bluuebunny/crossref_metadata_2025 huggingface dataset:
Spinophilin Blocks Arrestin Actions in Vitro and in Vivo at G Protein-Coupled Receptors
Authors: Wang Qin , Zhao Jiali , Brady Ashley E. , Feng Jian , Allen Patrick B. , Lefkowitz Robert J. , Greengard Paul , Limbird Lee E.
Published: June 2004

Arrestin regulates almost all G protein–coupled receptor (GPCR)–mediated signaling and trafficking. We report that the multidomain protein, spinophilin, antagonizes these multiple arrestin functions. Through blocking G protein receptor kinase 2 (GRK2) association with receptor-Gβγ complexes, spinophilin reduces arrestin-stabilized receptor phosphorylation, receptor endocytosis, and the acceleration of mitogen-activated protein kinase (MAPK) activity following endocytosis. Spinophilin knockout mice were more sensitive than wild-type mice to sedation elicited by stimulation of α
2
adrenergic receptors, whereas arrestin 3 knockout mice were more resistant, indicating that the signal-promoting, rather than the signal-terminating, roles of arrestin are more important for certain response pathways. The reciprocal interactions of GPCRs with spinophilin and arrestin represent a regulatory mechanism for fine-tuning complex receptor-orchestrated cell signaling and responses.
Activation of Transcription Factor NF-κB Requires ELKS, an IκB Kinase Regulatory Subunit
Authors: Sigala Jeanette L. Ducut , Bottero Virginie , Young David B. , Shevchenko Andrej , Mercurio Frank , Verma Inder M.
Published: June 2004

The nuclear factor–kappa B (NF-κB) family of transcription factors plays a seminal role in inflammation, apoptosis, development, and cancer. Modulation of NF-κB–mediated gene expression in response to diverse signals is coordinated by the IκB kinase (IKK) complex. We identified ELKS, an essential regulatory subunit of the IKK complex. Silencing ELKS expression by RNA interference blocked induced expression of NF-κB target genes, including the NF-κB inhibitor
I
κ
B
α and proinflammatory genes such as
cyclo-oxygenase 2
and
interleukin 8
. These cells were also not protected from apoptosis in response to cytokines. ELKS likely functions by recruiting IκBα to the IKK complex and thus serves a regulatory function for IKK activation.
Real-Space Observation of Molecular Motion Induced by Femtosecond Laser Pulses
Authors: Bartels Ludwig , Wang Feng , Möller Dietmar , Knoesel Ernst , Heinz Tony F.
Published: June 2004

Femtosecond laser irradiation is used to excite adsorbed CO molecules on a Cu(110) surface; the ensuing motion of individual molecules across the surface is characterized on a site-to-site basis by in situ scanning tunneling microscopy. Adsorbate motion both along and perpendicular to the rows of the Cu(110) surface occurs readily, in marked contrast to the behavior seen for equilibrium diffusion processes. The experimental findings for the probability and direction of the molecular motion can be understood as a manifestation of strong coupling between the adsorbates' lateral degrees of freedom and the substrate electronic excitation produced by the femtosecond laser radiation.
Carbon Monoxide: To Boldly Go Where NO Has Gone Before
Authors: Ryter Stefan W. , Morse Danielle , Choi Augustine M. K.
Published: June 2004

The discovery that nitric oxide (NO) has powerful vasoactive properties identical to those of endothelial-derived relaxing factor spawned a vast body of research investigating the physiological actions of small gas molecules. NO, which arises endogenously through the action of nitric oxide synthase (NOS) enzymes, is a highly reactive gas that plays important roles in the regulation of vascular and immune function. Carbon monoxide (CO), a similar yet much more chemically stable gas, occurs in nature as a product of the oxidation or combustion of organic materials. CO also arises in cells and tissues as a byproduct of heme oxygenase (HO) activity, which degrades heme to biliverdin-IXα. Like NO, CO acts as a vasorelaxant and may regulate other vascular functions such as platelet aggregation and smooth muscle proliferation. CO has also been implicated as a neurotransmitter in the central nervous system. HO-1, the inducible form of HO, confers cytoprotection against oxidative stress in vitro and in vivo
.
CO, when applied at low concentration, exerts potent cytoprotective effects mimicking those of HO-1 induction, including down-regulation of inflammation and suppression of apoptosis. Many of the effects of CO depend on the activation of guanylate cyclase, which generates guanosine 3′,5′-monophosphate (cGMP), and the modulation of mitogen-activated protein kinase (MAPK) signaling pathways. This review highlights new advances in the interaction of CO with cellular signaling processes.
The Use of Spin-Labeled Ligands as Biophysical Probes to Report Real-Time Endocytosis of G Protein
–
Coupled Receptors in Living Cells
Authors: Shafer Aaron , Voss John
Published: June 2004

Recycling and degradation of plasma membrane receptors and transporters are fundamental mechanisms for regulating cell signaling and metabolic processes. For many membrane proteins, endocytosis reduces the number of molecules available for transport or signal transduction, providing an attenuation response. Fluorescent reporters attached to either the receptor or ligand have been used to monitor the trafficking of internalization; however, these approaches provide poor resolution for the early endocytic response. Here, we describe the use of a spin-labeled ligand for a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor for measuring the kinetics of endocytosis in real time. Included are protocols for designing a nitroxide-labeled ligand and measuring receptor endocytosis in live cells using electron paramagnetic resonance (EPR) spectroscopy. Methods for the evaluation of the receptor binding and activation properties of modified ligands and the generation of a cell line stably expressing high receptor levels are also provided.
During the Respiratory Burst, Do Phagocytes Need Proton Channels or Potassium Channels, or Both?
Authors: DeCoursey Thomas E.
Published: June 2004

The NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase enzyme complex, a crucial component of innate immunity, produces superoxide anion (O
2
–
), which is a precursor to many reactive oxygen species. NADPH oxidase produces O
2
–
by transferring electrons from intracellular NADPH across the membrane to extracellular (or phagosomal) oxygen and is thus electrogenic. It is widely believed that electroneutrality is preserved by proton flux through voltage-gated proton channels. A series of recent papers have challenged several key aspects of this view of the "respiratory burst." The most recent study solidifies the proposal that O
2
-
and other reactive oxygen species produced by phagocytes are not toxic to microbes under physiological conditions. Further, an essential role for high-conductance, Ca
2+
-activated K
+
(maxi-K
+
) channels in microbe killing is proposed. Finally, the results cast doubt on the widely held view that H
+
efflux through voltage-gated proton channels (i) is the main mechanism of charge compensation, and (ii) is essential to continuous O
2
-
production by the NADPH oxidase. My analysis of the new data and of a large body of data in the literature indicates that the proposed role of maxi-K
+
channels in the respiratory burst is not yet credibly established. H
+
efflux through proton channels thus remains the most viable mechanism for charge compensation and continuous O
2
–
production. The important question of the toxicity of reactive oxygen species in phagocytes and in other cells, which has long been simply taken for granted, is a widespread assumption that deserves critical study.
Intracellular Chloride Channels: Determinants of Function in the Endosomal Pathway
Authors: Faundez Victor , Hartzell H. Criss
Published: June 2004

Endosomes, and related subcellular compartments, contain various Cl
−
channels in the ClC family. In this review, we describe the known roles of intracellular Cl
−
channels and also explore some of the functional implications of transmembrane Cl
−
flux in these organelles. Cl
−
influx acts to control intralumenal pH, both by shunting the effects of the proton pump on membrane potential and, possibly, through direct effects of Cl
−
on the proton pump. Changes in intralumenal pH likely help regulate membrane trafficking. We propose that changes in intralumenal Cl
−
concentration ([Cl
−
]) could theoretically play a direct role in regulating membrane trafficking and organellar function through effects on chloride-sensitive proteins in the vesicular membrane, which could transduce information about intralumenal [Cl
−
] to the outside of the vesicle and thereby recruit various signaling molecules. We present a model in which regulation of cytosolic [Cl
−
] and vesicular Cl
−
conductance could help control the amount or type of neurotransmitter stored in a particular population of synaptic vesicles.
Regulation of GABA Receptor Activity by Neurosteroids and Phosphorylation
Authors: Tasker Jeffrey
Published: June 2004

These two animations show two models for how neurosteroids regulate the flow of chloride ions (Cl
-
) through ionotropic gamma-aminobutyric acid (GABA) receptors. In the first model, binding of the neurosteroid allows a protein kinase C (PKC) phosphorylation site to become accessible. Phosphorylation of the channel increases flux through the channel. In the second model, phosphorylation by PKC allows the neurosteroid to bind and increase flux through the channel. The animations have two parts: (i) a diagrammatic representation of the sequence of events at the channel in the membrane and (ii) a representative current trace of data obtained using electrophysiological techniques. These animations would be useful in teaching how allosteric modulators (neurosteroids) and covalent modulators (kinases) can work together as regulators of protein activity.
Calcium Signaling: A Meeting of the Minds
Authors: Ray L. Bryan , Gough Nancy R. , Adler Elizabeth M.
Published: June 2004

STKE introduces a new feature called an "E-conference" within the Forums section of the site. The current E-conference is titled "Defining Calcium Entry Signals." With the outstanding assistance of conference chairs Don Gill and Randen Patterson, we have enlisted a group of leading experts on receptor-induced calcium entry signals to participate in a "virtual conference" at STKE. The E-conference is designed to focus upon current understanding on the function, control, and role of calcium entry signals in mammalian cells. One central question to be addressed is how calcium entry channels are activated in response to depletion of calcium from stores. Likewise, it is critical to know which channels contribute to such calcium entry. All STKE readers are encouraged to read the comments posted by the invited participants and to add their own comments and questions to the ongoing discussion.
CD40: A Growing Cytoplasmic Tale
Authors: Harnett Margaret M.
Published: June 2004

CD40, a member of the tumor necrosis factor (TNF) receptor family that is expressed on B cells, monocytes, dendritic cells, endothelial cells, and epithelial cells, as well as on B cell lymphomas and carcinomas, activates multiple signaling pathways. In B cells, the response to CD40 is complex and depends on the maturation status of the cell. It is well established that CD40 can promote cell survival through up-regulation of the expression of genes encoding antiapoptotic proteins. However, a new role for CD40 signaling is being recognized in promoting progression through the cell cycle. The roles of the phosphoinositide 3-kinase, mitogen-activated protein kinase, and nuclear factor κB pathways in mediating CD40 stimulation of the cell cycle are described.
Cold Shock Response and Adaptation at Near-Freezing Temperature in Microorganisms
Authors: Inouye Masayori , Phadtare Sangita
Published: June 2004

