bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of CaMKII at Thr253 occurs in vivo and enhances binding to isolated postsynaptic densities
Authors: Migues Paola V. , Lehmann Ingo T. , Fluechter Lisa , Cammarota Martin , Gurd James W. , Sim Alistair T. R. , Dickson Phillip W. , Rostas John A. P.
Published: May 2006

Autophosphorylation of Ca2+‐calmodulin stimulated protein kinase II (CaMKII) at two sites (Thr286 and Thr305/306) is known to regulate the subcellular location and activity of this enzyme in vivo. CaMKII is also known to be autophosphorylated at Thr253 in vitro but the functional effect of phosphorylation at this site and whether it occurs in vivo, is not known. Using antibodies that specifically recognize CaMKII phosphorylated at Thr253 together with FLAG‐tagged wild type and phospho‐ and dephospho‐mimic mutants of α‐CaMKII, we have shown that Thr253 phosphorylation has no effect on either the Ca2+‐calmodulin dependent or autonomous kinase activity of recombinant α‐CaMKII in vitro. However, the Thr253Asp phosphomimic mutation increased α‐CaMKII binding to subcellular fractions enriched in post‐synaptic densities (PSDs). The increase in binding was similar in extent, and additive, to that produced by phosphorylation of Thr286. Thr253 phosphorylation was dynamically regulated in intact hippocampal slices. KCl induced depolarisation increased Thr253 phosphorylation and the phospho‐Thr253‐CaMKII was specifically recovered in the subcellular fraction enriched in PSDs. These results identify Thr253 as an additional site at which CaMKII is phosphorylated in vivo and suggest that this dynamic phosphorylation may regulate CaMKII function by altering its distribution within the cell.
Conserved residues in juxtamembrane region of the extracellular domain of nicastrin are essential for γ‐secretase complex formation
Authors: Walker Emily S. , Martinez Maribel , Wang Jun , Goate Alison
Published: May 2006

The Alzheimer's disease‐linked protein, presenilin, forms the active site of the γ‐secretase enzyme complex. However, three other proteins, nicastrin (NCT), PEN‐2 and APH‐1, are required for enzyme activity. This complex is responsible for cleaving the β‐amyloid precursor protein to produce amyloid β and the intracellular domain (AICD). Although much research has focused on the regions of presenilin that are important for γ‐secretase function, less is known about NCT. To further our understanding of the role of NCT in γ‐secretase activity and complex formation, we have undertaken a systematic evaluation of conserved residues in the juxtamembrane region of the extracellular domain of NCT. Two mutants, S632A and W648A, greatly reduce γ‐secretase activity, as seen by a reduction in amyloid β and AICD levels. Several lines of evidence suggest that these mutations result in reduced γ‐secretase activity because they affect the ability of NCT to stably associate with the other γ‐secretase components. Since NCT and APH‐1 must first bind in order for presenilin and PEN‐2 to stably join the complex, we propose that S632 and W648 are essential for a stable interaction with APH‐1.
The neurotrophic effects of PACAP in PC12 cells: control by multiple transduction pathways
Authors: Ravni Aurélia , Bourgault Steve , Lebon Alexis , Chan Philippe , Galas Ludovic , Fournier Alain , Vaudry Hubert , Gonzalez Bruno , Eiden Lee E. , Vaudry David
Published: May 2006

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are closely related members of the secretin superfamily of neuropeptides expressed in both the brain and peripheral nervous system, and they exhibit neurotrophic and neurodevelopmental effectsin vivo. Like the index member of the Trk receptor ligand family, nerve growth factor (NGF), PACAP promotes the differentiation of PC12 cells, a well‐established cell culture model, to investigate neuronal differentiation, survival and function. Stimulation of catecholamine secretion and enhanced neuropeptide biosynthesis are effects exerted by PACAP at the adrenomedullary synapsein vivoand on PC12 cellsin vitrothrough stimulation of the specific PAC1 receptor. Induction of neuritogenesis, growth arrest, and promotion of cell survival are effects of PACAP that occur in developing cerebellar, hippocampal and cortical neurons, as well as in the more tractable PC12 cell model. Study of the mechanisms through which PACAP exerts its various effects on cell growth, morphology, gene expression and survival, i.e. its actions as a neurotrophin, in PC12 cells is the subject of this review. The study of neurotrophic signalling by PACAP in PC12 cells reveals that multiple independent pathways are coordinated in the PACAP response, some activated by classical and some by novel or combinatorial signalling mechanisms.
Comparison of the binding pockets of two chemically unrelated allosteric antagonists of the mGlu5 receptor and identification of crucial residues involved in the inverse agonism of MPEP
Authors: Malherbe Pari , Kratochwil Nicole , Mühlemann Andreas , Zenner Marie‐Thérèse , Fischer Christophe , Stahl Martin , Gerber Paul R. , Jaeschke Georg , Porter Richard H. P.
Published: May 2006

