bluuebunny/crossref_metadata_2025 huggingface dataset:
RXR gamma null mice are apparently normal and compound RXR alpha +/-/RXR beta -/-/RXR gamma -/- mutant mice are viable.
Authors: Krezel W , Dupé V , Mark M , Dierich A , Kastner P , Chambon P
Published: July 2012

The RXR gamma (RXR, retinoid X receptor) gene was disrupted in the mouse. Homozygous mutant mice developed normally and were indistinguishable from their RXR gamma +/- or wild-type littermates with respect to growth, fertility, viability, and apparent behavior in the animal facility. Moreover, RXR alpha -/-/RXR gamma -/- and RXR beta -/-/RXR gamma -/- mutant phenotypes were indistinguishable from those of RXR alpha -/- and RXR beta -/- mutants, respectively. Strikingly, RXR alpha +/-/RXR beta -/-/RXR gamma -/- triple mutants were viable. Thus, it appears that RXR gamma does not exert any essential function that cannot be performed by RXR alpha or RXR beta, and one copy of RXR alpha is sufficient to perform most of the functions of the RXRs.
Three-dimensional model of the potyviral genome-linked protein.
Authors: Płochocka D , Wełnicki M , Zielenkiewicz P , Ostoja-Zagórski W
Published: July 2012

The full sequence of the genome-linked viral protein (VPg) cistron located in the central part of potato virus Y (common strain) genome has been identified. The VPg gene codes for a protein of 188 amino acids, with significant homology to other known potyviral VPg polypeptides. A three-dimensional model structure of VPg is proposed on the basis of similarity of hydrophobic-hydrophilic residue distribution to the sequence of malate dehydrogenase of known crystal structure. The 5' end of the viral RNA can be fitted to interact with the protein through the exposed hydroxyl group of Tyr-64, in agreement with experimental data. The complex favors stereochemically the formation of a phosphodiester bond [5'-(O4-tyrosylphospho)adenylate] typical for representatives of picornavirus-like viruses. The chemical mechanisms of viral RNA binding to VPg are discussed on the basis of the model structure of protein-RNA complex.
Cleavage of cyclin A at R70/R71 by the bacterial protease OmpT
Authors: Yam Cain H. , Siu Wai Yi , Kaganovich Daniel , Ruderman Joan V. , Poon Randy Y. C.
Published: July 2012

Previous work has shown that cyclin A can be cleaved at Arg-70/Arg-71 by a proteolytic activity present in anin vitro-coupled transcription/translation system by using rabbit reticulocyte lysate programmed by plasmid DNA encoding p27KIP1, a cyclin-dependent kinase inhibitor, but not by plasmid DNAs encoding other cyclin-dependent kinases inhibitors. Here we report that cyclin A is also cleaved by translation product programmed by plasmid DNA encoding cyclin B. Several findings indicate that the cleavage activity in this assay is provided by the bacterial protease OmpT, which cofractionates with cyclin B and p27KIP1plasmid DNAs and is thus carried over into the coupledin vitrotranscription/translation reactions. (i) Cleavage activity appeared even when transcription or translation of the cyclin B or p27KIP1was blocked. (ii) Activity resembling OmpT, a serine protease that cleaves between dibasic residues, routinely copurifies with p27KIP1and cyclin B plasmid DNAs. (iii) Both cyclin A cleavage activity and OmpT activity are heat stable, resistant to denaturation, and inhibited by Zn2+, Cu2+, or benzamidine. (iv) Cyclin A cleavage activity is detected when using lysates or DNAs prepared fromEscherichia colistrains that contained OmpT but not with strains lacking OmpT. (v) Purified OmpT enzyme itself cleaves cyclin A at R70/R71. These data indicate that OmpT can be present in certain DNA preparations obtained by using standard plasmid purification protocols, and its presence can potentially affect the outcome and interpretation of studies carried out usingin vitro-translated proteins.
Cells adapted to the proteasome inhibitor 4-hydroxy- 5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically active proteasomes for continued survival
Authors: Princiotta Michael F. , Schubert Ulrich , Chen Weisan , Bennink Jack R. , Myung Jayhyuk , Crews Craig M. , Yewdell Jonathan W.
Published: July 2012

The proteasome is the primary protease used by cells for degrading proteins and generating peptide ligands for class I molecules of the major histocompatibility complex. Based on the properties of cells adapted to grow in the presence of the proteasome inhibitor 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone (NLVS), it was proposed that proteasomes can be replaced by alternative proteolytic systems, particularly a large proteolytic complex with a tripeptidyl peptidase II activity. Here we show that NLVS-adapted cells retain sensitivity to a number of highly specific proteasome inhibitors with regard to antigenic peptide generation, accumulation of polyubiquitinated proteins, degradation of p53, and cell viability. In addition, we show that in the same assays (with a single minor exception), NLVS-adapted cells are about as sensitive as nonselected cells to Ala-Ala-Phe-chloromethylketone, a specific inhibitor of tripeptidyl peptidase II activity. Based on these findings, we conclude that proteasomes still have essential proteolytic functions in adapted cells that are not replaced by Ala-Ala-Phe-chloromethylketone-sensitive proteases.
High-throughput development and characterization of a genomewide collection of gene-based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Authors: Buetow Kenneth H. , Edmonson Michael , MacDonald Richard , Clifford Robert , Yip Ping , Kelley Jenny , Little Daniel P. , Strausberg Robert , Koester Hubert , Cantor Charles R. , Braun Andreas
Published: July 2012

