Assembly and Infectivity
Authors: Blanchet Matthieu , Sureau Camille
Published: November 2006

The hepatitis B virus (HBV) envelope proteins have the ability to assemble three types of viral particles, (i) the empty subviral particles (SVPs), (ii) the mature HBV virions, and (iii) the hepatitis delta virus (HDV) particles, in cells that are coinfected with HBV and HDV. To gain insight into the function of the HBV envelope proteins in morphogenesis of HBV or HDV virions, we have investigated subdomains of the envelope proteins that have been shown or predicted to lie at the cytosolic face of the endoplasmic reticulum membrane during synthesis, a position prone to interaction with the inner core structure. These domains, referred to here as cytosolic loops I and II (CYL-I and -II, respectively), were subjected to mutagenesis. The mutations were introduced in the three HBV envelope proteins, designated small, middle, and large (S-HBsAg, M-HBsAg, and L-HBsAg, respectively). The mutants were expressed in HuH-7 cells to evaluate their capacity for self-assembly and formation of HBV or HDV virions when HBV nucleocapsid or HDV ribonucleoprotein, respectively, was provided. We found that SVP-competent CYL-I mutations between positions 23 and 78 of the S domain were permissive to HBV or HDV virion assembly. One mutation (P29A) was permissive for synthesis of the S- and M-HBsAg but adversely affected the synthesis or stability of L-HBsAg, thereby preventing the assembly of HBV virions. Furthermore, using an in vitro infection assay based on the HepaRG cells and the HDV model, we have shown that particles coated with envelope proteins bearing CYL-I mutations were fully infectious, hence indicating the absence of an infectivity determinant in this region. Finally, we demonstrated that the tryptophan residues at positions 196, 199, and 201 in CYL-II, which were shown to exert a matrix function for assembly of HDV particles (I. Komla-Soukha and C. Sureau, J. Virol.
80:
4648-4655, 2006), were dispensable for both assembly and infectivity of HBV virions.
Theiler's Murine Encephalomyelitis Virus Leader Protein Amino Acid Residue 57 Regulates Subgroup-Specific Virus Growth on BHK-21 Cells
Authors: Takano-Maruyama Masumi , Ohara Yoshiro , Asakura Kunihiko , Okuwa Takako
Published: November 2006

Strains of Theiler's murine encephalomyelitis virus (TMEV) are divided into two subgroups, TO and GDVII. TMEV strains show subgroup-specific virus growth and cell tropism and induce subgroup-specific diseases. Using site-directed mutagenesis, we demonstrated that the amino acid at position 57 of the leader protein (L
57
), which is located at the most N-terminal part of the polyprotein, regulates subgroup-specific virus growth on BHK-21 cells. Further study suggested that L
57
may regulate viral RNA encapsidation, although it does not affect the synthesis of viral proteins or the assembly of viral intermediates.
Indolopyridones Inhibit Human Immunodeficiency Virus Reverse Transcriptase with a Novel Mechanism of Action
Authors: Jochmans Dirk , Deval Jérôme , Kesteleyn Bart , Van Marck Herwig , Bettens Eva , De Baere Inky , Dehertogh Pascale , Ivens Tania , Van Ginderen Marcia , Van Schoubroeck Bertrand , Ehteshami Maryam , Wigerinck Piet , Götte Matthias , Hertogs Kurt
Published: November 2006

We have discovered a novel class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone compounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed “hot spots” for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-specific footprinting experiments revealed that INDOPY-1 traps the complex in the posttranslocational state, preventing binding and incorporation of the next complementary nucleotide. The novel mode of action translates into a unique resistance profile. While INDOPY-1 susceptibility is unaffected by mutations associated with NNRTI or multidrug NRTI resistance, mutations M184V and Y115F are associated with decreased susceptibility, and mutation K65R confers hypersusceptibility to INDOPY-1. This resistance profile provides additional evidence for active site binding. In conclusion, this class of indolopyridones can occupy the nucleotide binding site of HIV RT by forming a stable ternary complex whose stability is mainly dependent on the nature of the primer 3′ end.
Effect of Adenovirus-Mediated RNA Interference on Endogenous MicroRNAs in a Mouse Model of Multidrug Resistance Protein 2 Gene Silencing
Authors: Narvaiza Iñigo , Aparicio Oscar , Vera María , Razquin Nerea , Bortolanza Sergia , Prieto Jesús , Fortes Puri
Published: November 2006

