bluuebunny/crossref_metadata_2025 huggingface dataset:
Antioxidant activity of Ferrozine–iron–amino acid complexes
Authors: Berlett Barbara S. , Levine Rodney L. , Chock P. Boon , Chevion Mordechai , Stadtman Earl R.
Published: July 2012

Amino acid–Fe(II)–chelator complexes exhibit strong
antioxidant activity. Taking advantage of the unique spectral
characteristics of the complexes formed when Ferrozine (Fz) is used as
the chelator, we now show that the primary blue complex
(ɛ
max
at 632 nm) decomposes by two independent pathways:
(
i
) a nonoxidative pathway involving dissociation of the
amino acid component and formation of a purple complex
(ɛ
max
at 562 nm) and (
ii
) an oxidative
pathway leading to Fe(III) and colorless products. Quantitative
conversion of the blue to purple complex yields an isosbestic point
(i.p.) at 601 nm, whereas no i.p. is formed during quantitative
oxidation of the blue complex. However, under some experimental
conditions, decomposition of the blue product occurs by both pathways,
leading to occurrence of a clean i.p. at wavelengths varying from 601
to 574 nm. Results of simulation experiments, confirmed by direct
analysis, demonstrate that shifts in the i.p. reflect differences in
the fractions of blue compound that decompose by the oxidative and
nonoxidative pathways. Indeed, the fraction of blue that is converted
to the purple complex is readily deduced from the wavelength of the
i.p. These results suggest that identification of a physiological
chelator that can replace Ferrozine in amino acid–iron complexes might
have important physiological and pharmacological applications.
Stepwise unfolding of titin under force-clamp atomic force microscopy
Authors: Oberhauser Andres F. , Hansma Paul K. , Carrion-Vazquez Mariano , Fernandez Julio M.
Published: July 2012

Here we demonstrate the implementation of a single-molecule force
clamp adapted for use with an atomic force microscope. We show that
under force-clamp conditions, an engineered titin protein elongates in
steps because of the unfolding of its modules and that the waiting
times to unfold are exponentially distributed. Force-clamp measurements
directly measure the force dependence of the unfolding probability and
readily captures the different mechanical stability of the I27 and I28
modules of human cardiac titin. Force-clamp spectroscopy promises to be
a direct way to probe the mechanical stability of elastic proteins such
as those found in muscle, the extracellular matrix, and cell adhesion.
An
Agrobacterium
VirE2 channel for transferred-DNA transport into plant cells
Authors: Dumas Fabrice , Duckely Myriam , Pelczar Pawel , Van Gelder Patrick , Hohn Barbara
Published: July 2012

Transferred DNA (T-DNA) transfer from
Agrobacterium
tumefaciens
into eukaryotic cells is the only known example of
interkingdom DNA transfer. T-DNA is a single-stranded segment of
Agrobacterium
's tumor-inducing plasmid that enters the
plant cell as a complex with the bacterial virulence proteins VirD2 and
VirE2. The VirE2 protein is highly induced on contact of
A.
tumefaciens
with a plant host and has been reported to act in
late steps of transfer. One of its previously demonstrated functions is
binding to the single-stranded (ss) T-DNA and protecting it from
degradation. Recent experiments suggest other functions of the protein.
A combination of planar lipid bilayer experiments, vesicle swelling
assays, and DNA transport experiments demonstrated that VirE2 can
insert itself into artificial membranes and form channels. These
channels are voltage gated, anion selective, and single-stranded
DNA-specific and can facilitate the efficient transport of
single-stranded DNA through membranes. These experiments demonstrate a
VirE2 function as a transmembrane DNA transporter, which could have
applications in gene delivery systems.
Regulating ankyrin dynamics: Roles of sigma-1 receptors
Authors: Hayashi Teruo , Su Tsung-Ping
Published: July 2012

Ankyrin is a cytoskeletal adaptor protein that controls important
cellular functions, including Ca
2+
efflux at inositol
1,4,5-trisphosphate receptors (IP
3
R) on the endoplasmic
reticulum. The present study found that sigma-1 receptors (Sig-1R),
unique endoplasmic reticulum proteins that bind certain steroids,
neuroleptics, and psychotropic drugs, form a trimeric complex with
ankyrin B and IP
3
R type 3 (IP
3
R-3) in NG-108
cells. The trimeric complex could be coimmunoprecipitated by antibodies
against any of the three proteins. Sig-1R agonists such as pregnenolone
sulfate and cocaine caused the dissociation of an ankyrin B isoform
(ANK 220) from IP
3
R-3. This effect caused by Sig-1R
agonists was blocked by a Sig-1R antagonist. The degree of dissociation
of ANK 220 from IP
3
R-3 caused by Sig-1R ligands correlates
excellently with the ligands' efficacies in potentiating the
bradykinin-induced increase in cytosolic free Ca
2+
concentration. Immunocytohistochemistry showed that Sig-1R, ankyrin B,
and IP
3
R-3 are colocalized in NG-108 cells in perinuclear
areas and in regions of cell-to-cell communication. These results
suggest that Sig-1R and associated ligands may play important roles in
cells by controlling the function of cytoskeletal proteins and that the
Sig-1R/ANK220/IP
3
R-3 complex regulating
Ca
2+
signaling may represent a site of action for
neurosteroids and cocaine.
LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes
Authors: Laffitte Bryan A. , Repa Joyce J. , Joseph Sean B. , Wilpitz Damien C. , Kast Heidi R. , Mangelsdorf David J. , Tontonoz Peter
Published: July 2012

