bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Phosphorylation Sites Confer Reproducibility of the Rod's Single-Photon Responses
Authors: Doan Thuy , Mendez Ana , Detwiler Peter B. , Chen Jeannie , Rieke Fred
Published: July 2006

Although signals controlled by single molecules are expected to be inherently variable, rod photoreceptors generate reproducible responses to single absorbed photons. We show that this unexpected reproducibility—the consistency of amplitude and duration of rhodopsin activity—varies in a graded and systematic manner with the number but not the identity of phosphorylation sites on rhodopsin's C terminus. These results indicate that each phosphorylation site provides an independent step in rhodopsin deactivation and that collectively these steps tightly control rhodopsin's active lifetime. Other G protein cascades may exploit a similar mechanism to encode accurately the timing and number of receptor activation.
Activated Signal Transduction Kinases Frequently Occupy Target Genes
Authors: Pokholok Dmitry K. , Zeitlinger Julia , Hannett Nancy M. , Reynolds David B. , Young Richard A.
Published: July 2006

Cellular signal transduction pathways modify gene expression programs in response to changes in the environment, but the mechanisms by which these pathways regulate populations of genes under their control are not entirely understood. We present evidence that most mitogen-activated protein kinases and protein kinase A subunits become physically associated with the genes that they regulate in the yeast (
Saccharomyces cerevisiae
) genome. The ability to detect this interaction of signaling kinases with target genes can be used to more precisely and comprehensively map the regulatory circuitry that eukaryotic cells use to respond to their environment.
Mutational Analysis of the Cytoplasmic Tail of Jaagsiekte Sheep Retrovirus Envelope Protein
Authors: Hull Stacey , Fan Hung
Published: July 2006

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a transmissible lung cancer in sheep, ovine pulmonary adenocarcinoma. JSRV is unique in that the envelope protein functions as an oncogene, since it can morphologically transform fibroblast and epithelial cells in culture and can induce lung tumors in mice. Previous studies indicated that the transmembrane (TM) protein is essential for transformation, and particular attention has focused on a YXXM motif in the cytoplasmic tail. In this study, we carried out systematic mutagenesis of the cytoplasmic tail of JSRV Env. Alanine scanning mutagenesis revealed four classes of mutants: mutants in which transformation was abrogated, those in which transformation was not affected, those with reduced transformation, and those with increased transformation (supertransformers). In general, the alanine mutations did not affect Env protein production or its localization to the plasma membrane. Three functional domains of the cytoplasmic tail were identified: an amphipathic helix at the N-terminal (juxtamembrane) side, a nonessential C-terminal region, and an internal region (including the YXXM motif) where mutations resulted in abrogation, decreases, or increases in transformation. Alanine mutations in the amphipathic helix in both the hydrophobic and hydrophilic faces generally abolished transformation. The mutation R591A showed partial transformation that was consistent with loss of signaling through the Akt-mTOR pathway and signaling predominantly through the Ras-Raf-MEK1/2-extracellular signal-regulated kinase 1/2 pathway. The supertransforming mutants generally showed increased signaling through Akt and reduced activation of p38 MAPK that is inhibitory for transformation. These mutants provide further insight into the role of the TM cytoplasmic tail in JSRV transformation.
Global Changes in Cellular Gene Expression during Bacteriophage PRD1 Infection
Authors: Poranen Minna M. , Ravantti Janne J. , Grahn A. Marika , Gupta Rashi , Auvinen Petri , Bamford Dennis H.
Published: July 2006

Virus-induced changes in cellular gene expression and host physiology have been studied extensively. Still, there are only a few analyses covering the entire viral replication cycle and whole-host gene pool expression at the resolution of a single gene. Here we report changes in
Escherichia coli
gene expression during bacteriophage PRD1 infection using microarray technology. Relative mRNA levels were systematically measured for over 99% of the host open reading frames throughout the infection cycle. Although drastic modifications could be detected in the expression of individual genes, global changes at the whole-genome level were moderate. Notably, the majority of virus-induced changes took place only after the synthesis of virion components, indicating that there is no major reprogramming of the host during early infection. The most highly induced genes encoded chaparones and other stress-inducible proteins.
Viral Suppression and Immune Restoration in the Gastrointestinal Mucosa of Human Immunodeficiency Virus Type 1-Infected Patients Initiating Therapy during Primary or Chronic Infection
Authors: Guadalupe Moraima , Sankaran Sumathi , George Michael D. , Reay Elizabeth , Verhoeven David , Shacklett Barbara L. , Flamm Jason , Wegelin Jacob , Prindiville Thomas , Dandekar Satya
Published: July 2006