Fenobam [N‐(3‐chlorophenyl)‐N′‐(4,5‐dihydro‐1‐methyl‐4‐oxo‐1H‐imidazole‐2‐yl)urea], a clinically validated non‐benzodiazepine anxiolytic, has been shown to be a potent and non‐competitive metabotropic glutamate (mGlu)‐5 receptor antagonist. In the present study, we have used the site‐directed mutagenesis coupled with three‐dimensional receptor‐based pharmacophore modelling to elucidate the interacting mode of fenobam within the seven‐transmembrane domain (7TMD) of mGlu5 receptor and its comparison with that of 2‐methyl‐6‐(phenylethynyl)pyridine (MPEP), the prototype antagonist. The common residues involved in the recognition of MPEP and fenobam include Pro6543.36, Tyr6583.40, Thr7806.44, Trp7846.48, Phe7876.51, Tyr7916.55and Ala8097.47. The differentiating residues between both modulators' interacting modes are Arg6473.29, Ser6573.39and Leu7435.47. Our data suggest that these chemically unrelated mGlu5 antagonists act similarly, probing a functionally unique region of the 7TMD. Using [3H]inositol phosphates accumulation assay, we have also identified the critical residues involved in the inverse agonist effect of MPEP. The mutation W7846.48A completely blocked the inverse agonist activity of MPEP; two mutations F7876.51A and Y7916.55A, caused a drastic decrease in the MPEP inverse agonism. Furthermore, these three mutations led to an increased efficacy of quisqualate without having any effect on its potency. The fact that the residues Trp7846.48and Phe7876.51are essential equally in antagonism and inverse agonism effects emphasizes again the key role of these residues and the involvement of a common transmembrane network in receptor inactivation by MPEP.
American Indian Education
Authors: Spack Ruth
Published: May 2006

This article examines history writing on American Indian education to show its movement from a focus on federal policy to studies that incorporate Native people’s perspectives. The article discusses the benefits and challenges of using oral histories, interviews, and autobiographies for historical analysis and points to the value of a multi‐voiced, interdisciplinary approach.
THE SMALLEST DINOFLAGELLATE GENOME IS YET TO BE FOUND: A COMMENT ON LAJEUNESSE ET AL. “SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES”1
Authors: Lin Senjie
Published: May 2006

LaJeunessse and colleagues (LaJeunesse et al. 2005) have recently documented small genome sizes of Symbiodinium and concluded that Symbiodinium is a dinoflagellate lineage with the smallest genome. The conclusion is inconsistent with recent discoveries of picoplanktonic dinoflagellates. The search for the smallest genome and the effort to understand the evolutionary history of dinoflagellate genome should be an area of research in the years to come, which can be greatly aided by an understanding on the current hypotheses regarding mechanisms of genome size evolution. Even the smallest dinoflagellate genome documented to date is too large to be sequenced with current technology, but sequencing of chromosomes or expressed genes of key representative species is feasible and can be very insightful for understanding genome composition and function in this important lineage of eukaryotes.
HEMIFLAGELLOCHLORIS KAZAKHSTANICA GEN. ET SP. NOV.: A NEW COCCOID GREEN ALGA WITH FLAGELLA OF CONSIDERABLY UNEQUAL LENGTHS FROM A SALINE IRRIGATION LAND IN KAZAKHSTAN (CHLOROPHYCEAE, CHLOROPHYTA)1
Authors: Watanabe Shin , Tsujimura Shigeo , Misono Takahiro , Nakamura Shogo , Inoue Hiroshi
Published: May 2006

A coccoid green alga, Hemiflagellochloris kazakhstanica S. Watanabe, S. Tsujimura, T. Misono, S. Nakamura et H. Inoue, gen. et sp. nov., was described from soil samples of a saline irrigation land in Ili River basin, Kazakhstan. This alga had a parietal chloroplast with a pyrenoid, which was covered with starch segments and penetrated with thylakoid membranes. Reproduction occurred by the formation of aplanospores and zoospores. The aplanospores frequently formed tetrad aggregations in a mother cell. The zoospores were covered by a single‐layered cell wall and lacked stigmata. The zoospores had two flagella of considerably unequal lengths; the longer flagellum was 17–19 lm in length and the shorter one was 9–10 lm. The flagellar apparatus architecture was of the clockwise orientation group type in the Chlorophyceae. Molecular phylogenetic analysis using 18S and 28S rDNA sequence data resolved this organism in a separate clade from the green algae that had flagella of slightly unequal lengths. It was suggested that features such as inequality in flagellar lengths, parallel exsertion of basal bodies, and subapical position of the flagellar apparatus were sporadically evolved.
GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE LIBRARY CONSTRUCTION1
Authors: Varela‐Álvarez Elena , Andreakis Nikos , Lago‐Lestón Asunción , Pearson Gareth A. , Serrão Ester A. , Procaccini Gabriele , Duarte Carlos M. , Marbá Nuria
Published: May 2006

A method for isolating high‐quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 6–10 μg genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A260/A280 ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum. The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.
Two Methods for Predicting Limited Life Expectancy in Nursing Homes
Authors: Wallace J. Brandon , Prevost Suzanne S.
Published: May 2006

Purpose: To compare two methods of identifying nursing home residents with a life expectancy of 6 months or less.

