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JOURNAL OF VIROLOGY, Nov. 2005, p. 13538–13547
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 21
?-Adrenoreceptors Reactivate Kaposi's Sarcoma-Associated
Herpesvirus Lytic Replication via PKA-Dependent
Control of Viral RTA
Margaret Chang,1Helen J. Brown,2Alicia Collado-Hidalgo,3,6Jesusa M. Arevalo,3
Zoran Galic,3Tonia L. Symensma,2,4Lena Tanaka,4Hongyu Deng,2
Jerome A. Zack,1,3,5Ren Sun,2,5and Steve W. Cole3,5,6*
Departments of Microbiology, Immunology and Molecular Genetics,1Molecular and Medical Pharmacology,2and
Medicine,3David E. Geffen School of Medicine, University of California, Los Angeles, California; Department
of Biological Sciences, Mount Saint Mary's College, Los Angeles, California4; and Jonsson Comprehensive Cancer Center,5
UCLA AIDS Institute, and UCLA Molecular Biology Institute5and Norman Cousins Center,6
University of California, Los Angeles, California
Received 25 April 2005/Accepted 5 August 2005
Reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication is mediated by the viral
RTA transcription factor, but little is known about the physiological processes controlling its expression or
activity. Links between autonomic nervous system activity and AIDS-associated Kaposi's sarcoma led us to
examine the potential influence of catecholamine neurotransmitters. Physiological concentrations of epineph-
rine and norepinephrine efficiently reactivated lytic replication of KSHV in latently infected primary effusion
lymphoma cells via ?-adrenergic activation of the cellular cyclic AMP/protein kinase A (PKA) signaling
pathway. Effects were blocked by PKA antagonists and mimicked by pharmacological and physiological PKA
activators (prostaglandin E2and histamine) or overexpression of the PKA catalytic subunit. PKA up-regulated
RTA gene expression, enhanced activity of the RTA promoter, and posttranslationally enhanced RTA's trans-
activating capacity for its own promoter and heterologous lytic promoters (e.g., the viral PAN gene). Mutation
of predicted phosphorylation targets at RTA serines 525 and 526 inhibited PKA-mediated enhancement of RTA
trans-activating capacity. Given the high catecholamine levels at sites of KSHV latency such as the vasculature
and lymphoid organs, these data suggest that ?-adrenergic control of RTA might constitute a significant
physiological regulator of KSHV lytic replication. These findings also suggest novel therapeutic strategies for
controlling the activity of this oncogenic gammaherpesvirus in vivo.
known as human herpesvirus 8 [HHV-8]) is a lymphotropic
gammaherpesvirus originally identified in the context of Ka-
posi's sarcoma (8) and subsequently implicated in primary
effusion lymphoma and multicentric Castleman's disease (6,
66). The KSHV genome most closely resembles herpesvirus
saimiri (60), but the virus also bears more distant structural
and functional similarities to Epstein-Barr virus (48, 49). Like
all herpesviruses, KSHV displays a bifurcating gene expression
program that allows it to defer lytic replication and enter a
protracted state of latency during which only a small minority
of viral genes are expressed (19, 21, 71, 78). KSHV establishes
latency in B lymphocytes and vascular endothelial cells, but it
must resume lytic replication to disseminate or colonize a new
host. Discovery of the physiological signals that control KSHV
reactivation is thus key to controlling its pathogenic potential.
Suppression of such signals could block the spread of infection,
and pharmacological induction of such signals might flush la-
tently infected cells into lytic replication for elimination by
nucleoside analogue drugs (31).
Impaired cellular immunity plays a critical role in allowing
sarcoma-associatedherpesvirus (KSHV;also
KSHV replication, but the physiological stimuli that positively
induce lytic gene expression are poorly understood. Chemical
agents such as phorbol esters or N-butyrate can reactivate
KSHV in vitro (45, 50, 57), and proinflammatory cytokines
have similar, though weaker effects (7, 44, 46). At the level of
the viral genome, lytic reactivation is mediated by the KSHV-
encoded transcription factor RTA. Overexpression of the RTA
gene is sufficient to trigger lytic replication in latently infected
B-cell lines (37, 70), and RTA mutations can block viral reac-
tivation in vitro (36). RTA protein can also autoactivate the
RTA promoter (16, 25, 62), but the cellular signals that initiate
RTA expression are not well understood.
Several recent studies have shown that high levels of auto-
nomic nervous system activity can accelerate the onset of
AIDS-defining conditions during human immunodeficiency vi-
rus type 1 infection (11, 12, 14). These effects have been at-
tributed to autonomic nervous system regulation of human
immunodeficiency virus type 1 replication (10, 13, 14), but it is
also conceivable that autonomic nervous system activity might
directly activate opportunistic pathogens such as KSHV. The
present studies examined that hypothesis, with an emphasis on
the cellular signal transduction pathways that might allow cat-
echolamine neurotransmitters from the autonomic nervous
system to regulate key molecular events in KSHV reactivation.
Results show that physiological concentrations of epinephrine
and norepinephrine can induce lytic replication of KSHV in
* Corresponding author. Mailing address: Department of Medicine,
Division of Hematology-Oncology, 11-934, Factor Building, David
Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1678.
Phone: 310-267-4243. Fax: 310-206-1318. E-mail: email@example.com.
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latently infected lymphoid cells via ?-adrenergic activation of
the cellular protein kinase A (PKA) signaling pathway. These
effects are mediated by increased expression of RTA and post-
translational enhancement of its trans-activating capacity. Re-
sults provide an endocrinological perspective on the control of
KSHV replication and suggest novel strategies for therapeutic
Cell culture. KSHV reactivation was analyzed in the primary effusion lym-
phoma (PEL) cell lines KS-1, BC3, and BCBL-1. DG75 and Ramos served as B
lymphoid cells uninfected with KSHV or Epstein-Barr virus. Cells were cultured
under standard conditions in RPMI plus 10% fetal bovine serum or in serum-
free X-VIVO 15 (Cambrex, East Rutherford, NJ) supplemented by a defined mix
of growth factors (MITO?, BD Biosciences, Bedford, MA). Reactivation exper-
iments were performed as described (70), using the PKC inducer phorbol-12-
myristate-13-acetate (PMA, also known as TPA; Calbiochem, San Diego CA),
the PKA inducer dibutyrl cyclic AMP (db-cAMP), norepinephrine ([?]-artere-
nol; Sigma-Aldrich, St. Louis, MO), or other indicated agents acting on the
?-adrenoreceptor (?AR)/cAMP/PKA signaling pathway (all from Sigma-Aldrich
or Calbiochem). Norepinephrine and PKA modulating agents were diluted in
phosphate-buffered saline and added to cultures in volumes ?1% of total culture
volume. Unstimulated controls received an equivalent volume of vehicle.
Lytic protein expression. Intracellular expression of KSHV ORF59 was as-
sayed by flow cytometric quantification of indirect immunofluorescence in per-
meabilized KS1, BC3, or BCBL1 cells; 2 ? 106cells were stained for 30 min at
4°C with 1 ?g of mouse-anti KSHV ORF59 monoclonal antibody (Advanced
Biotechnology Incorporated, Colombia, MD) in 50 ?l of BD Cytofix/Cytoperm
(BD Immunocytometry Systems, San Jose, CA), washed in 450 ?l of permeabi-
lization buffer, stained for 30 min with 0.5 ?g of fluorescein isothiocyanate-
conjugated rat anti-mouse immunoglobulin G2b Ab (BD Immunocytometry
Systems), and washed again in 450 ?l of permeabilization buffer. Fluorescence
intensity data were acquired on a FACScan flow cytometer (BD Immunocytom-
etry) with dead cells and debris excluded on the basis of forward- versus side-
scatter gating using CellQuest software. KSHV lytic proteins and cellular ?-actin
were also assayed by Western blot with enhanced chemiluminescence using
KSHV patient serum, as previously described (70).
Lytic gene expression. mRNA for KSHV gene products and human ?-adre-
noreceptor subtypes were quantified relative to cellular housekeeping genes
using real-time reverse transcription (RT)-PCR. Reactions utilized 1/10 of the
total DNase-treated (QIAGEN, Valencia, CA) RNA extracted from 106cells in
a one-step thermal cycling protocol (QIAGEN One-Step RT-PCR) with 30 min
of reverse transcription at 50°C, 15 min. of RT denaturation at 95°C, and 40
cycles of DNA amplification (15 s at 95°C, 60 s at 60°C). Reactions utilized
established primers and fluorescent detection probes for human glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) and KSHV K8.1, ORF29, ORF50,
ORF72, and ORF57 (21). Primers for human ?-adrenoreceptor subtypes were:
?1(forward: TCG GAA TCC AAG GTG TAG GG, reverse:TGG CTT TTC
TCT TTG CCT CG), ?2(forward: CAT GTC TCT CAT CGT CCT GGC CA,
reverse:CAC GAT GGA AGA GGC AAT GGC A), and ?3(forward: GGC
TTC TTG GGG AGT TTC TTA GG, reverse: TTC TGG AGG GTA GAG
TGT CAC AGC), derived from GenBank sequences ADRB1: J03019, ADRB2:
M15169, and ADRB3: X70811, respectively. mRNA expression was normalized
to GAPDH by subtraction of threshold cycles (Ct; normalized target Ct? target
Ct? GAPDH Ct) and quantified as a fold change relative to an unstimulated
baseline (fold change ? 2[stimulated normalized target Ct– baseline normalized
target Ct]).