Apolipoprotein E (apoE) secreted by macrophages in the artery
wall exerts an important protective effect against the development of
atherosclerosis, presumably through its ability to promote lipid
efflux. Previous studies have shown that increases in cellular free
cholesterol levels stimulate apoE transcription in macrophages and
adipocytes; however, the molecular basis for this regulation is
unknown. Recently, Taylor and colleagues [Shih, S. J., Allan, C.,
Grehan, S., Tse, E., Moran, C. & Taylor, J. M. (2000)
J. Biol. Chem.
275, 31567–31572] identified two
enhancers from the human apoE gene, termed multienhancer 1 (ME.1) and
multienhancer 2 (ME.2), that direct macrophage- and adipose-specific
expression in transgenic mice. We demonstrate here that the nuclear
receptors LXRα and LXRβ and their oxysterol ligands are key
regulators of apoE expression in both macrophages and adipose tissue.
We show that LXR/RXR heterodimers regulate apoE transcription
directly, through interaction with a conserved LXR response element
present in both ME.1 and ME.2. Moreover, we demonstrate that the
ability of oxysterols and synthetic ligands to regulate apoE expression
in adipose tissue and peritoneal macrophages is reduced in
Lxrα
−/− or
Lxrβ
−/− mice and
abolished in double knockouts. Basal expression of apoE is not
compromised in
Lxr
null mice, however, indicating that
LXRs mediate lipid-inducible rather than tissue-specific expression of
this gene. Together with our previous work, these findings support a
central role for LXR signaling pathways in the control of macrophage
cholesterol efflux through the coordinate regulation of apoE, ABCA1,
and ABCG1 expression.
Interaction of Gα12 and Gα13 with the cytoplasmic domain of cadherin provides a mechanism for β-catenin release
Authors: Meigs Thomas E. , Fields Timothy A. , McKee David D. , Casey Patrick J.
Published: July 2012

The G12 subfamily of heterotrimeric G proteins, comprised of the
α-subunits Gα12 and Gα13, has been implicated as a signaling
component in cellular processes ranging from cytoskeletal changes to
cell growth and oncogenesis. In an attempt to elucidate specific roles
of this subfamily in cell regulation, we sought to identify molecular
targets of Gα12. Here we show a specific interaction between the G12
subfamily and the cytoplasmic tails of several members of the cadherin
family of cell-surface adhesion proteins. Gα12 or Gα13 binding
causes dissociation of the transcriptional activator β-catenin from
cadherins. Furthermore, in cells lacking the adenomatous polyposis coli
protein required for β-catenin degradation, expression of
mutationally activated Gα12 or Gα13 causes an increase in
β-catenin-mediated transcriptional activation. These findings provide
a potential molecular mechanism for the previously reported cellular
transforming ability of the G12 subfamily and reveal a link between
heterotrimeric G proteins and cellular processes controlling growth and
differentiation.
Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects
Authors: Nishigori Hidekazu , Tomura Hideaki , Tonooka Naoko , Kanamori Masao , Yamada Shirou , Sho Kimie , Inoue Ituro , Kikuchi Nobuyuki , Onigata Kazumichi , Kojima Itaru , Kohama Tomoko , Yamagata Kazuya , Yang Qin , Matsuzawa Yuji , Miki Takashi , Seino Susumu , Kim Mi-Young , Choi Hueng-Sik , Lee Yoon-Kwang , Moore David D. , Takeda Jun
Published: July 2012

Mutations in several genes encoding transcription factors of
the hepatocyte nuclear factor (HNF) cascade are associated with
maturity-onset diabetes of the young (MODY), a monogenic form of
early-onset diabetes mellitus. The ability of the orphan nuclear
receptor small heterodimer partner (SHP, NR0B2) to modulate the
transcriptional activity of MODY1 protein, the nuclear receptor
HNF-4Î±, suggested SHP as a candidate MODY gene. We screened 173
unrelated Japanese subjects with early-onset diabetes for mutations in
this gene and found five different mutations (H53fsdel10,
L98fsdel9insAC, R34X, A195S, and R213C) in 6 subjects as well as one
apparent polymorphism (R216H), all present in the heterozygous state.
Interestingly, all of the subjects with the mutations were mildly or
moderately obese at onset of diabetes, and analysis of the lineages of
these individuals indicated that the SHP mutations were associated with
obesity rather than with diabetes. Therefore, an additional group of
101 unrelated nondiabetic subjects with early-onset obesity was
screened for mutations in the SHP gene. Two of the previously observed
mutations (R34X and A195S) and two additional mutations (R57W and
G189E) were identified in 6 subjects, whereas no mutations were
identified in 116 young nondiabetic lean controls
(
P
= 0.0094). Functional studies of the mutant
proteins show that the mutations result in the loss of SHP
activity. These results suggest that genetic variation in the SHP gene
contributes to increased body weight and reveal a pathway leading to
this common metabolic disorder in Japanese.
A genome survey indicates a possible susceptibility locus for bipolar disorder on chromosome 22
Authors: Kelsoe John R. , Spence M. Anne , Loetscher Erika , Foguet Montserrat , Sadovnick A. Dessa , Remick Ronald A. , Flodman Pamela , Khristich Jason , Mroczkowski-Parker Zofi , Brown John L. , Masser Diane , Ungerleider Sharon , Rapaport Mark H. , Wishart William L. , Luebbert Hermann
Published: July 2012