Although the gut-associated lymphoid tissue (GALT) is an important early site for human immunodeficiency virus (HIV) replication and severe CD4+T-cell depletion, our understanding is limited about the restoration of the gut mucosal immune system during highly active antiretroviral therapy (HAART). We evaluated the kinetics of viral suppression, CD4+T-cell restoration, gene expression, and HIV-specific CD8+T-cell responses in longitudinal gastrointestinal biopsy and peripheral blood samples from patients initiating HAART during primary HIV infection (PHI) or chronic HIV infection (CHI) using flow cytometry, real-time PCR, and DNA microarray analysis. Viral suppression was more effective in GALT of PHI patients than CHI patients during HAART. Mucosal CD4+T-cell restoration was delayed compared to peripheral blood and independent of the time of HAART initiation. Immunophenotypic analysis showed that repopulating mucosal CD4+T cells were predominantly of a memory phenotype and expressed CD11α, αEβ7, CCR5, and CXCR4. Incomplete suppression of viral replication in GALT during HAART correlated with increased HIV-specific CD8+T-cell responses. DNA microarray analysis revealed that genes involved in inflammation and cell activation were up regulated in patients who did not replenish mucosal CD4+T cells efficiently, while expression of genes involved in growth and repair was increased in patients with efficient mucosal CD4+T-cell restoration. Our findings suggest that the discordance in CD4+T-cell restoration between GALT and peripheral blood during therapy can be attributed to the incomplete viral suppression and increased immune activation and inflammation that may prevent restoration of CD4+T cells and the gut microenvironment.
Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection
Authors: Timpe Jennifer M. , Verrill Kristin C. , Trempe James P.
Published: July 2006

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the
E3
gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.
Mutation of Dileucine-Like Motifs in the Human Immunodeficiency Virus Type 1 Capsid Disrupts Virus Assembly, Gag-Gag Interactions, Gag-Membrane Binding, and Virion Maturation
Authors: Joshi Anjali , Nagashima Kunio , Freed Eric O.
Published: July 2006

The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein Pr55
Gag
drives the assembly and release of virus-like particles in the infected cell. The capsid (CA) domain of Gag plays an important role in these processes by promoting Gag-Gag interactions during assembly. The C-terminal domain (CTD) of CA contains two dileucine-like motifs (L
189
/L
190
and I
201
/L
202
) implicated in regulating the localization of Gag to multivesicular bodies (MVBs). These dileucine-like motifs are located in the vicinity of the CTD dimer interface, a region of CA critical for Gag-Gag interactions during virus assembly and CA-CA interactions during core formation. To study the importance of the CA dileucine-like motifs in various aspects of HIV-1 replication, we introduced a series of mutations into these motifs in the context of a full-length, infectious HIV-1 molecular clone. CA mutants LL189,190AA and IL201,202AA were both severely impaired in virus particle production because of a variety of defects in the binding of Gag to membrane, Gag multimerization, and CA folding. In contrast to the model suggesting that the CA dileucine-like motifs regulate MVB targeting, the IL201,202AA mutation did not alter Gag localization to the MVB in either HeLa cells or macrophages. Revertants of single-amino-acid substitution mutants were obtained that no longer contained dileucine-like motifs but were nevertheless fully replication competent. The varied phenotypes of the mutants reported here provide novel insights into the interplay among Gag multimerization, membrane binding, virus assembly, CA dimerization, particle maturation, and virion infectivity.
Betaherpesvirus-Conserved Cytomegalovirus Tegument Protein ppUL32 (pp150) Controls Cytoplasmic Events during Virion Maturation
Authors: AuCoin David P. , Smith Geoffrey B. , Meiering Christopher D. , Mocarski Edward S.
Published: July 2006

The
UL32 gene of human cytomegalovirus (CMV) encodes a prominent
betaherpesvirus-conserved virion tegument protein, called pp150 (basic
phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion
adjacent to the nucleus at late times during infection. Using a UL32
deletion mutant (ΔUL32-BAC) (where BAC is bacterial artificial
chromosome), we demonstrate that pp150 is critical for virion
maturation in the cytoplasmic compartment. Cotransfection of a pp150
expression plasmid with ΔUL32-BAC DNA led to complementation of
the replication defect with focus formation due to secondary spread.
Deletion of the amino terminus of pp150 or disruption of the
betaherpesvirus conserved regions, CR1 and CR2, revealed these regions
to be critical for replication. In contrast, deletion of the carboxyl
terminus only partially compromised maturation while disruption of
glycosylation sites had no effect. An African green monkey CMV UL32
homolog complemented ΔUL32-BAC replication but murine CMV M32
failed to complement, consistent with evolutionary divergence of rodent
and primate cytomegaloviruses. Infection with ΔUL32-BAC showed
normal expression of all kinetic classes of viral genes and replication
of viral DNA, with accumulation of viral DNA-containing particles in
the cytoplasm; however, mutant virus did not spread to adjacent cells.
In contrast to this block in virion infectivity, cell-to-cell transfer
of pp65-containing particles was observed, suggesting that release of
dense bodies continued in the absence of pp150. These observations
demonstrate that pp150 is critical for virion egress, possibly at the
stage of final
envelopment.
Expression of the Jaagsiekte Sheep Retrovirus Envelope Glycoprotein Is Sufficient To Induce Lung Tumors in Sheep
Authors: Caporale Marco , Cousens Christina , Centorame Patrizia , Pinoni Chiara , De las Heras Marcelo , Palmarini Massimo
Published: July 2006