DNA replication. Replicating KSHV DNA was assayed by Gardella gel anal-
ysis as previously described (23). Briefly, 2 ? 107KS1 cells were lysed during
electrophoresis through a 0.75% agarose gel for 3 h at 0.8 V/cm followed by 17 h
at 4.5 V/cm. Resolved DNA was transferred to a Hybond N? membrane (Am-
ersham-Pharmacia, Buckinghamshire England), UV cross-linked, and probed
with a 3-kb32P-labeled PCR product spanning the majority of the KSHV ORF50
locus. Supernatant particle-associated KSHV DNA was assayed by treating 1 ml
of 0.45-?m-filtered PEL cell supernatants with 2 ?g DNase (Worthington Bio-
chemical Corporation, Lakewood, NJ) and 10 mM MgCl for 15 min, followed by
extraction of particle-protected DNA (QIAGEN MiniElute virus spin kit) and
real-time PCR amplification of KSHV K8.1 DNA (45 cycles of 95°C for 15
seconds and 60°C for 60 seconds) with resolution of products on an 3% agarose
Infectious virus. PEL cell production of infectious KSHV was assayed by
suspending peripheral blood mononuclear cells (PBMC) from healthy donors in
PEL cell supernatants that had been filtered at 0.45 ?m and treated with DNase
as described above; 2 ? 106PBMC were stimulated with PHA for 72 h, washed,
and incubated for 1 h. in 2 ml of cell culture supernatant from PEL cells treated
with phorbol myristate acetate (PMA), norepinephrine, db-cAMP, or vehicle for
48 h. After extensive washing, PBMC were cultured for another 24 h and
establishment of KSHV genomic DNA in PBMC was assayed by PCR detection
of ORF50 (forward: AAC CAG AAG CCT CGG GCG AAG, reverse: GTG
CAC GCC ACG GAT GTC) or K8.1 (forward: AAA GCG TCC AGG CCA
CCA CAG A, reverse: GGC AGA AAA TGG CAC ACG GTT AC) in cellular
DNA (45 cycles of 95°C for 15 seconds and 60°C for 60 seconds, with SYBR
green real-time detection). Cellular RNA was also extracted from 8 ? 106
PBMC, treated with RNase-free DNase (QIAGEN RNEasy), and assayed for
expression of the latent gene product ORF72 by real-time RT-PCR as described
(21). Results were normalized to GAPDH DNA amplified in parallel and visu-
alized on a 2% agarose gel.
Overexpression of PKA catalytic subunit. PEL cells were transduced with a
self-inactivating lentiviral vector expressing a constitutively active form of PKA
(43) and an enhanced green fluorescent protein (EGFP) reporter gene under
control of a recombinant Rh-MLV promoter (34). Both sequences were trans-
lated from a single transcript bearing the PS3 internal ribosome entry site (IRES)
(73). cDNA for the human immunodeficiency virus type 1 central polypyrimidine
tract (77) was introduced into pSIN-18-Rh (34) at the XhoI site upstream of the
Rh murine leukemia virus promoter to produce pSIN18RhMLV-E-CPPT.
cDNA of the PKA catalytic subunit ? (PRKACA: X07767) was amplified with
primers bearing AgeI and SalI restriction sites and subcloned into pCR-Blunt
TOPO (Invitrogen, Carlsbad, CA), sequenced, released by digestion, purified,
and subcloned into AgeI and SalI sites of pSIN18RhMLV-E-CPPT to produce
pSIN18RhMLV-E-CPPT-PKA. The PS3 IRES-EGFP sequence was excised
from pDF-PS3 (73) and subcloned into the NotI site of a circularized pCRII-
TOPO vector (Invitrogen). The EcoRV site upstream of the subcloned fragment
was changed into a SalI site, and a SalI/XhoI fragment was subcloned into
pSIN18RhMLV-E-CPPT-PKA to produce pSIN18RhMLV-E-CPPT-PKA-PS3-
EGFP. The negative control vector pSIN18RhMLV-E-CPPT-EGFP included
EGFP sequences in the absence of upstream PRKACA-PS3 sequences. Vectors
were constructed by transfection into 293T cells (34), and target cells were
transduced by 1 h of incubation in vector-containing supernatants supplemented
with 10 ?g/ml Polybrene.
RTA promoter activity. Luciferase reporter assays utilized a 3-kb sequence
upstream of the RTA translation start site (pRpluc) (16), and three truncation
variants generated by restriction enzyme digestion of pRpluc (pRp1 cut at PstI,
pRp2 at NdeI, and pRp8 at KpnI) to delete six potential cAMP response
elements (CREs) detected by sequence-based bioinformatics (76); 4 ?g of each
reporter construct was electroporated along with 50 ng of the control pRLCMV
(Renilla luciferase driven by the cytomegalovirus [CMV] promoter) (Promega,
Madison, WI) and 6 ?g of empty pcDNA3 vector (10 ?g total) into 107KS-1,
BC3, BCBL-1, or DG75 cells (240 V, 125 ?, 950 ?F) or 5 ? 107Ramos cells
supplemented with 26 ?g/ml DEAE dextran (2 cycles of 320 V, 0 ?, 950 ?F),
using a BTX ECM 630 pulse generator. Dual luciferase assays (Promega) were
performed in triplicate, with firefly luciferase light units normalized to Renilla
luciferase light units prior to analysis of log fold change by Student's t test.
RTA trans-activating capacity. RTA protein was expressed in the context of
RTA promoter assays by replacing 4 ?g of pcDNA3 with pcDNA3/RTA (a
pcDNA3-based vector expressing genomic KSHV RTA under control of the
CMV promoter) (16) or pFLAG/RTA (C-terminally FLAG-tagged KSHV RTA)
(3, 65). To control for any effect of PKA on the CMV promoter (56), data were
normalized to Renilla luciferase levels driven by the CMV promoter (pRLCMV).
To assess RTA-mediated trans-activation of a heterologous KSHV promoter,
luciferase reporter assays were conducted as above after replacing pRpluc with
pLUC/-69, a pGL3-Basic vector expressing firefly luciferase from a 69-nucleotide
fragment of the KSHV PAN RNA promoter (64).
Expressed RTA protein levels were assessed in parallel using flow cytometry to
detect C-terminal EGFP-tagged ORF50 expressed under control of the same
promoter (a kind gift from Joonho Choe, Department of Biological Sciences,
Korean Advanced Institute of Science and Technology) (26). Fluorescence in-
tensity data were acquired on a FACScan flow cytometer (BD Immunocytom-
etry) with dead cells and debris excluded on the basis of forward versus side
scatter gating using CellQuest software. In each of three replicate experiments,
GFP expression was quantified as a percent change above background fluores-
cence levels, with statistical significance of differences assessed by paired t test.
RTA phosphorylation targets. The predicted amino acid sequence of RTA
(AF091348) was scanned by Phosphobase 2.0 (32) to identify consensus PKA
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phosphorylation sites (55). Functional significance of predicted PKA and PKC
phosphorylation candidates at serines 526 (S525) and 526 (S526) was assessed by
converting both codons to alanines through site-directed mutagenesis of
pcDNA3/RTA-FLAG, with PCR primers changing RTA nucleotides 1573 to
1574 from GC to AG to produce S525A; and nucleotide 1576 from G to T to
produce S526A. Mutations were verified by sequencing and characterized in
PAN promoter luciferase assays as above.
Overexpression of ?-adrenoreceptors. To assess the effects of ?-adrenorecep-
tor overexpression in the absence of exogenous ligands, PEL cells were electro-
porated as described above with 4 ?g of mFLAG-?-pcDNA3 (a gift from B.
Kobilka, Stanford University) or pcDNA3 (empty vector). At 24 and 48 h later,
cells were assayed for lytic protein expression by ORF59 flow cytometry as
described above.
Effect of catecholamines on latent KSHV infection. To de-
termine whether autonomic nervous system activity might re-
activate lytic replication of KSHV, we treated three latently
infected B-cell lines with physiological concentrations of the
catecholamine neurotransmitters epinephrine and norepi-
nephrine (18, 39, 58, 61) and assayed lytic protein expression
48 h later. Catecholamine concentrations ranging from 1 nM to
100 ?M activated lytic gene expression in KS-1, BC3, and
BCBL-1 cells when assessed at the level of protein (Fig. 1A)
and mRNA (Fig. 1B). All lytic gene classes were induced,
ranging from the immediate-early gene RTA/ORF50 to the
late lytic gene ORF29 (Fig. 1C). Similar effects were observed
for representative early genes (e.g., ORF59; data not shown).
Norepinephrine induced KSHV DNA replication (Fig. 1D),
and PCR analysis of cell culture supernatants confirmed an
increased concentration of particle-associated KSHV DNA
(Fig. 1E). Supernatants from norepinephrine-treated PEL
cells contained sufficient concentrations of viral particles to
permit de novo infection of primary PBMC (Fig. 1F). Thus,
physiological concentrations of norepinephrine can induce the
entire lytic replication cycle in lymphoid cells latently infected
with KSHV.