Bipolar disorder or manic depressive illness is a major psychiatric
disorder that is characterized by fluctuation between two abnormal mood
states. Mania is accompanied by symptoms of euphoria, irritability, or
excitation, whereas depression is associated with low mood and
decreased motivation and energy. The etiology is currently unknown;
however, numerous family, twin, and adoption studies have argued for a
substantial genetic contribution. We have conducted a genome survey of
bipolar disorder using 443 microsatellite markers in a set of 20
families from the general North American population to identify
possible susceptibility loci. A maximum logarithm of odds score of 3.8
was obtained at D22S278 on 22q. Positive scores were found spanning a
region of nearly 32 centimorgans (cM) on 22q, with a possible secondary
peak at D22S419. Six other chromosomal regions yielded suggestive
evidence for linkage: 3p21, 3q27, 5p15, 10q, 13q31-q34, and 21q22. The
regions on 22q, 13q, and 10q have been implicated in studies of
schizophrenia, suggesting the possible presence of susceptibility genes
common to both disorders.
Reduction in CD8
+
cell noncytotoxic anti-HIV activity in individuals receiving highly active antiretroviral therapy during primary infection
Authors: Stranford Sharon A. , Ong June C. , Martinez-Mariño Beatriz , Busch Michael , Hecht Frederick M. , Kahn James , Levy Jay A.
Published: July 2012

Recent advances in the ability to detect people at the early
stages of HIV infection now permit the initiation of antiretroviral
treatment before the full complement of antiviral immune responses has
evolved. However, the influence of early treatment interventions on the
developing anti-HIV immune response is unknown. This study investigates
the impact of standard highly active antiretroviral therapy (HAART)
during the primary stages of HIV infection on the plasma HIV-1 RNA
level, CD4
+
and CD8
+
lymphocyte counts, and the
CD8
+
cell anti-HIV response. Individuals treated with HAART
within 6 months of infection showed dramatic and rapid reductions in
HIV-1 RNA levels along with modest increases in CD4
+
cell
number and decreases in CD8
+
cell numbers. A significant
reduction in the level of CD8
+
cell noncytotoxic
suppression of HIV replication was observed over time in most
participants receiving HAART. Importantly, those individuals choosing
not to receive therapy maintained low but detectable HIV-1 RNA levels
and showed no reduction in their CD8
+
cell antiviral
response. These results suggest that either continued antigenic
challenge is required to sustain CD8
+
cell-mediated
anti-HIV activity, or that HAART has some inhibitory effect on this
important immunologic function during the early stages of infection.
A peripheral marker for schizophrenia: Increased levels of D
3
dopamine receptor mRNA in blood lymphocytes
Authors: Ilani Tal , Ben-Shachar Dorit , Strous Rael D. , Mazor Marina , Sheinkman Ala , Kotler Moshe , Fuchs Sara
Published: July 2012

Dopamine is a major neurotransmitter in the central nervous system,
and its receptors are associated with a number of neuropathological
disorders such as Parkinson's disease and schizophrenia. Although the
precise pathophysiology of schizophrenia remains unknown, the
dopaminergic hypothesis of the illness assumes that the illness results
from excessive activity at dopamine synapses in the brain. Because, at
present, the diagnosis of schizophrenia relies on descriptive
behavioral and symptomatic information, a peripheral measurable marker
may enable a simpler, more rapid, and more accurate diagnosis and
monitoring. In recent years, human peripheral blood lymphocytes have
been found to express several dopamine receptors (D
3
,
D
4
, and D
5
) by using molecular biology
techniques and binding assays. It has been suggested that these
dopamine receptors found on lymphocytes may reflect receptors found in
the brain. Here we demonstrate a correlation between the D
3
dopamine receptor on lymphocytes and schizophrenia and show a
significant elevation of at least 2-fold in the mRNA level of the
D
3
, but not of the D
4
, dopamine receptor in
schizophrenic patients. This increase is not affected by different
antipsychotic drug treatments (typical or atypical). Moreover,
nonmedicated patients exhibit the same pattern, indicating that this
change is not a result of medical treatment. We propose the
D
3
receptor mRNA on blood lymphocytes as a marker for
identification and followup of schizophrenia.
Serological detection of cutaneous T-cell lymphoma-associated antigens
Authors: Eichmüller Stefan , Usener Dirk , Dummer Reinhard , Stein Angelika , Thiel Daniela , Schadendorf Dirk
Published: July 2012

Cutaneous T-cell lymphomas (CTCL) are a group of skin neoplasms
that originate from T lymphocytes and are difficult to treat in
advanced stages. The present study is aimed at the identification of
tumor-specific antigens from a human testis cDNA library using human
sera known as the SEREX (serological identification of recombinantly
expressed genes) approach. A cDNA library from normal testicle tissue
was prepared and approximately 2 million recombinants were screened
with sera from Sézary Syndrome and Mycosis fungoides patients. A
total of 28 positive clones belonging to 15 different genes/ORFs were
identified, including five hitherto unknown sequences. Whereas control
sera did not react with most clones, 11–71% sera from CTCL patients
were reactive against the identified clones. Expression analysis on 28
normal control and 17 CTCL tissues by reverse transcription–PCR
(RT-PCR) and Northern blotting revealed seven ubiquitously distributed
antigens, six differentially expressed antigens (several normal tissues
were positive), and two tumor-specific antigens that were expressed
only in testis and tumor tissues: (
i
) A SCP-1-like
sequence, which has already been detected in various tumors, has been
found in one CTCL tumor and four sera of CTCL patients reacted with
various SCP-1-like clones and (
ii
) a new sequence named
cTAGE-1 (CTCL-associated antigen 1) was detected in 35% of CTCL tumor
tissues and sera of 6/18 patients reacted with this clone. The
present study unravels CTCL-associated antigens independent of the
T-cell receptor. The SCP-1-like gene and cTAGE-1 were shown to be
immunogenic and immunologically tumor-specific and may therefore be
candidates for immunotherapy targeting CTCL.
Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4
+
T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): Implications for HIV-1 infections of humans
Authors: Igarashi Tatsuhiko , Brown Charles R. , Endo Yasuyuki , Buckler-White Alicia , Plishka Ronald , Bischofberger Norbert , Hirsch Vanessa , Martin Malcolm A.
Published: July 2012

