bluuebunny/crossref_metadata_2025 huggingface dataset:
Double-Stranded RNA Binding by a Heterodimeric Complex of Murine Cytomegalovirus m142 and m143 Proteins
Authors: Child Stephanie J. , Hanson Laura K. , Brown Crystal E. , Janzen Deanna M. , Geballe Adam P.
Published: September 2006

In response to viral infection, cells activate a variety of antiviral responses, including several that are triggered by double-stranded (ds) RNA. Among these are the protein kinase R and oligoadenylate synthetase/RNase L pathways, both of which result in the shutoff of protein synthesis. Many viruses, including human cytomegalovirus, encode dsRNA-binding proteins that prevent the activation of these pathways and thereby enable continued protein synthesis and viral replication. We have extended these analyses to another member of the β subfamily of herpesviruses, murine cytomegalovirus (MCMV), and now report that products of the m142 and m143 genes together bind dsRNA. Coimmunoprecipitation experiments demonstrate that these two proteins interact in infected cells, consistent with their previously reported colocalization. Jointly, but not individually, the proteins rescue replication of a vaccinia virus mutant with a deletion of the dsRNA-binding protein gene E3L (VVΔE3L). Like the human cytomegalovirus dsRNA-binding protein genes TRS1 and IRS1, m142 and m143 are members of the US22 gene family. We also found that two other members of the MCMV US22 family, M23 and M24, encode dsRNA-binding proteins, but they do not rescue VVΔE3L replication. These results reveal that MCMV, like many other viruses, encodes dsRNA-binding proteins, at least two of which can inhibit dsRNA-activated antiviral pathways. However, unlike other well-studied examples, the MCMV proteins appear to act in a heterodimeric complex.
Murine Cytomegalovirus m142 and m143 Are both Required To Block Protein Kinase R-Mediated Shutdown of Protein Synthesis
Authors: Valchanova Ralitsa S. , Picard-Maureau Marcus , Budt Matthias , Brune Wolfram
Published: September 2006

Cytomegaloviruses carry the US22 family of genes, which have common sequence motifs but diverse functions. Only two of the 12 US22 family genes of murine cytomegalovirus (MCMV) are essential for virus replication, but their functions have remained unknown. In the present study, we deleted the essential US22 family genes, m142 and m143, from the MCMV genome and propagated the mutant viruses on complementing cells. The m142 and the m143 deletion mutants were both unable to replicate in noncomplementing cells at low and high multiplicities of infection. In cells infected with the deletion mutants, viral immediate-early and early proteins were expressed, but viral DNA replication and synthesis of the late-gene product glycoprotein B were inhibited, even though mRNAs of late genes were present. Global protein synthesis was impaired in these cells, which correlated with phosphorylation of the double-stranded RNA-dependent protein kinase R (PKR) and its target protein, the eukaryotic translation initiation factor 2α, suggesting that m142 and m143 are necessary to block the PKR-mediated shutdown of protein synthesis. Replication of the m142 and m143 knockout mutants was partially restored by expression of the human cytomegalovirus TRS1 gene, a known double-stranded-RNA-binding protein that inhibits PKR activation. These results indicate that m142 and m143 are both required for inhibition of the PKR-mediated host antiviral response.
RNA Silencing Suppression by a Second Copy of the P1 Serine Protease ofCucumber Vein Yellowing Ipomovirus, a Member of the FamilyPotyviridaeThat Lacks the Cysteine Protease HCPro
Authors: Valli Adrian , Martín-Hernández Ana Montserrat , López-Moya Juan José , García Juan Antonio
Published: September 2006

The P1 protein of viruses of the familyPotyviridaeis a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genusPotyvirususe to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the generaPotyvirus,Rymovirus, andTritimovirusthat have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome ofCucumber vein yellowing virus(CVYV), a member of the genusIpomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genusPotyvirusand show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenicNicotiana benthamianaline 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirusPlum pox virusshowed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.
trans
Regulation of Cap-Independent Translation by a Viral Subgenomic RNA
Authors: Shen Ruizhong , Rakotondrafara Aurélie M. , Miller W. Allen
Published: September 2006

Many positive-strand RNA viruses generate 3′-coterminal subgenomic
mRNAs to allow translation of 5′-distal open reading frames. It
is unclear how viral genomic and subgenomic mRNAs compete with each
other for the cellular translation machinery. Translation of the
uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic
RNA1 (sgRNA1) is driven by the powerful cap-independent translation
element (BTE) in their 3′ untranslated regions (UTRs). The BTE
forms a kissing stem-loop interaction with the 5′ UTR to
mediate translation initiation at the 5′ end. Here, using
reporter mRNAs that mimic gRNA and sgRNA1, we show that the abundant
sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in
vivo. This
trans
inhibition requires the functional BTE in the
5′ UTR of sgRNA2, but no translation of sgRNA2 itself is
detectable. The efficiency of translation of the viral mRNAs in the
presence of sgRNA2 is determined by proximity to the mRNA 5′
end of the stem-loop that kisses the 3′ BTE. Thus, the gRNA and
sgRNA1 have “tuned” their expression efficiencies via
the site in the 5′ UTR to which the 3′ BTE base pairs.
We conclude that sgRNA2 is a riboregulator that switches off
translation of replication genes from gRNA while permitting translation
of structural genes from sgRNA1. These results reveal (i) a new level
of control of subgenomic-RNA gene expression, (ii) a new role for a
viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated
regulation of
translation.
Human Cytomegalovirus UL84 Protein Contains Two Nuclear Export Signals and Shuttles between the Nucleus and the Cytoplasm
Authors: Lischka Peter , Rauh Claudia , Mueller Regina , Stamminger Thomas
Published: September 2006