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The expression of the JSRV envelope (Env) alone is sufficient to transform a variety of cell lines in vitro and induce lung cancer in immunodeficient mice. In order to determine the role of the JSRV Env in OPA tumorigenesis in sheep, we derived a JSRV replication-defective virus (JS-RD) which expresses
env
under the control of its own long terminal repeat (LTR). JS-RD was produced by transiently transfecting 293T cells with a two plasmid system, involving (i) a packaging plasmid, with the putative JSRV packaging signal deleted, expressing the structural and enzymatic proteins Gag, Pro, and Pol, and (ii) a plasmid which expresses
env
in
trans
for JS-RD particles and provides the genomes necessary to deliver JSRV
env
upon infection. During the optimization of the JS-RD system we determined that both R-U5 (in the viral 5′ LTR) and the
env
region are important for JSRV particle production. Two independent experimental transmission studies were carried out with newborn lambs. Four of five lambs inoculated with JS-RD showed OPA lesions in the lungs at various times between 4 and 12 months postinoculation. Abundant expression of JSRV Env was detected in tumor cells of JS-RD-infected animals and PCR assays confirmed the presence of the deleted JS-RD genome. These data strongly suggest that the JSRV Env functions as a dominant oncoprotein in the natural immunocompetent host and that JSRV can induce OPA in the absence of viral spread.
NSm and 78-Kilodalton Proteins of Rift Valley Fever Virus Are Nonessential for Viral Replication in Cell Culture
Authors: Won Sungyong , Ikegami Tetsuro , Peters C. J. , Makino Shinji
Published: July 2006

Rift Valley fever viruses carrying mutations of the M gene preglycoprotein region, one lacking NSm protein expression, one lacking 78-kDa protein expression, and one lacking expression of both proteins, were compared in cell culture. All of the mutants and their parent virus produced plaques with similar sizes and morphologies in Vero E6 cells and had similar growth kinetics in Vero, C6/36, and MRC5 cells, demonstrating that the NSm and 78-kDa proteins were not needed for the virus to replicate efficiently in cell culture. A competition-propagation assay revealed that the parental virus was slightly more fit than the mutant virus lacking expression of both proteins.
Virion-Associated Restriction Endonucleases of Chloroviruses
Authors: Agarkova Irina V. , Dunigan David D. , Van Etten James L.
Published: July 2006

Chloroviruses are large, double-stranded-DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae. The prototype of the genus is
Paramecium bursaria
chlorella virus 1 (PBCV-1). Chlorovirus genomes contain various amounts of methylated nucleotides due to virus-encoded DNA methyltransferases (MTases); about 25% of the MTases are associated with companion DNA site-specific (restriction) endonucleases (REases). These enzymes constitute virally encoded restriction-modification (R/M) systems. Although several of the chlorovirus R/M systems are characterized, their biological functions are unknown. The PBCV-1 proteome reveals that two virus-encoded REases, but not their companion MTases, are virion associated, suggesting that viral REases might help degrade the host DNA early in infection. To test this hypothesis, host chromosomal DNA from PBCV-1-infected cells was examined by pulsed-field gel electrophoresis. Initiation of host chromosomal DNA degradation occurred within 5 min postinfection (p.i.). The DNA degradation was insensitive to protein synthesis inhibitors or UV inactivation of virus particles, consistent with the agent being a small protein associated with the virion. Nuclease activities, including those of the two predicted REases and an uncharacterized general nuclease(s), were detected in disrupted PBCV-1 particles. The general nuclease(s) degraded both host and viral DNAs in vitro, although the viral DNA was not degraded in vivo, suggesting differential intracellular trafficking of the virion-associated nucleases. Infection with chloroviruses lacking an R/M system(s) resulted in either delayed host chromosomal DNA degradation or no detectable host chromatin changes. These immediate-early events associated with chlorovirus infections may facilitate rapid switching of the host transcriptional apparatus to viral transcription, which begins within 5 to 10 min p.i.
Role of CCL5 (RANTES) in Viral Lung Disease
Authors: Culley Fiona J. , Pennycook Alasdair M. J. , Tregoning John S. , Dodd Jonathan S. , Walzl Gerhard , Wells Timothy N. , Hussell Tracy , Openshaw Peter J. M.
Published: July 2006