RNA interference with viral vectors that express short hairpin RNAs (shRNAs) has emerged as a powerful tool for functional genomics and therapeutic purposes. However, little is known about shRNA in vivo processing, accumulation, functional kinetics, and side effects related to shRNA saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shRNAs against murine ATP-binding cassette multidrug resistance protein 2 (Abcc2), which is involved in liver transport of bilirubin to bile, and analyzed Abcc2 silencing kinetics. C57/BL6 mice injected with these viruses showed significant impairment of Abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of Abcc2 function correlated with a specific reduction of Abcc2 mRNA and with high levels of processed shRNAs targeting Abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shRNAs. This finding suggests that a minimal amount of processed shRNAs is required for efficient silencing in vivo. This system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery.
Efficient Reactivation of Latent Herpes Simplex Virus from Mouse Central Nervous System Tissues
Authors: Chen Shih-Heng , Yao Hui-Wen , Huang Wen-Yen , Hsu Kuei-Sen , Lei Huan-Yao , Shiau Ai-Li , Chen Shun-Hua
Published: November 2006

For decades, numerous ex vivo studies have documented that latent herpes simplex virus (HSV) reactivates efficiently from ganglia, but rarely from the central nervous systems (CNS), of mice when assayed by mincing tissues before explant culture, despite the presence of viral genomes in both sites. Here we show that 88% of mouse brain stems reactivated latent virus when they were dissociated into cell suspensions before ex vivo explant culture. The efficient reactivation of HSV from the mouse CNS was demonstrated with more than one viral strain, viral serotype, and mouse strain, further indicating that the CNS can be an authentic latency site for HSV with the potential to cause recurrent disease.
Stoichiometry of Murine Leukemia Virus Envelope Protein-Mediated Fusion and Its Neutralization
Authors: Ou Wu , Silver Jonathan
Published: November 2006

Envelope glycoproteins (Envs) of retroviruses form trimers that mediate fusion between viral and cellular membranes and are the targets for neutralizing antibodies. Understanding in detail how Env trimers mediate membrane fusion, and how antibodies interfere with this process, is a fundamental problem in biology with practical implications for the development of antiviral drugs and vaccines. We investigated the stoichiometry of Env-mediated fusion and its inhibition by antibody by inserting an epitope from human immunodeficiency virus for a neutralizing antibody (2F5) into the surface (SU) or transmembrane (TM) protein of murine leukemia virus Env, along with point mutations that abrogate SU and TM function but complement one another. We transfected various combinations of these Env genes and investigated Env-mediated cell fusion and its inhibition by 2F5 antibody. Our results showed that heterotrimers with one functional SU molecule were fusion competent in complementation experiments and that one antibody molecule was sufficient to inactivate the fusion function of a trimer when its epitope was in functional SU or TM. 2F5 antibody could also neutralize trimers with the 2F5 epitope in nonfunctional SU or TM, but less efficiently.
RNA- and Virus-Independent Inhibition of Antiviral Signaling by RNA Helicase LGP2
Authors: Komuro Akihiko , Horvath Curt M.
Published: November 2006

Antiviral innate immune responses can be triggered by accumulation of intracellular nucleic acids resulting from virus infections. Double-stranded RNA (dsRNA) can be detected by the cytoplasmic RNA helicase proteins RIG-I and MDA5, two proteins that share sequence similarities within a caspase recruitment domain (CARD) and a DExD/H box RNA helicase domain. These proteins are considered dsRNA sensors and are thought to transmit the signal to the mitochondrial adapter, IPS-1 (also known as MAVS, VISA, or CARDIF) via CARD interactions. IPS-1 coordinates the activity of protein kinases that activate transcription factors needed to induce beta interferon (IFN-β) gene transcription. Another helicase protein, LGP2, lacks the CARD region and does not activate IFN-β gene expression. LGP2 mRNA is induced by interferon, dsRNA treatments, or Sendai virus infection and acts as a feedback inhibitor for antiviral signaling. Results indicate that LGP2 can inhibit antiviral signaling independently of dsRNA or virus infection intermediates by engaging in a protein complex with IPS-1. Experiments suggest that LGP2 can compete with the kinase IKKi (also known as IKKε) for a common interaction site on IPS-1. These results provide the first demonstration of protein interaction as an element of negative-feedback regulation of intracellular antiviral signaling by LGP2.
Modulation of DNA Vaccine-Elicited CD8
+
T-Lymphocyte Epitope Immunodominance Hierarchies
Authors: Liu Jinyan , Ewald Bonnie A. , Lynch Diana M. , Nanda Anjali , Sumida Shawn M. , Barouch Dan H.
Published: November 2006