Mediating receptors. Most catecholamine effects on leuko-
cytes are mediated by ?-adrenergic receptors (63) (?AR), but
?-adrenoreceptors can also be expressed under certain condi-
tions (30). To identify the specific receptors mediating cate-
cholamine reactivation of KSHV, we pretreated PEL cells with
either the ? antagonist propranalol or the ? antagonist phen-
tolamine for 1 h prior to norepinephrine exposure. ?-Blockade
efficiently inhibited norepinephrine-mediated reactivation of
KSHV lytic protein (Fig. 2A) and mRNA (Fig. 2B). In con-
trast, ?-adrenergic blockade did not affect norepinephrine-
mediated reactivation of KSHV lytic gene expression (Fig. 2B).
Neither inhibitor had any effect on HHV-8 lytic replication in
the absence of exogenous norepinephrine (data not shown).
To define the subtype of ? receptor responsible for these
effects, we assayed mRNA for each of the three known ?AR
types by RT-PCR and found only ?1adrenoreceptor mRNA to
be detectable in appreciable quantities in PEL cells (Fig. 2C).
Catecholamine sensitivity profiles matched the known pharma-
cology of ?1ARs, with epinephrine triggering a low-range in-
crease in KSHV lytic gene expression (?1 nM range) in addi-
tion to paralleling norepinephrine-mediated induction over the
range of 100 nM to 10 ?M (Fig. 1B). ?-Adrenoreceptors re-
quired exogenous stimulation to activate KSHV lytic genes, as
shown by studies in which overexpression of ?-adrenoreceptors
in the absence of adrenergic ligands failed to induce ORF59
protein expression (data not shown; average 0.4% cells positive
by flow cytometry at 24 h versus 0.2% positive for the empty
vector control, difference P ? 0.625).
Role of the cAMP/PKA signaling pathway. In lymphoid cells,
?ARs signal primarily through G?s-mediated activation of the
adenylyl cyclase/cAMP/PKA signaling pathway (29, 63). Other
signaling pathways can potentially be activated (15, 38, 79), so
we sought to verify PKA's role by treating PEL cells with the
PKA antagonist KT5720 prior to catecholamine exposure.
KT5720 inhibited norepinephrine induction of ORF29 mRNA
by more than 90% (Fig. 2D), and had similar effects on RTA/
ORF50 (data not shown). In contrast, the protein kinase C
(PKC) antagonists chelerythrine chloride and bisindolylmale-
imide hydrochloride failed to block norepinephrine-mediated
induction of KSHV lytic genes (results for chelerythrine chlo-
ride shown in Fig. 2D, and similar data for bisindolylmaleimide
hydrochloride not shown). Both PKC antagonists blocked
KSHV reactivation by PMA, but KT5720 had no effect on
PMA-mediated lytic gene expression. Thus, PKA and PKC
represent functionally independent signaling pathways for
KSHV reactivation in PEL cells.
Direct activation of cAMP signaling with the cell-permeable
analogue db-cAMP also activated KSHV lytic gene expression,
and other physiological cAMP inducers such as prostaglandin
E2, and histamine (H2) had similar effects (Fig. 2E). Hormone-
induced reactivation was specific to ligands that stimulate G?s/
adenylyl cyclase/cAMP/PKA signaling. Hormones that signal
through other pathways failed to induce KSHV lytic gene ex-
pression (e.g., acetylcholine or the glucocorticoid receptor ag-
onist dexamethasone) (Fig. 2E).
Activation of PKA is the most common mechanism by which
cAMP regulates cell function, but other cAMP targets have
been identified (17). To determine whether PKA activity can
account for cAMP-induced KSHV reactivation, we expressed a
constitutively active form of PKA in KS-1 and BCBL-1 cells
using a self-inactivating lentiviral vector. The ? catalytic sub-
unit of PKA (PKAc) was expressed from a bicistronic mRNA
that included a C-terminal EGFP reporter sequence indepen-
dently translated via a synthetic internal ribosome entry site.
This vector transduced more then 50% of KS-1 and BCBL-1
cells (Fig. 2F). Expression of the reporter gene alone had
minimal impact on KSHV lytic gene mRNA levels, but expres-
sion of the reporter gene in conjunction with PKAcup-regu-
lated expression of both immediate-early (RTA) and late lytic
transcripts (ORF29) (Fig. 2G). Thus, PKA activity alone is
sufficient to induce lytic gene expression in lymphoid cells
latently infected with KSHV.
Effect of PKA signaling on RTA expression and activity.
KSHV reactivation from latency is controlled by RTA-medi-
ated trans-activation, and we sought to determine how ?AR/
PKA signaling might modulate that process. To evaluate direct
effects on the RTA promoter in the absence of any contribution
from viral gene products, uninfected DG75 or Ramos cells
were electroporated with a reporter construct expressing firefly
luciferase under the control of ?3 kb of KSHV genomic DNA
upstream of ORF50 (previously described in reference 16).
Cells were then stimulated with the PKA activator db-cAMP
or the PKC activator PMA, and luciferase reporter activity was
assessed 18 h later. Reporter gene activity increased by fivefold
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following PKA activation in DG75 cells (Fig. 3A) and 14-fold
in Ramos cells (not shown), indicating that cellular transcrip-
tion factors alone are sufficient to mediate PKA induction of
the RTA promoter. Similar effects emerged in the context of
BC3 and KS-1 PEL cells (Fig. 3A and data not shown, respec-
Several cellular transcription factors respond to PKA, in-
cluding the CREB/ATF family of basic leucine zipper proteins
(5, 40, 47, 53). Bioinformatic analysis of the 3-kb sequence
upstream of ORF50 revealed six putative cAMP response el-
ements (CREs) that could potentially support CREB/ATF-
mediated activation of the RTA promoter (Fig. 3B). To define
the functional significance of these sites, we compared PKA
responsiveness of the full-length promoter (?3.09 kb) with
FIG. 1. Effect of norepinephrine (NE) on KSHV reactivation from latency. (A) Expression of the KSHV lytic cycle protein ORF59 was assayed
by flow cytometry using indirect immunofluorescence in BC3 cells fixed and permeabilized 24 h after exposure to 20 ng/ml PMA, 10 ?M
norepinephrine, or an equivalent volume of vehicle. (B) Dose dependence of catecholamine effects on KSHV lytic gene expression were also
assessed by quantitative real-time RT-PCR detection of KSHV ORF29 mRNA expression (normalized to GAPDH mRNA) 24 h following
exposure of BCBL-1 cells to indicated concentrations of epinephrine or norepinephrine. Similar results were observed for ORF50/RTA mRNA
(data not shown). (C) Kinetics of norepinephrine-induced lytic gene expression were assessed for immediate-early ORF50/RTA and late lytic
ORF29 at the indicated time points by real-time RT-PCR (data represent mean ? standard error fold change in target mRNA concentration in
three replicates after normalization to cellular GAPDH). Norepinephrine induction of ORF50/RTA mRNA was statistically significant at all time
points (12 h, P ? 0.0001; 24 h, P ? 0.0011; 36 h, P ? 0.0004; 48 h, P ? 0.0027; all by t test on log-transformed induction values). Norepinephrine
induction of ORF29 was not significant by 12 h (P ? 0.2086), but was highly significant at all subsequent time points (24 h, P ? 0.0003; 36 h, P
? 0.0006; 48 h, P ? 0.0003). Similar kinetics were observed in BCBL-1 cells (data not shown). (D) Gardella gel analysis of replicating KSHV DNA
was carried out by electrophoresis of KS-1 cell-associated DNA and hybridization with a32P-labeled PCR amplicon from the KSHV ORF50 locus
to distinguish episomal genomic DNA from linear replicating DNA. Data show cells treated for 48 h with vehicle (Control), PMA, norepinephrine,
or the PKA activator db-cAMP. (E) KSHV particle-associated DNA was assayed by PCR amplification of KSHV K8.1 sequences in 20 ?l of
DNase-treated 0.45-?m-filtered supernatant from KS-1 cell cultures treated with vehicle, PMA, or norepinephrine for 48 h (positive control is KS-1
cell-associated DNA). (F) Presence of infectious KSHV in cell culture supernatants from E was assessed by incubating PHA-stimulated PBMC
in filtered/DNased supernatants for 1 h followed by washing. 24 h later, PBMC were assayed for KSHV K8.1 DNA and cellular GAPDH by
real-time PCR. PBMC not exposed to PEL cell supernatants served as negative controls, and KS-1 cells served as positive controls.