CCL5/RANTES is a key proinflammatory chemokine produced by virus-infected epithelial cells and present in respiratory secretions of asthmatics. To examine the role of CCL5 in viral lung disease, we measured its production during primary respiratory syncytial virus (RSV) infection and during secondary infection after sensitizing vaccination that induces Th2-mediated eosinophilia. A first peak of CCL5 mRNA and protein production was seen at 18 to 24 h of RSV infection, before significant lymphocyte recruitment occurred. Treatment in vivo with Met-RANTES (a competitive chemokine receptor blocker) throughout primary infection decreased CD4+and CD8+cell recruitment and increased viral replication. In RSV-infected, sensitized mice with eosinophilic disease, CCL5 production was further augmented; Met-RANTES treatment again reduced inflammatory cell recruitment and local cytokine production. A second wave of CCL5 production occurred on day 7, attributable to newly recruited T cells. Paradoxically, mice treated with Met-RANTES during primary infection demonstrated increased cellular infiltration during reinfection. We therefore show that RSV induces CCL5 production in the lung and this causes the recruitment of RSV-specific cells, including those making additional CCL5. If this action is blocked with Met-RANTES, inflammation decreases and viral clearance is delayed. However, the exact effects of chemokine modulation depend critically on time of administration, a factor that may potentially complicate the use of chemokine blockers in inflammatory diseases.
New Antiviral Target Revealed by the Hexameric Structure of Mouse Hepatitis Virus Nonstructural Protein nsp15
Authors: Xu Xiaoling , Zhai Yujia , Sun Fei , Lou Zhiyong , Su Dan , Xu Yuanyuan , Zhang Rongguang , Joachimiak Andrzej , Zhang Xuejun C. , Bartlam Mark , Rao Zihe
Published: July 2006

The unique coronavirus transcription/replication machinery comprised of multiple virus-encoded nonstructural proteins (nsp) plays a vital role during initial and intermediate phases of the viral life cycle. The crystal structure of mouse hepatitis virus strain A59 (MHV-A59) nsp15 is reported at 2.15-Å resolution. nsp15 is an XendoU endoribonuclease and is the first one from this family to have its structure unveiled. The MHV-A59 nsp15 monomer structure has a novel protein fold. Two nsp15 trimers form a back-to-back hexamer that is believed to be the functional unit. The structure reveals the catalytic site including the highly conserved residues His262, His277, and Lys317, which is supported by mutagenesis analysis. Gel filtration and enzyme activity assays confirmed that the hexamer is the active form for nsp15 and demonstrate the specificity of nsp15 for uridylate. The high sequence conservation of nsp15 in coronaviruses, including that of severe acute respiratory syndrome, suggests that this protein may provide a new target for the design of antiviral therapeutics.
Amino Acid Residues in the N-Terminal Region of the PA Subunit of Influenza A Virus RNA Polymerase Play a Critical Role in Protein Stability, Endonuclease Activity, Cap Binding, and Virion RNA Promoter Binding
Authors: Hara Koyu , Schmidt Florian I. , Crow Mandy , Brownlee George G.
Published: July 2006

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.
Insights into the Selective Pressures Restricting
Pelargonium Flower Break Virus
Genome Variability: Evidence for Host Adaptation
Authors: Rico Patricia , Ivars Pilar , Elena Santiago F. , Hernández Carmen
Published: July 2006

The molecular diversity of
Pelargonium flower break virus
(PFBV) was assessed using a collection of isolates from different geographical origins, hosts, and collecting times. The genomic region examined was 1,828 nucleotides (nt) long and comprised the coding sequences for the movement (p7 and p12) and the coat (CP) proteins, as well as flanking segments including the entire 3′ untranslated region (3′ UTR). Some constraints limiting viral heterogeneity could be inferred from sequence analyses, such as the conservation of the amino acid sequences of p7 and of the shell domain of the CP, the maintenance of a leucine zipper motif in p12, and the preservation of a particular folding in the 3′ UTR. A remarkable covariation, involving five specific amino acid sites, was found in the CP of isolates largely propagated in the local lesion host
Chenopodium quinoa
and in the progeny of a PFBV variant subjected to serial passages in this host. Concomitant with this covariation, up to 30 nucleotide substitutions in a 1,428-nt region of the viral RNA could be attributable to
C. quinoa
-specific adaptation, representing one of the most outstanding cases of host-driven genome variation for a plant virus. Globally, the results indicate that the selective pressures exerted by the host play a critical role in shaping PFBV populations and that these populations are likely being selected for at both protein and RNA levels.
Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production
Authors: McCown Matthew F. , Pekosz Andrew
Published: July 2006

The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.
Supramolecular Architecture of Severe Acute Respiratory Syndrome Coronavirus Revealed by Electron Cryomicroscopy
Authors: Neuman Benjamin W. , Adair Brian D. , Yoshioka Craig , Quispe Joel D. , Orca Gretchen , Kuhn Peter , Milligan Ronald A. , Yeager Mark , Buchmeier Michael J.
Published: July 2006