Previous studies defined pUL84 of human cytomegalovirus as an essential regulatory protein with nuclear localization that was proposed to act during initiation of viral-DNA synthesis. Recently, we demonstrated that a complex domain of 282 amino acids within pUL84 functions as a nonconventional nuclear localization signal. Sequence inspection of this domain revealed the presence of motifs with homology to leucine-rich nuclear export signals. Here, we report the identification of two functional, autonomous nuclear export signals and show that pUL84 acts as a CRM-1-dependent nucleocytoplasmic shuttling protein. This suggests an unexpected cytoplasmic role for this essential viral regulatory protein.
Induction of Innate Immunity against Herpes Simplex Virus Type 2 Infection via Local Delivery of Toll-Like Receptor Ligands Correlates with Beta Interferon Production
Authors: Gill Navkiran , Deacon Philip M. , Lichty Brian , Mossman Karen L. , Ashkar Ali A.
Published: September 2006

Toll-like receptors (TLRs) constitute a family of innate receptors that recognize and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Previous studies have demonstrated that ligands for TLR3 and TLR9 induce potent innate antiviral responses against herpes simplex virus type 2 (HSV-2). However, the factor(s) involved in this innate protection is not well-defined. Here we report that production of beta interferon (IFN-β) but not production of IFN-α, IFN-γ, or tumor necrosis factor alpha (TNF-α) strongly correlates with innate protection against HSV-2. Local delivery of poly(I:C) and CpG oligodeoxynucleotides induced significant production of IFN-β in the genital tract and provided complete protection against intravaginal (IVAG) HSV-2 challenge. There was no detectable IFN-β in mice treated with ligands for TLR4 or TLR2, and these mice were not protected against subsequent IVAG HSV-2 challenge. There was no correlation between levels of TNF-α or IFN-γ in the genital tract and protection against IVAG HSV-2 challenge following TLR ligand delivery. Both TNF-α
−/−
and IFN-γ
−/−
mice were protected against IVAG HSV-2 challenge following local delivery of poly(I:C). To confirm that type I interferon, particularly IFN-β, mediates innate protection, mice unresponsive to type I interferons (IFN-α/βR
−/−
mice) and mice lacking IFN regulatory factor-3 (IRF-3
−/−
mice) were treated with poly(I:C) and then challenged with IVAG HSV-2. There was no protection against HSV-2 infection following poly(I:C) treatment of IFN-α/βR
−/−
or IRF-3
−/−
mice. Local delivery of murine recombinant IFN-β protected C57BL/6 and IRF-3
−/−
mice against IVAG HSV-2 challenge. Results from these in vivo studies clearly suggest a strong correlation between IFN-β production and innate antiviral immunity against HSV-2.
Production of Infectious Hepatitis C Virus by Well-Differentiated, Growth-Arrested Human Hepatoma-Derived Cells
Authors: Sainz Bruno , Chisari Francis V.
Published: September 2006

Dimethyl sulfoxide (DMSO) has been shown to induce the differentiation of primary hepatocytes in vitro. When actively dividing poorly differentiated human hepatoma-derived (Huh7) cells were cultured in the presence of 1% DMSO, cells became cytologically differentiated and transitioned into a nondividing state, characterized by the induction of hepatocyte-specific genes. Moreover, these cells were highly permissive for acute hepatitis C virus (HCV) infection, and persistent long term infection of these cultures could also be achieved. As HCV naturally replicates in highly differentiated nondividing human hepatocytes, this system may more accurately mimic the conditions under which HCV replicates in vivo than previous models using poorly differentiated rapidly dividing hepatoma cells.
Quantification of Poly(I:C)-Mediated Protection against Genital Herpes Simplex Virus Type 2 Infection
Authors: Herbst-Kralovetz Melissa M. , Pyles Richard B.
Published: September 2006