We describe here a system for the rapid identification, assay
development, and characterization of gene-based single nucleotide
polymorphisms (SNPs). This system couples informatics tools that mine
candidate SNPs from public expressed sequence tag resources and
automatically designs assay reagents with detection by a chip-based
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry platform. As a proof of concept of this system, a
genomewide collection of reagents for 9,115 gene-based SNP genetic
markers was rapidly developed and validated. These data provide
preliminary insights into patterns of polymorphism in a genomewide
collection of gene-based polymorphisms.
In planta
sequential hydroxylation and glycosylation of a fungal phytotoxin: Avoiding cell death and overcoming the fungal invader
Authors: Pedras M. S. C. , Zaharia I. L. , Gai Y. , Zhou Y. , Ward D. E.
Published: July 2012

To facilitate plant colonization, some pathogenic fungi produce
phytotoxic metabolites that damage tissues; plants may be resistant to
a particular pathogen if they produce an enzyme(s) that catalyzes
detoxification of this metabolite(s). Alternaria blackspot is one of
the most damaging and significant fungal diseases of brassica crops,
with no source of resistance known within the
Brassica
species. Destruxin B is the major phytotoxin produced by the
blackspot-causing fungus,
Alternaria brassicae
(Berkley)
Saccardo. We have established that a blackspot-resistant species
(
Sinapis alba
) metabolized
14
C-labeled
destruxin B to a less toxic product substantially faster than any of
the susceptible species. The first metabolite, hydroxydestruxin B
(
14
C-labeled), was further biotransformed to the
β-
d
-glucosyl derivative at a slower rate. The structures
of hydroxydestruxin B and β-
d
-glucosyl
hydroxydestruxin B were deduced from their spectroscopic data
[NMR, high resolution (HR)-MS, Fourier transform infrared (FTIR)] and
confirmed by total chemical synthesis. Although these hydroxylation and
glucosylation reactions occurred in both resistant (
S.
alba
) and susceptible (
Brassica napus
,
Brassica juncea
, and
Brassica rapa
)
species, hydroxylation was the rate limiting step in the susceptible
species, whereas glucosylation was the rate limiting step in the
resistant species. Remarkably, it was observed that the
hydroxydestruxin B induced the biosynthesis of phytoalexins in
blackspot-resistant species but not in susceptible species. This
appears to be a unique example of phytotoxin detoxification and
simultaneous phytoalexin elicitation by the detoxification product. Our
studies suggest that
S. alba
can overcome the fungal
invader through detoxification of destruxin B coupled with production
of phytoalexins.
Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands
Authors: Jalali Shila , del Pozo Miguel A. , Chen Kuang-Den , Miao Hui , Li Yi-Shuan , Schwartz Martin A. , Shyy John Y.-J. , Chien Shu
Published: July 2012

The aim of this study is to elucidate the role of integrins
in transducing fluid shear stress into intracellular signals in
vascular endothelial cells, a fundamental process in vascular biology.
We demonstrated that shear stress activates specific integrins in
endothelial cells plated on substrates containing the cognate
extracellular matrix (ECM) ligands. The shear stress-induced
mechanotransduction, as manifested by integrin–Shc association, was
abolished when new integrin–ECM ligand interactions were prevented by
either blocking the integrin-binding sites of ECM ligands or
conjugating the integrins to immobilized antibodies. Our results
indicate that the dynamic formation of new connections between
integrins and their specific ECM ligands is critical in relaying the
signals induced by shear stress to intracellular pathways.
Cholesterol binding at the cholesterol recognition/ interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide
Authors: Li Hua , Yao Zhi-xing , Degenhardt Babett , Teper Gary , Papadopoulos Vassilios
Published: July 2012

We previously defined a cholesterol recognition/interaction amino
acid consensus (CRAC;
AT
VL
NY
Y
VW
R
DNS) in the carboxyl
terminus of the peripheral-type benzodiazepine receptor (PBR), an outer
mitochondrial membrane protein involved in the regulation of
cholesterol transport into the mitochondria, the rate-determining step
in steroid biosynthesis. We examined (i) the PBR–cholesterol
interaction by UV crosslinking of the C17 side-chain containing
progestin, promegestone, and (ii) the role of the CRAC domain of PBR in
Leydig cell steroidogenesis by using a transducible peptide composed of
the TAT domain of HIV and the CRAC domain of PBR.
[
3
H]Promegestone photoincorporated into recombinant PBR,
and this labeling was displaced by cholesterol.
[
3
H]Promegestone also photoincorporated into the TAT-CRAC
peptide. [
3
H]Promegestone crosslinking to TAT-CRAC could
be displaced by cholesterol and promegestone, with IC50 values of 1 and
200 μM, respectively. TAT-CRAC efficiently transduced into MA-10
Leydig cells and inhibited the hCG- and cAMP-stimulated steroid
production in a dose-dependent manner. TAT-CRAC did not affect the
hCG-induced cAMP synthesis and the 22R-hydroxycholesterol-supported
steroidogenesis. Mutated TAT-CRAC lost its ability to bind
[
3
H]promegestone and to inhibit the hCG-stimulated
steroidogenesis. These results show that TAT-CRAC binds cholesterol and
competes for cholesterol interaction with endogenous PBR,
suggesting that the cytosolic carboxyl-terminal domain of PBR is
responsible for taking up and bringing steroidogenic cholesterol into
the mitochondria.
Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction
Authors: Raghuram Viswanathan , Mak Don-On Daniel , Foskett J. Kevin
Published: July 2012