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FIG. 2. Role of the ?-adrenergic receptor and cAMP/PKA signaling pathway in norepinephrine-induced reactivation of KSHV. (A) To define
the role of ?-adrenergic receptors in norepinephrine-mediated reactivation of lytic gene expression, BCBL-1 cells were treated with indicated
concentrations of the ?-antagonist propranalol for 1 h before exposure to 10 ?M norepinephrine, and then assayed for KSHV lytic protein
expression by Western blot 48 h later (primary antibody: Kaposi's sarcoma patient serum). Results show induction of the major KSHV lytic protein
species at MW 49,800 which was the only band diagnostic of reactivation by the PMA positive control. (B) Specificity of adrenergic receptor
involvement was tested by treating KS-1 cells with the ?-antagonist phentolamine or the ?-antagonist propranalol for 1 h prior to norepinephrine
(NE) exposure; 24 h later, concentrations of mRNA for the immediate-early ORF50/RTA and late ORF29 were assessed by real-time RT-PCR
(values normalized to GAPDH and expressed as a ratio relative to vehicle-treated controls). (C) Expression of mRNA for ?1, ?2, and ?3adrenergic
receptors was assessed in untreated KS-1 and BCBL-1 cells by real-time RT-PCR. Products were resolved on a 3.5% agarose gel and compared
to positive control PCR products (parallel amplification of genomic DNA for ?1and ?3adrenergic receptors or a PBMC cDNA library for ?2
adrenergic receptors and GAPDH). To verify that RT-PCR results were free of contaminating DNA, BCBL-1 and KS-1 RNA samples were
amplified in parallel in the absence of reverse transcriptase (No RT). Data are representative of four independent experiments in which ?1
adrenergic receptors were consistently detected at high levels, and ?2and ?3receptors were not significantly expressed. (D) To evaluate the role
of PKA and PKC in norepinephrine activation of KSHV lytic gene expression, KS-1 cells were pretreated with the PKA antagonist KT5720 (1 ?M)
or the PKC antagonists chelerythrine chloride (Chel, 1 ?M) or bisindolylmaleimide HCl (300 nM) for 1 h prior to norepinephrine exposure.
Expression of mRNA for the late lytic gene ORF29 was assayed by real-time RT-PCR 24 h later. PKA blockade inhibited norepinephrine-induced
lytic gene expression by ?90%, but PKC inhibitors failed to block norepinephrine effects. Chelerythrine and bisindolylmaleimide (data not shown)
efficiently blocked PMA induction of ORF29, verifying that inhibitors were capable of blocking known PKC activators. KT5720 failed to block
PMA-mediated ORF29 expression, indicating that norepinephrine/PKA and PMA/PKC signaling pathways are functionally independent in PEL
cells. (E) To determine whether cAMP activity was sufficient to induce lytic gene expression, KS-1 cells were treated with graded doses of
pharmacological db-cAMP or with indicated concentrations of physiological cAMP inducers prostaglandin E2(PGE2) and histamine (H2).
cAMP/PKA specificity was evaluated using hormones that signal through alternative pathways, such as acetylcholine (ACh) or the glucocorticoid
dexamethasone (Dex). (F) To determine if PKA activity alone is sufficient to induce lytic gene expression, KS-1 cells were transduced with a
lentiviral vector (34) expressing the catalytic subunit of PKA (PKAc) and a downstream EGFP reporter sequence translated from a single mRNA
bearing the PS3 synthetic internal ribosome entry site (73). At 48 h following transduction with an empty vector, or vector bearing bicistronic EGFP
and PKAc, EGFP-positive cells were quantified by flow cytometry to assess transduction efficiency. Cells transduced with EGFP alone showed
transduction efficiencies comparable to comparable to EGFP plus PKAc(data not shown). (G) Also at 48 h posttransduction, concentrations of
mRNA for immediate-early ORF50/RTA and late lytic ORF29 were quantified by real-time RT-PCR (data normalized to GAPDH and expressed
as a ratio relative to empty vector controls). Data represent the mean (? standard error) of three independent experiments, with statistical
significance evaluated by paired t test. Similar effects were observed with BCBL-1 cells (data not shown).


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Arnold Bax is a neglected composer over all, but his works for choir are even more neglected than some of the rest of his compositions. [Read more...]
The Blog of Dr. James F. McGrath, Clarence L. Goodwin Chair in New Testament Language and Literature at Butler University, Indianapolis
Arnold Bax is a neglected composer over all, but his works for choir are even more neglected than some of the rest of his compositions. [Read more...]
I've been thinking that the Matrix reference in the name of my blog has become rather dated. And so I'm contemplating changing it. Any suggestions for a new name? Should I just call it "James McGrath's Blog," or would something else be better? Silly as well as serious suggestions are welcome. Of course, I could [Read More...]
The blog Two Friars and a Fool has a tongue-in-cheek list of things to watch out for when church shopping. I particularly liked this sign that you should give the church a wide berth: Has "Bible" in the Name Look, any church will have Bibles in it, as well as people who read those Bibles [Read More...]
There has been a lot of speculation, and many rumors, related to the next Doctor after Matt Smith. But today I find myself wondering whether there will be another Doctor. I wonder whether the BBC is not planning on ending the show's run. Trenzalore, we have learned, is the place where the Doctor dies and [Read More...]
I'm not entirely certain whether this is official, but it is still well done. Do you think they are still planning to do something with the Doctor's name along the lines of the hints we had received up until now, as highlighted in the video? Or is that all a red herring? Or is it [Read More...]
If you're expecting the episode "The Name of the Doctor" to make perfect logical sense, you must be new to Doctor Who. As it has from the beginning, Doctor Who skirts the boundary between science fiction and fantasy, and so some of the "science" is always little different from "magic." But on a storytelling and [Read More...]
We were told that, in the episode of Doctor Who "Nightmare in Silver," Neil Gaiman aimed to make the cybermen scary again. It is arguable that he accomplished that, not only by making the cybermen a little scarier, but also making them part of something even scarier than they are. Spoilers ahead! Some speculated about whether the [Read More...]
Tablet magazine has an article about Doctor Who from a Jewish perspective. It includes less well known details, such as the Jewish background of the show's creator Sydney Newman, as well as more familiar topics such as the depiction of Nazism in the form of the Daleks, and the seemingly ineffable name of the Doctor [Read More...]
I recently rewatched the double Doctor Who episode "Silence in the Library" and "Forest of the Dead." Having now seen so much of the later interaction between the Doctor and River Song, and having so many questions answered and details filled in, rewatching the episode where they first introduced River reinforces just how impressively the [Read More...]
Over on the blog A God-Sized Puzzle, Eruesso brought Doctor Who and the Tao Te Ching together and came up with this: This can lead to some interesting discussions at the intersection of religion and science fiction. Human beings hunger revelations, but they fail to satisfy in the long term. You can see this in [Read More...]
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Stroll down Memory Lane revisiting America's greatest generation during and after the war years. Delight once again in the sights of the 1940's - and oh, those incredible, unforgettable songs! The War Front, Home Front, Big Band beat, Swing, Radio and 40's Hollywood and Broadway share center stage in our loving tribute. This is one from the heart!
Veterans of all wars are admitted free - as our honored guests!
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This Holiday Inn is nextdoor to the Eisenhower Avenue Metro Station on the Yellow Line. There is direct service to Regan National Airport and into the city with the first in-city stop at L'Enfant station.
The hotel staff was friendly and helpful. One staff member saw that my father was ill and overheard us discussing going to look for a pharmacy. The staff member offered to let us use the hotel shuttle bus to get to a pharmacy. This was a really appreciated gesture and a sign of a great staff.
The rooms were clean and comfortable.
Parking was free.
I recommend this hotel for folks looking for a less expensive hotel, near the metro, outside of the city.
- Official Description (provided by the hotel):
- Welcome to the Holiday Inn - Alexandria - I-95 hotel at Telegraph Road, a full service hotel conveniently located near the Capital Beltway/I-95, and minutes from Old Towne Alexandria, Washington, DC, Reagan National Airport (DCA), and the Eisenhower Metro Station. Our hotel has many features designed with you in mind: well appointed guest rooms, free high-speed Internet access, complimentary airport shuttle, indoor pool, fitness center, and ample free parking. The Holiday Inn Alexandria also offers the Eisenhower Station Restaurant & Lounge where Kids Eat Free, meeting facilities able to accommodate up to 350 guests, and much more.Area attractions include: the Smithsonian museums; Mt. Vernon, home of George Washington; Washington DC area memorials such as the George Washington Masonic National Memorial, the Vietnam Memorial, the WWII Memorial, and the Holocaust Memorial Museum; Arlington National Cemetery; and the White House. This Alexandria hotel is approximately one mile from Michael Baker Engineering, the Federal Courthouse, the Hoffman Buildings, AMC theatres and Strayer University.We look forward to meeting all of your lodging needs when your business or pleasure travels bring you to the historic Alexandria, Virginia area and our nation's capital ... more less
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A-Rod Misses Yankees Team Picture
OK, simple job for you: find the one non-Yankee player or coach in this photo. Alright, I'll make it easier — Alex Rodriguez missed the Yankees team picture on Tuesday. Instead, team director of publications Alfred Santasiere III stood in for him and will later be photoshopped out. I have no idea why A-Rod missed…Read More


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Ultravox Tickets & Tour Dates 2015 / 2016
New Wave rockers Ultravox are one of the most celebrated gifts that the British electronic pop music movement of the 70s and 80s gave us. Having gone through 10 studio albums with another on the way, the band are still a big force 38 years on from their formation.
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What, you may well ask, was I doing at 3 o'clock yesterday afternoon, drinking beer with a half naked French farmer and his toothless wife?
It wasn't some sort of bizarre 'swinging' thing, although I am reliably informed (by several very unreliable people) that there is an active swinging scene around here. I'm deeply offended that I've never been asked to join!
Before I continue though, I should say that this couple, M et Mme de B are some of the nicest, sweetest people you'll ever meet. They are, however, true French agriculteurs with a rundown farm full of rusting machinery, countless half wild dogs and cats and lots of fleas!