Microorganisms that naturally encounter sharp temperature shifts must develop strategies for responding and adapting to these shifts.
Escherichia coli
, which are adapted to living at both warm temperatures inside animals and cooler ambient temperatures, respond to low temperatures (10° to 15°C) by adjusting membrane lipid composition and increasing the production of proteins that act as "RNA chaperones" required for transcription and translation and proteins that facilitate ribosomal assembly. In contrast, yeast, which are adapted to cooler temperatures, show a relatively minor cold shock response after temperature shifts from 30° to 10°C but respond with a dramatic increase in the synthesis of trehalose and a heat shock protein when exposed to freezing or near-freezing temperatures. This emphasizes the fact that different groups of microorganisms exhibit distinct types of cold shock responses.
Accumulation of Heterochromatin Components on the Terminal Repeat Sequence of Kaposi's Sarcoma-Associated Herpesvirus Mediated by the Latency-Associated Nuclear Antigen
Authors: Sakakibara Shuhei , Ueda Keiji , Nishimura Ken , Do Eunju , Ohsaki Eriko , Okuno Toshiomi , Yamanishi Koichi
Published: June 2004

In the latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), its 160-kb circularized episomal DNA is replicated and maintained in the host nucleus. KSHV latency-associated nuclear antigen (LANA) is a key factor for maintaining viral latency. LANA binds to the terminal repeat (TR) DNA of the viral genome, leading to its localization to specific dot structures in the nucleus. In such an infected cell, the expression of the viral genes is restricted by a mechanism that is still unclear. Here, we found that LANA interacts with SUV39H1 histone methyltransferase, a key component of heterochromatin formation, as determined by use of a DNA pull-down assay with a biotinylated DNA fragment that contained a LANA-specific binding sequence and a maltose-binding protein pull-down assay. The diffuse localization of LANA on the chromosomes of uninfected cells changed to a punctate one with the introduction of a bacterial artificial chromosome containing most of the TR region, and SUV39H1 clearly colocalized with the LANA-associated dots. Thus, the LANA foci in KSHV-infected cells seemed to include SUV39H1 as well as heterochromatin protein 1. Furthermore, a chromatin immunoprecipitation assay revealed that the TR and the open reading frame (ORF) K1 and ORF50/RTA genes, but not the ORF73/LANA gene, lay within the heterochromatin during KSHV latency. Taken together, these observations indicate that LANA recruits heterochromatin components to the viral genome, which may lead to the establishment of viral latency and govern the transcription program.
Characterization of a Unique Group-Specific Protein (U122) of the Severe Acute Respiratory Syndrome Coronavirus
Authors: Fielding Burtram C. , Tan Yee-Joo , Shuo Shen , Tan Timothy H. P. , Ooi Eng-Eong , Lim Seng Gee , Hong Wanjin , Goh Phuay-Yee
Published: June 2004

A novel coronavirus (CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV genome encodes the characteristic essential CoV replication and structural proteins. Additionally, the genome contains six group-specific open reading frames (ORFs) larger than 50 amino acids, with no known homologues. As with the group-specific genes of the other CoVs, little is known about the SARS-CoV group-specific genes. SARS-CoV ORF7a encodes a putative unique 122-amino-acid protein, designated U122 in this study. The deduced sequence contains a probable cleaved signal sequence and a C-terminal transmembrane helix, indicating that U122 is likely to be a type I membrane protein. The C-terminal tail also contains a typical endoplasmic reticulum (ER) retrieval motif, KRKTE. U122 was expressed in SARS-CoV-infected Vero E6 cells, as it could be detected by Western blot and immunofluorescence analyses. U122 is localized to the perinuclear region of both SARS-CoV-infected and transfected cells and colocalized with ER and intermediate compartment markers. Mutational analyses showed that both the signal peptide sequence and ER retrieval motif were functional.
Genetic Analysis of the Herpes Simplex Virus Type 1 UL20 Protein Domains Involved in Cytoplasmic Virion Envelopment and Virus-Induced Cell Fusion
Authors: Melancon Jeffrey M. , Foster Timothy P. , Kousoulas Konstantin G.
Published: June 2004

The herpes simplex virus type 1 UL20 protein (UL20p) is an important determinant for cytoplasmic virion morphogenesis and virus-induced cell fusion. To delineate the functional domains of the UL20 protein, we generated a panel of single and multiple (cluster) alanine substitutions as well as UL20p carboxyl-terminal truncations. The UL20 mutant genes could be broadly categorized into four main groups: Group I UL20 mutant genes complemented for both virus production and virus-induced cell fusion; Group II UL20 mutant genes did not complement for either virus-induced cell fusion or infectious virus production; Group III UL20 mutant genes complemented for virus-induced cell fusion to variable extents but exhibited substantially decreased ability to complement UL20-null infectious virus production; Group IV mutant genes complemented for infectious virus production but had variable effects on virus-induced cell fusion; this group included two mutants that efficiently complemented for gBsyn3, but not for gKsyn1, virus-induced cell fusion. In addition, certain recombinant viruses with mutations in either the amino or carboxyl termini of UL20p produced partially syncytial plaques on Vero cells in the absence of any other virally encoded syncytial mutations. These studies indicated that the amino and carboxyl termini of UL20p contained domains that functioned both in infectious virus production and virus-induced cell fusion. Moreover, the data suggested that the UL20p's role in virus-induced cell fusion can be functionally separated from its role in cytoplasmic virion morphogenesis and that certain UL20p domains that function in gB-syn3 virus-induced cell fusion are distinct from those functioning in gKsyn1 virus-induced cell fusion.
Contribution of Ebola Virus Glycoprotein, Nucleoprotein, and VP24 to Budding of VP40 Virus-Like Particles
Authors: Licata Jillian M. , Johnson Reed F. , Han Ziying , Harty Ronald N.
Published: June 2004