Design: Secondary analysis of Minimum Data Set (MDS) assessment data from 111 U.S. nursing homes was used in two approaches to compare the prognostic value of each approach. MDS assessment data were collected during 2003 and 2004, and secondary analyses were completed during 2004 and 2005.

Methods: A bivariate analysis was used to identify variables associated with death within 6 months and a summative index was produced. Second, logistic regression was performed to develop a formula for calculating a Probability of Death score.

Findings: Both methods were reasonably sensitive in identifying dying residents. In the total sample of 21,852 residents, 17.5% died within 6 months. When the bivariate analysis and summative index were used, 51.1% of residents who had a score of 4 or higher died within 6 months, and residents who scored 10 or higher had an 80% mortality rate. With use of the logistic regression and Probability of Death scores, 52% of residents who had scores of 0.4 or higher died within 6 months, and residents who scored 0.9 or higher had an 80% mortality rate.

Conclusions: MDS assessment data, collected in all U.S. nursing homes, can be useful to increase the specificity of identifying terminal residents. These statistical methods have high and similar prognostic values, but the summative index is easier to understand. It also is more user‐friendly and could be incorporated into the nursing home assessment process with minimal additional training, and with no additional hardware or changes in software programming.
Sexual Assault Nurse Examiner (SANE) Program Goals and Patient Care Practices
Authors: Patterson Debra , Campbell Rebecca , Townsend Stephanie M.
Published: May 2006

Purpose: To examine Sexual Assault Nurse Examiners (SANE) programs' goals and guiding philosophies and how they influence patient care practices for sexual assault victims.

Design: Data were collected from a national random sample of SANE to examine whether programs differed significantly in their goals and patient care practices.

Methods: Iterative cluster analysis was used to identify types of programs distinct in their stated goals.

Findings: Three types of emphasis in SANE programs were identified: (a) prosecution of cases as a primary goal; (b) attending to patients' emotional needs, supporting feminist values, empowering patients, and changing the community response to rape; and (c) least importance on prosecution of cases and average importance on the other goals. Programs that were more focused on prosecution goals provided less comprehensive patient services.

Conclusions: Programs focused on prosecution as a primary goal were less likely to provide comprehensive services, especially those involving patient education. Such information is important for patients because their health concerns (e.g., pregnancy, STIs) have long‐term implications for their well‐being. Historical and structural differences among SANE programs might explain these different patient care practices. Researchers should examine the underlying processes in SANE programs that shape both their goals and patient care approaches.
Replication and Examination of Research Data on Job Stress and Coworker Social Support With Internet and Traditional Samples
Authors: AbuAlRub Raeda Fawzi
Published: May 2006

Purpose: To replicate previous results about job stress and coworkers' support that were obtained from a Web‐based American sample with a sample of Jordanian nurses using traditional methods.

Design and Methods: The convenience sample of 300 Jordanian staff nurses completed the pencil‐and‐paper version of a questionnaire that included the Nursing Stress Scale, the McCain and Marklin Social Integration Scale, and the demographic form. The sample of the replicated study consisted of 263 American staff nurses who were accessible over the Internet and completed the Web‐based version of the questionnaire. Descriptive statistics, chi‐square, t test, and Pearson product‐moment correlations were used to analyze the data.

Results: The American and Jordanian samples differed significantly on all demographic variables. However, they had similar patterns of responses in regard to the association between job stress and social support from coworkers.

Conclusions: The predicted comparability of findings with different methods and samples lends credence to the reliability and validity of Internet‐based research.
Psychoanalytic Criticism of Shakespeare
Authors: Mikics David
Published: May 2006

Why has Shakespeare proved such profitable ground for psychoanalytic study? Part of the answer lies in the affinity between theatre, not just Shakespeare’s, and psychoanalytic practice, an affinity recognized by Freud when he adapted Aristotle’s idea of catharsis to his own “talking cure.” This essay traces psychoanalytic criticism of Shakespeare from its origins in the work of Freud and Ernest Jones through studies by Jung, Rank, Lacan, Norman Holland, and others. In conclusion, the author presents a practical example of psychoanalytic Shakespeare criticism: a reading ofThe Tempestdrawing on the object relations school of D. W. Winnicott.
Excitation of rat hippocampal interneurons via modulation of endogenous agonist activity at the α7 nicotinic ACh receptor
Authors: Selina Mok M. H. , Kew James N. C.
Published: May 2006