The cystic fibrosis transmembrane conductance regulator (CFTR) is a
cAMP-dependent protein kinase- and ATP-regulated chloride channel, the
activity of which determines the rate of electrolyte and fluid
transport in a variety of epithelial tissues. Here we describe a
mechanism that regulates CFTR channel activity, which is mediated by
PDZ domains, a family of conserved protein-interaction modules. The
Na
+
/H
+
exchanger regulatory factor (NHERF)
binds to the cytoplasmic tail of CFTR through either of its two PDZ
(PDZ1 and PDZ2) domains. A recombinant fragment of NHERF (PDZ1–2)
containing the two PDZ domains increases the open probability
(
P
o
) of single CFTR channels in excised
membrane patches from a lung submucosal gland cell line. Both PDZ
domains are required for this functional effect, because peptides
containing mutations in either domain are unable to increase channel
P
o
. The concentration dependence of the
regulation by the bivalent PDZ1–2 domain is biphasic, i.e., activating
at lower concentrations and inhibiting at higher concentrations.
Furthermore, either PDZ domain alone or together is without effect on
P
o
, but either domain can competitively
inhibit the PDZ1–2-mediated stimulation of CFTR. Our results support a
molecular model in which bivalent NHERF PDZ domains regulate channel
gating by crosslinking the C-terminal tails in a single
dimeric CFTR channel, and the magnitude of this regulation is coupled
to the stoichiometry of these interactions.
Conserved interaction between distinct Krüppel-associated box domains and the transcriptional intermediary factor 1 β
Authors: Abrink Magnus , Ortiz José A. , Mark Charlotta , Sanchez Cecilia , Looman Camilla , Hellman Lars , Chambon Pierre , Losson Régine
Published: July 2012

The Krüppel-associated box (KRAB) domain, originally
identified as a 75-aa sequence present in numerous Krüppel-type
zinc-finger proteins, is a potent DNA-binding-dependent transcriptional
repression domain that is believed to function through interaction with
the transcriptional intermediary factor 1 (TIF1) β. On the basis of
sequence comparison and phylogenetic analysis, we have recently defined
three distinct subfamilies of KRAB domains. In the present study,
individual members of each subfamily were tested for transcriptional
repression and interaction with TIF1β and two other closely related
family members (TIF1α and TIF1γ). All KRAB variants were shown,
(
i
) to repress transcription when targeted to DNA
through fusion to a heterologous DNA-binding domain in mammalian cells,
and (
ii
) to interact specifically with TIF1β, but not
with TIF1α or TIF1γ. Taken together, these results implicate
TIF1β as a common transcriptional corepressor for the three distinct
subfamilies of KRAB zinc-finger proteins and suggest a high degree of
conservation in the molecular mechanism underlying their
transcriptional repression activity.
A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic
Authors: Cormont Mireille , Mari Muriel , Galmiche Antoine , Hofman Paul , Le Marchand-Brustel Yannick
Published: July 2012

The small GTPase Rab4 is implicated in endocytosis in all cell
types, but also plays a specific role in some regulated processes. To
better understand the role of Rab4 in regulation of vesicular
trafficking, we searched for an effector(s) that specifically
recognizes its GTP-bound form. We cloned a ubiquitous 69-kDa protein,
Rabip4, that behaves as a Rab4 effector in the yeast two-hybrid system
and in the mammalian cell. Rabip4 contains two coiled-coil domains and
a FYVE-finger domain. When expressed in CHO cells, Rabip4 is present in
early endosomes, because it is colocated with endogenous
Early Endosome Antigen 1, although it is absent from Rab11-positive
recycling endosomes and Rab-7 positive late endosomes. The coexpression
of Rabip4 with active Rab4, but not with inactive Rab4, leads to an
enlargement of early endosomes. It strongly increases the degree of
colocalization of markers of sorting (Rab5) and recycling (Rab11)
endosomes with Rab4. Furthermore, the expression of Rabip4 leads to the
intracellular retention of a recycling molecule, the glucose
transporter Glut 1. We propose that Rabip4, an effector of Rab4,
controls early endosomal traffic possibly by activating a backward
transport step from recycling to sorting endosomes.
A premature-termination mutation in the
Mus musculus
cyclin-dependent kinase 3 gene
Authors: Ye Xin , Zhu Cihui , Harper J. Wade
Published: July 2012

Our understanding of the mammalian cell cycle is due in large part
to the analysis of cyclin-dependent kinase (CDK) 2 and CDK4/6. These
kinases are regulated by E and D type cyclins, respectively, and
coordinate the G
1
/S-phase transition. In contrast, little
is known about CDK3, a homolog of CDK2 and cell division cycle kinase 2
(CDC2). Previous studies using ectopic expression of human CDK3 suggest
a role for this kinase in the G
1
/S-phase transition, but
analysis of the endogenous kinase has been stymied by the
low levels of protein present in cells and by the absence of an
identifiable cyclin partner. Herein we report the presence of a single
point mutation in the
CDK3
gene from several
Mus
musculus
strains commonly used in the laboratory. This mutation
results in the replacement of a conserved tryptophan (Trp-187) within
kinase consensus domain IX with a stop codon. The protein predicted to
be encoded by this allele is truncated near the T loop, which is
involved in activation by CDK-activating kinase. This mutation also
deletes motif XI known to be required for kinase function and is,
therefore, expected to generate a null allele. In stark contrast, CDK3
from two wild-mice species (
Mus spretus and Mus mus
castaneus
) lack this mutation. These data indicate that CDK3 is
not required for
M. musculus
development and suggest
that any functional role played by CDK3 in the G
1
/S-phase
transition is likely to be redundant with another CDK.
A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in
Phycomyces
Authors: Arrach Nabil , Fernández-Martín Rafael , Cerdá-Olmedo Enrique , Avalos Javier
Published: July 2012