The VP40 matrix protein of Ebola virus buds from cells in the form of virus-like particles (VLPs) and plays a central role in virus assembly and budding. In this study, we utilized a functional budding assay and cotransfection experiments to examine the contributions of the glycoprotein (GP), nucleoprotein (NP), and VP24 of Ebola virus in facilitating release of VP40 VLPs. We demonstrate that VP24 alone does not affect VP40 VLP release, whereas NP and GP enhance release of VP40 VLPs, individually and to a greater degree in concert. We demonstrate further the following: (i) VP40 L domains are not required for GP-mediated enhancement of budding; (ii) the membrane-bound form of GP is necessary for enhancement of VP40 VLP release; (iii) NP appears to physically interact with VP40 as judged by detection of NP in VP40-containing VLPs; and (iv) the C-terminal 50 amino acids of NP may be important for interacting with and enhancing release of VP40 VLPs. These findings provide a more complete understanding of the role of VP40 and additional Ebola virus proteins during budding.
The Phenylmethylthiazolylthiourea Nonnucleoside Reverse Transcriptase (RT) Inhibitor MSK-076 Selects for a Resistance Mutation in the Active Site of Human Immunodeficiency Virus Type 2 RT
Authors: Auwerx Joeri , Stevens Miguel , Van Rompay An R. , Bird Louise E. , Ren Jingshan , De Clercq Erik , Öberg Bo , Stammers David K. , Karlsson Anna , Balzarini Jan
Published: June 2004

The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 μM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 μM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC)·oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses.
Three Novel
cis
-Acting Elements Required for Efficient Plus-Strand DNA Synthesis of the Hepatitis B Virus Genome
Authors: Lee Jehan , Shin Myeong-Kyun , Lee Hye-Jin , Yoon Gyesoon , Ryu Wang-Shick
Published: June 2004

Synthesis of the relaxed-circular (RC) DNA genomes of hepadnaviruses by reverse transcriptase involves two template switches during plus-strand DNA synthesis. These template switches require repeat sequences (so-called donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. To determine
cis
-acting elements apart from the donor and acceptor sites that are required for plus-strand RC DNA synthesis by hepatitis B virus (HBV), a series of mutants bearing a small deletion were made and analyzed for their impact on the viral genome synthesis. We found three novel
cis
-acting elements in the HBV genome: one element, located in the middle of the minus strand, is indispensable, whereas the other two elements, located near either end of the minus strand, contribute modestly to the plus-strand RC DNA synthesis. The data indicated that the first element facilitates plus-strand RNA primer translocation or subsequent elongation during plus-strand RC DNA synthesis, while the last two elements, although distantly located on the minus strand, act at multiple steps to promote plus-strand RC DNA synthesis. The necessity of multiple
cis
-acting elements on the minus-strand template reflects the complex nature of hepadnavirus reverse transcription.
Isolation of Multiple TT Virus Genotypes from Spleen Biopsy Tissue from a Hodgkin's Disease Patient: Genome Reorganization and Diversity in the Hypervariable Region
Authors: Jelcic Ilijas , Hotz-Wagenblatt Agnes , Hunziker Andreas , zur Hausen Harald , de Villiers Ethel-Michele
Published: June 2004

We report the isolation of 24 novel genotypes of TT viruses from a surgically removed spleen of a patient with Hodgkin's disease. The sequence analysis of our 24 isolates revealed the remarkable heterogeneity of TT virus isolates not only from the same patient but also from the same biopsy material. These isolates belong to four phylogenetic groups of TT viruses. Nucleotide sequence analyses revealed five distinct genotypes (tth3, tth4, tth5, tth6, and tth7). The limited variation in sequence identity of the other isolates defines the latter as variants of four of these genotypes. A group of 6 isolates (the tth7 group) revealed a reorganization of open reading frame 1 (ORF1) leading to one larger and a varying number of smaller ORFs. The nucleotide difference of the full-length genomes was less than 1%. A variation of 69 to 97% in amino acids of a second group of 8 isolates (the tth3 group) was restricted to the hypervariable region of ORF1, indicating the existence of a quasi-species. These isolates differed by less than 2% in the remainder of their nucleotide sequences. An alignment of these isolates with 79 previously reported TT virus genotypes permits the proposal of TT virus genera and species within the family
Anelloviridae
in analogy to a previous proposal for the papillomaviruses (family
Papillomaviridae
).
Cellular and Viral Requirements for Rapid Endocytic Entry of Herpes Simplex Virus
Authors: Nicola Anthony V. , Straus Stephen E.
Published: June 2004