The α7 subtype of the nicotinic acetylcholine receptor (α7 nAChR) is prominently expressed in the hippocampus where it is thought to play a role in the regulation of cognitive function. In this study, we have investigated the effects of 5‐hydroxyindole (5‐HI), a positive modulator of the α7 nAChR, on GABAergic activity in hippocampal CA1 stratum radiatum interneurons in acute rat brain slices. Superfusion of 5‐HI (100 μm) increased the mean frequency and amplitude of spontaneous IPSCs (sIPSCs). The potentiation was occluded by pretreatment of slices with: (1) a high concentration of the broad‐spectrum agonist nicotine to desensitize the α7 receptor, (2) an α7 nAChR antagonist, and (3) tetrodotoxin to block action potential firing. These results indicate that facilitation by 5‐HI was mediated by the α7 nAChR and required neuronal excitation. In contrast, 5‐HI had no effect on sIPSCs recorded in hippocampal slices from younger animals, even though the expression of functional α7 nAChRs was confirmed by agonist application experiments. In these slices, 5‐HI only enhanced sIPSCs after pretreatment with the acetylcholinesterase inhibitor Bw284c51. Taken together, our results suggest that 5‐HI facilitates GABAergic transmission via excitation of the α7 nAChR, and that this effect requires the presence of the endogenous agonist ACh in the extracellular environment of the receptor.
Golfer’s dilemma
Authors: Gualtieri Marco , Tokieda Tadashi , Advis-Gaete L. , Carry B. , Reffet E. , Guthmann Claude
Published: May 2006

A ball rolling on a vertical cylinder exhibits a bizarre quasi-periodic motion. We explain this behavior in terms of Coriolis torque and study the phenomenon experimentally. The data confirm the predicted motion and the surprising invariance of the ratio between the horizontal and vertical frequencies. This result might account for the frustrating sight of a golf ball escaping from the hole an instant after it is putted in.
From baking a cake to solving the diffusion equation
Authors: Olszewski Edward A.
Published: May 2006

We explain how modifying a cake recipe by changing either the dimensions of the cake or the amount of cake batter alters the baking time. We restrict our consideration to the génoise and obtain a semiempirical relation for the baking time as a function of oven temperature, initial temperature of the cake batter, and dimensions of the unbaked cake. The relation, which is based on the diffusion equation, has three parameters whose values are estimated from data obtained by baking cakes in cylindrical pans of various diameters. The relation takes into account the evaporation of moisture at the top surface of the cake, which is the dominant factor affecting the baking time of a cake.
Falling chains
Authors: Wong Chun Wa , Yasui Kosuke
Published: May 2006

The one-dimensional fall of a folded chain with one end suspended from a rigid support and a chain falling from a resting heap on a table is studied. Because their Lagrangians contain no explicit time dependence, the falling chains are conservative systems. Their equations of motion are shown to contain a term that enforces energy conservation when masses are transferred between subchains. We show that Cayley’s 1857 energy nonconserving solution for a chain falling from a resting heap is incorrect because it neglects the energy gained when a link leaves a subchain. The maximum chain tension measured by Calkin and March for the falling folded chain is given a simple if rough interpretation. Other aspects of the falling folded chain are briefly discussed.
A simple optical demonstration of quantum cryptography using transverse position and momentum variables
Authors: Lemelle D. S. , Almeida M. P. , Souto Ribeiro P. H. , Walborn S. P.
Published: May 2006

We discuss a simple experiment illustrating the fundamentals of quantum cryptography. Our experiment is performed using novel complementary observables: the transverse position and momentum of photons. In the classical optics regime the experiment serves as an interesting and simple classroom demonstration of the principles of quantum key distribution.
Oceanographic Basis of the Global Surface Distribution of
Prochlorococcus
Ecotypes
Authors: Bouman Heather A. , Ulloa Osvaldo , Scanlan David J. , Zwirglmaier Katrin , Li William K. W. , Platt Trevor , Stuart Venetia , Barlow Ray , Leth Ole , Clementson Lesley , Lutz Vivian , Fukasawa Masao , Watanabe Shuichi , Sathyendranath Shubha
Published: May 2006

By using data collected during a continuous circumnavigation of the Southern Hemisphere, we observed clear patterns in the population-genetic structure of
Prochlorococcus
, the most abundant photosynthetic organism on Earth, between and within the three Southern Subtropical Gyres. The same mechanisms that were previously invoked to account for the vertical distribution of ecotypes at local scales accounted for the global (horizontal) patterns we observed. Basin-scale and seasonal variations in the structure and strength of vertical stratification provide a basis for understanding large-scale horizontal distribution in genetic and physiological traits of
Prochlorococcus
, and perhaps of marine microbial communities in general.
AXR4 Is Required for Localization of the Auxin Influx Facilitator AUX1
Authors: Dharmasiri S. , Swarup R. , Mockaitis K. , Dharmasiri N. , Singh S. K. , Kowalchyk M. , Marchant A. , Mills S. , Sandberg G. , Bennett M. J. , Estelle M.
Published: May 2006