Alternative strategies for controlling the growing herpes simplex virus type 2 (HSV-2) epidemic are needed. A novel class of immunomodulatory microbicides has shown promise as antiherpetics, including intravaginally applied CpG-containing oligodeoxynucleotides that stimulate toll-like receptor 9 (TLR9). In the current study, we quantified protection against experimental genital HSV-2 infection provided by an alternative nucleic acid-based TLR agonist, polyinosine-poly(C) (PIC) (TLR3 agonist). Using a protection quantification paradigm, groups of mice were PIC treated and then subdivided into groups challenged with escalating doses of HSV-2. Using this paradigm, a temporal window of PIC efficacy for single applications was defined as 1 day prior to (prophylactic) through 4 h after (therapeutic) viral challenge. PIC treatment within this window protected against 10-fold-higher HSV-2 challenges, as indicated by increased 50% infectious dose values relative to those for vehicle-treated controls. Disease resolution and survival were significantly enhanced by repetitive PIC doses. Using optimal PIC regimens, cytokine induction was evaluated in murine vaginal lavages and in human vaginal epithelial cells. Similar induction patterns were observed, with kinetics that explained the limited durability of PIC-afforded protection. Daily PIC delivery courses did not generate sustained cytokine levels in murine vaginal fluids that would be indicative of local immunotoxicity. No evidence of immunotoxicity was observed in selected organs that were analyzed following repetitive vaginal PIC doses. Animal and in vitro data indicate that PIC may prove to be a valuable preventative microbicide and/or therapeutic agent against genital herpes by increasing resistance to HSV-2 and enhancing disease resolution following a failure of prevention.
Growth Transformation of Human T Cells by Herpesvirus Saimiri Requires Multiple Tip-Lck Interaction Motifs
Authors: Heck Elke , Friedrich Ute , Gack Michaela U. , Lengenfelder Doris , Schmidt Monika , Müller-Fleckenstein Ingrid , Fleckenstein Bernhard , Ensser Armin , Biesinger Brigitte
Published: September 2006

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.
Isolation and Characterization of an Infectious Replication-Competent Molecular Clone of Ecotropic Porcine Endogenous Retrovirus Class C
Authors: Preuss Thomas , Fischer Nicole , Boller Klaus , Tönjes Ralf R.
Published: September 2006

Xenotransplantation of pig organs is complicated by the existence of polytropic replication-competent porcine endogenous retroviruses (PERV) capable of infecting human cells. The potential for recombination between ecotropic PERV-C and human-tropic PERV-A and PERV-B adds another level of infectious risk. Proviral PERV-C were characterized in MAX-T cells derived from
d
/
d
haplotype miniature swine. Three proviruses were cloned from a genomic library. Clone PERV-C(1312) generated infectious particles after transfection into porcine ST-IOWA cells. Electron microscopy revealed the same morphologies of virions in MAX-T cells and in ST-IOWA cells infected with cell-free PERV-C(1312) particles, indicating that MAX-T cells harbor one functional PERV-C provirus.
Packaging of Brome Mosaic Virus Subgenomic RNA Is Functionally Coupled to Replication-Dependent Transcription and Translation of Coat Protein
Authors: Annamalai Padmanaban , Rao A. L. N.
Published: September 2006

In
Brome mosaic virus
(BMV), genomic RNA1 (gB1) and RNA2 (gB2), encoding the replication factors, are packaged into two separate virions, whereas genomic RNA3 (gB3) and its subgenomic coat protein (CP) mRNA (sgB4) are copackaged into a third virion. In vitro assembly assays performed between a series of deletion variants of sgB4 and wild-type (wt) CP subunits demonstrated that packaging of sgB4 is independent of sequences encoding the CP open reading frame. To confirm these observations in vivo and to unravel the mechanism of sgB4 copackaging, an
Agrobacterium
-mediated transient in vivo expression system (P. Annamalai and A. L. N. Rao, Virology
338:
96-111, 2005) that effectively uncouples replication from packaging was used. Cultures of agrotransformants, engineered to express sgB4 and CP subunits either transiently (sgB4
Trans
and CP
Trans
) or in replication-dependent transcription and translation when complemented with gB1 and gB2 (sgB4
Rep
and CP
Rep
), were mixed in all four pair-wise combinations and infiltrated to
Nicotiana benthamiana
leaves to systematically evaluate requirements regulating sgB4 packaging. The data revealed that (i) in the absence of replication, packaging was nonspecific, since transiently expressed CP subunits efficiently packaged ubiquitous cellular RNA as well as transiently expressed sgB4 and its deletion variants; (ii) induction of viral replication increased specificity of RNA packaging; and most importantly, (iii) efficient packaging of sgB4, reminiscent of the wt scenario, is functionally coupled not only to its transcription via replication but also to translation of CP from replication-derived mRNA, a mechanism that appears to be conserved among positive-strand RNA viruses of plants (this study), animals (flock house virus), and humans (poliovirus).
Rabies Virus Infection of Primary Neuronal Cultures and Adult Mice: Failure To Demonstrate Evidence of Excitotoxicity
Authors: Weli Simon C. , Scott Courtney A. , Ward Christopher A. , Jackson Alan C.
Published: September 2006