The highly pathogenic simian immunodeficiency virus/HIV
type 1 (SHIV) chimeric virus SHIV
DH12R
induces a systemic
depletion of CD4
+
T lymphocytes in rhesus monkeys during
the initial 3–4 weeks of infection. Nonetheless, high levels of viral
RNA production continue unabated for an additional 2–5 months.
In situ
hybridization and immunohistochemical analyses
revealed that tissue macrophage in the lymph nodes, spleen,
gastrointestinal tract, liver, and kidney sustain high plasma virus
loads in the absence of CD4
+
T cells. Quantitative confocal
immunofluorescence analysis indicated that greater than 95% of the
virus-producing cells in these tissues are macrophage and less than 2%
are T lymphocytes. Interestingly, the administration of a potent
reverse transcriptase inhibitor blocked virus production during the
early T cell phase but not during the later macrophage phase of the
SHIV
DH12R
infection. When interpreted in the context of
HIV-1 infections, these results implicate tissue macrophage as an
important reservoir of virus
in vivo
. They become
infected during the acute infection, gradually increase in number over
time, and can be a major contributor to total body virus burden during
the symptomatic phase of the human infection.
Acid-sensing ion channel 3 matches the acid-gated current in cardiac ischemia-sensing neurons
Authors: Sutherland Stephani P. , Benson Christopher J. , Adelman John P. , McCleskey Edwin W.
Published: July 2012

Cardiac afferents are sensory neurons that mediate angina, pain
that occurs when the heart receives insufficient blood supply for its
metabolic demand (ischemia). These neurons display enormous acid-evoked
depolarizing currents, and they fire action potentials in response to
extracellular acidification that accompanies myocardial ischemia. Here
we show that acid-sensing ion channel 3 (ASIC3), but no other known
acid-sensing ion channel, reproduces the functional features of the
channel that underlies the large acid-evoked current in cardiac
afferents. ASIC3 and the native channel are both especially sensitive
to pH, interact similarly with Ca
2+
, and gate rapidly
between closed, open, and desensitized states. Particularly important
is the ability of ASIC3 and the native channel to open at pH 7, a value
reached in the first few minutes of a heart attack. The steep
activation curve suggests that the channel opens when four protons
bind. We propose that ASIC3, a member of the degenerin channel (of
Caenorhabditis elegans
)/epithelial sodium channel
family of ion channels, is the sensor of myocardial acidity that
triggers cardiac pain, and that it might be a useful pharmaceutical
target for treating angina.
Shaking up glycolysis: Sustained, high lactate flux during aerobic rattling
Authors: Kemper William F. , Lindstedt Stan L. , Hartzler Lynn K. , Hicks James W. , Conley Kevin E.
Published: July 2012

Substantial ATP supply by glycolysis is thought to reflect cellular
anoxia in vertebrate muscle. An alternative hypothesis is that the
lactate generated during contraction reflects sustained glycolytic ATP
supply under well-oxygenated conditions. We distinguished these
hypotheses by comparing intracellular glycolysis during anoxia to
lactate efflux from muscle during sustained, aerobic contractions. We
examined the tailshaker muscle of the rattlesnake because of its
uniform cell properties, exclusive blood circulation, and ability to
sustain rattling for prolonged periods. Here we show that glycolysis is
independent of the O
2
level and supplies one-third of the
high ATP demands of sustained tailshaking. Fatigue is avoided by rapid
H
+
and lactate efflux resulting from blood flow rates that
are among the highest reported for vertebrate muscle. These results
reject the hypothesis that glycolysis necessarily reflects cellular
anoxia. Instead, they demonstrate that glycolysis can provide a high
and sustainable supply of ATP along with oxidative phosphorylation
without muscle fatigue.
Myoglobin: A scavenger of bioactive NO
Authors: Flögel Ulrich , Merx Marc W. , Gödecke Axel , Decking Ulrich K. M. , Schrader Jürgen
Published: July 2012

The present study explored the role of myoglobin (Mb) in cardiac NO
homeostasis and its functional relevance by employing isolated hearts
of wild-type (WT) and myoglobin knockout mice.
1
H NMR
spectroscopy was used to measure directly the conversion of oxygenated
Mb (MbO
2
) to metmyoglobin (metMb) by reaction with NO. NO
was applied intracoronarily (5 nM to 25 μM), or its
endogenous production was stimulated with bradykinin (Bk;
10 nM to 2 μM). We found that infusion of authentic NO solutions
dose-dependently (≥ 2.5 μM NO) increased metMb formation in WT
hearts that was rapidly reversible on cessation of NO infusion.
Likewise, Bk-induced release of NO was associated with significant
metMb formation in the WT (≥1 μM Bk). Hearts lacking Mb reacted more
sensitively to infused NO in that vasodilatation and the
cardiodepressant actions of NO were more pronounced. Similar results
were obtained with Bk. The lower sensitivity of WT hearts to changes in
NO concentration fits well with the hypothesis that in the presence of
Mb, a continuous degradation of NO takes place by reaction of
MbO
2
+ NO to metMb + NO
3
−
, thereby
effectively reducing cytosolic NO concentration. This breakdown
protects myocytic cytochromes against transient rises in cytosolic NO.
Regeneration of metMb by metMb reductase to Mb and subsequent
association with O
2
leads to reformation of
MbO
2
available for another NO degradation cycle. Our data
indicate that this cycle is crucial in the breakdown of NO and
substantially determines the dose–response curve of the NO effects on
coronary blood flow and cardiac contractility.
Essential role of the small GTPase Rac in disease resistance of rice
Authors: Ono Eiichiro , Wong Hann Ling , Kawasaki Tsutomu , Hasegawa Morifumi , Kodama Osamu , Shimamoto Ko
Published: July 2012