It was recently demonstrated that herpes simplex virus (HSV) successfully infects Chinese hamster ovary (CHO) cells expressing glycoprotein D (gD) receptors and HeLa cells by an endocytic mechanism (A. V. Nicola, A. M. McEvoy, and S. E. Straus, J. Virol.
77
:5324-5332, 2003). Here we define cellular and viral requirements of this pathway. Uptake of intact, enveloped HSV from the cell surface into endocytic vesicles was rapid (
t
1/2
of 8 to 9 min) and independent of the known cell surface gD receptors. Following uptake from the surface, recovery of intracellular, infectious virions increased steadily up to 20 min postinfection (p.i.), which corresponds to accumulation of enveloped virus in intracellular compartments. There was a sharp decline in recovery by 30 min p.i., suggesting loss of the virus envelope as a result of capsid penetration from endocytic organelles into the cytosol. In the absence of gD receptors, endocytosed virions did not successfully penetrate into the cytosol but were instead transported to lysosomes for degradation. Inhibitors of phosphatidylinositol (PI) 3-kinase, such as wortmannin, blocked transport of incoming HSV to the nuclear periphery and virus-induced gene expression but had no effect on virus binding or uptake. This suggests a role for PI 3-kinase activity in trafficking of HSV through the cytosol. Viruses that lack viral glycoproteins gB, gD, or gH-gL were defective in transport to the nucleus and had reduced infectivity. Thus, similar to entry via direct penetration at the cell surface, HSV entry into cells by wortmannin-sensitive endocytosis is efficient, involves rapid cellular uptake of viral particles, and requires gB, gD, and gH-gL.
Direct Evidence for Natural Transmission of Small-Ruminant Lentiviruses of Subtype A4 from Goats to Sheep and Vice Versa
Authors: Shah Cyril , Huder Jon B. , Böni Jürg , Schönmann Marietta , Mühlherr Janine , Lutz Hans , Schüpbach Jörg
Published: June 2004

Small-ruminant lentiviruses (SRLV), which include the caprine arthritis-encephalitis and the maedi-visna virus, cause persistent inflammatory infections in goats and sheep. SRLV are mainly transmitted from mother to offspring through milk. Transmission after prolonged contact between adult animals has also been observed. The observation that certain SRLV subtypes are found in both goats and sheep suggests that interspecies transmission has occurred on several occasions in the past. We investigated seropositive goats and sheep that were kept together in small mixed herds. Phylogenetic analysis of long proviral sequences ingagandpol, combined with epidemiologic information, demonstrated natural sheep-to-goat transmission of the recently identified SRLV subtype A4 in two instances and goat-to-sheep transmission of the same subtype in one instance. In a further mixed cluster, the direction of the interspecies transmission could not be determined. These findings present for the first time direct evidence that natural interspecies transmission of SRLV is ongoing in both directions. The findings are of relevance to virus eradication programs in both species.
Phosphorylation of Epstein-Barr Virus ZEBRA Protein at Its Casein Kinase 2 Sites Mediates Its Ability To Repress Activation of a Viral Lytic Cycle Late Gene by Rta
Authors: El-Guindy Ayman S. , Miller George
Published: June 2004

ZEBRA, a member of the bZIP family, serves as a master switch between latent and lytic cycle Epstein-Barr virus (EBV) gene expression. ZEBRA influences the activity of another viral transactivator, Rta, in a gene-specific manner. Some early lytic cycle genes, such as BMRF1, are activated in synergy by ZEBRA and Rta. However, ZEBRA suppresses Rta's ability to activate a late gene, BLRF2. Here we show that this repressive activity is dependent on the phosphorylation state of ZEBRA. We find that two residues of ZEBRA, S167 and S173, that are phosphorylated by casein kinase 2 (CK2) in vitro are also phosphorylated in vivo. Inhibition of ZEBRA phosphorylation at the CK2 substrate motif, either by serine-to-alanine substitutions or by use of a specific inhibitor of CK2, abolished ZEBRA's capacity to repress Rta activation of the BLRF2 gene, but did not alter its ability to initiate the lytic cycle or to synergize with Rta in activation of the BMRF1 early-lytic-cycle gene. These studies illustrate how the phosphorylation state of a transcriptional activator can modulate its behavior as an activator or repressor of gene expression. Phosphorylation of ZEBRA at its CK2 sites is likely to play an essential role in proper temporal control of the EBV lytic life cycle.
RNA Sequences in the Moloney Murine Leukemia Virus Genome Bound by the Gag Precursor Protein in the Yeast Three-Hybrid System
Authors: Evans Matthew J. , Bacharach Eran , Goff Stephen P.
Published: June 2004

Encapsidation of the Moloney murine leukemia virus (MMLV) genome is mediated through a specific interaction between the major viral structural protein, Gag, and an RNA packaging signal, Ψ. Many studies have investigated this process in vivo, although the specific examination of the Gag-RNA interaction in this context is difficult due to the variety of other viral functions involved in virion assembly in vivo. The
Saccharomyces cerevisiae
three-hybrid assay was used to directly examine the interaction between MMLV Gag and Ψ. In this system, MMLV RNA regions exhibiting high-affinity Gag binding were mapped. All Gag-binding regions were located 3′ to the viral splice donor sequence of the viral RNA transcript. No single short RNA sequence within Ψ supported strong Gag interaction. Instead, an RNA comprised of nearly the entire Ψ region was necessary to demonstrate an appreciable Gag interaction in the yeast three-hybrid system. These finding support the notion that two stem-loops (C and D) are not sufficient to form a core MMLV encapsidation signal.
Rotavirus Replication: Plus-Sense Templates for Double-Stranded RNA Synthesis Are Made in Viroplasms
Authors: Silvestri Lynn S. , Taraporewala Zenobia F. , Patton John T.
Published: June 2004