Cultures derived from the cerebral cortices and hippocampi of 17-day-old mouse fetuses infected with the CVS strain of rabies virus showed loss of trypan blue exclusion, morphological apoptotic features, and activated caspase 3 expression, indicating apoptosis. The NMDA (
N
-methyl-
d
-aspartate acid) antagonists ketamine (125 μM) and MK-801 (60 μM) were found to have no significant neuroprotective effect on CVS-infected neurons, while the caspase inhibitor Ac-Asp-Glu-Val aspartic acid aldehyde (25 μM) exerted a marked neuroprotective effect. Glutamate-stimulated increases in levels of intracellular calcium were reduced in CVS-infected hippocampal neurons. Ketamine (120 mg/kg of body weight/day intraperitoneally) given to CVS-infected adult mice produced no beneficial effects. We have found no supportive evidence that excitotoxicity plays an important role in rabies virus infection.
Autorepression of the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer at Late Times of Infection Is Mediated by the Recruitment of Chromatin Remodeling Enzymes by IE86
Authors: Reeves Matthew , Murphy Jane , Greaves Richard , Fairley Jennifer , Brehm Alex , Sinclair John
Published: September 2006

The human cytomegalovirus major immediate-early protein IE86 is pivotal for coordinated regulation of viral gene expression throughout infection. A relatively promiscuous transactivator of viral early and late gene transcription, IE86 also acts during infection to negatively regulate its own promoter via direct binding to a 14-bp palindromic IE86-binding site, thecisrepression sequence (crs), located between the major immediate-early promoter (MIEP) TATA box and the start of transcription. Although such autoregulation does not involve changes in the binding of basal transcription factors to the MIEP in vitro, it does appear to involve selective inhibition of RNA polymerase II recruitment. However, how this occurs is unclear. We show that autorepression by IE86 at late times of infection correlates with changes in chromatin structure around the MIEP during the course of infection and that this is likely to result from physical and functional interactions between IE86 and chromatin remodeling enzymes normally associated with transcriptional repression of cellular promoters. Firstly, we show that IE86-mediated autorepression is inhibited by histone deacetylase inhibitors. We also show that IE86 interacts, in vitro and in vivo, with the histone deacetylase HDAC1 and histone methyltransferases G9a and Suvar(3-9)H1 and that coexpression of these chromatin remodeling enzymes with IE86 increases autorepression of the MIEP. Finally, we show that mutation of the crs in the context of the virus abrogates the transcriptionally repressive chromatin phenotype normally found around the MIEP at late times of infection, suggesting that negative autoregulation by IE86 results, at least in part, from IE86-mediated changes in chromatin structure of the viral MIEP.
Repression of HMGA2 Gene Expression by Human Cytomegalovirus Involves the IE2 86-Kilodalton Protein and Is Necessary for Efficient Viral Replication and Inhibition of Cyclin A Transcription
Authors: Shlapobersky Mark , Sanders Rebecca , Clark Charles , Spector Deborah H.
Published: September 2006

Human cytomegalovirus (HCMV) infection results in dysregulation of several cell cycle genes, including inhibition of cyclin A transcription. In this work, we examine the effect of the HCMV infection on expression of the high-mobility group A2 (HMGA2) gene, which encodes an architectural transcription factor that is involved in cyclin A promoter activation. We find that expression of HMGA2 RNA is repressed in infected cells. To determine whether repression of HMGA2 is directly related to the inhibition of cyclin A expression and impacts on the progression of the infection, we constructed an HCMV recombinant that expressed HMGA2. In cells infected with the recombinant virus, cyclin A mRNA and protein are induced, and there is a significant delay in viral early gene expression and DNA replication. To determine the mechanism of HMGA2 repression, we used recombinant viruses that expressed either no IE1 72-kDa protein (CR208) or greatly reduced levels of IE2 86-kDa (IE2 86) protein (IE2 86ΔSX-EGFP). At a high multiplicity of infection, the IE1 deletion mutant is comparable to the wild type with respect to inhibition of HMGA2. In contrast, the IE2 86ΔSX-EGFP mutant does not significantly repress HMGA2 expression, suggesting that IE2 86 is involved in the regulation of this gene. Cyclin A expression is also induced in cells infected with this mutant virus. Since HMGA2 is important for cell proliferation and differentiation, particularly during embryogenesis, it is possible that the repression of HMGA2 expression during fetal development could contribute to the specific birth defects in HCMV-infected neonates.
Characterization of a Novel Transferable CRM-1-Independent Nuclear Export Signal in a Herpesvirus Tegument Protein That Shuttles between the Nucleus and Cytoplasm
Authors: Verhagen Janneke , Donnelly Michelle , Elliott Gillian
Published: September 2006

A new group of nucleocytoplasmic shuttling proteins has recently been identified in the structural proteins encoded by several alphaherpesvirus UL47 genes. Nuclear import and export signals for the bovine herpesvirus type 1 UL47 protein (VP8 or bUL47) have been described previously. Here, we study the trafficking of bUL47 in detail and identify an import signal different from that shown before. It comprises a 20-residue N-terminal peptide that is fully transferable and targets a large, normally cytosolic protein to the nucleus. A conserved RRPRRS motif within this peptide was shown to be essential but not sufficient for nuclear targeting. Using interspecies heterokaryon assays, we further demonstrate that the export activity of the published leucine-rich nuclear export signal (NES) is also transferable to a large protein but is functionally weak compared to the activity of the HIV-1 Rev NES. We show that nuclear export dictated by this bUL47 NES is sensitive to leptomycin B (LMB) and therefore dependent on the export receptor CRM-1. However, nuclear export of full-length bUL47 is fully resistant to LMB, suggesting the presence of an additional NES. We go on to identify a second NES in bUL47 within a 28-residue peptide that is in close proximity to but entirely separable from the N-terminal import signal, and we use fluorescence loss in photobleaching to confirm its activity. This NES is resistant to leptomycin B, and therefore utilizes an export receptor other than CRM-1. As this new sequence bears little similarity to other export signals so far defined, we suggest it may be involved in bUL47 export from the nucleus via a novel cellular receptor.
Differential Signaling Networks Induced by Mild and Lethal Hemorrhagic Fever Virus Infections
Authors: Bowick Gavin C. , Fennewald Susan M. , Elsom Barry L. , Aronson Judith F. , Luxon Bruce A. , Gorenstein David G. , Herzog Norbert K.
Published: September 2006