Production of reactive oxygen intermediates (ROI) and a form of
programmed cell death called hypersensitive response (HR) are often
associated with disease resistance of plants. We have previously shown
that the Rac homolog of rice, OsRac1, is a regulator of ROI production
and cell death in rice. Here we show that the constitutively active
OsRac1 (
i
) causes HR-like responses and greatly reduces
disease lesions against a virulent race of the rice blast fungus;
(
ii
) causes resistance against a virulent race of
bacterial blight; and (
iii
) causes enhanced production
of a phytoalexin and alters expression of defense-related genes. The
dominant-negative OsRac1 suppresses elicitor-induced ROI production in
transgenic cell cultures, and in plants suppresses the HR induced by
the avirulent race of the fungus. Taken together, our findings strongly
suggest that OsRac1 has a general role in disease resistance of rice.
Proteins associated with the promyelocytic leukemia gene product (PML)-containing nuclear body move to the nucleolus upon inhibition of proteasome-dependent protein degradation
Authors: Mattsson Karin , Pokrovskaja Katja , Kiss Csaba , Klein George , Szekely Laszlo
Published: July 2012

Several recent findings have indicated that the promyelocytic
leukemia gene product (PML) oncogenic domains (PODs) are involved in
proteasome-mediated degradation of ubiquitinated proteins. We wanted to
examine the intracellular distribution of PML protein in the presence
of a proteasome inhibitor. We used high-resolution microscopy to study
the distribution of PML protein and other POD-associated proteins along
with the proteasomes themselves under normal conditions and in cells
treated with the proteasome inhibitor, MG132. Inhibition of the
proteasomes in MCF-7, HeLa, and IB-4 cell lines resulted in a radical
redistribution of the POD-associated proteins PML, Sp100, and SUMO-1.
After 6–10 h of MG132 treatment, PML, Sp100, and SUMO-1 were no longer
detectable in the PODs and accumulated mainly in the nucleolus.
Moreover, MG132 treatment changed the cellular distribution of the
proteasomes. Interestingly, this included the accumulation in
euchromatin areas of the nucleus and within the nucleoli. Several
non-POD-associated proteins did not change their cellular distribution
under the same conditions. The accumulation of POD-associated proteins
and proteasomes in the nucleoli of MG132-treated cells indicates that
these proteins may target the nucleoli under normal conditions and that
the nucleolus may have a function in the regulation of proteasomal
protein degradation.
Interaction of endostatin with integrins implicated in angiogenesis
Authors: Rehn Marko , Veikkola Tanja , Kukk-Valdre Eola , Nakamura Hitoshi , Ilmonen Maritta , Lombardo Christian R. , Pihlajaniemi Taina , Alitalo Kari , Vuori Kristiina
Published: July 2012

Endostatin, a fragment of collagen XVIII, is a potent antagonist of
angiogenesis and inhibitor of tumor growth in mouse models. At present,
the mechanism of action of endostatin is unknown. We show here that
recombinantly produced human endostatin interacts with
α
5
- and α
v
-integrins on the surface of
human endothelial cells. We further demonstrate that the
endostatin–integrin interaction is of functional significance
in vitro
, as we found that immobilized endostatin
supports endothelial cell survival and migration in an
integrin-dependent manner. Soluble endostatin in turn inhibits
integrin-dependent endothelial cell functions, such as cell migration.
Taken together, these results implicate integrins as potential targets
for endostatin function and support the importance of integrins in
endothelial cell biology and angiogenesis.
p53 accumulates but is functionally impaired when DNA synthesis is blocked
Authors: Gottifredi Vanesa , Shieh Sheau-Yann , Taya Yoichi , Prives Carol
Published: July 2012

p53 is required for the induction of a G
1
and/or
G
2
irreversible arrest after γ irradiation (IR), whereas
blocked DNA replication causes a p53-independent S-phase arrest. We
have examined the response to p53 when DNA synthesis is blocked by
hydroxyurea (HU) or aphidicolin or when DNA is damaged by γ IR.
Similarly to γ IR, blocked DNA synthesis induces high levels of
phosphorylated nuclear p53. Surprisingly, several (but not all) p53
transcriptional targets that are rapidly induced by γ IR are weakly
or not induced when DNA replication is blocked. Moreover, the p53
response to γ IR is inhibited by pretreatment of cells with HU or
aphidicolin, suggesting that blocked DNA replication prevents p53 from
being fully active as a transcription factor. HU-induced stabilization
of p53 neither requires functional ATM (ataxia telangiectasia mutated),
nor interferes with the γ IR-dependent activation of the ATM kinase.
Thus, stalled replication forks activate kinases that modify and
stabilize p53, yet act downstream of ATM to impair p53 transcriptional
activity. The ramifications of this novel regulation of p53 are
discussed.
Failure of ductus arteriosus closure and remodeling in neonatal mice deficient in cyclooxygenase-1 and cyclooxygenase-2
Authors: Loftin Charles D. , Trivedi Darshini B. , Tiano Howard F. , Clark James A. , Lee Christopher A. , Epstein Jonathan A. , Morham Scott G. , Breyer Matthew D. , Nguyen MyTrang , Hawkins Beau M. , Goulet Jennifer L. , Smithies Oliver , Koller Beverly H. , Langenbach Robert
Published: July 2012