Rotavirus plus-strand RNAs not only direct protein synthesis but also serve as templates for the synthesis of the segmented double-stranded RNA (dsRNA) genome. In this study, we identified short-interfering RNAs (siRNAs) for viral genes 5, 8, and 9 that suppressed the expression of NSP1, a nonessential protein; NSP2, a component of viral replication factories (viroplasms); and VP7, an outer capsid protein, respectively. The loss of NSP2 expression inhibited viroplasm formation, genome replication, virion assembly, and synthesis of the other viral proteins. In contrast, the loss of VP7 expression had no effect on genome replication; instead, it inhibited only outer-capsid morphogenesis. Similarly, neither genome replication nor any other event of the viral life cycle was affected by the loss of NSP1. The data indicate that plus-strand RNAs templating dsRNA synthesis within viroplasms are not susceptible to siRNA-induced RNase degradation. In contrast, plus-strand RNAs templating protein synthesis in the cytosol are susceptible to degradation and thus are not the likely source of plus-strand RNAs for dsRNA synthesis in viroplasms. Indeed, immunofluorescence analysis of bromouridine (BrU)-labeled RNA made in infected cells provided evidence that plus-strand RNAs are synthesized within viroplasms. Furthermore, transfection of BrU-labeled viral plus-strand RNA into infected cells suggested that plus-strand RNAs introduced into the cytosol do not localize to viroplasms. From these results, we propose that plus-strand RNAs synthesized within viroplasms are the primary source of templates for genome replication and that trafficking pathways do not exist within the cytosol that transport plus-strand RNAs to viroplasms. The lack of such pathways confounds the development of reverse genetics systems for rotavirus.
Comparison of Herpes Simplex Virus Reactivation in Ganglia In Vivo and in Explants Demonstrates Quantitative and Qualitative Differences
Authors: Sawtell N. M. , Thompson R. L.
Published: June 2004

The in vivo ganglionic environment directs the latent herpes simplex virus transcriptional program. Since stress-driven perturbations in sensory neurons are thought to play a critical role in the transition from latency to reactivation, a primary concern in the selection of a valid model of the molecular interactions leading to reactivation is the faithful recapitulation of these environments. In this study reactivation of latently infected ganglia excised and cultured in vitro (explanted) is compared to reactivation occurring in latently infected ganglia in vivo following hyperthermic stress. Three notable points emerged. (i) Neurons in explanted ganglia exhibited marked morphological changes within 2 to 3 h postexplant. DNA fragmentation in neuronal nuclei was detected at 3 h, and atypical expression of cell cycle- and stress-regulated proteins such as geminin, cdk2, cdk4, and cytochrome
c
became apparent at 2 to 48 h. These changes were associated with axotomy and explant and not with the initiation or progression of reactivation and were not observed in ganglia following in vivo hyperthermic stress. (ii) Despite these differences, during the first 22 h primary reactivation events were restricted to a very small number of neurons in vivo and in explanted ganglia. This suggests that at any given time only a few latently infected neurons are competent to reactivate or that the probability of reactivation occurring in any particular neuron is very low. Importantly, the marked changes detected in explanted ganglia were not correlated with increased reactivation, demonstrating that these changes were not associated with the reactivation process per se. (iii) Secondary spread of virus was evident in explanted ganglia within 36 h, an event not observed in vivo. We conclude that explant reactivation may provide an ancillary system for selected studies of the early events in reactivation. However, clear signs of neuronal degeneration within 2 to 3 h postexplant indicate that these ganglia are undergoing major physiological changes not associated with the reactivation process. This ongoing neurodegeneration could alter even the early virus-host interactions in reactivation, and thus caution in the extrapolation of results obtained in explants to the in vivo interactions initiating reactivation is warranted.
Functional Analysis of Late-Budding Domain Activity Associated with the PSAP Motif within the Vesicular Stomatitis Virus M Protein
Authors: Irie Takashi , Licata Jillian M. , Jayakar Himangi R. , Whitt Michael A. , Bell Peter , Harty Ronald N.
Published: June 2004

A PPPY motif within the M protein of vesicular stomatitis virus (VSV) functions as a late-budding domain (L-domain); however, L-domain activity has yet to be associated with a downstream PSAP motif. VSV recombinants with mutations in the PPPY and/or PSAP motif were recovered by reverse genetics and examined for growth kinetics, plaque size, and budding efficiency by electron microscopy. Results indicate that unlike the PPPY motif, the PSAP motif alone does not possess L-domain activity. Finally, the insertion of the human immunodeficiency virus type 1 p6 L-domain and flanking sequences into the PSAP region of M protein rescued budding of a PPPY mutant of VSV to wild-type levels.
Epstein-Barr Virus Latent Membrane Protein LMP-2A Is Sufficient for Transactivation of the Human Endogenous Retrovirus HERV-K18 Superantigen
Authors: Sutkowski Natalie , Chen Gang , Calderon German , Huber Brigitte T.
Published: June 2004