The family
Arenaviridae
includes several National Institutes of Allergy and Infections Diseases category A select agents which cause hemorrhagic fever. There are few vaccines available, and treatment is limited to ribavirin, which varies in efficacy. Development of new antiviral compounds has been hindered by a lack of understanding of the molecular basis of pathogenesis. We used two variants of Pichinde virus, one attenuated and one virulent in the guinea pig model, to delineate the host determinants which lead to either viral clearance or lethal disease. By analyzing protein level changes using pathway analysis, we have identified key intermediates which may be targets for therapeutic intervention.
CD127+CCR5+CD38+++CD4+Th1 Effector Cells Are an Early Component of the Primary Immune Response to Vaccinia Virus and Precede Development of Interleukin-2+Memory CD4+T Cells
Authors: Zaunders John J. , Dyer Wayne B. , Munier Mee Ling , Ip Susanna , Liu Jie , Amyes Elisabeth , Rawlinson William , De Rose Robert , Kent Stephen J. , Sullivan John S. , Cooper David A. , Kelleher Anthony D.
Published: September 2006

The stages of development of human antigen-specific CD4+T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++CD4+effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-γ)-producing vaccinia virus-specific CD4+T cells. The majority of these initial vaccinia virus-specific CD4+T cells were CD127+and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-γ and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++vaccinia virus-specific CD4+T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+T cells in healthy adults and their dysfunction in HIV-1 infection.
QUASI MONTE CARLO INTEGRATION IN GRID ENVIRONMENTS: FURTHER LEAPING EFFECTS
Authors: HOFBAUER HEINZ , UHL ANDREAS , ZINTERHOF PETER
Published: September 2006

The splitting of Quasi-Monte Carlo (QMC) point sequences into interleaved substreams has been suggested to raise the speed of distributed numerical integration and to lower the traffic on the network. The usefulness of this approach in GRID environments is discussed. After specifying requirements for using QMC techniques in GRID environments in general we review and evaluate the proposals made in literature so far. In numerical integration experiments we investigate the quality of single leaped QMC point sequence substreams, comparing the respective properties of Sobol', Halton, Faure, Niederreiter-Xing, and Zinterhof sequences in detail. Numerical integration results obtained on a distributed system show that leaping sensitivity varies tremendously among the different sequences and we provide examples of deteriorated results caused by leaping effects, especially in heterogeneous settings which would be expected in GRID environments.
PARALLEL COOPERATIVE SAVINGS BASED ANT COLONY OPTIMIZATION — MULTIPLE SEARCH AND DECOMPOSITION APPROACHES
Authors: DOERNER KARL F. , HARTL RICHARD F. , BENKNER SIEGFRIED , LUCKA MARIA
Published: September 2006

In this paper we study different parallel implementations of the Savings based Ant System algorithm developed for solving the Vehicle Routing Problem. We analyze the effects of low-level parallelization, multiple search strategies and domain decomposition approaches. For the different strategies speedup and efficiency as well as solution quality are reported. Different information exchanges are analyzed within the multiple search strategies.
N=1 SUPERSYMMETRIC YANG–MILLS THEORY IN d=4 AND ITS BATALIN–VILKOVISKY QUANTIZATION BY SPINOR SUPERFIELDS
Authors: DAYI ÖMER F.
Published: September 2006

Four-dimensional N=1 supersymmetric Yang–Mills theory action is written in terms of the spinor superfields in transverse gauge. This action is seemingly first order in spacetime derivatives. Thus, it suggests that the generalized fields approach of obtaining Batalin–Vilkovisky quantization can be applicable. In fact, generalized fields which collect spinor superfields possessing different ghost numbers are introduced to obtain the minimal solution of its Batalin–Vilkovisky master equation in a compact form.
TOPOLOGICAL MODELS FOR THE PREDICTION OF NEUTRAL ENDOPEPTIDASE AND ANGIOTENSIN-CONVERTING ENZYME INHIBITORY ACTIVITY OF MERCAPTOACYLDIPEPTIDES
Authors: LATHER VINEY , MADAN A. K.
Published: September 2006