The transition to pulmonary respiration following birth requires
rapid alterations in the structure of the mammalian cardiovascular
system. One dramatic change that occurs is the closure and remodeling
of the ductus arteriosus (DA), an arterial connection in the fetus that
directs blood flow away from the pulmonary circulation. A role for
prostaglandins in regulating the closure of this vessel has been
supported by pharmacological and genetic studies. The production of
prostaglandins is dependent on two cyclooxygenases (COX-1 and COX-2),
which are encoded by separate genes. We report here that the absence of
either or both COX isoforms in mice does not result in premature
closure of the DA
in utero
. However, 35% of
COX-2(−/−) mice die with a patent DA within
48 h of birth. In contrast, the absence of only the COX-1 isoform
does not affect closure of the DA. The mortality (35%) and patent DA
incidence due to absence of COX-2 is, however, significantly increased
(79%) when one copy of the gene encoding COX-1 is also inactivated.
Furthermore, 100% of the mice deficient in both isoforms die with a
patent DA within 12 h of birth, indicating that in COX-2-deficient
mice, the contribution of COX-1 to DA closure is gene dosage-dependent.
Together, these data establish roles for COX-1, and especially for
COX-2, in the transition of the cardiopulmonary circulation at birth.
PDX:PBX complexes are required for normal proliferation of pancreatic cells during development
Authors: Dutta Sanjoy , Gannon Maureen , Peers Bernard , Wright Chris , Bonner-Weir Susan , Montminy Marc
Published: July 2012

The homeobox factor PDX-1 is a key regulator of pancreatic
morphogenesis and glucose homeostasis; targeted disruption of the PDX-1
gene leads to pancreatic agenesis in
pdx-1(−/−)
homozygotes.
Pdx-1
heterozygotes develop normally, but
they display glucose intolerance in adulthood. Like certain other
homeobox proteins, PDX-1 contains a consensus FPWMK motif that promotes
heterodimer formation with the ubiquitous homeodomain protein PBX. To
evaluate the importance of PDX-1:PBX complexes in pancreatic
morphogenesis and glucose homeostasis, we expressed either wild-type or
PBX interaction defective PDX-1 transgenes under control of the PDX-1
promoter. Both wild-type and mutant PDX-1 transgenes corrected glucose
intolerance in
pdx-1
heterozygotes. The wild-type PDX-1
transgene rescued the development of all pancreatic lineages in
pdx-1
(
−/−
) animals, and these mice
survived to adulthood. In contrast, pancreata from
pdx-1(−/−)
mice expressing the mutant PDX-1
transgene were hypoplastic, and these mice died within 3 weeks of birth
from pancreatic insufficiency. All pancreatic cell types were observed
in
pdx-1(−/−)
mice expressing the mutant PDX-1
transgene; but the islets were smaller, and increased numbers of islet
hormone-positive cells were noted within the ductal epithelium. These
results indicate that PDX-1:PBX complexes are dispensable for glucose
homeostasis and for differentiation of stem cells into ductal,
endocrine, and acinar lineages; but they are essential for expansion of
these populations during development.
Extrafloral nectar production of the ant-associated plant,Macaranga tanarius,is an induced, indirect, defensive response elicited by jasmonic acid
Authors: Heil Martin , Koch Thomas , Hilpert Andrea , Fiala Brigitte , Boland Wilhelm , Linsenmair K. Eduard
Published: July 2012

Plant species in at least 66 families produce extrafloral nectar (EFN) on their leaves or shoots and therewith attract predators and parasitoids, such as ants and wasps, which in turn defend them against herbivores. We investigated whether EFN secretion is induced by herbivory and/or artificial damage, and thus can be regarded as an induced defensive response. In addition, we studied the underlying signaling pathway. EFN secretion by field-grownMacaranga tanariusincreased after herbivory, artificial leaf damage, and exogenous jasmonic acid (JA) application. Artificial damage strongly enhanced endogenous JA concentrations. The response in EFN production to artificial damage was much less pronounced in those leaves that were treated with phenidone to inhibit endogenous JA synthesis. Quantitative dose–response relations were found between the increase in nectar production and both the intensity of leaf damage and the amounts of exogenously applied JA. The amount of endogenously produced JA was positively correlated with the intensity of leaf damage. Increased numbers of defending insects and decreased numbers of herbivores were observed on leaves after inducing EFN production by exogenous JA treatment. Over 6 weeks, repeatedly applied JA or artificial damage resulted in a ten-fold reduction in herbivory. These results demonstrate that EFN production represents an alternative mechanism for induced, indirect defensive plant responses that are mediated via the octadecanoid signal transduction cascade.
Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca
2+
-permeable nonselective cation channel
Authors: González-Perrett Silvia , Kim Keetae , Ibarra Cristina , Damiano Alicia E. , Zotta Elsa , Batelli Marisa , Harris Peter C. , Reisin Ignacio L. , Arnaout M. Amin , Cantiello Horacio F.
Published: July 2012