In actual fact I was going to pick up an old school friend of DS who moved to French Guyana a few years ago, but comes back here to spend his summers. Wish I could find someone to have my little darlings for 2 months! The maximum security summer camp is only for a week.
So, we dutifully turned up at 3pm to pick him up from M et Mme de B, with whom he is staying. They invited us in and we sat down at the kitchen table. M, due to the incredible tropical heat we are experiencing, over 30 degrees in the shade yesterday, was stripped down to his shorts. That's the half naked bit sorted then.
We chatted away, or at least they did and I tried to decipher Mme's accent. Its the sort of local accent that you could use to cut granite and the lack of teeth doesn't help. Every other sentence I was whispering out of the corner of my mouth to DS 'whasshe saying?
We talked (I think) about how DS's friend was passing his 2 months in France, had we met up with his Mum when she was over in the Spring, wasn't the weather hot. All the while, waiting for the appearance of said friend. Eventually the son arrived (he's like a mini Sylvester Stallone) so we discussed the impending arrival of his partner's baby, her Braxton Hicks contractions, how difficult it was to be pregnant when it's so flaming hot! Still no friend.
I nodded my head, put in the odd 'oui', 'non' and 'd'accord' and apparently at some stage of the proceedings I said yes to a cold beer. I don't really drink and certainly not during the day in the heat so when it arrived, I explained my misunderstanding and said no to the beer. M de B was having none of it. 'Don't worry, there's no gendarmes around here' he said. Just as well because it's a standing joke among friends how I get stopped by every single gendarme I pass.
Only a few weeks ago I arrived at school with 3 carloads of gendarmes following me.
'Oh look' commented a friend wryly 'here come VLiF with her police escort'!
Eventually, after about 20 minutes of pleasant chat and still no sign of Friend from Guyane, I mouthed to DS 'he's not here is he?' 'No' replied DS.
I backtracked on the conversation and managed to deduce that he was most likely with a Croatian family in the village with an unpronounceable name.
'Ah, il est encore chez Draganononovinic' I slurred. (Told you I can't drink at lunchtime!) 'Oui' they answered in unison, not for one minute wondering why I was, therefore, sitting in their kitchen and not in the kitchen chez Dranogoanivinicic.
We bid our fond adieus, jumped up and down a few times to knock off the fleas and left to try and find chez Dargonvinicicic.
The trouble with Croatians is that they are like policemen, they go everywhere in twos, so we arrived at the little lotissement where live les Dragonvincicivivnics only to find that in our tiny little village, there are, in fact more than one. There are three.
Eventually we were reunited with Friend from Guyane, instantly recognisable by all the flea bits on his legs, which he politely insisted were mosquito bites, and all was well with the world.
Back at the homestead, Mellors the gardener was quietly expiring in the sub-tropical heat but godammit, he still had his shirt on. What's the point of employing a 22 year old gardener if he doesn't at least get his kit off (not all of it you understand)? Note to self to re-write his employment contract. Just my luck that the only bit of chest I've seen today belongs to an elderly French farmer with a paunch you could balance a tray on.
'Gosh, this is like the heatwave in England in 76' I comment. He looks at me blankly. The penny drops. 'You weren't even born then were you?' I ask. 'No ma'am' he replied. (OK, OK, he didn't say ma'am)
I went inside feeling every bit of my 40 something years.
Monday, November 15, 2010
Wednesday, October 7, 2009
Posted by The Accidental Author at 2:35 PM 2 comments:
Saturday, August 22, 2009
And now the end is near........
Well this is it folks, my last hurrah as Mme VLiF. The 24th August is fast approaching and it's time to draw my French blog to a close.
Best of times?
After almost 5 years here it's been a great adventure which I don't regret for a moment but it's time to move on. So, here are a few thoughts and ponderings about our time in France
What do I love?
Our valley
The views from the village
The weather in summer - not this summer. Too bloody hot!
My friends I've made here who I'll miss like mad but who hopefully are already planning their next trip to Wiltshire
Croissants from the lady in the village market on Sundays - but then they probably account for my expanding waistline
The fields when the wheat turns gold
The vast swathes of rape in Spring - apart from the coughing and sneezing it brings!
Sitting on our balcony watching the Milky Way and the shooting stars
What do I hate?
The nastiness that exists on some France forums
The weather for the rest of the year,
French customer services. Actually, that's something of an oxymoron, French and Customer Services!
French TV -- I really tried my best with it but it's pretty awful
The word 'integration'
People who say 'it's like the UK in the 50s' - what with malnutrition, polio, inequality of the sexes, backstreet abortions and hidden domestic violence? It's like France in the 2000s!
The word 'expats', we're not expats, we're immigrants. In fact, most of us are economic migrants really having moved to France so we could buy a bigger house. We are the Poles of France, except most don't actually have to work!
What drives me mad?
That you can't find things in the shops that have been available in the modern world for years - and I don't mean Marmite and Baked Beans, I mean, like ordinary things.
Paying for something in a shop and despite them having a drawer full of change they always want the odd centimes, even if there's 97 of them!
The lack of joined up thinking that seems so prevalent here
So not that much really!
What makes me smile?
The gallic shrug as the answer to everything.
The fact that Barko Sarko seems to be the world's biggest Francophobe, and he made Prez
Monsieur L's dancing
My lovely neighbour for whom I really hope life takes a turn for the better
What has been most suprising?
That despite everything you read it really is the same merde just different shaped bread. Maybe for the retired it's different but for me it just isn't. I'm quite convinced, as I have been for some time, that the authors of these 'Living the Dream' books are all taking mind-altering substances.
How few bilingual British people are actually employed in France.
How many people are prepared to live in absolute penury and accept things they absolutely wouldn't accept in their home countries just to live in a bigger house in France. I mean, it's great but .......
What has been most disappointing?
The education system. Dull, rigid, outdated. It's intended to produce drones who will work uncomplainingly for the ruling elite. So much for Egalité. One of the main reasons we are leaving is because our children, are so unhappy in the school system here. They can't wait to go back to 'proper' school in the UK. Let's hope it's not a disappointment.
Being ripped off by supposed friends. It can happen anywhere but it happened here.
The realisation that French couture doesn't really exist outside Paris. I can happily spend a whole day shopping and find absolutely nothing that I like. Shoes are the worst, hence my attachment to my Crocs
Hairdressers - I've lived in fear of coming out with aubergine or orange hair, or even both
The lack of opportunity for our bilingual British children. The French still seem to prefer to employ a French person with limited English than an English person with fluent French.
Best of times?
Christmas with some of our closest friends at the Chateau de L'hoste, Marchés Gourmands, time spent with friends, barbeque chez OnlyMe and Karlos Fandango, trying and largely failing to win the Quiz night (but we won this week, Allez les filles!!), the childrens' music concerts at Beauville Arts, listening proudly under the shade of the lime trees in Jon and Claire's garden, the High Security Music camp shows and singing my heart out with OnlyMe and of course, the Last Hurrah - our leaving party last weekend, of which I haven't had time to blog, sadly.
Worst of times?
Chopping wood in the winter, in fact anything to do with the winter!
Seeing good friends go through bad times and not being able to do anything to help.
Losing my beloved Mad Baz and Teabag and (whisper) accidentally running over DD's cat on the way to school one day. There.... I've said it. My name is Mme VLif and I've killed a family pet.
Telling the CH that I don't want to live here anymore because he loves it but he must love me more as he's agreed to move back. Aaaah!
Life in France vs Life in the UK?
UK for me I'm afraid. I try really hard to be a good 'expat' but I find after 4-5 years I realise just what a great place, despite everything, the UK really is and what opportunities exist for my children. France is, undoubtedly a great place to retire to but I'm not sure it's the place for families. All the reasearch I've done shows that most children brought up in France return to the UK. DD and DS are keen and very happy to return to the UK. There's lots I could add that has contributed to our decision to leave France but much of it is personal and not for sharing. All I would say is before passing judgement on someone's decision you need to walk in their shoes. I'm sure those of you desperate to leave the UK or whereever you live will think I'm bonkers but don't forget I was you 5 years ago. Sometimes it takes time away to make you realise what you had and also what you want.
Well, that's all folks, as they say. Thank you so much to everyone who has read and enjoyed my blog, to those who have commented and especially to all those clever people who's blogs I enjoy. I shall remain forever indebted to my Mum and Dad for encouraging me to start 'What French Dream'. It will serve as a permanent reminder of our French Adventure, assuming the Interweb doesn't get sucked into a black hole! Au revoir........
Posted by The Accidental Author at 7:53 AM 20 comments:
Labels: au revoir to all that
Wednesday, August 19, 2009
Easter Chicks..... (not for those who are sensitive about religion)
Once again our two cockerels have stepped up to the plate and performed their cockerelly duty so we have three new chicks. One black and two yellow ones that look like Easter chicks.
We decided to give them names that are synonymous with Easter so I give you......
Mary and Joseph!
Posted by The Accidental Author at 12:17 AM 3 comments:
Saturday, August 15, 2009
In which my equilibrium was unbalanced by the sight of so many men in Speedos.....
Eeeuwwww! The French and their speedos!
Today we went to a fabulous waterpark up in Dordogneshire. It was a last jolly for les enfants before we head back to the UK in just over a week
Now, in France they have these funny hygiene laws in swimming pools. Here you can use the toilet and not wash your hands, then go and handle the fresh fruit and veg, you can go to a Marché Gourmand where they will use the same utensils and chopping boards (wooden of course) to handle raw and cooked food, but you can't wear surf shorts in swimming pools. You have to wear speedos.