Superantigens are microbial proteins that strongly stimulate T cells. We described previously that the Epstein-Barr virus (EBV) transactivates a superantigen encoded by the human endogenous retrovirus, HERV-K18. We now report that the transactivation is dependent upon the EBV latent cycle proteins. Moreover, LMP-2A is sufficient for induction of HERV-K18 superantigen activity.
Molecular genetic analysis of FIH-1, FH, and SDHB candidate tumour suppressor genes in renal cell carcinoma
Authors: Morris M R , Maina E , Morgan N V , Gentle D , Astuti D , Moch H , Kishida T , Yao M , Schraml P , Richards F M , Latif F , Maher E R
Published: June 2004

Background:Overexpression of the hypoxia inducible factor 1 (HIF-1) and HIF-2 transcription factors and the consequent upregulation of hypoxia inducible mRNAs is a feature of many human cancers and may be unrelated to tissue hypoxia. Thus, the VHL (von Hippel-Lindau) tumour suppressor gene (TSG) regulates HIF-1 and HIF-2 expression in normoxia by targeting the α subunits for ubiquitination and proteolysis. Inactivation of the VHL TSG in VHL tumours and in sporadic clear cell renal cell carcinoma (RCC) results in overexpression of HIF-1 and HIF-2. However, RCC without VHL inactivation may demonstrate HIF upregulation, suggesting that VHL independent pathways for HIF activation also exist. In RCC, three candidate HIF activating genes exist—FIH-1 (factor inhibiting HIF), SDHB, and FH—which may be dependent or independent of VHL inactivation.

Aims:To investigate FIH-1, SDHB, and FH for somatic mutations in sporadic RCC.

Methods:Gene mutation was analysed in primary RCCs (clear cell RCCs, papillary RCCs, and oncocytomas) and RCC cell lines. SDHB mutation analysis was performed by denaturing high performance liquid chromatography followed by direct sequencing of aberrant PCR products. FH and FIH-1 mutation analysis were performed by single stranded conformational polymorphism and direct sequencing of PCR products.

Results:No mutations were identified in the three genes investigated.

Conclusions:There was no evidence to suggest that somatic mutations occur in the FH, FIH-1, or SDHB TSGs in sporadic RCCs.
The effect of statins versus untreated dyslipidaemia on renal function in patients with coronary heart disease. A subgroup analysis of the Greek atorvastatin and coronary heart disease evaluation (GREACE) study
Authors: Athyros V G , Mikhailidis D P , Papageorgiou A A , Symeonidis A N , Pehlivanidis A N , Bouloukos V I , Elisaf M
Published: June 2004

Background:Little is known about statins in the prevention of dyslipidaemia induced renal function decline. The secondary coronary heart disease (CHD) prevention GREACE study suggested that dose titration with atorvastatin (10–80 mg/day, mean dose 24 mg/day) achieves the national cholesterol educational programme treatment goals and significantly reduces morbidity and mortality, compared with usual care.

Aims:To report the effect of statin on renal function compared with untreated dyslipidaemia in both treatment groups.

Methods/Results:All patients had plasma creatinine values within the reference range < 115 µmol/litre (13 mg/litre). The on study creatinine clearance (CrCl), as estimated (for up to 48 months) by the Cockroft-Gault formula, was compared within and between treatment groups using analysis of variance to assess differences over time. Patients from both groups not treated with statins (704) showed a 5.2% decrease in CrCl (p < 0.0001). Usual care patients on various statins (97) had a 4.9% increase in CrCl (p  =  0.003). Structured care patients on atorvastatin (783) had a 12% increase in CrCl (p < 0.0001). This effect was more prominent in the lower two quartiles of baseline CrCl and with higher atorvastatin doses. After adjustment for 25 predictors of all CHD related events, multivariate analysis revealed a hazards ratio of 0.84 (confidence interval 0.73 to 0.95; p  =  0.003) with every 5% increase in CrCl.

Conclusions:In untreated dyslipidaemic patients with CHD and normal renal function at baseline, CrCl declines over a period of three years. Statin treatment prevents this decline and significantly improves renal function, potentially offsetting an additional factor associated with CHD risk.
Upper gastrointestinal cancer pathology reporting: a regional audit to compare standards with minimum datasets
Authors: King P M , Blazeby J M , Gupta J , Alderson D , Moorghen M
Published: June 2004

Aims:Accurate pathological (pTNM) staging of oesophageal and gastric cancer provides important prognostic information. The aim of this study was to compare the standard of pathology reporting of oesophageal and gastric cancer resections from a cancer network with standards set by the Royal College of Pathologists.

Methods:All reports for oesophageal and gastric cancer resections from the five hospitals in the cancer network in 2001 were collected. Individual items of information were compared with minimum datasets provided by the Royal College of Pathologists. Items were classified as “complete”, “partially complete”, or “absent”.

Results:One hundred and ten reports were audited (54 oesophageal and 56 gastric). Fourteen gastric and 17 oesophagectomy reports were over 75% complete. Clinically important missing data occurred most frequently for the pM component of TNM staging (pMx omitted in 87 reports) and completeness of resection expressed as a bold statement (absent in 50 reports). Twelve reports could not be classified because the specimen contained no residual tumour after neoadjuvant treatment.