The AUX1 and PIN auxin influx and efflux facilitators are key regulators of root growth and development. For root gravitropism to occur, AUX1 and PIN2 must transport auxin via the lateral root cap to elongating epidermal cells. Genetic studies suggest that AXR4 functions in the same pathway as AUX1. Here we show that AXR4 is a previously unidentified accessory protein of the endoplasmic reticulum (ER) that regulates localization of AUX1 but not of PIN proteins. Loss of AXR4 resulted in abnormal accumulation of AUX1 in the ER of epidermal cells, indicating that the
axr4
agravitropic phenotype is caused by defective AUX1 trafficking in the root epidermis.
Viruses: Making Friends with Old Foes
Authors: Douglas Trevor , Young Mark
Published: May 2006

The study of viruses has traditionally focused on their roles as infectious agents and as tools for understanding cell biology. Viruses are now finding a new expanded role as nanoplatforms with applications in materials science and medicine. Viruses form highly symmetrical monodisperse architectures and are ideal templates for engineering multifunctionality, including multivalent display of surface ligands and encapsulation of inorganic and organic materials. These developments assure that viruses will find applications as versatile nanoscale materials.
Nanoassembly of a Fractal Polymer: A Molecular "Sierpinski Hexagonal Gasket"
Authors: Newkome George R. , Wang Pingshan , Moorefield Charles N. , Cho Tae Joon , Mohapatra Prabhu P. , Li Sinan , Hwang Seok-Ho , Lukoyanova Olena , Echegoyen Luis , Palagallo Judith A. , Iancu Violeta , Hla Saw-Wai
Published: May 2006

Mathematics and art converge in the fractal forms that also abound in nature. We used molecular self-assembly to create a synthetic, nanometer-scale, Sierpinski hexagonal gasket. This nondendritic, perfectly self-similar fractal macromolecule is composed of
bis
-terpyridine building blocks that are bound together by coordination to 36 Ru and 6 Fe ions to form a nearly planar array of increasingly larger hexagons around a hollow center.
Simultaneous Negative Phase and Group Velocity of Light in a Metamaterial
Authors: Dolling Gunnar , Enkrich Christian , Wegener Martin , Soukoulis Costas M. , Linden Stefan
Published: May 2006

We investigated the propagation of femtosecond laser pulses through a metamaterial that has a negative index of refraction for wavelengths around 1.5 micrometers. From the interference fringes of a Michelson interferometer with and without the sample, we directly inferred the phase time delay. From the pulse-envelope shift, we determined the group time delay. In a spectral region, phase and group velocity are negative simultaneously. This means that both the carrier wave and the pulse envelope peak of the output pulse appear at the rear side of the sample before their input pulse counterparts have entered the front side of the sample.
RNA Recognition and Cleavage by a Splicing Endonuclease
Authors: Xue Song , Calvin Kate , Li Hong
Published: May 2006

The RNA splicing endonuclease cleaves two phosphodiester bonds within folded precursor RNAs during intron removal, producing the functional RNAs required for protein synthesis. Here we describe at a resolution of 2.85 angstroms the structure of a splicing endonuclease from
Archaeglobus fulgidus
bound with a bulge-helix-bulge RNA containing a noncleaved and a cleaved splice site. The endonuclease dimer cooperatively recognized a flipped-out bulge base and stabilizes sharply bent bulge backbones that are poised for an in-line RNA cleavage reaction. Cooperativity arises because an arginine pair from one catalytic domain sandwiches a nucleobase within the bulge cleaved by the other catalytic domain.
Advocating Science for the People
Authors: de Charadevian Soraya
Published: May 2006

J. D. Bernal
. The Sage of Science.
By Andrew Brown
. Oxford University Press, Oxford, 2005. 576 pp. $34.95, £25. ISBN 0-19-851544-8.

The author sketches the colorful life of the polymath Marxist whose work on x-ray crystallography helped lay the foundations of molecular biology and whose intellectual and political efforts during and after World War II made him one of the 20th century's most influential scientists.
Varicella-Zoster Virus Infection of Human Foreskin Fibroblast Cells Results in Atypical Cyclin Expression and Cyclin-Dependent Kinase Activity
Authors: Leisenfelder Stacey A. , Moffat Jennifer F.
Published: May 2006

In its course of human infection, varicella-zoster virus (VZV) infects rarely dividing cells such as dermal fibroblasts, differentiated keratinocytes, mature T cells, and neurons, none of which are actively synthesizing DNA; however, VZV is able to productively infect them and use their machinery to replicate the viral genome. We hypothesized that VZV alters the intracellular environment to favor viral replication by dysregulating cell cycle proteins and kinases. Cyclin-dependent kinases (CDKs) and cyclins displayed a highly unusual profile in VZV-infected confluent fibroblasts: total amounts of CDK1, CDK2, cyclin B1, cyclin D3, and cyclin A protein increased, and kinase activities of CDK2, CDK4, and cyclin B1 were strongly and simultaneously induced. Cyclins B1 and D3 increased as early as 24 h after infection, concurrent with VZV protein synthesis. Confocal microscopy indicated that cyclin D3 overexpression was limited to areas of IE62 production, whereas cyclin B1 expression was irregular across the VZV plaque. Downstream substrates of CDKs, including pRb, p107, and GM130, did not show phosphorylation by immunoblotting, and p21 and p27 protein levels were increased following infection. Finally, although the complement of cyclin expression and high CDK activity indicated a progression through the S and G
2
phases of the cell cycle, DNA staining and flow cytometry indicated a possible G
1
/S blockade in infected cells. These data support earlier studies showing that pharmacological CDK inhibitors can inhibit VZV replication in cultured cells.
Role of the Stable Signal Peptide and Cytoplasmic Domain of G2 in Regulating Intracellular Transport of the Junín Virus Envelope Glycoprotein Complex
Authors: Agnihothram Sudhakar S. , York Joanne , Nunberg Jack H.
Published: May 2006