Now, it's apparently for hygiene reasons, though every time I've asked the pools to clarify this, they can only spout the pre-printed script, but then that's very French.
Now, wearing speedos is fine if you look like this...
Trouble is, most of them look more like this......
Now, it's not easy to eat a baguette when confronted with bulging men's genitals stuffed into lycra and after the first hour I felt that my equilibrium had been well and truly upset. Now, with some men, the promise of what lies beneath might be quite nice but with the majority, what lies beneath is definitely not for my eyes - or anyone else's.
To be honest, I think men's genitals were one of God's little jokes. When left with three small pieces of whatever it is that man is made from, he thought to himself 'now what can I do with these that would be really silly? I know......'
But of course he hadn't banked on a Scotsman emigrating to Australia and thinking, now what can I make out of this little bit of nylon, or on French men embracing the end result with such fervour.
You could tell the French men in speedos. They were the ones who strode around, puffing out their chests saying 'I'm French and proud of my baguette' while the British men, forced into such revealing swimwear, crept around looking and feeling slightly foolish, generally with hands cupped protectively, or maybe just to shield their manhood from prying eyes.
The trouble is, regardless of how unattractive the man, when confronted with 'Man in Speedos' ones eyes seem naturally drawn downwards only to arrive 'youknowhwere', at which point you are suddenly overcome with nausea and horror because to be honest, you don't really want to be reminded that these men.... well... you know what I mean don't you ladies?
Recently, a Muslim lady was banned from wearing a burkhini in a public swimming pool under the same hygiene rules. Personally, I'd like to see all the fat men in speedos forced to wear one in public so I can enjoy my packed lunch in peace.
What do you think?
Posted by The Accidental Author at 1:25 PM 5 comments:
Labels: "life in France", burkhini, speedos
Wednesday, August 5, 2009
A lovely blog award
How ungrateful am I? Hadriana over at Hadriana's Treasures gave me this award last month and I clean forgot about it.
So I would now, in the final weeks of my blog, like to do as she did and pass it on to some of those who have commented on my blog. These will be my final awards so enjoy them!
So, here goes......
Not Waving but Drowning, French Fancy, A Fly in the Web, Completely Alienne, Shakespeare's Housekeeper, @eloh, Expateek, Random Ramblings and Recipes, Working Mum, Le Laquet, DnD, Galli, Just Me Really, Us in France
Take the award and pass it on......
Posted by The Accidental Author at 1:36 PM 9 comments:
Labels: blog awards
Sunday, August 2, 2009
What is it with men.....?
We're now in the final throes of our French adventure and with only three weeks left here we have a mountain of things to do for the lovely tenants who are taking over our house.
Rooms to repaint, airing cupboards to make, kitchen worktops to replace and...... oh God, I can't even bear to think about it..... the cellar to sort out. That in itself will take several days, as we have to sift through our impressive collection of 'hors-service' printers, children's schoolwork dating back to the days when we could legally send them down mines, empty boxes, christmas decorations, halloween decorations, duvets, dead mice and other assorted miscellania - or rubbish some less enlightened folk might say.
Every day I'm up at 7.30am, packing boxes, sorting out stuff for charity or dechetterie, or persuading DD that she really doesn't need to take all of her 180 books, of which she claims to be reading 'every single one'!
On top of that we have holiday guests until the day before we leave so pools still need to be cleaned, laundry to be done, guests to be looked after, hells teeth.. .I'm wearing myself out just typing it!
Around 9.30am I kick the CH out of bed (note that I've already been up an at it for 2 hours) whereup he makes a pot of coffee then spends the rest of the day doing the flaming garden!
Now, you have to bear in mind that we've had no significant rainfall since April and the garden already looks like the aftermath of some mad dictator's 'scorched earth' policy or even the Somme without the trenches. There's barely a thing alive. The grass is a fetching shade of yellow, the trees have a certain autumnal hue about them and the garden is ankle deep in dead leaves, dropped walnuts and windfall plums.
What little colour there was has either been dug up by Prudence the golden non-retriever, in her constant search for a cool place to lie, or eaten by the chickens, who are testing my patience to the maximum at the moment. On the promise of rain last night I put all my window pots of geraniums on the lawn to soak up the (as it turned out) non-existent precipitation. By this morning I had an attractive array of stalks. I can also boast dahlia stalks, petunia stalks and verbena stalks if you're interested.
So I ask you, what's the point? It will all grow back in the end.
I know the CH doesn't want to leave France, but he seems to be in total denial and I foresee a mad, crazy panic in three weeks time.......
Posted by The Accidental Author at 4:07 PM 9 comments:
Labels: "life in France", leaving France
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Clifford Smith
Clifford Barrington (Barrie) Smith, daughter of the late Evaline N. and Milton G. Smith, Jr., died May 28, 2014 in her home. She was the last of her immediate family.
Barrie was born in Tryon, N.C. At the age of 3 months, she and her parents moved from Tryon to Greenville. Her childhood schooling was in Donaldson Elementary, Hughes Middle School, and Greenville High School. Her intense interest in music, particularly voice, was inspired by Mrs. Virginia Uldrick, who was the director of Choral Music at Greenville High School. She began studying in the Preparatory Music Department at Furman University before her graduation. A Greenville School District report in 1991 listed her among distinguished graduates of the Greenville High School.
She won a scholarship to study voice under Dr. John McCrae at Converse College. She received a Bachelor of Music degree with Distinction in Performance. During her years at Converse she spent summers at the Brevard Music Center where she sang many leading roles, and became acquainted with numerous professional singers who were guest artists.
While at Converse she received a scholarship to continue graduate studies at the Juilliard School. There she received a master's degree in voice and opera. During her first year at Julliard she became a member of the American Opera Center, the professional opera company housed at Julliard. That same year she was invited to join the company of the Metropolitan Opera Studio, a touring wing of the Metropolitan Opera.
Barrie was one of only 25 students, from a field of several hundred who auditioned, accepted into the Maria Callas Master Classes. Mme. Callas chose to work with her as a soprano rather than a mezzo soprano, and as a result they became intimate friends. Barrie and Mme. Callas remained friends until Mme. Callas' death.
Among her awards was her acceptance to the Santa Fe Opera Apprentice Artist Program, a grant from the Metropolitan Opera to attend the famed Glyndebourne Festival in England, a grant from the Metropolitan Opera for European Study, a grant from the William Matheus Sullivan Foundation, a Martha Baird Rockefeller grant, and a two year grant from the National Opera Institute.
In 1974 she accepted the position of leading soprano with the Texas Opera Theatre, a new touring wing of the Houston Grand Opera. In 1975 she became the soprano in residence for Houston Grand Opera. In 1976 she was asked to sing the national anthem for the official opening of the South Carolina Bicentennial celebration. She appeared with Bob Hope, Martha Ray, and Minnie Pearl.
While in Houston, Barrie acquired a Master of Social Work degree from the University of Houston. She quietly retired from singing and took a position as counselor to parents of disabled children with the United Cerebral Palsy of Greater Houston. She also opened her own private psychotherapy practice at that time. Before returning to Greenville to care for her mother, she was employed by the Children's Museum of Houston as a consultant and writer while continuing to maintain her private practice.
She was a member of the Augusta Road Baptist Church, the Greenville Historical Society, the Crescent Music Club, the Huguenot Society of SC, and the National Society of the Daughters of the American Revolution, The National Association of Social Workers, and the Phi Kappa Phi, honorary national scholastic society.
Private graveside burial services were conducted at Springwood Cemetery. Memorials may be made to The Augusta Road Baptist Church, 1803 Augusta Road, Greenville, SC 29605, or the Humane Society of Greenville, SC 29605. Cremation Society of South Carolina is serving the Smith family.