The relationship between the topological indices and the Neutral Endopeptidase (NEP) inhibitory activity and Angiotensin-Converting Enzyme (ACE) inhibitory activity of mercaptoacyldipeptides has been investigated. Three topological indices — the Wiener index (a distance-based topological index), the molecular connectivity index (an adjacency-based topological index), and the eccentric connectivity index (an adjacency-cum-distance-based topological index), were presently used for investigation. A data set comprising 39 differently substituted mercaptoacyldipeptides was selected for the present study. The values of the Wiener index, molecular connectivity index, and eccentric connectivity index for each of the 39 compounds comprising the data set were computed using an in-house computer program. Resultant data were analyzed and suitable models were developed after identification of the active ranges. Subsequently, a biological activity was assigned to each compound using these models, and the biological activity was then compared with the reported NEP and ACE inhibitory activity of each compound. Accuracy of prediction up to a maximum of ~91% was obtained using these models.
PROTON AND ALKALI METAL IONS ATTACHED TO ALKYLPEROXYL RADICALS: STRUCTURE AND STABILITY OF M+-CnH2n+1O2 (M = H, Li, Na, K; n = 1 ~ 3) COMPLEXES
Authors: CHENG SHUANG , LI HAIYANG , LIU YING , LIANG JUN , PEI KEMEI , NIU DONGMEI
Published: September 2006

The structure and stability of the still experimentally unknown M +- C n H 2n+1 O 2 ( M = H , Li , Na , K ; n = 1 ~ 3) complexes were theoretically investigated via density function theory at the B3LYP/6-311+G(3df,2p)//B3LYP/6-311G(d,p) level. The addition of alkali metal ions ( Li , Na , and K ) to C n H 2n+1 O 2 are found to form only one stable structure, while proton transfer reactions with C n H 2n+1 O 2 produce two isomers except for CH 3 CH 2 CH 2 O 2. The optimized geometries and Mulliken population analysis indicate that the M + ( M = Li , Na , K )- C n H 2n+1 O 2 complexes exist as ion-dipole molecules. Our prediction for the affinity energies of M + to C n H 2n+1 O 2 are 165.4, 178.0, 181.9 and 176.8 kcal/mol ( H +); 34.3, 36.4, 37.5, and 38.4 kcal/mol ( Li +); 24.4, 26.1, 26.9, and 27.5 kcal/mol ( Na +); and 17.5, 19.2, 19.6, and 20.0 kcal/mol ( K +), respectively. Thus, these values suggest that C n H 2n+1 O 2- M + ( M = H , Li , Na , K ) complexes could be detected as stable species in gas phase at room temperature by ion attachment mass spectrometry.
High-calcium diet modulates effects of long-term prolactin exposure on the cortical bone calcium content in ovariectomized rats
Authors: Charoenphandhu Narattaphol , Tudpor Kukiat , Thongchote Kanogwun , Saengamnart Wasana , Puntheeranurak Supaporn , Krishnamra Nateetip
Published: September 2006

High physiological prolactin induced positive calcium balance by stimulating intestinal calcium absorption, reducing renal calcium excretion, and increasing bone calcium deposition in female rats. Although prolactin-induced increase in trabecular bone calcium deposition was absent after ovariectomy, its effects on cortical bones were still controversial. The present investigation, therefore, aimed to study the effect of in vivo long-term high physiological prolactin induced by either anterior pituitary (AP) transplantation or 2.5 mg/kg prolactin injection on cortical bones in ovariectomized rats. Since the presence of prolactin receptors (PRLR) in different bones of normal adult rats has not been reported, we first determined mRNA expression of both short- and long-form PRLRs at the cortical sites (tibia and femur) and trabecular sites (calvaria and vertebrae) by using the RT-PCR. Our results showed the mRNA expression of both PRLR isoforms with predominant long form at all sites. However, high prolactin levels induced by AP transplantation in normal rats did not have any effect on the femoral bone mineral density or bone mineral content. By using45Ca kinetic study, 2.5 mg/kg prolactin did not alter bone formation, bone resorption, calcium deposition, and total calcium content in tibia and femur of adult ovariectomized rats. AP transplantation also had no effect on the cortical total calcium content in adult ovariectomized rats. Because previous work showed that the effects of prolactin were age dependent and could be modulated by high-calcium diet, interactions between prolactin and these two parameters were investigated. The results demonstrated that 2.0% wt/wt high-calcium diet significantly increased the tibial total calcium content in 9-wk-old young AP-grafted ovariectomized rats but decreased the tibial total calcium content in 22-wk-old adult rats. As for the vertebrae, the total calcium contents in both young and adult rats were not changed by high-calcium diet. The present results thus indicated that the adult cortical bones were potentially direct targets of prolactin. Moreover, the effects of high physiological prolactin on cortical bones were age dependent and were observed only under the modulation of high-calcium diet condition.
Hormone, cytokine, and nutritional regulation of sepsis-induced increases in atrogin-1 and MuRF1 in skeletal muscle
Authors: Frost Robert A. , Nystrom Gerald J. , Jefferson Leonard S. , Lang Charles H.
Published: September 2006