Enveloped viruses utilize the membranous compartments of the host cell for the assembly and budding of new virion particles. In this report, we have investigated the biogenesis and trafficking of the envelope glycoprotein (GP-C) of the Junín arenavirus. The mature GP-C complex is unusual in that it retains a stable signal peptide (SSP) as an essential component in association with the typical receptor-binding (G1) and transmembrane fusion (G2) subunits. We demonstrate that, in the absence of SSP, the G1-G2 precursor is restricted to the endoplasmic reticulum (ER). This constraint is relieved by coexpression of SSP in
trans
, allowing transit of the assembled GP-C complex through the Golgi and to the cell surface, the site of arenavirus budding. Transport of a chimeric CD4 glycoprotein bearing the transmembrane and cytoplasmic domains of G2 is similarly regulated by SSP association. Truncations to the cytoplasmic domain of G2 abrogate SSP association yet now permit transport of the G1-G2 precursor to the cell surface. Thus, the cytoplasmic domain of G2 is an important determinant for both ER localization and its control through SSP binding. Alanine mutations to either of two dibasic amino acid motifs in the G2 cytoplasmic domain can also mobilize the G1-G2 precursor for transit through the Golgi. Taken together, our results suggest that SSP binding masks endogenous ER localization signals in the cytoplasmic domain of G2 to ensure that only the fully assembled, tripartite GP-C complex is transported for virion assembly. This quality control process points to an important role of SSP in the structure and function of the arenavirus envelope glycoprotein.
Characterization of a Permissive Epitope Insertion Site in Adenovirus Hexon
Authors: McConnell Michael J. , Danthinne Xavier , Imperiale Michael J.
Published: May 2006

A robust immune response is generated against components of the adenovirus capsid. In particular, a potent and long-lived humoral response is elicited against the hexon protein. This is due to the efficient presentation of adenovirus capsid proteins to CD4+T cells by antigen-presenting cells, in addition to the highly repetitive structure of the adenovirus capsids, which can efficiently stimulate B-cell proliferation. In the present study, we take advantage of this immune response by inserting epitopes against which an antibody response is desired into the adenovirus hexon. We use a B-cell epitope fromBacillus anthracisprotective antigen (PA) as a model antigen to characterize hypervariable region 5 (HVR5) of hexon as a site for peptide insertion. We demonstrate that HVR5 can accommodate a peptide of up to 36 amino acids without adversely affecting virus infectivity, growth, or stability. Viruses containing chimeric hexons elicited antibodies against PA in mice, with total immunoglobulin G (IgG) titers reaching approximately 1 × 103after two injections. The antibody response contained both IgG1 and IgG2a subtypes, suggesting that Th1 and Th2 immunity had been stimulated. Coinjection of wild-type adenovirus and a synthetic peptide from PA produced no detectable antibodies, indicating that incorporation of the epitope into the capsid was crucial for immune stimulation. Together, these results indicate that the adenovirus capsid is an efficient vehicle for presenting B-cell epitopes to the immune system, making this a useful approach for the design of epitope-based vaccines.
Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles
Authors: Johnson Reed F. , McCarthy Sarah E. , Godlewski Peter J. , Harty Ronald N.
Published: May 2006

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions.
Anti-V3 Humanized Antibody KD-247 Effectively Suppresses Ex Vivo Generation of Human Immunodeficiency Virus Type 1 and Affords Sterile Protection of Monkeys against a Heterologous Simian/Human Immunodeficiency Virus Infection
Authors: Eda Yasuyuki , Murakami Toshio , Ami Yasushi , Nakasone Tadashi , Takizawa Mari , Someya Kenji , Kaizu Masahiko , Izumi Yasuyuki , Yoshino Naoto , Matsushita Shuzo , Higuchi Hirofumi , Matsui Hajime , Shinohara Katsuaki , Takeuchi Hiroaki , Koyanagi Yoshio , Yamamoto Naoki , Honda Mitsuo
Published: May 2006