Coronavirus particles are enveloped and pleomorphic and are thus refractory to crystallization and symmetry-assisted reconstruction. A novel methodology of single-particle image analysis was applied to selected virus features to obtain a detailed model of the oligomeric state and spatial relationships among viral structural proteins. Two-dimensional images of the S, M, and N structural proteins of severe acute respiratory syndrome coronavirus and two other coronaviruses were refined to a resolution of ∼4 nm. Proteins near the viral membrane were arranged in overlapping lattices surrounding a disordered core. Trimeric glycoprotein spikes were in register with four underlying ribonucleoprotein densities. However, the spikes were dispensable for ribonucleoprotein lattice formation. The ribonucleoprotein particles displayed coiled shapes when released from the viral membrane. Our results contribute to the understanding of the assembly pathway used by coronaviruses and other pleomorphic viruses and provide the first detailed view of coronavirus ultrastructure.
Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on SpeciesD
Authors: Ruzsics Zsolt , Wagner Markus , Osterlehner Andrea , Cook Jonathan , Koszinowski Ulrich , Burgert Hans-Gerhard
Published: July 2006

Until
recently, adenovirus (Ad)-mediated gene therapy was almost exclusively
based on human Ad type 5 (Ad5). Preexisting immunity and the limited,
coxsackievirus and adenovirus receptor-dependent tropism of Ad5
stimulated attempts to exploit the natural diversity in tropism of the
other 50 known human Ad serotypes. Aiming in particular at
immunotherapy and vaccination, we have screened representative
serotypes from different Ad species for their ability to infect
dendritic cells. Ad19a, an Ad from species D, was selected for
development as a new vector for vaccination and cancer gene therapy. To
clone and manipulate its genome, we have developed a novel methodology,
coined “exposon mutagenesis,” that allows the rapid and
precise introduction of virtually any genetic alteration (deletions,
point mutations, or insertions) into recombinant Ad bacterial
artificial chromosomes. The versatility of the system was exemplified
by deleting the E3 region of Ad19a, by specifically knocking out
expression of a species-specific E3 gene, E3/49K, and by reinserting
E3/49K into an E3 null Ad19a mutant. The technology requires only
limited sequence information and is applicable to other Ad species.
Therefore, it should be extremely valuable for the analysis of gene
functions from any Ad species. In addition, a basic,
replication-defective E1- and E3-deleted Ad19a vector expressing GFP
(Ad19aGFP) was generated. This new vector based on species D Ads
exhibits a very promising tropism for lymphoid and muscle cells and
shows great potential as an alternative vector for transduction of cell
types that are resistant to or only poorly transduced by conventional
Ad5-based
vectors.
Eclipse Phase of Herpes Simplex Virus Type 1 Infection: Efficient Dynein-Mediated Capsid Transport without the Small Capsid Protein VP26
Authors: Döhner Katinka , Radtke Kerstin , Schmidt Simone , Sodeik Beate
Published: July 2006

Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-ΔVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-ΔVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-ΔVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin.
Sudden death in a patient newly diagnosed with diabetes having hyperosmolar non-ketotic acidosis with sickle cell trait
Authors: Biedrzycki O , Gillespie H , Lucas S
Published: July 2006

Sickle cell trait (SCT) has a UK urban population rate estimated at 3.2%. The condition may remain unrecognised, but sudden death is a recognised feature. The sudden death of a 51-year-old man with SCT who presented with hyperosmolar non-ketotic acidosis (HONK) as his first presentation of diabetes is reported here. After admission to hospital, a rapid deterioration was seen in his condition, with loss of cardiac output, leading to death. He was found to have had acute-chest syndrome and hepatosplenic infarction on postmortem examination. The pathophysiology of sudden death in patients with SCT and the role of HONK in the patient’s demise is discussed here.
“Occult” mastocytosis with activatingc-kitpoint mutation evolving into systemic mastocytosis associated with plasma cell myeloma and secondary amyloidosis
Authors: Sotlar K , Saeger W , Stellmacher F , Stahmer J , Jäckle S , Valent P , Horny H-P
Published: July 2006

A case of a 70-year-old man presenting with exsudative enteropathy due to light-chain-associated amyloidosis is reported. The diagnosis of systemic mastocytosis associated with IgG/λ plasma cell myeloma and secondary generalised amyloidosis was carried out by morphological evaluation of bone marrow biopsy. Thec-kitpoint mutation D816Y was detected by molecular analysis. Two years before, a cystadenolymphoma of the left parotid gland had been removed. A moderate increase of loosely scattered spindle-shaped mast cells, a subpopulation of them expressing CD25, an antigen that is not expressed by normal or reactive mast cells, was shown by retrospective analysis carried out on an intraparotideal lymph node. Thec-kitmutation D816Y was shown by the molecular analysis of the lymph node. In summary, the notion that systemic mastocytosis may very rarely be associated with B cell neoplasms and that neoplastic mast cell infiltrates may be obscured because of only a minimal increase of atypical mast cells, which are outnumbered by other non-neoplastic cells in the same tissue, is supported by this case. This finding was preliminarily termed “occult” mastocytosis.
Role of colonoscopic biopsy in distinguishing between Crohn’s disease and intestinal tuberculosis
Authors: Kirsch R , Pentecost M , de M Hall P , Epstein D P , Watermeyer G , Friederich P W
Published: July 2006

Background:The histological differential diagnosis of Crohn’s disease and intestinal tuberculosis can be very challenging, as both are chronic granulomatous disorders with overlapping histological features.