Funeral Home
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Published in The Greenville News on June 8, 2014
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Forced to work in a studio at the age of nine, Prakash Tilokani picked the nuances of the art to emerge as a leading wedding photographer having top business families and Bollywood celebs as clients. Kavita Kanan Chandra chronicles his life
Executing water conservation projects in over 4,200 locations is no joke. But Ayyappa Masagi has done that and more to save water, says Ruchita S, who drove 119 km to find out why the much honoured man is called a 'Doctor of Dry Borewells'
A family car, a pickup truck, a mini-generator and more - all rolled into one for a genre called Personal Utility Vehicle. Partho Burman checks out the one of a kind vehicle, Multix MX, that's been launched to meet the needs of entrepreneurs
Music therapy is fast catching up in India with specialists helping patients with a range of ailments from depression to cancer overcome their problems. Nishant Arora tunes in to the trend
Leaders of the women's vigilante group in Manipur that launched a protest after the rape and murder of Thangjam Manorama by armed forces in 2004, want a policy change, says Anjulika Thignam
The latest American dream is dreamt by the Americans and it is to lure more Indian tourists to their nation. Indians visiting US are expected to touch1.85 million by 2021, says Aparajita Gupta
A young crop of photographers in Kashmir is presenting the valley to others through social media, with a new perspective, focusing on the diverse culture, issues and conflict, says Ruwa Shah
Close to the Taj Mahal in Agra is another living monument exemplifying the courage of women scarred by acid attack. Prashant Kumar visits Sheroes Hangout café which is set to start branches
Contrary to popular perceptions, couples in India prefer girls over boys, at least when they have to adopt a child. Azera Parveen Rahman has the statistics proving that girls had more takers
Yana Ngoba has set her sight on the ramps of Paris and Milan after marking presence at London with her designs, showcasing the handloom weaves of northeastern India, says Natalia Ningthoujam
As the first woman from the new state of Telangana to represent India at World Billiards Championship, Aakansha Singh is excited. Mohammed Shafeeq talks to her as she is all set for Adelaide
As a living example of reservation empowering women, a demure housewife Vandana Bahadur Maida turned decision maker in Khankhadvi of Madhya Pradesh and is now plotting the village's growth
Niladri Kumar, who invented the zitar by combining the sitar with guitar, is now taking a plunge into electronic dance music. Ankit Sinha tells us about his new single, 'Head to the Heart'
Where witch hunting really means violence and violation of the dignity of women, Birubala Rabha stood for those branded, only to be killed. Indrani Raimedhi pays tribute to the tribal savior
Savouring the scenic beauty of Bhutan, which is an island of culture, tradition and modernity, Preetha Nair is amazed by the red rice, which was similar to the one she grew up with in Kerala
As India votes, The Weekend Leader's Friends of Positive Journalism Forum gets a new team of office bearers with outgoing Chairman P Saravanaraja handing over the baton to Madan A Sendhil
Photographer Media Ramu captures some of the memorable moments from The Weekend Leader – VIT Chennai Person of the Year Award Function held at VIT Chennai. The Award went to Balasaheb Darade
A first year engineering student, Aditya Bali, used 6500 waste tetra packs, and loads of sawdust for building couple of sound proof rooms at the St. Mary's School, Dwarka, says Deeksha Bhatia
Fossilised dinosaur bones and eggs of gigantic creatures that measured around 18-20 metres and weighed 10-13 tonnes have been discovered in Salbardi area near Amravati in eastern Maharashtra
Fashion designer Jaskeerat Bedi is participating in one of the world's toughest mountain biking events, not to win any title but to spread awareness about breast cancer, says Vishal Gulati
A 15-minute boat ride through the Poovar River took Sanu George to a resort where there were a row of floating cottages and a floating restaurant. The place is 20 km from Thiruvananthapuram
A drone built by a computer science student in Kolkata is capable of camera surveillance, weather forecasting, and even attack and defence, but the DRDO is not responding, says Sahana Ghosh
The unanimous decision of the UPA government and the Congress Working Committee to grant statehood to Telangana sets right a historic blunder committed by Nehru 57 years ago, says Sam Rajappa
Rayna Srivastava, a 16-year-old high school student from Texas, USA, spent her summer vacation teaching English at a girls' home in Mumbai, even skipping a trip to Brazil, says Krishnaraj Rao
Forty sightless people, who are themselves waging a grim battle for survival, have formed an association in West Bengal to extend help to those less fortunate than them, says Ritwik Bagchi
The story of Raj Kumar Gupta reads more like a fairytale. From a mill worker to a millionaire, he has scaled great heights, starting with an apartment building in Hooghly district at a time when no one ever sold ownership flats, says G Singh
Claiming that there is a link between increasing incidence of rapes and other acts of sexual perversion to pornography, Kamlesh Vaswani, a lawyer from Indore, is waging a battle to get a ban on porn websites. Partho Burman spoke to the man
Irrigation wells, schools, a weavers' cooperative and many such schemes have transformed villages in Maharashtra's Sangli and Kolhapur districts. But Arun Chavan, the man behind it all, is not known outside the area, says Kavita Kanan Chandra
Former President A P J Abdul Kalam not just asked the youth to dream but also motivated people, helping them realise their dreams. P C Vinoj Kumar has the story of former army driver V Kathiresan becoming assistant professor, thanks to Kalam
The new 225 feet long bridge across river Ghaggar, linking the villages of Panihari and Alikan in Haryana, is a model project. Costing Rs 1.5 crore, the bridge is an example of a people's initiative, funded by the public, says Partho Burman
From a 'crying bed' that alerts mom to change baby's diaper to 'auto-safe' helmet making distress call in the event of an accident, Rudra Narayan Mukherjee has fabricated a plethora of devices from a tin-roof lab, says Santosh H K Narayan
Once a domestic help, it is just natural that Anuradha Bhosale dedicated her life to protect children from exploitation. Kavita Kanan Chandra tells us how she charted her path out of poverty through education and work among child labourers
"The Weekend Leader not only gives a glimpse of the better things happening around us but also tells stories of people who made it possible."


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Oct 10, 2014
LONDON [MENL] -- Satellite images have confirmed that a leading Iraniannuclear facility was bombed.
Oct 06, 2014
NICOSIA [MENL] -- Iran has reaffirmed plans to launch indigenous satellites.
Sep 22, 2014
WASHINGTON [MENL] -- The United Arab Emirates has launched a communicationssatellite project.
Aug 21, 2014
WASHINGTON [MENL] -- The United States has allowed private contractors tooffer military-grade satellite images.


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Handmade Upcycled Mantle Clock
An inspiring wedding gift from Alex Weiderman.posted in: member junk
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Girl's Day Out in Lanesboro, MN
Come for the day or better yet come for the whole weekend. There will be plenty of fun for all! October 23-25, 2015.posted in: blog, projects
For the latest news and postings, sign up for our junkmail e-letter or subscribe to our rss feeds
It's Fall Color Time!
Let the Fall fun begin in beautiful Minnesota bluff country!posted in: blog, projects
Grate-ful Junk Decor
This is a great grate idea for all you junkers. Insanely simple! Insanely beautiful! Gotta love this kind of junking insanity!posted in: blog, member junk
Hello JMS!
"To invent, you need a good imagination and a pile of junk." -Thomas A. Edison I have incredibly been inspired by the creativity of all on this site! So I thought I'd present my fall centerpiece...posted in: member junk
Uncorked JUNKMARKET Style, Holiday Edition
November 5-7, 2015 at Four Daughters Vineyard and Winery, Spring Valley, MNposted in: blog
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Old Window + Bed Posts + Wood = Plant Stand
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Ph.D. Graduate Scarlet Bowen publishes "The Politics of Custom in Eighteenth-Century British Fiction"
Wed, December 1, 2010
The Politics of Custom in Eighteenth-Century British Fiction breaks new ground in the history of the novel by revealing both the persistent influence of popular culture and of an older, patrician model of social relations. Bowen demonstrates that this "customary culture" had effects not just on novelistic representation, but on the British imagination as a whole. Resisting the view of the novel's rise as one of increasing refinement and politeness, Bowen draws from a variety of popular sources, such as the criminal broadside, ballad, graphic prints, and pantomimes to foreground the eighteenth-century novel's cultural and social hybridity. This book further argues that representations of popular and laboring culture serve as repositories of traditional social values, strategically mobilized by authors such as Defoe, Richardson, Smollett, and Godwin in order to both impede and make palatable Britain's transition to a modern, capitalist and imperial state.
"Bowen makes theories of class relevant to the eighteenth century by understanding them in terms of culture rather than identity. The theoretical and historical gains of this shift are enormous: 'plebian,' 'middling,' and 'patrician' are transformed from static categories into dynamic terms, relational to a concept of English national culture early in the century and responding to radical politics as they change into the nineteenth century."—Kristina Straub, Professor of Literary and Cultural Studies, Carnegie Mellon University
"At last! A book on eighteenth-century fiction that acknowledges and investigates what the novel borrowed from the chapbooks and broadside ballads of customary culture. Bowen shows us how the laboring class popular forms authorized and infused the fledgling novel."—Ruth Perry, Professor of Literature, Massachusetts Institute of Technology
About the Author
Scarlet Bowen is Assistant Professor of English at the University of Colorado at Boulder, where she specializes in eighteenth-century British literature and culture. Her articles have appeared in Eighteenth-Century Life, Studies in Eighteenth-Century Culture, and Women's Studies Quarterly.
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Need tips for surviving a cross country plane ride!
Next week my two kids and I will be flying from CA to NC to go visit grandparents for two weeks. Now my 5 yr old son I'm not worried about because he will be more than happy to...
Started by Amy on 06/02/2011 in Moms Of Babies Born In October 2009
Last update on 06/15/2011 by Lindsay


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Hotel Le Jura
No prices found through our partners. Please contact the business directly or check some of our recommended alternatives.
How has this hotel rated in the past?
- Free High-Speed Internet
- Room service
- Bar/lounge
- Wheelchair accessibility
- Pets allowed
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TripAdvisor Reviews Hotel Le Jura Dijon
Travel Blogs from Dijon
Day 20: Avignon and Beaune
Today started in a very disappointing way, as we walked in to have our breakfast we were greeted with many hungry people. We found very little juice, no bread or hot food, very little cereal (many eating on plates and with forks due to no bowls or spoons), and one of our tour party clearing tables so many of us could sit down. Apparently having two groups eating close to each other didn't work, and the hotel didn't cope very well. On the plus side our tour director did say as ...