In an accompanying report (Y. Eda, M. Takizawa, T. Murakami, H. Maeda, K. Kimachi, H. Yonemura, S. Koyanagi, K. Shiosaki, H. Higuchi, K. Makizumi, T. Nakashima, K. Osatomi, S. Tokiyoshi, S. Matsushita, N. Yamamoto, and M. Honda, J. Virol. 80:5552-5562, 2006), we discuss our production of a high-affinity humanized monoclonal antibody, KD-247, by sequential immunization with V3 peptides derived from human immunodeficiency virus type 1 (HIV-1) clade B primary isolates. Epitope mapping revealed that KD-247 recognized the Pro-Gly-Arg V3 tip sequence conserved in HIV-1 clade B isolates. In this study, we further demonstrate that in vitro, KD-247 efficiently neutralizes CXCR4- and CCR5-tropic primary HIV-1 clade B and clade B′ with matching neutralization sequence motifs but does not neutralize sequence-mismatched clade B and clade E isolates. Monkeys were provided sterile protection against heterologous simian/human immunodeficiency virus challenge by the passive transfer of a single high dose (45 mg per kg of body weight) of KD-247 and afforded partial protection by lower antibody doses (30 and 15 mg per kg). Protective neutralization endpoint titers in plasma at the time of virus challenge were 1:160 in animals passively transferred with a high dose of the antibody. The antiviral efficacy of the antibody was further confirmed by its suppression of the ex vivo generation of primary HIV-1 quasispecies in peripheral blood mononuclear cell cultures from HIV-infected individuals. Therefore, KD-247 promises to be a valuable tool not only as a passive immunization antibody for the prevention of HIV infection but also as an immunotherapy for the suppression of HIV in phenotype-matched HIV-infected individuals.
Influence of Pseudorabies Virus Proteins on Neuroinvasion and Neurovirulence in Mice
Authors: Klopfleisch Robert , Klupp Barbara G. , Fuchs Walter , Kopp Martina , Teifke Jens P. , Mettenleiter Thomas C.
Published: May 2006

Neurotropism is a distinctive feature of members of the
Alphaherpesvirinae
. However, its molecular basis remains enigmatic. In the past, research has been focused mainly on the role of viral envelope proteins in modulating herpesvirus neuroinvasion and neurovirulence (T. C. Mettenleiter, Virus Res. 92:192-206, 2003). To further analyze the molecular requirements for neuroinvasion of the alphaherpesvirus pseudorabies virus (PrV), adult mice were infected intranasally with a set of single- or multiple-deletion mutants lacking the UL3, UL4, UL7, UL11, UL13, UL16, UL17, UL21, UL31, UL34, UL37, UL41, UL43, UL46, UL47, UL48, UL51, US3, US9, glycoprotein E (gE), gM, UL11/US9, UL11/UL16, UL16/UL21, UL11/UL16/UL21, UL11/gE, UL11/gM, UL43/gK, UL43/gM, or UL43/gK/gM genes. Neurovirulence was evaluated by measuring mean survival times compared to that after wild-type virus infection. Furthermore, by immunohistochemical detection of infected neurons, the kinetics of viral spread in the murine central nervous system was investigated.
LC16m8, a Highly Attenuated Vaccinia Virus Vaccine Lacking Expression of the Membrane Protein B5R, Protects Monkeys from Monkeypox
Authors: Saijo Masayuki , Ami Yasushi , Suzaki Yuriko , Nagata Noriyo , Iwata Naoko , Hasegawa Hideki , Ogata Momoko , Fukushi Shuetsu , Mizutani Tetsuya , Sata Tetsutaro , Kurata Takeshi , Kurane Ichiro , Morikawa Shigeru
Published: May 2006

The potential threat of smallpox as a bioweapon has led to the production
and stockpiling of smallpox vaccine in some countries. Human monkeypox,
a rare but important viral zoonosis endemic to central and western
Africa, has recently emerged in the United States. Thus, even though
smallpox has been eradicated, a vaccinia virus vaccine that can induce
protective immunity against smallpox and monkeypox is still invaluable.
The ability of the highly attenuated vaccinia virus vaccine strain
LC16m8, with a mutation in the important immunogenic membrane protein
B5R, to induce protective immunity against monkeypox in nonhuman
primates was evaluated in comparison with the parental Lister strain.
Monkeys were immunized with LC16m8 or Lister and then infected
intranasally or subcutaneously with monkeypox virus strain Liberia or
Zr-599, respectively. Immunized monkeys showed no symptoms of monkeypox
in the intranasal-inoculation model, while nonimmunized controls showed
typical symptoms. In the subcutaneous-inoculation model, monkeys
immunized with LC16m8 showed no symptoms of monkeypox except for a mild
ulcer at the site of monkeypox virus inoculation, and those immunized
with Lister showed no symptoms of monkeypox, while nonimmunized
controls showed lethal and typical symptoms. These results
indicate that LC16m8 prevents lethal monkeypox in monkeys, and they
suggest that LC16m8 may induce protective immunity against
smallpox.
Diversity of the CD8+T-Cell Response to Herpes Simplex Virus Type 2 Proteins among Persons with Genital Herpes
Authors: Hosken Nancy , McGowan Patrick , Meier Amalia , Koelle David M. , Sleath Paul , Wagener Felecia , Elliott Mark , Grabstein Ken , Posavad Christine , Corey Lawrence
Published: May 2006