Previous complementation and mapping of mutations that change the
usual yellow color of the Zygomycete
Phycomyces
blakesleeanus
to white or red led to the definition of two
structural genes for carotene biosynthesis. We have cloned one of these
genes,
carRA
, by taking advantage of its close linkage
to the other,
carB
, responsible for phytoene
dehydrogenase. The sequences of the wild type and six mutants have been
established, compared with sequences in other organisms, and correlated
with the mutant phenotypes. The
carRA
and
carB
coding sequences are separated by 1,381
untranslated nucleotides and are divergently transcribed. Gene
carRA
contains separate domains for two enzymes,
lycopene cyclase and phytoene synthase, and regulates the overall
activity of the pathway and its response to physical and chemical
stimuli from the environment. The lycopene cyclase domain of
carRA
derived from a duplication of a gene from a common
ancestor of fungi and
Brevibacterium linens
; the
phytoene synthase domain is similar to the phytoene and squalene
synthases of many organisms; but the regulatory functions appear to be
specific to
Phycomyces
.
Untargeted mutation of the maternally derived mouse hypervariable minisatellite allele in F
1
mice born to irradiated spermatozoa
Authors: Niwa Ohtsura , Kominami Ryo
Published: July 2012

Length change mutation at the Ms6hm hypervariable mouse
minisatellite locus was analyzed in C57BL/6N × C3H/HeN
F
1
mice and the F
1
of the reciprocal cross born
to irradiated male parents. Spontaneous mutant frequencies were 8.4%
and 9.8% for the paternally derived and maternally derived C3H/HeN
alleles, respectively. The mutant frequencies for the paternally
derived allele increased to 22% and 19% when the male parents were
irradiated with 6 Gy at the postmeiotic spermatozoa stage and the
spermatogonia stage, respectively. These increases in the mutant
frequency were at least 10 to 100 times higher than those expected from
the frequency of hits to the 3- to 4-kb allele, suggesting that the
length change mutation at this minisatellite locus was not a targeted
event due directly to DNA damage in the region. Further analysis
demonstrated that the mutant frequency increased also at the maternally
derived C3H/HeN allele to 20% when the male parents were irradiated
at the spermatozoa stage. This increase in the maternal allele mutation
was not observed in F
1
born to irradiated spermatogonia.
The present study suggests that introduction of DNA damage by
irradiated sperm triggers genomic instability in zygotes and in embryos
of subsequent developmental stages, and this genomic instability
induces untargeted mutation in cis at the paternally derived
minisatellite allele and in trans at the maternally derived
unirradiated allele. Untargeted mutation revealed in the present study
defines a previously unnoticed genetic hazard to the maternally derived
genome by the paternally introduced DNA damage.
Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism
Authors: Tang Hong , McLachlan Alan
Published: July 2012

Hepatotropism is a prominent feature of hepatitis B virus (HBV)
infection. Cell lines of nonhepatic origin do not independently support
HBV replication. Here, we show that the nuclear hormone receptors,
hepatocyte nuclear factor 4 and retinoid X receptor α plus peroxisome
proliferator-activated receptor α, support HBV replication in
nonhepatic cells by controlling pregenomic RNA synthesis, indicating
these liver-enriched transcription factors control a unique molecular
switch restricting viral tropism. In contrast, hepatocyte nuclear
factor 3 antagonizes nuclear hormone receptor-mediated viral
replication, demonstrating distinct regulatory roles for these
liver-enriched transcription factors.
Maintenance of Epstein–Barr virus (EBV)
oriP
-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1
Authors: Hung Siu Chun , Kang Myung-Soo , Kieff Elliott
Published: July 2012

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting
viral DNA element,
oriP
, enables plasmids to persist in
dividing human cells as multicopy episomes that attach to chromosomes
during mitosis. In investigating the significance of EBNA-1 binding to
mitotic chromosomes, we identified the basic domains of EBNA-1 within
amino acids 1–89 and 323–386 as critical for chromosome binding. In
contrast, the EBNA-1 C terminus (amino acids 379–641), which includes
the nuclear localization signal and DNA-binding domain, does not
associate with mitotic chromosomes or retain
oriP
plasmid DNA in dividing cell nuclei, but does enable the accumulation
of replicated
oriP
-containing plasmid DNA in transient
replication assays. The importance of chromosome association in episome
maintenance was evaluated by replacing EBNA-1 amino acids 1–378 with
cell proteins that have similar chromosome binding characteristics.
High-mobility group-I amino acids 1–90 or histone H1–2 could
substitute for EBNA-1 amino acids 1–378 in mediating more efficient
accumulation of replicated
oriP
plasmid, association
with mitotic chromosomes, nuclear retention, and long-term episome
persistence. These data strongly support the hypothesis that mitotic
chromosome association is a critical factor for episome maintenance.
The replacement of 60% of EBNA-1 with cell protein is a significant
step toward eliminating the need for noncellular protein sequences in
the maintenance of episomal DNA in human cells.
Disfacilitation and active inhibition in the neocortex during the natural sleep-wake cycle: An intracellular study
Authors: Timofeev Igor , Grenier François , Steriade Mircea
Published: July 2012