Generating broad cellular immune responses against a diversity of viral epitopes is a major goal of current vaccine strategies for human immunodeficiency virus type 1 (HIV-1) and other pathogens. Virus-specific CD8
+
T-lymphocyte responses, however, are often highly focused on a very limited number of immunodominant epitopes. For an HIV-1 vaccine, the breadth of CD8
+
T-lymphocyte responses may prove to be critical as a result of the need to cover a wide diversity of viral isolates in the population and to limit viral escape from dominant epitope-specific T lymphocytes. Here we show that epitope modification strategies can alter CD8
+
T-lymphocyte epitope immunodominance hierarchies elicited by a DNA vaccine in mice. Mice immunized with a DNA vaccine expressing simian immunodeficiency virus Gag lacking the dominant D
b
-restricted AL11 epitope generated a marked and durable augmentation of responses specific for the subdominant D
b
-restricted KV9 epitope. Moreover, anatomic separation strategies and heterologous prime-boost regimens generated codominant responses against both epitopes. These data demonstrate that dominant epitopes can dramatically suppress the immunogenicity of subdominant epitopes in the context of gene-based vaccines and that epitope modification strategies can be utilized to enhance responses to subdominant epitopes.
Nonneutralizing Human Rhinovirus Serotype 2-Specific Monoclonal Antibody 2G2 Attaches to the Region That Undergoes the Most Dramatic Changes upon Release of the Viral RNA
Authors: Hewat Elizabeth A. , Blaas Dieter
Published: November 2006

The monoclonal antibody 2G2 has been used extensively for detection and quantification of structural changes of human rhinovirus serotype 2 during infection. It recognizes exclusively A and B subviral particles, not native virus. We have elucidated the basis of this selectivity by determining the footprint of 2G2. Since viral escape mutants obviously cannot be obtained, the structures of complexes between Fab fragments of 2G2 and 80S subviral B particles were determined by cryoelectron microscopy. The footprint of the antibody corresponds to the capsid region that we predicted would undergo the most dramatic changes upon RNA release.
Spleen-Dependent Turnover of CD11b Peripheral Blood B Lymphocytes in Bovine Leukemia Virus-Infected Sheep
Authors: Florins Arnaud , Gillet Nicolas , Asquith Becca , Debacq Christophe , Jean Geneviève , Schwartz-Cornil Isabelle , Bonneau Michel , Burny Arsène , Reichert Michal , Kettmann Richard , Willems Luc
Published: November 2006

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.
Construction and Mutagenesis of an Artificial Bicistronic Tick-Borne Encephalitis Virus Genome Reveals an Essential Function of the Second Transmembrane Region of Protein E in Flavivirus Assembly
Authors: Orlinger Klaus K. , Hoenninger Verena M. , Kofler Regina M. , Mandl Christian W.
Published: November 2006

Flaviviruses have a monopartite positive-stranded RNA genome, which serves as the sole mRNA for protein translation. Cap-dependent translation produces a polyprotein precursor that is co- and posttranslationally processed by proteases to yield the final protein products. In this study, using tick-borne encephalitis virus (TBEV), we constructed an artificial bicistronic flavivirus genome (TBEV-bc) in which the capsid protein and the nonstructural proteins were still encoded in the cap cistron but the coding region for the surface proteins prM and E was moved to a separate translation unit under the control of an internal ribosome entry site element inserted into the 3′ noncoding region. Mutant TBEV-bc was shown to produce particles that packaged the bicistronic RNA genome and were infectious for BHK-21 cells and mice. Compared to wild-type controls, however, TBEV-bc was less efficient in both RNA replication and infectious particle formation. We took advantage of the separate expression of the E protein in this system to investigate the role in viral assembly of the second transmembrane region of protein E (E-TM2), a second copy of which was retained in the cap cistron to fulfill its other role as an internal signal sequence in the polyprotein. Deletion analysis and replacement of the entire TBEV E-TM2 region with its counterpart from another flavivirus revealed that this element, apart from its role as a signal sequence, is important for virion formation.
Role of Ran Binding Protein 5 in Nuclear Import and Assembly of the Influenza Virus RNA Polymerase Complex
Authors: Deng Tao , Engelhardt Othmar G. , Thomas Benjamin , Akoulitchev Alexandre V. , Brownlee George G. , Fodor Ervin
Published: November 2006