Aim:To evaluate selected clinical and histological parameters in colonic biopsy specimens for their ability to discriminate between Crohn’s disease and intestinal tuberculosis.

Methods:25 patients with Crohn’s disease and 18 patients with intestinal tuberculosis were selected for this study on the basis of established clinical, radiological and histological criteria. Clinical data and selected histological parameters in colonoscopic biopsy specimens were assessed retrospectively. A total of 103 and 41 biopsy sites were evaluated in patients with Crohn’s disease and intestinal tuberculosis, respectively.

Results:Clinical parameters helpful in differentiating intestinal tuberculosis from Crohn’s disease included chest radiographic features of tuberculosis (56%v0%), perianal fistulae (0%v40%) and extraintestinal manifestations of Crohn’s disease (0%v40%). Histopathological features that seemed to reliably differentiate between intestinal tuberculosis and Crohn’s disease included confluent granulomas, ⩾10 granulomas per biopsy site and caseous necrosis (in biopsy samples of 50%, 33% and 22% of patients with intestinal tuberculosis, respectively,v0% of patients with Crohn’s disease). Features that were observed more often in patients with intestinal tuberculosis than in those with Crohn’s disease included granulomas exceeding 0.05 mm2(67%v8%), ulcers lined by conglomerate epithelioid histiocytes (61%v8%) and disproportionate submucosal inflammation (67%v10%).

Conclusion:Clinical features and selected histological parameters in colonoscopic biopsy specimens can help in differentiating between Crohn’s disease and intestinal tuberculosis.
Subspecialisation in cellular pathology in the DGH setting: the Warwick experience
Authors: Sanders D S A , Carr R A , Stores O P , Chachlani N , Simon J
Published: July 2006

A successful move towards subspecialised reporting in cellular pathology in the setting of a small to medium-sized district general hospital staffed by five consultant pathologists is described. This move has been facilitated by implementation of a prospective workload allocation system. Perceived benefits in service delivery and career progression are highlighted.
STUDY ON PROPERTIES OF ENERGY SPECTRA OF THE MOLECULAR CRYSTALS
Authors: PANG XIAO-FENG , CHEN XIANG-RONG
Published: July 2006

The energy-spectra of nonlinear vibration of molecular crystals such as acetanilide have been calculated by using discrete nonlinear Schrödinger equation appropriate to the systems, containing various interactions. The energy levels including higher excited states are basically consistent with experimental values obtained by infrared absorption and Raman scattering in acetanilide. We further give the features of distribution of the energy-spectra for the acetanilide. Using the energy spectra we also explained well experimental results obtained by Careri et al..
q-DEFORMED FERMIONS: ALGEBRA, FOCK SPACE AND THERMODYNAMICS
Authors: NARAYANA SWAMY P.
Published: July 2006

We investigate an algebra describing q-deformed Fermion oscillators. We establish the nature of the basic numbers which follow from this algebra, study the Fock space of these generalized Fermions and determine the Jackson Derivative appropriate for these Fermions. The statistical thermodynamics of these Fermions is examined and it is demonstrated that a consistent formulation preserving its full Legendre structure is accomplished by the use of Jackson Derivatives in place of the ordinary thermodynamic derivatives. We determine the important thermodynamic functions and illustrate the thermodynamic properties of q-Fermions.
FERMI PAIRING IN DILUTE 3He-HeII MIXTURES
Authors: AL-SUGHEIR M. K. , GHASSIB H. B. , JOUDEH B. R.
Published: July 2006

In this paper the Galitskii–Migdal–Feynman (GMF) formalism is applied to dilute 3He-HeII mixtures. In particular, the effect of the hole-hole scattering on pairing in these systems is investigated. To this end, the relative phase shifts incorporating many-body effects based on both Brueckner–Bethe–Goldstone (BBG) and GMF formalisms are calculated. In the GMF formalism, the S-wave phase shift at zero relative momentum is –π and has a cusp at the Fermi momentum; while in the BBG formalism, this phase shift has zero values up to the Fermi momentum. From these results we conclude that hole-hole scattering plays a crucial role in any possible fermion-fermion pairing in these systems.
MAGNETIC ENTROPY CHANGE IN A TWO-DIMENSIONAL ANTIFERROMAGNETIC SQUARE LATTICE
Authors: GENCER H.
Published: July 2006