Earlier extracellular recordings during natural sleep have shown
that, during slow-wave sleep (SWS), neocortical neurons display
long-lasting periods of silence, whereas they are tonically active and
discharge at higher rates during waking and sleep with rapid eye
movements (REMs). We analyzed the nature of long-lasting periods of
neuronal silence in SWS and the changes in firing rates related to
ocular movements during REM sleep and waking using intracellular
recordings from electrophysiologically identified neocortical neurons
in nonanesthetized and nonparalyzed cats. We found that the silent
periods during SWS are associated with neuronal hyperpolarizations,
which are due to a mixture of K
+
currents and
disfacilitation processes. Conventional fast-spiking neurons
(presumably local inhibitory interneurons) increased their firing rates
during REMs and eye movements in waking. During REMs, the firing rates
of regular-spiking neurons from associative areas decreased and
intracellular traces revealed numerous, short-lasting, low-amplitude
inhibitory postsynaptic potentials (IPSPs), that were reversed after
intracellular chloride infusion. In awake cats, regular-spiking neurons
could either increase or decrease their firing rates during eye
movements. The short-lasting IPSPs associated with eye movements were
still present in waking; they preceded the spikes and affected their
timing. We propose that there are two different forms of firing rate
control: disfacilitation induces long-lasting periods of silence that
occur spontaneously during SWS, whereas active inhibition, consisting
of low-amplitude, short-lasting IPSPs, is prevalent during REMs and
precisely controls the timing of action potentials in waking.
Expression of γ-aminobutyric acid ρ1 and ρ1Δ450 as gene fusions with the green fluorescent protein
Authors: Martínez-Torres Ataúlfo , Miledi Ricardo
Published: July 2012

The functional characteristics and cellular localization of
the γaminobutyric acid (GABA) ρ1 receptor and its
nonfunctional isoform ρ1Δ450 were investigated by expressing them
as gene fusions with the enhanced version of the green fluorescent
protein (GFP). Oocytes injected with ρ1-GFP had receptors that gated
chloride channels when activated by GABA. The functional
characteristics of these receptors were the same as for those of
wild-type ρ1 receptors. Fluorescence, because of the chimeric
receptors expressed, was over the whole oocyte but was more intense
near the cell surface and more abundant in the animal hemisphere.
Similar to the wild type, ρ1Δ450-GFP did not lead to the expression
of functional GABA receptors, and injected oocytes failed to generate
currents even after exposure to high concentrations of GABA.
Nonetheless, the fluorescence displayed by oocytes expressing
ρ1Δ450-GFP was distributed similarly to that of ρ1-GFP. Mammalian
cells transfected with the ρ1-GFP or ρ1Δ450-GFP constructs showed
mostly intracellularly distributed fluorescence in confocal microscope
images. A sparse localization of fluorescence was observed in the
plasma membrane regardless of the cell line used. We conclude that
ρ1Δ450 is expressed and transported close to, and perhaps
incorporated into, the plasma membrane. Thus, ρ1- and ρ1Δ450-GFP
fusions provide a powerful tool to visualize the traffic of GABA type C
receptors.
Estrogen receptor α, not β, is a critical link in estradiol-mediated protection against brain injury
Authors: Dubal Dena B. , Zhu Hong , Yu Jin , Rau Shane W. , Shughrue Paul J. , Merchenthaler Istvan , Kindy Mark S. , Wise Phyllis M.
Published: July 2012

Estradiol protects against brain injury, neurodegeneration,
and cognitive decline. Our previous work demonstrates that
physiological levels of estradiol protect against stroke injury and
that this protection may be mediated through receptor-dependent
alterations of gene expression. In this report, we tested the
hypothesis that estrogen receptors play a pivotal role in mediating
neuroprotective actions of estradiol and dissected the potential
biological roles of each estrogen receptor (ER) subtype, ERα and
ERβ, in the injured brain. To investigate and delineate these
mechanisms, we used ERα-knockout (ERαKO) and ERβ-knockout
(ERβKO) mice in an animal model of stroke. We performed our studies
by using a controlled endocrine paradigm, because
endogenous levels of estradiol differ dramatically among
ERαKO, ERβKO, and wild-type mice. We ovariectomized ERαKO,
ERβKO, and the respective wild-type mice and implanted them with
capsules filled with oil (vehicle) or a dose of 17β-estradiol that
produces physiological hormone levels in serum. One week later, mice
underwent ischemia. Our results demonstrate that deletion of ERα
completely abolishes the protective actions of estradiol in all regions
of the brain; whereas the ability of estradiol to protect against brain
injury is totally preserved in the absence of ERβ. Thus, our results
clearly establish that the ERα subtype is a critical mechanistic link
in mediating the protective effects of physiological levels of
estradiol in brain injury. Our discovery that ERα mediates protection
of the brain carries far-reaching implications for the selective
targeting of ERs in the treatment and prevention of neural dysfunction
associated with normal aging or brain injury.
A second-generation vaccine protects against the psychoactive effects of cocaine
Authors: Carrera M. Rocío A. , Ashley Jon A. , Wirsching Peter , Koob George F. , Janda Kim D.
Published: July 2012