Instalment 1 of our trip
... weeks of sun, cheese, baguettes, pate, wine, and all things French. We travel down to the South of France in 2 weeks to visit my Italian cousin (who has 2 girls (more girls!!) ages 3 1/2 and 18 months) who are holidaying near Arles (where Van Gogh lived and painted his famous 'Cafe at Night') and my Aunt who lives near Uzes a really pretty French town not far from Arles.
and so, that's all for now. Keep well. Xxx
Beautiful Burgundy
... call centre number was too fast, so I rang the international help number while we were in Lyon...closed on Sunday! On Monday I reached someone but he couldn't recharge it, I had to ring the French number. No phone! Arrived in Dijon and got a message saying national security required me to identify myself as user of the phone but language skills meant I could not o this. Offto SFR shop in Dijon, , English speaking girl. No she couldn't help me because I bought the SIM card at a tabac ...
Finding Mr. Mustard in Dijon
... appeared to be, permanent locations for their respective businesses. Functioning like a supermarket with competing multiple vendors. Throughout the market many different non-food items were for sale but it was about five grades above the typical flea market in the US.
Notre Dame of Dijon was close by. Built around 1230 in Gothic style, it is much smaller than the Notre Dame ...
Dijon - Not so hot, but very very tasty.
... a shared dessert of 'crepe Normade' (sweet crepe with carmel ice-cream, caramelised apple and Chantilly cream) – OMG+. I'll be dreaming of this meal for years to come!
After lunch we had a little bit of a wander admiring various gourmet shops stocked with regional specialities/epicuries. We were beckoned into a patisserie/chocolaterie by the truly exceptionally exquisite range of desserts. Too embarrassed to take photos sadly ...


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Wallabies face biggest challenge so far
Chris Dutton 5:38 AM A "gutted" Michael Hooper has backed Sean McMahon and Ben McCalman to stand tall in his absence.
White puts hand up for England job
Chris Dutton 5:55 AM Former ACT Brumbies coach and Wallabies candidate Jake White has declared his interest in "the biggest job in world rugby" as the sharks circle for Stuart Lancaster's England job.
Vice-captain Hooper suspended
Injured Folau may miss Wales clash
Live scores - Fiji v Uruguay
Romania come from behind to beat Canada
Ledesma in for long-haul from intern to guru
Editor's picks
Full 2016 Super Rugby Draw
Draw is packed with highlights for Australian Rugby fans.
If your team was a band....
James Polson Still haven't been able to pick a second (or first) side for the World Cup? From ABBA to Anthrax, consult our scientific guide to find out which musical act your pick best resembles.
Your ultimate Cup teams guide
Richard Shute, James Polson, Phil Lutton and Rupert Guinness We bring you all the predictions for every World Cup pool, viewing times for television and betting on all 20 nations contesting the rugby championship tournament to be held in England and Wales.
Wales may not believe enough
Rupert Guinness 6:41 PM Wales have defied a horrendous run of injuries to win their three World Cup pool games so far and shore up a quarter-final berth with their last pool game to go against Australia on Sunday AEDT.
Wallabies prepare for tricks
Chris Dutton 4:00 PM Wallabies lineout general Rob Simmons is preparing for the unexpected, including staying on the lookout for a 13-man lineout at Twickenham.
Henry stunned by England trash talk
David Sygall 1:53 PM World Cup winning coach says he can't believe the 'garbage' that England came up with before their fatal loss to Australia.
Why Australia are the favourites
Liam Napier 11:19 AM After rolling the All Blacks in Sydney some knew then that Australia were genuine World Cup contenders. Now they've blown all their cover.
Beware the wounded dragon
Chris Dutton Beware of the wounded beast and the master coach. That's Michael Cheika's warning to the Wallabies.
Vunipola denies Burgess sledge
Gavin Mairs England's humiliating World Cup exit has plunged the Rugby Football Union into turmoil.
Cheika praises Horne's grunt
Chris Dutton Winger Rob Horne's World Cup fate will be decided in the coming days.
Wales before big Wallabies clash
10:26 AM Welsh preparations for their Rugby World Cup clash with Australia at Twickenham took a spooky twist on Monday with ghostly tales and talk of former King of England Henry VIII.
Insight & Analysis
Giffin cheers , but spares thought for England
Rupert Guinness 3:32 PM David Giffin was as happy as anyone to see the Wallabies beat England last Sunday.
English woes will last for years
Paul Cully A thousand glasses will be raised to the name of Bernard Foley after the Wallabies' superb 33-13 against England on Saturday night.
Douglas stars in sibling soap opera
Rupert Guinness Kane Douglas is one tough fella who provides the Wallabies World Cup squad some old fashioned grunt and plays with a bit of grit, but don't expect to hear him swear on the field.
Wallabies could be down and out
Paul Cully It's been fun watching England agonise over last weekend's loss to Wales but if the Wallabies do not match the hosts' desperation at Twickenham on Saturday their own World Cup campaign could be hanging by a thread.


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Boston Ghosts and Gravestones Tour
Join other adventure seekers and become an apprentice grave digger on an entertaining tour through the darker side of Boston. A scary, fun and informative frightseeing experience that is truly one of a kind.
- Ghosts and Gravestones Tour
- Step aboard the trolley of the doomed and join your host, a 17th-century grave digger
- Step inside two of the oldest burying grounds in Boston
- Visit Copp's Hill Burying Ground, the final resting place of Cotton Mather, a man famous for his "intimate" involvement with the Salem Witch Trials
Recent Photos of This Tour
Our guidePhoto by: Wallace B
Boston Ghosts and Gravestones ...Photo by: Kevan B
10 2014
Boston Ghosts and Gravestones ...Photo by: Kevan B
10 2014
The escapee??Photo by: Sara G
09 2013
Among the grateful deadPhoto by: Wallace B
Spirits in the NightPhoto by: Wallace B
Anthrax stalks his prey.Photo by: Glenn P
Anthrax Makes ContactPhoto by: Glenn P
Anthrax is PossessedPhoto by: Glenn P
What You Can Expect
Step aboard the trolley of the doomed and join your host, a 17th-century grave digger, cursed with a sentence of eternal damnation. This wretched creature is doomed to travel the streets of Boston and relay the stories of the unfortunate souls he encountered and the macabre events he had been witness to during his purgatory.
Hear about Boston's most sinister characters - from the Angel of Death to the king of all killers - the Boston Strangler. Learn about all the haunts that call the "Hub" their home. But that's not all! Step inside two of the oldest burying grounds in Boston for a walking tour you will never forget. Learn the significance of much of the imagery found in these 17th century burial grounds and the famous and not so famous (but even more interesting) occupants therein.
Visit Copp's Hill Burying Ground in the North End, nestled atop the tallest point in one of Boston's oldest historic districts. Here you will visit the final resting place of Cotton Mather, a man famous for his "intimate" involvement with the Salem Witch Trials. Then to the Granary Burying Ground where you will visit the final resting places of many of the famous heroes of the American Revolution including John Hancock, Paul Revere and Samuel Adams.
Remember that half of the tour is walking, so comfortable shoes are recommended (so you don't become "dead" on your feet)!
Customer Reviews
September 2015
This was a blast! It was scary but in a fun way. Our tour guide had me in stitches. He was funny and dramatic and really knew the history of the cemeteries and the dark side of Boston.
August 2015
Please ask for Venora. she interacted with all of my tour !!!!
August 2015
What fun - what amazing information - what amazing 'actors'. Our guides were something else! Scared my granddaughter and she loved it! The facts brought to our attention were very interesting. We went at 7pm which as far as I'm concerned, was the ideal time. You could still see everything, and it was just getting dark. I will recommend this to anyone travelling to Boxton
June 2015
This tour was amazing!
January 2015
This was such a fun tour. We enjoyed it very much.
December 2014
This tour was amazing!
Barry H
October 2014
very informative and enjoyable experience.. good way to learn interesting facts about historic Boston...Anthrax was very entertaining......recommend
July 2014
This was a very fun and informative tour. The tour guides made it even more fun by staying in ghoulish character! In this tour you learned many things about Boston that may not have been obvious. The trolleys were well maintained and overall this was a very interesting tour and easy to do with only a bit of walking.
July 2014
We really enjoy discover Boston with this tour. Thanks
July 2014
Excellent tour both in humor and historical info. Fiona was our tour guide and she was absolutely fantastic !


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With a red and purple 'Love' intarsia, Sonia by Sonia Rykiel's black and cream color-block cotton T-shirt will make a sweetly chic statement. Wear it tucked into a striped skirt for a covetable Left Bank look. Shown here with See by Chloé shoes, Anya Hindmarch bag, Dominic Jones ring, Sonia by Sonia Rykiel skirt and a Miu Miu bracelet.
Black and cream knitted cotton color-block T-shirt with a red and purple 'Love' intarsia at front. Sonia by Sonia Rykiel T-shirt has a scoop neck, short sleeves, white stitching, ribbed trims and simply slips on. 100% cotton. Dry clean.
Product code: 105062 - Need help? Contact us


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Northside Hospital offers a full range of outpatient weight management and nutrition services designed to help you achieve optimal health and feel your best. For more information call 404-236-8036.
Whether you simply want to learn to eat healthy or you have specific dietary requirements, our staff will conduct an individualized nutritional assessment and create a plan just for you.
Services include:
Outpatient Nutrition Services
993 D Johnson Ferry Road
Suite 250
Atlanta, GA 30342
Centralized scheduling:


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