In this study, we investigate magnetic entropy variation and magnetocaloric properties as a function of frustration by considering a spin-1/2 antiferromagnetic Heisenberg model on a square lattice with nearest (J1) and next-nearest neighbor exchange interaction (J2). We show that the degree of frustration in the square lattice increases with α=J2/J1. While the square lattice is unfrustrated for α = 0, it becomes fully frustrated for α=0.5. Numerical results show that the entropy of the square lattice approaches zero for the unfrustrated case at T=1 K. In contrast, finite entropy can survive in the frustrated case at the same temperature. We also calculate the magnetic entropy change (ΔSm) in the square lattice and show that the maximum value of ΔSm increases with increasing α. These results indicate that the magnetic entropy change and consequently the magnetocaloric effect can be enhanced by increasing the degree of frustration. We conclude that the enhanced magnetocaloric effect is related to quantum fluctuations and disordered ground state present in the frustrated square lattice.
SYNTHESIS OF DOUBLE-WALL CARBON NANOTUBES ON MESOPOROUS SILICA: INFLUENCES OF CATALYST PRETREATMENT ON THE NANOTUBE GROWTH
Authors: RAMESH P. , SATO K. , OZEKI Y. , KISHI N. , SUGAI T. , SHINOHARA H.
Published: July 2006

Experimental conditions that govern the double-wall carbon nanotube (DWNT) growth during alcohol catalytic chemical vapor deposition (CCVD) have been optimized for mesoporous silica (MCM41) support materials. Catalyst pretreatment and preparation methods influence the quality of as-grown carbon nanotubes (CNTs). Catalyst pretreatment at low pressure leads to high-quality CNTs concomitant with an increase in the DWNT yield. Impregnation of metal precursors using methanol as the solvent is found to be more suitable than the aqueous solution. Influence of the precursor anion/metal ion on the DWNT yield is also studied under these optimized conditions. Thermogravimetric analysis (TGA), transmission electron microscopy (TEM) and Raman spectroscopic methods have been employed to estimate the quality of the CNTs. It is observed that the present optimized method yields high-quality DWNTs with low amounts of side-wall defects and other carbon impurities.
STRUCTURES OF Ni(II) OCTAETHYLPORPHYRIN AND C60 AT Au(111) SURFACE INVESTIGATED BY STM
Authors: PAN GE-BO , YAN HUI-JUAN , WAN LI-JUN
Published: July 2006

In situ scanning tunneling microscopy (STM) was employed to investigate the adsorption of C 60 on a Ni(II) octaethylporphyrin ( NiOEP ) adlayer at liquid/ Au (111) interface. It was found that C 60 molecules tend to form small islands without defined coordination to the underlying NiOEP array. Moreover, increasing the surface coverage of C 60 resulted in the formation of multilayers. The mixture of C 60 and NiOEP prepared in solution formed separated domains. The results suggested that the C 60– C 60 interaction is stronger than that of C 60– NiOEP and dominated the adsorption of C 60 on NiOEP -modified Au (111) surface. This work provides a direct evidence at molecular scale for the coordination of C 60 with NiOEP in a two-dimensional assembly, demonstrating that immobilizing C 60 at the geometric center of NiOEP on an Au (111) surface in solution is complicated.
BORON NANOWIRES AND NOVELTUBE–CATALYTIC PARTICLE–WIREHYBRID BORON NANOSTRUCTURES
Authors: XU TERRY T. , NICHOLLS ALAN W. , RUOFF RODNEY S.
Published: July 2006

Catalyst-assisted growth of boron nanowires and novel tube–catalytic particle–wire hybrid boron nanostructures were achieved by pyrolysis of diborane at 820–890°C and ~ 200 mTorr in a quartz tube furnace. Electron microscopy imaging and diffraction analysis reveal that most of the nano-structures are amorphous. Elemental analysis by EELS and EDX shows that the nanostructures consist of boron with a small amount of oxygen and carbon. Possible growth mechanisms for the tube–catalytic particle–wire hybrid boron nanostructures are discussed.
FABRICATION OF CARBON NANOSTRUCTURES BY LASER ASSISTED CHEMICAL REACTION
Authors: PARK JONGBOK , JEONG SUNGHO , CHA OK HWAN , JEONG MUN SEOK , KO DO-KYEONG , LEE JONGMIN
Published: July 2006

In this work, a simple method is reported for the synthesis of bundles of carbon nanostructures under room temperature and atmospheric pressure. A pulsed Nd:YAG laser (355 nm, 10 Hz) is focused into the mixture of ferrocene and xylene solutions to produce the nanostructures in which ferrocene plays the role of a catalyst while xylene is the carbon source for nanostructure growth. During the period of irradiation, the color of solution turns into dark brown from transparent orange. Upon the completion of irradiation, typically for an hour, a variety of bundles of carbon nanostructures are found in the solution. Raman spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are used to investigate the nanostructures.
DETECTING AND MANIPULATING SINGLE MOLECULES WITH STM
Authors: HOU J. G. , ZHAO AIDI
Published: July 2006