The effects of immunization with the second-generation cocaine
immunoconjugate GND-keyhole limpet hemocyanin (KLH) or with the
anti-cocaine mAb GNC92H2 were assessed in a model of acute
cocaine-induced locomotor activity. After i.p. administration of
cocaine⋅HCl (15 mg/kg), rats were tested in photocell cages, and
stereotypy was rated to determine preimmunization drug response
(baseline). Experimental animals were subjected to an immunization
protocol with GND-KLH or treated with the mAb GNC92H2. Rats were then
challenged with systemic cocaine, and their locomotor responses were
again measured. Active immunization with GND-KLH produced a 76%
decrease in the ambulatory measure (crossovers) in the experimental
group and a 12% increase in the control group compared with baseline
values. Also, stereotypic behavior was significantly suppressed in the
vaccinated animals. Decreases in both measures were seen in the
experimental group on two subsequent challenges. The maximum effect was
observed at the time of the second challenge with a dramatic 80%
decrease in crossovers. Treatment with GNC92H2 resulted in a 69%
decrease in crossovers compared with baseline. This effect persisted
across two additional challenges over 11 days with decreases of
46–47%. In contrast, the control group showed increases of up to
28%. Significant differences between groups were observed in the
stereotypic measure in all three challenges. The results indicate that
these immunopharmacotherapeutic agents have significant
cocaine-blockade potential and therefore may offer an effective
strategy for the treatment of cocaine abuse.
A MEASUREMENT OF SECONDARY COSMIC MICROWAVE BACKGROUND ANISOTROPIES WITH TWO YEARS OF SOUTH POLE TELESCOPE OBSERVATIONS
Authors: Reichardt C. L. , Shaw L. , Zahn O. , Aird K. A. , Benson B. A. , Bleem L. E. , Carlstrom J. E. , Chang C. L. , Cho H. M. , Crawford T. M. , Crites A. T. , de Haan T. , Dobbs M. A. , Dudley J. , George E. M. , Halverson N. W. , Holder G. P. , Holzapfel W. L. , Hoover S. , Hou Z. , Hrubes J. D. , Joy M. , Keisler R. , Knox L. , Lee A. T. , Leitch E. M. , Lueker M. , Luong-Van D. , McMahon J. J. , Mehl J. , Meyer S. S. , Millea M. , Mohr J. J. , Montroy T. E. , Natoli T. , Padin S. , Plagge T. , Pryke C. , Ruhl J. E. , Schaffer K. K. , Shirokoff E. , Spieler H. G. , Staniszewski Z. , Stark A. A. , Story K. , van Engelen A. , Vanderlinde K. , Vieira J. D. , Williamson R.
Published: July 2012

We present the first three-frequency South Pole Telescope (SPT) cosmic microwave background (CMB) power spectra. The band powers presented here cover angular scales 2000 < ℓ < 9400 in frequency bands centered at 95, 150, and 220 GHz. At these frequencies and angular scales, a combination of the primary CMB anisotropy, thermal and kinetic Sunyaev–Zel'dovich (SZ) effects, radio galaxies, and cosmic infrared background (CIB) contributes to the signal. We combine Planck/HFI and SPT data at 220 GHz to constrain the amplitude and shape of the CIB power spectrum and find strong evidence for nonlinear clustering. We explore the SZ results using a variety of cosmological models for the CMB and CIB anisotropies and find them to be robust with one exception: allowing for spatial correlations between the thermal SZ effect and CIB significantly degrades the SZ constraints. Neglecting this potential correlation, we find the thermal SZ power at 150 GHz and ℓ = 3000 to be 3.65 ± 0.69 μK2, and set an upper limit on the kinetic SZ power to be less than 2.8 μK2 at 95% confidence. When a correlation between the thermal SZ and CIB is allowed, we constrain a linear combination of thermal and kinetic SZ power: D
tSZ
3000 + 0.5D
3000
kSZ = 4.60 ± 0.63 μK2, consistent with earlier measurements. We use the measured thermal SZ power and an analytic, thermal SZ model calibrated with simulations to determine σ8 = 0.807 ± 0.016. Modeling uncertainties involving the astrophysics of the intracluster medium rather than the statistical uncertainty in the measured band powers are the dominant source of uncertainty on σ8. We also place an upper limit on the kinetic SZ power produced by patchy reionization; a companion paper uses these limits to constrain the reionization history of the universe.
AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway
Authors: Dores Michael R. , Paing May M. , Lin Huilan , Montagne William A. , Marchese Adriano , Trejo JoAnn
Published: July 2012

The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.
RedoxDB—a curated database for experimentally verified protein oxidative modification
Authors: Sun Ming-an , Wang Yejun , Cheng Han , Zhang Qing , Ge Wei , Guo Dianjing
Published: July 2012

Summary: Redox regulation and signaling, which are involved in various cellular processes, have become one of the research focuses in the past decade. Cysteine thiol groups are particularly susceptible to post-translational modification, and their reversible oxidation is of critical role in redox regulation and signaling. With the tremendous improvement of techniques, hundreds of redox proteins along with their redox-sensitive cysteines have been reported, and the number is still fast growing. However, until now there is no database to accommodate the rapid accumulation of information on protein oxidative modification. Here we present RedoxDB—a manually curated database for experimentally validated redox proteins. RedoxDB (version 1.0) consists of two datasets (A and B, for proteins with or without verified modified cysteines, respectively) and includes 2157 redox proteins containing 2203 cysteine residues with oxidative modification. For each modified cysteine, the exact position, modification type and flanking sequence are provided. Additional information, including gene name, organism, sequence, literature references and links to UniProt and PDB, is also supplied. The database supports several functions including data search, blast and browsing. Bulk download of the entire dataset is also available. We expect that RedoxDB will be useful for both experimental studies and computational analyses of protein oxidative modification.