Conclusion:The use of a standard proforma for reporting upper gastrointestinal cancers based on a minimum dataset provided by the Royal College of Pathologists is recommended, with modifications to allow for specimens with no tumour after neoadjuvant treatment.
Chromosome in situ hybridisation, Ki-67, and telomerase immunocytochemistry in liquid based cervical cytology
Authors: Cheung A N Y , Chiu P M , Tsun K L , Khoo U S , Leung B S Y , Ngan H Y S
Published: June 2004

Aims:To assess the potential value of chromosome in situ hybridisation (CISH), Ki-67, and telomerase immunocytochemistry in liquid based cervical cytology to help detect carcinoma cells and precursors.

Method:Sixty ThinPrep processed cervical cytology samples were studied: 23 cases within the normal limit, 13 low grade squamous intraepithelial lesions (LSILs), 10 high grade squamous intraepithelial lesions (HSILs), six squamous cell carcinomas, three endocervical adenocarcinomas, two cervical adenosquamous cell carcinomas, and three endometrial adenocarcinomas. CISH was performed with DNA probes specific for the pericentromeric regions of chromosome 11 and 16. Hybridisation signals were visualised with the streptavidin–biotin peroxidase technique. The monoclonal MIB1 and polyclonal TRT-H231 antibodies were used to detect Ki-67 and telomerase immunoreactivity, respectively.

Results:Non-specific background staining was almost absent in CISH slides. Normal squamous and glandular cells showed a diploid chromosomal pattern. A relative gain in chromosomes 11 and 16 (aneusomy) was seen in HSIL and the carcinomas (p<0.0001). In MIB1 stained smears, normal cells and koilocytes showed inconspicuous immunoreactivity, whereas strongly immunoreactive nuclei were found in cancer cells and HSIL (p<0.0001). Not only carcinoma and HSIL cells, but also some normal cells, showed cytoplasmic staining for telomerase.

Conclusions:These preliminary results indicate that ThinPrep processed cervical smears are suitable for CISH and immunocytochemical studies. The neoplastic squamous and glandular cells were easily identified based on nuclear aneusomy and strong Ki-67 immuoreactivity in the context of abnormal nuclear morphology. This is the first study to apply CISH in cervical cytology using an immunoenzymatic approach.
Blood cells with fetal haemoglobin (F-cells) detected by immunohistochemistry as indicators of solid tumours
Authors: Wolk M , Martin J E , Constantin R
Published: June 2004

Aims:Fetal hemoglobin (HbF) is an established serological indicator of cancer. However, its distribution in tumour tissues is rarely investigated. Therefore, HbF was studied immunohistologically in different cancers characterised by high blood HbF concentrations.

Methods:Anti-HbF was immunoaffinity purified and used to study HbF immunohistochemically in the following cancers: germ cell tumour (GCT), trophoblastic disease (TD), lymphoma, myelodysplastic syndrome (MDS), multiple myeloma (MM), and ovarian adenocarcinoma (OA).

Results:In GCT a distinction was made between tumours substantially without HbF positive red blood cells (F-RBC) and those with F-RBC. Those without F-RBC were non-metastatic mature teratomas and dermoid cysts. Those containing F-RBC were mainly embryonal carcinomas and metastatic teratomas. HbF positive myeloid cells (F-MLC), HbF positive normoblasts (F-NBS), and F-RBC were common in the bone marrow and in the lymphoid tissues of lymphoma, MDS, and MM. In TD, normal and nucleated F-RBC were seen in the trophoblastic villi in one case with incomplete molar pregnancy (ICM) but not in other cases of ICM and complete molar pregnancy. However, F-RBC and F-MLC were seen in the decidua of both types of TD. Generally, F-cells were observed either within blood vessels or concentrated in certain areas of the neoplastic tissue.

Conclusions:HbF was evaluated as an inducible marker within different tumour tissue blood cells. The dual distribution of these cells—circulating in the blood or concentrated in areas of the neoplastic tissues—might reflect the two independent serological indicators of HbF: one in whole blood and the other in plasma of patients with cancer.
The diagnosis of hepatosplenic candidiasis by DNA analysis of tissue biopsy and serum
Authors: Kirby A , Chapman C , Hassan C , Burnie J
Published: June 2004

Hepatosplenic candidiasis is traditionally diagnosed by blood culture, magnetic resonance imaging (MRI), and histological analysis. The limitations of these methods include: low sensitivity (blood culture) and failure to isolate the organism (MRI/histology). This report describes a case of hepatosplenic candidiasis diagnosed by simultaneous polymerase chain reaction (PCR) analysis of liver biopsy and serum. PCR of biopsy and/or serum may offer a sensitive and specific diagnostic test for hepatosplenic candidiasis. Candida species can be identified, which helps guide antifungal chemotherapy, an important aspect in this difficult to treat infection.
Gonococcal endocarditis: a rare complication of a common disease: Figure 1
Authors: Shetty A , Ribeiro D , Evans A , Linnane S
Published: June 2004

This report describes a case of gonococcal endocarditis in a 28 year old male patient with no history of previous valvular heart disease. More cases of gonococcal endocarditis (a rare complication of gonorrhoea) may occur with the increase in the incidence of gonorrhoea and the increase in resistance to ciprofloxacin, which is currently used as the first line agent for the treatment of gonorrhoea in 75% of genitourinary medicine clinics.