The influenza A virus RNA-dependent RNA polymerase is a heterotrimeric complex of polymerase basic protein 1 (PB1), PB2, and polymerase acidic protein (PA) subunits. It performs transcription and replication of the viral RNA genome in the nucleus of infected cells. We have identified a nuclear import factor, Ran binding protein 5 (RanBP5), also known as karyopherin β3, importin β3, or importin 5, as an interactor of the PB1 subunit. RanBP5 interacted with either PB1 alone or with a PB1-PA dimer but not with a PB1-PB2 dimer or the trimeric complex. The interaction between RanBP5 and PB1-PA was disrupted by RanGTP in vitro, allowing PB2 to bind to the PB1-PA dimer to form a functional trimeric RNA polymerase complex. We propose a model in which RanBP5 acts as an import factor for the newly synthesized polymerase by targeting the PB1-PA dimer to the nucleus. In agreement with this model, small interfering RNA (siRNA)-mediated knock-down of RanBP5 inhibited the nuclear accumulation of the PB1-PA dimer. Moreover, siRNA knock-down of RanBP5 resulted in the delayed accumulation of viral RNAs in infected cells, confirming that RanBP5 plays a biological role during the influenza virus life cycle.
Feline Model of Acute Nipah Virus Infection and Protection with a Soluble Glycoprotein-Based Subunit Vaccine
Authors: Mungall Bruce A. , Middleton Deborah , Crameri Gary , Bingham John , Halpin Kim , Russell Gail , Green Diane , McEachern Jennifer , Pritchard L. Ian , Eaton Bryan T. , Wang Lin-Fa , Bossart Katharine N. , Broder Christopher C.
Published: November 2006

Nipah virus (NiV) and Hendra virus (HeV) are paramyxoviruses capable of causing considerable morbidity and mortality in a number of mammalian species, including humans. Case reports from outbreaks and previous challenge experiments have suggested that cats were highly susceptible to NiV infection, responding with a severe respiratory disease and systemic infection. Here we have assessed the cat as a model of experimental NiV infection and use it in the evaluation of a subunit vaccine comprised of soluble G glycoprotein (sG). Two groups of two adult cats each were inoculated subcutaneously with either 500 or 5,000 50% tissue culture infective dose(s) (TCID
50
) of NiV. Animals were monitored closely for disease onset, and extensive analysis was conducted on samples and tissues taken during infection and at necropsy to determine viral load and tissue tropism. All animals developed clinical disease 6 to 9 days postinfection, a finding consistent with previous observations. In a subsequent experiment, two cats were immunized with HeV sG and two were immunized with NiV sG. Homologous serum neutralizing titers were greater than 1:20,000, and heterologous titers were greater than 1:20,000 to 16-fold lower. Immunized animals and two additional naive controls were then challenged subcutaneously with 500 TCID
50
of NiV. Naive animals developed clinical disease 6 to 13 days postinfection, whereas none of the immunized animals showed any sign of disease. TaqMan PCR analysis of samples from naive animals revealed considerable levels of NiV genome in a wide range of tissues, whereas the genome was evident in only two immunized cats in only four samples and well below the limit of accurate detection. These results indicate that the cat provides a consistent model for acute NiV infection and associated pathogenesis and an effective subunit vaccine strategy appears achievable.
Activation of Microglia by Borna Disease Virus Infection: In Vitro Study
Authors: Ovanesov Mikhail V. , Sauder Christian , Rubin Steven A. , Richt Jürgen , Nath Avindra , Carbone Kathryn M. , Pletnikov Mikhail V.
Published: November 2006

Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to the certain neuronal populations. Since persistent BDV infection of neurons in vitro is noncytolytic and noncytopathic, activated microglia have been suggested to be responsible for neuronal cell death in vivo. However, the mechanisms of activation of microglia in neonatally BDV-infected rat brain have not been investigated. To address these issues, activation of primary rat microglial cells was studied following exposure to purified BDV or to persistently BDV-infected primary cortical neurons or after BDV infection of primary mixed neuron-glial cultures. Neither purified virus nor BDV-infected neurons alone activated primary microglia as assessed by the changes in cell shape or production of the proinflammatory cytokines. In contrast, in the BDV-infected primary mixed cultures, we observed proliferation of microglia cells that acquired the round morphology and expressed major histocompatibility complex molecules of classes I and II. These manifestations of microglia activation were observed in the absence of direct BDV infection of microglia or overt neuronal toxicity. In addition, compared to uninfected mixed cultures, activation of microglia in BDV-infected mixed cultures was associated with a significantly greater lipopolysaccharide-induced release of tumor necrosis factor alpha, interleukin 1β, and interleukin 10. Taken together, the present data are the first in vitro evidence that persistent BDV infection of neurons and astrocytes rather than direct exposure to the virus or dying neurons is critical for activating microglia.