Availability: The database is freely available at: http://biocomputer.bio.cuhk.edu.hk/RedoxDB.

Contact:  djguo@cuhk.edu.hk

Supplementary Information:  Supplementary data are available at Bioinformatics Online.
Oligogalacturonides: Novel Signaling Molecules in Rhizobium-Legume Communications
Authors: Moscatiello Roberto , Baldan Barbara , Squartini Andrea , Mariani Paola , Navazio Lorella
Published: July 2012

Oligogalacturonides are pectic fragments of the plant cell wall, whose signaling role has been described thus far during plant development and plant-pathogen interactions. In the present work, we evaluated the potential involvement of oligogalacturonides in the molecular communications between legumes and rhizobia during the establishment of nitrogen-fixing symbiosis. Oligogalacturonides with a degree of polymerization of 10 to 15 were found to trigger a rapid intracellular production of reactive oxygen species in Rhizobium leguminosarum bv. viciae 3841. Accumulation of H2O2, detected by both 2′,7′-dichlorodihydrofluorescein diacetate–based fluorescence and electron-dense deposits of cerium perhydroxides, was transient and did not affect bacterial cell viability, due to the prompt activation of the katG gene encoding a catalase. Calcium measurements carried out in R. leguminosarum transformed with the bioluminescent Ca2+ reporter aequorin demonstrated the induction of a rapid and remarkable intracellular Ca2+ increase in response to oligogalacturonides. When applied jointly with naringenin, oligogalacturonides effectively inhibited flavonoid-induced nod gene expression, indicating an antagonistic interplay between oligogalacturonides and inducing flavonoids in the early stages of plant root colonization. The above data suggest a novel role for oligogalacturonides as signaling molecules released in the rhizosphere in the initial rhizobium–legume interaction.
A Polyketide Synthase Gene, ACRTS2, Is Responsible for Biosynthesis of Host-Selective ACR-Toxin in the Rough Lemon Pathotype of Alternaria alternata
Authors: Izumi Y. , Ohtani K. , Miyamoto Y. , Masunaka A. , Fukumoto T. , Gomi K. , Tada Y. , Ichimura K. , Peever T. L. , Akimitsu K.
Published: July 2012

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including β-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, β-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.
PRESCRIBING AIDS FOR GENTAMICIN
Authors: MAWER G.E. , AHMAD R. , DOBBS SYLVIA M. , McGOUGH J.G. , LUCAS S.B. , TOOTH J.A.
Published: July 2012

A nomogram and a digital computer program have been developed to calculate dosage schedules of gentamicin for individual patients. The minimum input data consist of the patients' age, sex, body weight and serum creatinine concentration.

These prescribing aids have been evaluated in 36 patients with severe Gram negative infections. Renal function ranged from normal to complete anuria. Nomogram dosage schedules gave serum concentrations of gentamicin within the chosen therapeutic limits. Physician dosage schedules gave serum concentrations which sometimes exceeded and sometimes fell below these limits. The validity of the computer program was demonstrated by its ability to predict serum concentrations of gentamicin whatever the dosage schedule.

Half the patients recovered from the bacterial infection but seven remained infected and eleven died. Pseudomonas aeruginosa was the most difficult organism to eradicate.

Four of the patients who survived developed ataxia and two developed hearing loss at high frequencies. The risk of ototoxicity was a function of mean trough serum gentamicin concentration and duration of treatment. Ototoxicity was only detected in patients with serum creatinine concentrations above 3 mg/100 ml who tended to have higher trough concentrations. When treatment was prolonged beyond 8–10 days the risk of ototoxicity was increased without evidence of further substantial therapeutic benefit.
ACUTE EFFECTS OF VARYING DOSES OF PROPRANOLOL UPON OXYGEN HAEMOGLOBIN AFFINITY IN MAN
Authors: BRAIN M.C. , CARD R.T. , KANE J. , LYONNAIS J. , DOLLERY C.T.
Published: July 2012

The effect of propranolol 2 h following doses of 10, 30, or 100 mg upon the blood oxygen‐haemoglobin dissociation curves has been studied in four normal subjects.

The pO2 at 50% saturation (p50) was not changed significantly under control conditions or after 10 and 100 mg doses. There was a just significant increase of 2 mmHg in the p50 value after the 30 mg dose.

There was no significant change in red cell adenosine triphosphate or 2, 3‐diphospho‐glycerate nor in plasma phosphate. 4 There was no correlation between plasma propranolol concentration and changes in blood p50.
AVAILABILITY OF METHAQUALONE FROM COMMERCIAL PREPARATIONS: in vitro AND in vivo STUDIES IN MAN
Authors: WILLIAMS M.E. , KENDALL M.J. , MITCHARD M. , DAVIS S.S. , POXON R.
Published: July 2012

The plasma profiles of methaqualone obtained from different commercially available preparations have been compared. Tablets were absorbed more efficiently than capsules. Mandrax preparations achieved much higher plasma levels than Melsedin tablets or the Melsed or Sedaquin capsules. These differences appear to be due to formulation factors.