Various atrophic stimuli increase two muscle-specific E3 ligases, muscle RING finger 1 (MuRF1) and atrogin-1, and knockout mice for these “atrogenes” display resistance to denervation-induced atrophy. The present study determined whether increased atrogin-1 and MuRF1 mRNA are mediated by overproduction of endogenous glucocorticoids or inflammatory cytokines in adult rats and whether atrogene expression can be downregulated by anabolic agents such as insulin-like growth factor (IGF)-I and the nutrient-signaling amino acid leucine. Both atrogin-1 and MuRF1 mRNA in gastrocnemius was upregulated dose and time dependently by endotoxin. Additionally, peritonitis produced by cecal ligation and puncture increased atrogin-1 and MuRF1 mRNA in gastrocnemius (but not soleus or heart) by 8 h, which was sustained for 72 and 24 h, respectively. Whereas the sepsis-induced increase in atrogin-1 expression was completely prevented by IGF-I, the increased MuRF1 was not altered. In contrast to the IGF-I effect, the sepsis-induced increased mRNA of both atrogenes was unresponsive to either acute or repetitive administration of leucine. Whereas exogenous infusion of TNF-α increased atrogin-1 and MuRF1 in gastrocnemius, pretreatment of septic rats with the TNF antagonist TNF-binding protein did not prevent increased expression of either atrogene. Similarly, whereas dexamethasone increased atrogene expression, pretreatment with the glucocorticoid receptor antagonist RU-486 failed to ameliorate the sepsis-induced increase in atrogin-1 and MuRF1. Thus, under in vivo conditions in mature adult rats, the sepsis-induced increase in muscle atrogin-1 and MuRF1 mRNA appears both glucocorticoid and TNF independent and is unresponsive to leucine.
Effects of glucose-dependent insulinotropic peptide on osteoclast function
Authors: Zhong Qing , Itokawa Takashi , Sridhar Supriya , Ding Ke-Hong , Xie Ding , Kang Baolin , Bollag Wendy B. , Bollag Roni J. , Hamrick Mark , Insogna Karl , Isales Carlos M.
Published: September 2006

Acute nutrient ingestion leads to a rapid inhibition of bone resorption while effects on makers of bone formation are less marked or absent, suggesting that there is a transient shift toward skeletal accretion in the immediate postprandial period. The cellular bases for these effects are not clear. Glucose-dependent insulinotropic peptide (GIP), a known modulator of glucose-induced insulin secretion, is secreted from intestinal endocrine cells in response to nutrient ingestion. In addition to the effect of GIP on pancreatic β-cells, GIP receptors are expressed by osteoblastic cells in bone, suggesting a role for this incretin hormone in bone formation. To determine whether GIP also plays a role in the anti-resorptive effect of nutrient ingestion, osteoclasts were analyzed for the presence of GIP receptors by PCR, immunohistochemical and immunocytochemical analyses of bone tissue, and freshly isolated mature osteoclasts and osteoclast-like cells cultured in vitro. Osteoclast function was assessed by fetal long bone resorption assay and by use of the Osteologic disc assay. Our results demonstrate that GIP receptor transcripts and protein are present in osteoclasts. In addition, with the use of an in vitro organ culture system and mature osteoclasts, GIP was found to inhibit bone resorption in the organ culture system and the resorptive activity of mature osteoclasts. These data are consistent with the hypothesis that GIP inhibits bone breakdown through a direct effect on osteoclast-resorptive activity and suggest one mechanism for the postprandial reduction in markers of bone breakdown.
Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice
Authors: Chattopadhyay Naibedya , Jeong Kyeong-Hoon , Yano Shozo , Huang Su , Pang Jian L. , Ren Xianghui , Terwilliger Ernest , Kaiser Ursula B. , Vassilev Peter M. , Pollak Martin R. , Brown Edward M.
Published: September 2006

The factors controlling the migration of mammalian gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus are not well understood. We studied whether the extracellular calcium-sensing receptor (CaR) promotes migration/chemotaxis of GnRH neurons. We demonstrated expression of CaR in GnRH neurons in the murine basal forebrain and in two GnRH neuronal cell lines: GT1-7 (hypothalamus derived) and GN11 (olfactory bulb derived). Elevated extracellular Ca2+concentrations promoted chemotaxis of both cell types, with a greater effect in GN11 cells. This effect was CaR mediated, as, in both cell types, overexpression of a dominant-negative CaR attenuated high Ca2+-stimulated chemotaxis. We also demonstrated expression of a β-chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CC motif receptor-2 (CCR2), in the hypothalamic GnRH neurons as well as in GT1-7 and GN11 cells. Exogenous MCP-1 stimulated chemotaxis of both cell lines in a dose-dependent fashion; the effect was greater in GN11 than in GT1-7 cells, consistent with the higher CCR2 mRNA levels in GN11 cells. Activating the CaR stimulated MCP-1 secretion in GT1-7 but not in GN11 cells. MCP-1 secreted in response to CaR stimulation is biologically active, as conditioned medium from GT1-7 cells treated with high Ca2+promoted chemotaxis of GN11 cells, and this effect was partially attenuated by a neutralizing antibody to MCP-1. Finally, in the preoptic area of anterior hypothalamus, the number of GnRH neurons was ∼27% lower in CaR-null mice than in mice expressing the CaR gene. We conclude that the CaR may be a novel regulator of GnRH neuronal migration likely involving, in part, MCP-1.
Activation of glycogen synthase in myocardium induced by intermittent hypoxia is much lower in fasted than in fed rats
Authors: Wu Yangsong , Wang Hong , Brautigan David L. , Liu Zhenqi
Published: September 2006