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of
unknown function, is a major cause of autosomal dominant polycystic
kidney disease (ADPKD), whose manifestation entails the development of
fluid-filled cysts in target organs. Here, we demonstrate that
polycystin-2 is present in term human syncytiotrophoblast, where it
behaves as a nonselective cation channel. Lipid bilayer reconstitution
of polycystin-2-positive human syncytiotrophoblast apical membranes
displayed a nonselective cation channel with multiple subconductance
states, and a high perm-selectivity to Ca
2+
. This channel
was inhibited by anti-polycystin-2 antibody, Ca
2+
,
La
3+
, Gd
3+
, and the diuretic amiloride. Channel
function by polycystin-2 was confirmed by patch-clamping experiments of
polycystin-2 heterologously infected Sf9 insect cells. Further,
purified insect cell-derived recombinant polycystin-2 and
in
vitro
translated human polycystin-2 had similar ion channel
activity. The polycystin-2 channel may be associated with fluid
accumulation and/or ion transport regulation in target epithelia,
including placenta. Dysregulation of this channel provides a mechanism
for the onset and progression of ADPKD.
Polymer-based gene delivery with low cytotoxicity by a unique balance of side-chain termini
Authors: Putnam David , Gentry Christine A. , Pack Daniel W. , Langer Robert
Published: July 2012

Protein expression after delivery of plasmid DNA to the cell nucleus depends on the processes of transcription and translation. Cytotoxic gene-delivery systems may compromise these processes and limit protein expression. This situation is perhaps most prevalent in current nonviral polycationic gene-delivery systems in which the polycationic nature of the delivery system can lead to cytotoxicity. To approach the problem of creating nontoxic but effective gene-delivery systems, we hypothesized that by optimizing the balance between polymer cationic density with endosomal escape moieties, effective gene transfer with low cytotoxicity could be created. As a model system, we synthesized a series of polymers whose side-chain termini varied with respect to the balance of cationic centers and endosomal escape moieties. Specifically, by polymer-analogous amidation we conjugated imidazole groups to the ɛ-amines of polylysine in varying mole ratios (73.5 mol % imidazole, 82.5 mol % imidazole, and 86.5 mol % imidazole). The primary ɛ-amine terminus of polylysine served as a model for the cationic centers, whereas the imidazole groups served as a model for the endosomal escape moieties. These polymers condensed plasmid DNA into nanostructures <150 nm and possessed little cytotoxicityin vitro. Transfection efficiency, as measured by luciferase protein expression, increased with increasing imidazole content of the polymers in a nonlinear relationship. The polymer with the highest imidazole content (86.5 mol %) mediated the highest protein expression, with levels equal to those mediated by polyethylenimine, but with little to no cytotoxicity.
Complete switch from Mdm2 to human papillomavirus E6-mediated degradation of p53 in cervical cancer cells
Authors: Hengstermann Arnd , Linares Laetitia K. , Ciechanover Aaron , Whitaker Noel J. , Scheffner Martin
Published: July 2012

The E6 oncoprotein of human papillomaviruses (HPVs) that are
associated with cervical cancer utilizes the cellular
ubiquitin–protein ligase E6-AP to target the tumor suppressor p53 for
degradation. In normal cells (i.e., in the absence of E6), p53 is also
a target of the ubiquitin–proteasome pathway. Under these conditions,
however, p53 degradation is mediated by Mdm2 rather than by E6-AP. Here
we show in a mutational analysis that, surprisingly, the structural
requirements of p53 to serve as a proteolytic substrate differ between
E6 proteins derived from different HPV types and, as expected, between
Mdm2 and E6 proteins
in vitro
and
in
vivo
. Stable expression of such mutants in HPV-negative and
HPV-positive cell lines demonstrates that in HPV-positive cancer cells,
the E6-dependent pathway of p53 degradation is not only active but,
moreover, is required for degradation of p53, whereas the
Mdm2-dependent pathway is inactive. Because the p53 pathway was
reported to be functional in HPV-positive cancer cells, this finding
indicates clearly that the ability of the E6 oncoprotein to target p53
for degradation is required for the growth of HPV-positive cancer
cells.
Odor-evoked calcium signals in dendrites of rat mitral cells
Authors: Charpak Serge , Mertz Jerome , Beaurepaire Emmanuel , Moreaux Laurent , Delaney Kerry
Published: July 2012

Mitral cell dendrites do more than passively integrate and convey
synaptic potentials to the soma, they release transmitter onto local
interneurones to mediate recurrent and lateral inhibition. Several
mechanisms may control the level of dendritic intracellular calcium
([Ca
2+
]) and define timing for dendritic release. Here we
investigated
in vivo
, how odor controls calcium dynamics
in mitral cell dendrites by combining intracellular recording and
two-photon microscopy imaging of [Ca
2+
]. During odor
stimulation, two types of [Ca
2+
] changes accompany
membrane potential oscillations that are phase-locked with the
respiratory cycle: (
i
) one is graded and parallels the
membrane potential, even below the threshold for action potential
firing; (
ii
) a second is transient, triggered by sodium
action potentials that invade the entire dendritic tree. These results
indicate that mitral cell dendritic compartments are synchronized by
action potentials and suggest that the efficacy of dendritic synapses
is finely tuned by odor-evoked graded changes in [Ca
2+
].
Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method
Authors: Wilson Gary G. , Karlin Arthur
Published: July 2012