The efficacy of the different drugs as hypnotics corresponded well with the blood levels achieved.

There was a significant correlation between the in vitro 1/t50% and the in vivo peak plasma levels. Thus in vitro dissolution studies can be used to predict the efficiency of absorption of different formulations in man.
INDOMETHACIN OR AMYLOBARBITONE SODIUM FOR SLEEP IN RHEUMATOID ARTHRITIS, WITH SOME OBSERVATIONS ON THE USE OF SEQUENTIAL ANALYSIS
Authors: HUSKISSON E.C. , GRAYSON M.F.
Published: July 2012

A double blind crossover trial in patients with rheumatoid arthritis showed that indomethacin given at night was superior to a hypnotic in providing sleep and relieving morning stiffness.

In a second trial, the combination of a hypnotic with indomethacin was not superior to indomethacin alone as judged by patients' preference, but further analysis suggested that the combination improved sleep without affecting morning stiffness.

It is argued that the use of a small closed sequential design produced too small a sample in the first trial. In the second trial, the sample was relatively large but the result was inconclusive; measurement of the response in terms of preference alone obscured the effect of treatment on one aspect of the symptomatology studied.
RATE OF ELIMINATION OF TRACER DOSES OF PHENYTOIN AT DIFFERENT STEADY‐STATE SERUM PHENYTOIN CONCENTRATIONS IN EPILEPTIC PATIENTS
Authors: HOUGHTON G.W. , RICHENS A.
Published: July 2012

Serum phenytoin concentration, the serum half‐life of a tracer dose of carbon‐labelled phenytoin, and the ratio of the major metabolite of phenytoin to unchanged drug in urine (p‐HPPH: DPH ratio) were measured in epileptic patients on chronic anticonvulsant therapy.

A significant correlation was found between serum phenytoin concentration and half‐life, the slope of the regression line being dose dependent.

A significant negative correlation was found between serum phenytoin concentration and p‐HPPH: DPH ratio.

Increasing the daily dose of phenytoin lead to a lengthening of the half‐life and a reduction in the p‐HPPH: DPH ratio. The reverse occurred on lowering the dose.

These changes indicate that phenytoin hydroxylation is saturable.

Difficulty in achieving a stable serum phenytoin concentration within the therapeutic range may result.
THE EFFECTS OF SALBUTAMOL AND TERBUTALINE ON PHYSIOLOGICAL TREMOR, BRONCHIAL TONE AND HEART RATE
Authors: WATSON JUDITH M. , RICHENS A.
Published: July 2012

The effects of oral doses of the bronchodilator drugs, salbutamol (4 and 8 mg) and terbutaline (5 and 10 mg), on physiological tremor, bronchial tone, heart rate and blood pressure were studied in six normal subjects.

Both drugs produced a significant increase in physiological tremor, compared with placebo. Terbutaline, but not salbutamol, produced a dose related response. No significant differences were observed between drugs.

Terbutaline (5 and 10 mg) and salbutamol (8 mg) produced a significant bronchodilatation compared with placebo. Dose related responses were obtained with both drugs, although this reached significance only with terbutaline.

Both doses of each drug produced a tachycardia, although this was only statistically significant with the high doses.

No significant change in blood pressure was observed with either drug.

Peak effects of salbutamol occurred between 1 and 3 h and terbutaline between 1 and 4 hours. Both drugs were still active at 6 hours.
THE INFLUENCE OF URINARY pH ON THE ELIMINATION OF MEXILETINE
Authors: KIDDIE MOIRA A. , KAYE CM. , TURNER P. , SHAW T.R.D.
Published: July 2012

The plasma elimination half‐life of mexiletine was measured in four subjects when the urine was rendered (a) acidic and (b) alkaline.

Urinary acidification (pH 5.0) was associated with a plasma elimination half‐life of 2.8 h and 57.5% of the intravenous dose was excreted in the urine within 48 hours. When the urine was alkaline (pH 8.0) the half‐life increased to 8.6 h with negligible amounts of drug appearing in the urine.

Urinary pH varies widely in cardiac patients and should be controlled or monitored to provide better therapeutic precision with mexiletine.
THE EFFECT OF PREDNISONE TREATMENT ON THE HALF‐LIFE OF INTRAVENOUS HYDROCORTISONE
Authors: SHENFIELD GILLIAN M. , PATERSON J.W. , COSTELLO J.F. , IJADUOLA O.
Published: July 2012

The half‐life of hydrocortisone (Cortisol) has been measured in six patients before and after a three‐week course of prednisone given for asthma.

There was considerable variation in peak plasma level of Cortisol both within and between patients, but on both occasions the plasma half‐lives were the same.

It was concluded that short courses of prednisone have no effect on the handling of hydrocortisone.
THE PERSISTENCE OF ADRIAMYCIN IN MAN AND RAT
Authors: WILKINSON P.M. , MAWER G.E.
Published: July 2012

Adriamycin was administered as a single intravenous dose. The plasma concentration in man and rat decayed as a double exponential function of time.

A two compartment model was used to predict plasma and tissue concentrations in man and the rat. The validity of the tissue concentration predictions was confirmed experimentally in the rat.

Tissue components had a high capacity for the drug. There was evidence in the rat of preferential accumulation in the liver, spleen and bone marrow.

The large tissue capacity and the prolonged half time for elimination predispose to accumulation. This may be responsible for the greater toxicity of treatment schedules with short intervals between consecutive doses.