Obstructive sleep apnea is characterized by intermittent obstruction of the upper airway, which leads to intermittent hypoxia. Myocardial glycogen is a major energy resource for heart during hypoxia. Previous studies have demonstrated that intermittent hypoxia rapidly degrades myocardial glycogen and activates glycogen synthase (GS). However, the underlying mechanisms remain undefined. Because sleep apnea/intermittent hypoxia usually happens at night, whether intermittent hypoxia leads to GS activation in the postabsorptive state is not known. In the present study, male adult rats were studied after either an overnight fast or ad libitum feeding with or without intermittent ventilatory arrest (3 90-s periods at 10-min intervals). Hearts were quickly excised and freeze-clamped. Intermittent hypoxia induced a significant decrease in myocardial glycogen content in fed rats and stimulated GS in both fasted and fed rats. However, the portion of GS in the active form increased by ∼38% in fasted rats compared with a larger, ∼130% increase in fed rats. The basal G-6- P content was comparable in fasted and fed animals and increased approximately threefold after hypoxia. The basal phosphorylation states of Akt and GSK-3β and the activity of protein phosphatase 1 (PP1) were comparable between fasted and fed control rats. Hypoxia significantly increased Akt phosphorylation and PP1 activity only in fed rats. In contrast, hypoxia did not induce significant change in GSK-3β phosphorylation in either fasted or fed rats. We conclude that hypoxia activates GS in fed rat myocardium through a combination of rapid glycogenolysis, elevated local G-6- P content, and increased PP1 activity, and fasting attenuates this action independent of local G-6- P content.
Extracellular pH alkalinization by Cl−/HCO3−exchanger is crucial for TASK2 activation by hypotonic shock in proximal cell lines from mouse kidney
Authors: L'Hoste S. , Barriere H. , Belfodil R. , Rubera I. , Duranton C. , Tauc M. , Poujeol C. , Barhanin J. , Poujeol P.
Published: September 2006

We have previously shown that K+-selective TASK2 channels and swelling-activated Cl−currents are involved in a regulatory volume decrease (RVD; Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P. J Gen Physiol 122: 177–190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol 284: F812–F828, 2003). The aim of this study was to determine the mechanism responsible for the activation of TASK2 channels during RVD in proximal cell lines from mouse kidney. For this purpose, the patch-clamp whole-cell technique was used to test the effect of pH and the buffering capacity of external bath on Cl−and K+currents during hypotonic shock. In the presence of a high buffer concentration (30 mM HEPES), the cells did not undergo RVD and did not develop outward K+currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH (pHi) and increased the external pH (pHe) in wild-type but not in cftr−/−cells. The inhibitory effect of DIDS suggests that the acidification of pHiand the alkalinization of pHeinduced by hypotonicity in wild-type cells could be due to an exit of HCO3−. In conclusion, these results indicate that Cl−influx will be the driving force for HCO3−exit through the activation of the Cl−/HCO3−exchanger. This efflux of HCO3−then alkalinizes pHe, which in turn activates TASK2 channels.
Increased renal phosphodiesterase-5 activity mediates the blunted natriuretic response to ANP in the pregnant rat
Authors: Knight Sarah , Snellen Harold , Humphreys Michael , Baylis Chris
Published: September 2006

Normal rat pregnancy is characterized by plasma volume expansion due to renal sodium retention and is associated with a blunted response to natriuretic stimuli, such as atrial natriuretic peptide (ANP). ANP signals via cGMP, and phosphodiesterases (PDE) inactivate cGMP and terminate the natriuretic response. We previously reported that increased medullary PDE-5 activity occurs in rat pregnancy, which may be the mechanism of the blunted natriuretic effect of ANP. Here, we used anesthetized 16-day pregnant and virgin rats to investigate whether intrarenal infusion of a selective PDE-5 inhibitor, sildenafil, would reverse the blunted response to ANP in pregnancy. We measured blood pressure, renal clearances using inulin and p-aminohippuric acid, and electrolyte excretion at baseline and during an ANP infusion. ANP caused a fall in mean arterial pressure in all groups, and sildenafil induced a further reduction. We observed an increase in sodium excretion with ANP in all rats, but this was blunted in the vehicle-infused pregnant rats. This could not be explained by differences in renal hemodynamics and was of tubular origin, as reflected by the reduced rise in fractional excretion of sodium with ANP in the pregnant rat given vehicle (45 ± 11 vs. 204 ± 49%; P < 0.05). However, intrarenal sildenafil increased the natriuretic response and the rise in fractional excretion of sodium to the virgin value (226 ± 23 vs. 245 ± 73%; not significant), whereas the blunting persisted in the contralateral kidney. This demonstrates that increased intrarenal PDE-5 mediates the blunted natriuretic response to ANP during pregnancy and may contribute to the physiological volume expansion.