Scanning tunneling microscope (STM) is a powerful and unique tool for study single molecules. We review recent advances in single-molecule characterizations including direct STM imaging and I–V spectroscopy, dI/dV spectroscopy and mapping, and d2I/dV2spectroscopy and mapping. Some recent experiments of STM-excited single-molecule light emission are also introduced. In the final part, recent developments of single-molecule manipulation with the STM as well as the applications are discussed.
THE EFFECT OF SINGLE WALL CARBON NANOTUBES ON THE DIPOLE ORIENTATION AND PIEZOELECTRIC PROPERTIES OF POLYMERIC NANOCOMPOSITES
Authors: KANG JIN HO , PARK CHEOL , GAIK STEVEN J. , LOWTHER SHARON E. , HARRISON JOYCELYN S.
Published: July 2006

Recently, a series of single wall carbon nanotube (SWNT) polyimide nanocomposites were developed since the demand of electroactive polymeric materials as sensors and actuators for use in high temperature applications has been growing. Adding SWNTs into electroactive polyimides enhanced their electrostrictive strain as well as their mechanical integrities and chemical stability. Although an increase in piezoelectricity resulting from the incorporation of SWNTs could be expected, there has been no systematic study detailing the effect of SWNTs on piezoelectricity. In this article, the effects of various types and concentrations of SWNT on the dipole orientation and piezoelectricity were investigated using a thermally stimulated current (TSC) technique and a modified Rheovibron. It was found that the barely modified SWNTs led to a more substantial increase in the remanent polarization (Pr) than the highly modified SWNTs did. As the loading level of SWNTs increased, Pr increased. However, excessive loading of SWNTs showed a reduction in Pr since the actual poling field decreased due to a large leakage of current. The trend of the piezoelectric strain coefficient, d31, was consistent with that of Pr. The increase in interfacial polarization caused by adding SWNT was believed to be primarily responsible for the increase of Pr and d31.
Pharmacological and biophysical isolation of K+ currents encoded by ether-à-go-go-related genes in murine hepatic portal vein smooth muscle cells
Authors: Yeung Shuk Yin M. , Greenwood Iain A.
Published: July 2006

Previous studies have shown that murine portal vein myocytes express ether-à-go-go related genes (ERGs) and exhibit distinctive currents when recorded under symmetrical K+ conditions. The aim of the present study was to characterize ERG channel currents evoked from a negative holding potential under conditions more pertinent to a physiological scenario to assess the possible functional impact of this conductance. Currents were recorded with ruptured or perforated patch variants of the whole cell technique from a holding potential of −60 mV. Application of three structurally distinct and selective ERG channel blockers, E-4031, dofetilide, and the peptide toxin BeKM-1, all inhibited a significant proportion of the outward current and abolished inward currents with distinctive “hooked” kinetics recorded on repolarization. Dofetilide-sensitive currents at negative potentials evoked by depolarization to +40 mV had a voltage-dependent time to peak and rate of decay characteristic of ERG channels. Application of the novel ERG channel activator PD-118057 (1–10 μM) markedly enhanced the hooked inward currents evoked by membrane depolarization and hyperpolarized the resting membrane potential recorded by current clamp and the perforated patch configuration by ∼20 mV. In contrast, ERG channel blockade by dofetilide (1 μM) depolarized the resting membrane potential by ∼8 mV. These data are the first record of ERG channel currents in smooth muscle cells under quasi-physiological conditions that suggest that ERG channels contribute to the resting membrane potential in these cells.
ApoE deficiency leads to a progressive age-dependent blood-brain barrier leakage
Authors: Hafezi-Moghadam Ali , Thomas Kennard L. , Wagner Denisa D.
Published: July 2006

Previously, we reported a defect in the blood-brain barrier (BBB) of apolipoprotein E-deficient (apoE−/−) mice ( 24 ). Here, we investigate BBB permeability in wild-type (WT) and apoE−/− mice as a function of age. Both WT and apoE−/− mice showed significantly increased cortical BBB leakage with age. However, in apoE−/− mice, the leakage increased at a 3.7× higher rate compared with WT mice. Surprisingly, the cerebellum showed significantly more leakage than other brain regions across age, while there was no difference between the two hemispheres. To determine the contribution of tissue- vs. blood-borne apoE to vascular permeability, we generated chimeric mice by bone marrow transplantation and measured their BBB leakage. These experiments suggest that both blood- and tissue-derived apoE are equally important for BBB function. In sum, we find an age-dependent defect in the BBB that is exacerbated in apoE−/− mice. Since vascular defects are found in patients with age-related neurodegenerative diseases, such as Alzheimer's, age-related BBB leakage could underlie these defects and may thus be an important contributor to the cumulative neuronal damage of these diseases.