Cytolytic T cells play a major role in controlling herpes simplex virus type 2 (HSV-2) infections in humans. In an effort to more thoroughly evaluate the response to HSV-2 directly, ex vivo, we developed an enzyme-linked immunospot (ELISPOT) assay that utilized pools of overlapping synthetic peptides presented by autologous dendritic cells to purified CD8+T cells. Donor response rates to individual open reading frames (ORFs) ranged from fewer than 5% responding to as many as 70% responding, with the greatest frequency of responses (by ORF) being directed against UL39, UL25, UL27, ICP0, UL46, and UL47 in descending order of frequency. HSV-2-seropositive subjects responded to as few as 3 or as many as 46 of the 48 ORFs tested, with a median of 11 ORFs recognized. HLA-B*07 expression correlated with stronger responses overall that were directed primarily against UL49 and UL46. Cumulative precursor frequencies in the blood ranged from 500 to almost 6,000 HSV-2 spot-forming units/106CD8+T cells. The magnitude and breadth of the response in the infected population were greater than previously appreciated. Whether this variability in the CD8+T-cell response within individuals is associated with the frequency of viral reactivation warrants further study.
Evidence for Functional Protein Interactions Required for Poliovirus RNA Replication
Authors: Teterina Natalya L. , Levenson Eric , Rinaudo Mario S. , Egger Denise , Bienz Kurt , Gorbalenya Alexander E. , Ehrenfeld Ellie
Published: May 2006

Poliovirus protein 2C contains a predicted N-terminal amphipathic helix that mediates association of the protein with the membranes of the viral RNA replication complex. A chimeric virus that contains sequences encoding the 18-residue core from the orthologous amphipathic helix from human rhinovirus type 14 (HRV14) was constructed. The chimeric virus exhibited defects in viral RNA replication and produced minute plaques on HeLa cell monolayers. Large plaque variants that contained mutations within the 2C-encoding region were generated upon subsequent passage. However, the majority of viruses that emerged with improved growth properties contained no changes in the region encoding 2C. Sequence analysis and reconstruction of genomes with individual mutations revealed changes in 3A or 2B sequences that compensated for the HRV14 amphipathic helix in the polio 2C-containing proteins, implying functional interactions among these proteins during the replication process. Direct binding between these viral proteins was confirmed by mammalian cell two-hybrid analysis.
Separate Basic Region Motifs within the Adeno-Associated Virus Capsid Proteins Are Essential for Infectivity and Assembly
Authors: Grieger Joshua C. , Snowdy Stephen , Samulski Richard J.
Published: May 2006

Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 (
120
QAKKRVL
126
) and BR2 (
140
PGKKRPV
146
) had minor effects on viral infectivity (∼4- and ∼10-fold, respectively), whereas BR3 (
166
PARKRLN
172
) and BR4 (
307
RPKRLN
312
) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.
A Novel Assay for Viral MicroRNA Function Identifies a Single Nucleotide Polymorphism That Affects Drosha Processing
Authors: Gottwein Eva , Cai Xuezhong , Cullen Bryan R.
Published: May 2006

MicroRNAs (miRNAs) are a class of ∼22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3′ untranslated region of the
Renilla
luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the
Renilla
luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells,
Renilla
luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.
Genetic diversity in colonial bentgrass (Agrostis capillarisL.) revealed byEcoRI–MseI andPstI–MseI AFLP markers
Authors: Zhao Han , Bughrara Suleiman S , Oliveira Jose Alberto
Published: May 2006

Colonial bentgrass (Agrostis capillaris L.) is a potential source for genetic improvement of resistance to environmental stress and disease for other bentgrass species (Agrostis spp.). To conserve and study the existing genetic resources of colonial bentgrass for use in breeding, genetic diversity was investigated using amplified fragment length polymorphism (AFLP) markers. Included in this study were 22 accessions from US Department of Agriculture germplasm collected from 11 countries, in conjunction with 14 accessions from northern Spain and 3 commercial cultivars. Ten EcoRI–MseI and 6 PstI–MseI AFLP primer combinations produced 181 and 128 informative polymorphic bands, respectively. Cluster analysis of genetic similarity estimates revealed a high level of diversity in colonial bentgrass species with averages of 0.51 (EcoRI–MseI) and 0.63 (PstI–MseI). Greater genetic diversity was detected by the EcoRI–MseI AFLP primer combinations. A low but significant positive correlation (r = 0.44, p = 0.0099) between the 2 Jaccard similarity matrices was obtained by the Mantel test. Commercial cultivars of bentgrass showed a narrow genetic background. The assessment of genetic diversity among colonial bentgrass accessions suggested the potential value of the colonial bentgrass germplasm in turfgrass cultivar improvement.Key words: colonial bentgrass, genetic diversity, AFLP, cluster analysis.