The nicotinic acetylcholine (ACh) receptors cycle among classes of
nonconducting resting states, conducting open states, and nonconducting
desensitized states. We previously probed the structure of the
mouse-muscle ACh receptor channel in the resting state obtained in the
absence of agonist and in the open states obtained after brief exposure
to ACh. We now have probed the structure in the stable desensitized
state obtained after many minutes of exposure to ACh. Muscle-type
receptor has the subunit composition α
2
βγδ. Each
subunit has four membrane-spanning segments, M1–M4. The channel lumen
in the membrane domain is lined largely by M2 and to a lesser extent by
M1 from each of the subunits. We determined the rates of reaction of a
small, sulfhydryl-specific, charged reagent, 2-aminoethyl
methanethiosulfonate with cysteines substituted for residues in αM2
and the αM1–M2 loop in the desensitized state and compared these
rates to rates previously obtained in the resting and open states. The
reaction rates of the substituted cysteines are different in the three
functional states of the receptor, indicating significant structural
differences. By comparing the rates of reaction of extracellularly and
intracellularly added 2-aminoethyl methanethiosulfonate, we previously
located the closed gate in the resting state between αG240 and
αT244, in the predicted M1–M2 loop at the intracellular end of M2.
Now, we have located the closed gate in the stable desensitized state
between αG240 and αL251. The gate in the desensitized state
includes the resting state gate and an extension further into M2.
Dopamine-dependent synaptic plasticity in striatum during
in vivo
development
Authors: Tang Ka-Choi , Low Malcolm J. , Grandy David K. , Lovinger David M.
Published: July 2012

The neurotransmitters dopamine (DA) and glutamate in the striatum
play key roles in movement and cognition, and they are implicated in
disorders of the basal ganglia such as Parkinson's disease. Excitatory
synapses in striatum undergo a form of developmental plasticity
characterized by a decrease in glutamate release probability. Here we
demonstrate that this form of synaptic plasticity is DA and DA D2
receptor dependent. Analysis of spontaneous synaptic responses
indicates that a presynaptic mechanism involving inhibition of
neurotransmitter release underlies the developmental plasticity. We
suggest that a major role of DA in the striatum is to initiate
mechanisms that regulate the efficacy of excitatory striatal synapses,
producing a decrease in glutamate release.
Contribution of cytoskeleton to the internalization of AMPA receptors
Authors: Zhou Qiang , Xiao Min-Yi , Nicoll Roger A.
Published: July 2012

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) receptors (AMPARs) at synapses has been suggested to play
an important role in the expression of synaptic plasticity. Both the
regulated and the constitutive trafficking of synaptic AMPARs are
thought to involve the insertion and removal of receptors by means of
an exocytotic and endocytotic process, respectively. In contrast,
N
-methyl-
d
-aspartate (NMDA) receptors
(NMDARs), which are colocalized with AMPARs at excitatory synapses,
appear to be much less dynamic. Here, we present evidence supporting
the idea that synaptic AMPARs turn over through a constitutive
endocytotic process and that glutamate application greatly enhances
this turnover of AMPARs. The glutamate-induced internalization of
AMPARs requires a rise in postsynaptic Ca
2+
. The AMPAR
internalization is mimicked by latrunculin A, a drug that selectively
depolymerizes actin and is blocked by jasplakinolide, a drug which
stabilizes actin filaments. The rate of endocytosis is not altered by
glutamate application, whereas a clear enhancement is observed with
insulin application. We propose a model in which the glutamate-induced
dissociation of AMPARs from their anchor on the postsynaptic membrane
involves actin depolymerization, which allows the released AMPARs to
segregate from the NMDARs and diffuse to a presumably perisynaptic
site, where they become available to an endocytotic machinery and
are selectively internalized.
Control of eye movements and spatial attention
Authors: Moore Tirin , Fallah Mazyar
Published: July 2012

Several lines of evidence suggest that planning eye movements and
directing visuospatial attention share overlapping brain mechanisms.
This study tested whether spatial attention can be enhanced by altering
oculomotor signals within the brain. Monkeys performed a spatial
attention task while neurons within the frontal eye field, an
oculomotor area within prefrontal cortex, were electrically stimulated
below the level at which eye movements are evoked. We found that we
could improve the monkey's performance with microstimulation when, but
only when, the object to be attended was positioned in the space
represented by the cortical stimulation site.
Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor
Authors: Yao Jia-Long , Dong Yi-Hu , Morris Bret A. M.
Published: July 2012

Fruit development in higher plants normally requires pollination and fertilization to stimulate cell division of specific floral tissues. In some cases, parthenocarpic fruit development proceeds without either pollination or fertilization. Parthenocarpic fruit without seed has higher commercial value than seeded fruit. Several apple (Malus domestica) mutants (Rae Ime, Spencer Seedless and Wellington Bloomless) are known to produce only apetalous flowers that readily go on to develop into parthenocarpic fruit. Through genetics, a single recessive gene has been identified to control this trait in apple. Flower phenotypes of these apple mutants are strikingly similar to those of theArabidopsismutantpistillata(pi), which produces flowers where petals are transformed to sepals and stamens to carpels. In this study, we have cloned the applePIhomolog (MdPI) that shows 64% amino acid sequence identity and closely conserved intron positions and mRNA expression patterns to theArabidopsis PI. We have identified that in the apetalous mutantsMdPIhas been mutated by a retrotransposon insertion in intron 4 in the case of Rae Ime and in intron 6 in the case of Spencer Seedless and Wellington Bloomless. The insertion apparently abolishes the normal expression of theMdPIgene. We conclude that the loss of function mutation in theMdPIMADS-box transcription factor confers parthenocarpic fruit development in these apple varieties and demonstrates another function for the MADS- box gene family. The knowledge generated here could be used to produce parthenocarpic fruit cultivars through genetic engineering.